neuron, vol. 9, 749-758, october, 1992, copyright 0 1992...

Neuron, Vol. 9, 749-758, October, 1992, Copyright 0 1992 by Cell Press

Inhibitors of Protein and RNA Synthesis Block Structural Changes That Accompany Long-Term Heterosynaptic Plasticity in Aplysia

Craig H. Bailey,* Piergiorgio Montarolo,*+ Mary Chen,* Eric R. Kandel,*+ and Samuel Schacher* *Center for Neurobiology and Behavior and New York State Psychiatric Institute +Howard Hughes Medical Institute College of Physicians and Surgeons Columbia University New York, New York 10032

Summary

Synaptic connections between the sensory and motor neurons of Aplysia in culture undergo long-term facilitation in response to serotonin (5-HT) and long-term depression in response to FMRFamide. These long-term functional changes are dependent on the synthesis of macromolecules during the period in which the transmitter is applied and are accompanied by structural changes. There is an increase and a decrease, respectively, in the number of sensory neuron varicosities in response to 5-HT and FMRFamide. To determine whether macromolecular synthesis is also required for the structural changes, we examined in parallel the effects of inhibitors of protein (anisomycin) or RNA (actinomycin D) synthesis on the structural and functional changes. We have found that anisomycin and actinomycin D block both the enduring alterations in varicosity number and the long-lasting changes in synaptic potential. These results indicate that macromolecular synthesis is required for expression of the long-lasting structural changes in the sensory cells and that this synthesis is correlated with the long-term functional modulation of sensorimotor synapses.

Introduction

Long-term memory storage in both vertebrates and invertebrates shares two features: a requirement for protein synthesis (Davis and Squire, 1984; Montarolo et al., 1986; Matthies, 1991) and structural changes within the central nervous system that are most often manifested by an alteration in the number of synaptic connections (Greenough and Chang, 1985; Creenough and Bailey, 1988; Wallace et al., 1991; Bailey, 1991, 1992). However, the relationship between these two features and the relative contribution each makes to the long-term process are not known. To address these issues, we have utilized a relatively simple behavioral system-thegill-and siphon-withdrawal reflex of Aplysia. This reflex exhibits two elementary forms of nonassociative learning--habituation and sensitization, both of which can exist in a short-term form, lasting minutes to hours (Pinsker et al., 1970; Carew et al., 1971), and a long-term form, persisting for days to weeks (Carew et al., 1972; Pinsker et al., 1973).

A particularly well-studied component of this reflex is the monosynaptic connection between mechanore- ceptor sensory neurons and their interneuronal and motor neuron follower cells. Although this component accounts for only part of the behavioral modification measured in the intact animal, its simplicity has allowed reduction of the analysis of the long-term memory for both habituation and sensitization to the cellular and molecular levels. The long-term memory for sensitization and habituation is accompanied by an enduring alteration in the strength and structure of the sensory to motor synapse. Long-term sensitization is characterized by an increase in synaptic efficacy(Frostetal.,1985)and bythegrowthof newsynap- tic connections, resulting in an increase in the total number of sensory neuron varicosities (Bailey and Chen, 1988a) as well as a parallel remodeling of their active zones (Bailey and Chen, 1983, 1988b). By contrast, long-term habituation is accompanied by a decrease in the strength of the sensorimotor connection (Castellucci et al., 1978) and a corresponding reduction in the number of sensory neuron synapses (Bailey and Chen, 1983,1988a, 1988b). Quantitative analysis of the time course over which these anatomical changes occur during long-term sensitization has further demonstrated that only alterations in the number of sensory neuron varicosities and active zones persist in parallel with the behavioral retention of the memory (Bailey and Chen, 1989a).

Similar changes in synaptic effectiveness and in the structure of the monosynaptic sensory-to-motor neuron synapse can be elicited in dissociated cell culture by the application of specific neurotransmitters. Re- peated applications of serotonin (5-hydroxytrypta- mine, 5-HT), a neurotransmitter normally released by sensitizing stimuli in the intact animal, evoke a long- term (24 hr) facilitation of the synapse (Montarolo et al., 1986; Dale et al., 1988; Schacher et al., 1990) that is characterized by a long-lasting enhancement in the amplitude of the sensorimotor excitatory postsynaptic potential (EPSP) as well as an increase in the number of sensory neuron varicosities contacting the postsynaptic motor cell (Glanzman et al., 1990). By contrast, repeated presentation of the neuropeptide Phe-Met- Arg-Phe-amide(FMRFamide), which produces presynaptic inhibition of the synaptic connections between sensory and motor neurons, results in a long-term decrease in the size of the excitatory postsynaptic potential (Montarolo et al., 1988) and a corresponding

reduction in the number of sensory neuron varicosities (Schacher and Montarolo, 1991). Moreover, studies in the semi-intact animal (Castellucci et al., 1989) and in primary cell culture (Montarolo et al., 1986, 1988) have shown that the functional changes-the long-term facilitation and depression of the connection between the sensory and motor neurons-can be selectively blocked by inhibitors of transcription or

nV

ns

OHr 24Hr - Figure 1. Long-Term Effects of 5-HT or FMRFamide on the Amplitude of the EPSP and Structure of the Sensory Neuron

(A) 5-HT evokes long-term facilitation and an increase in the number of fluorescently labeled sensory neuron varicosmes. The EPSPs evoked before (0 Hr) and after (24 Hr) treatment are illustrated in the insets for (Al) and (A2). Treatment with five applications of 5-HT results in a significant enhancement in the amplitude of the sensorimotor EPSP when measured 24 hr later. Repeated 5-HT treatment also evokes structural changes in the sensory neuron, resulting in a net increase of 2 varicosities in this view area. The two montages represent fluorescence micrographs of the same regions of sensory neurites contacting the major axons of L7 before (Al) and 24 hr

Inhibitors Block Long-Term Structural Plasticity 751

translation when these are present during the acquisi- tion phase.

Do the structural changes that accompany long- term facilitation and inhibition of sensorimotor synapses also require new protein and RNAsynthesis? To address this question, we have examined in parallel the effects of inhibitors of protein (anisomycin) or RNA(actinomycin D) synthesison the long-term modulation of the sensorimotor synaptic connection, as well as on the long-lasting alterations in the number of sensory neuron varicosities. We have found that both the long-term structural and functional modulation of sensorimotor synapses in dissociated cell culture by 5-HT (facilitation) or FMRFamide (inhibition) are blocked by inhibitors of translation or transcription present during the period in which the transmitter is applied. Our results suggest that macromolecular synthesis is required for the long-term structural changes in the sensory cell and that this synthesis is coupled with the persistent functional modulation of the sensory-to-motor neuron synapse.

Results

The physiological and structural changes associated with long-term behavioral modifications in Aplysia can be reconstituted in dissociated cell cocultures of sensory neurons and the gill motor neuron L7. The experimental accessibility of this monosynaptic connection facilitates the study of mechanisms that un- derlie long-term synaptic plasticity. We first deter- mined the long-term effects of repeated application of a modulatory transmitter on the physiological efficacy of the sensorimotor synapse. This was accom- plished by measuring the amplitude of the EPSP in L7 evoked by stimulation of the sensory neuron, both before and 24 hr after the cultures were treated with either five 5 min applications at 20 min intervals of 5-HT (2.5 PM), or four 5 min applications at 30 min intervals of FMRFamide (1.0 PM). We also labeled each cell with twoseparateinjectionsofthefluorescentdye 5(6)-carboxyfluorescein and imaged sensory neuron neurites contacting the motor cell L7 with low light epifluorescence video microscopy both before and 24 hr after treatment with each transmitter. Finally, we counted the number of fluorescently labeled sensory neuron varicosities contacting the initial segment and major axons of L7. Control cocultures were treated identically to experimental cultures, except the trans- mitters were omitted.

As previously reported (Montarolo et al., 1986,1988), we found that repeated exposure to 5-HT evoked a significant increase in the amplitude of the EPSP when retested 24 hr later (Figure IA) and that repeated exposure to FMRFamide resulted in a significant decrease in EPSP size (Figure IB). In addition to the long-term effects on the functional strength of the sensorimotor synapse, repeated presentations of each transmitter evoked long-term changes in the structure of the sensory neurons when reimaged 24 hr later (Glanzman et al., 1990; Schacher and Montarolo, 1991). 5-HTelicited an overall increase in the number of fluorescently labeled sensory neuron varicosities contacting the major axons of L7 (Figure IA), and FMRFamide evoked a corresponding decrease in the number of varicosities (Figure IB). To determine whether the long-term modulation of the synapse between the sensory and motor neurons might bedependenton macromolecular synthesis, we examined whether inhibitorsof protein (anisomycin) or RNA (actinomycin D) synthesis would interfere with the transmitter-induced alterations in the amplitude of the EPSP and the corresponding changes in the number of sensory neuron varicosities. We found that both anisomycin and actinomycin D blocked the long-term structural and functional changes produced by 5-HT (Figure 2) and FMRFamide (Figure 3; Figure4). The inhibitors alone had no significant effect on sensory cell structure or the EPSP.

Figure 5 summarizes the long-term changes in the EPSP and in the number of varicosities evoked by5-HT (Figure 5A) and by FMRFamide (Figure 58) and illustrates the blockade of these changes by anisomycin and actinomycin D. Figure 5 also summarizes the effects of the inhibitors by themselves compared with control treatments (Figure 5C). A one-factor analysis of variance indicated a significant effect of treatments on both the amplitude of the EPSP (df = 8,87, F = 10.836, p < 0.001) and the number of sensory varicosities (df = 8,87, F = 12.139, p < 0.001). Control (un- treated) cocultures showed little change in the amplitude of the synaptic connections between the sensory neuron and the motor neuron L7 (7% + 10, n = 13) or in the number of sensory cell varicosities contacting L7 (2% f 6%, n = 13). By contrast, repeated applications of 5-HT produced a significant long-term increase in the amplitude of the synaptic potential in L7 (61% f 10, n = 15, Dunnett’s t = 4.88, p < 0.01) and a corresponding increase in the number of sensory cell varicosities contacting the major axons of L7 (46% + 7%, n = 15, t = 5.82, p < 0.01). Conversely, repeated

after (AZ) treatment. Arrowheads illustrate examples of varicosities present 1 day after 5-HT treatment. Both portions of each montage are composed of superimpositions of sensory neurites from two adjacent focal planes. As a result, the shape of individual varicosities may be obscured. (B) FMRFamide evokes long-term synaptic depression and a corresponding decrease in the number of sensory neuron varicosities. Repeated treatment with FMRFamide results in a significant reduction in the amplitude of the sensorimotor EPSP (insets in [Bl] and [BZ]). Four applications of FMRFamide also resulted in a net decrease in 3 varicosities along sensory neurites contacting the major axons of L7 in this view area. Arrows in (Bl) illustrate some varicosities not present 24 hr later (82). The montages in (B) represent superimpositions of sensory neurites from two adjacent focal planes.

For (A) and (B), areas of diffuse fluorescence are fluorescence images (neurites and varicosities) in other focal planes. Bar, 15 urn.

251

?lV

0 Hr 24Hr - Figure 2. Effects of Inhibitors of Protein and RNA Synthesis on the SHT-Induced Long-Term Modulation of Sensory Neuron Synapses

(A) The 5HT-induced long-term facilitation of the sensorimotor EPSP is blocked by 10 uM anisomycin present during the transmitter application (insets in [Al] and [A2]). Anisomycin also blocks the increase in sensory neuron varicosities evoked by repeated presentation of 5-HT. In this example, there was no net change in the number of varicosities. (8) Exposure toactinomycin D (50 uglmbadded 1 hr before the first recording session also blocks the 5-HT-induced long-term facilitation (insets in [Bl] and [B2]). The inhibitor of RNA synthesis also blocks the increase in the number of sensoty neuron varicosities evoked by repeated presentations of 5-HT (net increase of 1 varicosity in this view area). Bar, 15 urn.


application of FMRFamide produced a long-term reduction in the amplitude of the synaptic potential I-41% + 4%, n = 10, t = 3.90, p < 0.01) and in the number of sensory cell varicosities (-26% + 2%, n = 10, t = 3.44, p < 0.01).

The presence of either a translational or a transcriptional inhibitorduringtheapplicationsoftransmitters not only blocked the long-term alteration in synaptic strength, but also blocked the long-term structural changes (Figure 5). Anisomycin and actinomycin D blocked the actions of 5-HT (Figure 5A) on the long- term enhancement of the EPSP in L7 (-11% + 9%, n = 12, t = 1.52, p > 0.25; and 16% + IO%, n = 6, t = 3.636, p > 0.5, respectively) and on the increase in sensory neuron varicosities (-6% + 8%, n = 12, t = 1.01, p > 0.3; and 5% f 5%, n = 6, t = 0.291, p > 0.75, respectively). The presence of the inhibitors blocked the long-term changes in EPSP amplitude and sensory cell structure evoked by FMRFamide (Figure 5B). Ani- somycin and actionmycin D blocked the long-term depression of the EPSP in L7 (-6% f II%, n = 10, t = 1.08, p > 0.25; and 2% + 3%, n = 7, t = 0.367,

Figure3. Effects of Anisomycin on the FMRFamide-Induced Long-Term Modula- tion of Sensory Neuron Synapses

Anisomycin present during theapplication of FMRFamide blocks the decrease in the amplitude of the sensorimotor EPSP measured 24 hr later (insets in [A] and [B]). Ani- somycin also blocks the long-term decrease in the number of sensory neuron varicosities (a net change of +I). The arrow in (A) illustrates a varicosity not present 24 hr later (B). Arrowheads in (B) point to varicosities present 24 hr after repeated FMRFamide treatment. Bar, 15 pm. Inset scale bars, 10 mV and 25 ms.

Ohr

p > 0.75, respectively). They also blocked the corresponding decrease in the number of sensory cell varicosities by FMRFamide (-6% & 4”/, n = 10, t = 1.00, p > 0.25; and 5% + 6%, n = 7, t = 0.258, p > 0.75, respectively). By themselves (Figure 5C), anisomycin and actinomycin D had no significant effect either on the change in EPSP (1% + 8%, n =: 11 and -1% f 5, n = 12, respectively) or on the change in sensory cell varicosities (3% * 5%, n = 11; 0% * 4%, n = 12, respectively). As previously reported, the inhibitors had no significant effect on the short-term changes evoked by a single application of either 5-HT or FMRFamide. Moreover, the inhibitors had no significant effect on the resting potentials, the shape of the action potentials, or the input resistances of both the sensory and motor cells (Montarolo et al., 1986,1988; Dale et al., 1987; Schacher et al., 1988).

We have used multiple regression analysis (Figure 6) to determine the statistical relationship between the long-term changes in the amplitude of the EPSP and the changes in the number of sensory neuron varicosities evoked by repeated presentations of 5-HT

Figure 4. Effects of Actinomycin D on the FMRFamide-Induced Long-Term Modula- tion of Sensory Neuron Synapses

Acinomycin D blocks both the long-term depression of the EPSP (inset) and the long-lasting reduction in the number ot sensory neuron varicosities evoked by repeated applications of FMRFamide (net change of +I). Arrows in (A) point to varicosities present at 0 hr and not present 24 hr later (B). Arrowheads in(B) illustrate sensory neuron branches and varicosities that have appeared 1 day after FMRFamide application. Bar, 15 pm. Inset scale bars, 10 mV and 25 ms.

and FMRFamide. We also used this analysis to exam- ine the effectiveness of the blockade of these changes byanisomycin or actinomycin D. An analysisof covari- ante indicated that the changes in the amplitude of the EPSP in L7 correlate significantly with the measured changes in the number of sensory neuron varicosities (R* = 0.307, df = 2,93, F = 20.55, p < 0.001; see also Schacher and Montarolo, 1991).

Discussion

Among the most distinctive features of long-term

memory are its stability and duration. Yet most of the molecular constituents of the nervous system have limited lifespans and turn over at regular intervals (Crick, 1984). One candidate for generating a persistent memory, in spite of the limited lifespan of the molecules, is altered gene expression (Goelet et al., 1986; Montarolo et al., 1986; Squire, 1987).

The importance of new macromolecular synthesis for long-term memory was first suggested bythe studies of Flexner et al. (1963), Agranoff (1967), and Baron- des (1975). A similar requirement for protein synthesis has now been demonstrated repeatedly for a variety of different learning paradigms, in species ranging in complexity from higher invertebrates (Montarolo et al., 1986; Crow and Forrester, 1987, Sot. Neurosci., abstract) to mammals (Davis and Squire, 1984).

How do these newly synthesized proteins contrib- ute to the induction or maintenance of long-term memory? In Aplysia, it is possible to study the repre- sentation of memory on the cellular level by exploring one component of the gill-withdrawal reflex, the connections between sensory and motor neurons. Here, we have found that macromolecular synthesis seems to have a double function. First, it leads to persistent activity of the CAMP-dependent protein kinase (Ber- gold et al., 1990), resulting in persistent protein phos-


EPSP Amplitude Number of Varicosities A 75

1 T

B 25

-50 ’

ci$ 25 5 6 tl: 0

Figure 5. Summary of the Long-Term Changes Evoked by the Neuromodulators and the Blockade of These Changes by the Inhibitors

(A) The 5-HT-induced increases in the amplitude of the EPSP and the number of sensory neuron varicosities are blocked by anisomycin and actinomycin D. (B) The FMRFamide-induced decreases in the EPSP amplitude and varicosity number are blocked byanisomycin and actinomy- tin D. (C)The inhibitors by themselves have no effect on the amplitude of the EPSP or on varicosity number. For the functional changes, the height of each bar is the mean + SEM of the percent change in the amplitude of the EPSP retested 24 hr after each treatment. For the structural changes, the height of each bar represents the mean f SEM of the percent change in the number of fluorescent varicosities per sensory neuron reexamined 24 hr later.

phorylation (Sweatt and Kandel, 1989), changes in excitability (Dale et al., 1987), and enhanced transmitter release during long-term facilitation (Montarolo et al. 1986). Second, thereare structural changes in sensory neuron synapses (Bailey and Chen, 1983,1988a, 1988b, 1989a, 1989b; Clanzman et al., 1990; Nazif et al., 1991; Nazif et al., 1991, Sot. Neurosci., abstract).

In the present study we have shown that macromolecular synthesis is required for the synaptic growth that accompanies long-term facilitation. In addition, aur work demonstrates that the retraction of synaptic connections associated with long-term depression

also requires the synthesis of proteins and mRNAs. Thus, independent of the sign of the synaptic change, structural changes dependent on macromolecular synthesis seem to be the signature of long-term memory processes. Moreover, the retractions produced by FMRFamide are similar to the pruning and synapse elimination characteristic of the later stages of neuronal development (for review, see Purves and Licht- man, 1985).

Not all aspects of structural modifications seem to require new protein synthesis. Thus, in some cases, the early and more transient aspects of synaptic structural plasticity-those that are less resistant to disrup- tive agents-may be limited to interactions involving preexisting macromolecules and may not require altered gene expression or altered transport of structural proteins to the site of synaptic remodeling. Some evidence for this notion comes from studies of long- term facilitation at the crayfish neuromuscular junction, where rapid (0.5-I hr) morphological transforma- tions involving modifications of preexisting active zones as well as insertion of new release sites occur in the absence of the neuronal cell body and protein synthesis (Atwood et al., 1989; Wojtowicz et al., 1989).

Even more rapid changes follow the induction of long-term potentiation in the rat hippocampus, in which Lee et al. (1980,198l) and Chang and Creenough (1984) have reported the formation of new synapses within IO-15 min of the onset of long-term potentiation. Such rapid synaptic restructuring and resultant synaptogenesis might be triggered by self-assembly from preexisting components. This would permit the local control of synaptic morphology and allow the initial structural changes to take place in minutes rather than hour or days (see, for example, Kelly, 1991, and Han et al., 1991). The stabilization of these initial and perhaps local structural changes might require

an additional set of cellular processes, including altered macromolecular synthesis, which would then facilitate the further elaboration of the newly formed synaptic connections and allow them to persist for the duration of the long-term memory.

In contrast to the studies of learning and memory in the vertebrate central nervous system, the work on Aplysia provides an example of learning-related synaptic modification in which behavioral, physiological, morphological, and molecular data can be directly correlated at the level of a single identified synapse. These advantages have allowed us to demon- strate that the formation and elimination of synaptic connections which accompany long-term heterosynaptic plasticity are dependent on altered macromolecular synthesis. By combining video-enhanced imaging of the living terminal, thin-section electron microscopy, specific molecular probes, and gene transfer methods, it should now be possible to exam- ine the subcellular and molecular mechanisms re- sponsible for the structural changes characteristic of long-term memory.

NeWOn 756

W Serotonin A FMRFamide - - -

0 Sero + Aniso 0 FMRF+Aniso A Actinomycin D 0 Sero + Actino + FMRF + Actino

-100 ! I I I 1 I I I I I I -60 -40 -20 0 20 40 60 80 100 120 140

SN Varicosities (% Change)

Figure 6. Multiple Regression Analysis of the Relationship between the Amplitude of the EPSP and the Changes in the Number ot Sensory Neuron Varicosities

The analysis of covariance indicates that the change in EPSP is correlated with the change in varicosities (see text for details).

Experimental Procedures

Cell Culture Aplysia sensory neurons were isolated from either the abdominal ganglion or pleural ganglia dissected from adult animals (70- 120 g) and cocultured with the gill motor neuron L7 isolated from the abdominal ganglion of juvenile animals (l-3 g; Howard Hughes Medical Institute) as described previously (Schacher, 1985; Rayport and Schacher, 1986). Individual cells were isolated from the ganglia with a segment (200-800 pm) of their axons intact (Schacher and Proshansky, 1983). Each culture contained a single sensory neuron and a single motor cell. Cocultures of sensory neurons and L7wereallowed togrow for5days to permit the formation of stable connections and synaptic contacts. Previ- ous studies demonstrated that the amplitude of the EPSP recorded in L7 gradually increases over the first 4 days in culture before stabilizing on day 5 (Montarolo et al., 1986; Dale et al., 1988; Schacher et al., 1990). Sensory neuron neurites contacting the major axons of L7, a region of the motor cell that is a preferred substrate for the growth of new sensory neuron varicosities containing transmitter release sites, also reach a level of stability by 5 days and remain relatively stable over the next 24-48 hr (Glanzman et al., 1989, 1990). We have therefore used 5-day-old cocultures of sensorimotor synapses to determine the effects of inhibitors of protein (anisomycin) and RNA (actinomycin D) synthesis on the long-lasting, transmitter-induced structural changes in the sensory neurons and their relationship to the long-term regulation of the EPSP evoked in the motor cell L7.

Electrophysiology and Application of Neuromodulators The stimulation and recording techniques and perfusion medium have been described (Dale et al., 1988; Montarolo et al., 1988).

The motor cell was impaled intracellularly with a glass mi- croelecrode (15-25 Ma) containing 2.0 M KCI and held at a potential of -30 mV below resting level to permit accurate measure- ment of the amplitude of the EPSP. EPSPs were evoked in L7 by stimulating each sensory cell with a brief (0.2-0.5 ms) depolariz- ing pulse using an extracellular electrode filled with perfusion medium placed near the cell body of the sensory neuron. One EPSP was evoked in L7 before and after the various treatments. The initial EPSP amplitudes ranged from 6 to 46 mV.

To produce long-term facilitation, theexperimental cocultures received five 5 min applications of 5-HT as previously described

(Glanzman et al., 1990). 5-HT (creatinine sulfate salt; Sigma) was added to the bath to a final concentration of 2.5 PM. After 5 min the 5-HT was gradually washed out by perfusion with 20 cc of 50% L15 culture medium (Flow Laboratories) and 50% artificial seawater (pH 7.6). Washout of each 5-HTapplication took 15 min.

To produce long-term depression, each culture was perfused with 5 ml of 1 PM FMRFamide (5 min) (Peninsula Laboratories) followed by 15 ml of wash at a rate of 1.5 mllmin. This was repeated four times at 30 min intervals. After treatment the cocultures were returned to the culture medium and placed back into an incubator operating at 18OC. Control cocultures were given four applications of perfusion medium used to dissolve the neuromodulators. The same cocultures were reexamined approxi- mately 24 hr later.

Application of Inhibitors of Protein and RNA Synthesis We examined the effects of transcriptional and translational inhibitors on long-term facilitation and long-term depression fol- lowing a protocol previously described (Montarolo et al., 1986). Anisomycin (IO PM) was added to the cocultures 1 hr before the initial recording and was present continuously until 30 min after the last neuromodulator application. Actinomycin D (50 pg/ml) was added 1 hr before the first recording session, after which cells were perfused with drug-free medium. Actinomycin D was not perfused continuously, since a previous study had demonstrated that a 1 hr exposure irreversibly blocked total RNA synthesis in Aplysia neurons by more than 90% (Montarolo et al., 1986). Cocultures treated with the inhibitor alone received identi- cal protocols, but no 5-HT or FMRFamide.

Dye Injection and Cell Imaging To label sensory neurons fluorescently, cells were impaled with glass micropipettes filled with a 6% sterile-filtered (Gelman 0.2 vrn Acrodisc 13 filter) solution of 5(6)-carboxyfluorescein (chro- matographically purified; Molecular Probes) in 0.44 M KOH (pH 7.0, resistances 50-90 MO) and injected into each sensory neuron with 0.4-0.6 nA hyperpolarizing current pulses (500 ms duration at 1 Hz) for 4-6 min as described previously (Glanzman et al., 1989, 1990). Sensory cells were injected with dye immediately after measuring the amplitude of the EPSP on each day to exam- ine the long-term effects of 5-HT, FMRFamide, or the control apphcation. Phase or Nomarski contrast and fluorescence images of the same view areas were taken both before and approx- imately 24 hr after treatment on a Zeiss IM 35 microscope


equipped with conventional phase-contrast and epilfuorescence (FITC filter set) optics with a silicon-intensified target (SIT) video camera (Hamamatsu C2400) or on a Nikon Diaphot microscope with a Dage 66 SIT camera. Video images were recorded and enhanced with a monochrome optical disc recorder (Panasonic) or by a Dell 310 computer with a PC Vision Plus frame grabber, using an image processing software library from ITEX, and stored on a Storage Dimension optical disc drive. Alignment of the live view area on the second day with the recorded image of the first day was performed by hand. Illumination used for obtaining the fluorescence images was kept as low as possible to prevent photo damage, but was of sufficient intensity to permit sharp computer-enhanced views. To minimize differences in imaged structures that might arise as a result of differences in the amount and extent of dye filling, the view area was illuminated with an appropriate light intensity and by subsequent computer enhancement of the images. Light intensities used on the second daywereadjusted to match the light intensityofthestored image taken on thefirstday. Black and white fluorescence micrographs of the images were made with a Mitsubishi P6OU or Sony video copy processor.

Quantification of Structural Change Measurements were obtained from fluorescence images of sensory neuron neurites as they extended on the axon hillock and major axons of L7 until the distal axon stumps (300-500 urn). Only those sensory neuron varicosities in apposition to the initial segment and major axons of L7 were counted. Previous studies (Glanzman et al., 1989,199O; Schacher and Montarolo, 1991) have indicated that this postsynaptic surface of L7 is the most favor- able for sensory neuron regrowth and contains most of the neurite outgrowth, varicosities, and release sites of the sensory neuron. Since the initial segment of L7 and the most proximal parts of its major axons are relatively thick structures, it often required up to four different focal planes to image all of the labeled sensory neuron neurites fully. For each area, each focal plane imaged after treatment was matched to that viewed before treatment. Furthermore, to minimize slight differences in focus that could obscure varicosities and processes, we used computer- assisted superimposition of the various focal planes (two, three, or four, depending upon the complexity of the outgrowth) onto one two-dimensional image. The fluorescence images of each focal plane along with the superimpositions for the two time points were compared, and the total number of varicosities was counted. We measured the net change in varicosity number for each coculture and did not statistically compare whether treatment influenced the loss and/or gain of specific varicosities. Vari- cosities were identified according to criteria previously estab- lished for sensory neurons (Bailey et al., 1979; Bailey and Chen, 1983, 1988a; Glanzman et al., 1990; Schacher and Montarolo, 1991) and included all slightly elongated spheres of approxi- mately 3 urn or more in diameter. The counting of varicosity number in the fluorescence micrographs was performed blind, i.e., the individual counting did not know the amplitude of the EPSPs before or after treatment or the nature of the treatment.

Analysis of Data All data are represented as the mean change f SEM (unless specified). A one-factor analysis of variance and Dunnett’s t test (two-tailed) were used to measure significance of the long-term changes produced by various treatments. The graph in Figure 6 presents the values for each culture in each treatment group (n = 96cuItures).Theregressionlinesdrawn tofitthedatapoints are based on the equation, y = J.& + J&T, + Bzx, in which B0 is the y axis intercept; T, is the treatment variable, and 8, and BZ are the values for the B coefficient for the treatment variable and x axis variable (x), respectively. The F value given in the text indicates the significanceof R*when both independent variables are used.

Acknowledgments

We thank H. Ayers and A. Krawetz for typing the manuscript. This research was supported by NIH grant MH37134 to C. H. B.,

NSF grant BNS 9021612 to S. S., and Program Project Grant CM32099 as well as support from the Howard Hughes Medical Institute to E. R. K.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Sec- tion 1734 solely to indicate this fact.

Received May 22,1992; revised July 21, 1992.

References

Agranoff, B. W. (1967). Agents that block memory. In The Neuro- sciences: A Study Program, C. C. Melnechuk, T. Schmitt, and F. 0. Schmitt, eds. (New York: The Rockefeller University Press), pp. 756-764.

Atwood, H. L., Dixon, D., and Wojtowicz, J. M. (1989). Rapid introduction of long-lasting synaptic changes at crustacean neuromuscular junctions. J. Neurobiol. 20, 373-385.

Bailey, C. H. (1991). Morphological basis of short- and long-term memory in Aplysia. In Perspections on Cognitive Neuroscience, R.G. ListerandH.1. Weingartner,eds.(NewYork:OxfordUniver- sity Press), pp. 76-92.

Bailey, C. H. (1992). Morphological basis of learning and memory: invertebrates. In Encyclopedia of Learning and Memory, 1. Squire, ed. (New York: Macmillan), in press.

Bailey, C. H., and Chen, M. (1983). Morphological basis of long- term habituation and sensitization in Aplysia. Science 220, 91-93.

Bailey, C. H., and Chen, M. (1988a). Long-term memory in Aplysia modulates the total number of varicosities of single identified sensory neurons. Proc. Natl. Acad. Sci. USA 85, 2372-2377.

Bailey, C. H., and Chen, M. (1988b). Long-term sensitization in Aplysia increases the number of presynaptic contacts onto the identified gill motor neuron L7. Proc. Natl. Acad. Sci. USA 85, 9356-9359.

Bailey, C. H., and Chen, M. (1989a). Time course of structural changes at identified sensory neuron synapses during long-term sensitization in Aplysia. J. Neurosci. 9, 1774-1780.

Bailey, C. H., and Chen, M. (198913). Struciural plasticity at identified synapses during long-term memory in Aplysia. J. Neurobiol. 20, 356-372.

Bailey, C. H., Thompson, E. B., Castellucci, V. F., and Kandel, E. R. (1979). Ultrastructure of the synapses of sensory neurons that mediate the gill-withdrawal reflex in Aplysia. J. Neurocytol. 8, 415-444.

Barondes, S. H. (1975). Protein synthesis dependent and protein synthesis independent memory storage processes. In Short- Term Memory, D. Deutsch and J. A. Deutsch, eds. (New York: Academic Press, Inc.), pp. 379-390.

Barzilai, A., Kennedy, T. E., Sweatt, J. D., and Kandel, E. R. (1989). 5-HT modulates protein synthesis and the expression of specific proteins during long-term facilitation in Aplysia sensory neurons. Neuron 2, 1577-1586.

Bergold, P. J., Sweatt, J. D., Winicov, I., Weiss, K. R., Kandel, E. R., and Schwartz, J. H. (1990). Protein synthesis during acquisi- tion of long-term facilitation is needed for the persistent loss of regulatory subunits of the Aplysia CAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 87, 3788-3791.

Carew, T. J., Castellucci, V. F., and Kandel, E. R. (1971). An analysis of dishabituation and sensitization of the gill-withdrawal reflex in Aplysia. Int. J. Neurosci. 2, 79-98.

Carew, T. J., Pinsker, H. M., and Kandel, E. R. (1972). Long-term habituation of a defensive withdrawal reflex in Aplysia. Science 175, 451-454.

Castellucci, V. F., Carew, T. J., and Kandel, E. R. (1978). Cellular analysis of long-term habituation of the gill-withdrawal reflex of Aplysia californica. Science 202, 1306-1308.

Castellucci, V. F., Blumenfeld, H., Coelet, P., and Kandel, E. R. (1989). Inhibitorof protein synthesis blocks long-term behavioral

NellrOn 7%

sensitization in the isolated gill-withdrawal reflex of Aplysia. J. Neurobiol. 20, l-9.

Chang, F. F., and Greenough, W. T. (1984). Transient and enduring morphological correlates of synaptic activity and efficacy change in the rat hippocampal slice. Brain Res. 309, 35-46.

Crick, F. (1984). Memory and molecular turnover. Nature 372, 101-102.

Dale, N., Kandel, E. R., and Schacher, S. (1987). Serotonin produces long-term changes in the excitability of Aplysia sensory neurons in culture that depend on new protein synthesis. J. Neu- rosci. 7, 2232-2238.

Dale, N., Schacher, S., and Kandel, E. R. (1988). Long-term facilitation in Aplysia involves increases in transmitter release. Science 239, 282-285.

Davis, H. P., and Squire, L. R. (1984). Protein synthesis and memory: a review. Physiol. Bull. 96, 518-559.

Flexner, J. B., Flexner, L. B., and Stellar, E. (1963). Memory in mice as affected by intracerebral puromycin. Science 747, 567-569.

Frost, W. N., Castellucci, V. F., Hawkins, R. D., and Kandel, E. R. (1985). Monosynaptic connections made by the sensory neurons of thegill and siphon-withdrawal reflex in Aplysia participates in the storage of long-term memory for sensitization. Proc. Natl. Acad. Sci. USA 82, 8266-8269.

Clanzman, D. L., Kandel, E. R., and Schacher, S. (1989). Identified target motor neuron regulates neurite outgrowth and synapse formation of Aplysia sensory neurons in vitro. Neuron 3, 44- 450.

Clanzman, D. L., Kandel, E. R., and Schacher, S. (1990). Target- dependent structural changesaccompanying long-term synaptic facilitation in Aplysia neurons. Science 249, 799-802.

Goelet, P., Castellucci,V. F., Schacher, S.,and Kandel, E. R. (1986). The long and the short of long-term memory-a molecular frame- work. Nature 322, 419-422.

Greenough, W. T., and Bailey, C. H. (1988). The anatomy of a memory: convergence of results across a diversity of tests. Trends Neurosci. 77, 142-147.

Greenough, W. T., and Chang, F.-L. F. (1985). Synaptic structural correlates of information storage in mammalian nervous sys- tems. In Synaptic Plasticity, C. W. Cotoran, ed. (New York: Guil- ford Press), pp. 335-372.

Han, H.-Q., Nichols, R. A., Rubin, M. R., Blhler, M., and Creengard, P. (1991). Induction of formation of presynaptic ter- minals in neuroblastoma cells by synapsin Ilb. Nature 349,697- 700.

Kelly, R. B. (1991). A system for synapse control. Nature 349,650- 651.

Lee, K. S., Schottler, F., Oliver, M., and Lynch, C. (1980). Brief bursts of high-frequency stimulation produce two types of structural change in rat hippocampus. J. Neurophysiol. 44, 247-258.

Lee, K. S., Oliver, M., Schottler, F., and Lynch, C. (1981). Electron microscopic studies of brain slices: the effects of high-frequency stimulation on dendritic ultrastructure. In Electrophysiology of Isolated Mammalian CNS Preparations, G. A. Kerkut and H. V. Wheal, eds. (New York: Academic Press, Inc.), pp. 189-211.

Matthies, H. (1991). Long-lasting maintenance of long-term potentiation and macromolecular synthesis. In Long-Term Potenti- anon-A Debate of Current Issues, M. Baudryand J. L. Davis, eds. (Cambridge, Massachusetts: MIT Press), pp. 233-243.

Montarolo, P. G., Goelet, P., Castellucci,V. F., Morgan, J., Kandel, E. R., and Schacher, S. (1986). A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia. Science 234, 1249-1254.

Montarolo, P. G., Kandel, E. R., and Schacher, S. (1988). Long-term heterosynaptic inhibition in Aplysia. Nature 333, 171-174.

Nazif, F., Byrne, J. H., and Cleary, L. J. (1991). CAMP induces long-term morphological changes in sensory neurons of Aplysia. Brain Res. 539, 324-327.

Pinsker, H., Kupfermann, I., Castellucci, V. F., and Kandel, E. R. (1970). Habituation and dishabituation of thegill-withdrawal reflex in Aplysia. Science 767, 1740-1742.

Pinsker, H. M., Hening, W. A., Carew, T. J., and Kandel, E. R. (1973). Long-term sensitization of a defensive withdrawal reflex in Aplysia. Science 782, 1039-1042.

Purves, D., and Lichtman, J. W. (1985). Principles of Neural Devcl- opment (Sunderland, Massachusetts: Sinauer Associates).

Rayport, S. G., and Schacher, S. (1986). Synaptic plasticity in vitro: cell culture of identified neurons mediating short-term habituation and sensitization. J. Neurosci. 6, 759-763.

Schacher, S. (1985). Differential synapse formation and neurite outgrowth at two branches of the metacerebral cell of Aplysia in dissociated cell culture. j. Neurosci. 5, 2028-2034.

Schacher, S., and Montarolo, P. C. (1991). Target-dependent structural changes in sensory neurons of Aplysia accompany long-term heterosynaptic inhibition. Neuron 6, 679-690.

Schacher, S., and Proshansky, E. (1983). Neurite regeneratron by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment. J. Neurosci. 3, 2403-2413.

Schacher, S., Castellucci, V. F., and Kandel, E. R. (1988). <AMP evokes long-term facilitation in Aplysia sensory neurons that requires new protein synthesis. Science 240, 1667-1669.

Schacher, S., Montarolo, P. G., and Kandel, E. R. (1990). Selective short- and long-term effects of serotonin, small cardioactive pep- tide, and tetanic stimulation on sensorimotor synapses ofAplys/a in culture. J. Neurosci. 10, 3286-3294.

Squire, L. R. (1987). Memory and Brain (NewYork: Oxford Univer- sity Press).

Sweatt, J. D., and Kandel, E. R. (1989). Persistent and tran- scriptionally-dependent increase in protein phosphorylation upon long-term facilitation in Aplysia sensory neurones. Nature 339, 51-54.

Wallace, C. S., Hawrylak, N., and Greenough, W. T. (1991). Stud- ies of synaptic structural modifications after long-term potentiation and kindling: context for a molecular morphology. In Long- Term Potentiation-A Debate of Current Issues, M. Baudry and J. L. Davis, eds. (Cambridge, Massachusetts: MIT Press), pp. 189- 232.

Wojtowicz, J. M., Marin, L., and Atwood, H. L. (1989). Synaptjc restructuring during long-term facilitation at the crayfish neuromuscular junction. Can. J. Physiol. Pharmacol. 67, 167-171.

neuron, vol. 9, 749-758, october, 1992, copyright 0 1992...

Documents