burst firing in midbrain dopaminergic neurons

7/30/2019 Burst Firing in Midbrain Dopaminergic Neurons

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.Brain Research Reviews 25 1997 312334

Full-length review

Burst firing in midbrain dopaminergic neuronsP.G. Overton ), D. Clark

Neuropsychopharmacology Laboratory, Department of Psychology, Uniersity of Wales, Singleton Park, Swansea SA2 8PP, UK

Accepted 9 September 1997

Abstract

.Midbrain dopaminergic DA neurons fire bursts of activity in response to sensory stimuli, including those associated with prim

reward. They are therefore conditional bursters the bursts conveying, amongst other things, motivationally relevant information to

forebrain. In the forebrain, bursts give rise to a supra-additive release of dopamine, and possibly favour the release of co-locali

neuropeptides. Evidence is presented that in rat DA neurons, bursts are engendered by the activity of cortically-regulated affere .Certain factors are identified which, in combination, lead to burst production: 1 A burst of activity in EAAergic afferents to DA neu

.arising from non-cortical sources, but controlled by the medial prefrontal cortex; 2 N-methyl-D-aspartate receptor activation, produ .a slow depolarising wave in the recipient neuron; 3 activation of a high threshold, dendritically located calcium conductance w

.produces a plateau potential; 4 activation of a calcium-activated potassium conductance, which terminates the burst. These factors

argued to operate in the context of an optimal level of intracellular calcium buffering for bursting. Other factors which appear to

involved in bursting in other systems, in particular a low threshold calcium conductance, are rejected as being necessary for burstin

DA neurons. The factors which do play a crucial role in burst production in DA neurons are integrated into a theory from which aris

series of hypotheses amenable to empirical investigation. Additional factors are discussed which may modulate bursting. These may ei .act indirectly through changes in membrane potential or intracellular calcium concentration , or they may act directly through

interaction with certain conductances, which appear to promote or inhibit burst firing in DA neurons. q 1997 Elsevier Science B.V

Keywords: Ventral tegmental area; Substantia nigra pars compacta; Plateau potential; Slow EPSP; Calcium activated potassium conductance; Rat

Contents

1. Natural burst firing in midbrain dopaminergic neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Functional aspects of burst firing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. Afferent control of burst firing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4. The mechanism of burst firing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.1. Distal influences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2. Proximal influences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.1 Synaptic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.2 Intrinsic membrane properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.3 Role of intracellular calcium buffering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. A theory of natural burst firing in dopaminergic neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6. Endogenous modulatory influences on bursting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.1. Indirect modulatory influences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.2. Direct modulatory influences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

) .Corresponding author. Fax: q44 1792 295679; E-mail: [email protected]


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( )P.G. Oerton, D. ClarkrBrain Research Reiews 25 1997 312334

7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Natural 1 burst firing in midbrain dopaminergic

neurons

The review will be mainly concerned with dopaminer- .gic DA neurons belonging to the cell groups A9 and

A10. Cell group A9 is located mainly in the substantia .nigra pars compacta SNPc , whilst cell group A10 is

located mainly in the ventral tegmental area VTA, which

includes the paranigral nucleus and parabrachial pigmentedw x.nucleus, according to Paxinos and Watson 132 . The

third group of midbrain DA neurons, A8, located in the

retrorubral field, will receive little attention, primarily

because far less is known about the electrophysiological

characteristics of these cells. Again, primarily due to the

preponderance of studies, the review will be mainly con-

cerned with rat DA neurons. However, information gleaned

from the study of DA neurons in other species will be

included wherever relevant. Although bursts in DA neu-

rons of diverse species are almost certainly homologues of

those in the rat, nonetheless some caution should be excer-

cised when species barriers are crossed.

A9 and A10 DA neurons of rats anaesthetised with

chloral hydrate discharge action potentials in a pattern

which consists of irregular single spikes, or bursts of

spikes with short interspike intervals. Spikes within the

bursts exhibit a progressively decreasing spike amplitude

and a progressively increasing spike duration and inter-spike interval. The burst is followed by a quiescent period

w x .before spiking recommences 184,49,25,55,125 Fig. 1A .

This bursting pattern is exhibited by approximately 18% of

A9 and 73% of A10 cells in the chloral hydrate anaes-w xthetised rat 55 . However, the picture is different in

paralysed, locally anaesthetised animals. Here, A9 cellsw xonly rarely show bursts 12,109 and A10 cells show

w xlittle or no bursting 12 . This contrasts markedly with

the bursting seen in cells recorded in animals under chloral w x.hydrate anaesthesia in the same studies e.g. Ref. 12 , and

suggests that this anaesthetic increases bursting in rat DA

neurons. This contention is supported by the cat literature,where an increase in bursting has been reported in DA

neurons when chloral hydrate was administered in a pre-w xdrugrpost-drug comparison 163 .

1We will use the term natural bursts to denote those events which

DA cells exhibit as part of their normal physiological repertoire. The

alternative term spontaneous bursts, which is used by some investiga-

tors, is rejected because it implies that the events are acausal, which we

consider to be misleading.

In contrast to the low level of bursting in loc

anaesthetised, paralysed rats, in freely moving rats level of bursting is substantially higher. Over 82% of

.neurons cell group unknown were reported to burst w xin the freely moving rat by Miller et al. 113 . Likew

over 90% of A10 neurons exhibited burst firing in w xfreely moving rat in Freeman and Bunney 36 . The

crepancy between the level of bursting in paralysed,

cally anaesthetised rats, and freely moving rats sugg

that bursting is related either to the degree of sensstimulation which should be greater in the freely mov

.animal , or to an interaction between movement and sti

lation.

The activity pattern which is characteristic of burst

DA neurons in the rat has also been found in DA neurw x of the mouse 144 , guinea-pig Overton and Greenfi

. w xunpublished observations , cat 163,176,164,177,166 w x w xmonkey 147 . Interestingly, Schultz and Romo 147

port that, in the pentobarbitone-anaesthetised monk

only very rarely DA neurons were recorded that

charged in burstlike patterns as described for DA cell

the rat. However, in the behaving monkey, as in

freely moving rat, the level of bursting is substanti .higher see Section 2 .

2. Functional aspects of burst firing

The studies above were concerned with the burst ac

ity of DA neurons under basal conditions, i.e. in sit

tions where no attempt was made to correlate burst activ

with the occurrence of particular stimuli or movem .where relevant in the animal. However, a number

studies in rats and other species have demonstrated

bursting is responsive.

In the rat, the first hint of a correlation between burst

and stimuli or movements appeared in a paper by Millew x

al. 112 . Rats were required to press a lever concomitawith the presentation of a visual, auditory or compo

stimulus. Although no quantitative data were presen

one A10 cell shown in Fig. 2 of their paper gave a burs

spikes 120 ms after the presentation of the discrimina

stimulus. The relationship between the response and mo

ment was not explored. Again, rather anecdotally, in w xfreely moving rat, Freeman et al. 35 reported

w x bursts . . . appeared to be associated with an orien

response elicited by a whistle or by intrusion of

experimenters hand into the recording chamber. T


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Fig. 1. Bursts in midbrain DA neurons of the rat under chloral hydrate anaesthesia. In each case, spikes included in the burst are bracketed together. A

.natural burst, consisting of a series of action potentials with increasing interspike intervals and decreasing action potential amplitudes. B : a time-loburst during an IE response elicited by single-pulse electrical stimulation of the mPFC. Stimulus delivery is indicated by arrow head. As in the case of

natural burst, this burst consists of a series of action potentials with increasing interspike intervals and decreasing action potential amplitudes. The b

was preceded by a single-spike excitation.

initial observation was expanded upon by Freeman andw xBunney 36 , who reported that small movements of the

.vibrissae and snout as in mild sniffing were associated

with the emission of a burst of spikes. Bursts were also

seen during manual stimulation of vibrissae, where they

were produced in a discrete manner one burst per

stimulus. In addition, transient increases in burst firingwere seen in association with orienting responses to audi-

tory stimuli. However, since the animals always exhibited

an orienting response to the auditory stimulus, it could not

be determined whether the burst was movement or stimu-

lus related in this case.

The same is true of the cat, where orientation in re-sponse to a stimulus opening the door to the experimental

room in which the cats chamber was housed, or the.appearance of the experimenter in the field of vision has

been reported to be associated with a short approximately. w x200 ms burst of unit activity 164 . Again, the relation-

ship between the burst response and movement was notexplored. However, the bursting response engendered byw xvibrissal stimulation in Freeman and Bunney 36 suggests

that movement is not necessary for bursting. This conclu-w xsion is reinforced by the work of Diana et al. 31 , where

DA neurons were recorded whilst rats walked on a circular

treadmill. They found that the cells exhibited an increase

in bursting after circular walking. It must also be remem-

bered that bursting is exhibited by DA neurons under

general anaesthesia, and, to some extent, in the paralysed .animal see Section 1 .

The conclusions derived from the rat work above

garding the stimulus-bound nature of bursting in DA n

rons are supported by work in the monkey. In 1w x .Schultz 146 reported that many 55% DA neurons in

behaving monkey responded to a behavioural trigger st

ulus in an operant task with a short burst of impulse

Although no formal comparison was made between characteristics of these bursts and those in the rat, some

the cells shown in the figures exhibited a clear post-b

quiescent period. The stimulus was the opening of the d

of a food box into which the animal would subsequen

reach for a food reward. Each stimulus produced a sin

burst temporally correlated with its presentation. Analy

of the neuronal response suggested that it was not rela

to overt aspects of task performance, like reaching for

food. For example, 9r11 neurons were activated by

opening of an accessory box which did not contain foand therefore did not trigger a limb movement or EM

.activity . Although most of the cells belonged to thegroup, cells belonging to groups A10 and A8 were a

recorded. There were no qualitative differences betw

the responses of neurons from the three cell groups.

These findings were confirmed and extended in l

studies. During self-initiated movements in the monke

burst of impulses was found to occur when the anim

hand touched a morsel of food located inside a food bw x143 . The responses appeared to be related to the app

tive properties of the object being touched, since most

neurons were not activated by the touch of non-f


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objects in the box when the monkey entered the box inw xabsence of food 143 . During stimulus-triggered move-

ments, DA neurons discharged a burst of impulses in

response to the opening of the food box door, but failed to

respond to the touch of food in the box. Numerous subse-

quent studies demonstrated that DA neurons respond to

conditioned stimuli once task performance is established w xfor example in a go-no go task 148 or a delayed

w xalternation task 91 . This suggests that the response of theDA neuron is not immutably associated with primary

reinforcers, but can transfer under appropriate circum-

stances to conditioned stimuli. This issue was more fullyw xinvestigated by Schultz et al. 149 , who examined learning

vs. established performance in three operant tasks. They

found that more DA neurons responded to delivery of

liquid reward during learning than after task performance

was established. Once established, neurons responded to

the conditioned stimuli used in the tasks.

Although the studies by Schultz and colleagues show

that DA neurons in the monkey produce a burst response

in the presence of appetitive stimuli, which is unrelated to .limb movement or EMG activity , or eye movements,

certain findings suggest that the burst response is notw xlimited to appetitive stimuli. In Schultz and Romo 148

animals performed a go-no go task, consisting of twodoors, one rewarded animals reached for food when it

. opened and one unrewarded animals refrained from.reaching . Dopaminergic neurons were activated by open-

ing of both go and no go doors. However, in a variation on

the task, a light was illuminated 23 s before door open-

ing. In one of the two monkeys, 38% of DA neurons were

activated by the light. Importantly, responses were present

when the task was used infrequently, but largely disap-

peared with more regular testing. This suggests that DAneurons may respond to novel stimuli with properties

uncertain to the animal. Subsequent studies have con- .firmed that some DA neurons exhibit a phasic response

w x.to novel stimuli e.g. Ref. 92 . Hence, although bursting

in the monkey appears to be stimulus-related, it appears to

signal the occurrence of ethologically significant or salient

stimuli, rather than simply appetitive stimuli. Having saidthis, DA neurons are not activated by aversive but non-

. w xnoxious stimuli 115 , suggesting that bursts signal salient

stimuli with a positive valence.

The fact that bursting in DA neurons is stimulus-bound

in vivo suggests that burst activity in these cells is underthe control of afferent inputs. Indeed, in the slice where.the cells lack medium and long-length afferents DA neu-

rons exhibit a highly regular pacemaker-like firing patternw x w x136,157,50 ; but see 111 . Hence, DA neurons are condi-

tional rather than intrinsic or constitutive bursters.

Many conditional bursting cells have been identified invertebrates e.g. neurons of the guinea-pig subthalamic

w x. nucleus 126 and invertebrates e.g. the anterior burster

neuron of the stomatogastric ganglion of the spiny lobsterw x.60 .

Burst firing in DA neurons is almost certainly of fu

tional significance, since electrical stimulation of the ax

of these cells in a pattern which resembles natural bu

firing produces significantly greater dopamine release

the forebrain than is observed with stimuli delivered at

same overall frequency but with a constant inter-stimuw xinterval 41 . This is probably the result of a build up

w calcium in the terminal during closely spaced spikes 1

which produces an increase in the probability of relew x165 . Hence, not only does the burst elevate transmi

release in the forebrain, but it also decreases the unw xtainty inherent in neurotransmission 89 .

The pattern of cell firing has been found to be import

in other systems for the release of co-localised pep

neurotransmitters. In particular, high frequency firing,

pecially in bursts, can produce greater peptide relew x98,71 . Given that dopamine is co-localised with cholec

.tokinin CCK or neurotensin in some A9 and A10 w xneurons in the rat 63,64 , it is possible that preferen

release of these peptides occurs during bursts. Howe

the preferential release of peptides during bursts has been demonstrated in the ascending dopamine system

the rat. Furthermore, the extent to which DA neur

contain co-localised peptides may be species-specific

that CCK mRNA appears to be lacking in the humw xsubstantia nigra 130 .

In summary, bursts seem to signal the occurrence .salient stimuli with a positive valence . This implies

highly processed information is being passed, via an

known afferent, to the DA neurons, and that the bu

have some effect in the forebrain which is related

motivation. Given that the DA systems innervate, inalia, forebrain areas concerned with motor control e.g.

.neostriatum , bursts seem to communicate motivationrelevant information to forebrain structures involved

response execution. However, the functional aspects of

postsynaptic actions of transmitter release during bu

will not be addressed here, since this would involve a

examination of the function of dopamine in the forebr

which is beyond the scope of this review. Instead, we w

now turn our attention to the source of the afferent in

which produces bursts in DA neurons.

3. Afferent control of burst firing

It is likely that the afferent in question utilises .excitatory amino-acid EAA , since iontophoretic appl

.tion of the competitive N-methyl-D-aspartate NMDA . .tagonists CPP 3- " -2-carboxypiperazin-4-yl propy. .phosphonic acid and AP-5 " -2-amino-5-phospho

. w xpentanoic acid reduce the level of bursting 125,20 .

various issues surrounding the selectivity of this effect

fully discussed below in Section 4.2.1.

Major EAAergic inputs to DA neurons arise from .frontal cortex, subthalamic nucleus STN and the ped

.culopontine nucleus PPN . The frontal cortex has b


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w xshown to send a projection to the SNPc 119 , which isw xglutamatergic 17,83 , and an aspartatergic projection to

w xthe VTA 24 , which synapses directly with tyrosine hy- . w xdroxylase-positive presumably DA dendrites 150 . The

w xPPN projects to both the SNPc and the VTA 72 and thew xSTN directly to the SNPc 81 , and indirectly to the VTA

w xvia a projection to the PPN 72 . Stimulation of either

nucleus produces excitation in a high proportion of respon-

w x w xsive DA neurons 145,141 . Both the PPN 26 and thew xSTN 3 contain neurons which appear to be glutamatergic,

and the excitatory action of the PPN on the activity of DAw xneurons is blocked by an EAA antagonist 145 .

In addition to the frontal cortex, STN and PPN there are

other more minor sources of EAAergic afferents to the

ventral midbrain. For example, several subdivisions of the

amygdaloid complex project to the VTA, including thew xbasolateral subdivision 133 , some neurons of which have

been shown to be immunoreactive for glutamate androrw xaspartate 103 . Likewise, the laterodorsal tegmental nu-

w xcleus contains glutamate immunoreactive neurons 26 andw xprojects to the SNPc 43 . Finally, monosynaptic EAA-

mediated excitations have been recorded in A10 neuronsw xfollowing stimulation of the habenula nucleus 99 .

Hemisections at the level of the anterior STN or just

anterior to the STN significantly increases the regularity ofw xneuronal firing in both A9 and A10 cells 192 . Bursting

.expressed as the percentage of spikes occurring in bursts

is reduced in both A9 and A10 cells by this manipulation,

which implies that some regulatory control over bursting

comes from structures which are anterior to STN. The

major EAAergic source anterior to STN is the frontal

cortex.

An increasingly large amount of evidence implicates the

.medial prefrontal area of the frontal cortex mPFC in theinduction of burst firing in midbrain DA neurons. The

mPFC is the medial projection cortex of the mediodorsal

nucleus of the thalamus, consisting of areas CG1, CG2 andw xCG3 as demarcated by Zilles 194 , anterior and dorsal to

w x w x.the genu of the corpus callosum see Refs. 86 and 6 .w xCooling the mPFC 167 or injection of a local anaesthetic

w x w x.into this area 118 but see Ref. 156 has been reported

to reduce burst activity of A10 DA neurons, whilst chemi-

cal stimulation of the mPFC has been found to increasew xbursting 118 .

The mPFC receives extensive inputs from the limbic

w xsystem rat; reviewed by Neafsey et al. 120 ; primate;w xreviewed by Uylings and van Eden 179 . In the rat, the

mPFC sends projections to brainstem and spinal structures

involved in autonomic control, and to forebrain structures w x.directly connected to autonomic centres e.g. Ref. 69 .

Stimulation of the mPFC produces, amongst other things,w xpressor and depressor responses 13 , activation of the

w x w xsplanchnic nerve 181 and effects on gastric motility 70 .

Given these factors, it is not surprising that this area of the

cortex has been referred to as visceral motor cortexw x120 , and it is clear that it plays a role in the somatic

processes which underlie motivation. As stated abo

bursts of activity in DA neurons seem to signal the occ

rence of salient stimuli. Clearly, the mPFC is in a g

position to supply such motivationally relevant infor

tion.

If the mPFC is involved in the production of b

activity, then stimulation of the mPFC should be ass

ated with a stimulus-bound excitatory event which be

some resemblance to natural bursts. Until recently, o w x.one short manuscript Ref. 40 had focused on the eff

of electrical stimulation of the mPFC on the burst fir

activity of DA neurons. The authors reported that acti

tion of the mPFC produced bursts in a small numbe

A10 and A9 cells in the rat, although the details

unclear. In particular, the relationship between these bu

and natural bursts in DA neurons was not explored.

A more comprehensive analysis by the present auth

investigated the effects of single pulse electrical stimu .tion 0.25 and 1 mA of the mPFC on the extracell

w xactivity of A10 and A9 DA neurons 173 . The majority

cells were responsive to the stimulation, and two m .patterns of activity were elicited; 1 responses chaterised by an initial excitation E responses; 42% of

sponses at 0.25 mA and 27% at 1 mA; cell gro. .combined , and 2 responses characterised by excita

following an initial inhibition IE responses; 43% of

sponses at 0.25 mA and 57% at 1 mA; cell gro.combined . Analysis of the excitatory phases of E and

responses revealed that approximately one third contai

events which resembled natural bursts in DA neurons, which were closely time-locked to the stimulus Fig. 1

Further investigation revealed that the excitatory phase

IE responses was not causally related to the preced

w xinhibition 128 ; instead, the excitatory phases of IE anresponses, and the time-locked bursts which they cont

are probably homologous.

Given that natural bursts are produced by the activit

EAAergic afferents, if mPFC-induced time-locked bu

are homologues of natural bursts, EAA antagonists sho

attenuate them. Hence, we applied the competitive NMantagonist CPP and the AMPA "-a-amino-3-hydroxy

.methylisoxazole-4-propionic acid rkainate antago .CNQX 6-cyano-7-nitroquinoxaline-2,3-dione by

tophoresis to DA neurons exhibiting time-locked buw xduring mPFC stimulation 174 . CPP produced a sign

cant reduction in time-locked bursting. In contrast, CN .at currents which antagonised AMPA responses did

These effects of CPP and CNQX on time-locked burst

mirror the effects previously reported for these drugs

natural bursting. Since natural bursting and bursting

duced by mPFC stimulation are both blocked selectiv

by CPP, these results increase the degree of anal

between the two burst phenomena.

Indeed, the degree of analogy between natural bu

and time-locked bursts in DA neurons is high. Time-loc

bursts consist of two or more spikes with increas


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w xinterspike intervals and decreasing spike amplitudes 173 . w xFig. 1B , as do natural bursts 49 . Furthermore, there is a

positive correlation between the characteristics spikes per

burst, burst interspike interval and duration of the post-burst.quiescent period of natural bursts and time-locked bursts

w xexhibited by the same neuron 173 . In addition to these

similarities in the characteristics of the two burst phenom-

ena, they respond in a similar manner to certain pharmaco-

logical agents. Hence, both natural bursts and time-lockedbursts are increased by picrotoxin although in the latter

.case only in the lower dose range , and decreased byw xdopamine agonists 128 . Coupled with the fact that the

cortex is involved in producing both time-locked and

natural bursting, we believe that the above similarities

make time-locked bursts induced by cortical stimulation a

valid model of natural bursting in DA neurons.

The simplest explanation for the time-locked bursts is

that they are produced by the excitatory action of the

monosynaptic input to DA neurons from the mPFC, whichw xhas been demonstrated anatomically 150,119 . The princi-

pal problem for this hypothesis is the latency of the burst .events. Some E responses and occasional IE responses

showed a short-latency single-spike excitation before the .main response Fig. 1 , which exhibited a reasonable de-

.gree of temporal invariance, and a latency median 25 ms

which was within the realms expected for a monosynaptic

event. However, the bursts themselves had a median la-

tency of around 150 ms, which is well in excess of that

normally expected for monosynaptic events. Cortico-w xnigralrVTA fibres are very thin 119 , and therefore con-

duction velocity is low. However, although Thierry et al.w x171 found velocities as low as 0.7 mrs for fibres project-

ing from the mPFC to the substantia nigra, with an approx-

imate interelectrode distance in our study of 10 mm, amedian burst latency of 150 ms would give a conduction

velocity of 0.07 mrs ten times slower than the slowestw xfibres reported by Thierry et al. 171 . Given this, and the

degree of temporal variation in the latency of each burst,

we hypothesised that the bursts were polysynaptically gen-w xerated 173 .

The most straightforward scenario is that stimultion of

the mPFC affects pathways which ultimately result in the

activation of EAAergic afferents to the DA neurons, origi-

nating for example from the STN or PPN. This could be

achieved with comparatively few synapses, since the mPFC

has been shown to send a substantial projection to the STNw x w xin the rat 15 , which is excitatory 82 . There is also

evidence of a cortical projection to the PPN, at least in thew xcat 116 . In this regard it is interesting that ibotenic acid

lesions of the STN reduce the number of cells in the

substantia nigra pars reticulata which show excitatory re-w xsponses to cortical stimulation 37 . Hence, in the induc-

tion of natural burst activity in DA neurons, the mPFC

could be operating indirectly through the STN.

This conclusion is supported by the fact that lesions of

STN reduce bursting in DA neurons of the lateral SNPc

w xwithout affecting firing rate 160 . Likewise, local injec

of a GABA agonist into the STN decreases burstingAw xnigral DA neurons 160 . Conversely, local injection o

GABA antagonist into the STN increases bursting, agAw xwithout affecting firing rate 160 , and this increase

bursting is blocked by the NMDA antagonist AP-5, butw xby the AMPArkainate antagonist CNQX 21 . These

sults indicate that the STN can influence bursting

certain DA neurons, and that it plays a role in natbursting in these cells. This conclusion receives fur

support from the fact that stimulation of STN produce

burst-like event in 35% of A9 DA neurons, consisting

25 spikes exhibiting decreasing amplitude and increw xing duration 160 . Significantly, as with mPFC stim

tion in our own work, these events were long-latenlong-duration excitations although other similarities

tween the two burst phenomena remain to be establish

Involvement of non-cortical sources in the burst acti

of DA neurons may also be inferred from cell cul

studies. Bursting DA neurons have been reported in p

mary cultures derived from the mesencephalon of w x w xmouse 22,23 and the ventral midbrain of the rat 16 . O

possibility is that these cultures contain EAAergic cells

the SNPc of the guinea-pig, STN neurons appear tow xintermixed with DA neurons 127 . The same may be

for the mouse and rat. In cultured mouse DA neuro .excitatory post-synaptic potentials EPSPs are obser

w x23 , although the nature of the transmitter which under

them is unknown. However, the fact that EAA-media .excitatory post-synaptic currents EPSCs have b

w xrecorded in DA neurons in rat nigral slices 110 ,

NMDA-mediated bursts have been reported in DA neurw xin slices from immature rats 111 , suggests that there m

be an EAAergic cell population in the SNPc of orodent species besides the guinea-pig. Although the e

tence of a population of local, burst generating neur

appears to raise the possibility that non-cortical EAAer

sources can exert autonomous control over bursting in

neurons, i.e. independent of the cortex, it is difficul

ascertain how cultured neurons or those in the slice wo

perform in the presence of full circuitry in vivo.

4. The mechanism of burst firing

The observation that bursting is linked to specific st

uli in vivo suggests that the phenomenon is afferent drivHowever, this still leaves open the question of how

afferents responsible for bursting induce the phenomen

Broadly speaking, there are two levels at which fac

could exist which ultimately lead to the production

bursts in DA neurons. Firstly, bursts could arise a

consequence of activity patterns in pathways afferen

DA neurons, referred to here as distal influences. S

ondly, bursts could arise as a consequence of factors at

level of the cell itself, either arising from the propertie

certain synapses impinging upon the cells, or as a con


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quence of intrinsic membrane properties of the cells. These

factors are referred to here as proximal influences. Of

course, these different mechanisms may work in concert to

generate bursts. Furthermore, it is important to realise that

burst firing in DA neurons can be produced by more than w x.one mechanism see Ref. 87 . Hence, certain factors may

produce bursting in DA neurons, but this bursting may

have little relevance to the natural burst activity of these

cells. We must stress that we are ultimately interested inthe mechanism which generates natural bursts in DA neu-

rons.

4.1. Distal influences

In other systems, bursts can arise as an emergent prop-

erty of a neuronal network, for example the bursting of

lumbar motoneurons of the rat in vitro induced by thew xapplication of bicuculline and strychnine 14 . Perhaps the

most straightforward example of a network influence

would be a scenerio in which the EAAergic cells which

innervate DA neurons and which are responsible for burst

induction themselves burst fire. Bursts may be produced in

DA neurons because the requisite cell population which

innervates them is bursting. In effect, DA cells simply

follow a distal lead.

Evidence suggests that single-pulse stimulation of

EAAergic afferent fibres in the slice does not elicit burstsw x in DA neurons 73,110,57 although in the first two

studies holding current may have been applied to the cell.during afferent stimulation . This deficiency cannot be

attributed to a general inability of single-pulse stimulation

to elicit bursts via afferent stimulation in the slice, since

bursting can be elicited by this means in some systems, for

example the cortex, where single pulse activation of affer-

.ent fibres produces bursting in intrinsic bursting IBw xneurons in a number of areas 28,101,19 .

However, the response of DA neurons is very different

when EAAergic afferents in the slice are stimulated in aw xburst-like manner. Hence, Mercuri et al. 108 used a

.train of pulses 40200 ms, 5 20 stimuli applied to

EAAergic afferents in VTA. This train elicited a shortlatency, short duration depolarisation presumably

.AMPArkainate mediated followed by a long latency,

long-duration depolarisation. The latter was found to be

mediated by NMDA receptor activation, since it wasblocked by the NMDA antagonists ketamine and AP-5 the

AMPArkainate antagonist CNQX was ineffective against.the depolarisation . The fast and slow depolarisations were

separated by a GABA-mediated hyperpolarisation, and

GABA antagonists were found to enhance the late excita-

tion. In some cases, the fast depolarisation was not seen,

and the response began with the hyperpolarisation. A

similar finding was recently obtained by Shen and Johnsonw x153 , who reported that a train of pulses applied to the

VTA using stimulation parameters very similar to Mercuriw x.et al. 108 elicited a slow EPSC, which was partly

NMDA receptor mediated. In their study, a large compo-

nent of the slow EPSC was mediated by the activation .metabotropic glutamate mGlu receptors.

w xThe findings of Mercuri et al. 108 in vitro para

very closely those obtained by our group in vivo dur

mPFC-induced time-locked burst production in DA n

rons. Hence, stimulation of the mPFC elicts a long-late

excitation, a large part of which is mediated by the acti

tion of NMDA receptors. Activation of AMPArkain

w xreceptors does not appear to play a role 174 . In socells, the long-latency NMDA-mediated excitation is p

w x ceded by another short-latency excitation 173 Fig. 1

Furthermore, in the majority of responsive cells, the lo

latency excitation follows a period of inhibition, and ministration of a GABA antagonist in the lower dA

. w xrange enhances the late excitation 128 . These paraw xbetween our results and those of Mercuri et al. 108

quite striking. Importantly, in our study, burst-like eve

were elicited which closely resembled natural bursts in

neurons. Since these bursts appear to be a valid mode

natural bursts, this raises the possibility that natural bu

are also dependent upon a burst of activity in relevEAAergic afferent sources.

We argued above, based on results derived from

model of bursting, that time-locked bursting in DA n

rons produced by stimulation of the mPFC arises f

activity in non-cortical EAAergic afferents. If we hypo

sise that a bursting pattern of activity is required in

relevant EAAergic afferent pathways to produce burst

DA neurons, single-pulse stimulation of the mPFC m

ultimately lead to a burst of activity being generated in

most likely EAAergic afferent sources, the STN and

PPN. STN neurons are capable of generating burst

response to synaptic activation, largely via the activat

of a low threshold calcium conductance, producing a w x .threshold calcium spike 126 LTS . This makes it theo

ically possible that cortical stimulation could lead toafferent-induced burst response in STN cells. Some n

.cholinergic PPN neurons exhibit an LTS which can

activated by depolarising or hyperpolarising current puw x75 . However, as far as we are aware, the ability

afferent activity to generate bursts in PPN neurons has

been demonstrated directly.

Although there is evidence from our work that cort

control of bursting may occur via an indirect route,

long-latency of the monosynaptic NMDA-mediated de

w xlarising envelope in Mercuri et al. 108 and Shen w xJohnson 153 suggests that time-locked bursts do

necessarily have to be generated via a polysynaptic ro

It is possible that some are produced directly, via

monosynaptic pathway from the mPFC to A10 and

neurons which has been demonstrated anatomicw x150,119 . As we mentioned above, activation of affer

fibres to cortical neurons using single-pulse stimula

can produce bursts in IB neurons in various areas of w xcortex, including the anterior cingulate cortex 101 , wh

is part of the mPFC. Intrinsic bursting neurons are pyra


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w xdal neurons 101 , some of which project subcorticallyw x186 . This suggests that during single-pulse stimulation of

the cortex, stimulation of afferent fibres to IB neurons in

the vicinity will produce bursts in these neurons, some of

which may project directly to the ventral midbrain. The

high degree of latency variability of the time-locked bursts

in individual cells suggests that in most cases the phe-

nomenon is generated polysynaptically. However, there

may be a subpopulation of cells where bursting is pro-duced by this more direct route.

4.2. Proximal influences

4.2.1. Synaptic factors

As we stated above, evidence suggests that activation of

the NMDA receptor plays a crucial role in the burst

activity of DA neurons. Hence, one important proximal

influence appears to be the properties of this receptor.

Before we examine this issue more fully, it will be instruc-

tive to look more closely at the study which originally

demonstated the involvement of the NMDA receptor in the

burst activity of DA neurons, namely Overton and Clarkw x125 . The results of this study were clear-cut, in that they

did not encounter certain interpretational difficulties which

must be borne in mind when assessing the literature on

burst production in DA cells. .Most extracellular studies of bursting in DA neurons

use simple temporal criteria to discriminate burst from

non-burst events. These criteria, based on the work of w x.Grace and colleagues e.g. Ref. 47 , consider burst onset

to be indicated by an interspike interval of 80 ms or less,

and burst termination to be indicated by an interspike

interval of 160 ms or more. Other burst characteristics are

usually ignored. This means that simple changes in firing

.rate could lead to apparent spurious changes in bursting.Furthermore, since firing rate is proportional to membrane

w xpotential in DA neurons 157 , changes in firing rate may

reflect changes in membrane potential. Bursting in DA

neurons appears to be influenced by membrane potential .see below , and any effect on bursting produced by a drug

which affects firing rate may occur indirectly via a changew xin membrane potential. In Overton and Clark 125 , the

burst reduction produced by CPP was not accompanied by

a firing rate change.

The second interpretational difficulty which is encoun-

tered in the literature on bursting is engendered by the use

of intravenous drug administration. Where changes inbursting are produced when a drug is given intravenously,

the site of action of the drug relative to the cell beingw xstudied is unknown. In Overton and Clark 125 , CPP was

administered directly onto the cell under investigation

using iontophoresis. Hence, given the fact that the locus of

action was known, and the fact that CPP selectively attenu-

ated the bursting firing pattern, the study provides a cate-

gorical demonstration that bursting in DA neurons is pro-

duced by the activation of NMDA receptors located on DA

neurons.

This finding was confirmed and extended by Cherguw xal. 20 . Here, bursting was blocked by local applica

.iontophoresis and pressure ejection of the NMDA an

onist AP-5 but not by the AMPArkainiate antago w x.CNQX also see Ref. 174 . A role for NMDA in the b

activity of DA neurons is supported by fact that NMw xproduces burst firing in DA neurons 125 and in o

systems, for example rat abducens motoneurons in v

w x w x32 , rat supraoptic nucleus neurons in vitro 67 , w xnucleus basalis neurons in vitro 78 and cat cau

w xneurons in vivo 61 .

How specific is the involvement of NMDA receptor

the burst activity of DA neurons? DA neurons rece

afferents from some non-EAAergic sources which

excitatory. Is there any evidence for the involvemen

these transmitters in bursting? From the comparativ

little data that exists, the answer appears to be negat

For example, substance P, which is contained in afferew ximpinging on neurons of the A9 cell group 11 , some

w xwhich it excites 137 , does not appear to play a role

bursting, since intravenous administration of CP 96,345antagonist at the neurokinin-1 receptor at which subtancacts, leaves basal bursting unaffected in A9 and A

w xneurons 114 .

However, it does not simply appear to be the distinc

between non-EAAergic afferents and EAAergic affer

which is important for bursting in DA neurons, beca

only one subtype of EAA receptor appears to be involv

namely the NMDA receptor. The reason for this is unw xtain at present. In the study by Chergui et al. 20 , not o

did the AMPArkainate antagonist CNQX fail to decre

bursting in DA neurons, but it also failed to affect fir

rate. This raises the possibility that there may be a lack

endogenous activity at AMPArkainate receptors on neurons under normal circumstances. However, this se

unlikely, given that DA neurons have functional subsynw xtic AMPArkainate receptors 73 . Indeed, AMPArkain

receptor-mediated EPSCs have been detected in DA nw xrons in the slice 110 . Hence, the selective involvemen

the NMDA receptor in the burst activity of DA neur

does not appear to result from a lack of endogen

activity at AMPArkainate receptors on these cells. Rat

it may arise as a consequence of the properties of

NMDA receptor itself.

This opinion is reinforced by the findings of Chergu

w xal. 20 , where iontophoretic application of NMDA tsubpopulation of slow, non-bursting DA cells, produ

bursting whereas similar application of other EAA agon .quisqualate, kainate did not. Although NMDA may h

been producing bursting indirectly via a depolarisationthe membrane potential see above; NMDA did incre

.firing rate , this possibility is made less likely by the

that NMDA, but not quisqualate or kainate, produ

bursting even though the latter two compounds increa

firing rate. Other studies have also found that the action .NMDA or NMDA agonists is very different from tha


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other EAA receptor agonists. Hence, in the rat supraoptic

nucleus in vitro, bath application of NMDA producesw xbursting whereas AMPA does not 67 . Likewise, in the cat

caudate nucleus in vivo, iontophoretically applied NMDA .and the NMDA receptor agonist quinolinate produces

bursting, whereas glutamate, aspartate and quisqualate dow xnot 61 . Finally, in A10 DA neurons recorded in vitro,

w xJohnson et al. 74 found that burst firing was produced by

bath application of NMDA, but not kainate or quisqualate. .This latter study is important. One apparent problem

for the NMDA hypothesis of bursting in DA neurons was

that initial evidence suggested that NMDA did not producew x w xbursting in the slice 151 . However, Johnson et al. 74

reported that bursting could be produced in DA neurons

during bath application of NMDA, if hyperpolarising cur-

rent was injected into the cells to maintain spike threshold

at predrug levels and the bee venom toxin apamin was

added to the medium. In subsequent papers, apamin was

reported to be unnecessary for bursting in this preparation w x.e.g. Ref. 152 .

At first sight, there may seem to be a problem with

continuous application of NMDA in these studies. Why

does continuously applied NMDA substitute for NMDA

receptor activation by synaptic inputs? The latter occurs

during the discrete, time-limited release of transmitter

which takes place during synaptic activation. After all,bursts in DA neurons can occur as isolated events see

.Section 2 . However, the effects of continuous application

and discrete release during synaptic transmission may be

closer than they superficially appear. Natural bursts in DA

neurons appear to be delimited by intrinsic membraneproperties of the cell evidence indicates that a calcium-

activated potassium conductance is involved; see Ref.

w x.49 . Given that this is the case, after recovery from theprocess which forcibly terminates the burst, the cell be-

comes susceptible again to agonist, whether this is deliv-

ered synaptically in vivo, or has been present throughout in

the bath in vitro. Therefore, continuous application of a

drug which produces bursting may not be as unnatural as

it may at first appear. On a related point, in the lampreyw xspinal cord 183 , continuously applied NMDA produces

membrane potential oscillations in the majority of neurons,

which produce bursts of action potentials in the absence of . w xtetrodotoxin TTX . Wallen and Grillner 183 point out

that, in locomotion, afferent pathways will substitute for

bath applied drug, the assumption being that the two routescan achieve qualitatively similar effects.

However, simply assuring ourselves that bath applied

and synaptically applied NMDA receptor agonists can insome senses be considered equivalent ignoring the fact

that non-synaptic and subsynaptic receptors will be stimu-

lated by bath application, whereas only the latter are.stimulated during normal synaptic transmission , does not

w xmean that the bursts elicited by Johnson et al. 74 are

homologues of natural bursts in DA neurons. Indeed, therew xare serious problems with the bursts in Johnson et al. 74 .

Firstly, their bursts or at least the membrane poten.oscillation which underlies them are not calcium dep

dent, whereas natural bursts in DA neurons are criticw xdependent on intracellular calcium 49 . Instead, the bu

w xin Johnson et al. 74 are sodium dependent. These sod

dependent bursts may arise as a consequence of the f

that sodium is the principal charge carrier through

NMDA receptor-associated ionophore in their studies;

cium makes little contribution to the current see Rw x.191 . This is aberrant given the high calcium:sod

permeability ratio of NMDA receptor-associa w x.ionophores in other systems e.g. Ref. 100 , and the

w xthat others 105 have found that the current produced

bath applied NMDA in DA neurons is reduced by low

ing the extracellular calcium concentration.

Secondly, the NMDA-induced bursting in DA neurw xin Johnson et al. 74 required the injection of hyperpola

ing current. This would imply that, if this in vitro burs

was homologous to natural bursting, concurrent activat

of an inhibitory influence was necessary for natural bu

ing in DA neurons. Yet drugs which depolarise the mebrane potential of DA neurons, for example cholew x w xtokinin-8 190 , enhance burst firing in vivo 159 . H

w xever, Wang et al. 185 found that bath application

NMDA produced a burst-like pattern in DA neurons w

out the injection of hyperpolarising current if prolon .exposure 13 min to the agonist was allowed, wh

suggests that the injection of holding current may not

an absolute requirement for the occurrence of NM

induced bursting in vitro. How natural bursts are wh

evolve over such a time-frame is uncertain, especi

when their endogenous counterparts are produced ov

millisecond time-frame via receptor agonism during neu

transmission.The dissimilarity between natural bursts in DA neur

w xand those reported in Johnson et al. 74 and Wang etw x185 can perhaps be inferred from the fact that the cha

teristics of the elicited bursts differ from those of natu

bursts. Hence, the post-burst quiescent period is extremw xlong compared to that of natural bursts 74 . Furtherm

w xjudging by Fig. 2 of Johnson et al. 74 , spike height d

not appear to reduce within the burst as it does in natu w x.bursts see Ref. 49 . Likewise, the bursts in Wang e

w x185 were characterised by a progressive decrease in

interspike interval rather than a progressive increase a

w x.seen in natural bursts see Ref. 49 . However, Johnsow x w xal. 74 and Wang et al. 185 aside, the NMDA recepto

clearly implicated in the generation of natural burst ac

ity in DA neurons. What properties of NMDA recept

might be involved in burst firing in DA neurons?

Firstly, the NMDA receptor is a calcium ionophw x100 , and as mentioned above, bursting in DA neuron

w xdependent on intracellular calcium 49 . AMPA recepwon DA neurons have a low calcium permeability

However, bursts can be produced by the simple injectw xof depolarising pulses in vivo 49 and in DA neuron


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w xprimary cell cultures 23 . Burst-like events can also be

produced by depolarising pulses in DA neurons in the slicew x under certain circumstances 139 also see Fig. 5 of Kita

w xet al. 80 ; although most slice workers have been unable

to elicit bursts using depolarising pulses; for example, seew x.Grace and Onn 50 . Given that bursts can be elicited by

depolarising pulses and by NMDA receptor activation, this

implies that there must be some common property shared

by NMDA receptor activation and depolarising pulseswhich is relevant to bursting, and that this property is

specific to NMDA receptors. Since the depolarisation pro-

duced by current injection is unlikely to raise intracellular

calcium levels more effectively than burst-ineffective EP-SPs produced by AMPArkainate receptor activation for

.example , the critical property shared by NMDA receptor

activation and current pulses is unlikely to be calcium

entry per se.

The second property of the NMDA receptor which may

be crucial for bursting in DA neurons is that, as in many

neuronal systems, activation of NMDA receptors on DA

neurons produces a region of negative slope conductance

in the currentvoltage relationship measured under voltagew xclamp 105,191 , due to the blockade of the ion channel by

magnesium at relatively negative membrane potentials e.g.w x. .Ref. 100 . A negative slope region NSR seems to play a

fundamental role in burst firing in certain neuronal sys-

tems. EPSCs in DA neurons which are generated by

NMDA receptor activation have a NSR between y70 andw xy30 mV 110 ; i.e. the current associated with synaptic

activation of NMDA receptors has a NSR which spans theresting membrane potential of the cell active DA cells in

vivo have a resting membrane potential of y55"2.9 mVw x.48 . Hence, when NMDA receptors are activated synapti-

cally in vivo, it is likely that the membrane potential at thetime will be in the NSR of the currentvoltage relation-

ship.

A NSR appears to be a prerequisite for burst firing inw xmolluscan neurons 161 . Furthermore, rhythmical burst

firing in spinal interneurons of the rat induced by certain .pharmacological agents including NMDA appears to be

w xassociated with the enhancement of a NSR 79 . Likewise

for NMDA induced bursts in cat neocortical neurons.

Here, iontophoretic NMDA induces membrane potential .oscillations depolarising shifts surmounted by bursts of

w xaction potentials 34 . These cells already show a degree of

negative slope conductance, produced by the activation ofa persistent, non-inactivating sodium current. NMDA can

w xenhance the NSR 34 . However, all of these examples

concern the generation of rhythmic bursting. In what way

might the NSR be important for burst firing in DA neu-

rons?

It may increase the rate of rise of the depolarisation

associated with NMDA receptor activation. The NSR ef-

fectively turns the depolarisation into a regenerative spike.Clearly, one aspect of depolarising current pulses which

.elict bursts in DA neurons is that they have a rapid rate of

rise. In spinal interneurons, where NMDA and other ag

produce membrane potential oscillations and burstw xKiehn et al. 79 propose that intrinsic membrane pro

ties may shape the response in some cells. With an N

the rate of rise of the depolarisation will be stee

However, NMDA receptor mediated EPSPs in the h

pocampus take considerably longer to reach a peak tw xnon-NMDA receptor mediated EPSPs 27 . Effects m

ated via the NMDA receptor also seem to have slokinetics than those mediated by AMPArkainate recep

in DA neurons. In particular, the rate of rise of NMw xreceptor-mediated EPSCs are much slower 110 .

So, if the rate of rise of the NMDA receptor media

EPSP in DA neurons is actually slower than that

non-NMDA mediated EPSPs, this cannot be the crit

determinant underlying the role of the NMDA recepto

burst firing. It thereby calls into question the the neces

of the NMDA-receptor mediated NSR for bursting in th

neurons. What other properties are shared by NM

receptor activation and the depolarising pulses used

elicit bursts? One remaining parameter is duration. pulses used to elicit bursts in DA neurons in culture, in

slice and in vivo, were all long-duration events. Indew xGrace and Bunney 49 found that long pulses were ef

tive whereas short pulses were ineffective. As we

cussed above, the activation of NMDA receptors on

neurons can produce a prolonged depolarisation, wh

AMPArkainate receptor activation does not appearw xproduce 108 . The NMDA receptors on nigral DA neur

show the slowest rate of deactivation found amongw xrange of basal ganglia neurons 42 . Evidence revie

.below Section 4.2.2.4 suggests that long-duration de

larisations are necessary for evoking burst-relevant cell

phenomena, and hence we consider that it is the abilitythe NMDA receptor to generate long-duration depolar

tions which is critical for bursting in DA neurons.

4.2.2. Intrinsic membrane properties

As mentioned above, not only could bursts arise a

consequence of synaptic factors, but they may also aris

a consequence of intrinsic membrane properties of

cells. Below, we consider certain intrinsic membr

properites which have either been found to be import

for bursting in other systems, or for which there may

evidence of involvement in the burst activity of DA nrons themselves.

4.2.2.1. Low threshold calcium conductance. Cells inw x w xthalamus 30 , inferior olive 93,94 and elsewhere h

been found to possess a low threshold calcium cond

tance which produces a regenerative depolarising poten .the low threshold calcium spike or LTS at the term

tion of hyperpolarising pulses. The LTS itself gives ris w x.a burst of sodium spikes see Ref. 30 . In vitro,

underlying conductance is largely inactivated at the rest


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membrane potential of the cells, and is revealed by thew xinjection of hyperpolarising current 93,94 . In vivo, this

low threshold calcium conductance seems to be deinacti-

vated to some extent even at the resting membrane poten-

tial. Thus, thalamic cells recorded intracellularly in the

whole animal produce a burst of spikes riding on a slow

depolarisation at the termination of brief hyperpolarisingw xpulses, in the absence of current injection 33 . The pres-

ence of a similar conductance in rat DA neurons has beenw xclearly demonstrated by Kang and Kitai 76,77 .

There are a number of systems in which bursts elicited

by the intracellular injection of current pulses appears to w xbe due to the activation of T-channels e.g. STN 126 ;

w x w x.dorsal root ganglia 96 ; neocortex 19 . However, in-

volvement of T-channels in the natural burst activity of

cells remains unproven. In a number of bursting systems,

cells behave as if an LTS underlies the burst activity. For

example, IB cells in the guinea-pig neocortex in vitro.

About 50% of IB neurons exhibit bursting at the resting

membrane potential. In these cells, intracellular injection

of constant depolarising current leads to an increase in

burst frequency, and finally to a change in firing mode to asingle-spike pattern, as the current and hence level of

.depolarisation is gradually increased. Depolarising agents .e.g. noradrenaline and acetylcholine also cause a shift in

w xfiring mode from bursting to single spike activity 186 .

These responses to depolarisation parallel those expected

of a system in which T-channels underlie the burst activity,

which gives prima facie evidence for the involvement of

an LTS in the natural burst activity of these cells. Naturalburst activity in the reticular nucleus of the thalamus the

.cells of which exhibit an LTS also responds to changes inw xmembrane potential in the appropriate way 102 .

There is a certain amount of evidence in favour of LTSinvolvement in the natural burst firing of DA neurons.

Firstly, the LTS is a calcium spike, and natural bursts have

been shown to be critically dependent on intracellularw xcalcium levels 49 . Secondly, the low threshold calcium

conductance does not appear to be involved in the genera-w xtion of single-spike activity in DA neurons 38,76,77 , and

hence it is, in a sense, a conductance looking for a

function. Finally, many aspects of the morphology of

natural bursts in DA neurons can be accounted for by an

LTS. Natural bursts consist of a series of action potentials

of decreasing amplitude and increasing interspike interval,

which ride on a slow depolarisation. At the termination ofthe burst, there is evidence of the activation of a calcium-

. w xactivated potassium conductance GK 49 . An LTS canCagive rise to an almost identical set of phemonena see Ref.

w x.126 .

However, there are a number of problems for an LTS

theory of bursting in DA neurons. Firstly, bursts cannot be

elicited in the slice in situations where an LTS can be w x w x.demonstrated e.g. Ref. 50 ; but see Ref. 39 . Indeed, the

LTS exhibited by DA neurons in the slice seems to be only

a short, low amplitude depolarisation, rather than the

long-duration depolarisation found in some neuronal s w x.tems e.g. STN 126 . This may, however, be a prob

with dendritic transection. In some cell types, the L

seems to be generated by a conductance which is locaw xin the dendrites 95,117,121 . Dopaminergic neurons h

w xbeen shown to possess very long dendrites 121,168 , so

of which may be transected in the slice, presumably

moving a proportion of the channels which underlie

conductance, reducing the size of the LTS. If this was case, the LTS would be expected to be larger in vivo.

In support of this contention, DA neurons in cultuw xderived from embryonic mice 22,23 and neonatal

w x 16 burst fire both naturally, and in the former casew xresponse to current pulses 23 . The dendrites of these cw xare intact and extensive 22,23,16 , and hence resem

those of cells recorded in vivo. However, the LTS elic

from these cells is of a comparable size to the LTS elic w in the slice see Fig. 6A in Chiodo and Kapatos 2

Interestingly, when the degree of dendritic transec

inflicted on DA neurons during the preparation of coro w x.

slices used, for example, by Grace and Onn 50 has bw xformally examined 138 , it has been concluded that l

dendrite is actually removed. This small amount of d

dritic damage is unlikely to be of significance for burst

in DA neurons, since 10% of DA neurons in culturesw xdissociated adult cells burst fire 56 , even though ther

evidence of significant dendritic truncation in this prepw tion. Furthermore, in a recent extracellular study 1

some DA neurons in coronal slices from immature

were found to exhibit burst activity.

The fact depolarising pulses can elicit bursts from .neurons in primary cultures but usually not from

neurons in the slice, yet the LTS appears to be of a sim

magnitude in the two preparations, suggests that an LTnot involved in a simple way in the burst activity of

neurons, although it does not preclude involvement. Ho

ever, further observations make it unlikely that an L

plays any role. Firstly, if an LTS was involved in natu

bursting, it is unclear why agents which depolarise

membrane potential of DA neurons should increase bu .ing see Section 4.2.1 or why sustained injection

w xdepolarising current increases bursting 49 . Depolarisa

should lead to increased inactivation of T-channels. S

ondly, it is unclear why agents such as apomorph

which hyperpolarise the membrane potential of DA n

w x w xrons 47 , reduce burst firing 35 . Hyperpolarisation shodeinactivate T-channels. Finally, flunarazine, which blo

w x.the T-type calcium channel see Ref. 104 , does

reduce burst firing in DA neurons when given invenously Overton and Clark, unpublished observatio

Instead, it leads to an increase in bursting firing, probaby virtue of its action as a dopamine antagonist see R

w x.104 . Hence, in spite of prima facie evidence for

involvement of an LTS in the natural burst activity of

neurons, the weight of evidence does not support

contention.


12/23


4.2.2.2. Calcium-actiated potassium conductance. Inw x1988, Shepard and Bunney 154 reported that apamin

produced bursts in DA neurons recorded extracellularly in

the slice. The bursts consisted of between 2 and 12 spikes,

with a progressive increase in interspike interval and re-

duction in spike amplitude. The smaller amplitude action

potentials towards the end of the bursts exhibited pro- .longed initial segment-somatodendritic IS-SD breaks and

were generally broader than spikes occurring earlier in theburst. These reported characteristics of apamin-induced

bursts closely resemble those of natural bursts in DA

neurons. The earlier finding was expanded upon in anw xintracellular study by the same authors in 1991 155 .

Bursts produced by apamin consisted of 312 spikes,

superimposed on a depolarising envelope, the termination

of which was triggered by a spontaneous repolarisation .or application of a brief hyperpolarising pulse .

Apamin acts as an antagonist at one type of calcium-

activated potassium channel, the small conductance, SK Caw xchannel 18 . Apamin blocks SK channels in many cellCa

w x w xtypes 7 , including DA neurons 155 . This raises the

possibility that SK channels might play a role in theCanatural burst activity of DA neurons. This possiblility is

supported by the fact that one of the differences between

burst and non-burst events elicited by depolarising

pulses in vivo is that in the former there is less spike w x.accommodation see Fig. 8 in Grace and Bunney 49 .

SK channels appear to play a role in spike accommoda-Ca w x.tion in many neuronal systems see Ref. 62 . Although it

has been argued that apamin does not affect spike accomo-w xdation in DA neurons in the slice 155 , DA neurons in the

slice exhibit substantially less accomodation than DA neu-w xrons in vivo 45 , and hence a floor effect may prevent

apamin from exerting clear effects on accomodation.The involvement of a GK has been implicated in theCa

burst activity of other systems. Hence, in turtle motoneu-

rons, 5-HT produces bursting by revealing a plateau

potential mediated by calcium. 5-HT appears to work byw xblocking a GK 66 , although the identity of the GK isCa Ca

not known. The fact that the characteristics of apamin-in-

duced bursts in DA neurons resemble those of natural

bursts, and the fact that a burst-producing blockade of a

GK can occur via the action of a transmitter substance inCaother systems, suggests that apamin-induced bursts in DA

neurons may not be as unnatural as they may at first

seem. This point is reinforced by the observation that thevoltage excursions produced by apamin, upon which the

bursts of spikes ride, are abolished by the dihydropyridine .DHP nifedipine, an L-type calcium channel antagonistw x122 , suggesting that they are calcium dependent. As we

mentioned earlier, natural bursts in DA neurons are criti-w xcally dependent on intracellular calcium 49 .

However, although many of the reported characteristics

of apamin-induced bursts resemble those of natural bursts

in DA neurons, the example of an intracellularly recordedw xapamin burst which appears in Shepard and Bunney 155

shows a burst whose interspike interval decreases as w x.burst progresses see Fig. 3A of Ref. 155 , rather t

increases as in natural bursts. The same appears to be t

of the apamin burst illustrated in Fig. 4B of Ping w xShepard 135 . This suggests that apamin-induced bu

and natural bursts are not entirely homologous.

4.2.2.3. Voltage-gated potassium channels. The spec

relationship between the blockade of a GK and natCabursting in DA neurons is further called into question

the fact that a phenomenon which resembles apamin

duced bursting can be produced by the potassium chanw xblocker caesium 107 , even though SK channels Ca

w xpermeable to caesium ions 131 . Potassium channel blo

ade with intracellular caesium produces bursts in

neurons recorded in vitro, which consist of a depolarisw xplateau giving rise to 210 action potentials 107 .

though the mean interspike interval within the burst

shorter than that of natural bursts in vivo, the intersp

interval increases within the burst and the spike he

reduces. So, apamin and intracellularly applied caesboth produce events in DA neurons which are simila

natural bursts. What is the relationship between th

seemingly diverse compounds? The one factor which t .have in common is that they alter or are likely to alter

.post-spike afterhyperpolarisation AHP .

As we discussed above, apamin blocks SK chanCaw x155 . One consequence of this is that it reduces the A

w xin DA neurons 155 . Although caesium does not bl

SK channels, it interacts with another current whCacontributes to the AHP in DA neurons. Of the curr

which contribute to the AHP, one of the most importan

the transient outward rectifier, I . This potassium currAw xwhich DA neurons exhibit 158 , is produced by a cond

.tance GK which is deinactivated by hyperpolarisatAfor example during the AHP. The subsequent activatio

this conductance during the depolarising phase of the s .oscillatory potential SOP which underlies spike prod

w xtion in DA neurons 76 will oppose the depolarisat w x.prolonging the AHP see Ref. 58 . Although extern

applied caesium has no effect on the GK -like condAw x w xtance 58 or the AHP 59 in DA neurons, when CsCl-fi

recording electrodes are used, GK is blocked in moAw xand rat DA neurons in primary culture 23,90 . Interstin

bursting has also been reported in guinea-pig DA neur

recorded in vitro during the bath application of barium .in the absence of calcium . Externally applied bar

w xblocks a GK -like conductance in these cells 58 .AIn dorsal raphe neurons, the a -adrenoceptor ago1

w xphenylepherine blocks GK 2 . Phenylepherine increAthe discharge rate of dorsal raphe neurons by reducing

duration of the AHP. The decay of the AHP is accelerat

whilst the initial amplitude of the AHP is unaffec

These changes occur in the absence of a change in mew xbrane potential 180 . We propose that interference w

the AHP of DA neurons by caesium and apamin allows


13/23


spike frequency elicited by a given depolarisation to be

increased sufficiently to saturate the intracellular calcium

buffer and produce the classical burst characteristics. In

the slice in many laboratories this is necessary because .evidence suggests see Section 4.2.3 that intracellular

calcium buffering is supra-optimal for bursting. .In addition to caesium and barium , other divalent

cations also affect GK . However, rather than blocking theA

conductance, they change channel gating, such that theactivation and inactivation curves are shifted to more

depolarised levels. This also appears to be true for GK inA w x.DA neurons see Ref. 158 . Changes to GK may ex-A

plain why intracellular calcium injection leads to burstingw xin non-bursting DA neurons in vivo 49 . In these cells,

intracellular calcium administration accelerates the decay w x.of the AHP see Fig. 10C of Grace and Bunney 49 . A

similar change is seen in dorsal raphe neurons when GK A w x.is blocked see Ref. 180 . In the case of DA neurons,

accelerated decay of the AHP presumably arises because

GK is not activated until more depolarised levels ofAmembrane potential are achieved by the depolarising phase

of the SOP. This may decrease spike accomodation which.is very marked in these cells and allow the emergence of

the classical burst characteristics in response to an appro- .priate depolarising event see Section 4.2.3 . An action on

GK may also explain why intracellular injection of tetra-A .ethylammonium TEA ions leads to bursting in non-burst-w xing cells in vivo 49 . External TEA does not block SK Ca

channels or the channels which underlie GK in DAAw xneurons 158 . However, in invertebrate nerve cells, GK A

is much more sensitive to blockade by internal TEA thanw x w xexternal TEA 84,123 . Although Grace and Bunney 49

state that the burst-enhancing effects of intracellularly

injected TEA precede blockade of the AHP, no quantita-tive data are presented which might confirm or refute the

possibility that the AHP had undergone a shape change

consistent with the blockade of GK .AA role for GK in the burst activity of DA neurons wasA

w xanticipated by Grace and Bunney 49 , who argued that a

voltage-gated potassium conductance inactivates during

the burst, based on the progressive action potential broad-

ening which occurs during the burst. According to Silva etw xal. 158 , GK is the only voltage-gated potassium conduc-A

tance exhibited by DA neurons which shows any degree of

inactivation. However, in reality, the substantive evidence

for the gradual inactivation of a voltage-gated potassiumconductance during the burst is actually quite limited.

Although spike broadening does occur during the burst, as

is apparent in extracellular recordings, there is a disassoci-ation of the IS and SD components of the spike see Grace

w x .and Bunney 49 , Fig. 8D . This will lead to an apparent

broadening of the action potentials in the absence of

inactivation. It may also make a contribution to the reduc-

tion in spike height which ocurs during the burst, since the

disassociation will disrupt the additive effects of depolari-

sation arising from the two compartments. However, since

the SD component of the action potential seems to largw xconsist of a high threshold calcium spike 44 , reductio

spike height during the burst probably reflects calcium .duced inactivation of the calcium conductance s wh

w x.underlie the SD component of the spike see Ref. 49

To reiterate, we propose that, by altering the AHP

DA neurons, apamin and caesium allow the spike

quency elicited by a given depolarisation to be increa

sufficiently to saturate the intracellular calcium buffer produce the classical burst characteristics. Howe

apamin and caesium must have other burst-relevant eff

additional to interference with the AHP, since they

produce plateau potentials even when spike productiow xblocked 122,107 . We propose that the agents bloc

conductance which is involved in repolarising the S

Evidence for this proposition comes from the fact that

plateau potentials produced by apamin seem to be tw x w xgered by the SOP 135 . In Nedergaard et al. 122 , apa

transformed the SOP into a voltage plateau, an ac

most simply explained by blockade of the hyperpola

ing phases necessary for recycling the SOP. There relatively few findings concerning the conductances

volved in repolarising the SOP in DA neurons. Howev

intracellular EGTA prolongs the decay time-course of

SOP, suggesting the possible involvement of a GK Caw xthe repolarisation 76 . This is a possible target for apam

4.2.2.4. High threshold calcium conductance. Applicaof apamin to DA neurons in presence of TTX and TE

leads to replacement of the SOP with a ramp-like de

larisation followed by a prolonged plateau phase duw xwhich time the membrane potential remain s in a r

w xtively depolarised state 135 . As we discussed above,

voltage excursions induced by apamin which undeapamin-induced bursting in DA neurons are blocked by

w xDHP calcium channel antagonist nifedipine 122 .

bursting and plateau potentials in DA neurons which w xproduced by intracellular caesium 107 exhibit sim

properties. The calcium channel antagonists nifedipine

nimodipine reduce the amplitude and duration of

plateaus, and reduce the number of spikes in the bu

which they generate. The plateaus are blocked comple

by a zero calcium medium.

The plateau potentials seen in DA neurons with caesi

filled electrodes can be elicited by a short-duration de

w x larising pulse 107 in the presence of TTX and TEThe plateau potential consists of two components:

early, DHP-insensitive component and a later compon

which is sensitive to DHPs. The former has to be at le

150 ms long before the latter is observed. This sugge

that during rhythmic bursting in the presence of caesi .which prolongs the SOP; see Section 4.2.2.3 , the p

longed SOP has to achieve a certain duration before

true plateau potential is triggered. This rather string

requirement suggests that the underlying conductanc

located at a site which is electrotonically remote from


14/23


DHP-insensitive conductance. The SOP appears to be gen-w xerated at the cell soma or close by 50,121 . Hence, the

DHP-sensitive conductance is probably located in the den-

drites. This hypothesis is supported by the fact that the

DHP-sensitive component of the plateau potential can onlyw xbe elicited in the presence of TEA 107 , which increases

w x.the electrotonic length of the neuron see Ref. 95 .

Evidence suggests that plateau potentials can be trig-

gered in DA neurons in the absence of apamin or caesium,by depolarisation. Hence, in DA neurons in the slice

w xrecorded under voltage clamp, DeFazio and Walsh 29

report that voltage steps from y70 mV to greater than

y30 mV evoke a large amplitude inward current. These

plateau-like currents often had durations of several hun-w xdred ms after the termination of the voltage clamp pulse.

The authors hypothesise that the current was carried by

calcium, and that it was dendritically located, since it was

difficult to clamp effectively. Dopaminergic neurons have

been shown to exhibit a very slowly inactivating, high w x.threshold calcium conductance see Ref. 77 . Hence,

evidence suggests that under normal circumstances i.e..in the absence of apamin or caesium , DA neurons are

capable of generating calcium dependent plateau potentials

via a dendritically located conductance in response to

depolarisation. Many neurons can generate calcium depen-

dent plateau potentials. For example, 5-HT reveals a plateau

potential in the isolated turtle spinal cord, which is carried

in part by a non-inactivating nifedipine sensitive calciumw xcurrent 68 .

Involvement of high threshold calcium channels in the

burst activity of DA neurons in vivo cannot be assessed

extracellularly because DHPs inhibit action potential gen-w xeration in DA neurons 107,178 . The issue has not been

formally examined intracellularly, although in naturalbursts recorded intracellularly, when fast spikes have inac-

tivated, a plateau-like depolarisation is clearly visible seew x .Grace and Bunney 47 , Fig. 5B . Evidence reviewed

above suggests that long-duration depolarisations in vitro .under certain conditions can trigger the generation of

plateau potentials mediated by high threshold calcium

channels. In this regard it is interesting that short-duration

depolarising pulses do not elicit bursts in bursting DAw xneurons in vivo 49 , suggesting that natural bursting and

plateau potentials may be related. This contention is sup-

ported by the fact that, in one of the few published

instances of a burst-like phenomenon being elicited in aDA neuron in the slice by a depolarising pulse in normal

w x.medium Kita et al. 80 , depolarising pulses also elicited

plateau potentials in these cells. A similar phenomenon has

not been reported in other slice papers.

4.2.3. Role of intracellular calcium buffering

Vertebrate neurons possess a series of different mecha- .nisms for controlling the level of buffering cytosolic

free calcium, including calcium binding proteins, seques-

tration in intracellular organelles and extrusion via the

w xsodiumrcalcium exchanger 8 . In the case of DA neur

there are indications that intracellular calcium bufferin

often disturbed in the slice, such that buffering is m

effective in the slice that it is in vivo.Firstly, spike accomodation i.e. a progressive incre

.in the interspike interval in response to the injection

depolarising pulses is much more prominent in vivo.wdeed, there is often little accomodation in the slice

Accomodation usually involves the activation of a GK w x.see Ref. 62 . Hence, its absence in the slice can

interpreted as reflecting the fact that the calcium wh

enters the neuron through voltage-gated channels dur

each spike is being buffered so efficiently that it produ

less activation of the GK .CaSecondly, the AHP following spikes elicited usin

depolarising pulse is much smaller in the slice than

vivo. In vivo, the amplitude of the AHP is proportionaw xnumber of spikes elicited 48 . In the slice, the AHP se

to be simply a continuation of the AHP following w xspike in train 50 . The AHP following a series of spike

attenuated by the intracellular injection of the calcw xchelator EGTA 48 , suggesting that it reflects a calc

dependent mechanism, most likely the activation o

GK by calcium influx through voltage-gated calcCachannels during the spikes. The attenuation of the AHP

the slice implies that there is no calcium build up.

Over-efficient calcium buffering in DA neurons in

slice may explain why bursts are very rarely elicited

depolarising pulses in this preparation. The SD compon

of the action potential in DA neurons is largely a hw xthreshold calcium spike 44,107 . Reduction in spike he

within the burst may reflect calcium-induced inactiva w x.of a calcium conductance see Ref. 49 . If buffering w

too strong, such inactivation may not take place, becathe calcium would be buffered instead of being allowe

accumulate. Given this, it may be possible to re-c

acterise the action of apamin and caesium on DA neur

in the slice as substituting for the effects of disrup

calcium buffering. These manipulations may allow delarisation the prolonged SOP and plateau potential com

.nation to elicit a high enough spike frequency to satu

the over-efficient intracellular calcium buffer, such

calcium-induced inactivation of the calcium spike occ

and hence the classical burst charateristics are manifes

At the same time, these agents may also allow the trigg

ing of a GK to terminate the burst. It is possible thatCaGK which contributes to the termination of the bursCadistinct from the apamin-sensitive GK which generCathe post-spike AHP. Dopaminergic neurons possess at l

w two GK s, only one of which is apamin sensitive 1CaA change in calcium buffering may explain the differ

tial effectiveness of depolarising pulses at eliciting bu

in DA neurons in vivo and in the slice. However, calci

buffering also plays a role in the natural burst activity

some systems, in so far as the level of calcium bufferin

negatively correlated with burst activity. For example


15/23


.the rat supraoptic nucleus SON , in continuously firing

neurons, antibodies targetted at one calcium binding pro-

tein, calbindin-D28k, induces phasic firing. Conversely,

bursting SON neurons lose their phasic firing pattern withw xintracellular calbindin-D28k injection 88 . Additional evi-

dence suggests that the level of calbindin-D28k in neurons .of the SON and paraventricular nucleus PVN correlates

with their tendency to burst fire. Hence, bursting neurons

of the ventral SON and lateral PVN express lower levelsof calbindin-D28k mRNA than non-bursting neurons of

w xthe dorsal SON and medial PVN 182 . It is interesting that

not only does anti-calbindin induce burst firing in non-

bursting SON neurons, but it also reveals calcium depen-w xdent plateau potentials in these neurons 88 . Likewise,

bursting SON neurons lose their ability to generate plateauw xpotentials when injected with calbindin-D28k 88 .

These findings raise the possibility that the distinction

between bursting and non-bursting DA neurons in vivo

may also resolve to differences in calcium buffering.

Dopaminergic neurons contain varying levels of the cal-w xcium bin

burst firing in midbrain dopaminergic neurons

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