Altered neuronal responsiveness to biogenic amines in rat cerebral cortex after serotonin denervation or depletion

Brain Research, 231 (1982) 93-108 Elsevier Biomedical Press

93

A L T E R E D N E U R O N A L RESPONSIVENESS TO B I O G E N I C A M I N E S IN RAT C E R E B R A L C O R T E X A F T E R S E R O T O N I N D E N E R V A T I O N OR DEPLETION*

ANDRE FERRON **, LAURENT DESCARRIES and TOM/~S A. READER*** Centre de recherche en sciences neurologiques, D~partement de physiologie, Facultd de mddecine, Universitd de Montrdal, Montr&d, Qudbec, H3C 3T8 (Canada)

(Accepted June 25th, 1981) Key words: dopamine - - noradrenaline - - serotonin -- supersensitivity - - hyposensitivity- - micro-

iontophoresis -- cerebral cortex

SUMMARY To further investigate monoaminergic mechanisms in cerebral cortex, responsiveness of cortical neurons to microiontophoretic applications of serotonin (5-HT), dopamine (DA) or noradrenaline (NA) was examined in the frontoparietal region of control, 5,7-dihydroxytryptamine (5,7-DHT)- and p-chlorophenylalanine (PCPA)treated rats anesthetized with urethane. As a rule, 100 nA applications of either one of these biogenic amines induced marked slowings or total interruptions of'spontaneous' firing overlasting the 30 s periods of ejection. Given the large amounts of monoamines ejected, it could be inferred that such microiontophoretic applications produced a maximal activation of receptors. In control rats, the responses to 5-HT, DA and N A were of approximately equal duration (_~ 5 rain). Two to 4 weeks after denervation with 5,7-DHT, most neurons (75 ~ ) exhibited greatly prolonged responses to 5-HT ( _~ 14 min), and marked depressions of firing could be induced by small ejection currents (-~ 2 hA) having little or no effect in the controls. In addition, 85 ~o of the units supersensitive to 5-HT showed considerably shortened responses to D A and NA (_~ 1 rain). After 2 14 days of depletion with PCPA, there was no change in the responsiveness to 5-HT in spite of a 9l ~ lowering of cortical 5-HT content equivalent

* Presented in part at the 9th and 10th Annual Meetings of the Societyfor Neuroscience (1979, 1980) and the Colloque International du CNRS 'The Serotoninergic Neuron' (1980) ** Present address: Groupe NB, Inserm U-114. CollAgede France, 11, Place Marcelin-Berthelot, 75231, Paris Cedex 05, France. *** Scholar of the Medical Research Council of Canada. 0006-8993/82/0000-0000/$02.75 '.~ Elsevier Biomedical Press

94 to that measured after denervation. Nevertheless, responsiveness to DA and NA was again diminished in a majority (80'.~i) of the units tested. In control or P('PA-treated rats. acute administration of tire 5-HT re-uptake blocker fluoxetine increased thc duration of depressions induced by 100 nA applications of 5-HT but did m)t enhance responsiveness to low ejection currents. This suggested that, after 5-HT dencrvation. the suppression of re-uptake was mainly responsible for the prolongation of 5-1-t~I' responses ('presynaptic' component of supersentivity), whereas a modification oi" 5HT receptors accounted for the greater efficacy of small doses of 5-HT (~po~tsynaptic component). Responsiveness to the microiontophoretic application of phcnylephrinc (PHE), a noradrenergic ¢~-agonist, was comparable with that to NA in PCPA- and 5.7DHT-treated as well as in control rats. Therefore, the hyposensitivity to I)A and NA appeared indicative of a desensitization of catecholamine receptors caused by the absence of 5-HT. Such a desensitization may be viewed as an adaptive change resulting from an increased release of endogenous DA and NA. This interpretation would in turn imply that, normally, 5-HT regulates catecholamine release in the ncccoHcx.

INTRODUCTION The monoaminergic afferents of mammalian cerebral cortex have been well characterized from the neuroanatomical point of view, especially ill tire rat (for review, see ref. 33). The dopamine (DA) innervation~.4v, 94 takes origin in the mesencephalic tegmentum, mainly from the cell body groups A-9 and A-10 of the substantia nigra and ventral area of Tsai, and distributes preferentially to certain regions of the cortex, where it predominates in deep layers 12,43,47,59,60 The noradrenatine (NA) innervation ~1 arises essentially from the nucleus locus coeruleus (A-6) ',~(; and spreads to all regions and all layers of the iso- as well as aiM-cortex, with some predilection for the molecular layer 14,42,a
95 serving the action of DA, NA and 5-HT in cerebral cortex 1,16,a4,49,fa,65,67,ga,l°°A°a, have also come to bear on this notion. The present microiontophoretic study was aimed at analyzing eventual changes in the biogenic amine responsiveness of cortical neurons which would follow the removal of one of their monoaminergic inputs. It was chosen to manipulate the 5-HT innervation since this input could be either destroyed selectively with the cytotoxic drug 5,7-dihydroxytryptamine (5,7-DHT), or depleted of its endogenous stores with the 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA). MATERIALS AND METHODS Adult male Sprague-Dawley rats (body weight: 250-350 g) were used. 5-HT denervation was achieved by intraventricular injection of 200 #g (F. B.) of 5,7-DHT 5,'m (5,7-dihydroxytryptamine creatinine sulfate, Regis) 1 h after desipramine pretreatment 6,13 (25 mg/kg i.p.). The neurotoxic drug was administered in 20 #1 of saline containing 1 ~ ascorbic acid and delivered in 4.5 min into the left lateral ventricle (stereotaxic coordinates: A, 5.4 mm; L, 2.4 mm; H, +2.75 mm, according to De GrootZT). 5-HT depletion was obtained by daily administration of the synthesis inhibitor PCPA '51 (p-chlorophenylalanine methyl ester hydrochloride, Regis), at a dose of 400 mg/kg i.p., for 2-14 days. The biogenic amine content (5-HT, DA, NA) of the neocortical region to be examined by microiontophoresis was measured in 10 rats from each group, which, in the case of treated rats, were sacrificed two weeks after 5,7-DHT or following 2-4 days of PCPA administration. The cortical samples were removed as described previously 79,82, and assayed with the radioenzymatic methods of Saavedra et al. vs,s4 and Coyle and Henry 22 for 5-HT and catecholamines, respectively. The microiontophoretic experiments were performed under urethane anesthesia (I.25 g/kg i.p.). The rats were placed in a stereotaxic head frame, a large opening was made in the fronto-parietal skull on one side, the dura mater was retracted, and a thin layer of Agar (4 ~,i in saline) deposited on the cortex. Access was thus given to an area corresponding to the cytoarchitectonic zones 1-4, 6 and 10, according to Krieg's map,~e. Five-barrel micropipettes (tip diameter: 4-9 /,m) were utilized. The central barrel was filled 76 with a 4 M NaC1 solution for recording, and one side barrel with 2 M NaC1 for automatic current balancing. The other barrels contained 0.1 M serotonin oxalate, 0.5 M noradrenaline-HCl, and 0.5 M dopamine-HC1 solutions, made in 0.1 ~ ascorbic acid and adjusted to pH 4 with 0.1 N NaOH. Extracellular unitary activity was amplified and displayed on an oscilloscope. Action potentials were separated from background noise by means of a spike amplitude discriminator, and the frequency of discharge integrated over 10 s intervals with a ratemeter:7, sl. Only neurons exhibiting spontaneous firing and a relatively stable rate of discharge were studied. These cells were found 400 1600 ~um below the cortical surface. All drugs were applied with ejection currents of + 100 nA during 30 s, except

96 in a few instances where a minimal ejection current ( ~_ '- 2 hA) was used. Between applications, a retention current of ..... 10 nA was maintained to prevent drug leakage. Under these conditions, all 3 biogenic amines consistently induced marked depressions or total interruptions of firing s~. The very few units which showed an increased frequency of discharge were not included in the analysis oi ~he data. Neurons failing to show a reduction of at least 301).;, of their firing iate were categorized as unresponsive. Any response without subsequent return to 80 ~ or more of the pretest rate of firing was excluded from the analysis. The ell'cots ol" the treatments on the responsiveness to the biogenic amines were assessed in ~x~,~ways: ( 1 ) by determining the proportion of responsive and unresponsive units; (2) by measuring the duration of depressions from the onset of microiontophoretic administration. Two series of complementary experiments were carried out. First, the 5-HT ~cuptake blocker fluoxetine a°5 was administered systemically (20 mg/kg i~i3) during some recording sessions, mainly in PCPA-treated rats. Second, in control. 5~7-DHTand PCPA-treated rats, the effects of the noradrenergic ¢t-agonist phcnylephrine (PHE) l°z were tested by microiontophoresis (0.5 M, pH 4). RES U LTS

Re.sponses to biogenic amines in control rat,s As reported earlier sa, all 3 biogenic amines, 5-HT, DA and NA, depressed the firing rate of a vast majority of 'spontaneously' active neurons in the fronto-parietaJ cortex of urethane-anesthetized rats (Fig. I). The percentage of cells which showed a 30 ~ or greater reduction of their frequency of discharge is given in Table 1. With ejection currents of 100 nA applied during 30 s, the mean duration of these responses was 5.6:~ 1 . 2 m i n f o r 5 - H T , 5.2 i 1 min for DA and 5.5 ~: 1 . 4 m i n t \ ~ r N A ( F i g . l, first trace, and Figs. 2 and 3). Responses to 5-HT after denervation with 5,7-DHT Two weeks after 5,7-DHT, the cortical 5-HT content was lowered to 9"~, of control values (32 : : 8 vs 360 [ 62 ng/g wet weight). DA was also markedly diminished (52 F 4 vs 160 ~ 32 ng/g), whereas NA content was unaffected (309 :i 17 vs 330 21 ng/g). Two to 4 weeks after 5,7-DHT, the proportion of spontaneously firing neurons depressed by 5-HT was unchanged (Table l). However, the duration i~f 5-HT responses assumed a bimodal distribution: 75~.!i~, of the units exhibited greatly prolonged responses to 5-HT (Fig. l, second trace) averaging 14.7 2.2 rain, whereas the 5-HT-induced depressions of the remaining units were of similar duration to those in the controls (Fig. 2). Marked depressions of spontaneous firing could also be induced with small currents of 5-HT (Fig. 1, third trace) never found to liave such an effect in the controls. Responses to 5-HT after depletion with PCPA After 2-4 days of PCPA treatment, cortical 5-HT content was again reduced to

97

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Fig. I. Ratemeter records of the firing rate of 4 cortical neurons, showing the responses to microiontophoretically applied 5-HT, DA or NA in control, 5,7-DHT- and PCPA-treated rats. The frequency of discharge was integrated over 10 s intervals. Ejections of 5-HT, D A or NA (30 s) are indicated below the traces, with the currents in hA. Control: 5-HT, NA and DA tested on the same unit induce comparably long depressions of spontaneous firing. 5,7-DHT: after this treatment, there is a denervation supersensitivity to 5-HT, as evidenced by the longer duration of the response to a 100 nA application (second trace) and the marked depression induced by a 2 nA ejection of 5-HT (third trace). Concomitant hyposensitivity to NA and DA is manifested by shortened responses to these catecholamines (third trace). PCPA (4 days of treatment): prior to the injection of the 5-HT re-uptake blocker fluoxetine, the duration of 5-HT responsiveness is comparable to that in control rats, but the depressions by NA and DA are shortened as after denervation with 5,7-DHT. After fluoxetine (arrow), the duration of the depression induced by 5-HT is markedly increased (the dotted baseline represents 10 rain of spontaneous activity cut out from the recording). TABLE I

Percentage of spontaneously firing cortical neurons depressed by microiontophoretic applications of 5-HT, DA, NA or P H E (100 nA during 30 s) Many of these units were tested with all three biogenic amines.

Group of rats

5-HT

DA

NA

PHE

Control 5,7-DHT PCPA

92 97 94

97 93 91

96 91 91

95 72 83

98 9 it,~oo f c o n t r o l values (32 :L 2 ng/g), b u t DA content was unchanged (t3~ ~ 45 ng/gi a n d N A only slightly d i m i n i s h e d (207 :i 30 ng/g). In these rats, there was no obvious difference in the n u m b e r o f units depressed by 5 - H T (Table 1) n o r in the average d u r a t i o n o f their responses, as c o m p a r e d to c o n t r o l s (Fig. 1, f o u r t h trace, and Fig. 2i. Similar o b s e r v a t i o n s were m a d e in rats treated with P C P A f o r 14 days. In P C P A - t r e a t e d rats, as well as in a few controls, fluoxetine lengthened the depressions induced by 100 nA a p p l i c a t i o n s o f 5 - H T to an average d u r a t i o n c o m p a r a b l e with t h a t m e a s u r e d after 5 , 7 - D H T (Fig. l, f o u r t h trace, ~nd Fig. 2). Nevertheless, it did n o t seem to enhance the effects o f small ejection curr~:.m~.

Responses to DA and NA after denervation with 5,7-DHT and depletion wilh t ' ( T A In 5 , 7 - D H T a n d P C P A - t r e a t e d rats, the p r o p o r t i o n o f n e u r o n s responding to D A or N A r e m a i n e d a p p r o x i m a t e l y the same as in the controls ('Fable I ) t--lov~ever. t h e d u r a t i o n o f depressions induced by D A or N A was often m a r k e d l y diminished (Fig. I, t h i r d and f o u r t h traces, a n d hatched c o l u m n s in Fig. 3). Every unit responding to b o t h c a t e c h o l a m i n e s exhibited c o m p a r a b l e hyposensitivity to D A and NA. In 5,7D H T - t r e a t e d rats, 85 o~ o f the n e u r o n s supersensitive to 5 - H T showed the shortened

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Fig. 2. Duration of depressions induced by microiontophoretically applied 5-HT (100 nA foJ: 30 s) in the cortex of control, 5,7-DHT- and PCPA-treated rats. The number of units tested is given in brackets. The histograms relate the percentage of cells depressed with the duration of the!r responses. The duration of depressions was measured from the onset of microiontophoretic ejection, The means :k S.D. are given for each histogram. After 5,7-DHT, 75 % of the units showed a prolongation of the effects of 5-HT. In PCPA-treated rats, the average duration of 5-HT depressions was comparable to that in the controls (solid histogram). In these rats, the administration of fluoxetine markedly increased the duration of the depressions induced by 5-HT (dotted histogram).

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Fig. 3. Duration of depressions induced by DA, NA and PHE (100 nA for 30 s) in the cortex of control, 5,7-DHT- and PCPA-treated rats. In 5,7-DHT-treated rats, the hatched histograms depict neurons which were supersensitive to 5-HT. The duration of responses to DA and NA was comparable to that in the controls in only 4 such units. In PCPA-treated rats, hyposensitivity to DA, NA or PHE was at least as frequent as after denervation with 5,7-DHT.

responses to D A a n d / o r N A (Fig. 3). The mean d u r a t i o n o f these reduced depressions was 1.3 :t 0.7 and 1.6 ~ 0.8 rain for D A a n d N A , respectively (Fig. 1, fourth trace, and Fig. 3). The remaining 5-HT supersensitive units, as well as the neurons failing to show p r o l o n g e d responsiveness to 5-HT, exhibited depressions by D A or N A o f the same average d u r a t i o n as in the controls (Fig. 3). A f t e r depletion with P C P A , shortened responses to b o t h D A and N A were also observed in a large m a j o r i t y (80 ?~) o f the neurons tested, a n d these depressions averaged 1. I -L 0.7 and 1.1 - - 0.6 rain, respectively (Fig. 1, f o u r t h trace, and Fig. 3). Fluoxetine had no effect on the mean d u r a t i o n o f these shortened responses to D A and N A .

Re.sponses to P H E in controls, 5,7-DHT- and PCPA-treated rats The effects o f the n o r a d r e n e r g i c ~-agonist P H E were examined by systematic c o m p a r i s o n with those o f N A on the same neurons, in controls, P H E depressed s p o n t a n e o u s firing in a c o m p a r a b l e p r o p o r t i o n o f the units tested (Table 1) and for a similar average d u r a t i o n (5.1 ± 1.1 rain) as N A (Figs. 3 and 4, first trace). Every cell r e s p o n d i n g to P H E was depressed by N A .

100

CONTROL PHE

NA

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30 1

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Fig. 4. Effects of NA and PHE in control, 5,7-DHT- and PCPA-treated rats. Same condimms as in Fig. 1. Responsiveness to PHE always paralleled that to NA. After denervation with 5,7-DHT (second trace) or depletion with PCPA (third trace), it appears markedly decreased.

In both 5,7-DHT-denervated and PCPA-depleted cortex, all neurons hyposensitive to NA, and only these, showed a parallel diminution of their responsiveness to PHE (Figs. 3 and 4, second and third traces). In a few such units, responses to PHE were totally absent. The average duration of the shortened depressions b? PH E was 0.9 ! 0.6 and 1.3 :~- 0.8 min after denervation and depletion, respectively (Fig. 3). DISCUSSION

This study has demonstrated an altered neuronal responsiveness to microiontophoretically applied 5-HT, DA and NA in cerebral cortex deprived of its serotoninergic afferents or depleted of its endogenous 5-HT content. Supersensitivity to 5-HT was found after 5,7-DHT denervation but not after PCPA depletion; whereas hyposensitivity to DA and NA developed in both experimental models: Complementary data obtained with the 5-HT re-uptake blocker fluoxetine and the noradrenergic a-agonist PHE shed some light on the possible nature of these effects. Control responsiveness to the biogenic amines Numerous investigations have already indicated that, in the cerebral cortex. 5-HT, DA and NA may depress 'spontaneous' neuronal firing for periods of time outlasting the duration of their microiontophoretic ejection 17,31,39,z3'~s'7~'~'s~''9°~ :% Relatively long depressions of unitary activity in the neocortex have also been reported

101 upon electrical stimulation of the midbrain raphe69, 87 and of the locus coeruleus 31,7°, 74, which respectively contain the cell bodies of origin of coTtical serotoninergic and noradrenergic afferents. It cannot be ascertained at present how closely these effects actually mimick the physiological action of the biogenic amines. Under the present experimental conditions, however, it may be inferred, that the observed responses resulted from a maximal activation of biogenic amine receptors. Indeed, the amounts ejected iontophoretically during a single 30 s application were probably equivalent to the whole content of several thousand 5-HT or N A axonal varicosities*. Moreover, given the relatively sparse monominergic innervation of the neocortex v,l~ 56, the re-uptake mechanisms were presumably saturated for at least several minutes after such microiontophoretic ejections, until diffusion brought back local amine concentration near physiological levels. Radioligand studies with tritiated LSDS-10, 37, 5-HT10,.~v,G7,s~, haloperidol and dopamine 19, domperidone 4, spiroperidol 6a,va, norepinephrine 97, alprenolol 2, dihydroalprenolol 2°, dihydroergocryptine 64 and prazosin 4s have suggested the existence of distinct binding sites for 5-HT, DA and NA in cerebral cortex. Furthermore, microchemical, fluorescence-histochemical, radioautographic and immunocytochemical investigations have shown the presence ot these 3 types of nerve endings within the deep layers of the neocortex (e.g. refs. 7, 12, 56-58, 72, 78, 79). The observation that most 'spontaneously' active units in the deep cortical layers were depressed by either one of the biogenic amines is therefore consistent with a ubiquitous distribution of 5HT, DA and NA receptors in cortex and/or with the coexistence of such receptors on the same target cells. This latter supposition is strengthened by the occurrence of opposite changes in the responsiveness of the same units to 5-HT and DA or NA following denervation with 5,7-DHT. Supersensitivity to 5 - H T After deafferentation with 5,7-DHT, two observations witnessed increased responsiveness to 5-HT: (1) an almost 3-fold lengthening of depressions induced by 100 nA applications and (2) marked depressions of spontaneous firing induced by small currents having little or no effect in control rats. Denervation supersensitivity to 5-HT has been previously documented by microiontophoresis in the amygdala lm, the lateral geniculate 101 and the facial nucleus 6'~. Interestingly, it does not develop in the hippocampus deafferented with 5,7-DHT 2s, either because of regional differences in the pattern of 5-HT innervation and/or in the mode of action of 5-HT. Denervation supersensitivity to 5-HT in the neocortex was seemingly due to the absence of 5-HT afferents rather than that of 5 - H T itself 36. Indeed, no supersensitivity * The amount of biogenic amine ejected from our micropipettes was measured in in vitro experiments with tritiated 5-HT and NAs0 : 5 pg of NA and 8 pf of 5-HT were ejected by 100 nA currents applied for 30 s. The mean biogenic amine content of 5-HT and NA axonal varicosities in cortex has been previously estimated to be 3.5 × l0 -4 pg7 and 2.33 × 10 3 pg~G,respectively. The present ejections were, therefore, equivalent to the whole biogenic amine content of some 14,500 5-HT and 3400 NA varicosities.

102 could be detected following equally severe depletion of endogenous 5-HI store., b}. PCPA. A similar finding has already been made by Wang et al. in the amygdala H~ These results are consistent with the lack of 5-HT supersensitivity reported alter PCPA in behavioral experiments 95. Alternatively, it is possible that, following 5,7-DH] deafferentation, the 90 I~, lowering of cortical 5-HT represented a total toss of this type of nerve endings around certain neurons but not others, whereas after PCPA no cells were completely deprived of 5-HT input. Hence, it cannot be excluded that, a|ier PCPA, a minimal release of 5-HT, with or without augmented turnover, suffices to maintain a normal sensitivity to this biogenic amine. But, whether triggered by the absence of 5-HT fibers or by that of 5 - H I itself. two mechanisms were seemingly involved in this denervation supersensitivity: the suppression of 5-HT re-uptake into nerve terminals ('presynaptic' component), and a modification of 5-HT receptors on the target neurons ('postsynaptic' componentL ()~ the one hand, the prolongation of 5-HT responses by fluoxetine, which w~s found in PCPA-treated as well as in control cortex, was similar to that observed after denervation, suggesting that it was due to increased availability o[' 5-}tT in the extracellular space. On the other hand, the fact that alter deafferentatlor, x~ith 5,7DHT, but not after fluoxetine, small ejection currents of 5-HT were sufficient to induce marked depressions of firing indicated an increased sensitivity of 'postsynaptic ~ 5-Hq receptors. It remains to be determined if such a compensatory supersensitivity to 5-H~[ reflects an augmentation in the density and/or affinity of the 5-HT receptors and/or a modification of their transducing mechanisms. In this regard, Fuxe et al; haxe reported that, in cerebral cortex, both the number of LSD-binding sites and the affinity for 5H T are augmented 14 days after 5,7-DHT lesioning of the ascending 5-HT pathways 4~. Presumed 5-HT receptor changes following 5-HT denervation have also been postulated by Wang et al., who found a significant increase in neuronal responsivenes~ to microiontophoretically applied 5-HT and LSD in the amygdala and ventral lateral geniculate nucleus after 5,7-DHT I°j. Analogous receptor changes were also demonstrated in slices of cerebral cortex fi'om rats previously treated with 0-hydro×ydopamine, using the ~-adrenergic antagonist iodohydroxybenzylpindolol~ ttyposensitiviO' to DA and NA

Markedly diminished responsiveness lo DA and NA t~llowed either 5-HT deafferentation with 5,7-DHT or depletion with PCPA. The comparable decrease in the responsiveness to PHE, which has little affinity for re-uptake 18, indicated that this altered responsiveness was mainly imputable to the development of a catecholaminc receptor hyposensitivity. Earlier investigations of cAMP production after chronic administration of reuptake blockers 4°,88,9'~, or MAO inhibitors 98, have led to the notiot~ that ~m 'overstimulation' of catecholamine receptors may result in a reduction of their sensitivity (for reviews, see 46, 89). In homogenates of cerebral cortex, chronic treatment with desipramine, an inhibitor of NA re-uptake, has been found to decrease the density of fl-adrenergic receptors as measured by the binding of the antagonist iodohydroxybenzylpindoloU04. Recent data obtained in the peripheral nervous system

103 suggest that such changes could in turn be secondary to a decreased sensitivity of ~presynaptic' NA receptors which inhibit the release of NA z4. In this context, it is plausible that the decreased DA and NA responsiveness here observed in cortex was caused by an augmentation of catecholamine release. An alternate explanation would be that, in normal cortex, the effects of microiontophoretically applied DA or NA are always observed in summation with those of a tonic or catecholamine-induced liberation of 5-HT. Experiments involving the acute administration of a specific blocker of 5-HT release in control rats, or the coiontophoresis of small amounts of 5-HT along with DA or NA in PCPA-treated rats, would be needed to substantiate this theoretical possibility. In considering mechanisms by which 5-HT might regulate the release of DA and NA in cortex, it should be pointed out that temporary increases in the activity of the biosynthetic enzyme tyrosine hydroxylase have been measured following 5-HT denervation of the locus coeruleus with 5,6-DHT vS,s3, or depletion of its 5-HT content with PCPA 23. As in the present experiments, these effects were already detectable after 48 h of PCPA depletion and persisted one month after denervation. In view of such findings, secondary changes in the turnover of catecholamines might be envisaged as being responsible for the decreased responsiveness to DA and NA occurring in cortex. However, the present hyposensitivity to DA and NA could also be indicative of a local regulation of DA and NA release by 5-HT, perhaps involving 'presynaptic' 5-HT receptors on catecholamine nerve terminals a6. Interactions between 5-HT and catecholamines have already been documented in other regions of the central nervous system, such as the neostriatum s5 and the amygdala 1°1. In the amygdala, sensitivity to microiontophoretically applied NA has been shown to increase after deafferentation with 5,7-DHT, hinting at some regional specificity of the interregulations between biogenic amines. More generally, it may be concluded that the function of a given biogenic amine system, especially in the neocortex, might be largely dependent on its interactions with the other monoaminergic systems. In this sense, as well as through their more direct effects, the cortical biogenic amines truly fulfil the current definitions of neuromodulators 33,aa. It would be of interest to understand why 5-HT, DA and NA, which seemingly exert comparable effects on cortical neurons, should influence one another. The answer to this question, as well as a more precise elucidation of the functional role of the biogenic amines in cerebral cortex, awaits better knowledge of the type of information conveyed by each of these afferent systems. ACKNOWLEDGEMENTS The authors thank Ms. Chantal Mercure for her technical assistance with the biochemical determinations, and Messrs. Giovanni Battista Filosi, Daniel Cyr and Edward Rupnik for their graphic and photographic work. They are also grateful to Dr. C. De Montigny for his detailed, critical revision of the manuscript. This study was supported by Grants MT-3544 (L.D.) and MA-6244 (T.A.R.) from the Medical Research Council of Canada.

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