Neuroscience Letters, 157 (1993) 53 56 53 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00

NSL 09639

Prefrontal cortex regulates burst firing and transmitter release in rat mesolimbic dopamine neurons studied in vivo

S u m i o M u r a s e a'l, J o h a n G r e n h o f f "'2, G u y C h o u v e t b, F ranqo i s G. G o n o n b a n d T o r g n y H. Svensson a

"Department of Pharmacology, Karolinska Institute, Stockholm (Sweden) and bINSERM U171 et CNRS URA 1195, Centre hospitalier Lyon-Sud, Pierre B~nite (France)

(Received 5 October 1992; Revised version received 7 April 1993; Accepted 13 April 1993)

Key words: Accumbens; Glutamate; Hypofrontality; Lidocaine; Single cell recording; Ventral tegmental area; Voltammetry

The influence of the medial prefrontal cortex (PFC) on mesolimbic dopamine activity was studied with electrophysiological techniques and in vivo voltammetry in the chloral hydrate-anesthetized male rat. Glutamate injected into the PFC selectively increased burst firing of single dopamine cells in the ventral tegmental area and enhanced the release of dopamine from nerve terminals in the nucleus accumbens. PFC injection of the local anesthetic lidocaine produced the opposite effects on burst firing and terminal release. This selective modulation of the dynamic activity of mesolimbic dopamine neurons by the prefrontal cortex might be important in motivation, learning and schizophrenia.

Several clinical studies have demonstrated a correla- tion between dysfunction of the prefrontal cortex (PFC), so-called hypofrontality, and negative symptoms in schizophrenia, e.g. flattening of affect, lack of initiative and motivated behavior as well as anhedonia [11]. In addition, these symptoms have been found to correlate to low dopamine (DA) activity, as indicated by measure- ments of DA metabolites in cerebrospinal fluid and urine of patients [13].

The PFC, i.e. the frontal region receiving afferents from the thalamic mediodorsal nucleus, is critically in- volved in working memory and other aspects of the tem- poral organization of behavior in humans, other pri- mates and rats [6], and recent clinical data support a behavior-specific relationship between PFC activity and DA function [21]. An afferent input from the PFC to the ventral tegmental area (VTA), the origin of the mesolim- bocortical DA system, has recently been shown to make synaptic contact with DA neurons in the VTA [16]. The present study was undertaken to analyse the effects of functional activation or inactivation of the PFC on the neuronal activity of the VTA-DA cells, in particular the temporal organization of discharge. In view of the docu-

Correspondence." T.H. Svensson, Department of Pharmacology, Karo- linska Institutet, P.O. Box 60400, S-104 01 Stockholm, Sweden. ;Present address: Department of Physiology, Mie University School of Medicine, Edobashi, Tsu, Mie 514, Japan. 2Present address: Vollum Institute, Oregon Health Sciences University, Portland, OR 97201, USA.

mented role for incentive behavior of the DA nerve ter- minals in the nucleus accumbens, a major VTA-DA cell projection area [2], the effects of altered activity in the PFC on DA release in this brain region was also as- sessed. Thus, single cell recording techniques and in vivo vol tammetry methodology were used in the present ex- periments, in which activation or inactivation of the me- dial PFC was achieved by means of local microinfusions of glutamate or lidocaine, respectively.

Male Sprague-Dawley rats (200-350 g) were anesthe- tized with chloral hydrate (0.4 g/kg i.p. initially) and maintained under surgical anesthesia by additional doses. At the end of the experiment, the animal was killed by an overdose of chloral hydrate. Body tempera- ture was maintained at 37-38°C. The rat was mounted in a stereotaxic apparatus and injection cannulae, record- ing electrodes or vol tammetry electrodes were inserted at coordinates determined from an anatomical atlas [14]. Surface coordinates for local injection in relation to bregma were 2.7 m m anterior and 0.5 m m lateral in elec- trophysiological experiments, and 3.2 m m anterior and 0.5 m m lateral in vol tammetry experiments. Depth of injection was 2.8 mm from dura mater. In the electro- physiological experiments, a 23-gauge guide cannula was fixed 2.3 m m ventral to dura mater with dental cement 1 h before the injection. A 30-gauge injection cannula filled with artificial cerebrospinal fluid (ACSF; 147 mM NaC1, 3.0 m M KC1, 1.3 m M CaC12, 1.0 m M MgCI2, 1.0 mM NaH2PO4, pH 7.4) mixed with Fast green was inserted

54

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140

120

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firing

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~z , z ~z

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var iat ion coe f f i c ien t

rate burst f i r ing

Fig. 1. Effects of local microinfusion of glutamate (~, 25 nmol, n = 7) compared to vehicle (u, n = 10) in medial PFC on the firing of VTA- DA neurons. Burst firing is increased by glutamate while the other firing parameters are unaffected. Baseline values (glutamate/vehicle): firing rate 4.76 _+ 0.39/4.99 + 0.5 Hz, variation coefficient 61.3 + 5.3/ 74.1 _+ 7.1%, burst firing 21/34% (not different between groups). Values are given as mean + S.E.M., except for burst firing values which devi- ated from a normal distribution. Statistical analysis of firing rate and variation coefficient values was performed with Student's t-test, and with Mann-Whitney U test for burst firing values. A two-tailed

P < 0.05 was considered significant. *P < 0.05.

10-20 min before the injection. L-Glu tamate (BDH,

Poole, UK; 50 m M in A C S F ) or l idocaine HC1 (Astra ,

S6dert~ilje, Sweden: 75 m M in A C S F ) was injected in a

volume o f 0 .5 /a l over 2 min. Con t ro l inject ions were

made with equal volumes o f A C S F vehicle. Af te r the ex-

per iment , cannula p lacement was verified by means o f a

green-s ta ined area in his tological sections.

Single cell record ing me thods and D A cell identifica-

t ion cri ter ia were as previous ly descr ibed [10]. Single cell

act ivi ty recorded 5 min before infusion was c o m p a r e d to

act ivi ty recorded the first 2 4 min after injection. Ac t ion

potent ia ls were ana lyzed by the Spike2 p r o g r a m (Cam-

br idge Electronic Design, Cambr idge , UK) , which col-

lected all t ime intervals between spikes and enabled

quant i ta t ive analysis of neurona l firing rate, burs t firing

and regular i ty o f firing. The c o m p u t e r recognized a burs t

onset at an interval shor ter than 80 ms, and a burs t ter-

mina t ion at the next interval longer than 160 ms. The

burs t firing value o f a cell is the ra t io between in t ra -burs t

intervals and to ta l number of intervals over a per iod o f

time, expressed as a percentage. Regula r i ty o f firing was

assessed f rom the var ia t ion coefficient, i.e. the ra t io be-

tween the s t anda rd devia t ion and mean value o f an inter-

spike t ime interval h i s togram of 500 consecut ive inter-

vals. The locat ion o f the recording site in the VTA was

verified by means of a Pon tamine Sky blue dye spot in

his tological sections.

Differential normal pulse vo l t ammet ry was uti l ized in

pa rgy l ine -pre t rea ted (75 mg/kg i.p., 1 h before anesthe-

sia) rats with e lec t rochemical ly t rea ted ca rbon fiber elec-

t rodes ( 1 2 / a m thick and 0.5 m m long), reference and

auxi l iary electrodes as previously descr ibed [8]. The elec-

t rode was loca ted in the nucleus accumbens at 1.7 m m

anter ior and 1.5 m m lateral to b regma, 6.5--7.5 m m ven-

tral to du ra mater . Different ia l no rma l pulse vo l tam-

m o g r a m s were recorded every minute. In animals t rea ted

with pargyl ine (to suppress endogenous DOPAC) , the

ox ida t ion peak appea r ing at +85 mV entirely corre-

sponded to ext racel lu lar DA. In vi t ro ca l ibra t ions were

pe r fo rmed with the e lect rode af ter the exper iment for es-

t ima t ion o f the ext racel lu lar D A concent ra t ion . At the

end o f the exper iment , an electrolyt ic lesion (5 V, 3 s) was

made th rough the ca rbon fiber e lec t rode for his tological

verif ication.

Spon taneous ly active D A neurons in the VTA dis-

p layed firing rates o f 1 -10 Hz, with a var ied firing pat-

tern consis t ing bo th o f single spikes and bursts. Micro in-

fusion o f g lu t ama te (25 nmol) into the P F C specifically

a l tered the t empora l o rgan iza t ion o f the V T A - D A cell

discharge, i.e. the firing pat tern . Thus, burs t firing was

selectively increased, while firing rate and regular i ty o f

firing were unaffected (Fig. 1 ). In contras t , micro infus ion

o f l idocaine (38 nmol) into the P F C selectively reduced

burs t firing, while firing rate and regular i ty o f firing were

unaffected (Fig. 2). The effects o f g lu t ama te and lido*

caine were reversible and burs t firing re turned to basel ine

values within 5-15 rain.

e- , i

..a

a,e

120

100

80

60

40

20

0

T I

~ x x

f i r ing rate

T

KX×~ x x x ~ y y K x x ~

K X X N

var ia t ion burst f i r ing coe f f i c i en t

Fig. 2. Effects of local microinfusion of lidocaine (E, 38 nmol, n = 6) compared to vehicle (L, n = 10) in the medial PFC on the firing of VTA-DA neurons. Burst firing is decreased by lidocaine while the other parameters are unaffected. Single cell activity was recorded within 5 min before infusion and compared to the activity recorded 8 12 min alter administration of tidocaine or vehicle. Baseline values (lidocaine): firing rate 3.87 + 0.53, variation coefficent 75.7 _+ 7.5%, burst firing

27% (not different from vehicle-treated group). *P < 0.05.

60

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i l ~ i~!~i i !~i !

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Time {rain}

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Fig. 3. Effects o f local microinfusion of glutamate (e, 25 nmol; n = 3-6)

compared to vehicle (o, n = 3-5) into the medial PFC on DA levels in

the nucleus accumbens measured with in vivo voltammetry. The shaded

area corresponds to the infusion period. Basal DA peaks were stable and corresponded to a DA concentration of 25.3 + 1.2 nM in vitro

(n = 4). DA peaks 0-3 min after injection were higher in glutamate- treated rats compared to vehicle-treated rats (repeated measures two-

way analysis of variance, P < 0.05).

Microinfusion of glutamate into the PFC significantly increased the DA oxidation peak in voltammograms re- corded from the nucleus accumbens (Fig. 3). This effect appeared already during the ongoing infusion. In con- trast, microinfusion of lidocaine into the PFC signifi- cantly reduced the DA oxidation peak (Fig. 4). The ef- fects of glutamate and lidocaine were reversible, and the extracellular DA concentration in the nucleus accum- bens returned to baseline values within 5-15 min.

The results show that the PFC exerts a tonically active, specific control of the temporal organization of VTA- DA cell discharge, i.e. burst firing of the neurons. Previ- ous studies have shown that stimulation of brain DA neurons in bursts causes a dramatically increased termi- nal release of DA and the co-existing peptide neuroten- sin, when compared with the release induced by regularly spaced stimulation [1, 8]. The present results indicate that changes also in physiological burst activity of VTA- DA cells can alter terminal DA release. Since some evi- dence suggests that the VTA receives an excitatory amino acid (EAA)-containing input from the PFC [5], and EAAs increase burst firing in midbrain DA cells [17], the stimulatory effect of PFC activation on burst firing in the VTA-DA cells may be mediated via such an input. Moreover, since intracerebroventricular or somatoden- dritic application of EAA antagonists, including selective N-methyl-D-aspartate (NMDA) receptor antagonists, have been shown to block spontaneous burst firing in midbrain DA neurons, this firing mode seems critically dependent on a tonically active EAA input [3, 4, 9]. Thus,

55

the decreased burst firing in the VTA-DA cells associ- ated with PFC inactivation may be mediated through reduced activity in the above-mentioned, excitatory input from the PFC (see also ref. 20). This interpretation is supported by the finding that electrical stimulation of the PFC can induce burst firing in some midbrain DA cells [7], although only in a few percent of the neurons. The more general effect observed here may be explained by more widespread and uniform stimulation produced by the microinfusion of glutamate. Moreover, local cool- ing of the PFC has been shown to cause suppression of burst firing in the VTA-DA cells, associated with regu- larization of their firing pattern in spite of an unaltered firing rate [18]. The present results indicate that the effect of cooling on burst firing is related to functional inactiva- tion of the PFC. The difference in magnitude between the effect of cooling of the PFC [18], and the present findings with local PFC infusion of lidocaine may pertain to the more selective and discrete nature of the present proce- dure. Taken together the above results suggest that the PFC selectively controls burst firing in the VTA-DA neurons, probably via modulation of an EAA-contain- ing input.

Although the PFC-induced changes in burst firing of DA cells can explain the concomitant alterations in DA release in the nucleus accumbens, these changes in re- lease might also be mediated through pathways from the PFC to the nucleus accumbens. In a recent ultrastructu- ral study, afferents from the PFC and dopaminergic nerves were shown to converge on accumbens neurons [16]. Even though no axo axonic synapses were ob-

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- 40 , , . . . . , j , ,

- 7 - 5 - 2 0 5 10 15

Time (rain)

Fig. 4. Effects of local microinfusion of lidocaine (e, 38 nmol; n = 8) compared to vehicle (o, n -- 3-5) into the medial PFC on DA levels in

the nucleus accumbens measured with in vivo voltammetry. The shaded area corresponds to the infusion period. DA peaks 0-10 min after injec-

tion were lower in lidocaine-treated rats compared to vehicle-treated rats (P < 0.05).

56

served, a local non-synaptic interaction between the two types of afferent nerves might exist.

The present results clearly show that changes in DA release can occur in the absence of altered firing rate of' the DA neurons. Still, they may be caused by altered temporal organization of the neuronal discharge, i.e. al- tered firing pattern of the DA cells. Recent experiments combining recording of midbrain DA cells with measure- ments of DA release in nucleus accumbens show that burst firing induced by somatodendritic application of N M D A strongly enhances nerve terminal DA release [171.

A significant result per se of this study is the demon- stration that altered activity in the PFC such as hy- pofrontality causes reduced release of DA from termi- nals in the accumbens nucleus. DA transmission in this terminal area appears critical for the reward value at- tributed to different stimuli [12]. Moreover, recordings in behaving animals have shown that midbrain DA neu- rons respond specifically to reward-related environ- mental stimuli with a rapid burst of impulses [15]. Conse- quently, an impairment of burst activity in the VTA-DA cells with a concomitant reduction in nerve terminal re- lease, as induced by reduced functional activity in the PFC, may well explain the loss of motivated behavior or the anhedonia often associated with clinical hypofrontal- ity, as well as the reduction in CSF DA metabolites in conjunction with negative symptoms in schizophrenia. Additional support for this notion is provided by our previous findings that drugs such as ritanserin or am- perozide [10, 19], drugs which have been reported to an- tagonize negative symptoms in schizophrenics, both can maintain burst firing of VTA-DA cells in spite of cold inactivation of the PFC.

Financial support was provided by the Swedish Medi- cal Research Council (project 4747), Torsten och Ragnar S6derbergs Stiftelser and the Karolinska Institute. Dr. Murase was supported by grants from the Ministry of Education, Science and Culture, Japan, and the Karo- linska Institute. We thank Monica Marcus for expert technical assistance. ,

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