NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO.1 41

Electrophysiologic Evidence for Desensitization of u2-Adrenoceptors on Terminals Following Long-Term Treatment with Drugs Increasing Synaptic Concentration Raymond Mongeau, M.Sc., Claude de Montigny, M.D., Ph.D., and Pierre Blier, M.D., Ph.D.

Previous results from our laboratory have indicated that or saline using subcutaneous osmotic minipumps small intravenous doses of the u2- (removed 48 hours before the experiment). No significant increase serotonin (5-HT) neurotransmission by change in the effect of the small dose of clonidine (10 attenuating the release of endogenous norepinephrine Jjglkg, IV) was found following the befloxatone, the (NE), as a result of the activation of u2-adrenergic , or the treatments. The reduction of autoreceptor on NE neurons, and that high doses of 5-HT neurotransmission by the high dose of clonidine clonidine decrease 5-HT neurotransmission by directly (400 Jjglkg, IV) was no longer present in rats treated activating u2-adrenergic heteroreceptors on 5-HT with nisoxetine or befloxatone, but was unaltered in those terminals. The aim of the present study was to assess treated with paroxetine. Furthermore, in rats pretreated whether treatments that increase the with the NE neurotoxin 6-hydroxydopamine, a long-term synaptic concentration of NE or 5-HT alter the ability of treatment with befloxatone failed to alter the reducing clonidine to modulate 5-HT neurotransmission through effect of the high dose of clonidine but abolished the these two u2-adrenoceptors. Rats were treated for 3 weeks reducing effect of the low dose of clonidine. These results with 0.75 mglkg per day of befloxatone (a reversible suggest that antidepressant drugs that increase NE inhibitor of A), 10 mglkg per day of synaptic concentration induce a desensitization of nisoxetine (a selective NE ), 10 mglkg u2-heteroreceptor on 5-HT terminals. per day of paroxetine (a selective 5-HT reuptake inhibitor) [Neuropsychopharmacology 10:41-51, 1994]

KEY WORDS: u2-Adrenergic autoreceptors; u2-Adrenergic Several studies have documented the possibility that heteroreceptors; Antidepressant drugs; Norepinephrine presynaptic u2-adrenergic autoreceptors become desen­ reuptake inhibitors; Serotonin reuptake inhibitors; sitized following long-term antidepressant treatments Monoamine oxidase inhibitors that increase the synaptic concentration of norepineph­ rine (NE) (Crews and Smith 1978; Svensson and Usdin 1978; McMillen et al. 1980; Spyraky and Fibiger 1980; From the Department of Psychiatry, Neurobiological Psychiatry Cohen et al. 1982; Finberg and Tal 1985; Lacroix et al. Unit, McGill University, Montreal, Quebec, Canada Address all correspondence to: Raymond Mongeau, M.Sc., 1991). Furthermore, the density of high-affinity state Department of Psychiatry, Neurobiological Psychiatry Unit, Mc­ u2-adrenoceptors has been shown to be increased on Gill University, 1033 Pine Avenue West, Montreal, Quebec, Canada platelets of depressed patients, a condition normalized H3A 1A1. Received June 29, 1993; revised September 17, 1993; . accepted by long-term treatment with antidepressant drugs September 26, 1993. (Garcia-Sevilla et al. 1986, 1987; Doyle et al. 1985;

© 1994 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 0893-133X/94/$7.00 42 R. Mongeau et al. NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO. 1

Takeda et al. 1989; Piletz et al. 1991). However, not all atone (Curet et al. 1992a), 10 mg/kg per day of the selec­ antidepressant treatments share the property of de­ tive 5-HT reuptake inhibitor paroxetine (Thomas et al. creasing these u2-adrenoceptors (Willner 1985). In con­ 1987), 10 mg/kg per day of the selective NE reuptake trast, several classes of antidepressant treatments have inhibitor nisoxetine (Fuller et al. 1979), or saline deliv­ been shown to alter serotonin (5-HT) neurotransmis­ ered by osmotic minipumps (ALZA, Palo Alto, CAl in­ sion (for review, see Blier et aI., 1990). serted subcutaneously. The minipumps were removed The capacity of u2-adrenergic ligands to modulate 48 hours before the experiments began to allow elimi­ the evoked release of [3H]5-HT in vitro has long been nation of the drugs. Lesions of NE neurons were per­ known to be exerted by u2-adrenoceptors located on formed under chloral hydrate anesthesia (400 mg/kg 5-HT terminals (Starke and Monte11973; Gothert and IP) by injecting 6-0HDA intracerebroventricularly (120 Huth 1980; Frankhuyzen and Mulder 1980,1982; Goth­ �g free base in 20 �l of 0.9% N aCl and 0.1% ascorbic ert et al. 1981; Maura et al. 1982). Using an in vivo elec­ acid) 1 hour after an injection of (10 mg/kg, trophysiologic paradigm, it was recently shown that IP) administered to protect the 5-HT system from the these u2-adrenergic heteroreceptors in the rat hip­ neurotoxic action of 6-0HDA. One week after this pro­ pocampus are tonically activated by endogenous NE cedure, the rats were treated with 0.75 mg/kg per day (Mongeau et al. 1993), as is the case in the human brain of befloxatone for 3 weeks. At the time of the experi­ (Galzin et al. 1992; Feuerstein et al. 1993). Small doses ments, the rats (weighing between 325 and 375 g) were (2 �g/kg and 10 �g/kg IV) of the u2- anesthetized with an injection of 400 mg/kg (IP) of chlo­ clonidine enhance 5-HT neurotransmission, an effect ral hydrate. that is abolished by a 6-hydroxydopamine (6-0HDA) pretreatment and blocked by the u2-adrenergic an­ Recording from Dorsal Hippocampus CA3 tagonist (0.1 mg/kg IV). These results indi­ Pyramidal Neurons cate that a low dose of clonidine preferentially activates u2-adrenergic autoreceptors on NE neurons, reducing Extracellular unitary recording and microiontophore­ the tonic inhibitory action of endogenous NE on U2- sis onto pyramidal neurons in the CA3 region of the adrenergic heteroreceptors on 5-HT terminals and, con­ dorsal hippocampus were conducted with five-barrelled sequently, increasing 5-HT release. However, at higher micropipettes, pulled conventionally with the tip bro­ doses (100 �g/kg and 400 �g/kg IV), clonidine decreases ken to a diameter of 9 �m to 11 �m. The central barrel, 5-HT neurotransmission through a direct activation of used for recording, was tilled with a 2 moliL NaCl so­ u2-adrenergic heteroreceptors, as this effect is not lution. The side barrels contained the following solu­ affected by NE denervation and is blocked by yohim­ tions: 5-HT creatinine sulfate (0.5 mmoliL in 200 rnrnoliL bine (1 mg/kg IV; Mongeau �t al. 1993). NaCl, pH 4), NE bitartrate (20 mmollL in 200 mmoliL In the present study, this invivo modulation of 5-HT NaCl, pH 4), chloride (20 mmol/L in 200 neurotransmission by clonidine via u2-adrenergic auto­ mmoliL NaCl, pH 4), and a 2-moliL NaCl solution used and heteroreceptors was used to investigate the effect for automatic current balancing. The 5-HT and NE so­ of long-term treatments that increase the synaptic con­ lutions were retained with a - 7 nA current between centration of NE or 5-HT. The changes in the synaptic ejections. Pyramidal neurons were identified by their concentration of the neurotransmitters were induced large amplitude (0.5 mV to 1.2 mV) and long-duration by either selective reuptake blockade or monoamine ox­ (0.8 msec to 1.2 msec) simple spikes alternating with idase A (MAO-A) inhibition. This electrophysiologic complex spike discharges (Kandel and Spencer 1961). paradigm was considered most appropriate to inves­ These characteristics readily allow the differentiation tigate the modulation of 5-HT neurotransmission by the of pyramidal neurons from interneurons. A small cur­ NE system because, as described before, it allows the rent or leak of acetylcholine (- 1 nA to 5 nA) was used in vivo assessment of the responsiveness of the U2- to activate silent or slowly discharging pyramidal neu­ adrenergic heteroreceptors on 5-HT terminals and of rons to a physiologic firing rate (8 Hz to 12 Hz), because the level of tonic inhibitory action of NEacting on them. most of these cells do not discharge spontaneously in chloral hydrate-anesthetized rats. The responsiveness to micro iontophoretic application of 5-HT and NE was METHODS evaluated from the number of spikes suppressed/nA calculated by an on-line computer with a lOa-milli­ Animals and Treatments second discrimination. Male Sprague-Dawley rats, maintained on a 12-hour/12- To evaluate the effectivenessof the 6-0HDA lesion, hour light-dark cycle with free access to food and wa­ the recovery of the fIring activIty of the pyramidal neu­ ter, were treated over a course of 3 weeks with 0.75 rons following the microiontophoretic application of NE mg/kg per day of the reversible MAO-A inhibitor beflox- was assessed by determining the recovery time 50 NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO.1 Desensitization of u2-Adrenergic Heteroreceptors 43

(RTso). This parameter is defined by the time (in sec­ Drugs onds) required for the firingrate to recover by 50% from The following drugs were used: befloxatone (Dela­ the termination of the microiontophoretic application lande, Rueil-Malmaison, France); paroxetine (Smith­ (de Montigny et al. 1980). Kline Beecham, Harlow, England); nisoxetine and fluoxetine (Eli Lilly, Indianapolis, IN); clonidine HCl, Stimulation of the 5-HT Pathway 5-HT creatinine sulfate, NE bitartrate, 6-0HDA HCl, and acetylcholine chloride (Sigma Chemical, St. Louis, To activate the 5-HT projections to the dorsal hip­ MO); and chloral hydrate (American Chemicals, Mon­ pocampus, a bipolar electrode (NE-I00; David Kopf, treal, Quebec, Canada). Tujunga, CA) was implanted on the midline with a 100 backward angle in the ventromedial tegmentum, 1 mm anterior to lambda, and 8.3 mm below the cortical sur­ Statistical Analyses face. Two hundreds square pulses of 0.5 milliseconds All results are expressed as means plus/minus standard were delivered by a stimulator (Model 58800; Grass, error of the mean. The statistical significance of the Quincy, MA) at a frequency of 1 Hz and at an intensity difference between the effectsof the stimulation of the of 300 J.l.A. The stimulation pulses and the firing activ­ 5-HT pathway before and after the administration of ity of the neuron recorded were fed to a computer a drug in the same rat was assessed using the paired equipped with a Tecmar interface. Peristimulus time Student's t-test. Possible differencesin the magnitude histograms were generated to determine the duration of the effects of clonidine between saline- and drug­ of suppression of firing, measured to absolute silence treated rats were tested for statistical significance by value (SIL, in milliseconds; Chaput et al. 1986). This covariance analysis. Differences in the efficacy of the parameter is obtained by dividing the total number of stimulation of the 5-HT pathway and neuronal respon­ events suppressed with the stimulation by the mean siveness to microiontophoretic application of NE and frequency of firing of the neuron recorded. It thus 5-HT between saline- and drug-treated rats were as­ represents an estimate of the duration of the suppres­ sessed for statistical significanceusing the unpaired Stu­ sion of firing corrected for the mean prestimulation dent's t-test. frringfrequency. Several lines of evidence indicate that the effectof the electrical stimulation of the ascending 5-HT pathway is due to the release of 5-HT into the syn­ RESULTS aptic cleft. First, it is virtually abolished by a pretreat­ Effect of Long-Term MAO Inhibition on the ment with the 5-HT neurotoxin 5,7-dihydroxytrypta­ Modulation of 5-HT Neurotransmission mine (Blier and de Montigny 1983, 1985). Second, it is by Clonidine blocked by the acute intravenous injection of the 5-HT1A antagonist BMY 7378 (Yocca et al. The suppressant effect of the electrical stimulation of 1987; Chaput and de Montigny 1988). Third, it is en­ the ascending 5-HT pathway on the firing rate of CA3 hanced by terminal 5-HT autoreceptor blockade pyramidal neurons of the dorsal hippocampus was as­ (Chaput et al. 1986). Fourth, it is reduced by terminal sessed following long-term drug treatments. The peri­ 5-HT autoreceptor activation (Chaput and de Montigny stimulus time histograms in Figure lA show that 10 J.l.g/ 1988). kg IV of clonidine enhanced, while 400 J.l.g/kg IV re­ In most neurons, following the suppression of firing duced, the duration of suppression of firing in the same induced by the electrical stimulation there is a transient neuron of a saline-treated rat. Following the adminis­ increase of the probability of firing, the exact nature of tration of 10 J.l.g/kg and 400 J.l.g/kg IV of clonidine, the which is presently unknown. However, this transient mean duration of suppression of firing of the saline­ increase was shown to be unaffectedby 5-HT reuptake treated group (Fig. IB) was increased by 37% ± 8% and inhibitorsor MAO inhibitors (Blier et al. 1988). The rel­ decreased by 25% ± 7%, respectively. These incremen­ ative activation was calculated by dividing the total tal and decremental effects of clonidine were statisti­ number of supplementary events by the mean pres tim­ cally significant at p < .01 and p < .001, respectively, ulation firing frequency. using the paired Student's t-test. In contrast, the facilita­ The effectof stimulating the ascending 5-HT fibers tory effect on the firing rate of pyramidal neurons in­ was determined while recording from the same neu­ duced by the electrical stimulation was not significantly ron before and after the successive intravenous injec­ changed by either dose of clonidine (Table 1). tions of 10 J.l.g/kg and 400 J.l.g/kg of clonidine. The time To induce a sustained increase in the synaptic con­ elapsed between the control stimulation periods and centration of 5-HT and NE, MAO-A was blocked over those following the administration of clonidine was ap­ a 3-week period with 0.75 mg/kg per day of befloxa­ proximately 15 minutes. tone. The minipumps were removed 48 hours before 44 R. Mongeau et al. NEUROPSYCHOPHARMACOLOGY 1994- VOL. 10, NO.1

A B

Prior to 24

16 70

8

o 60 *

Following clonidine (10 j.l.g/kg) 16 - 50 :z m ::'2 a: 8 w UJ c... 40 , o Cf) :z� w +1 ** > w (/) Following clonidine (400 j.l.g/kg) 30 ---E 32 ....J 24 C/) 20

16 10 8

o 11 0 -5 0 o 50 100 150 CTL CLO CLO TIME (ms) 10 Ilg/kg 400 Ilg/kg

Figure 1. Effectof systemic injections of c10nidine on the efficacyof the electrical stimulation of the ascending 5-HT path­ way in suppressing the fIringactivity of CA3 pyramidal neurons of hippocampus in the saline-treated group. A: Represen­ tative peristimulus time histogr1.ms illustrating the effects of c10nidine (10 Jlg/kg and 400 Jlg/kg IV) in a single experiment. Each peristimulus time histogram was constructed from 200 pulses of 0.5 msec delivered at 1 Hz at time 0 with an intensity of 300 JlA. Bin width is 2 msec. B: Histograms showing the effectsof the high and the low dose of c10nidine in saline-treated rats. The number of neurons tested is given at the bottom of the open column. * p < .01, ** P < .001, using a two-tailed paired Student's t-test, comparing prior to and following c10nidine administration.

Table 1. Effect of Long-Term Drug Treatments with Befloxatone, Nisoxetine, or Paroxetine on the Late Excitatory Effect of the Firing Activity of Dorsal Hippocampus Pyramidal Neurons Induced by the Electrical Stimulation Before and After the Intravenous Administration of Clonidinea

After Clonidine Before No. of Treatmentb Clonidinec 10 Jlg/kg 400 Jlg/kg Neurons

Saline 102 ± 25 97 ± 23 140 ± 25 11 Befloxatone 89 ± 22 117 ± 15 167 ± 61 9 6-0HDAd + befloxatone 135 ± 37 171 ± 34 162 ± 42 6 Nisoxetine 130 ± 54 131 ± 35 155 ± 42 9 Paroxetine 172 ± 69 134 ± 37 123 ± 36 8

a One neuron per rat was studied before and after the administration of clonidine. b Befloxatone: 0.75 mg/kg per day; nisoxetine and paroxetine: 10 mg/kg per day. e Data are expressed as the relative activation in milliseconds ± the standard error of the mean. d One-hundred twenty micrograms free base of 6-0HDA was injected intracerebroventricularly 1 week before the initiation of the befloxatone treatment. NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO. 1 Desensitization of u2-Adrenergic Heteroreceptors 45

A B

Prior to

16 70 * 8

o 60

-

z Following clonidine (10 J..Lg/kg) � 50 iii a: 24 UJ w n.. (J) � 16 40 z +1 w (j) T 8 i:ij E ...... " 30 o ....J (J)

Following clonidine (400 J..Lg/kg) 20

16 10 8

9 o 0 -50 o 50 100 150 CTL CLO CLO TIME (ms) 10 Jl�g 400 Jl�kg

Figure 2. Effect of systemic injections of clonidine on the efficacyof the electrical stimulation of the ascending 5-HT path­ way after a long-term treatment with 0.75 mg/kg per day of befloxatone. The minipumps containing the drug were removed 48 hours before the experiment began. A: Representative peristimulus time histograms illustrating the effects of clonidine (10 Ilg/kgand 400 Ilg/kg IV) in a single experiment. 8: Histograms showing the effectsof the high and the low dose of cloni­ dine in befloxatone-treated rats. The number of neurons tested is given at the bottom of the open column. p < .01, using a two-tailed paired Student's t-test, comparing prior to and following clonidine administration.

the experiments began to regain full enzymatic activity accompanied by any change in the responsiveness of (Curet et al. 1992a). The results in Figure 2A are repre­ pyramidal neurons of the CA3 region of hippocampus sentative peristimulus time histograms of the effectof to microiontophoretic applications of 5-HT and NE.The c10nidine in befloxatone-treated rats. The small dose of suppressant effectsof the microiontophoretic applica­ c10nidine (10 J..Lg/kg IV) produced a 72% ± 21% increase tions of 5-HT and NE on the nring rate of the pyrami­ of the duration of the suppression of frring in the dal cells after the befloxatone treatment (5-HT: 322 ± befloxatone-treated group (Fig. 2B). Although the am­ 37 spikes/nA, n = 13; NE: 407 ± 43 spikes/nA; n = 13) plitude of the effect of the small dose of clonidine ap­ were not different from those of the saline-treated group

peared greater than that of the saline group (37% ± 8%; (5-HT: 376 ± 62 spikes/nA, n = 11; NE: 400 ± 71 Fig. IB), the difference between the two groups did not spikes/nA, n = 11). reach statistical signifIcanceusing covariance analysis. The assumption that the increased synaptic con­ Thesubsequent administration of the high dose of cloni­ centration of NE, but not that of 5-HT, is responsible dine (400 J..Lg/kg IV) decreased the duration of suppres­ for the decrease in the responsiveness of the Q2-adreno­ sion of bring (Fig. 2B); however, unlike the controls (Fig. ceptors mediating the effect of the high dose of cloni­ IB), it was not reduced below the initial value. This de­ dine was assessed by investigating the effectof the same creased responsiveness of the Q2-adrenoceptors medi­ befloxatone treatment in NE-denervatedrats. Intracere­ ating the effect of the high dose of clonidine was not broventricular injections of the neurotoxin 6-0HDA (1 46 R. Mongeau et al. NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO.1

A B

70 I * 70 60

60 *

50

50 � ui cri 40 :e 40 +1 0 1-10 en a: ..s 30 30 -I Ci5

20 20

10 10

6 0 0 SAL 6-0HDA CTL CLO CLO 10 Ilg/kg 400 Ilglkg Figure 3. Effect of long-term treatment with 0.75 mg/kg per day of befloxatone in NE-denervated rats. The intracerebroven­ tricular injection of 120 Jlg of 6-0HDA was given 1 week before the minipump containing the drug was installed. The minipumps were removed 48 hours before the experiment began. A: Neuronal recovery from the microiontophoretic application of NE in saline (SAL) and 6-0HDA + befloxatone-pretreated rats. The RTso value is the time required for the neuron to re­ cover its fIring activity by 50% from the end of the application. The number of neurons tested is given at the bottom of

each column. Data are expressed as mean ± SEM. * P < .001, two-tailed Student's t-test comparing control and 6-0HDA­ pretreated rats. B: Histograms showing the effectsof systemic injections of clonidine on the efficacyof the electrical stimula­

tion of the ascending 5-HT pathway after a long-term treatment with befloxatone in six 6-0HDA-pretreated rats. * p < .01, using a two-tailed paired Student's t-test, comparing prior to and following clonidine administration.

hour after a pretreatment with the 5-HT reuptake in­ IV) was still present and was even of a greater ampli­ hibitor fluoxetine) were performed 1 week before initi­ tude than that in the saline group (saline: 25% ± 5%,

ating the befloxatone treatment. The effectiveness of n = 11; 6-0HDA: 39% ± 9%, n = 6; P = .06, using covar­ the lesions was verifIed by assessing the RTso of the iance analysis). fIring rate of CA3 pyramidal neurons following the The efficacy of the stimulation of the 5-HT pathway microiontophoretic application of NE. In keeping with in the saline group was compared to those in the drug­ the notion that intact NE fIbersare required for the NE treated groups using the unpaired Student's t test. The reuptake process to allow a prompt recovery of the fIring duration of suppression of fIring in the 6-0HDA plus activity of these neurons (de Montigny et al. 1980), the befloxatone group was increased by 71% (SIL values:

marked increased (327%) in the RTso of 6-0HDA­ saline 38 msec ± 3 msec, n = 11; 6-0HDA + befloxa­

treated rats (Fig. 3A) confIrmed an adequate lesioning tone 65 msec ± 7 msec, n = 6; P < .01; Figs. 1 and 3). of the NE terminals (except in two rats that were ex­ However, the befloxatone treatment did not by itself cluded from statistical analyses). In these conditions, change the efficacy of the stimulation in suppressing the enhancing effectof the small dose of clonidine (10 the fIringactivity of the pyramidal neurons (Figs. 1 and J.lg/kgIV) was completely abolished, as previously ob­ 2). It is noteworthy that in the two rats pretreated with served (Mongeau et al. 1993). On the other hand, the 6-0HDA plus befloxatone for which the NE denerva­ decreasing effectof the high dose of clonidine (400 J.lg/kg tion failed (as indicated by the unchanged RTso), the NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO. 1 Desensitization of u2-Adrenergic Heteroreceptors 47

increase in 5-HT transmission was not observed, and A the effectsof clonidine were similar to those observed PAROXETINE for the befloxatone group (data not shown). Finally, 70 none of the above treatments signiflcantlychanged the

late excitatory effect on the flring rate induced by the * electrical stimulation (Table 1). 60

;-50 Effect of Long-Term Monoamine Uptake Inhibition on the Modulation of 5-HT Neurotransmission by ill 1 Clonidine uj 40 ** +1 For two reasons, it was deemed crucial to test whether monoamine reuptake inhibitors could also affect the u2-adrenoceptors that modulate 5-HT transmission. First, selective 5-HT reuptake inhibitors have long been known to have antidepressant efficacy, and, second, 20 5-HT and NE reuptake inhibitors have been shown to increase the synaptic concentration of 5-HT and NE, respectively (L'Heureux et al. 1986; Bel and Artigas 10 1992; Curet et al. 1992b). The effect of a sustained in­ crease in the synaptic concentration of either NEor 5-HT 7 was investigated using speciftcreuptake inhibitors. Fol­ o CTL CLO CLO lowing long-term treatment with the selective 5-HT re­ 10 ).lg/kg 400 ).lg/kg, uptake inhibitor paroxetine (10 mg/kg per day for 21 B days), the effectsof the low and the high dose of cloni­ NISOXETINE dine on 5-HT transmission were unchanged. After the paroxetine treatment, the small dose of clonidine (10 70 J,lg/kg IV) increased the efficacy of the stimulation of ** the 5-HT pathway by 29% ± 9% whereas the high dose 60 (400 J,lg/kg IV) decreased it by 25% ± 7% (Fig. 4A). In contrast, the 21-day treatment with the selective NE reuptake inhibitor nisoxetine (10 mg/kg per day) 50 � produced effects similar to those obtained in the w befloxatone-treated rats. The small dose of clonidine uj I 40 produced a 30% ± 8% increase (Fig. 4B), which was +1 similar to that of the saline group (Fig. 18). The subse­ CIl E quent administration of the high dose of clonidine de­ - 30 creased the duration of suppression of flring; however, -l it was not reduced below the initial value (Fig. 4B). Fur­ (f) thermore, as observed in the befloxatone group (Fig. 20 2B), there was no change in the responsiveness of py­ ramidal neurons of the CA3 region of hippocampus to microiontophoretic applications of 5-HT and NE in the 10 nisoxetine-treated rats. The suppressant effects of the micro iontophoretic applications of 5-HT and NE on the 9 frring rate of the pyramidal neurons after the nisoxe­ o CTL CLO CLO tine treatment (5-HT: 332 ± 66 spikes/nA, n = 11; NE: 10 ).lg/kg 400 f,lg/kg 378 ± 71 spikes/nA, n = 11) were not different from those of the saline-treated group (5-HT: 376 ± 62 Figure 4. Histograms showing the effectof systemic injec­ spikes/nA, n = 11; NE: 400 ± 71 spikes/nA, n = 11). tions of clonidine on the efficacyof the electrical stimulation The paroxetine and nisoxetine treatments did not of the ascending 5-HT pathway after a long-term treatment signiftcantIy modify the efficacy of the stimulation of with 10 mg/kg per day of paroxetine (A) and 10 mg/kg per the 5-HT pathway in suppressing the flringactivity of day of nisoxetine (B). The number of rats tested is given in­ side the columns. * p < .05, ** P < .01 using a two-tailed paired pyramidal neurons of the dorsal hippocampus (Fig. 4). Student's t-test, comparing prior to and following clonidine In addition, the late excitatory effect ofthe stimulation administration. 48 R. Mongeau et al. NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO. 1

observed on these same neurons was not changed by (Lacroix et al. 1991). However, despite an these treatments (Table 1). attenuated responsiveness of the somatodendritic NE auto receptors of neurons to clonidine following this treatment, a 10-J.1g/kg IV dose of cloni­ dine still substantially decreased (85% instead of 100%) DISCUSSION the fIringrate of these neurons. It can thus be concluded The present electrophysiologic data show that long­ that the desensitization of the (12-adrenergic autorecep­ term treatment with pharmacologic agents that increase tors would not hinder the enhancement of 5-HT neu­ the synaptic concentration of NE, but not that of 5-HT, rotransmission that results from a decrease in the syn­ decrease the inhibitory action of a high dose of cloni­ aptic concentration of NE when these autoreceptors are dine (400J.1g/kg IV) on 5-HT neurotransmissionand that activated by the 10-J.1g/kgIV dose of clonidine. In fact, the enhancing effectof a low dose of clonidine (10 J.1g/kg a desensitization of (12-adrenergic auto receptors could IV) is not affected by such treatments (Figs. 2, and 4B). even increase the inhibitory tone of NE on 5-HT neu­ These changes observed in the befloxatone and the rotransmission, because the synaptic concentration of nisoxetine groups are most likely related to the sus­ NE is normally expected to be increased when the nega­ tained increase in NE concentration in the synapse, be­ tive feedback autoregulation is decreased. cause similar changes were not produced either by long­ It is also interesting that the high dose of clonidine term 5-HT reuptake blockade or by long-term MAO-A in both nisoxetine- and befloxatone-treated rats brought inhibition in NE-denervated rats (Figs. 3B and 4B). None back the effectivenessof the stimulation to initial value of the long-term treatments produced any signifIcant (Figs. 2B and 4B), suggesting that these receptors still changes in the enhancing effectof the low dose of cloni­ possess residual capacity to modulate 5-HT release af­ dine (Figs. 2 and 4). However, the lesion of the NE neu­ ter the befloxatone or the nisoxetine treatment. The rons abolished, as expected, this enhancing effect of tonic inhibitory action of endogenous NE on the (12- clonidine (Fig. 3B). adrenergic heteroreceptors at baseline was probably In a previous study, the incremental and decremen­ maximal because the high dose of clonidine merely re­ tal effects of clonidine have been fully characterized established the initial duration of suppression of fIring using the present electrophysiologic paradigm (Mon­ (Figs. 2B and 4B). geau et al. 1993). On the one hand, it was concluded These observations lead us to the following in­ that the reducing effect of the high dose of clonidine terpretation: normally 5-HT neurotransmission is de­ is most likely mediated by the activation of (12-adrener­ creased in the presence of an increased NE output. Af­ gic heteroreceptors on 5-HT terminals, which results ter long-term exposure to a high concentration of in a decrease in the amount of 5-HT released per action synaptic NE, there is a desensitization of (12-adrenergic potential. On the other hand, low doses of clonidine heteroreceptors. Although the paradigm used in the enhance the suppression of fIring by preferentially ac­ present study does not provide clues about the cellular tivating (12-adrenergic autoreceptors, thereby reducing mechanisms involved, it might result from a decrease the concentration of endogenous NE tonically activat­ in the density of the receptors, a change in the coup­ ing (12-adrenergic heteroreceptors on 5-HT terminals ling of the receptors with their G proteins, or an altera­ (Mongeau et al. 1993). Thus, the present results sug­ tion in second messenger function. This desensitiza­ gest that although the sensitivity of (12-adrenergichet­ tion would hinder the tonic inhibitory action of the NE eroreceptors is reduced by long-term treatment with system on 5-HT neurotransmission, more so in the con­ nisoxetine and befloxatone, an inhibitory action of en­ dition of a high NE output. dogenous NE on 5-HT neurotransmission still appears No change in the efficacyof 5-HT neurotransrnission to be present. was observed withlong-term treatmentwith befloxatone, The magnitude of the effect of the small dose of paroxetine, or nisoxetine. However, in the 6-0HDA­ clonidine on 5-HT neurotransmission does not provide pretreated rats, the long-term befloxatone treatment a direct estimate of the responsiveness of the (12- signifIcantly increased the duration of suppression of adrenergic autoreceptors, because its magnitude is also fIring. This increase most likely resulted from the deple­ determined by the level of activation of (12-adrenergic tion of the endogenous NE that would normally exert heteroreceptors by endogenous NE. Thus, the un­ a tonic inhibitory action on 5-HT neurotransmission altered effect of the low dose of clonidine after these through (12-adrenergicheteroreceptors. Finally, the in­ treatments is not an indication that the (12-adrenergic crease in the magnitude of the effect of the high dose autoreceptors on the cell body and terminalsof NE neu­ of clonidine in these rats was expected and is consis­ rons are normosensitive. Previous investigations per­ tent with our previous study (Mongeau et al. 1993). formed in our laboratory have shown that terminal (12- It was most likely related to a supersensitivity of the adrenergic autoreceptors are desensitized following a (12-adrenergic heteroreceptors resulting from the NE long-term treatment with the NE reuptake inhibitor depletion. NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO. 1 Desensitization of u2-Adrenergic Heteroreceptors 49

To our knowledge, no study has directly assessed the concentration-effect curve to the right of the U2- the modulation of 5-HT neurotransmission by u2-adre­ adrenergic agonist UK 14.304 on electrically induced nergic ligands following antidepressant treatments in release of [3H]5-HT in guinea pig slices an in vivo paradigm. Nevertheless, this has been pre­ (Blier and Bouchard 1992; Blier et al. 1993). Hence, these viously investigated in in vitro studies. Some have failed results suggest that u2-adrenergic heteroreceptors are to detect a reduction in the efficacy of u2-adrenergic endowed with the capacity to become desensitized af­ heteroreceptors function following MAO inhibition of ter a sustained increase in NE synaptic concentration. NE reuptake blockade. GroB et al. (1987) reported no Although the antidepressant property of befloxa­ change in the potency of clonidine to inhibit the elec­ tone has not yet been documented in humans, all po­ trically evoked overflow of [3H]5-HT in preloaded rat tent MAO-A inhibitors, specific or not, that bind irre­ brain cerebral cortex slices when superfusing through­ versibly or not to the (clorgyline, , out the experiment 1 �M of the NE reuptake inhibitor , , , and tol­ . This result suggested that short-term ex­ oxatone) increase 5-HT and NE synaptic concentration posure to an elevated synaptic concentration of NEdoes and are effective in major . Furthermore, not desensitize u2-adrenergic heteroreceptors on 5-HT minalcipran which, like nisoxetine, selectively blocks terminals. However, long-term NE reuptake blockade the reuptake of NE (Blier et al. 1993), has been reported with desipramine or long-term MAO inhibition with to be an effective antidepressant in humans (Macher MDL 72394 or has not been reported to et al. 1989; von Frenckell et al. 1990). It is thus possible change the ability of u2-adrenergic heteroreceptors to that the desensitization of u2-adrenergic heterorecep­ reduce the electrically induced release of [3H]5-HT in tors on 5-HT terminals contributes to the therapeutic rat cortical and hippocampal slices (Schlicker et al. 1982; action of these drugs. Schoffelmeerand Mulder 1982; Palfreyman et al. 1986). Finally, another study using rat brain synaptosomes re­ vealed that the attenuation of K + -stimulated release of PH]5-HT by clonidine was diminished following a ACKNOWLEDGMENTS long-term treatment with the irreversible MAO inhibi­ tor clorgyline but not by desipramine (Ellison and This work was supported in part by the Medical Research Campbell 1986). This effectof clorgyline was, however, Council of Canada (MRC) Grants MT-6144 and 11014 and the Fonds de la Recherche en Sante du Quebec (FRSQ). R. Mon­ attributable to a competition between clonidine and the geau is the recipient of a studentship from FRSQ and P. Blier increased NEin the biophase. 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