Brain Research, 92 (1975) 291-306 291 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

INHIBITION OF BOTH N O R A D R E N E R G I C AND SEROTONERGIC NEURONS IN BRAIN BY THE a -ADRENERGIC AGONIST CLONIDINE

T. H. SVENSSON*, B. S. BUNNEY AND G. K. AGHAJANIAN

Departments of Psychiatry and Pharmacology, Yale University School of Medicine and Connecticut Mental Health Center, New Haven, Conn. 06508 (U.S.A.)

(Accepted February 15th, 1975)

SUMMARY

By means of single unit recording techniques it was found that a small systemic- ally administered (intravenous) dose of the a-adrenergic agonist clonidine inhibited the spontaneous firing of brain norepinephrine (NE)-containing neurons in the locus coeruleus. In addition, the NE neurons were consistently inhibited by the direct (microiontophoretic) application of minute amounts of NE or clonidine. Intravenous clonidine also inhibited the firing of the great majority of 5-hydroxytryptamine (5- HT) neurons in the midbrain dorsal raphe nucleus. However, this action does not appear to be a direct one since clonidine (and NE) had relatively weak or variable effects when applied microiontophoretically to raphe neurons. The clonidine-induced depression of raphe firing may be secondary to an impairment in adrenergic trans- mission since (1) the depression could be reversed by the NE-releasing agents D- and L-amphetamine, (2) high doses of clonidine itself (which have been reported to have postsynaptic a-agonistic activity) reversed the depression produced by a low dose of clonidine and (3) prior destruction of NE neurons by 6-hydroxydopamine (7-12 days) rendered raphe neurons insensitive to the depressant effect of i.v. cloni- dine. Dopaminergic (substantia nigra, zona compacta) neurons did not respond to either low or high doses of clonidine. These results are consistent with previous data showing that clonidine decreases NE and 5-HT but not dopamine turnover. We conclude that systemically administered clonidine inhibits the firing of brain NE neurons by acting directly upon adrenergic receptors located on or near the soma of these neurons but that the concomitant inhibition of 5-HT neurons is an indirect effect (possibly secondary to an impairment in noradrenergic transmission).

* Present address: Department of Pharmacology, University of GSteborg, Fack S-400 33, G~Steborg 33, Sweden.

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I N T R O D U C T I O N

Clonidine is an antihypertensive agent which can stimulate peripheral a- adrenergic receptors 14,2~,4°. However, it appears that the various cardiovascular and

behavioral effects of clonidine are mediated primarily through an action within the central nervous system (e.g., on central a-receptors)15-17,36,42,46,48,51,67, 69 71,73,85,

ss. Studies on central transmitter substances show that low doses of clonidine (e.g., 30 #g/kg) diminish norepinephrine (NE) and serotonin (5-HT) turnover; this is indicated by a deceleration in the disappearance of both amines in the rat brain after inhibition of synthesis s,63. Moreover, the drug slows the rate of formation of NE

from labeled tyrosine 6z and reduces the brain level of the N E metabolite 3-methoxy- 4-hydroxyphenylglycol (MOPEG) is. Furthermore, at a dose of 0.5 mg/kg, clonidine

reduces the concentration of the 5-HT metabolite 5-hydroxy-3-indoleacetic acid (5-HIAA) in rat brain 68. The latter effect was not found at a higher dose level, 1 mg/

kgSS. The above biochemical findings with clonidine are all compatible with the

hypothesis that clonidine, at low doses, reduces the firing rate of both brain NE and 5-HT neurons since (1) variations in the concentrations of MOPEG in the rat brain are directly related to changes in physiological activity of brain NE neurons ~3, sg, (2) variations in the firing rate of 5-HT neurons in the raphe nucleus are correlated with variations in brain 5-HT turnover7, 55,7~ and (3) changes of rates of turnover or synthesis of monoamines in general have been found to reflect changes in the firing rate of the neurons containing monoamines3,ZL To test the above hypothesis directly we investigated whether clonidine has any effect on the firing rate of brain NE neurons in the locus coeruleus 27 and 5-HT neurons in the dorsal raphe nucleus 1°,27

by means of single cell recording techniques. We used dopamine (DA)-containing neurons in the substantia nigra (zona compacta) as control cells since previous data indicate that clonidine has little or no effect on brain DA turnover s. Cells in the reticular formation were also used as control cells. Clonidine was administered both intravenously (i.v.) and directly by means of microiontophoresis. In order to further analyze whether the effect of clonidine on raphe neurons might be direct or indirect, NE neurons were destroyed by intraventricular administration of 6-hydroxydopa- mine s7 and the activity of the raphe neurons was subsequently recorded during clonidine administration.

METHODS

Single unit recording and microiontophores& Male albino (Sprague-Dawley) rats weighing 210-290 g were used. The rats

were anesthetized with chloral hydrate (400 mg/kg, i.p.); additional injections were given as needed. The animals were mounted in a stereotaxic apparatus and a 3 mm burr hole was drilled at varying coordinates as follows: (1) locus coeruleus, lateral - - I . 1 mm, posterior --1.1 mm; (2) dorsal raphe nucleus, anterior--0.35 mm, lateral - -0 .0 ram; (3) substantia nigra, anterior - - 2.9 ram, lateral - -2 .2 mm; (4) reticular

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formation, anterior --0.35 mm, lateral - - 1.0--2.0 mm, (all extrapolated from K6nig and Klippe152). In most animals, the extracellular recording technique was similar to one previously described 2°. Briefly, micropipettes with a tip diameter of 1 #m were used. They were filled with 2 M NaC1 saturated with fast green. In vitro impe- dances were typically 3-7 Mf~, measured at 60 Hz. The electrode signals were passed through a high input-impedance amplifier into an oscilloscope, and finally into an electronic rate meter triggered by individual neuronal spikes. Integrated firing rate was graphically recorded as consecutive 10 sec samples from the analog output of the counter. The signals from the oscilloscope also drove an audiomonitor.

In the microiontophoretic studies, 5-barrel micropipettes were used according to techniques previously described 38. Briefly, the tips of the micropipettes were broken back to 4-5/zm. Impedances were typically 4-6 Mf~ in the recording barrel. The drug solutions were directly injected into barrels which contained a few strands of fiber glass to allow rapid filling of the tips by capillary action sl. All ejection periods were 30 sec. The central (recording) barrel was filled with 2 M NaCI saturated with fast green. One barrel (the 'balance' channel 65) was filled with 4 M NaCI. The other 3 contained 0.01 M clonidine hydrochloride in 0.1 M NaCI, pH 4.0 (Boeh- ringer, Ingelheim, G. F. R.), 0.001 M D-lysergic acid diethylamide (LSD) bitartrate in 0.1 M NaCI, pH 4.0 (cf. Haigler and Aghajanian as) and 0.1 M NE bitartrate, pH 4.0. Current balance was kept within d: 2.0 nA. A retaining current of 10 nA was maintained between periods of ejection. In the microiontophoretic experiments electrode signals were recorded as described above except that 1 sec sample intervals were chosen. The body temperature of the animals was kept at 36--37 °C. Intravenous injections of drugs, the doses of which are expressed in terms of their salt, were administered via a tail vein.

Under the above conditions the transport number (an indicator of the efficiency of iontophoretic ejection) should be considerably larger for NE than for clonidine since the clonidine (0.01 M) was ionically diluted 10-fold by NaC1 (0.1 M). The ionic dilution of the clonidine in the iontophoretic studies was necessitated by the prelimi- nary observation of an extreme sensitivity of noradrenergic neurons to the direct application of this drug.

Identification of cells The recording sites were marked at the end of each experiment by passing a

20/zA cathodal current through the recording barrel for 5 min thereby depositing a green spot (,~ 50/~m) at the tip of the electrode 82. The rats were then perfused with 10~ buffered formalin and serial frozen sections (50 /zm) of the brain were cut, mounted and stained with neutral red. The localization and neurophysiological characteristics of the different cell groups were similar to those previously described for noradrenergicaS, 54, serotonergic4, 5 and dopaminergic (zona compacta, sub- stantia nigra) neurons 2°. An additional criterion used for identification of 5-HT neurons in the dorsal raphe nucleus was that of inhibition by a small ( ~ 20/tg/kg, i.v.) dose of LSD 4,~.

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Intraventrieular injections The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted

in a stereotaxic apparatus. A 3 mm burr hole was made at the following coordinates: anterior --7.9 mm, lateral --1.3 mm (extrapolated from K6nig and Klippe152). 6-Hydroxydopamine dihydrobromide (6-OHDA, Regis) in a concentration of 5 #g/ ml (free base) in distilled water containing ascorbic acid (0.1 mg/ml) was injected at a rate of 5/A/min (total amount 100 #g) through a 50 #1 Hamilton syringe (Whittier, Calif.) which had been lowered to a depth of 3.5-4.0 mm from skull surface. Only freshly made solutions were used. An aspiration test (i.e., slight rise in fluid meniscus in syringe) was always performed prior to the injection to confirm ventricular loca- tion. The lesioned animals were used for the clonidine experiments 7-12 days later. The extent of the 6-OHDA induced destruction of NE neurons, especially the ter- minals surrounding the raphe nuclei, was checked by histoftuorescence or biochemical methods.

Histofluorescenee Tissue slabs from the pons and midbrain were cut and quickly frozen in liquid

propane-propylene (cooled by liquid N2) in preparation for fluorescence microscopy according to a slight modification I of the Falck-Hillarp formaldehyde condensation technique 29.

Biochemical determination of NE and 5-HT Rats were decapitated and their brains were rapidly removed and placed on a

block of dry ice. The brains were weighed and homogenized in 15 ~ trichloracetic acid. For the NA assay, an alumina column separation followed by a potassium ferricyanide oxidation procedure, was performed according to a previously described method.12,64.

For 5-HT determination, brains were homogenized in 0.1 N HCI and the subsequent assay followed that of Bogdanski et al. 13. All data were corrected for recovery.

RESULTS

Effects of intravenous clonidine on the firing rate of NA and 5-HT neurons Pilot experiments showed that clonidine at a very low dose, rapidly inhibited

ceils in the locus coeruleus. Thus, in the subsequent experiments the objective was to establish the dose required to bring the firing of the cells to a full stop for a short period of time (about 1.5 min). Under the experimental conditions employed, single cells in the locus coeruleus (n = 10) were firing at a relatively slow rate (14/sec) and typically responded to noxious stimuli (e.g., toe-pinch) by a transient increase in firing rate (el Korf et al.54). Clonidine was administered in small incremental i.v. doses at intervals of about 1 min until a total inhibition of firing was just attained. Using this approach a cumulative dose of 6.5 :L 0.5 #g/kg (mean ± S.E.M.) con- sistently and rapidly inhibited the firing of these cells for a short time (mean 103 sec;

295

v

Z

\

locus coeruleus

CC

oil LSD

1 IOMIN

, i raphe

Fig. 1. Inhibition by clonidine (C) of a noradrenergic cell in the locus coeruleus (top trace) and a serotonergic cell in the dorsal raphe nucleus (bottom trace). Clonidine was given intravenously (i.v.). The locus cell was inhibited by 4 + 2/~g/kg, and the raphe neuron by 4 + 4/~g/kg. In contrast to cells in the locus coeruleus (see Results) the raphe neuron was also inhibited by LSD, 13 #g/kg, i.v.

Fig. 1, upper panel). When higher doses were given (up to 2 mg/kg) the cells were also inhibited but for a longer time, indicating the effect to be dose-dependent. In some additional recordings from locus cells (n = 4), LSD in a dose sufficient to inhibit cells in the dorsal raphe nucleus (20/~g/kg) 4,5 was administered before clonidine. LSD caused either a slight increase in or had no effect on the firing rate of locus cells; subsequently the inhibitory response to clonidine was unaffected.

Cells in the dorsal raphe nucleus (n = 32) had a regular rhythm and slow rate (0.5-2 spikes/sec) which is characteristic of histochemically identified serotonergic neurons s. After the intravenous administration of a low dose of clonidine (10.9 q- 0.9 ~g, mean 4- S.E.M.) the raphe cells in most cases (27/32) were inhibited for a short period of time (mean 106 sec; Fig. 1, lower panel). The dose necessary to stop these raphe units thus was significantly higher (P < 0.005) than that required to stop the firing of locus coeruleus cells for a similar period. By histological localization, the raphe units were found throughout the dorsal raphe nucleus with a majority near the center of the nucleus. Several raphe cells were encountered (n ---- 5) which did not respond at all to clonidine (16-80 #g/kg).

The effect of i.v. clonidine on the firing rate of neurons in certain other areas was also determined. Unidentified cells in the reticular formation (n ---- 7) and dopa- mine-containing cells of the zona compacta, substantia nigra (n = 8; area A9, DahlstrSm and Fuxe 2v) were used. The dopaminergic cells of group A9, should, because of the structural similarity between DA and NE, provide additional evidence pertaining to the specificity of the effects of clonidine. In the substantia nigra, the

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TABLE I

RESPONSE OF NE NEURONS IN LOCUS COERULEUS AND 5-HT NEURONS IN DORSAL RAPHE TO IONTOPHORE- TICALLY APPLIED CLONIDINE (C), NE AND LSD

The 3 drugs were not all applied to all cells because some cells were lost before this was accomplished. n ~- total number of cells tested in each area. The vertical columns list the number of units that responded as indicated.

Area Drug Total Partial Excitation No effect Ejection inhibition inhibition current (nA)

Locuscoeruleus C 9 0 0 0 2- 8 NE 6 3 0 0 6-20

(n 9) LS D 0 I I 2 10-20

Dorsal raphe C 1 5 I 3 2-20 NE 0 2 3 4 1 0-20

(n :- 10) LSD 4 0 0 0 8-10

neurons in most cases (n = 7) showed no response to c lonidine (20-800/~g/kg, i.v.).

One cell showed a slight increase in rate. In accordance with ear l ier studies the D A

cells were also not sensitive to L S D (20 #g/kg , i.v.) 2°. Cells in the re t icular fo rma t ion

showed a var iable response to c lonidine (20-1200 #g/kg, i.v.) i.e., either an increase,

a decrease or no effect on firing rate. LSD (10-30 #g/kg , i.v.) also had a var iable

effect on these units, as repor ted previously 4.

Microiontophoretic studies

When appl ied micro ion tophore t ica l ly at low ejection currents c lonidine con-

sistently inhibi ted the firing of cells in the locus coeruleus (Fig. 2, upper pane l ;

Table I). The ejection current needed to s top the firing for a shor t per iod o f t ime was

as low as 2 nA; higher currents p roduced a longer inhibi t ion. Character is t ica l ly there

was a delay o f 0.5-1.5 min before maximal effect was ob ta ined (Fig. 2, upper panel).

This was in cont ras t to the a lmos t immedia te response to ion tophore t ica l ly appl ied

NE, which also reduced the firing ra te o f all locus coeruleus units tested (Table I,

Fig. 2). When LSD was appl ied iontophore t ica l ly , at ejection currents which consis-

tently inhibi t raphe units0, ~8, the locus coeruleus units showed litt le or no response

(Table I). In no case was a reduc t ion in ac t ion potent ia l size observed.

When either c lonidine or N E were appl ied mic ro ion tophore t i ca l ly to raphe

units in a dose range s imilar to or higher than tha t used previously on the locus, the

response was var iable , and not dose-dependent (Table I). N o consis tent paral le l was

found between the responses to c lonidine and NE. In con t ras t with the la t ter drugs,

L S D appl ied mic ro ion tophore t i ca l ly at low ejection currents (8-10 nA) p roduced a

rap id and consis tent inhibi t ion o f all r aphe units tested (Table 1). A typical exper iment is shown in Fig. 2, lower panel.

6-OHDA Lesions: effects on the clonidine-induced depression o f raphe units

The in t ravent r icu lar inject ion of 6 - O H D A (7-12 days) p roduced a reduct ion

NE 16

c 2

LSD lO

c 4

297

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NE C LSD 166 4 8_.

JOCUS coeruleus

raphe I I 5MIN

Fig. 2. Comparison of the response of a neuron in the locus coeruleus (top trace) and that of a cell in the dorsal raphe (bottom trace) to the microiontophoretic administration of norepinephrine (NE), clonidine (C) and LSD. The numbers above bars indicate ejection currents in nA. All ejections lasted 30 sec, indicated by the length of the horizontal bars. The noradrenergic cell was inhibited both by NE, 16 nA, and clonidine, 2 and 4 nA, but not by LSD, 10 nA. In contrast, the serotonergic cell showed a slight increase in firing rate after NE, 16 nA, possibly a slight increase after clonidine, 4 nA, but was completely inhibited by LSD, 8 nA.

in whole b ra in N E concent ra t ion by abou t 80 ~o; the concent ra t ion of 5-HT was

considerably less affected (13 ~o; Table II). In other animals , examined by the histo-

fluorescence method, the N E terminals within the dorsal raphe nucleus as well as

those in adjacent areas, e.g. , the ventrolateral central gray, were no longer detectable.

The N E cell bodies in the locus coeruleus appeared damaged, whereas the ventral N E

pathway 86 was largely intact. The 5-HT cell bodies in the dorsal raphe nucleus appear-

ed normal .

The spontaneous firing rate of raphe neurons 7-12 days after 6 - O H D A was

not not icably different f rom that in untreated rats. However, at earlier t imes after

TABLE II

DIFFERENTIAL EFFECTS OF INTRAVENTRICULARLY ADMINISTERED 6-OHDA ON BRAIN CONCENTRATIONS (/~g/g) OF NE AND 5-HT

Number of animals in each group is 5. The means ~ S.E. are shown. 6-OHDA (6-hydroxydopamine) 100 ~tg was given (as hydrobromide) 8 days before decapitation.

Treatment NA 5-HT

Control 0.35 ! 0.01 0.45 4- 0.01 6-OHDA 0.08 d- 0.01"* 0.40 4- 0.01"

* Significance of difference from control, P < 0.02. * * Significance of difference from control, P < 0.001.

298

Z w x,,

8 0 "

C LSD

I IOMIN I

Fig. 3. Lack of effect of intravenously administered clonidine (C) on the firing rate of a neuron in the dorsal raphe nucleus in a rat pretreated 1 week previously with 6-hydroxydopamine (intraventricular- ly, 100/~g). Clonidine was injected via a tail vein in increasing doses (4 ~- 4 ~- 40 4- 40 -L 120 + 120 "k 120 4- 120 /~g; total dose 568/tg) with no effect. In contrast, LSD (10 + 5/tg/kg) completely inhibited the firing of the cell.

6-OHDA (24 48 h) the firing of raphe neurons was first erratic and then abnormally slow. When clonidine (12-1500 mg/kg) was administered intravenously to the 6- OHDA lesioned rats ( n ~ 14) no marked effect on the firing rate of the raphe units was obtained, although the neurons displayed their previously described sensitivity to a small dose of LSD (Fig. 3).

Experiments were also performed to investigate whether impairment of nor- adrenergic transmission by means of synthesis inhibition might affect the firing rate of raphe units. Rats were pretreated at 3 h intervals with a-methyl-p-tyrosine methyl ester (a-MT), 100 mg/kg, i.p. × 3. Single unit recordings were performed 2 h after the last injection. A sample of 24 raphe cells showed a mean firing rate of 0.66

18 ̂

CCC jI-A-.~ LSD 1.1 .r ,,1

Z

o,.. ,..o

lOoU 1

C C 35 400 LSD

Fig. 4. Reversal of L-amphetamine (L-A) or a large dose of clonidine (C) of the inhibition of firing of neurons in the dorsal raphe nucleus by a small dose of clonidine. L-Amphetamine 0.5 mg/kg, i.v. reversed the inhibition by clonidine (4 + 4 + 4 / tg /kg, i.v.; top trace). Subsequent injections of L-amphetamine (0.5 + 0.5 ~ 0.5 mg/kg) caused no further increase in the firing rate of the cell. The cell was subsequently inhibited by LSD, 12/~g/kg, i.v. Following inhibition of a raphe cell by a small dose of clonidine (35/~g/kg, i.v. ; bottom trace) a large dose of clonidine 400/~g/kg, i.v., re- versed the inhibition of the cell, which was subsequently inhibited by LSD, 12 + 8/~g/kg, i.v.

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0.05 (mean 4- S.E.M.) spikes/sec vs. 1.27 ± 0.10 spikes/sec in control rats (cell sample: n = 14). The latter rate is typical for raphe cells 7 but the former was signif- icantly (P < 0.001) depressed.

Reversal of the depressant effect of clonidine on raphe units Following the inhibition of raphe units by intravenous clonidine (n = 10),

L-amphetamine sulfate (n = 8) or D-amphetamine sulfate (n = 2) each at a dose of 0.5 mg/kg, was injected intravenously. In all raphe units tested both treatments caused a rapid reversal of the clonidine-induced depression of firing rate up to, or slightly above, baseline rate (Fig. 4, upper panel). All cells subsequently could be inhibited by the usual low dose of LSD (10-20 #g/kg).

In other experiments, an attempt was made to antagonize the inhibitory effect on raphe units of a low dose of clonidine by a high dose of the same drug since previous data s indicate that high doses of the drug (e.g., 0.1-1.0 mg/kg) have a postsynaptic NE receptor stimulating action. When a large dose (400 #g/kg) of clinidine was given following inhibition of raphe units by a small dose of clonidine (Fig. 4, lower panel), the firing rate of raphe cells rapidly returned to baseline levels. At this high dose level there was piloerection and increased respiratory rate. Following the large dose of clonidine, the raphe units were still inhibited by the usual small dose of LSD.

DISCUSSION

A very small dose of clonidine (e.g., 6.4/tg/kg, i.v.) similar to that which re- duces blood pressure in animals and humans, inhibited the firing of neurons in the locus coeruleus. In the rat, the latter nucleus consists exclusively of cell bodies of NE-containing neurons 27. The effect of clonidine was potent and dose-dependent, in contrast to weak or absent responses of control cells (e.g., DA neurons of the substantia nigra). These results were paralleled in the microiontophoretic experiments in which ejection of clonidine at very low currents (e.g., 2-8 nA) in an ionically diluted solution (0.01 M clonidine in 0.1 M NaC1) produced a dose-dependent, uniform inhibition of units in the locus coeruleus. This was clearly not a local anesthetic effect since no reduction in spike amplitude was seen. When clonidine was applied in similar amounts to neurons in the dorsal raphe no consistent response was ob- tained. Similar results have been reported showing that cells in the cerebral cortex and the reticular formation are relatively insensitive to clonidine (e.g., 60-80 nA required for inhibition) 9. The NE neurons in the locus coeruleus were also consistently in- hibited by NE at low ejection currents. When similar amounts of NE were applied to units in the dorsal raphe, variable responses were obtained, in agreement with previous data6, 26.

Evidence from the peripheral adrenergic system has suggested the existence of a local synaptic feedback mechanism regulating NE release ~7. There is evidence that an inhibition of release of NE by clonidine is mediated locally via activation of a presynaptic or prejunctional a-receptor56, 75. In the central nervous system evidence for similar presynaptic or 'auto-receptors' (i.e., receptors on the soma, dendrites,

300

and/or terminals of a neuron which are responsive to its own transmitter) has been derived from studies on DA2,22,30, 47, 5-HT24,as, 62 as well as on NE neurons62,74, 76.

Thus, one interpretation of the present data is that clonidine inhibits NE neurons in the locus coeruleus via activation of adrenergic autoreceptors on or near NE cell bodies. An alternative interpretation of our data would be that clonidine and NE are acting upon receptors related to adrenergic afferents to locus coeruleus neurons. There is in fact recent evidence for an adrenaline neuronal input to the locus coerule- us 41. In view of the fact that clonidine is so highly effective by the local, iontophoretic route, it seems unlikely that the inhibition of firing of locus coeruleus units is due to a systemic effect of the drug. The clonidine-induced inhibition of firing of brain NE neurons may account for the decrease in turnover of brain NE (of Introduction) seen after systemic administration of the drug.

In contrast to its effect on NE neurons, the inhibitory effects of systemically administered clonidine on 5-HT neurons on the dorsal raphe nucleus is probably indirect. This view is supported by the lack of a sensitive or uniform response of raphe units to iontophoretically applied clonidine (and NE). Further support for this view comes from the fact that pretreatment with 6-OHDA eliminated the inhibitory effect of intravenously administered clonidine, whereas the response to the directly acting drug LSD was unaffected. Assuming that intraventricular 6-OHDA, in the amounts used, selectively destroys catecholaminergic neurons, the inhibitory effect of clonidine in the dorsal raphe may require mediation by an intact NE system. In other studies, pretreatment with 6-OHDA (intracisternal) has been found to attenuate the hypo- tensive effects of intravenous or intracisternal clonidine, suggesting that this effect is also dependent on the integrity of central catecholaminergic neurons 2s. However, an opposite view has been proposed based on experiments showing a depletion of endogenous catecholamines by reserpine and a -MT does not interfere with the hypotensive action of clonidine 49.

The decreased firing rate of raphe units following a-MT treatment, which prob- ably impairs noradrenergic transmission, lends additional support to the view that acute impairment of NE transmission reduces the activity of 5-HT neurons in the dorsal raphe. However, in a recent abstract, it has been reported that the activation of brain NE neurons may exert an inhibitory influence on 5-HT neurons in the cat dorsal raphe nucleus 61. Monophasic electrical stimulation of the locus coeruleus caused an inhibition of firing of the raphe units. It remains to be determined, however, whether NE neurons were selectively activated by the stimulation or whether another pathway was involved. Moreover, there is data within the present study which in- directly supports the view that clonidine inhibits raphe neurons via impairment of noradrenergic transmission. I f this is the mechanism by which clonidine is acting one would expect that an increased release or reduced reuptake of NE produced by amphetamine t2,zS,34,S9,6°,66,79,84'91 would reverse the effects of clonidine. In fact, it has been shown at various peripheral adrenergic sites that o-amphetamine counter- acts the block in adrenergic transmission induced by clonidine 77. Similarly we have found a reversal of clonidine effects on all 5-HT units tested with either D- or L- amphetamine. There is much recent evidence indicating that D- and L-amphetamine

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have a similar action on brain NE neurons, but that D-amphetamine is much more potent than L-amphetamine in its effects on DA neurons19,81, 39,8°,sa,9°. Thus, the fact that small doses of L- and D-amphetamine were equally effective in reversing the clonidine-induced inhibition of 5-HT neurons, is consistent with a NE-mediated effect. However, further experiments will be required to establish the equality of D- and L-amphetamine in this system.

The reversal of the raphe inhibitory action of a low dose of clonidine by a high dose of clonidine (e.g., 400 mg/kg, i.v.) appears paradoxical. However, there is evidence that clonidine at a relatively high dose level (0.1-1.0 mg/kg) has a stimulatory action on presumably postsynaptic NE receptors in the central nervous system 8. After the high doses of clonidine, signs of sympathomimetic stimulation, such as piloerection, exophthalmos and increased respiratory rate appeared. A postsynaptic NE receptor activation is likely to account for these effects as well as the initial rise in blood pressure seen after the administration of high doses of this drug 50. In any event, the reversal of the inhibition of 5-HT units by a low dose of clonidine was obtained by two chemically and pharmacologically different drugs, amphetamine and clonidine (at a high dose). The one common mechanism providing the simplest ex- planation for the reversal by the two drugs would be that they either directly or indirectly facilitate NE transmission. However, it is also possible that the high dose of clonidine acts by producing a desensitization of adrenergic receptors on the NE neurons.

In conclusion, our results show an excellent correlation between the effects of systemically administered clonidine on the firing of monoaminergic neurons and the reported effects of this drug on the turnover of monoamines 8. Clonidine reduced the firing rate of both NE and 5-HT neurons, paralleling the reduction of NE and 5-HT turnover produced by this drug. On the other hand, clonidine did not alter the firing of DA neurons, which correlates well with its previously reported lack of effect on DA turnover. The iontophoretic results suggest that NE neurons in the locus coeruleus possess sensitive adrenergic receptor on or near their soma ('autorecep- tors'). A direct activation of these receptor sites by clonidine could account for the inhibition of the activity of these NE neurons. The dose of clonidine which produces inhibition of brain NE neurons is similar to that which causes a reduction in blood pressure. Thus, a direct inhibitory action on central NE neurons may contribute to the hypotensive or sedative effects of this drug (cf. Dollery and Reid28). On the other hand, the effect of low doses of clonidine on 5-HT (raphe) neurons does not appear to be through a direct action. We have, therefore, considered the possibility that the raphe inhibiting effect of systemically administered clonidine is secondary to its action on NE neurons (see above). There are a number of biochemical studies that also suggest the occurrence of interactions between brain NE and 5-HT systems TM

43-44,78. A possible anatomical basis for such interactions is provided by histofluores- cence observations, showing NE nerve terminals in the raphe nuclei and in the adja- cent ventrolateral central gray of the midbrainaa, 86. The NE projection to the raphe region appears to be derived from the NE neurons of the locus coeruleus 57. How- ever, when NE is applied microiontophoretically to 5-HT neurons, weak and/or

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variable responses are obtained 6,26, thus arguing against a direct uniform synaptic

NE input. Nevertheless, even if there is no direct NE input , there may be in terneurons

mediat ing a physiologically significant influence of brain NE neurons on raphe

(5-HT) neurons. An alternative to a neuronal explanat ion, would be that the c?onidine-

induced depression of 5-HT neurons results from a systemic effect of the drug (e.g.,

hypotension). However, it should be noted that raphe neurons are rather insensitive

to wide fluctuations in blood pressure a~. Clearly our present data are insufficient to

discriminate between the above alternatives and the question as to the mechanism by

which the firing of 5-HT neurons is inhibi ted by clonidine remains unresolved at this

time.

ACKNOWLEDGEMENTS

This work was supported by P. H. S. Grants MH-17871, MH-14459, a Scottish

Rite Schizophrenia Research Grant , and the State of Connecticut .

T. H. Svensson was supported by travel grants from the Swedish Medical

Research Council (SMF B74-04R-4185) and from the Parkinson 's Disease Founda -

t ion, Inc., New York and by a J. Hudson Brown Fellowship.

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