Effect of short-term serotonin depletion on the efficacy of serotonin neurotransmission: Electrophysiological Studies in the Rat central Nervous System

SYNAPSE 6:328-337 (1990)

Effects of Short-Term Serotonin Depletion - on the Efficacy of Serotonin

Neurotransmission: Electrophysiological Studies in the Rat Central Nervous System

YVES CHAPUT, PHILIPPE LESIEUR, AND CLAUDE DE MONTIGNY Neurobiological Psychiatry Unit, De artment of Psychiatry, McGill University,

Montreal, Queb)ec, Canada H3A IAI

KEY WORDS p-Chlorophenylalanine, 5-HT, Dorsal hippocampus, Dorsal raphe, 5 , 7 -Dihydroxytryp tamine

ABSTRACT The effects of short-term serotonin (5-HT) depletion by p-chlorophenylalanine (PCPA) on the firing activity of dorsal raphe nucleus 5-HT neurons, on the responsiveness of dorsal hippocampus pyramidal neurons to microiontophoretically applied 5-HT and on the efficacy of the electrical stimulation of the ascending 5-HT pathway in suppressing the firing activity of CA, dorsal hippocampus pyramidal neurons were assessed in chloral hydrate-anesthetized rats. PCPA (250 mg/kg/day i.p. for 2 days) reduced the 5-HT content of the dorsal hippocampus by 90%. However, the number of spontaneously active 5-HT neurons per microelectrode trajectory through the dorsal raphe or their average rate of firing was unaltered. The effect of afferent 5-HT pathway stimulation was reduced in only 40% of treated rats, whereas the sensitivity of CA, pyramidal neurons to microiontophoretic 5-HT was not modified. The function of the terminal 5-HT autoreceptor was assessed using methiothepin, an autoreceptor antagonist. Methiothepin (1 mg/kg, i.v.) significantly enhanced the efficacy of the stimulation in PCPA-treated rats, although the degree of enhancement was much less than in controls. A greater reduction of the effectiveness of the stimulation was obtained by increasing the dose of PCPA (350 mg/kg/day i.p. for 2 days). This regimen reduced the 5-HT content of the dorsal hippocampus by 95%. In these rats, the sensitivity of the terminal 5-HT autoreceptor was assessed by increasing the frequency of the stimulation from 1 to 5 Hz. This procedure reduced to a similar extent the efficacy of the stimulation in treated and control rats, suggesting that the reduced effectiveness of methiothepin in enhancing 5-HT synaptic transmission in PCPA-treated rats is due to a lower degree of activation of the terminal 5-HT autoreceptor. The present results showing that the 350 mglkglday regimen of PCPA, but not the 250 mg/kg/day regimen, reduced the efficacy of the stimulation of the ascending 5-HT pathway suggest that a greater than 90% depletion is required to affect 5-HT neurotransmission significantly. The reduced level of activation of terminal 5-HT autoreceptors in rats treated with the lower dose of PCPA may facilitate the release of the remaining 5-HT per stimulation-triggered action potential, ensuring a virtually unaltered synaptic efficacy.

INTRODUCTION The clinical observation by Shopsin et al., (1975,

1976) of a rapid reversal of the therapeutic effect of two chemically distinct antidepressants (imipramine or tranylcypromine) by the short-term administration of the serotonin (5-HT) synthesis inhibitor para-chlorophenylalanine (PCPA) (Jequier et al., 1967; Koe and Weiss- man, 1966) to remitted patients constitutes strong evidence that the 5-HT system may play a pivotal role in mediating the therapeutic effect of antidepressant treatments. The recent report by the rapid mood lowering effect tryptophan in 0 1990 WILEY-LISS, INC.

patients oints to the same conclusion. There is a sub- stantial gody of clinical evidence in support of this contention, such as the therapeutic efficacy of selective 5-HT reuptake blockers and of 5-HT precursors in major depression (Asberg et al., 1986; Bech, 1988; Kaneko et al., 1979; Moller et al., 1980;Van Praag, 1981) as well as the potentiation of antidepressant treatments by L-tryptophan (Ayuso Guttirez and Lopez-Ibor, 1971; Coppen et al., 1963; Glassman and Platman, 1969; Pare, 1963; WAlinder et al., 1976) or by the addition of lithium

Received August 15,1989 accepted in revised form May 22,1990

EFFECT OF PCPA ON 5-HT NEUROTRANSMISSION 329

(de Montigny et al., 1981, 1983, 1985; Heninger et al., 1983; Nelson and Byck, 1982; Nelson and Mazure, 1986; Ryan et al., 1988).

Electrophysiological investi ations of the effects of

treatments in the rat suggest that an enhancement of 5-HT neurotransmission might be a common denomina- tor of treatments as diverse as tricyclic, tetracyclic, and atypical antidepressants; selective 5-HT reuptake blockers; monoamine oxidase inhibitors; and electro- convulsive shocks (for reviews, see Blier et al., 1987a; Willner, 1985). Furthermore, the time course of this potentiation of 5-HT neurotransmission is consistent with the delayed clinical efficacy of these treatments in ma'or de ression.

i h e efkcts of 5-HT depletion by the trypto han hy-

transmission in the rat hippocampus has never been extensively investigated. Segal(1975) reported that the short-term administration of PCPA to rats partially abolished one of two inhibitory effects of the electrical stimulation of either the dorsal or medial raphe nuclei on the firing activity of dorsal hip ocampus pyramidal

nent that was not modified by PCPA pretreatment was one characterized by a short latency and a short duration (10 and 40 msec). This latter effect is similar in its characteristics to the suppression of the firing activity of hippocampus pyramidal neurons produced by the electrical activation of the ascending 5-HT athway in

shown to be a sensitive index of changes in the efficacy 5-HT synaptic transmission (Blier and de Montigny, 1983; Blier et al., 1987b; Chaput et al., 1986a,b). We therefore undertook to investigate, using an electrophysiological paradigm, the effects of short-term 5-HT depletion by PCPA on 5-HT neurotransmission in chloral h drate-anesthetized rats. Three major constituents

activity of 5-HT-containing neurons of the dorsal raphe nucleus; 2) the efficacy of 5-HT synaptic transmission, as assessed by the effect of the electrical activation of the ascending 5-HT pathway on the firing activity of postsyna tic CA, dorsal hippocampus pyramidal neu-

5-HT applied microiontophoretically.

the repeated administration o 7 various antidepressant

droxylase inhibitor PCPA on the efficacy of 5- I! T neuro-

neurons. Interestingly, however, t K e inhibitory compo-

the ventromedial tegmentum (VMT), whic E has been

of 5- B T neurotransmission were examined: 1) the firing

rons; an Lp 3) the responsiveness of the same neurons to

MATERIAL AND METHODS Treatments

PCPA Male Sprague-Dawley rats (225-250 g) were admin-

istered either saline or PCPA (250 or 350 mgkglday i.p.) for 2 days. Electrophysiological experiments were carried out under chloral hydrate anesthesia (400 mgkg, i.p.), 24 hr after the second injection of saline or PCPA.

5,7-Dihydroxytryptamine Male Sprague-Dawley rats (200-225 g) were anesthe-

tized with chloral hydrate (400 mgkg i.p.) and mounted in a stereotaxic ap aratus, 1 hr following an intraperi-

tered to rotect noradrenergic neurons rom the neuro- f toneal injection o P desipramine (25 m kg), adminis-

toxic e ff ect of 5,7-dihydroxytryptamine (5,7-DHT). A

burr hole was drilled 1 mm lateral and 1 mm posterior to bre a and 200 pg free base of 5,7-DHT in 20 pl of 0.9%

lateral ventricle over a 5 min eriod. Control rats were

were carried out under chloral hydrate anest esia also, 10-14 days following the intracerebroventricular injection of 5,7-DHT or saline.

Na t? 1 and 0.1% ascorbic acid was injected into the right

injected with saline. Electrop R ysiological ex eriments g

Recording from 5-HT-containing neurons of the nucleus raphe dorsalis

Sin le-barrelled glass micropipettes, preloaded with

ulled in the conventional manner and filled with a 2 M K aC1 solution saturated with fast green FCF (Fisher). A burr hole, 3 mm in diameter encompassing lambda, was drilled, and the micropipette was descended 1 mm anterior to lambda on the midline, into the dorsal raphe, usual1 attained at a depth of between 4.5 and 5.5 mm from t e surface of the brain.

Dorsal raphe 5-HT neurons were identified according to the criteria of Aghajanian (1978). Each 5-HT neuron encountered was recorded for a proximately 1 min in

electrode descent in the dorsal ra he consisted in a 1

electrode descents were performed: one 1 mm anterior to lambda on the midline, two 200 pm lateral to the first tract on each side, and two 200 pm rostra1 and caudal to the first electrode tract. A fast green deposit was left at the last recording site by passing a -26 pA current through the microelectrode for 15 min for subsequent histological verification of the location of recording sites.

fiberg 7 ass strands to promote capillary filling, were

order to obtain a measure of its 1 asal firing rate. Each

mm tract from the floor of the Sy P vius aqueduct. Five

Recording from CA, dorsal hippocampus pyramidal neurons

Five-barrelled glass micro ipettes, preloaded with

manner and their tips broken back to approximately 10 pm under microscopic control. The central barrel, used for extracellular unitary recordings, was filled with a 2 M NaCl solution saturated with fast green. The side barrels, used for microionto horesis, were filled with

40 mM in 200 mM NaC1, pH 4; Sigma), acetylcholine chloride (ACh; 20 mM in 200 mM NaC1, pH 4; Sigma). The fourth barrel, used for automatic current balancing, was filled with a 2 M NaCl solution.

The microelectrode was lowered 4.2 mm lateral and 4.2 mm anterior to lambda into the CA, region of the dorsal hip ocampus. Pyramidal neurons were identified accorzing to the criteria of Kandel and Spencer (1961). Since these latter neurons demonstrate little spontaneous activity under chloral hydrate anesthesia, a leak or small ejection current of ACh (0-8 nA) was used to activate them within their hysiological range

midal neurons to 5-HT and to norepinephrine (NE) was assessed using the I.T,, method, i.e., by determining the charge in nC (current in nA X time in sec) required to obtain a 50% depression of firing rate from baseline (de Montigny and Aghajanian, 1977).

fiberglass strands, were pu P led in the conventional

the following solutions: 5-H 8 creatinine sulfate (0.5 or

(10-15 Hz). The responsiveness of K ippocampus pyra-

330 Y. CHAPUT ET AL

Electrical stimulation of the ascending 5-HT pathway

A bipolar concentric electrode NE-100 (David Kopf Instruments) was positioned with a lo" backward angle in the VMT, 1 mm anterior to lambda on the midline at a depth of 8 mm. Two stimulation paradigms were used. First, square 0.5 msec pulses of 300 p,A were delivered at a frequency of 1 Hz approximating that of the spontaneous firing activity of dorsal raphe 5-HT neurons under chloral hydrate anesthesia while recording the firing activity of CA3 dorsal hippocampus pyramidal neurons. This control stimulation was chosen, because it has been shown to produce a detectable suppression of the firing activity of postsynaptic neurons in 93% of trials (Chaput et al., 1986a). These stimulation parameters were chosen to carry out the present study because they result in a submaximal response of postsynaptic neurons. Indeed, the duration of the suppression of postsynaptic neuron firing activity can be prolonged by increasing the intensity of the current to 640 FA (Chaput et al., 1986a), by terminal 5-HT autoreceptor blockade (Blier et al., 1988; Chaput and de Montigny 1988; Chaput et al., 1986a,b), or by reducing the stimulation frequency from 1 to 0.5 Hz (Chaput and de Montigny, 1988). A second paradigm, whereby stimulations were carried out using a frequency of 5 Hz, was performed to assess the function of the terminal 5-HT autoreceptor. It has been observed in vivo that increasing the frequency of the stimulation of the 5-HT pathway from 1 to 5 Hz decreases its effectiveness (Blier et al., 1988; Chaput et al., 1986a). Presumably, the higher frequency of the stimulation results in a greater degree of activation of the terminal 5-HT autoreceptor at the time of arrival of the subsequent stimulation- triggered action potential. Therefore, each pulse a t the 5 Hz frequency releases a smaller amount of 5-HT. Fur- thermore, the effect of increasing the frequency of the stimulation from 1 to 5 Hz is reduced following long- term treatment of rats with drugs which decrease the function of the terminal 5-HT autoreceptor (Blier et al., 1988). This suggests that this approach provides a useful index of terminal 5-HT autoreceptor sensitivity.

Pulses were delivered through a direct-coupled isola- tion unit (SIU 478A, Grass). The unitary activity and the stimulation pulse were recorded on-line with an IBM-PC computer equipped with a Tecmar interface. Peristimulus time histograms of dorsal hippocampus pyramidal neuron firing activity were generated to measure the suppression of firing produced by the stimulation. The latter was quantified by computer as the absolute silence value (SIL), defined as the theoret- ical duration of a total suppression of firing that would represent the number of events suppressed by the stimulation. This latter value was calculated by computer by dividing the total number of events suppressed by the stimulation by the mean prestimulation frequency of firing of the neuron recorded. Thus the SIL value represents the duration of the suppression of firing of these neurons normalized for their firing activity.

Biochemical analysis Brains were quickly removed and frozen on dry ice.

Coronal 300-~m-thick sections were cut in a cryostat at - 15°C. The CA, region was microdissected and punched

out. Tissue pellets were homogenized with a hand mi- crohomogenizer a t 4°C in 150 pl of 0.4 N HCl,,. Fifteen microliters were collected for protein determination (Lowry et al., 1951). The remaining homogenates were centrifu ed for 90 sec and the clear supernatant was collecte and analyzed for 5-HT by high- ressure liquid chromatography coupled with electroc emical detec- tion, as previously described (Chiew et al., 1982).

Statistical analysis One-way analysis of variance was used for the statis-

tical comparison of I.TSo values or SIL values obtained in PCPA- or 5,7-DHT-pretreated rats to those obtained in corresponding control rats. The statistical signifi- cance of the difference between the SIL values for the same hippocampus pyramidal neuron when the stimulation was carried out a t 1 and 5 Hz or prior to and following the acute intravenous injection methiothepin (MET) or 5-hydroxytryptophan (5-HTP) was assessed using the paired Student's t test. All results are ex- pressed as means ? SEM.

RESULTS Effect of PCPA treatment on 5-HT neuron

firing activity To obtain an index of the activity of 5-HT neurons

following PCPA, systematic electrode descents were performed into the dorsal raphe nucleus in control rats and in rats treated with PCPA (350 mg/kg/day for 2 days). The PCPA treatment modified neither the number of spontaneously active 5-HT neurons encountered per tract nor their average discharge frequency (Tables I and 11). This confirms the finding of Wang and Aghaja- nian (1978) who observed a normal firing activity of dorsal raphe 5-HT neurons following a two-day PCPA pretreatment. Effect of PCPA treatments on the efficacy of 5-HT

synaptic transmission The 300 pA stimulation of the ascending 5-HT ath-

way at a frequency of 1 Hz in control rats resulte B in a suppression of hippocampus pyramidal neuron firing activity of 42 2 3.5 msec (Figs. 1A and 21, with a latency from the time of the stimulation to the onset of suppression of 6 5 0.3 msec. The 5-HT content in the CA, region of the dorsal hippocampus of seven of these control animals was 10.3 2 0.6 ng/mg protein.

R f

TABLE I . Number of spontaneously active 5-HT neurons (k SEM) per 1 mm microelectrode track through the dorsal raphe nucleus

No. of 5-HT neurondtract No. of tracts

Control PCPA'

6.1 i 0.5 6.5 + 0.5

18 23

'Rats treated for 2 days with p-chlorophenylalanine (PCPA) at dose of 250 mg/kg/day.

TABLE II. Spontaneous firing activity (Hz f SEM) of 5-HT neurons in the dorsal raphe nucleus

No. of neurons recorded Firing activity

Control 1.07 i 0.07 109 PCPA' 1.12 i 0.08 148 'Rats treated for 2 days with PCPA at a dose of 250 mg/kg/day.

EFFECT OF PCPA ON 5-HT NEUROTRANSMISSION

100-

80 - - 5 60-

+I

J - 40-

07

v : 07

20 -

33 1

A - CONTROL

161

- PCPA (350 mglkgldayX2 days)

I

C - 5,7-DHT (200 pg, i.c.v.)

-50 -25 25 50 75 100

TIME (ms)

Fig. 1. Peristimulus time histograms obtained in a control rat (A), in a rat treated with p-chorophenylalanine (PCPA) (B), and in a rat pretreated with an intracerebroventricular injection of 5,7-dihydroxytryptamine (C). Each histogram was constructed from 150 stimulations of the ascending 5-HT pathway. The pulses, of an intensity of 300 p4 and of a duration of 0.5 msec, were delivered at 1 Hz in the ventromedial tegmentum at time 0 during the recording of a postsynaptic neuron in the CA, region of the dorsal hippocampus.

Pretreatment of rats with PCPA (250 mgkglday for 2 days) reduced the 5-HT content in the CA, region of the dorsal hippocampus by 90% 2 1% (1.03 2 0.01 nglmg; n = 16), in close agreement with previously published data using the same regimen of PCPA (Lighton et al., 1984). However, such a treatment did not significantly reduce the efficacy of the stimulation of the ascending 5-HT pathway (35 2 7 msec; n = 17; Fig. 2). Interest- ingly, the PCPA-treated animals did not represent a homogeneous group. Rather, rats could be divided into two subgroups, according to the duration of the response

+

+ + +

+

+ * k

0 @ CONTROL PCPA PCPA

250 m g l k g 350 m g l k g

Fig. 2. Scatto ams of the SIL values obtained in control rats and in rats treated witreither 250 or 350 mg/kg/day of PCPA for 2 days. The SIL value represents the duration of the su pression of dorsal hi PO campus pyramidal neuron firing activity !y the stimulation o?the ascendinf 5-HT pathway. The stimulation parameters were the same as those escribed in the legend to Fi re 1 Each point represents the mean SILvalue for one rat, from whicrone to four neurons weretested. The shaded area represents the mean k SEM value for each group. *P < 0.05 compared to control values (one-way analysis of variance).

of postsynaptic neurons to the stimulation of the ascending 5-HT pathway: a nonresponder subgroup (NR- PCPA) in which the SIL values (5 * 2 msec; range 0-9 msec; n = 7) were all below the lowest SIL value of control rats (range 12-83 msec), and a responder subgroup (R-PCPA) whose average SIL value (50 % 7 msec; range 32-93 msec; n = 10) was in the range of the values of control rats (Fig. 2). No difference in the mean microiontophoretic ejection current of ACh required to activate hippocampus pyramidal neurons to a physiological frequency (10-15 Hz) was found between control and PCPA-treated rats (5.5 & 0.5 nA; n = 29, vs. 4.3 t 0.5 nA; n = 17, respectively).

To exclude the possibility that the PCPA treatment induced a postsynaptic supersensitivity that might account for the unaltered efficacy of the 5-HT pathway stimulation when the PCPA group was taken as a whole, the responsiveness of hi pocampal pyramidal

mM in 200 mM NaC1) was assessed in control and PCPA-treated rats. The res onsiveness of these latter

not modified following the two-day treatment with PCPA (Fig. 3, Table 111). Furthermore, no difference in the responsiveness of hippocampus pyramidal neurons to exogenous 5-HT was found when I . T,, values obtained in the NR-PCPA rats were compared to those of the R-PCPA animals (Table 111).

neurons to microiontophoretica P ly-applied 5-HT (0.5

neurons to 5-HT, as assesse a by the I . T,, method, was

200 -

ln 0 --. 2 100- Y - 0

- a

0-

53 17 107 + 11 PCPA' NR-PCPA3 108 i 20 25 7 R-PCPA3 103 i 15 28 10

'The I.T5o value represents the charge (in nC) required to depress by 50W the firing activity of the neuron recorded. 2Rats treated for 2 days with PCPA a t a dose of 250 mg/kg/day. 3PCPA-treated rats were divided a posteriori in two subgroups: the NR-PCPA subgroup showing a markedly reduced response to electrical stimulation of the ascending 5-HT pathway and the R-PCPA showinga normal response (see text for details).

200 -)

ln 0 7

LLI 100- Y a m -

0-

(I) '' 40 2 - i (I) -

20

0 CONTROL PCPA

250 mg/kg


mg/kg/day, i.p. for 2 days (n = 29). The 5-HT content of the CA, hippocam a1 region was determined in eight of

manipulation during the electrophysiological experi- ment: There was a 95% reduction of the 5-HT content (0.5 2 0.1 ng/mg rotein). This regimen of PCPA si if-

ascending 5-HT pathway (Figs. 1B and 2). However, as can be appreciated from the scattograms of Figure 2, there was a large variation in the efficacy of the stimulation of the ascending 5-HT pathway in this group of PCPA-treated rats also.

To assess terminal 5-HT autorece tor function follow-

increasin the frequency of the stimulation (from 1 to 5

sion was assessed in treated rats, where 5-HT pathway stimulation had yielded SIL values comparable to those obtained in the control rats (SIL values 3 30 msec). This ost-hoc analysis was based on the assumption

responsible for the unaltered efficacy of the stimulation in these treated rats, then increasing the frequency of the stimulation from 1 to 5 Hz should no longer reduce its efficacy, as it does in control animals as a result of an increased level of activation of the 5-HT terminal autorece tor by endogenously released 5-HT (Blier et al.,

data, increasing the stimulation frequency from 1 to 5 Hz in control rats significantly reduced its efficacy (Fig. 5). A comparable degree of reduction was obtained at 5 Hz in the subgroup of PCPA-treated rats, which had a normal response to the 1 Hz stimulation. This suggests that the unaltered res onse to afferent 5-HT pathway stimulation in this su ! group of rats could not be ex- plained on the basis of an alteration in 5-HT terminal autoreceptor sensitivity (Fig. 5).

these rats in whic R there had been no pharmacological

icantly decrease cp the efficacy of the stimulation o F-l the

ing treatment with 350 mgkg of f: CPA, the effect of

Hz) in re P ucing the efficacy of 5-HT synaptic transmis-

that i P 5-HT terminal autoreceptor desensitization were

1988; 8 haput et al., 1986a). In agreement with previous

r +I E v)

1 20 cn

0 CONTROL PCPA

350 rnglkg

0 1 Hz 5 Hz

Fig. 5. Degree of suppression of CA, dorsal hippocampus pyramidal neuron firing activity by stimulations at 1 and 5 Hz of the ascending 5-HT athway in control and in rats treated with PCPA for 2 days. The stimufat ion parameters were the same as those presented in legend to Figure 1. The number at the bottom of each pair of columns represents the number of neurons tested. *P < 0.05, **P < 0.005 compared to corresponding value at 1 Hz (paired Student’s t test).

To determine whether, in rats treated with PCPA (350 mgkg) in which the stimulation of the ascending 5-HT pathway resulted in a markedly reduced SIL value (8 ? 2 msec; n = 9), the efficacy of the stimulation could be augmented by the immediate 5-HT precursor 5-hydroxyt tophan (5-HTP), stimulations of the as-

following, the intravenous injection of 5-H P (combined with carbidopa, 1.5 mgkg, to prevent its peripheral decarboxylation). 5-HTP (5 mgkg i.v.) produced a two- fold increase of SIL values (Fi . 6), the maximal effect

In an attempt to determine whether a comparable increase in the SIL value could be obtained by applying 5-HT in the vicinity of the soma of CA, dorsal hippocampus yramidal neurons, stimulations of the ascending

the microiontophoretic application of 5-HT with an avera e ejection current of 55 nA, for an average duration of255 sec (range 150-1000 sec), using iontophoretic solutions of 5-HT of 0.5 or 40 mM in 200 mM NaCl. No modification of the SIL value could be detected (prior to 7 i 1 msec; following 9 2 2 msec; n = 15).

Effect of 5,7-dihydroxytryptamine To compare the effect of the PCPA-induced depletion

of 5-HT to that of lesioning 5-HT neurons, the response of hippocampus pyramidal neurons to the microiontophoretic a plication of 5-HT and to the stimulation of

following the intracerebroventricular administration of

.F cending 5-H ?l! pathway were carried out rior to, and

being observed about 20 min a fg ter the injection.

5-H 4 pathway were carried out prior to, and following,

the ascen B ing 5-HT pathway was assessed 10-14 days

0

PRIOR TO 5-HTP FOLLOWING 5-HTP (5 mg/kg, i.v.)

Fig. 6. Degree of suppression of the firing activity of CA, dorsal hi ocampus pyramidal neurons by the stimulation of the ascending 5% pathway prior to, and following, the intravenous administration of 5-hydroxyt to han (5-HTP) in rats treated for 2 days with PCPA (350 mg/kg/d3. !. timulation parameters were the same as those described in the legend to Fi re 1 The number at the bottom of the columns represents the numgr ofneurons tested. *P < 0.05 (paired Student’s t test).

334 Y. CHAPUT ET AL.

either 5,7-DHT7 a 5-HT neurotoxin, or saline. The 5,7- DHT pretreatment reduced the 5-HT content in the CA, region of the dorsal hippocampus by 90% (1 t 0.1 ng/mg protein; n = 10). As previously observed (de Montigny et al., 1980), there was no difference in the I * T,, value for the microionto horetic application of 5-HT between

indicating that the latter pretreatment had not induced a postsynaptic supersensitivity in this region of the brain. The efficacy of the stimulation of the ascending 5-HT pathway was reduced by 60% relative to control rats (Fig. 7).

DISCUSSION The present results showing that the 350 mg/kg/day

regimen of PCPA, but not the 250 mg/kg/day regimen, reduced significantly the efficacy of the stimulation of the ascending 5-HT pathway (Fig. 2) suggest that a greater than 90% depletion of the dorsal hippocampus 5-HT content is required to affect the 5-HT synaptic efficacy in this region of the brain. Interestingly, this conclusion is similar to that reached by Curet and de Montigny (1988) concerning the degree of depletion of the dorsal hippocampus NE content to produce a decrease of the efficacy of the stimulation of the locus coeruleus. However, before considering the possible substratum for this apparent remarkable homeostatic capacity of the 5-HT synapse, several other possibilities must be considered.

First, the limited efficacy of PCPA in reducing the effect of the stimulation of the VMT on the firing activity of dorsal hippocampus pyramidal neurons raises the issue of the validity of this paradigm as an index of the

control and 5,7-D H T-pretreated rats (data not shown),

0

' 0 ° 1

0 I 0 0

0

AAA A

0 CONTROL 5, 7-DHT

Fi 7 Scattograms of the degree of suppression of the firing activity of C f 3 dorsal hi ocampus pyramidal neurons by the stimulation of the ascending5-% pathway in control rats and in rats pretreated with 5,7-DHT. Stimulation parameters were the same as those described in the legend to Figure 1. Each value represents the mean value for one rat, from which two to four neurons were tested. The shaded area represents the mean ? SEM value for the group. *P < 0.01 (one-way analysis of variance).

function of 5-HT synaptic transmission. That this paradigm could be unsuitable to assess 5-HT synaptic function appears unlikely in light of the following electrophysiological observations: 1) The electrical stimulation the VMT antidromically activates 5-HT-containing neurons of the dorsal and median raphe nuclei, an effect blocked by a 5,7-DHT, but not a 6-hydroxydopamine, pretreatment into the VMT (Wang and Aghajanian, 197713,1978); 2) the latency of the effect of the stimulation on postsynaptic neurons is entirely compatible with the slow conduction velocity of small unmyelinated 5-HT axons and the distance from the tip of the stimulation electrode to the hippocampus (Blier and de Mon- tigny, 1983,1985; Chaput et al., 1986a); 3) the effect of the stimulation is markedly reduced by the systemic administration of a selective 5-HTlA receptor antagonist (Chaput and de Montigny, 1988), the 5-HTIA receptor being in large part responsible for the suppressant effect of 5-HT in the CA1 and CA, regions of the rat hippocampus (Andrade and Nicoll, 1987; Beck, 1989; Chaput and de Montigny, 1988; Colino and Halliwell, 1987); 4) the terminal 5-HT autoreceptor blockade, which augments the stimulus-evoked release of F3H15- HT in vitro (Moret, 1985), enhances the efficacy of the stimulation in vivo (Blier et al., 1988; Chaput and de Montigny, 1988; Chaput et al., 1986a,b; present results), and, to the contrary, terminal 5-HT autoreceptor activation, which has been shown to decrease 5-HT release in vivo (Martin and Marsden, 19861, decreases the SIL value (Chaput and de Montigny, 1988). Taken together, these data strongly suggest that the SIL value obtained with the VMT stimulation is a sensitive and accurate index of 5-HT synaptic transmission. That the effect of the stimulation was virtually abolished in more than one-half of the 5,7-DHT-treated rats (Fig. 7), in keeping with the report of Blier et al. (1987b), and its enhancement by small doses of 5-HTP in PCPA-treated animals (Fig. 6) support this contention. The inability of prolonged microiontophoretic applications of 5-HT onto CA, pyramidal neurons to restore the efficacy of the stimulation in PCPA-treated rats is most likely due to fact that most 5-HT terminals are found within the stratum radiatum (Azmitia and Segal, 1978), whereas electrophysiological recordings are obtained some distance away, in the stratum pyramidale. Alternatively, that this failure might have been due to the activation, by microiontophoresed 5-HT7 of 5-HT terminal autoreceptors would appear less likely in that prolonged microiontophoretic applications of 5-HT does not alter the efficacy of the stimulation in control animals (unpub- lished observations).

A second possibility is that 5-HT depletion by PCPA resulted in postsynaptic 5-HT receptor supersensitivity, cancelling out the reduction of the synaptic efficacy expected to result from the depletion. Indeed, an increase in [3H15-HT specific binding to crude rat fore- brain membrane preparations has been reported to occur following short-term PCPA administration (Bennet and Snyder, 1976; Fleisher et al., 1979; Steigrad et al., 1978). In addition, Fleisher et al. (1979) have reported that PCPA, as well as its metabolite p-chlorophenyl- acetic acid, can increase the affinity of L3H]5-HT binding in vitro. However, this increased binding, as Steigrad et al. (1978) proposed, is more likely the result of a decreased 5-HT content leading to a reduced competi-


tion for the binding sites rather than of an increased number of receptors. This is supported by the finding of a normal sensitivity of postsynaptic 5-HT receptors following PCPA in both behavioral (Dourish et al., 1986; Stewart et al., 1976; Trulson et al., 1976) and electrophysiological paradigms (Ferron et al., 1982; Wang et al., 1979; present study).

A third possibility for interpreting the rather limited efficacy of PCPA in reducing the effect of the VMT stimulation despite a marked 5-HT depletion may pertain to either the coexistence of another neurotransmitter within 5-HT-containing neurons or the existence of non-5-HT fibers emanating from both the dorsal and median raphe nuclei. Indeed, it has been suggested that nond-HT neurons [possibly catecholaminergic or pepti- dergic (Steinbush and Nieuwenhuys, 1983)l in the dorsal and median raphe nuclei contribute to their ascending fiber tracts, including those afferent to the hippocampus (Kohler and Steinbush, 1982; Kohler et al., 1982; Kosofsky and Molliver, 1987; O’Hearn and Molliver, 1984). Therefore, the possibility that the stimulation of the ascending 5-HT tracts within the VMT might also activate nond-HT axons cannot be excluded. On the other hand, coexistence has been most extensively investigated in the rat medullary raphe nuclei projecting to the spinal cord, where substance P (SP) and thyrotropin-releasing hormone (TRH) immunore- activity have been detected in 5-HT-containing soma (for review, see Hokfelt et al., 1987). Prior treatment of cats with PCPA has been shown to enhance markedly the excitatory effect of the microiontophoretic application of SP and as well as that of 5-HT onto dorsal horn neurons, without altering SP content of the spinal cord (Jeftinija et al., 1986). In the rat, an increase in SP content of the midbrain raphe nuclei has been reported following short-term PCPA administration (Savard et al., 1984), although in these latter regions SP immu- nostaining may reflect terminal projections of SP-containing neurons originating from outside the raphe (Hokfelt et al., 1987; Neckers et al., 1979). Both the midbrain raphe nuclei and the hippocampus of the rat have been shown by radioimmunoassay to contain mod- erate amounts of TRH (Lighton et al., 1984). Further- more, TRH recognition sites have been reported in the hip ocampus (Funatsu et a]., 19851, and microinjection

PCPA-induced muricidal behavior in rats (Pucilowski et al., 1988). However, lesioning with 5,7-DHT markedly reduced the 5-HT, but not TRH, content of the median and dorsal raphe nuclei (Lighton et al., 1984). Given both in vivo and in vitro evidence in the rat spinal cord that TRH exerts a postsynaptic effect similar to that of 5-HT and that SP enhances 5-HT release via terminal 5-HT autoreceptor blockade (Barbeau and Be- dard, 1981; Mitchell and Fleetwood-Walker, 1981; Hansen et al., 1983), it could be speculated that an analogous situation exists in the hippocampus account- ing for the maintenance of 5-HT transmission despite severe 5-HT depletion. However, such an interpretation would ap ear premature given the paucity of evidence

projections of the midbrain raphe nuclei to the hippocampus.

Although the uptake of L3H]5-HT rat hypothalamic synaptosomes does not appear to be altered by pretreat-

of f RH into this region has been shown to inhibit

for the co Y ocalization of either neuropeptide within the

ment of rats with PCPA (Pazdernik and Tuomisto, 19831, McBride et al. (1976) have reported a marked increase in the specific activity of 5-hydroxyindole acetic acid (5-HIAA) over that of 5-HT following D,Ld-HTP administration in PCPA-pretreated rats, suggesting an increased release of 5-HT. Galzin et al. (1985), using rat hypothalamic slices, have shown that the electrically evoked release of [3H]5-HT is enhanced by the short- term treatment of rats with PCPA. Furthermore, in this latter study, the efficacy of MET in enhancing, but not that of terminal 5-HT autoreceptor agonists in decreasing, the stimulus-evoked release of L3H15-HT was reduced, suggesting that the enhanced release of L3H15- HT was due to a lesser degree of activation of the terminal 5-HT autoreceptor. Consistent with these data, in the present study, the effectiveness of MET in enhancing the efficacy of the VMT stimulation in rats treated with PCPA was reduced, whereas there was no reduction in the effectiveness of increasing the frequency of the stimulations from 1 to 5 Hz. These data suggest that the terminal 5-HT autoreceptor is acti- vated to a lesser degree in rats pretreated with PCPA and that this degree of activation can be readily increased by reducing the time interval between stimulation-trig ered action potentials. Therefore, the possibil-

R-PCPA animals derives from a lower level of activation of the 5-HT terminal autoreceptors by endogenous 5-HT as a result of neurotransmitter de letion within 5-HT

mechanism might explain, a t least partly, the normal response to afferent 5-HT pathway stimulation of most pyramidal neurons in rats treated with the low dose (250 mg/kg/day) of PCPA. Indeed, a lower level of tonic activation of 5-HT autoreceptors prior to the beginning of the stimulation would allow a relatively greater release of neurotransmitter per stimulation pulse.

Numerous methodological differences (stimulation sites; pulse duration, intensity, and frequency, pooling of the responses of CA, and CAB neurons) exist between the present study and that of Segal (19751, which ren- ders a comparison of the effects of PCPA treatment on 5-HT synaptic transmission difficult. In the latter study, the responses of pyramidal neurons to dorsal or median raphe stimulation were much more heteroge- neous than those in the present study. However, the short-latency inhibition of pyramidal neurons predomi- nantly observed by Segal(1975) with the median raphe stimulation closely resembles the effect studied here in both its temporal characteristics (latency, duration) and its resistance to PCPA treatment. In contrast, Wang and Aghajanian (1977a) reported that, in the rat, the inhibitory response of amygdaloid neurons to electrical stimulation of the dorsal raphe nucleus in vivo was reduced by 75% by a single dose of PCPA (400 mgkg). A possible explanation for the discrepancy between the effectiveness of PCPA in the latter and present study may pertain to differences in the degree of 5-HT depletion obtained between discrete regions of the rat CNS (Fleisher et al., 1979; Reader and Gauthier, 1984; Val- zelli et al., 1983).

The present observation that the 5,7-DHT pretreatment, but not the regimen of 250 mg/kg/day of PCPA, significantly reduced the efficacy of the stimulation of the ascending 5-HT pathway (Figs. 2 and 71, despite the

ity that t a e unaltered efficacy of the stimulations in the

terminals must be entertained. 8 uch a homeostatic

336 Y. CHAPUT ET AL.

fact that both produced a similar (90%) depletion of the 5-HT content in the dorsal hippocampus. It might be speculated that the PCPA treatment most likely decreases the 5-HT content per terminal, whereas the 5,7-DHT neurotoxine reduces the number of terminals but presumably not the 5-HT content of the few remaining terminals. If this is the case, the mechanism whereby the 5-HT synapse might maintain its homeo- stasis in PCPA-treated rats (i.e., a reduced activation of terminal 5-HT autorece tors) might not be operative in 5,7-DHT-treated rats. T K is would account for the differential effect of the PCPA and the 5,7-DHT pretreat- ments on the efficacy of the stimulation of the ascending 5-HT pathway.

The present results demonstrate that the efficacy of 5-HT synaptic transmission is significantly reduced following short-term administration of a high dose of PCPA in the rat. The rapid reversal of pharmacological remission by PCPA, at a dose of approximately 40 mgkg, in patients treated with various antidepressants (Shopsin et al., 1975, 19761, and the rapid mood-lowering effect of decreasing plasma tryptophan in depressed patients in pharmacological remission (Delgado et al., 1990) suggest a role for 5-HT in mediating the therapeutic effect of antidepressant drugs in major affective disorders. However, there is an apparent discrepancy between the dose of PCPA required in the present study to affect 5-HT neurotransmission in the rat and that inducing a relapse in pharmacologically remitted depressed patients. Obviously, a s ecies difference might well exist. Nonetheless, in lig R t of the enhancement of 5-HT neurotransmission by various types of antidepressant treatments in the rat (see Blier et al., 1987a; Willner, 19851, it would be most interesting to investigate the possibility that the 5-HT system is much more sensitive to the disruptive effect of PCPA following an antidepressant treatment as a result of an alteration of the properties of the 5-HT synapse.

ACKNOWLEDGMENTS This work was supported in part by the Canadian

Medical Research Council (MRC) grant MT-6444 (to C.de M.) and by a Mental Health Research Network Grant from the Fonds de la Recherche en Sante du Quebec. Y.C. is in receipt of an MRC Fellowship. We thank M. Boulay for his technical assistance. L. Caille for writing the programs, G. Filosi for the illustrations, and T. Romano for her secretarial assistance.

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Effect of short-term serotonin depletion on the efficacy of serotonin neurotransmission: Electrophysiological Studies in the Rat central Nervous System