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SEQUENTLAL CEIANGES IN SYNAPTIC STRUCTURE FOLLOWING
LONG-TERM POTENTIATION IN THE RAT DENTATE CYRUS
Andrew Charles Watson Weeks
A Thesis submitted in conformity with the requirements for the Degree of
Doctor of Philosophy, Graduate Department of Psychology
at the University of Toronto
O Copyright by Andrew C.W. Weeks, 2000
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SEQUENTIAL CHANGES LN SYNAPTIC STRUCTURE FOLLOWING
LONGTERM POTENTLATION IN T m RAT DENTATE GYRUS
Andrew Charles Watson Weeks
A Thesis submitted in conformity with the requirements for the Degree of Doctor of
Philosophy, Graduate Departrnent of Psychology at the University of Toronto
O Copyright by Andrew C.W. Weeks, 2000
ABSTRACT
Long-terni potentiation (LTP) continues to be one of the most compelling models of learning and memory. While LTP has been shown to be associated with changes in synaptic morphology, the nature of these changes over the electrophysiological time course of the enhanced response, has not been determined. The current research involved a detailed electronmicroscopic examination of synaptic structure in the rat dentate gyms at various time points following the induction of LTP.
The total number of synapses per neuron, synaptic curvature, the proportion of perforated synapses, and the maximum length of synapses were examined. No overall change in the number of synapses per neuron was observed in the LTP tissue at any of the time intervals. At 24 h, however, the degree of LTP expressed was associated with the number of synapses per neuron. in the LTP animals, the proportion of irregular shaped synapses was increased at 1 h, concave shaped synapses were increased at 24 h (not apparent under phannacological blockade), and no overall changes in shape were observed at 5 days. The proportion of perforated synapses was increased at 1 h but did not differ h m controls at later time periods. The increase in perforated synapses at 1 h was particularly apparent in the proportion of concave perforated synapses and these concave perforated synapses were also more prevalent at 24 h and 5 days. Synapses were larger overall at 1 h and 5 âays but not different at 24 h. These differences in length were particularly evident in concave shaped synapses which were longer at 1 h, shorter at 24 h (no change under phannacological blockade), and longer at 5 days.
Stimulateà synapses appear to initially lengthen and become more perforated (1 b), then become more concave in shape and divide or form new smailer concave synapses (24 h). Finally, synapses appear to grow in length and a higher proportion of concave perforated synapses is maintaiad (5 days). These results describe a sequence of changes in synaptic morphology that accompany and rnay support the neural plasticity that underlies LTP in a structure tbat is associated with leaming and memory.
ACKNOWEDGMENTS
Many individuais have contributed to the work described in the following dissertation
and to my academic success in general. 1 welcome this opportunity to acknowledge their
invaluable assistance. During every stage of this mearch project Dr. Ted Petit has provided
guidance and support buhiore h&xtantly he has allowed me to grow as a Neuroscientist by
providing the opportunity for me to take on a leadership role in the laboratory. By treating
me more as a colleague than a student, Dr. Petit has demonstrated his genuine interest in the
professional development of his students which extends beyond the laboratory. 1 would also
like to thank Dr. Robert McDonald and Dr. Alison Fleming for their guidance and
suggestions throughout the research process and the writing of this dissertation.
Another individual to whorn 1 am truly gratefùl is Ms. Janelle LeBoutillier. Janelle
has always taken that extra step to support the development of my laboratory skills. Her
patience and quiet guidance have helped me get through the day to day trials inhercnt to the
laboratory setting. 1 will always value her friendship. 1 am also indebted to Ramond Or,
Lucy Pickering, Brenda Brown, Dr. Colin MacLeod and countless others at the University of
Toronto at Scarborough..
To my parents Dr. Ronald Weeks and Mrs. Barbara Weeks, your love and support
dwing these past several years and throughout m y entire litè have helped me complete this
degree and have truiy made me who I am. While it is impossible for me to thank you
properly here, 1 want you to always know how much 1 appreciate everything you have done
and how much you both mean to me. To my new parents MY. Vasco Benevides and Mrs.
Lucia Benevides you have added so much to my life and I thank you for al1 of your support.
Finally and most importantly, 1 would like to thank my wife Tina. Without your
limitless love, patience and sacrifice 1 wouid not have been able to fulfill my d.rea.cn of
attaining a graduate education. From the numerous brain storming, proof reading, and
editing sessions to the many words of encouragement, your support has been tremendous. 1
look forward to spending the rest of my life with you as we continue to pursue both of our
drearns together.
iii
................................................................................................................. GENERAL INTRODUCTIOS.. 1
ROLE OF THE HIPPOCAMPUS IN LEAWP~NG AND MEMORY ............................................................................... 2 ... JI; ....................................................................................... LEARNING AND MEMORY: PHYS~OLOGICAL MODELS 3 .................................................................................................................................. Other Leorning Models 3 ............................................................................................................................. LONGTERM POTEN~ATION 4
........................................................................ LONGTERM POTENTIATION: ELE~PHYSIOLOGICAL ISSUES 5 .......................................................................................... CELLULAR AND MOLECULAR MECHAN~SMS OF LTP 7
............................................................................................................................................ induction of LTP 7 MAINTENANCE OF LTP ................................................................................................................................ 10
Conclusions ................................................................................................................................................. 14 SYNAPTIC STRUCTURAL CHANGES ASSOCIATED WITH LTP ............................................................................. 14
...................................................................................................................................... Synaptic Curvahwe I5 Denhitic Spines .......................................................................................................................................... 16 Spinules ........................................................................................................................................................ I8 Perfoated Synopses .................................................................................................................................... 18 Length. Area (and Volume) ofJLnuptic Elements ....................................................................................... 20
WEARCH GOALS AND RATIONALE ................................................................................................................. 22
....................................................................................................................... GENERAL METHODOLOGY 23
ANIMALS ..................................................................................................................................................... 24 SURGERY .......................................................................................................................................................... 24 ST~MULATION AND RECORDING ....................................................................................................................... 25
....................................................................................................................................... TISSUE PREPARATION 26 TrSsuE SECTIONING ........................................................................................................................................ 26 ELECTRON MlCROSCOPY .................................................................................................................................. 27
.......................................................................................................................................... S Y N A ~ C NUMBER 28 SYNAPTfC STRUmRE .................................................................................................................................... 28 STATISTICAL ANALYSIS OF STRUCTURAL PROFILE .......................................................................................... 29 CORRELA~ONAL ANALYSIS ......................................................................................................................... 30
................................................ .......................... GENERAL ELECTROPHYSIOLOGICAL RESULTS .. 30
...................................... SYNAPTIC STRUCTURAL PROFILE AT 1 HOUR POST-LTP INDUCTION 31
~NTRODUCflON ................................................................................................................................................. 3I .............................................................................................................................. MATERIALS AND METHODS 31
Animals ........................................................................................................................................................ 31 Stimulation ................................................................................................................................................... 32
RESULTS ...............................+................I. ....................................................................... 32 ......................................................................................................................................... SUnqptic Nunrbe K 32
Synaptic Cwyame ...................................................................................................................................... 32 *-tic Perjio~cations .................................................................................................................................. 33
.......................................................................................................................................... Synoptic Length 34 ................................................................................................................................. Correlational Analysis 35
Drscussio~ ...................................................................................................................................................... 46 ................................................................................................................... Methodological Consi&rations 46
............................................... ................................... Synaptic Number , 47 ............................................................. ............................ Synaptic Curvature .. 47
Synaptic Perforations .................................................................................................................................. 49 Length of Synapses .................... ..., ........................................................................................................... 49 CorteIutional Analysis ................................................................................................................................. 50
Dynamic Interactions .................................................................................................................................. 50 Functional Relevawe of Observed Strucn~al Changes.. ........................................................................ 52
.................................................................. Conclusions: Synaptic Remodefling at I h Post-LTP Indrrction 53
.... ............... SYNAPTIC STRUCTURAL PROFILE AT 24 HOUR POST-LTP INDUCTION ......... ......... 54
INTRODUC~ON ................................................................................................................................................. 54 ... .............................................................................................................................. MATERIALS AND M ~ O D S 55 .. - . .C . ' Animah ................... ........................ .......................................................................................................... 55
................................................................................................................................................... Stimulation 55 RESULTS ........................................................................................................................................................... 55
.......................................................................................................................................... Synaptic Number 55 Synaptic Curvature ...................................................................................................................................... 56 Syruptic Pcrrforu f ions .................................................................................................................................. 56
........................................................................................................................ ................ Synaptic Lengths .. 57 ................................................................................................................................. Correlational Anafysis 58
...................................................................................................................................................... D~scussro~ 67 Methodological Considerations ................................................................................................................. 67 Synaptic Nurn ber ......................................-................................................................................................... 68 Symptic Curvature ..................................................................................................................................... 69 *naptic Per/ortions .................................................................................................................................. 69 Length ofSynapses ........................................~.........~.................................................................................. 70
................................................................................................................................. Correlational Analysis 71 Dynamic Interactions ................................................................................................................................... 72 Co~~:lusions: Synaptic Remodelling at 24 h Post-LTP Induction ................................................................ 72
SYNAPTIC STRUCTURAL PROFILE AT 24 HOUR POST-LTP INDUCTION UNDER KETAMINE ........................................................................................................... PIIARMACOLOCICAL BLOCKADE 73
................................................................................................................................................. INTRODUCTION 73 .............................................................................................................................. MATERIALS AND METHODS 73
....................................................................................................................................................... Animals 73 Stimulation ................................................................................................................................................... 74
........................................................................................................................................................... ~ U L - r s 74 Electrophysiologicai Results ......................... ... ...................................................................................... 74
......................................................................................................................................... S y ~ p t i c Number 74 ...................................................................................................................................... Synaptic Cumal~re 75
.................................................................................................................................. Synaptic Pe florafions 75 .......................................................................................................................................... Synaptic Lengths 75
DWJSS~ON ........................................~...............................-............................................................................. 81 Methodofogicul Cowi&rations ..............~.................................................................................................... 81 Synaptic Number .................................................................-..................................................................... 82 Synaptic Curvature ..........~...........................~............................................................................................... 82 Synaptic Perforations .............................................................................................................................. 83 Length of Synapses ............................~......~.................................................................................................. 83 C01y:lwions: LTPSpeciflc versus Genera! Stirnufation Eflects ................................................................. 84
SYNAPTIC STRUCTURAL PROFlLE AT S DAYS POST-LTP INDUCTION ....ee.....,....,...e.....e.....e..... 85
I M R O D U a O N ................................................................................................................................................. 85 MATERIALS AND METHODS ........................................................--.-.................................................................. 86
AnimaIs ........................................................................................................................................................ 86 Stimuhtiun .~............~..................~...~..........................-...........~......-.......~........~............~............~.~.................. 86
RESULTS ........................................................................................................................................................... 86 ......................................................................................................... Synoptic Counts ............................... ,.. 86
Synaptic Curvature ..................................................~................................................................................... 87 Synaptic Perjiorations .......................................~...................................................................................... 87 Symptic Lengths .......................................................................................................................................... 87 Correlational Ana fysb ...................~..~~.....................................bb...............................................................~.~ 88
Discussio~ ...................................................................................................................................................... 94 Methodoogical Consideratiom ................................................................................................................... 94 Symptic Number .......................................................................................................................................... 95
...................................................................................................................................... 2$wptic Curvuhrre 95 Synaptic Perjorationr ................................................................................................................................. 95 Length of 2&twpses .................. ..... ......................................................................................................... 96 Correlcrromf AnalysrS ................ a .............................................................................................................. 97
............................................................. Co~~:Iusions= S)Inqptic RemOIjeflingüt 5 &ys Pm-LTP Induction 98
.................................... ................ GENERAL DISCUSSION -.. . e ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ o H ~ H o ~ ~ ~ e ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ o ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ w ~ ~ ~ o ~ ~ ~ ~ ~ n .. 99
....................................................................... SUMMARY OF CHANGES IN SYNAPTIC STRUC~URE OVER TIME 99 METHODOLOGICAL CONSIDERATIONS .............................................................................................................. 99 Sm~mrc NUMBER ........................................................................................................................................ LOI DYNAMIC MERA ACTIONS ........................................................................................................................... 101 CORRELATION AL FINDINGS .................................................................................................................... 102 SYNAPTIC TAGGING ....................................................................................................................................... 102 LTP, REACTlVE SYNAF~OGENESIS, LURNNG, AND MEMORY: IS THE= A COMMON STRUCTURAL MECHANISM? ................................................................................................................................................. 103 ARE LTP AND LEARNING EQUIVALENT IN THE HIPPOCAMPUS? ................................................................... 106
........................ CONCLUSIONS: SYNAPTIC REMODELLWG OVER TlME FOLLOWWG THE INDUCTION OF LTP 107
REFERENCES .. ....................... .....................e............................................................................................... 112
GENERAL, INTRODUCTION
Discoverhg and describing the neural mechanisms tbat underlie leaniing and memory
continues to be an area of intensive study within psychological neuroscience. Learning and .. - . ----
memory are known to arise fkom the flexibility or plasticity of the brain but the specific
nature of the changes that occur during various forms of leaming and memory remains
unclear. Ramon y Cajal (1893) initially theorized that the plasticity of the brain resulted
pnmarily fiom changes in the synapses between neurons. While Cajal hypothesized that
learning and memory resulted fiom the formation of new synapses, Tanzi (1 893) suggested
that existing synapses simply change in strength to increase or decrease neural signais.
nK issue of the mie nature of synaptic plasticity resisted M e r substantial
advancement until Donald Hebb (1 949) postulated that one neuron's involvement in firing a
second neuron would strengthen the connection (or synapse) between the two. Interesting ly ,
experimental support for this view did iiot corne until technological advanc~s allowed for
more minute study of the nervous system. Today the study of neural plasticity pivots on the
dynamic nature of the synapse (Malenka and Nicoll, 1993). The synapse's plasticity
involves both biochemical and morphologicai changes. While biochemical changes will be
discussed, the structural changes that occur in synapses are the focus of the research that
makes up this dissertation. Of specific interest are the changes in synaptic structure over
time in the dentate gynis of the rat hippocarnpus following electrophysiological stimulation.
These synapses were selected because the hippocampus is known to be involved in leaniing
and memory (Eichenbaum and Otto, 1992).
Role of the Hippocampus in Learning and Memory
nie hippocampus was first dewtively linlced to human learning and memory
following the operation which removed much of the medid temporal lobes of a patient
known as H.M. (Scoville-and MifDet, 1957). Profound amnesia was an unexpected
copsequence of this surgery intended to d u c e epileptic seizures. It was soon discovereà
that H.M. had retained some i d n g abiiity as he couid master repetitive motor leanllng
tasks such as tracing while looking in a mirror (Milner et al., 1968).
From these initial studies, animais models were developed to fùrther explore the role
of the hippocampus in leaming and memory (see Eichenbaum and Otto, 1992 for review).
Eichenbaum and Otto sumrnarize this body of research by stating that, "Across species and
across leaming materials the hippocampal system is critical to declarative membi ji. This
form of memory is identified by its essentiaily relational representation and its
npresentational flexibility" (Eichenbaum and Otto, 1 992, p. 29).
In the rat, this type of learning is exemplified by spatial maze leanllng (Morris, 1 984;
McDonald and White, 1993). Research has shown that the rat hippocampus can assume
neural activity which produces a representation of the space around the animals (Nadel,
1991). The rat then uses this representation to successfully navigate and solve tasks such as
the water m m . In this task, escape requires learning and remembering the location of a
undawater platform (Moms, 1984). When the hippocampus is mnoved or its activity is
temporarily blocked, the animais can not leam to solve the water maze ta& (Morris et al.,
1990). While the limits of the mle of the hippoccunpus continue to be defineci in various
species, it is clear that this structure contributes to certain types of learning and memory
(Malenka and Nicoll, 1993).
Learning and Memo y: Physiologieal Modeb
The search for physiological mechanisms in the brain that support the behavioural
changes observed during leaming has led to the development of several models. Bliss and 4 ' '
Lomo (1 973) are credited-with thèdiscovery of long-terni potentiation (LTP) which has
proven to be one of the most popular of these models. LTP was the k t mode1 to include a
lasring change in symptic efficacy which is required to explain long ïerm memory. "LTP
exhibits many of the properties that make it an ideal synaptic mechanism for memory
storage" (Malenka and Nicoll, 1993, p. 522).
The possible involvement of LTP in leaming was illuminatecl when rodents' spatial
leaming in a water maze was disturbed by a chernical intervention known to block LTP
(Morris et al., 1986). More recently, LTP-like potentiation enects have bcen discovered
following fear conditioning (Rogan et al. 1997; McKeman and Shimick-Gallagher, 1997)
and radial arm maze training (Ishihara et al., 1997) which m e r supports the argument that
LTP mechanisms are involved in memory formation. Some researchers have, however,
been able to dissociate the ability of a neural system to express LTP fiom its ability to
express leaming (McEachem and Shaw, 1996) which indicates that the relationship
between L W and l d n g is complex (sec General Discussion).
Other Learning Modes
LTP differs h m shori-term potcntiation (Sn) in the leagth of potentiation and the
mechanisms involved. STP is potentiation that lasts minutes, to tens of minutes which rnay
account for processes like short-tem memory. LTP, however, has ken shown to last for
seved &ys to weeks (Malenka and Nicoil, 1993). Also, enzymatic processes and protein
synthesis (see below) are required for LTP but not for STP formation (Bliss and
Collingridge, 1993). It would appear fiom these results that LTP is not simply a temporal
extension of STP.
Another model that involves lasting synaptic change i s long-terrn depression (LTD).
LTD involves a la-g nduction in synaptic efficacy that changes more quickiy and goes a'
beyond what occurs d u ~ g nonn8i.decay of potentiation (Artola and Singer. 1993). Purkinje
neurons in the cerebellum exhibit LTD and have k e n studied extensively (Linden and
Cornor, 1991; Ito, 1989). Linden and Comor hypothesized that LTD may contribute to the
role the cerebellum is thought to play in leaming new body movements. Interestingly, the
mechanisms for LTD induction in the hippocampus resemble those of LTP. LTD is usually
induced when the fkequency of stimulation does not reach the threshold required for LTP
(Artola and Singer, 1993). LTD is an attractive learning model because specific depression
of certain neural pathways could be as usehl a mechanisrn as specific potentiation (Ito,
1989). That is, a coniplex neural signal can &se from the potentiation of a subset of fibers
or the depression of a subset of fibers.
Long-Term Potentiation
LTP may be defined as the sustaincd increase (hours, days, or weeks) in the
amplitude of the response evoked in a cell, or population of cells, by a test pulse delivered
to an afZerent pathway following tetaaic stimulation of that pathway (Landfield and
Deadwy ler, 1988). LTP has been studied in the thm excitatory afTerent pathways of the rat
hippocampus. These pathways Uiclude the perforant path axons to the dendrites of the
granule cells of the dentate gyrus (the patbway examined in the c m t rescarch), the mossy
fibers of the granule celis to the dendrites of the CA3 region neurons, and the ScMcr
collaterais fiom the CA3 to the dendntes of the pyramidal cells of the CA1 region
(McNaughton and Miller, 1 986; Amaral and Witter, 1 989).
It is important to note that LTP is not a unitary process acrcss each of these intriasic
hippocampai connections. Unlike LTP in the dentate gynis and area CA1 , LTP in ana
CA3 is not dependent upon NMDA receptor activation (Otani and Ben-An, 1993; see
below for discussion). LTP also-&cm in other neural structures includuig, but not limited
to, the rat cerebral cortex (Lee, 1982), the rat olfactory coiter (Patineau and Stripling,
1992). the rai sympaihetic gangiia (Brown and McAffet, 1982), the cat motor cortex
(Sakamoto et al., 1986). and the sensory motor pathways of the aplysia (Walter and Byrne,
1985; Bailey et al., 1992).
Long-term Poteatiation: Electrophysiological Issues
The electrophysiological properties of LTP have been extensively researched and a
detailed discussion of this research is beyond the scope of this thesis. There are several
issues, however, that are directly relevant to this thesis. It is important to differentiate
between the potentiation of individual cells and potentiation of a population spike.
Individual ce11 potentiation is measured intracellularly by observing excitatory post synaptic
potentiais (EPSP). Conversely, the potentiation of a population of cells is rneasured
extracellularly by o b s e ~ n g the population spike. The population spike is created by the
synchnous fiiing of the group of neurons from which you are ncording. In the c m n t
research, the population spike amplitude was measured extracellularly as an indicator of
LW. Measuring the population @ce is appropnate in the current nsearch because the
bippwampi were intact and the animals were âeely moving during the induction of LTP.
The population spike is dso known to be a diable measurement of enhancement in this
system (Pugliese et al., 1994).
An extension of the issue of single cells versus populations of ce11 involves the
distinction between in vivo and in vitro LTP. The majority of research conducted on LTP in
the dentate g y m has w d in vitro slice preparations because of the accessibility of the tissue
for phanriawlogical manipulation (Otani and Ben-An, 1993). While more accessible, this
preparation does not provide a situation in which the observed nsults can be easily extended - * -
to the intact hippocampus; - h d h n d Witter (1989) describeci the many excitatory and
uihibitory extrinsic connections that affect hippocarnpd activity. In a slice preparation this
complexiiy of input is lost. Another concem is that Kirov et al. (1999) found that slice
preparatiom naturaily exhibit up to 90% more synapses than the same tissue in vivo. Thus,
the current research involved delivering electrophysiological stimulation to fieely moving
animals with intact hippocarnpi to mon accunitely reflect the system that is involved in
hippocampus dependent learning.
Extensive research has indicated that LTP can be readily induced in the dentate gyms
by delivering stimulus trains via an electrode irnplanted in the perforant path. These trains
usually consist of several pulses at approximately 400 Hz (Racine, Moore, & Wicks, 199 1).
The recording electrode is placed in the granule cells of the dentate g p s . This amount of
stimulation has ken shown to resdt in significant potentiation that decays to half strength in
three days (Racine et al., 199 1). If, however, the tetanic stimulation was repeated, the
potentiation would strctch to approximately Uvce weeks (Landfield and Deadwyler, 1988).
In the cumnt research, repeated tetanization was delivered to induce the longer lasting form
of potentiation.
Earlia (Racine et al., 1983) and mon recent (Bolshakov et al., 1997; Winder et al.,
1998) rrsearch has indicated that LTP is not a simple uniîary temporal process but may
involve up to three overlapping phases which rely on digerent mechanisms. The cellular and
molecular mechanisms that are thought to underlie the various phases of LTP are discussed
in the next section.
CeUuîar and Molecular Mechanisms of LTP
Induction of L TP
LTP is thought to occur &stages, where the initial induction of the potentiation gives
way to two or more maintenance phases in which the potentiation continues for relatively
long periods of time (Winder et al., 1998). Otani and Ben-An (1993) suggested that at any
particular tirne point the phses of LTP overlap so it is dificuit to cleaily delineate between
the mechanisms involved with each stage. Qne of the unresolved issues is whether the
observed potentiation is achieved and maintained by presynaptic or postsynaptic
mechanisms. The most likely scenario is a combination of these two possibilities (Bliss and
Lynch, 1988).
The finding that calcium (Ca2+) influx in the postsynaptic element plays a role in the
induction of LTP is well documented (Dunwiddie and Lynch, 1979). Support cornes from
experiments that have either rendered the calcium ions inactive with chelators or blocked
receptor c h e l s which greatly reduced calcium entry (Wigstom and Gustafsson, 1984). In
these cases, LTP was reduced or eliminated without adequate intracellular calcium.
Sharply increased calcium concentrations are thought to be achieved mainly via the
voltage dependent NMDA receptor channefs (Baudry et al., 1993). Voltage dependence
refers to the necessity for postsynaptic excitation by way of non-NMDA receptor activation
to depolarizc the postsynaptic membrane. In the dentate gym, this non-NMDA receptor
excitation is thought to occur via AMPA receptor chamels (Jenisalinsky et al., 1992). If the
membrane depolarization, via AMPA receptor or other activîty, reaches a threshoid level, a
M~*' ion which blocks the NMDA chanacl at the resting potential is ejected. With the M ~ ~ +
ion removed, activation of the NMDA recepton by glutamate (endogenously) allows
7
significantiy more calcium to enter the postsynaptic element. During LTP the tetanic
stimulation of the &ennt pathway is thought to provide both of the requirements for NMDA
channel activation. . . Further evidence for the in%olvement of the NMDA receptor in LTP cornes from
studies of the cornpetitive NMDA antagonists APS (Collingridge et al., 1983) and the non-
cornpetitive NMDA antagonist MK-80 1 (Gilbert and Mack, 1990). When these antagonists
were administered pnor to tetanidon, LTP was not induced. Recent gene deletion studies
have also implicated the NMDA receptor in the induction of LTP. A CA1 restricted gene
deletion of the NMDA receptor 1 (an essential subunit of the receptor complex) lead to the
inability of this system to exhibit LTP (Tsien et al., 1996). Thenfore, NMDA receptors
semis to play a critical role in the induction of LTP.
The role(s) that the elevated calcium levels play in the postsynaptic element has not
yet b e n resolved. It is iiwolved in the activation of the second messenger protein kinase C
(PKC; Malenka et al., 1989) aiid the activation of calcium-calmodulin kinase (Malinow et
ai., 1989). One important consequence of elevated PKC and calcium-calmodulin kinase
levels is that they change the properties of sodium and potassium conductance at the AMPA
receptor mediated channels in the postsynaptic membrane (Orover and Teyler, 1990).
Essentially, the recepton and channels now respond to the neurotransmitters in a heightened
manna. This makes the synapses mon effective at depolarizing the postsynaptic ce11
membrane which in tuni creates a larger number of, and mon frequent, action potentids and
this enhamernent in action pottntials can then k measureâ in the population of neurons.
Retrograde Messengers. The finding that the changes in receptor sensitivity
d e d b e d above are relatively short lived (Grover and Teyler, 1990), leads to the necessity
for other processes to maintain the induced pctentiation. One possible mechanism for this
maintenance is a sustained increase in presynaptic neumtransmitter release. The difficulty
with this theory is the lack of a communication mechanisrn between the postsynaptic element
which has experienced a change in receptor sensitivity and the presynaptic element when the
vesicular release occurs. A suitable substance for this retrograde communication would be
membrane permeable, produceci or released by the postsynaptic element, and would be able
to affect processes in the presynaptic elernent that mediate tnuisrnitter release.
Nitric oxide (NO) was first identified as a potential retrograde messenger candidate
by Bohme et al. (199 1 ) who blocked the induction of LTP with an NO inhibitor. NO readily
crosses the ce11 membrane and it is thought that NO is synthesized fiom L-arginine by the
calcium-calmodulin dependent enzyme NO synthase (Fazelli, 1992). High calcium levels
which lead to elevated levels of calcium-calmodulin kinase, may produce elevated NO
synthase levels and fmally production of NO (Fazelli, 1992). Evidence against NO
involvement in the hippocampus includes the fmding that some LTP induction can exist
when NO is Uihibited (Shuman and Madison, 199 1).
While the role of NO and other retrograde rnessengers in hippocarnpal LTP remains
unclear, NO is known to play a critid role in some foms of leaming. Benabeu et al.
(1995) found that intrahippocampal infusion of the NO releaser S-nitr0so-N-aminopenicillin
causes memory facilitation in a hippocampal dependent leaming task. in the same study a
NO synthase inhibitor causcd rctrograâe ammsia for the same ta& (Banabeu et al., 1995).
It appears thet while retrograde messengcrs may play some role in the induction of LTP it is
more clearly implicated in the formation of memory in hippocarnpal leaniing tasks.
Maintenance of LTP
Eariy and Intermediate Maintenance. An increase in transmitîer release may
sontribute to the initiai maintenance (1 h to 48h pst-LTP induction) of the potentiation -'-
observed during induction @lissa al., 1985). Increased release has been assesscd using
quantal analysis where the nurnber of quantuns or vesicles released d u h g depolarization
was measured (Malinow and Tsien, 1990). niese mearchers found that an increase in
transmitter release accompanied LTP. However, subsequent work has found no increase in
release during LTP in granule cells (Yamamoto et al., 1992).
If an increase in transmitter release does occur with LTP, it would be important to
understand the processes that cause transmitter release and how changes in synaptic
chemistry could bring about an increase in this release. The cascade of events leading to
release have been identified in the simple nervous system of the aplysia (Kandel, et al.,
199 1). This cascade is thought to result in an influx of calcium ions into the presynaptic
element of the synapse. This influx, however, is not achieved with NMDA receptors and
channels. Cyclic AMP (CAMP; a second messenger) levels increase in the presynaptic
terminal due to excitatory stimulation fiom axo-axonal intemeuron synapses. CAMP levels
may also rise due to the prcsence of a remgracie messenger substance. CAMP then activates
a protein kinase that phosphorylates proteins associated with specific ion channels.
Presynaptic calcium channels an thought to be altercd to allow more calcium ions to enter
the presynaptic element.
Higher calcium-levels activate more calcium-calmodulin kinase, which in tum may
fiee more synaptic vesicles by breaking d o m the cytoskeletal bonds that hold the vesicles in
place. Calmodulin is also involved in the docking and fusing of the vesicles tc th=
presynaptic membrane which causes neurotransmitter release (Hawkins et al., 1993). A
10
retrograde messenger dnven increase in CAMP concentrations at the beginning of this
cascade may lead to an eventual increase in trammitter release. This increase would then
sustain the potentiated response across the synapse observed during early LTP maintenance. -*.
There is also evidence, h ~ e v e r ; h t LTP is not associated with enhanceci transmitter release
(Yamamoto et al., 1992; Baudry et ai.,1993).
Resent research has shown that there are processes specific to an intennediate
maintenance phase of LTP (Winder et al., 1998). Winder et ai. created transgenic mice that
overexpressed a truncated form of caicineurin (an enzyme thought to be the first step in a
calcium-dependent cascade of phosphatases). This intennediate fom of LTP is thought to
differ from early maintenance because it's induction requires multiple trains of stimulation
and is dependent on CAMP-dependent protein kinase (PU). This form of LTP also differs
from longer-term maintenance as it is not dependent upon protein synthesis (Winder et ai.,
1998).
Long-Term M&?I~M~C~. The sustained altered suite of the synapse several days
after LTP has been inducecl is known to require protein synthesis (Rosenzweig and Bennet,
1984). Several studies have inhibird the production of different proteins and found this
interferes with LTP maintenance (Deadwyler et al, 1987). By using the protein inhibitor
anisomycin, Bailey et al. (1 992) blocked the lasting potentiation nomally observed in the
sensory motor newons of the aplysia. Protcins, once produced, could provide a longer
lasting change in the nature of the preqmaptic element both chemically and physically.
Physical changes in synaptic structure will be discused bclow.
Protein synthesis rnay also affect the nature of the postsynaptic element during the
long-tenn maintenance of LTP. Protein producing polyribosomes are often found near the
base of the dendritic shaft (Steward and Levy, 1982; Greenough et al., 1985). These
researchers speculated that these polyribosomes may contribute to the maintenance of LTP
by creating locally effective pïoteins. Support for this came fiom Desmond and Levy (1990)
who found that high fieqaency stimulation of the perforant path only changed the state of the
polyribosomes in the dendntic layer that was directly sthulated. Protein synthesis is a
relatively slow process and is therefore thought tu contribute to LTP maintenance by
replacing partially cleaved proteins that were initiaily phosphorylated to create the earlier
enhancernent (Desmond Br Levy, 1990). This would maintain the structurai and functional
changes for which the proteins were responsible.
Gene transcription l a d s to protein synthesis and has, therefore, also k e n linked to
the late phase of LTP maintenance. Transcription appears to have a critical period of activity
that supports the long lasting maintenance phase of LTP. Nguyen et al. (1994) found that
blocking RNA production with the transcriptionai inhibitors actinomycin D (Act-D) and $6-
dichloro-l OB-D-ribofuranosyl benzimidazole (DRB), disnipted the maintenance of LTP.
Interestingly, they ody did so if the inhibitors were applied during the first two houn afler
LTP was induced. If the inhibitors were applied after two hours the LTP remained intact.
These resdts appear to support the concept of early and late LTP proposed initially by
Racine, et al. (1983).
Gene expression, uniike transcription, involves the switchhg on of certain genetic
material by messengers t&at resuît from activity in the all. Once switchd on, this DNA
creates RNA which could crcate long-tmn changes in the chernistry and sûucnue of the
synapse. The main potential limitation of this process is its lack of specificity. One neuron
can have huche& of synapses and the genetic expression for al1 synapses occurs in the
soma. How can the potentiating effects be specinc to only the active synapses? One
response to this conceni has corne fiom Greenough et al. (1985) who clah that because each
synapse ofien has its own ribosomes, protein synthesis rnay be genetically driven but locally c' .
expressed. Recent researciiby F&y and Morris (1997) indicated that specificity may be
achieved via synaptic tagging. Frey and Moms theorized that tetanic stimulation wouid not
only initiate somatic gene expression but could cause a specific change in only the synapses
that were active during the inductive process. These taggeâ synapses would then
preferentially utilize the proteins created in the soma. The pnsence of phosphorylated
kinases may act as the synaptic tag (Frey and Moms, 1997).
Immediate early genes (IEG's) eg. FOS, Jun-B etc., seem to be responsible for
stimulating changes in gene expression. Calcium entry and other membrane signals have
ken shown to increase c-fos production which is thought to travel to the soma and affect
gene expression (Dragnow et al., 1989). Dragnow et al. speculated that since c-fos seems to
be involved in the long-term phase of LTP, tetanization in the absence of c-fos would result
in a faster decay of the potentiation. This tumed out not to be the case in a recent study by
this group (Jeffery et al., 1990). In another study, however, the arnount of IEG induction
~ m l a t e d highly with the amount of LTP stimulation given and an increase in RNA present
severai days after LTP induction correlated highly with the amount of potentiation that
remained in the late phase of LTP (Abraham, et al., 1993). Due to these conflicting resdts,
the mle of IEG's in the late phase of LTP remallis unclear.
Ail of the genetic mechaaisms discussed nfer to changes in individual synapses.
These individual changes are thought to combine to fonn the potentiation observed in a given
neural pathway. Another possible mechanism for potentiation is an increase in the total
number of synapses. This possibility was discussed by Kandel and his colleagues. "A
second consequence of gene activation is the growth of new synaptic connections . . . researchers have found that in eained animals the semory neurons had twice es many
postsynaptic temiinals than in untrained animals" (Kandel, et al., 199 1, p. 10 16).
Conclusions
While the research discussed represent only a sampie of the large number of studies
conducted in this are% two conclusions seem clear. First, difierent cellular and molecular
processes are involved in different phases of LTP induction and maintenance. Second, long-
term maintenance of LTP seems to rely on protein synthesis which may lead to aitered
synaptic structure. Altered synaptic structure would provide a mechanism for potentiation
which lasts longer than several days. A longer lasting mechanism is essential because al1 of
the cellular and molecular changes described above return to near baseline levels of activity
well before the electrophysiological potentiation ceases (Otani and Ben-Ari, 1993). Synaptic
structural change, therefore, may support the persistence of the electrophysiological
potentiation.
Synaptic Structural Changes hsoclateâ with LTP
The morphologicai charac~ristics of synapses have been shown to change following
the induction of LTP (Geinisman et al., 1992,1993,1996; Desmond and Levy, 1983,1986;
Weeks et al., 1998,1999,2000). Specific resuits h m these W e s appuirs to depend on the
mode1 system used and the length of delay between induction and sacrifice. Anothei issue is
what role, if any, these changes play in the enhanceci neural signal observed during the
induction and maintenance of LTP? While LTP has not ken associated with an overall
change in the number synapses in a system, many categories of synapses an known to
change in number following the induction of LTP.
Synaptic Curvature
Changes in synaptic curvature are known to occur following various forms of
synaptic activation (Petit, 1995). -.. Markus and Petit (1 989) evaluated synaptic curvature using .- * . -%
four possible States; concave (presynaptic element protnides into the postsynaptic element),
Bat (no cwatwe), convex (postsynaptic element protmdes into the presynaptic element),
and irregular (w-shaped or more than one curvature). Several hypothesis have been proposed
to account for changes in the cwature of synapses. The increase in neurotransmitter release
following tetanization may enlarge the amount of material associated with the presynaptic
membrane as spent vesicles collapse and this could result in an invagination of the
presynaptic element into the postsynaptic membrane (Markus and Petit, 1989).
Another possibility is that plastic cytoskeletal networks rnay create changes in
curvatw in response to calcium influx (Fifkova, 1987). Fifkova suggests that the intemal
actin and myosin cytoskeleton, which are broken down by calcium, may be responsible for
postsynaptic shape change following activity. Fifkova (1985) found that cytoskeletal
networks that contain oniy highly plastic actin structures are found exclusively in developing
neurons and mature dendritic spines (see below for a discussion on dendritic spines). Finally,
curvanire may be inherent to different subsets of synapses which contact dendritic spines or
shah in a different location or direction, thereby changing the curvature that is observed
(Markus and Petit, 1989). Synaptic activation, therefore, may lead to the formation of
synapses on different amas of the denclritic arbour which display different curvatures.
The numùer of concave shaped synapses has ken s h o w to incfease as early as 2
minutes and until at least 60 minutes following the induction of LTP in the rat dentate g y w
(Desmond and Levy, 1983). Contrary evidence has corne from Chang and Greenough (1984)
who did not confimi this finding. However, Chang and Greenough were snidying the CA1
region of the hippocampus and not the dentate gynis. The disctepaacy may have also
remlted fiom diffennces in stimulation technique. . "*.
Functiondly, cuivature islhought to effect synaptic eficacy by increasing contact
area and probability of the transrnitters reaching their target (see Markus and Petit, 1 989 for
review). Further, changes in curvature affects the proximity of receptor sites to the dendntic
shafi. The closer the receptor channels are to the dendritic shaft the larger the synapse's
potential eEect on the postsynaptic neuron. Another functional consequence of curvature,
such as a concave shape, is that such a change may confine the diffusion of presynaptic
intracellular calcium leading to higher calcium concentrations and an increased probability of
vesicular release (Ghaffhri et al., 1997).
Dendritic S ' n e s
Desmond and Levy (1986) reported increases in spine head size and spine stem
parameters (length and thickness), changes in spine head shape, and more axo-spinous
synapses 60 minutes following the induction of LTP in the dentate gynis. Baudry et ai.
(1993) pointed out that we do not know whether the observed changes are due to the
transformation of the existing spine structures h m one configuration to another or whether
they represent the formation of new synaptic contacts. Functionally, Baudry et al. stated tbat
a change in spine sbape could d u c e spint neck mistance and iacrease the flow of cumnt
fiom the synapse to the dendntic shatt. This case of conductance would dlow for
potentiation in the absence of incrrased receptor scasitivity or increased transmitter nlease.
Another role of the dendritic spine was suggested by H a d s and Kater (1 994) who
stated that the denâritic spine may fonn a locaîized cornpartment in the vicinity of the
synapse. This allows the localized calcium level to rise instead of diffushg to a
16
concentration that would not drive the changes necessary for potentiation. When this
calcium level is sustained it allows these changes to stabilize into a more permanent state.
Interestïngly, evidence in support of this hypothesis cornes h m new-bom animals who cm - * *
not establish LTP that lasis longePthan 2.5 hours (Harris and Kater, 1994). In that study
these LTP deficient young animals had equai numbers of thin, mushroorn, and stubby shaped
dendritic spines. By the age of 48-60 days, however, the rnajority of the spines were small
and thin. These findings suggest that a specific number of spines with restricted necks are
required for persistent LTP.
Dendritic spines have also been shown to be highly motile which could allow for
rapid alterations in spine shape. Hayashi and Shirao (1 999) demonstrated that drebrin, a
destabilizing actin-binding protein, nguiates spine shape by competing with other stabilizing
actin binding proteins to allow for structwal change. Further, using a fluorescent imaging
technique they showed that spines with higher levels of bound drebrin were longer than those
with less bound drebrin. Interestingly, while drebrin is found throughout the developing
brain (axons, dendritic shafts etc.) it is found only in dendritic spines in the adult brain. This
provides a potential mechanism for rapid structurai plasticity of dendritic spines in the adult
brain.
Chang and Greenough ( 1 984) studied dendntic spines following the induction of LTP
in the rat hippocampal slice and found an incnase in the number of sha. synapses and sessile
syuapses (synapses on stubby, h d e s s spines) which persisteci for 8 h. Due to the lack of an
overail increase in spiae number, thcse results suggest that the induction of LTP is associated
with a shortening of dendritic spines. Geinisman et ai. (1996) found more numerous axo-
denciritic synapses (synapse is directly on dendntic shaft) 1 3 days following the induction of
LTP in the rat dentate gyrus. This result a& support to the movement of spines towards
shorter and wider configurations following stimulation. Functionally, these axodendritic
synapses may have a more direct effect on the soma and could therefore combine to create a
potentiated signal (see H s s andkater, 1994 for discussion). t
SjMu1e.s
A synaptic spinule is a postsynaptic invagination into the presynaptic efement.
Schuster, et al. (1990) found that in spinules the postsynaptic membrane is devoid of dense
material associated with the PSD. Followhg the induction of LTP, Schuster et al.
determincd the density of axo-spinous synapses and counted the number of axo-spinous
synapses containhg spinules. They found that the number of spinules significantly increased
8 h and 48 h after the induction of LTP. The increase is thought to reflect a process of
synaptic turnover which enhances the overall cfficacy of the synapse. Spinule are also
thought to be involved with synaptic perforations which are discussed in the next section.
P e fit-ated Synapses
A perforated synapse can be defined as any synapse with discontinuities or segmented
PSD (Geinisman et al., 1993). Perforated synapses are thought to include a presynaptic
perforation as well but most studies have defined this type of synapse by the distinctive break
in the PSD (Calverly and Jones, 1990). The huictional importance of this type of synapse
was initially reaîized when i n d numbers of perforated synapses were obsewed in rats
following exposure to an enriched environment (ûreenough et al., 1978). Subsequently,
perforated synapses have been increasingly implicated in synaptic temodelling and turnover
(Geinisman et al. 1989).
Jones et al. (1 99 1) assessed the number of perforated synapses during the
development of the rat neocortex using early 3D reconstructive techniques. They found that
as the brain developed the total number of synapses decrease but the relative number of
perforateû synapses increased (Jones et al. 1991). When they reconstructeà the synapses,
severai shapes of the PSDs were observed. Carlin and Siekevitz (1983) proposed that at
some optimal size the p s t synap6c material would pedorate, eventuaily segment, and new
simple synapses would be formed. The shapes observed by Jones et al. (1 99 1) comspond to
interinediate steps fiom a simple to a perforated synapse. The shapes inciuded a donut, then
a borseshoe, followed by a dumbbell shape, and finally split into separate segments within
the same terminal. Geinisman and colleagues (1993; 1996) proposed a similar mode1 of
s p p t i c transformation.
Geinisman et al. (1 99 1,1993) found an increase in the number of perforated synapses
per neuron following the induction of LTP in the nit dentate gyrus. This observation was
made 1 h after the last LTP tetanization but followed 4 days of LTP inducing stimulation.
Importantiy, the significant increase in pedorated synapses observed at I h post-induction
was no longer evident at 13 days pst-induction (Geinisman et al., 19%). Buchs and Muller
(1996) utilized calcium accumulation markers to identify activated synapses and found that
these activated synapses were more perforated following LTP induction. In other work from
that laboratory, Toni a al. (1998) reported an increase in synaptic paforations in activated
synapses during the £kt 30 minutes following in vitro LTP induction in the rat CA1 region.
At 60 minutes pst-induction, they found an incnase in the proportion of double synapses
(tw spines comccted to the same presynaptic terminal) suggesting that pafomted synapses
rnay eventually split to form new simple synapses.
Synaptic perforations may play a fiinctional role in the maintenance of LTP (Petit,
1995). Perforations may indicate synapses ready to divide and this may lead to more
nurnerous synapses and a larger neural signal (Toni et al., 1998). Altemtively, perforations
may allow calcium charnels to be located closer to the vesicular release apparatus which
could result in greater neurotransmitter release (Jones et al., 1992). .'*
There is, however; évidence against the notion that synapses split to form new simple
synapses. Jones et al. (1992) found few double headed spines, axo-spinous non-perforated
synapses lying adjacent to one another, and spinules completely travershg the presynaptic
terminais of perforated synapses in the intact dentate gynis. Al1 of these observations, would
provide evidence for the splitting hypothesis because they represent the missing steps
between perforations and new autonomous synapses. The fact that Jones et al. did not find
these structures in suscient numbers casts doubt on the division hypothesis.
Contrary evidence has corne fiom a report of a new synaptic subtype that rnay
account for the missing steps (Geinisman, 1993). Using three dimensional reconstruction,
Geinisman discovered synapses that exhibit partitions that emanate fiom the postsynaptic
spine in the perforated synapse. These partitions invaginate the presynaptic axon terminal
and divide its portion contacted by the spine into distinct protnisions. This provides a barrier
between discrete transmission zones, each one being formed by a separate axon terminal and
corresponding segment of PSD. This could be an intemediate stage in a process of synaptic
division. Visdly, these partitions are very similar to spinules (described above) which also
incxease in number following the induction of LTP (Schuster et al., 1990).
Length, Area (id Volume) of SVMptic Elements
Beyond the increasc in n e d signal that would most likeiy result h m more synaptic
connections, or changes in cwature or perforations, most researchers have theorized that a
bigger synapse is more effective at exciting the postsynaptic cell. Conceivably, more
neurotransmitter could be released (presynaptically), more postsynaptic receptors could be
20
created, and more conductance through the spine stems could occur (Petit, L 995). A recuit
study u d cultured cortical murons to study the mle of synaptic size in the quantal capacity
of the synapse (Mackenzie et al., 1999). By irnaging ca2+ and then conducting
morphological analyses'on ihe &é synapse, Mackenzie et al. concluded that synaptic size
cornlates positively with the amplitude (measured in miniature calcium transients) of the
postsynaptic response. This suggests that larger synapses express a greater number of
NMDA receptoa and thenfore cmte a larger synaptic signal.
The one dimensional length and two dimensional area of the synaptic elements are
known to correlate with their three dimensionai volume and therefore to their size and
potentially, their efficacy. General increases in synaptic size following LTP have been
reported (Chang and Greenough, 1984, Desmond and Levy, 1988). These studies found an
enlargement of the presynaptic and postsynaptic elements, and an increase in length of the
presynaptic thickening and postsynaptic density (PSD).
These changes in synaptic size often interact with changes in the proportion of
synapses with different curvatures and changes in the number of synapses with perforations.
Desmond and Levy (1986) reported a significant increase in the total postsynaptic surface
ara per unit volume for only concave shaped synapses. Concave shaped synapses have also
been shown to increase in size while convex shaped synapses decreased in size following
synaptic activation (Petit et al., 1989). Based on their results, Desmond and Levy (1 988)
pmposed that a rapid structurai interconvasion occurs following the induction of synaptic
potentiation, w h m synapses change h m non-concaw to concave and subsequently grow in
size. Synaptic perforations also comlate highly with increases in overall synaptic size (Jones
and Calverly, 1 99 1 ).
Other Structural Changes. ne total number of synaptic vesicles and particularly the
number near the active zones bas been show to increase with LTP (Meshul and Hopkins,
1990). This movement in vesicle location may have fùnctional implication for the efficacy . . of the synapse as more ~Ürkrousb&sicles would be in a favourable position for release.
Associated with an increase in vesicles and potentially related to synaptic curvature was the
finding of an overall increase in the average presynaptic membrane length following LTP
(Geinisman, et al., 1990).
It seems clear that changes in synaptic structure are associated with the induction and
maintenance of LTP. Further research is needed to complete the search for these structural
changes and to plot the ways synapses change over the tirnecourse of the electrophysiological
potentiation.
Research Goals and Rationale
Harris and Kater (1 994) point out that a great deal of interest remains in the search for
the significant structurai mechanisms of LTP and as the tools of observation improve, new
and more precise findings can emerge. The central goal of this research project was to
conduct an extensive examination of the time course of synaptic stnictural change associated
with LTP. This research used transmission electronmicroscopy to make precise observations
of synaptic characteristics at three separate ps t induction time periods. These analyses
detennined whether changes in synaptic number, synaptic shape, synaptic perforations, and
/or synaptic length o e c d at the various time points following LTP induction. In addition
to a non-stimulateci implanted conûol group, an additional group was added which received
the LTP induchg tetanization under pharmacological blockade. This group was included to
define LTP-specific vs general stimulation-iaduced synaptic changes. Further, al1 synaptic
structural results were correlated with the degree of LTP expressed by each individual
animal. These additional analyses were Uicluded to search for the structural changes that
appear to be critical for the degree of LTP expressed.
While previous research has indicated that changes in synaptic morphology are b -
associated with LTP, these jxeviob studies have used different LTP induction protocols,
considered different synaptic structural characteristics and have waited for varying post-
induction time internais before perfusing the animais. The current research addressed al1 of
these issues to provide a clear p i c m of how synapses change over time. The various
findings of previous research suggested that a sequence of changes in synaptic structure may
emerge fiom the cunent experiments.
The need for continued investigation into the mechanisms underlying LTP was
summed up by Collingridge and BIiss (1995) who listed the aspects of LTP that continue to
resist solution. They asked the following questions: "Are the cellular mechanisms that
support the persistent expression of LTP located presynaptically or postsynaptically? Are
there changes in morphology or in the number of synapses? . . . What (if anything) is the role
of LTP in learning?" @p. 54). The current research attempts to answer the second question
raised by Collingridge and Bliss.
GENERAL METHODOLOGY
The following procedures represent those methods which were comrnon to each of
the four experiments described below (1 h, 24 h, ketarnine, and 5 days). Any deviations fiom
these procedures are described in the sections for each individual experirnent.
AU clectmphysiological procedures were carried out in the labontory of Ronald J.
Racine at McMaster University, while al1 morphological procedures were carried out in the
laboratory of Ted L. Petit at the University of Toronto. To insure that the research was
conducted blind, the animals nom ail groups were coded at McMaster University and the
code was not released to the Petit laboratory until al i data were collected, entered, and reaày
Adult male Long-Evans hooâed rats were supplied by the Racine Laboratory
Breeding Colony (McMaster University) for this research projeci. MI rats weighed between
300 and 500 g at the tirne of surgery. They were maintainecl on an ad lib feeding schedule
and were on a 12 h onIl2 h off light cycle. It is important to note that six of the experimental
animals in the 24 h expriment (see below) and six of the control animals examined in these
experiments had also been used in a previous shidy (Weeks, 1995: Master's Thesis).
SurWY
Twisted biplar electrodes were prepared with Teflon-coated stainless steel wire,
insulated except for the tips. The stimulating and recording electrodes were constructeci from
wire 300 pm and 200 pm in diameter, respectively. The poles of these bipolar electrodes
were separated such that one pole was about 500 pn longer than the other. This allowed for
stimulation of a large number of fibers and differential recording (see Racine, et al., 1991).
Animais were anaesthetizeâ with soâium pentobarbitol(65 mgkg) for
physiologically controlled electrode placement. The stereotaxic CO-ordinates selected from
Paxinos a d Watson (1986) wcre (a)prrfrantparh: 7.6 mm posterior h m Bregma, 4.1 mm
laterd h m the midine, and 3.3 mm ventrai firom the s M 1 surface; and (b) de~ute gynu:
3.5 mm posterior h m Bngma, 2.2 mm latcral to the midline (ipsilateral to the perforant path
electrode), and 3.3 mm ventral h m skuil surface. Electrodes were adjusted to maximize the
EPSP and population spike amplitude.
Electmdes were comected to male arnphenol pins that were inserted into a %pin plug
that was mounted to the skull with dental cernent and anchored with stainless steel jewellers'
screws. A jewellers' screw soldered to a single Teflon-coated wire with an amphenol pin ,S.
attached was used as a grciund. ~ileast two weeks were allowcd for ncovery before testing
began.
Stimulation and Recording
Stimulation pulses were delivered via a Grass S88 stirnulator and photoelecnic
isolation units (Grass SIU6B). Input /output (I/O) curves were constructed for al1 animals to
establish a baseline. To create the VO curves, pulses of increasing intensity were delivered to
the perforant path at a fkequency of 0.1 Hz. Ten responses, 35 ms in duration, were evoked,
arnplified, digitized, and averaged at each of 16 logarithmically spaced intensities (1 6,25,32,
40,63,79, 100, 126, 1 59,25 1,398,50 1.63 1,794, 1000, and 1259 mA). Responses were
stored on a cornputer hard drive for off line anaiysis. Using the baseline curves, animals
were divided into two groups based on response morphology.
Half amplitudes were determineci for dl experirnental animals and were used as the
stimulation intensity for delivery of traîx~s. Trains consisted of pairs of 400 Hz bursts with
durations of 27 ms (10 pulses), separated by 200 ms. Non-stimulated, irnplanted control
animals received oniy the UO tests. A single control group of eleven animais was used as a
basis of cornparison for each experimentai group. Baseline responses were retested after al1
trains had ban dclivered to the experimcntal animals to ensure that LTP had been Uidud.
Baseluie responses were aiso rtcorded irnmediately before sacrifice et the various time
intervals. This provided a measwement of the degree of LTP king express at the time of
sacrifice and subsequent synaptic analysis. Animals were anaesthetized and perfwd in
preparation for electron microscopic examination.
25
Tissue Preparation
Ali animals wen anaesthetized at McMaster University by sodium pentobarbitol
injection and perfused intracardially, using a Masterfiex pump, with 30 ml phosphate b d e r
followed by fixative (2% parafoimddehyde, 2.5% glutaraldehyde in a 0.1 M phosphate
buffer, pH 7.3). The brains were then removed and placed in the same fixative. Following
transfer to the University of Toronto, the ipsilaterai hippocampus was dissected and two 1
mm sections were taken h m the rostral face, 1.5 and 2.5 mm caudal to the septal pole of the
hippocampus (an area 1 mm caudal from the recording electrode).
The hippocarnpal slices remained in fixative for an additional 12 h befon being
placed in three 1 h washes of phosphate bufTer. Four tissue blocks were randomly dissected
(started fiom a randornly chosen distance dong the septo-temporal extent) fiom the central
segment (immediately ventral to the CA1 region) of the dentate gynis and placed in I %
osmium tetroxide in a 0.1 M phosphate buffer for 1 h. The tissue was dehydrated in a graded
series of ethanol solutions and embedded in Spurr's embedding medium (Ladd Research
Industries, Burlington, Vermont).
Tissue Sectioning
Excess embedding medium was trimmed away and thick sections were taken from
one randomly selected tissue block per animal using an Ultracut microtome. Completely
trimmed blocks includeâ the entire granule ce11 layer (GCL), the inner molecdar layer, and
half of the middle rnolecular layer for more efficient edge orientation during elcctron
microscopy. Ultra-thin sections (80 nm, determincd by a silver-gold reflection) were cut
using a diamond knife and placed on Fomvar dot grids yielding approxirnately 50 thin serial
sections per block. The sections were subsequently counterstained with uranyl acetate and
lead citrate.
Electron Microscopy
Synapses in the inner (ML) aud middle (MML) thirds of the dendntic molrcular
layer of the dentate gynis, ipsilateral to the recordkg electrode, were examined. Synapses
fiom the MML of LTP stimulateci animais were used for the experimenial group measures,
while control group measures were taken fiorn synapses fiom the IML of LTP stimulated
animals (not-directly stimulated controls), and fiom the MML of non-stimuiated animals
(implanted controls).
The rniddle third of the dentate molecular layer (MML) was used as the area of
primary interest in the LTP animals because the MML has been show to receive the
rnajority (-90%) of its afSerent neural contacts fiom perforant path axons. Conversely, the
IML served as one of our control sites because it receives pnmarily commissural input rather
thaa direct perforant path innervation (Amad and Witter, 1989). Although previous
experiments have used the IML as a control site for MML measures (e.g. Geinisman, et al.,
1992), there is a M e r potential confound in this procedure. Kindling and LTP have both
been show to induce sprouting in the mossy fiber pathway (Sutula et al., 1988; Adams et al.,
1997; Escobar et al., 1997). In at least some of these experiments, there was an indication
that spmuting is direct4 into the M L (Sutula et ai., 1988; Adams et al., 1997). Therefore,
the IML may not be an appropriate control for the MML measures. Consequently, we have
added the implanted wntrol group for k t w a n group comparisons.
Two areas in each of t& molecular layers werr selccted randomly and photographed
on each successive section ushg morphological landmarks to capture the sarne m a on each
section. The molecular layers were photographed at a magnification of 18,360X
magnification. The GCL was also photographed (needed for the double dissector counting
technique, see below) in the first and 1st section of each series at a magnification of 9OOX.
Synaptic Number
An estimate of the aumber of synapses was detemllned using the unbiased dissector
technique employed by Gcinisman et al. (1 99 1 ; please ser the Appendix for a discussion on .@.
the nezessity of using uiibiased coLunting techniques). This technique produces an estimate of
the total number of synapses per neuron in each region. Synapses were counted by
comparing adjacent sections, one a reference section and the other a look-up section
immediately above it in the series. Synapses were identified in the rnicrographs by the
presence of synaptic vesicles, dense material in a presynaptic axon terminal, and an
accompany ing PSD. Synapses were sampled (counted) if they were observed in a reference
section micrograph within the area limited by the unbiased sampling frame of Gundersen
(1977). but not observed in the corresponding look-up section. This provided an unbiased
estirnate of synaptic number as synapses were oniy counted once regardless of the number of
sections in which they appeared.
Granule newons were sampled if the nucleus of the ce11 appeared in the last section of
each series but not in the first. The formula n/N = [(q' A k)/Qa)] (w/W) was employed to
estimate the number of synapses per neuron (Braendgaard and Gundenen, 1986). In this
formula, Q- and q' indicate nurnbers of newons and synapses sampled in an area A or a,
respectively; k is the number of sections in a series minus one; w designates the width of the
middk or h i e r diird of the ML; and W represcnts the width of the GCL (Geinisman et al.,
1993).
Synaptic Structure
Following the analysis of synaptic number, al1 synapses identified in each series were
categorized according to cwature (presynaptically concave, presynaptically convex, flat or
inrgular) and the presence or absence of perforations was noted. For defining synaptic
28
curvatun, a d e r was placed dong the length of the active zone, and synapses were
clarsified into one of the cuwahue categories based on deviation from a straight line. Any
deviation fiom a flat configuration was classified as a curved synapse, and in the cases where
the degree of curvature 'coiild not be clearly categorized as curved, the synapses were defined
as flat. Synapses that contained more than one cwature configuration were classified as
irregular.
A perforated synapse was defined as having a visible break in the PSD on any of the
two dimensional profiles associated with that synapse. The Bioquant OS12 BQ-microVoxe1
Image Analysis System was used to digitize the photographie negatives and electronically
muisure the maximal length of each of these synapses. The maximai length was attained by
movhg forward and backward through the tissue series to the specific section where a
particular synapse was largest. In perforated synapses the maximal length of the synapse was
measured in two distinct ways. First, the maximum overall length was obtained (starting
fiom one end of the PSD across the perforation to the other end). Second, the length of the
perforation (gap) was measured and this value was subtracted fiom the total length to yield
the cumulative active zone length.
Statistical Analysb of Smietural Profile
Following the synaptic anaiysis, the code was partially broken and the data separated
into pups such that the Petit laboratory rernained blhd to which group had receiveû trains
to induce LTP and which pst-induction delay had been used. The unbiased estimate of the
overall numkr of synapses per neuron in each molecular layer of the two groups was
analyzeâ using a general factorial analysis of variance (ANOVA). The remaining data was
analyzed using the ANOVA and Chi-squared non-paramettic test programs fiom the
Statistical Package for the Social Sciences (SPSS) for Windows 6.0.
29
The varying proportions of synapses with different synaptic cwatures was analyzed
ushg the non-paramettic Chi-square test. The proportion of perfomted synapses was also
anaiyzed using the Chi-square test. The total synaptic length and active wne length were - * -
analyzed using a general faCtoria1-ANOVA. Subsequent cross over analysis was conducted
on the length of perforateci synapses and the length of synapses with different curvatures.
When the M O V A reached signifiuuice @<O.OS), post-hoc analysis was carricd out using
the Duncan test. At the completion of the statistical analysis the group identity code that
blinded the Petit laboratory was broken.
Correlational Analysis
In the three groups where LTP was induced, the structurai chatacteristics descnbed
above from individual animais were correlated with the degree of potentiation that particular
animal expresscd at the time of sacrifice. The degree of potentiation was denved fiom a
composite measurement based on a standardized mean of the population spike amplitudes
evoked fiom the middle 12 intensities. nK multiple regression prognun of SPSS was
employed to ascertain which structural aspects were significantly associated with the degree
of potentiation.
GENERAL ELECTROP WSIOLOGICAL RESULTS
Analyses of the electrophysiological results were conducted using the ANOVA
program h m the Windows version of Statistica. The population spike amplitude evoked
from the middle 12 intensities (32-794 pA) were analyzed. A statistical analysis conducted
on eacb group prior to tetanization revealed no differences between the experimental and
control animals in population spike amplitudes. Al1 animals in the LTP stimulation groups
showed significantly enhanced population spike amplitudes directly before sacrifice. Group
mean values of potentiation revealed significant increases in population spike amplitude at 1
h (pa.OS), 24 h (pd).Ol), and 5 days @<0.01). The degree of potentiation values were
derived Utdividuaily for all animais in the LTP groups (raaged fiorn 2.6% tu 1 12.2% 6 .
- . potmtiation).
SYNAPTIC STRUCTURAL PROFILE AT 1 HOUR POST-LTP INDUCTION
Introduction
The majority of research on the synaptic structural correlates of LTP have used short
pst-induction time intervals. For example, finding increased numbers of perforated
synapses per neuron was denved fiom synaptic analysis 1 h d e r the final tetanization
(Geinisman et al., 1992,1993). Further, the increase in concave synapses reported above
was observed at 1 h pst-LTP induction (Desmond & Levy, 1983, 1986).
Many of the biochemical mechanisms discussed in the generai introduction begin to
affect synaptic fbnction and preswnably synaptic structure hed ia te ly following
tetanization. Calcium influx via activated NMDA receptor channels and many other foms of
synaptic activation are known to produce changes in curvatute within 1 h of stimulation (see
Markus and Petit, 1989 for review). Given the importance of the inductiodearly
maintenance phase of LTP, the current expriment was conducted to examine synaptic
structure 1 h following the induction of LTP.
Materials and Mtthoàs
Animals
Twenty addt male Long-Evans hooded rats were suppiied by the Racine Labotatory
B reeding Colony (McMaster University) for this stud y. Nine animals received LTP inducing
tetanization and eleven animals served as implant-only controls.
Stimularion
Trains wnsisted of pairs of 400 Hz bursts with durations of 27 ms (10 pulses),
separated by 200 ms, with an inter-train intemai of 8.5 sec. A series of 30 stimulation trains +'.
were delivered to the perforant path over 30 minutes, for a total of 225 trains. These protocol
modifications were needed to ensun significant LTP at 1 h pst-LTP induction. Baseline
responses were retestcd 1 h d e r the beginning of tetanization to ensure that LTP had k e n
induced. Al1 other procedues did not differ from those described in the General
Methodology section.
Results
Synaptic Number
The unbiased estimate of the number of synapses pet neuron for both molecular
layers was used as the initial unit of anal ysis. An ANOVA did not reveaî a significant
difference in the number of synapses per neuron between the LTP and control groups. The
mean number of synapses per neuron in the MML of the LTP and irnplanted control animals
was 2928 and 2933 respectively. There was a main effect of location @=0.003) with the
MML having a mean of 293 1 synapses per newon, while the IML mean was 228 1 synapses
per neuron. The LTP group by location interaction was not significant.
SY)aptic Curvature
There are naturally occwing differences (i.e. within the control animals) between the
two molecular layers in the dative proportion of synapses of different shapes. Specifically,
there are clifferences between the proportion of concave and convex shaped synapses in the
two molecular layers. Significantly more convex synapses were found in the IML compared
to the proportion of convex synapses in the MML @<0.001), while significantly more
concave synapses were found in the MML compared to the proportion of concave synapses
32
in the IML @<0.001, see Fig. 1.1). As a result of this apriori difference in synaptic
cwature proportions. analysis of curvature differences in the cumnt reseamh, compared the
LTP-MML synapses to implanted control MML synapses, while LTP-IML synapses were * D r
compared to implanted control IML synapses.
Analysis of cwature revealed a significant increase in the proportion of irregular
shaped synapses Q~û.006) and a significant decrease in the proportion of convex shaped
synapses @=0.003 1) in the directly stimulated tissue (LTP-MML) relative to the MML of
control animais @10.006 ; see Fig. 1.2). The proportion of irregular synapses was 56%
higher and the proportion of convex synapses was 42% lower in the MML of the LTP
animals than in the MML of controls. In the not directly stimulated IML, LTP was
associated with a significant decrease (-20%) in the proportion of convex synapses (p=0.036)
and a significant increase (1 9%) in flat synapses (p4HKN) compared to implanted control
IML tissue (see Fig. 1.3).
Syfiapiic Perfrtaaiions
The proportion of perforated synapses was significantly higher (+19%) in the
potentiated LTP-MML compared to the control MML (pQ.041). Unlike the MML, the
proportion of perforated synapses was not significantly different between the not directly
stimulated LTP-IML tissue and the conaol IML (see Fig . 1.4).
Perfomted synapses with spccific curvantres wem also analyzed In the LTP-MML.
thcre was a significant incrcasc in the proportion of jdorated synapses that wmc concave
@<0.001), and a significant decrease in the proportion of perforatecl synapses that wen flat
m.003) compared to the control MML (see Fig. 1 S). There wete no significant
differences in the proportion of perforated synapses of different shapes in the IML.
An analysis of non-perforated (simple) synapses revealed several significant findings.
In the MML, LTP was associated with a significant decrease ki the proportion of convex
synapses (p=û.OiS) and a significant hcrease in the proportion of irregular shaped synapses 4..
(pKO.001). In the IML, the= w&%so a decrease in the proportion of non-perforated convex
synapses in the LTP animals @=0.034) and an incruise in the proportion of non-perforated
fiat synapses @<0.00 1 ; see Fig. 1.6).
S y ~ p i c Lengths
An ANOVA of total synaptic length (including the length of any synaptic
perforations) revealed a significant interaction between LTP group and location (p=0.02 1).
Post-hoc analysis revealed that the LTP-MML synapses were significantly longer than the
control-MML synapses (see Fig. 1.7). Analysis of active zone length (excluding the length
of any synaptic perforatinm) did not reveal significant differences.
The total synaptic length of syiiapses with different curvatures was aiso analyzed. In
the MML, LTP was associated with a significant increase in the length of concave synapses
(p<O.00 1 ; see Fig. 1.8). Active zone length by shape analyses were not significant. When
perforated synapses were analyzed separately, an ANOVA did not reveal any significant
diffemices in either the total synaptic length or active zone length between any groups. In
non-perjorafed synapses, the cornparisons between LTP and control animals were also not
significant in either layer. Analysis of the maximal perforation (gap) length revealed a main
effect of LTP group (p4I.00 1). with the mean length of the perforations increasing by 2 1.8
nrn or 28% in the LTP corn@ to the control animals.
Combining synaptic cwature with the perforated versus non-perforated distinction
for synaptic length revealed several additional fmdings. The significant increase in the total
length of al1 concave shaped synapses in the LTP-MML reported above was found only in
34
the non-perforated synapses Qd.045; i.e. the lengh of perforated concave synapses were
not significantiy different between LTP and contml animals; see Fig. 1.9). In the ML, the
opposite effect on the non-perforated concave synapses was observed: non-perforated . . concave shaped synapsesin the Lm-ML were significantiy smaller than the non-perforated
concave synapses in the control IML Q~û.024; see Fig. 1.9).
Correlationd Analysis
The correlations between morphological and electrophysiologicai measures, taken
from the multiple regression anaiysis, are presented in Table 1A for the MML and in Table
1B for the ML. In the stimdated MML, no structural characteristics were significantly
associated with the degree of potentiation. Severd structurai measurements, such as concave
shape etc., were significantly correlated with other structurai measurments, such as synaptic
length etc., but none of these measures were significantly correlated with the degree of LTP
expressed. In the IML, there were also no significant associations between any stnictural
measures and the degree of LTP. As in the MML. several structurai measures were
correlated with each d e r .
MIDDLE INNER
MOLECULAR LAVER
CONCAVE CONVEX FLAT IRREGULAR
Figure 1.1 The proportion of synapses with different curvature in the MML and IML of the implanted control animals. expressed as a a percent of the total syna ses. "a' indicates sign. different from "bu; 'cm sisn. different /= rom "dm (p~0.05)
CONCAVE CONVEX SHAPE
FLAT IRREGULAR
Figure 1.2 The proportion of synapses nith different curvatwes in the MML of the LTP and implanted control animals. '*' indicates a sign. difference between the LTP and control group (pg0.05)
O CONTROL
CONCAVE CONVEX FLAT SHAPE
IRREGULAR
Figum î.3 The proportion of synapses with difîbmk N v r t v s s in the M, af the LTP and imp1urbed control animrh. '*' indides a si- âffemnce bhhmn the Lfe and control gmups (p<0.05)
PROPORTION OF PERFORATED SYNAPSES ( X )
MIDDLE INNER LOCATION
TOTAL SYNAPTIC LENGHT (nm) NON-PERFORATED CONCAVE
B. MVER M O L E C U W LAYER
TABLE 1
CORRELATIONS BETWEEN THE DEGREE OF POTENTUTION AND THE VARIOUS STRUCTURAL h W U R E S AT 1 HOUR POST-LTP INDUCTION
A. MIDDLE MOLECULAR LAYER
#
Curvatun Concave (CC)
CC 1
Curvature Concave (CC)
I
Convcx (CX)
Flat (FU (negulm (IR) Active Zone Lemgth (Len) Perfonted Synapses Qert) Synapses Per Neuroa (S per N) Dtgrvc of LTP (LTP)
1 * * I
. e
L
e
I
4
. e
+
+
Convex (CX) Flat (FL) I m g a (W Active Zone Lnngth ( ' 0 )
Perfoirtcd Syaapscs (Pctf) Synapsea Per Neuron (S per N) Degree of LTP (LTP)
P a f 0.36
CX 1 FL
CC Perf' S per N LTP IR
IR 6.15
0.01 0.87* 0.44 0.33
1
-0.29
Len
4.30 1 I
* I
Len 0.25
S perN 0.16 0.28
CX 1 ~ - 0 . 2 2 6 . 2 7 ~ ~
0.28 0.17 0.24
1 L
8
LTP '
-0.38 0.22 0.06 -0.30 6.21
FL
0.33 4.35 -0.23 -0.21 '
I
e
I
I
- +
1
-0.1 1 1 0.20
1
8
I
I
L
0.06 1
w I
I
I
-0.05 0.42 0.20 0.32
1
4
-0.04 1
-0.01 0.35
1 I
I
I
-0.09 1 4
0.01 0.28
1 e
L
-0.33 0.46
1
0.36 4.14 -0.1 1 0.25 0.09
1 I
4.14 -0.25 0.26 '
4.04 -0.41 0.09
1
1
Discussion
No signifiant change in the number of synapses per neuron was found during the
induction / early maintenance phase of LTP (1 h pst-LTP induction). There were, however, -'* .
significant alterations in the s t r u c h of existing synapses. In the MML, LTP was associated
with: (1) an increase in the proportion of perforated synapses (due primarily to concave
perforated synapses), (2) a larges proportion of irreguiar shaped synapses and fewer convex
shaped synapses, (3) an increase in the length of non-perforated synapses (due pnmarily to
concave non-perforated synapses). In the UIL, LTP was associated with: (1) a decrease in
the proportion of convex synapses, (2) a concomitant increase in flat synapses, and (3) a
decrease in the length of non-perforated concave synapses. The length of synaptic
perforations was found to be larger in both the MML and IML following LTP induction.
Methodoogicd Considerations
The middle third of the dentate molecular layer (Ma) was used as the area of
primary interest in the LTP animals because the MML has ken showri to receive the
majority of its afSerent neural contacts from perforant path axons. Conversely, the IML
served as one of the control sites because it receives primarily commissural input rather than
direct perforant path innervation (Amaral and Witter, 1989). Due to numerous a priori
morphological differences between the layers and previous findings of significant synaptic
structural alterations in the IML following LTP induction, it was important to examine the
IML of LTP animals and both the IML and MML of implanted-only control animsls.
The use of "induction / e d y maintenance" to desaibe the post-induction time pcriod
of 1 h, attempts to differentiate this time point fiom later intermediate (approx. 24 h) and
long-term (days) phases of maintenance. These phases may compliment the
electrophysiological stages of LTP previously described (Racine et al., 1983; Adams et al.,
46
1997). Furthemore, various synaptic structural changes have also been found by other
mearchers at different pst-induction t h e periods (e.g. k m o n d and Levy, 1983, 1986;
Geinisman et al., 1993, 1996). This terminology, however, is meant only as a general d e -
description of the pst-Lm i n d d o n phases.
Synaptic Number
No overdl change in the number of synapses per neuron was observed in the present
research. Desmond and Levy (1 983, 1 WO), also did not find an increase in the total number
of synapses per unit volume fiom 1 to 6 h pst-LTP induction, and Geinisman et al. (1 991,
1993) did not fud a significant change in the number of synapses per neuron (using unbiased
stereological techniques). Lee et al. ( 1980, 198 1 ), however, found a significant increase in
the number of axo-dendritic (shaft) synapses 10 min &et the completion of the LTP
stimulations. Increased numbers of axodendritic synapses at 10 min., 2 h, and 8 h post-
induction were also found by Chang and Greenough (1 984). While these changes are not
overall differences in synaptic number, they are mentioned here because they represent
changes in a distinct category of synaptic contacts (Le. axo-denciritic venus axo-spinous)
present in the dentate g y n ~ ~ (Amaral and Witter, 1989). The changes discussed below
involve curvatwe, perforation, and length diffmnces in al1 synapses prwnt with the
majocity king axo-spinous ( A m d and Witter, 1 989).
S y ~ p t i c Cuwatwe
Concave shaped synapses, whiie not increasing overail, were integrai to the present
fïndings. Non-perforated concave synapses were found to increase in length and more
perforated synapses were concave following LTP (see below for M e r discussion).
Desmond and Levy (1983) observed an increase in the number of concave synapses in
animals sacrifced 10 and 60 min following tetanization. Further, Petit et al. (1989) observed
47
more concave synapses in the hippocampal CA1 region 2 h following kanic acid induced
synaptic activation. Increaseâ concavity in synapses has also been observed following the
induction of kindling (Cronin et al., 1987). Therefore, an increase in concave synapses (or in d B *
the present experiment, c&icavé&h$orated synapses) appears to be a consistent fmding after
synaptic activation.
In the present study, LTP was also associated with an increase in the proportion of
imgdar shaped synapses and a decrease in convex shaped synapses in the MML. Irregular
and concave shaped synapses have been found to change their relative proposions in the
same direction following neural stimulation (Petit et ai., 1989). Imgular shaped synapses are
also known to be the largest and most ofien perforated of the synaptic curvature subtypes
(Desmond and Levy, 1986; Markus et al., 1994). Finding an increased proportion of
imgular synapses foilowing LTP in the present study suggests a movement towards more
numerous large complex perforated synapses, and is consistent with previous findings of
increased synaptic length and perforations following LTP (Desmond and Levy, 1986;
Geinisman et al., 1 99 1, 1993).
A decrease in convex shaped synapses may represent a reduction in the number of
synapses that have lower efficacy (Petit et al., 1989). If concave synapses represent
potentiated synapses, it may be that synapses which appear in the inverse curvature (convex)
reprcsent synapses with lower synaptic efficacy (sa beiow for a discussion of cwature and
synaptic hction). Fewer convex synapses in the MML may, thereforc, lead to greatcr
potentiation in the system. Changes w m also obsmed in the IML tissue between the LTP
and conml animals. It is difficult to draw any specific conclusions fiom these results
because the ways in which the IML synapses affect the level of potentiation in the dentate
gynis are not yet known. These findings do, however, add support to the contention that
synapses in the IML are also afliected by the induction of LTP.
Synaptic P erforatioon <"
LTP was associateâ-with aaignificant increase in the propartion of perforated
synapses in the present expriment. GeUiisrnan et al. (1 99 1,1993) also found an increase in
the proportion of perforated synapses 1 h after the f d LTP tetankation (aithough their
stimulation protocol involved 4 days of LTP inducing sessions). Buchs and Muller (1996)
utilized calcium accumulation markers to identiQ activated synapses and found that these
activated synapses were more perforated 1 h following LTP induction.
Perforated synapses have also been show to increase following kindling (Geinisman
et al., 1 W), and other types of synaptic plasticity (Greenough et al. 1978, see Jones and
Harris, 1995 for review). At longer pst-induction tirne intervals, the significant increase in
perforated synapses is no longer observed (Geinisman et al., 1996). Therefore, the increased
proportion of perforated synapses would appear to be unique to early pst-induction time
intervais.
Length of Synapses
In the MML, overall synaptic length was increased following LTP in the present
shidy. While no overall change in synaptic length has previously been observed in the
dentate gynis, synaptic apposition length bas been show to change in the CA1 region of the
hippocampus foilowhg LTP (Chang and Greenough, 1984; Fifkova, 1985). Interestingly,
the apposition length of CA1 synapses was found to decrrase following LTP in the first d y
(Chang and Greenough, 1984), while an increase in length was observed in the second study
(Fifkova, 1985). Thus, alterations in synaptic length following LTP may not be a unitary
phenornenon; indeeâ, the research to date suggests that there may be a complex interaction
between synaptic shape, perforations, and synaptic length over t h e following LTP induction
(see below for discussion).
Correiationul Analysis
N o synaptic structural chaiacteristics were significantly correlated with the degree of
potentiation king expressed in either the directly stimulated MML or the IML of the LTP
animds. This findings appears to suggest that whiie overail structural changes may have
begua to occur by 1 h pst-LTP induction, these changes are not significantly related to the
degree of potentiation king expressed by each animal. One explanation for this result is the
large contribution of the molecular and cellular mechanisms described above. For example,
elevated kinase levels are known to remain 1 h after tetanization ( 0 t h and Ben-Ari, 1993)
and this elevation no doubt contributes to the degree of potentiation expressed at 1 h post-
LTP induction. Results from later time peiods dernonstrated that as these molecular levels
subside, structurai correlates do becorne significantly associated with the degree of
potentiation (see below) so the structural changes observed at 1 h may represent the initial
stage of functionally relevant structural alterations.
Dynamic Interucf ions
In this series of experiments, several aspects of synaptic structure (e.g. synaptic
cwature, length or perforations), while not showing overall changes following LTP, were
altered within specific synaptic subtypes. These interactions may ais0 be important in
understanding the synaptic structurai b i s of LW.
Chnges in the Direct& Stimuhted MUL. The ciimnt msults suggest an interaction
baween synaptic curvature, perforations, and length While the proportion of concave
synapses did not change overall, concave perforated synapses were the only synaptic type to
show a significant proportional increase following LTP. Further, a signifïcant overall
50
increase in length occurred ody in the non-perforated concave synapses. The present resdt
of incrrased length in the non-perforateci concave synapses is in agreement with Desmond
and Lew (1986) who also found that the PSD's of concave synapses became larger at 10 to ,*'
60 min following LTP indiction.*'~s mention4 above, these data support the importance of
concave shaped synapses and point out the necessity of considering the complex interactions
which occur among the various structural characteristics.
Changes in the NUI-Directly Stimuiated IML. The proportion of convex synapses
was decreased and the proportion of Bat synapses increased in the IML following LTP.
Differences in cwa tun have not often k e n studiad in the IML following LTP or other
foms of neural activation. In this series of experiments, later time points did not involve
differences in curvature in the IML (see below). Thexfore, the decrease in convex synapses
and concomitant increase in flat synapses, which may represent an interconversion fiom
convex to flat shaped synapses, appears to reflect a change unique to this early post-LTP
induction phase.
LTP-associated changes in synaptic morphology in the IML may be related to the
observation that mossy fibers can be induced to sprout following perforant path activation
(Adams, et al., 1997; Escobar, et al., 1997). Another possible explanation for the changes in
the IML is that polysynaptic activation may be sac ien t to induce alterations in synaptic
structure in the excitatory U o r inhibitory fecdback cirîuitry in the dentate gyw. It is also
possible that the changes observed in the IML spapses arc part of a total reaction of the
denciritic arbor ta selective activation of a specific synaptiddendritic region (Le. the MML).
These results reinforce the importance of examinhg the entire denciritic extent of the neuron
following tetanization and suggests that alterations in synaptic structure (possibly in different
directions) in parts of the neuronal denclritic structure, that are not being directiy stimulated,
may play an integral role in the induction and maintenance of LTP.
Functional Relevonce of Obserwd Structural Changes ,** .
The ways in which chengSin curvaturz effect synaptic hct ion are not yet Mly
understood. Synaptic curvature rnay alter synaptic efficacy by changing the proximity of
receptor channels to the dendritic shaft or by increasing the amount of synaptic contact area
(see Markus and Petit, 1989; Petit, 1988, 1995). Presynaptically, cwature rnay effect
calcium concentrations leading to altered probability of transmitter release. By using a
computer mode1 to simdate the activity of synapses with different curvature types, Ghaffki
et al. (1997) showed that synapses with a concave or imgular shape and perforations have a
higher probability of transmitter release. Conversely, convex shaped synapses may lead to a
decreased probability of release. Therefore, the alterations in the proportion of concave and
convex synapses in the MML and IML tissue in the cuirent study rnay play a critical role in
the potentiated response.
Functionally, perforated synapses are thought to represent large, mature, and highly
efficacious synapses that could affect the amount of potentiation observed (Calverly and
Jones, 1990; see Jones and Harris, 1995 for discussion). These mearchers have theorized
that the perforations rnay allow for a greater proximity of the presynaptic calcium c h a ~ e i s to
the vesicular release sites and thercfore a gratter probability of transmitter release. As
suggested by Petit (1995), Geinisman et al. (1993), and ouias, perforated synapses may also
reprisent an intermediate structurai configuration. With continued stimulation these
perforated synapses rnay continue towards division and the formation of two new
independent synapses. The increased proportion of perforated synapses in the present mdy
may, therefore, support the potentiation observed.
Synaptic length, also found to increase in the present experiment. may be associated
with synaptic efficacy as larger synapses may have mon comective strength (see Petit, 1988
for discussion). Recent support for this hypothesis has corne fiom an expriment by
Mackenzie et al. (1999). By ushg calcium imaging they measured the NMDA receptor-
mediated miniature synaptic calcium transients amibuted to the release of single trammitter
quanta. They proceeded to compare the ultrastructure of the various synapses that had been
imaged to the number of miniatures and found that synapse size correlates positively with the
amplitude of the postsynaptic response. They suggested from these results that larger
synapses express a greater number of NMDA receptors and therefore have greater comective
strength.
Conclusions: Synaptic Remodelling at I h Post-LTP Induction
The 1 h pst-LTP induction tirne period is associated primarily with an increased
proportion of concave shaped perforated synapses and increased length of non-perforated
concave synapses. As outlined above, these results are similar to pnvious research at early
pst-induction time periods (Geinisman et al., 1991, 1993; Desrnond and Levy, 1983, 1986).
Toni et ai. (1998) found more perforations in activated synapses initially following LTP
induction, which was later replaceâ by an increase in the proportion of double synapses (two
spines connected to the same presynaptic terminal). These results provide additional support
for the possibility tbat activated synapses may pcrforate and divide following the induction of
LTP. Some data suggest, howevet, that pcrforated synapses do not represent an intemediate
stage of synaptic division (Jones et al., 1992; Dyson and Jones, 1984).
The present fmdings of increased concave perforated synapses and larger non-
perforated concave synapse may represent an early step in a morphological transformation
where potentiated synapses move toward a concave shape, grow in length, and become more
perforated. Cledy, the synaptic remodelling associated with LTP is a dynaniic and complex
pmcess involving alterations in various morphologid characteristics which are ôoth
denciritic region and pst-induction time interval dependent. The present experiment . . represents the earliest post-LTP u;huction t h e p e n d in a series of studies exarnining this
sequence of events. A general discussion of this synaptic structural sequence follows
detailed expianations of the other post-LTP induction time points.
SYNAPTIC STRUCTURAL PROFILE AT 24 HOUR POST-LTP INDUCTION
Introduction
The majority of experimentation on the mechanisrns underlying LTP have examined
the cellular and molecular processes that are thought to support the electrophysiological
potentiation. In most cases, these changes in chernicd and protein leveis are known to revert
to baseline levels well before the potentiation of the system ends (Otani and Ben-%, 1 993).
As discussed above. one candidate for the continued synaptic enhancement is a more lasting
change in the stmcture of potentiated synapses. Most of the previous experiments have
examined synapses following short pst-LTP induction delays (se+ Wallace et al., 1991 for
review). The present experiment examined the intermdiate maintenance phase of LTP 24 h
after induction and was conducted to build upon the previous results ai earlier post-induction
time periods. This experiment was particularly relevant due the lack of previous
experimentation during this phase of LTP maintenance when the electrophysiological
Materials and Methods
Animals
Fourteen adult d e Long-Evans h d e d nits were supplied by the Racine Laboratory
Breeding Colony (McMaster ~~&msit-y) for this study. These expimental anirnals were
compared to the 1 1 coatrol anirnals described above.
Stimulation
Trains consisted of pairs of 400 Hz bursts with durations of 27 ms (IO pulses),
separated by 200 ms, with an interotmin interval of 10 sec. A senes of 9 stimulation trains
were delivered to the perforant path once an hour for 5 h, for a total of 45 trains. Baseline
responses were retested 24 h after al1 trains had been delivered to ensure that LTP had been
induced and the animals were perfhed for synaptic anaîysis. Al1 other procedures did not
differ from those described in the General Methodology section.
Results
Synaptic Nurn ber
The unbiased estimate of the nurnber of synapses per neuron for both molecular
layea was used as the initial unit of analysis. An ANOVA revealed a non-significant
incrase of 1 1% in the total nmber of synapses per neuron in the MML between the LTP
and implanted control group following LTP. The MML of the LTP tissue had an average of
3 127 synapses per ne- while the implantecl control MML tissue averaged 28 16 synapses
per neunm. A main effect of location reveded that overall there were significantly more
synapses in the MML compareci to the IML @<O.ûûl) in ôoth the LTP and implanted control
group, but the LTP group by location interaction was not significant.
Synaptic Cutvahve
Overall anaiysis of curvature rcvealed a simcant increase in concave synapses in . .
the dirrctly stimulateci tissue (L%MML) relative to the MML of control animals @<0.001;
see Fig. 2.1). The proportion of concave synapses was 34% in the MML of the LTP animals
while only 22% in the MML of controls. nie proportions of the other shapes did not differ
significantly in either the MML or the IML between the groups.
Synaptic Perforations
The proportion of synapses that were pefiorated did not differ significantly between
the LTP-MML tissue (25.5%) and the implanted control-MML tissue (24.9%). The
proportion of perforated synapses was also not significantly different between the LTP-IML
Ussue (19.8%) and the implanted contml-IML tissue (20.6%). There was, however, a
significant main effect of location 6~3.026) whexe the IML had a significantly lower mean
proportion of perforated synapses (20.1%) compared to the MML (24.9%) but this difference
was not LTP specific.
Men the proportion of perforated synapses with specific cwatures was analyzed, a
signifiant change in the number of concave and flat pedorated synapses was observed in the
LTP tissue (see Fig. 2.2). The percent of concave perforated synapses (as a proportion of ail
perforateci synapses) was 38% in the MML of the LTP animals cornpareci to 19% in the
MML of controls m.012). The proportion of flat perforateci synapses significantly
declined tiom 49% to 2% in the MML of LTP animais compareci to implanted controls
@<0.001). Interestingly, the significant 19% increase of perforated concave synapses is
larger than the significant 8% overaîl increase in concave synapses and the nonsignificant
0.6% overall increase in perforated synapses. No other changes in the proportions of
56
perforated synapses with specifc curvatures, including those in the IML, were significant.
Synaptic Lengths
An ANOVA of synaptic length revealed a significant interaction between LTP group
and location @=0.004). ' Post-hocanalyses revealed that in the ML, synapses were
significantly longer in the LTP animals compared to the implanted controls. The LTP-IML
synapses were also found to be significuitly longer than synapses in the MML of both the
LTP and implanted controls. It is important to reiterate that the LTP-IML tissue was not
directly stimulated. The mean length of the LTP-ML synapses was 220 nm which was
approximately 10% larger than the synapses in the other three amdgroups (see Fig. 2.3).
No other significant differences were observeâ.
When synaptic length was analyzed in relation to synaptic shape, concave synapses
were found to be significantly srnaller in the LTP-MML tissue cornpared to implanted
control-MML synapses @=O.04; see Fig. 2.4). No other changes in lengths of different
shapes were significant. In perforated synapses, the length of the perforation (gap) was not
found to be significantly different between any of the groups. Further, the length of
perforated synapses did not differ significantly following LTP. An ANOVA exarnining only
perforated synapses rcvealcd that perforatd synapses of different shapes did not differ
significantly in length in the MML as a result of LTP. When only the non-perforated
synapses were examinai, the concave synapses were found to be significantly srnaller in the
LTP-MML tissue comparecl to irnplanted control-MML tissue Q~û.0085; see Fig. 2.5).
Thercfore, the overall decrease in length of concave synapses described above nsultcd
primarily fiom changes in the length of non-perforated concave synapses.
Correkatio~l Anulysis
The correiations between morphoIogicÛi anci electrophysiologicai measures, taken
fiom the multiple regressioa d i s i s , are presented in Table 2A for the MML and in Table
2B for the IML. in the stimulated MML there was a significant association between the
degree of potentiation and the numbcr of synapses per neuron Q~û.046). Higher levels of
potentiation were associated with a greater number of synapses per neuron and while this
association was significant in the stimulated MML, it was not significant in the IML (see Fig.
2.6). There were no significant associations bctween the degree of potentiation and any other
synaptic structural measure.
The observed association between synaptic number and LTP could have been due to
either an LTP-induced increase in synaptic number or because animals with a higher pre-
existing number of synapses show greater potentiation. To explore these two possibilities, a
descriptive anaiysis was conducted on the distribution of synaptic number per neuron in the
LTP and control aaimals. This analysis revealed that the mean number of synapses per
neuron was higher and the variance larger in the LTP animals compared to the non-
potentiated controls. These increascs w a found to be specific to the MML (see Fig. 2.7).
CONCAVE CONVEX FLAT
SHAPE
LTP n
CONTROL
IRREGULAR
Figure 2.1 The proportion of synapses with different shapes in the MML of LTP and implanted control animais. .*= represents a sign. difference between the LTP and contrd group (p*0.05)
MIDDLE
LTP
fl CONTROL
LOCATION INNER
LTP
CONCAVE CONVEX FL AT SHAPE
C] CONTROL
IRREGULAR
Figure 2.4 T h average maximum Iength of synapses w i t h dîfferent shapes in the W l of the LTP and implantad control animls.
Inacates a dgn. differcnce betneen the LTP and conttd groups (0~0.05)
LTP
CONCAVE CONVEX FLAT
O CONTROL
IRREGULAR SHAPE
Figure 2.5 Tha average maximum synaptic kngth of non-puforatd synapses w i U i ditfannt shapes in the MML of the LTP and implanbâ conkol urbnala inâicttss a sign. d i f fume betneen the LTP and contrd mwm b<O8O5)
A. MIDDLE MOLECULAR LAYER
B. INNER MOLECULAR LAYER
Figure 2.6 Tbe mmcirtion bctweea the d q n c of potc11tiatka (LW) and t h number of synapses pet neuron im (A) the middk molceulrr lryer and (6) the iancr mokculrr Isyer of tbe denîate gyrus. Thc regression liac in part A represcnts the sign. (pdl.05) carrelation bctwun the d e g m of LTP rad the nurnbtr of synapses pcr neuroa.
Fïurc 2.7 The scatter plot of the iiumbcr o f 'Y-(wa pcr reuron in the MML oltk L m and imphnted contrai groupa Tbe brr npnrcab îhe nmge of d u u without the highcrt and lowest outlyiog scores.
MIDDLE MOLECULAR UYER
t I I
CONTROL LTP
GROUP
INNUI MOLECULAR LAVER
CONTROL LTP
TABLE 2
CORRELATIONS BETWEEN THE DECREE OF POTENTIATION AND THE VARIOUS STRUtXuRAL MEASURES AT 24 HOURS POST-LTP INDUCTION
A MiDDLE MOLECtILAR LAYER
- --
I ~ l a ~ (FL) I * I *
Concave (CC)
Convex (CX)
B. lNNER MOLECULM LAYER
1
Active Zone Lcngtb (Lcn)
Pedorated Synapses (Perf)
Synapses Pet Ncuron (S per N)
Dcgiut of LTP (LTP)
1 Active Zone Lcagîh (Lm) 1 * 1 1
4.40
1
1 Degrec of LTP (LTP) 1 ' 1 ' 1 '
L
i
Discussion
The present data indicate that there are changes in the synaptic structural profile 24 h
&r the final LTP tetanizatioa. This alteration does not involve a signifiant change in the
total number of synapses per neuron in either the MML or the IML of the LTP animals.
T h was, however, a direct correlation between the degree of potentiation expressed by
eûch animal and their correspondhg number of synapses per neuron in the MML. LTP was
associated with a significant overall increase in the proportion of concave shaped synapses in
the MML and these concave synapses were also found to be significantly smaller. This
difference in length was limited, however, to non-perforated concave synapses. Conversely,
synapses in the IML of LTP animals were found to be significantly longer than ail other
groups. While there was not an overail increase in the proportion of perforated synapses,
more of the perforated synapses were concave in the directly stimulated LTP-MML tissue.
Methodological Corniderations
It is important to recall that there are pre-existing differences between the IML and
the MML and that these differences necessitate the consideration of both molecular layers in
the tetanized and implanted-only animals. In the present study, the significant increase in
synaptic length in the IML following LTP m e r points out the importance of examining
both thirds of the dentate molecular layer in stimulated and implant-only animals. As
discussed above, it has becorne increasingly acceptai that alterations to one part of the
neuron tend to induce changes in the synaptic p d l e in othu neuronal areas. For example,
Anthes et al. (1993) found that affetcnt denmation of the p r b u y MML inputs by adult
entorhinal cortex lesions also induced changes in the IML. These changes included a
signifiant decrease in synaptic nurnber as well as alterations in several aspects of synaptic
The use of the terni 'intermediate' to describe the pst-induction time period of 24
hours, attempts to differentiate between early maintenance (see above), intermediate
maintenance (approx. 24 h) and long-tenn maintenance (days). As mentioneâ, these phases I.
may compliment the electrophysioiogical stages of LTP previously described (Racine et al.,
1983; Adams et al., 1997). Since previous experimentation had not considered the 24 h post-
induction time period, uiere are no direct cornparisons for the present findings. The results,
therefore, will be discussed in relation to the result described above at 1 h pst-induction and
the other relevant results from earlier or later tirne points.
Synup~ic Number
Similar to the 1 h experiment, the present study did not find a significant change in
the overall number of synapses per neuron at 24 h post-LTP induction. As mentioned, Chang
and Greenough (1984) found an increase in axo-dendntic (shaft) synapses at 10 min., 2 h,
and 8 h pst-induction. Further, a significant increase in sessile spine synapses was found in
that study, but the increase in both of these synaptic sub-types lead only to a non-significant
5% overail increase in synaptic number (Chang and Greenough, 1984). The non-
significant 1 1% overall increase in the number of synapses per neuron in the MML of LTP
animais in the prcscnt experiment may be important if the selective nature of LTP is
considered. For example. McNaughton et al. (1 98 1) estimated that activation of only 1 -5%
of the synaptic contacts in the MML of the dentate gynis is sufficient to evoke a granule ceil
discharge. Further, -nt work on CA1 slices found that oaly 15% of synapses becorne
labelled as potentiatcd (based on calcium accumulation) following the induction LTP (Buchs
and Muller, 1996). The present experiment also reveaied a significant positive correlation
between the degree of potentiation and the number of synapses per neuron within the MML
of potentiated animals at 24 h pst-LTP induction (see below for discussion). Taken
68
together, these findings suggest that synaptic number may be important for the maintenance
of LW. Further delineation of the role of synaptic nurnber may occur as more specific
electron microscopie Iiibelling techniques becorne available.
Synopric Cww f w e
One of the major changes observed in the present study was an increase in the
proportion of concave synapses in the potentiated tissue. While not increasing overdi,
concave synapses were involved in differences at 1 h post-induction (see above). As
mentioned, previous research by Desrnond and Levy (1 983) aiso examined synaptic
cwature following the induction of LTP and found an increase in the number of concave
synapses in the dentate gynis in animals sacrificed at 10 and 60 min following tetanization.
Petit et al. (1 989) observed an increase in concave synapses in the hippocampal CA1 region 2
h following kanic acid induced synaptic activation and more concave synapses have dso
been observed following kindling (Cronin et al., 1 987). It appears that the movement of axa-
spinous synapses towards a concave configuration is a consistent result of synaptic activation
(see 1 h Discussion above for potential hctional consequences of curvature changes).
Synaptic Perforations
Unlike the 1 h group, the= was not a signifiant inc- in the overall proportion of
perforated synapses at 24 h post-LTP induction. As mentioned, a nurnber of previous studies
have found an incttase in the numkr ofpatoratui synapses following LTP (Geinisman et
al., 199 1 ; 1993; Buchs and Muller, l996), kindling (Geinisman et al., 1992). and o t k types
of synaptic plasticity (Greenough et al. 1978, se Jones and Harris, 1995 for revkw). More
recently, Geinisrnan's gmup did not h d an increase in perforated synapses 13 days
following the last tetanization (Geinisman et al., 1996). Due to diffemncts in stimulation
protocol and pst-induction delay, the current resdts, rather than contradicting previous
69
research, suggest that the number of perforated synapses observed may be depcndent u p n
the phase of LTP maintenance.
Length of SyMpes
Unlike the 1 h p u p , the& was no signifiiant overall change in synaptic leiigth in the
LTP-MML compared to the implanted control MML at 24 h post-LTP induction. The lack of
change in overall length, while suggesting that LTP is not associated with a universal change
in synaptic length, does not d e out the possibility of altered length within specific synaptic
subpopulations. As in the 1 h group, concave shaped synapses were involved in changes of
synaptic length at 24 h post-LTP induction. In the present experiment concave synapses
were found to be significantly smaller in the MML of the LTP animals. This finding
represents a reversal of the Uicrease in concave synapse length observed at I h in this series
and at 1 h and 6 h in previous research (Desmond and Levy, 1986).
Overall, synapses in the not-directly stimulated LTP-IML were significantly longer
than in d l other groups. It is intereshg that al1 synaptic curvature types increased in length
in the IML, while only specific cwature types were afTected in the MML. As mentioned,
changes in synaptic morphology in the IML, in mponse to LTP induction, are consistent
with the observation that mossy fibers, which send projections back to the IML, can be
induced to sprout following perforant path activation (Adams, et al., 1997; Escobar, et al.,
1997). Polysyneptic activation in the MML, as inducd in die pnsmt study, may be
sufncient to induce aitcrations in synaptic structure in the excitatory andor inhibitory
feedback cimlltry in the dentatc gynu. Another possibility is tint the strong @orant path
activation induced a compensatory heterosynaptic enhancement of inhibition affecthg IML
projections. This explanation seems less likely, because one member of our research group
(Racine, unpublished results) has found no evidence for changes in paired pulse inhibition in
70
this system following the induction of LTP.
The comlational analysis nvealed a significant positive association between the , .
degree of potentiation and the number of synapses pcr nemon in the MML of the LTP
a n a s . This association was not observeci in the IML and no other synaptic parameters
were associated with the degree of potentiation in either rnolecuiar layer. The main issue is
whether the positive association between synaptic number and the amount of potentiation
expressed reflects an active increase in the nurnber of synapses after the LTP was induced or
a preexisting condition where animals with more synapses can express a greater degree of
potentiation. If the latter explanation were me, the potentiated and control animals shodd
display a similar distribution of synapses per neuron. This does not appear to be the case as
the mean number of synapses per nemn is 1 1% higher and the variance is larger in the LTP
group. Although this increase in synaptic nwnber is not statistically significant, the
differences in the synaptic distributions seem to support the suggestion that new synapses
have focmed by 24 h pst-LTP induction.
Further support for the formation of new synapses &ses fiom the correlational results
h m the 1 h gmup. If the diffetcnces in LTP w m the m l t of pre-existing diffmnces in
the number of synapses, the 1 h poup should aiso display a positive association between the
numbct of synapses and the degtee of potentiation expresseci. This, however, was not the
case as the association was not signifiant at 1 h pst-LTP induction. It would appear that
changes in the number of synapses per nemn arc associated with the degree of potentiation
expressed b y individual animals.
DyMmic Interactions
As noted above, the cunent resdts demonstrated b t concave synapses in the LTP-
MML were significantiy smaller 24 h d e r the last tetanization which is the reverse of
changes observed at earlier pst-induction tirne points. Previous work in this laboratory
found that newly formed adult synapses are smdler than pre-existing synapses and simiiar to
the synapses observed during development (Anthes et al., 1993; Markus and Petit, 1987). If
LTP results in the de-novo formation of new concave synapses, this would provide one
possible explanation for the present finding that these synapses are smaller. Another possible
explanation is that synaptic activation, including the induction of LTP, involves a series of
changes in synaptic structure wbich leads to the division of activated synapses (see Generai
Discussion).
Conclusions: Synaptic Remodelling at 24 h Post-LTP Induction
The 24 h pst-LTP induction time period is associated primarily with an increased
proportion of concave shaped synapses and decreased length of non-perforated concave
synapses. Further, the degree of potentiation was found to be significantly and positively
associated with the number of synapses pcr nemn in individual animals. Together with the
observations fiom 1 h post induction, these mults provide support for the possibility that
activatcd synapses may becorne concave, perforate and then dinde into smaller more
numemus synapses foiiowing the induction of LW.
The pnsent findings may represent an intemediate step in the morphologicai
transformation. As stated, the synaptic remodelling associated with LTP appears to be a
dynamic and complex process uivolving alterations in various morphological characteristics
which are both dendritic region and pst-induction time interval dependent. The present
72
experiment represents the second pst-LTP induction time period in a senes of studies
examining this sequence of events. A general discussion of this synaptic structurai sequence
follows detailed explanations of the last pst-LTP induction tirne points. . ..
* -
SYNAPTIC STRUCTURAL PROFILE AT 24 HOUR POST-LTP INDUCTION UNDER KETAMLNE PHARMACOLOGICAL
BLOCKADE
Introduction
When considering electrophysiologicai models such as LTP, one major issue is
whether any changes observed are due to LTP specifically or result merely fiom the electricai
stimulation. One way of addressing this concern is to apply the electrophysiological
stimulation in the presence of a phamiacological agent known to block the development of
the potentiation. As mentioned above, the NMDA receptor plays a critical role in the
production of LTP. This receptor, therefore, provides an ideal target for iriterfering with the
induction of LTP. Ketamine is a cornpetitive NMDA antagonist which cornpetes for the
glutamate recognition site on the NMDA receptor wmplex (Izquierdo and Medina, 1997).
When applied prior to tetanization, ketamine has been found to block the formation of LTP
(Otani and Ben-An, 1993). This experiment was conducteâ to replicate the initial
expriment at 24 h under phamacological blockade to identiQ LTP-specific versus general-
stimulation derived changes in synaptic structure.
Materials and Methods
Ten adult male Long-Evans hooded rats were supplied by the Racine Laboratory
Breeding Colony (McMaster University) for this study. These experimental animals were
compared to the 1 1 control animals descnbed above and the 14 LTP potentiated 24 h
animals.
Stimulation
Stimulation trains mnsistcd of pairs of 400 Hz butsts with durations of 27 rns (10
pulses), separated by 200 ms. witii an inter-ûain interval of 10 sec. A series of 9 stimulation
trains were delivered to the perforant path once an hou for 5 h, for a total of 45 trains.
Approximately 15 minutes before each stimulation session, the experimental animals
received an interperitoneal injection of the cornpetitive NMDA receptor antagonist ketamine
at 70 mgkg. Baseline responses were retested 24 h after al1 trains had ken delivered to
assess the level of potentiation and the animais were perfused for synaptic analysis. Al1 other
procedures did not differ fiom those described in the General Methodology section.
Results
EZectrophysiological Remh
The final assessrnent of badine responses directly prior to sacrifice confirmed that,
despite receiving tetanic stimulation, no potentiation had occumd in any of the animais
included in the ketamhe group.
Synopiic Nurnber
The unbiased estimate of the number of synapses pet neuron for both moleculair
layers was used as the initial unit of analysis. An ANOVA revealed no significant changes in
the total nwnber of synapses pet newn in the MML or IML between the ketarnint group,
Unplanteci control group, or the LTP 24 h animais. The MML of the ketamine tissue had an
average of 27 1 1 synapses per neuron, the implanted control MML tissue averaged 28 16
synapses per neuron, and the LTP 24 h gmup averaged 3 127 synapses per neuron.
syMpic Curvaiure
Overail analysis of curvahve did mt reveal any significant differences in the hIML of
the ketamine animals compared to the MML of controi anirnals. The ketamine group did,
however, differ fiom the 24 h group as the proportion of concave synapse was 26% in the
ketamine group and 34% in the 24 h group (p4.0024). niere wen aiso no differences in the
IML synapses.
Synuptic Perforations
The proportion of synapses that were perforated did not di ffer signi ficantl y between
the ketamine-MML tissue (22.5%), the implanted control-MML tissue (24.9%), and the 24 h
group (25.5%). The proportion of perforated synapses was also not significantly different
between any of the groups in the ML. When the proportion of perforated synapses with
specific curvatures was analyzed, the proportion of concave perforated synapses differed in
the MML across the three groups (see Fig. 3.1). The percent of concave perforated synapses
(as a proportion of ail perforated synapses) was 26% in the ketamine animals which was not
significantly different fiom the 19% in the MML of controls, but was significantly different
h m the 38% obsewed in the 24 h group Q~û.0073). In the IML, the proportion of
perforated convex synapses was 9% in the ketamine group which was significantîy lower
tbaa the wntrols at 19% and the 24 h group at 27% (pK0.00 1).
Synaptic hngths
An ANOVA of total synaptic length did not reveai any signïficant overall differences
between the ketamine, control, or 24 h groups in either molecular layer. Analysis of the
maximum active zone length revded a significant main effect of ketamine @=0.0 16) but no
group by location interaction. The ketamine group's rnean active zone length collapsed
across molecular layers was 193 nm which was significantly larger than the control groups
mean length of 184 nm but did not differ h m the 24 h group which had a mean length 192
nm.
An ANOVA of length also revealed a significant main effect of ketamine
0 . 0 4 9 ) . The mean perforation length of the Icetamine perforated synapses collapsed
across molecular layers was 8.8 nm or 13% smaller than in the control perforated synapses
and 6 nm or 10% smaller than the 24 h group. When perforation length was analyseci in
synapses with different cwatwes, an ANOVA revealed a significant ketamine gmup by
shape interaction (p=0.0 14). Post-hoc analyses revealed that perforations were signi ficantl y
smaller in concave perforated synapses in the MML of the ketamine group compared to the
control and the 24 h groups (see Fig. 3.2).
When total synaptic length was anaiyzed in relation to synaptic shape, post-hoc
analyses revealed that irregular shaped synapses were significantly larger in the IML of the
ketamine group (mean=359 nm) compared to the control (mean=297 nm) and 24 h groups
(mean=261 nm). No other changes in the length of synapses with different shapes were
significant .
An ANOVA examining the total length of only perforated synapses revealed a
signifiant main effect of group @=0.02) but no group by location interaction. Ketamine
caused an o v e d incnase of 38 nm or 14% in the total synaptic length of perforatcd
synapses compared to the conid group and an increase of 36 nm or 13% compared to the 24
h group mem. When the active zone length was examined in perforated synapses, the same
main effect was observed (p4.001). When only the ncn-perforated synapses were
examined, no significant differences in total synaptic length were observed.
When the !engths of pedorated versus non-perforated synapses with different
curvatures wen examine& the only significant finding was that ketamine was associated
with a signifrcmt increase in the length of inegular shapeâ non-perforated synapses in the *'*
IML @<O.OS). These irregular shiped synapses were an average of 102 MI or 48% longer in
the ketamine IML tissue when compared to control IML synapses but only 50 nm or 25%
longer than the non-perforated irregdar shaped synapses in the 21 h group (see Fig. 3.3).
Unlike non-perforated synapses, perforated synapses with different shapes did not differ in
total length or active zone length.
PROPORTION OF PERFORATED CONCAVE SYNAPSES IN MML ( X )
CONTROL KETAMINE LTP
GROUP Figure 3.2 The average kngth of synaptic prn#rtlan In the p(KfOrPtsd synapses in the MML of the LW, ketamine, and bnplanted cocrtrol groups. '*a mpmsents sign. âiffennt from LTP and contm1 groupr (p<O*OS)
CONTROL KETAMINE LTP
GROUP Figwe 3.3 The wemge maximum length of non-pertbntad synapses with an imegular shape in the IML of the CTP, ketmine, and impîuikd control animals. 'an npresents sign. different fm the LTP and contrd group (p<O.OS)
Discussion
The present data indicate that while no electrophysiological potentiation was
observed, some changes in the synaptic structural profile persisted under ketaMm blockade .*-
at 24 h pst-LTP induction. Th&-changes, however, were not the same as in the LTP 24 h
group and the overall pattern was more similar to the control group. Uniike the marpinaily
significant 1 1% increase Ui synapses per neuron observed in the 24h group, ketamine was not
associated ~6th any change in the total number of synapses per neuron. 'ïhere were also no
changes in the overall proportion of synapses with different curvatures or in the proportion of
peiforated synapses in the ketamine animais compared to the control group. Ketamine was,
however, associated with a significantly lower proportion of concave shaped synapses when
compared to the 24 h group.
A consideration of the proportion of perforated synapses with different curvanires
revealed that the ketamine group was shilar to controls in displaying a lower proportion of
concave perforated synapses than the 24 h group. Ketarnine was not associated with an
overall change in total synaptic length but was associated with a significant increase in active
zone length, although this difference was not molecular layer specific. The length of other
subgroups, particularly irregular shaped synapses, also changed following ketamine
bloc kade.
Methodoiogical Conriderutions
As described above, kctsmine is known to block the formation of LTP (Otani and
Ben-Ari, 1993). One conceni, however, is that pharmacoIogical blockade! may cause 0th-
extnuieous compensatory changes in the neural system. Revious research has shown that
besides blocking the activity of the NMDA receptor, MK-80 1 (a non-cornpetitive NMDA
antagonist) can cause compensatory excitotoxicit. and neural death (Weeks et al., 1993).
81
The current f'indings also demonstrate the need for caution in interpretirig the synaptic
structural results following the use of a pharmacological blocket. Despite the lack of
potentiation, novel changes in synaptic structure were observed 24 h pst-LTP induction
under ketamine blockade. The foiiowing sections discuss these changes and compares thern
to the structurai profile observed in the control animals and the potentiated 24 h group.
S'ynopric Nimber
As in the 1 h and 24 h group, this experiment did not show a significant change in the
overall number of synapses per neuron at 24 pst-LTP induction under ketamine blockade.
Based on the finding of a significant positive correlation between the degree of potentiation
and the number of synapses per neuron within the MML of potentiated animals at 24 h, it is
particularly interesthg to h d no increase in synaptic number in the ketamine group which
also displayed no electrophysiological potentiation despite receiving the same amount of
tetanization. Taken together these results suggest a role for synaptic nurnber in the degree of
potentiation displayed at 24 h pst-LTP induction.
Synaptic Curvatwe
One of the major changes observed in the LTP 24 h experiment was an increase in the
proportion of concave synapses in the potentiated tissue. No overall curvature changes were
observed in either rnolecular layer in the ketamine animals. This would appear to indicate
that the o v d l cwaturc changes observeci at 24 h an due to the induction and maintenance
of LTP and not simply tetanbation. As d i s c d above, the prcsence of incrcased concavity
is a consistent hding of synaptic activation (Petit, 1995). The lack of this change in the
ketamine group suggests that changes in curvatwe may be LTP-specific.
Synaplic P e r f h i n s
As in the 24 h group, the ketamine anhals did not display an ovedl change in the
proportion of perforated synapses in either molecuiar layer. When the shape of the
perforated synapses was considered in the MML, the ketamine group did not differ h m the
control group. fmportantly, the increase in concave perforated synapses observed in the
potentiated 24 h group was not observed in the ketamine group. Perforations dong with
concavity have been consistently associated with synaptic activation and the lack of an
increase in this subgroup in the ketamine MML suggests that this change is also LTP
specific. No changes were observed in the proportion of non-perforated synapses with
different shapes.
In the IML, ketamine was associateâ with a decrease in proportion of convex shaped
perforated synapses compared to the control group and the potentiated 24 h group. It is
difficult to speculate on what importance this finding may have other than to reiterate that the
molecular layers interact and that ketamine or simply tetanization may be exerting an effect
on the structure of the IML synapses.
Length of Sympses
Ketamine was not associated with an overall change in total synaptic length but was
essociated with a signincant increase in active zone length in both the MML and IML. This
change in synaptic length rnay repmnt a general stimulation effect (as opposed to an LTP-
specific effect) due to the similarity betwecn the ketamine group and the potentiated 24 h
group. It appears that the synaptic active zone lengthens in a non-layer specific and non-
subgroup specific way following tetanization regardless of whether or not LTP was induced.
The length of the perforations in perforated synapses were found to be significantly
smaller in the both the MML and M L of the ketlmlle group cornparcd to the control and 24
h gmups and this was particularly evident in concave perforated synapses. If. as arped . . above, these perforated conixve sjhapses represent highly efficacious synapses, the
reductioa in the perforation size may reprisent a change (decrease) in the efficacy of these
synapses.
Men the length of synapses with different curvahires was analyzeà, irregular shaped
synapses were significantly larger in only the IML of the ketamine group and this was
particularly evident in non-perforated irregular synapses. As mentioned, it is dif'ficult to
speculate about the functional importance of synaptic structural changes that are specific to
the IML. One possibility is that these imgular shaped synapses are involved in commissural
communication and are altered during tetanization under ketamine blockade (Am& and
Witter, 1989).
Perforated synapses in general were also significantly larger in the MML and IML of
the ketamine group. Due to the lack of layer specificity of this finding it appears that
ketamine may induce a generai increase in the length of perforated synapses. Again the
functional nlevance of this finding is unclear. Perforated synapses are, however, a consistent
indicator of various forms of synaptic plasticity and the change in their length observed in
îhis experiment may represent an important aiteration (ûreenough et al., 1978; Geinisman et
al., 1991,1993; Jones et al., 1991).
Conclwions: LTP-Specific versus Generui StiwtuIution Eflects
It seems clear that most of the changes observed at 24 h pst-LTP induction do not
persist under ketamine blockade. Specifically, the increase in the number of concave shapad
synapses, the increase in perforated concave synapses, and the decrease in the length of non-
84
perforated concave synapses are not observed if LTP is not induced. It appears that these
changes are LTP-specific and do not mur simply in response to tetanbation.
Ketamine was associated with several novel structural changes in synaptic length and . .
imgular shaped synapses particulady which indicates that the use of ketsmine rnay cause
extraneou andor compensatory changes in the system. The fact that most of the structural
fhdiags were main effects (not Iayer specific) ais0 suggests that they resulted fiom the
ketamhe and not the layer specific stimulation The current results appear to indicate that
specific structurai changes occur during the maintenance of LTP.
SYNAPTIC STRUCTURAL PROFILE AT 5 DAYS POST-LTP INDUCTION
Introduction
The b t three experiments in this series have described the induction (1 h) phase of
LTP, the intermediate maintenance (24 h) phase of LTP, and LTP specific changes using
ketamine blockade. The electrophysiological potentiation following the fom of tetanization
used in these experiments is known to last at significant levels for at least one week (Maienka
and Nicoll, 1993). Like the 24 h post-LTP induction time point, synaptic structural change at
5 days may also help support the conhuing potentiation as molecular changes retum to
base fine.
As mentioned, almost al1 previous research examining synaptic stnichire associated
with LTP has foc& on short pst-induction time periods. Geinisman et al. (1996),
however, examined synaptic structure 13 days after the induction of LTP and found more
numerous axodendritic synaptic contacts but no change in axo-spinous synapses. Geinisman
et al. did not fmd the changes in the number of perforated synapses obmved at 1 h fiom the
end of tetankation (Geinisman et ai., 1992,1993). They concluded that long-term
85
mainte11811ce is associated with a unique synaptic structural profile.
This expriment was designed to examine synaptic structurai change associated with
the long-terni maintenance of LTP. The stimulation pmtocol and general methodology ..B.
rernained the same as in the previimsly described experiments. This ensureci the
comparability of the results h m the three different pst-induction time points. Added to the
results found at 1 h and 24 h post-induction, the current findings completed a description of
the time dependent sequence of synaptic structural change associateû with LTP.
Materiah and Methods
Animals
Nine adult male Long-Evans hooded rats were supplied by the Racine Labonitory
Breeding Colony (McMaster University) for this snidy. These experimental animals were
compared to the 1 1 control animals described above.
Stimula f ion
Trains consisted of pairs of 400 Hz b u t s with durations of 27 ms (10 pulses),
separated by 200 ms, with an inter-train interval of 10 sec. A series of 9 stimulation trains
were delivered to the perforant path once an hour for 5 h, for a total of 45 trains. Badine
nspo~scs were retested 5 days d e r dl trains had ken delivered to ensure that LTP had been
induced and the animais wen then perfbed for synaptic analysis. Ail other procedures did
not Mer h m those dedbed in the General Mehdology section.
Rcsults
Sywptic Counts
The unbiased estimate of the number of synapses per neuron for both molecdar
layers was used as the initial unit of analysis. An ANOVA did not reveal any significant
changes in the total number of synapses per neuron in the MML or the IML between the LTP
86
and implanted control group. In the MML, the LTP tissue had an average of 2684 sjmpses
per nemn while the implanted control MML tiswe averaged 28 16 synapses per neuron.
Synuptic Curvahrre
Overall analysisof curvature did not nvealed any significant differences in the hlML
of the LTP animals relative to the MML of eontrol animals. There were also no differences
in the IML synapses.
Synaptic Perfiations
The proportion of synapses that were perforated did not differ significantiy between
the LTP-MML tissue (25.5%) and the implanted control-MML tissue (24.9%). The
proportion of perforated synapses was also not significantly different between the LTP-ML
tissue (1 8.5%) and the implanted control-IML tissue (20.6%). When the proportion of
perforated synapses with specific curvatures was analyzed, significant changes in the
proportion of concave pedorated and flat perforated synapses were observed in the LTP
tissue (see Fig. 4.1). The percent of concave perforated synapses (as a proportion of al1
perforated synapses) was 3 1% in the MML of the LTP animals compared to 19% in the
MML of controls @<0.001). Flat perforateci synapses declined by 1 1% in the LTP tissue
wmpared to controls (pû.026). No other changes occumd in the MML or ML.
Synaptic Lengths
An ANOVA of total synaptic length mealed a significant LTP group by location
interaction (pû.011, sec Fig. 4.2). Post-hoc analyses indicated that the average total
synaptic length was significantiy larga in the directly stimdated LTP-MML tissue compared
to the control-MML tissue. In the LTP-MML tissue, the mean synaptic length was 223 nm
while the control-MML mean length was 204 am. Analysis of the maximum active zone
length revealed the same pattern of signincance (p4.001). In perforated synapses, no
changes in perforation length were obsewed.
Analysis of the total synaptic length in relation to synaptic shape revealed a
significant LTP group by location by shape interaction (p=û.009). Posthoc analyses * '*
revealed a significant incr- in the total length and active zone length of concave and
irregular shaped synapses in the LTP-MML tissue compared to the MML tissue ofcontrols
(see Fig. 4.3 for totai synaptic length). Imgular synapses were also found to be significantly
larger in the LTP-IML tissue (426 nm) compared to the IML of controls (297 nm).
When the totai synaptic length of perforated synapses was analysed, there was a main
effect of LTP group @=0.002) but no LTP group by location interaction. The LTP group
mean length collapsed across layers was 3 18 nm and the control group mean was 283 m.
Analysis of the active wne length in perforated synapses also revealed the same main effect
of LTP group (~M.004). When the totai synaptic length and active zone length of perforated
synapses with different synaptic shapes was considemi, post-hoc analyses revealed that the
imgular perforated synapses were significantly longer (total synaptic length +76 nm (20%)
and active zone +87 nm (35%)) in the LTP-IML tissue compared to the IML of controls. No
other groups of perforated synapses with various curvatures differed in the IML or MML.
Analysis of the totai length of non-paforated synapses did not reveal a significant
LTP group by location interaction. When the total synaptic length of non-perforated
synapses with different synaptic shapes was considered, post-hoc analyses mealed that the
irreguia non-perforated synapses wen significantly longer (totai synaptic length +159 nm
(75%)) in the LTP-iML tissue c o m p d to the IML of controls.
Correfational Analjais
The correlations between morphological and electmphysiological measures, taken
fiom the multiple regression analysis, are presented in Table 4A for the MML and in Table
4B for the IML. In the stimulated MML, no structural characteristics were significantly
associatbd with the degree of LTP expressed by eacb anUnal. In the IML, the degree of
potentiation was significantly and negatively associated with tht mean length of the synapses
@<O.OS). As the degree of ptentiation increased the length of synapses decreased.
CORRELATIONS BETWEEN THE DEGREE OF PO'I'ENTUTION AND THE VARIOUS STRUCTURAL MEASURES AT 5 DAYS POST-LTP INDUCTION
-4. MIDDLE MOLECüLAR LAYEW
B. INNER MOLECULAR LAVER
Curvaturv
Concave (CC)
Convcx (CX)
Ftat (FL) I
Irregular (IR)
' Active Zme Length (Lei)
Perforateci Synapses (Pen)
- Synapses Per Ncuron (S per N)
Degrcc of LTP (LTP)
indiata signifiant diffbmcu O . O S )
CC
1 ' a
I
8
I
I
Cunrture
Concave (CC)
Convex (CX)
Flat (FL)
Irrcgular (IR)
CX
-0.07
1
I
I
I
CC
1 I
CX
0.2 t - 1 1
Fi,
0.32
0.65+
1 I
s
1
t
Perforated Synapses (Perf) I I
Synapses Pcr Neuron (S pcr N) I
1 z
FL 1 IR
IR
-0.03
0.18
0.20
1
I
Len
-0.18
-0.01
-O.S8*
0.29
0.33
1
-7-
I
0.16038.-OJt. -0.02
1
4.39
-0.30
4.52*
1
I
Len
0.16
0.37 '
O S *
0.37
1
L
Petf
0.11
0.50
0.34
-0.24
-0.08
I 4.12
Pcrf
0.36
039+
0.89*
0.20
039*
1
I
+
S pcr N
-0.48
' -0.36
-0.72*
1
LTP
-0.11
0.06
0.1
0.28 0.43
S perN
-0.24
-0.23
6.37
-0.34
-0.51*
-0.44 -
1
4.02
LTP
4 .33
0.09
0.12
4.03
-0.14
-0.04 '
0.3 0
1
Discussion
The present data iadicate that there are changes in the synaptic structural profile 5
days afkr the induction of LTP. These alterations do not include a significant change in the
total number of synapses pet neuron or overall changes in curvahur in either the MML or the
M L of the LTP animds. While the number of perforated synapses did not change in overail
proportion, there were more concave perforateci synapses and fewer flat perforated synapses
in the MML of the LTP animals.
Another important fmding in this expriment was a MML specific overall increase in
total synaptic length and active zone length in the LTP group. Post-hoc analyses revealed
that this increase was due primarily to increases in the length of concave and irregular shaped
synapses. The total length and active zone length of perforated synapses were larger in both
the MML and M L of the LTP animals and this was particularly evident for ineguiar shaped
synapses in the ML. Finally, the degree of LTP was negatively associated with the length of
the IML synapses. That is, LTP was greatest in animals with smailer IML, synapses.
Methodological Consideraîions
Again it is important to recail that there are pre-existing differences between the IML
and the MML and that these diffames necessitate the consideration of both rnolecular
layers in the tetanized and implanted-only animals. This is particularly important as several
M L specific changes werc obscrved in the present experiment. The use of the tenn 'long-
term' to describe the pst-induction time penod of 5 days, attcmpts to diffetcntiate between
eariy maintenance (sec above), intenncdiate maintenance (approx. 24 h) and long-tcrm
maintenance (days) but is wed only as a general descriptive tem. Since previous
experimentation had not considered the 5 &y pst-induction time pend, there are no direct
comparîsons for the present findings. The results, therefore, will be discussed in relation to
94
the result described above at 1 h and 24 h pst-induction and the other relevant resdts from
d e r or later thne points.
Symaptic Number
The 5 &y pst-LTP induction phase was not associated with a change in the number
of synapses per neuron. The non-significant 1 1% overall increase in the number of synapses
per neuron in the MML of LTP animais at 24 h pst-induction was not obseived at 5 days as
the number of synapses was very similar to the 1 h and control groups. The positive
correlation between the degree of potentiation and the number of synapses per neuron within
the MML of potentiated animals at 24 h pst-LTP induction was also no longer present at 5
days post-induction Taken together, these findings suggea that synaptic nurnber may
change marginaily and transiently over the course of LTP maintenance and appears to only
be associated wirh LTP during the intemediate maintenance phase.
S ' i c Curvatwe
One of the important fuidings in the present experiment is that the overail increase in
the proportion of concave synapses observed at 24 h is no longer present at 5 days. While
not increasing overall, concave synapses continue to be involved in subgmup changes at 5
days (sec below). The pattern which begins to emerge fiom this finding is that many of the
overall changes observed at 1 h and 24 h have nverted to baseline levels by 5 days pst-LTP
induction. Thus, the signifiant potentiation which remains at 5 days rnay bc supported by
stmcnual changes in ceriein synaptic subgroups.
sLMpic Perforations
Unlike the 1 h group, but similar to the 24 h group, there was not a significant
increase in the overall proportion of worated synapses at 5 days pst-LTP induction. As
mentioned, Geiaisman et al. (1 996) did nof find an increase in perforated synapses 13 days
following the last tetanization. It would appear that ovedl incrases in the proportion of
perforated synapses are restricted to relatively bnef pst-LTP induction time periods.
Finding an increased proportion of concave perforated synapses in the MML of the
LTP group in the present experiment strengthens the potential importance of these synapses
to the maintenance of LTP. Concave perforated synapses were observed in greater numbers
at 1 h, 24 h, 5 days but wt under ketamine blockade. As discussed, these synapses may have
mater connective strength due to the potentially enhancing effects of concavity and synaptic
perforations (Ghaffari et al., 1997).
Length of Synapses
Similar to the 1 h group, and unlike the 24 h animals, long-term LTP maintenance
was associated with a significant overall increase in synaptic length. The potential fùnctional
importance of this finding seems clear as larger synapses may be stronger synapses (Petit,
1995). Since overall concavity and the number of perforated synapses has retwned to
baseline by 5 days pst-induction, this increase in length may npresent the primary stnicnual
change which persists into long-terni maintenance.
Altered length was also observed within specific synaptic subgroups. As in the 1 h
group, concave shaped synapses were larger in the MML of the LTP animais a! 5 days post-
induction. Irregular shaped synapses were also larger which indicates that synapses h m
both of the curvatw mbgroups thought to have hcnased efficacy are larger in thcse animals
(Markus and Petit, 1989). Interrstingly, these same concave synapses wm smaller at 24 h
pst-induction which suggests that this subgroup is particularly involved in the structural
plasticity associated with LTP.
It is difficult to explain the iacrrase in the length of perforated synapses in both the
IML and MML in the LTP group. If this was an LTP specific effect, it would presumably be
96
limited to the directly stimulated MML. However, there have ken numerous reports in this
series of experiments of main effects or IML specific changes associated with LTP. Ii is
possible that during the long-term maintenance of LTP the structural alteration of the
perforated synapses extends into the adjacent IML. If these synapses were excitatory, this
incnase in length couid M e r strengthen the potentiation which is ultimately expressed in
the granule cells (Coliingridge and Miss, 1995). It is aiso difficult to explain the IML
specific increase in the length of irreguiar shaped prrforated and imgular shaped non-
perforated synapses other than to speculate that this curvahue subgroup may be particularly
affected by polysynaptic eEects (Le. across the layers of the dendntic arboration).
Correlational Analysis
At 24 h pst-LTP induction, the degree of potentiation expressed was found to be
significantly associated with the number of synapses per neuron in the MML. In this
experiment at 5 days pst-LTP induction, no structural characteristics in the MML were
significantly associated with the degree of potentiation. There was, however, a negative
association between synaptic length in the IML and the degree of potentiation expressed.
While the IML is not dkctly stimulated during LTP induction, it's fibers are (as
mentioncd above) part of the dendritic arboration of the dentate gym granule cells. In the
rat, the IML is known to receive numerous inhibitory commissural and ineinsic synaptic
contacts (Amad and Witter, 1989). If the average size of these inhibitory synapses was
duced by pcemisting variation or active structurai processes, the amount of inhibition
applicà to the dendritic signal could also k d u d . This could lead to a larger amount of
expressed potentiation in those anùnals with smaller IML synapses. While it is not possible
in the present experiment to conclude whether the differences in synaptic length were pre-
existing or the result of active structural modification, this resdt does add support to the
97
contention that synapses in the IML, as well as the MML, are involved in LTP in dentate
gynis of the rat.
Conclusions: Sywptic Remodelling at 5 &ys PwLTP Induction . . The 5 day pst-LTP induction time period appeam to involve an overail increase in
synaptic length in the MML of the LTP animals. This increase was particulariy evident in
proportion of concave and irregular shaped synapses. Together with the observations fiom 1
h, and 24 h pst-induction, these results suggest that activated synapses may become
concave, grow in length, perforate and potentially divide into smaller more numerous
concave synapses. Later these concave synapses appear to grow in length again to support
the long-tenn maintenance of LTP. It is also evident that at 5 days the overall increases in
the proportion of perforated synapses or concave synapses no longer exists.
The present findings represent the long-term phase in the morphological
transformation associated with LTP. As stated, the synaptic remodelling associated with
LTP appears to be a dynarnic and complex process involving alterations in various
morphological characteristics which are both dendritic region and post-induction time
interval dependent. The present expriment represents the final pst-LTP induction time
period in a senes of studies examining this sequence of events. A generai discussion of this
synaptic structural sequence follows.
GENERAL DISCUSSION
Sumaa y of Changes in Synaptic Structure Over Time
The tirnecourse of synaptic structurai change described in this series of experiments is
present in schematic fom (see Fig. 5.0). No overail change in the number of synapses per
neuron was observed in the LTP tissue at any of the time intervais. There was, however, a
non-significant 1 1% increase in the number of synapses per neuron at 24 h pst-LTP
induction (this increase did not occur under pharmacological blockade). LTP was associated
with a significant increase in irregular shaped synapses at 1 h, an increase in concave shaped
synapses at 24 h (not apparent under pharmacological blockade), and no overail changes in
shape at 5 days. The proportion of perforated synapses was increased at 1 h post-LTP
induction but did not differ fkom comols at later tirne periods. The increase in perforated
synapses at 1 h was particularly apparent in the proportion of concave perfbrated synapses
and these concave perforated synapses were aiso more numerous at 24 h and 5 days post-
LTP induction.
Synapses were larger overall at 1 h and 5 days but not different at 24 h. These
differences in length were particularly evident in concave shaped synapses whiçh were
longer at 1 h, shorter at 24 h (no change under pharmacological blockade), and longer at 5
days. The degree of potentiation expressed by individual animals was significantiy
associateci with the number of synapses per newon at 24 h but not at 1 h or 5 days.
The main strength of the current series of experhents was that they used the same
stimulation parameters and quanti fied the same structural characte ristics during di fferent
pst-induction tirne periods. This consistency provided the opportunity to compare the
various patterns of results with confidence that the changes observed were due primarily to
the difftreace in the pst-induction intaval.
It is important to note that LTP is not o unitary process within the various circuits of
the hippocampus, within various neural structures, and bctween species (Otani and Ben-An,
1993). It is necessary, thenfore, to define the limits of the conclusions drawn fiom the
research that makes up this thesis. While many of the findings may extend to LTP in other
species, other neural structures, and the other circuits of the hippocampus, the cumnt nsult
describe changes associated with LTP in the rat dentate gynis.
It is also important to achowledge that randomly selecting tissue 1.5 mm to 3.5 mm
from the septal pole of the hippocampus in al1 four experiments does not represent random
sampling of the tissue fiom the entire hippocampus. This area was selected due to its
proximity to the recording electrode in an attempt to capture the tissue that was most likely
expressing any alterations in synaptic structure. This distance assured that the tissue was not
damaged by the recording electrode while maintaining a proximity that maximized the
correspondence between the electrophysiological recordings and the tissue responsible for its
production.
The differences between the IML and MML in all of the groups analyzed suggests
that it is not appropriate to use the IML as the only control for the MML profile in this type
of nsearch. The different amber of synapses per n e m and the different structurai pattems
bctwecn the two regions observeci in the control animals makes the examination of the MML
in implanteci animais necessary as a second control. Another concem with the suitability of
ushg the IML as a control region involves the potential for anatomicai or physiological
alterations being induced in the IML synapses following LTP.
Synaptic Number
Synapse number does not change during the induction phase of LTP. The number of
synapses is associated with the degreg of LTP expressed dwhg intemediate maintenance
and is not diaerent or associateci &th the degree of potentiation during long-term
maintenance. One explmation for this pattern of results is that a subset of synapses is
recnllted or tagged (see below) during the induction of LTP and these synapses evennially
divide duMg the intermediate maintenance phase yielding a marginal overdl increase in
synaptic number. The system appears to rem to a baseline number of synapses during
long-term maintenance whicb may involve the elimination of some of the synapses not
recruited during the induction phase. It is important to note that these results do not rule out
the possibility of de-novo synaptic formation during intermediate maintenance but some
subsequent reduction would still need to occur to explain the results h m the long-term
maintenance phase.
Dynamic Interactions
Al1 of the simple sequential changes in curvature, perforations, and length were
discussed above in the 5 day expriment discussion and will not be restatcd here. One issue
that does need to be revisited is the way that certain dynamic interactions mur across the
three pst-LTP induction time periods. The main interactions involved concave synapses
which interacted with perforations end synaptic length during each pst-induction tirne
perîod Initially, concave synapses aLalre the largest contribution to the inciew in the
proportion of pedorated synapses obscrved a 1 h pst-induction. These synapses may
represent those which were tagged for structural modification in association with LTP
maintenance. During intemediate maintenance the concave synapse appear to be smaller
which would suggest that they are newly h e d or are the result of synaptic division.
101
Findy, the long-term maintenance of LTP involves a r e m to larger concave synapses
which contirue to be more paforated than in contml animals. This pattern of results may be
explained as the initial growth and perforation, division, and subsequent growth of caicave "'* . -
synapses.
Correlational Findings
Taken together, the iesdts from 1 h, 24 h, and 5 days suggest that the number of
synapses present is not related to the amount of potentiation expressed during induction but is
associated with the amount of potentiation expressed during the intermediate maintenance of
LTP. Interestingly, the number of synapses is not related to long-term maintenance despite
the continuation of significant electrophysiological potentiation. One explanation for this
pattern of results involves a transient change in synaptic number at 24 h post-LTP induction.
To niterate, it is possible that the induction of LTP recruits or potentiates a subset of
synapses which begin to undergo stni1ctural modification. By 24 h many of these synapses
have perforateci and divided or new synapses have formed in proximity to the potentiated
synaptic sites. Those animals in which more synapses were initially recruited display more
synapses and more potentiation at 24 h poainduction. By 5 days, however, the MML in
general has mverted to a stable number of synapses overall (possibly via elimination of
inactive synapses) while those clusters of potentiated synapses remain in a more eficacious
state (concave, pepaforated, and larger).
Syniptic Tagging
The main potentid limitation of protein dependent changes such as synaptic growth
and division is the lack of synapse specificity. One neuron can have thousands of synapses
and the genetic expression for al1 synapses occurs in the soma. How can the potentiating
effects be specific to only the active synapses? Recent research by Frey and Moms (1997)
102
indicated that spccificity ma)- bc achieveâ via synaptic tagging. They theorized that tetanic
stimulation would not only initiate somatic gene expression but could cause a specific change
in only the synapses that were active during the inductive proccss. These tagged synapses
would then preferentially utilize the proteins created in the soma. The presence of
phosphorylated kinases may act as the synaptic tag (Frey and Morris, 1997). This provides a
mechanism for activated synapses to undergo the cwature, perforation, division, and
subsequent growth changes that have k e n described.
LTP, Reactive Synaptogenesb, Learning, md Memory: 1s There a Common Structural Mechanism?
The data required to make a detailed cornparison between the synaptic structurai
changes associated with LTP, reactive synaptogenesis, learning, and memory is far fiom
complete but it is tempting to speculate about a common synaptic mechanism. Do activated,
ncruited, or newly formed synapses undergo the same sequence of structural change and if
so how does this potentially support the observed potentiation, learning, or dtered neural
functioning ?
Reactive synaptogenesis in the hippocampus was examined by Anthes et al. (1 993).
Following ipsilateral entorhinal cortical lesions, synaptic counts and structurai features were
quantified in the rat dentate g y m at 3,6, 10, 15, and 30 days pst-lesion. Anthes et al. found
that the lesions caused an initial 88% synaptic loss in the MML at &y 3 which was followed
by rapid synaptogenesis h m day 6 through to &y 15. They speculated that following the
loss of entorhinal input, previously dormant or suppressed fibers and their synapses became
active in the absence of the primary innervation. Interestingly, synaptic size was found to
decrease during the phase of rapid synaptogenesis. As synaptogenesis returned to baseline
levels (approx. &y IS), synaptic size also rehimed to control or pn-lesion levels. Another
important finding was that the number of perforated synapses was greatest at the peak of
syiiaptogenesis (day 10-1 5) and retumed to conml leveb by day 30 pst-lesion.
Despite the Werences in the length of time psi-lesion, the sequence of synaptic
structural changes observecl by &thes et al. is very similar to ihat observeci in the current
series of experhents on LTP in the same tissue. In both cases activation caused synapses to
undergo mtmcturing which involved the formation of more perforated synapses, then
smailer more numemus synapses, followed by a retum to baseline structurai Ievels. It would
appear that despite king induced by very different neural interventions, LTP and reactive
synaptogenesis share a common sequence of synaptic structural change. The differences in
the timecourse of the structurai change (LTP=hours; Synaptogenesis4ays) may reflect the
nature of the activation in each case. From a fùnctional stand point the timecourse of the
changes associated vnth LTP provides a better mode1 for the plasticity needed to support
hippocampal dependent learning (Eichenbam et al., 1992).
S ynaptic structure has also ken show to change following learning. Reempts et al.
(1992) found changes in the shape of spine synapses in the rat dentate gyrus following 3 days
of avoidance training. Specifically, they found an increased number of concave perforated
synapses following the acquisition of a one-way active avoidance task. Both non-perforated
and perforated concave synapses were also found to k increased in size relative to controls.
This d t mirrors the cumnt structural findings at 1 h pst-LTP induction. Again the
Similarity in the sequence of structural change seems clcar. The diffennce in the timccourse
and quantity of synaptic change may k explainad by the relative stnngth of the input signal
during avoidance l e d g versus tbat received during LTP. In this case, however, leaniing
alone was suficient to induce perforation and size differences in the concave synapses.
Leaming in the cerebeliurn has also k e n associated with changes in synaptic
structure. Kieim et al. (1996) formd that motor ski11 learning caused synaptogenesis in the rat
cerebeilum. The cerekllar synapses of rats taught to navigate an aerial maze were compmd
to the synapses of rats that received the same amount of motor activity but did not Ieam the
task. They found that the number of synapses per pwkinje ce11 increased in the aerial maze
p u p minpared to the motor controls. Further, Uie number of multiple varicosities (clusten
of synapses) increased following learning as diâ the arnount of branchuig in the purkinje ceIl
processes. The length of these branches was also found to be increased following the
acquisition of the task. Finally and most important for a cornparison to the curent nsearch,
Kleim et al. found that as the number of synapses increased, their average size decreased.
While Kleim et al. did not conduct a detailed analysis of synaptic shape, the common
fmding of structural change involving a transient decrease in synaptic size as the number of
synapses increases following synaptic activation parallels the cunrnt observations following
LTP. While only three exarnples of synaptic structurai change outside of the research on
LTP, the preceding studies suggest that a comrnon structural mechanism of synaptic
plasticity may exist. This mechanism involves an initial increase in size and concavity, then
a decrease in size and increased synaptic number, followed by a retum to baseline levels.
One intriguing possibility which was suggested by the clusters of synapses observed
by Kleim et al. (1996) and by a similar mecdotal observation of grouped concave synapses in
the cumnt rtsearch, is that a subset of synapses becorne involved or recruitcd in the various
fonns of neural plasticity dernonstmted in differcnt neural structures. This subset of synapses
then follows the sequence of structural changes outlined in this dissertation to produce more
numemus and efficacious synapses within the groups of afferent fibers that were involved in
the initial neural signal.
Are LTP and Learahg Equivaitnt in the Rippocampus?
In the hippocampus the type of structurai mechanism described in this dissertation + ) '
rnay support the eahancement of minute circuits potentially involved in the production of
hippocampal learning. Given this possibility, a finai discussion on the relationship between
LTP and leaming is wmmted. Sutherland et al. (1993 j stated that there are three types of
research that have attempteà to link the processes underlying long-term potentiation and
leaming . These researc h types are: (1 ) New leaming of hippocampal-relevant behaviors
should cause synaptic potentiation at a subpopulation of hippocampal synapses; (2)
Widespread phamacological blockade of hippocampai LTP should block new hippocampal-
dependent learning; and (3) Experimentally induced LTP at a suficiently large number of
hippocampal synapses should interfere with normal hippocampal learning.
Early research linked LTP and learning in the second and third type of research
descnbed by Sutherland (Moms et al., 1986; McNaughton et al., 1986). Moms et al. (1986)
used the competitive NMDA antagonist AP5 to show that both LTP and spatial learning can
be disrupted using the same pharmacological agent. McNaughton et al. added hinher
eviduice for a leaming-LTP link by showhg that excessive LTP can saturate the plasticity
within the dentate gynis. They then found that following this prwess of saturation spatial
leaming is im- on the Barnes circuiar platform task (1 986). Both of thew studies
providecl a ground work for fiirther investigation which has largely fded to replicate thcsc
earlier findings (Sutherland et ai., 1993; Hoh et al., 1999).
The findings of Moms et al. (1986) were challengeci in a papa by Hoh et al. (1999).
In this experiment animals were able to aquire knowledge of the platform location in the
water maze despite competitive NMDA blockade using CGS. The essential difference
between this study and the eadier work by Moms et al. (1 986) was the addition of some
familiarization with the testing environment prior to learning the task under NMDA
blockade. Importantly, these animals were able to aquin the strategy necessary to solve the
task despite the NMDA blockade and the inability of the system to express LTP.
The findings of McNaughton et al. (1 986) have been contradicted by Sutherland et al.
(1 993). In this experiment LTP was induced bilaterally to asymptotic ievels in the perforant
path synapses of the dentate gynis. Place leaming in the water maze proceeded normally in
these task naïve animafs 24 hours after the final tetanization. This result seriously questioned
McNaughton et d.'s earlier findings and provides evidence for a dissociation between LTP
and learning.
Even more recently, however, support for McNaughton's original assertions has corne
fiom Moser et al. (1 998). Moser et al. found that the elimination of one of the hippocampi
and saturation of the remaining perforant path synapses using an array of stimulating
electrodes was sufficient to interfere with place leaming in the water maze. It may be that
the intact system has a very complex circuitry which allows for residual plasticity despite the
apparent saturation of the system and that this saturation does in fact interfere with learning
when one pathway is isolated (Moser et al. 1998).
Recentiy, evidence has emerged fiom the first type of research described by
Sutherland et al. (1993). This rtsca~ch has demonstrateci LTP-like potentiation following
leamhg (Rogan et al. 1997; lshihara et al., 1997). Specifidy, Rogan et al. (1 997) found
electrophysiological potentiation in the amygdala following feer conditionhg and Ishihara et
ai. (1997) found potentiation in the dentate gynis-CA3 mossy fiber pathway following
acquisition of the radiai a m mazc task. Despite many contradictory findings, evidence is
mounting in support of a strong link between hippocampal LTP and !earning.
Conclusions: Synaptic Remodeiiirg ûver Time Following the Induction of LTP
This research, together with pnvious research on the morphological underpuinings of
LTP, indicates that a series of synaptic structural profile changes occur following induction.
Stimuiated synapses appear to initially lengthen and becorne more perforated (1 h), then
becoming mon concave in shape and divide or fom new srnalier synapses (24 h). Finally,
synapses return to control levels with regard to curvature and perforations while naintainiog
length differences prirnarily in concave shaped synapses (5 days). Therefore, it seems
increasingly clear that the synaptic structurai profiles observed at the various time periods
represent a continuous sequence of sûuctural changes beginning at the initial tetanization and
continuing through to the decay of LTP (see Fig. 5.0). This sequence of structurai changes
may recruit and proliferate a subset of synapses which contribute to the potentiation
observed. As outlined above, this theory of synaptic stnictural turnover is similar to others in
the literatwe (Geinisman et al., 199 1; Desmond and Levy, 1986).
While the extent of the correspondence between the mechanisms of LTP and the
biologieal underpinnings of learning (memory) in the hippocampus remains unclear,
understanding the ways in which synapses change in structure following stimulation has the
potential to help define and explain the characteristics of hippocampai plasticity. This
understanding could assist in defuiing the contribution of the hippocampus to various
psychological processes including declarativc leaming and memory. These findings may
a b be added to the growing body of research on synaptic structure throughout the entire rat
b d n and the brains of other spccies. It rnay eventually be possible to describe sirnilarities
and differences in the way al1 synapses change in structure following stimulation and how
this süucturai plasticity may contribute to the bction of neural systems.
108
FIGURE 5 TIMECOURSE OF SYNAPTIC STRUCTURAL CHANGE
SYNAPTIC A r n A T I O N . Nh.lDA activation, calcium entry, and activation of second m a n g e r cascades.
INDUcTION/EARLY MAINTENANCE (1 HOURS) Activation of internai cytoskeletan and larger synapses with more perforations.
MTERMEDIATE MAINTENANCE (24 HOURS)
Aoduction of new synaptic pmteins, division andfor formation of mw smaller and more concave synapses.
LONC-TERM MAINTENANCE (5 DAYS) L8rgcr concave s y u a p s nnvia but systcm tends to rem to ôaselirîe structural levels.
w
Necessity of Unbiwd Countiig Techniqua
Deriving the overall number of synapses presed in the various layers of the dentate gynis is
central to any examination of synaptic structural change following LTP. Most uuly experiments,
however, employed biascd counting techniques. The main probiem arose h m counting the number
of synaptic profiles !km a group of pictures taken from a single two dimensional plane. Coggeshall
and Lekan (1996) point out that considering only one plane biases the synaptic counts because the
number of two dimensional synaptic profiks observed depends not only on the actual n u m k of
whole synapses but also on the size, shape, orientation, and many other variables (Coggeshall, 1992).
An exarnple of this biasing effect cm be observed when counting perforated synapses. If you
considered only one hvo dimensional plane. there would be no way of knowing whether the non-
perforated synaptic profiles you observed were actuall y non-perforated. That is, unless you followed
the synapses down through successive sections you would not be able to view al1 of the profiles of
each synapse. You could not, therefore, make a definite decision about whether the synapses were
penorated or not. A second problern with perforated synapses is that they are typically larger than
non-perforated synapses. Logically, larger objects would be mon I ike ty to appear in any given plane
through the tissue. The result would be an over estimation of the total number of perforated synapses.
Gundersen (1 977) pointed out that the ideal solution to these biasing effects of two
dimensional profile counting is to completely nconsûuct al1 of the objects of interest in three
dimensions. This would nmove the biases associated with size and orientation by allowing the
researcher to visualize each entire synapse. Completc rcconstmction of a large number of synapses
is however, prohibitively time consuming. Gundersen proposed the use of stereological counting
methods as an unbiased alternative to 3D reconstruction.
The Double Dissector Technique
As described in the General Methods section, synapses were counted by comparing adjacent
sections, one a reference section and the other a look-up section immediately above it in the series.
Synapses were identified in the micrographs by the presence of synaptic vesicles, dense material in a
presynaptic won terminal, and an accompanying PSD. Synapses were sampled (counted) if they
were observed in a referencc section micrognph within the area limitai by the unbiased sarnpling
h e of Gundersen (1977), but not observai in the comsponding look-up section. Gundenen's
sampling fiame was applied to the computer monitor so that synapses were counted if they partially
appeared on the included edge (left of screen) and not counted if they appeamd along the excluded
edge (right of screen). This also helped limit the degree of bias introduced by synapses of various
sises. Using a refennce and lookup section within Gundersen's counting frame provided an unbiased
estimate of synaptic number becaux synapses were only counted once regardless of the number of
sections in which they appeared. This half of the counting process is known as the stereological
dissector. The double dissector aspect involves counting the number of neurons in the granule cell
layer.
Granule neurons were sampled if the nucleus of the cell appeared in the last section of each
series but not in the tint. The formula n/N = [(q- A k)/Q-a)] (wMT) WU employed to estimate the
number of synapses per muron (Bnendgaard and Gundersen, 1986). In this fornula, Q' and q'
indicate numbers of neurons and synapses sarnpled in an area A or a, respectively; k is the number of
sections in a series minus one; w designates the width of the middle or inner third of the ML; and W
represents the width of the GCL. A description of the development of this formula is beyond the
scope of this appendix but the reasons for using this double dissector aspect are clear. One of the
main difficulties in any estimation method is defining the volume of space in which the synapses
were counted. This becomes especially difficult when you consider that there is variebility in the
thickness of each section within each xries. One solution is to describe the nurnber of synapses as a
ratio of the number of neurons to which the synapses belong (synapses per neuron).
By fotlowing the formula describcd and stating the number of synapses as a ratio, the volume
of tbe sprce in which you samplcd kcomcs unimpomt (the formula above does not requirc an
estimate of volume, only 2 dimensional layer widths, sunpling area, and numkrs of synapses and
ncwns). Due to the advantagcs descnbed above, the u n b i d double dis~ector technique was
employed in these experiments.
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