Modulatory role of serotonin on GABA-elicited inhibition of cerebellar Purkinje cells

Nrurosrirnw Vol. 30. No. Prmted in Great Britam

1, pp. 117-125,

1989

0306.4522189 $3.00 + 0.00 Pergamon Press plc 8, 1989 IBRO

MODULATORY GABA-ELICITED

ROLE OF SEROTONIN ON INHIBITION OF CEREBELLAR PURKINJE CELLS

J. C. STRAHLENDORF,* M. LEE and H. K. STRAHLENDORF Department of Physiology.

Texas Tech University Health Sciences Lubbock, TX 79430, U.S.A.

Center,

3601 4th Street.

Abstract-The present study was designed to directly examine the postsynaptic acttons of serotonin on GABA-mediated inhibition of cerebellar Purkinje cells. The findings indicate that serotonin at currents that produced minimal effects on the spontaneous tiring rates of Purkinje cells modified GABA effects in a biphasic manner. Serotonin initially decreased GABA-mediated inhibitions followed secondarily by either continued inhibition or, in the majority of cases, augmentation of GABA responses. When a comparison was made of the secondary effects of serotonin on GABA-mediated inhibition with the initial spontaneous firing rates of the Purkinje cells. the group in which serotonin augmented GABA actions had a significantly higher initial firing frequency than the group in which serotonin attenuated GABAmediated inhibition. Furthermore, with increasing firing rates. the proportion of cells showing augmentation of GABA inhibition increased, and the proportion of cells displaying attenuation of GABA effects decreased. Serotonin affected fi-alanine-mediated inhibitions in a manner stmilar to that seen with GABA.

whereas glycine was differentially altered. This study identifies another neuromodulatory role of serotonin on Purkinje cells in the cerebellum. Furthermore, the effects of serotonin on GABA inhibition seem to be governed by some intrinsic property of the Purkinje cell. which is apparently related to the firing rate of the cell.

Within the cerebellum4~“~14~“~45 as well as else5-HT) where, “N.*~x~~ serotonin (5-hydroxytryptamine, was shown to act as a modulator of various other neurotransmitter actions and to exert direct effects on neuronal firing. Serotonin was reported to inhibit significantly glutamate-induced excitations of most Purkinje cells while eliciting minimal changes in the spontaneous rate. I4 Similar inhibitory actions of 5-HT on glutamate excitations was shown on cortical’3 and thalamic’3.24~3x neurons. In contrast. serotonin facilitated glutamate actions on motoneurons.‘7,42 Direct effects of serotonin on various neurons are most frequently inhibitory although excitation and mixed effects were also reported.4.“.25~‘2.4’ In a previous study,” we showed that iontophoretically applied 5-HT elicited three different direct effects on Purkinje cells of the cerebellum: inhibition, a biphasic effect consisting of transient inhibition followed by excitation, and excitation only, effects that appear to be related to the baseline firing rate of the neuron. Purkinje cells that responded to serotonin with an increase in firing rate had a significantly stower predrug frequency than those cells that were suppressed by 5-HT. It was concluded from these studies that 5-HT may serve as a biaser, setting the cell at a preferred firing rate. The question remains whether the direct effects of 5-HT are separate from its neuromodulatory actions on other putative neuro-

*To whom correspondence should be addressed. Abbrrciations: 5-HT, 5_hydroxytryptamine, serotonin; norepinephrine.

transmitters in the cerebellum. Serotonin strongly suppresses glutamate-evoked excitation of Purkinje cells,” yet its action on GABA and related amino acid-mediated inhibitions of these cells has not been throughly investigated. Many investigators have suggested that GABA is the transmitter for various cells within the cerebellar cortex. including stellate, basket, Golgi and Purkinje cells (for review, see Ref. 5). Additional studies in our laboratory showed the inhibitory effects of iontophoretically applied serotonin on cerebellar Purkinje cells to be antagonized significantly by continuous application of the chloride ionophore antagonist picrotoxin.“.” Furthermore, iontophoretically and systemically administered pcntobarbital potentiatcd serotoninmediated inhibitions of Purkinjc cells. effects which were antagonized by the GABA antagonists pentylenetetrazole, picrotoxin and bicuculline.” These studies suggest a potential interplay between the cerebellar GABAergic and serotonergic systems on Purkinje neurons, possibly via a postjunctional interaction with the intrinsic GABAergic system. The present experiments were designed to directly examine the postsynaptic actions of 5-HT on GABA- and related amino acid-mediated inhibition of Purkinjc cells. EXPERIMENTAL PROCEDCRES

We conducted our experiments on male SpragueeDawley (Sasco Laboratories) rats (206350 g) anesthetized by injections of urethane. 1.2 g/kg. i.p. A proportionally controlled d.c. heating pad maintained body temperature between 36.5

NE.

117

and 37.5 C. A craniotomy exposing the cerebellum at the level of the fissura prima (which separates lobules V and VI) permitted the exploration of the anterior and posterior cerebellar vermis out to the lateral edge of the pars intermedia. To reduce movement artifacts and prevent the cerebellar surface from drying, we placed 3% agar in a saline solution on the exposed cerebellar cortex as necessary,

Rwording und mic,roionrophoresi.c For iontophoretic experiments, we used live-barrel micropipettes with 5dnrn tips to record extracellularly the spontaneous discharges of Purkinje cells. Purkinje cells were identified by their characteristic electrophysiology, including their complex discharges4 The outer drug barrels of each multibarrel pipette contained 5hydroxytryptamine creatinine sulfate (Sigma Chemical Co., 0.05 M, pH 4.5). GABA (Sigma Chemical Co., 0.2 M, pH 4.0). glycine (Sigma Chemical Co., 0.5 M, pH 4.0). b-alanine (Sigma Chemical Co., 0.5 M, pH 4.0), norepinephrine bitartrate (Regis Chemical Co., 0.5 M. pH 4.5), spiperone (RBI. 0.01 M. pH 4.0) ketanserin tartrate (Janssen Pharmaceuticals, 0.01 M, pH 3.0). methysergide maleate (Sandoz Pharmaceuticals, 0.01 M, pH 4.4), and sotalol hydrochloride (Mead Johnson Pharmaceuticals, 0.25 M, pH 4.5). all dissolved in double distilled water. We applied a retaining current of IOnA with appropriate polarity to all drug barrels between periods of ejection. Current effects that could influence cell discharges directly were minimized by an automatic balancing circuit that passed a current of equal magnitude and opposite polarity to the sum of all currents through one side barrel of the electrode filled with 4 M NaCI. Retention and ejection periods were sequenced automatically and maintained constant throughout each experiment by the use of an Apple He computer. Typically, ejection periods were set for 2&30 s and retention periods for 40-60 s. The computer served as an external clock for the ionotophoretic unit while constructing simultaneously integrated histograms on-line from the window discriminator output. The center barrel of the micropipette was filled with 4 M NaCl (335 MR impedance) for recording action potentials (simple spikes and complex discharges), which we amplified by conventional means, monitored on an oscilloscope, and converted to uniform voltage pulses by passage through a window discriminator. The pulses were summed by a rate meter over 1-s epochs (cumulative records) and displayed on a strip-chart recorder. The amplified signals were stored on a multichannel magnetic tape recorder (bandpass: 25 Hz-24 kHz). To control for non-specific artifacts (e.g.. current effects, pH effects or local anesthetic actions) occasionally inherent with iontophoretic ejection, we led action potentials into an analog delay. the output of which was displayed on another oscilloscope. Recordings that displayed artifactual responses to drug applications were eliminated from the study.

We assessed drug effects qualitatively by tabulating the total number of cells tested versus the number of cells evincing a prescribed response. Computer software designed for an Apple He was used to construct integrated histograms summed over 1 s epochs for periods of 3- 5 min. The program compared the total number of discharges of Purkinje cells during the ejection period with the number occurring during an equivalent period of time immediately prior to ejection. The data were expressed as a percentage change from control. In the present study, the computer averaged a number of individual responses (usually consisting of 3 4 individual trials); the reported percentage change from control was computed from the averaged response. For statistical analysis, a Student’s r-test was used to compare singularly two means. A correlation coefficient was calculated as an index of the strength of a particular relationship.

Churacterization GABA -mediated

of’ the .serotonergic, igjluenw inhibitions of Purkirzje neurons

on

To investigate the influence of 5-HT on GABAmediated inhibitions, pulsatile applications of GABA (l-20 nA) were delivered at regular intervals (20 s ejection, 40s retain). GABA produced a consistent decrease in the firing rates of Purkinje cells. Serotonin, at currents that produced minimal effects on the spontaneous firing rates, modified GABA effects in a biphasic manner. Serotonin initially decreased GABA-mediated inhibitions, followed in a few cases by a continued diminution or, most frequently. augmentation of the GABA response. For descriptive purposes, the biphasic response was subdivided into initial and secondary phases primarily on the basis of response character. The secondary phase began once augmentation was observed; from 3 to 16 min of 5-HT iontophoresis. Cells that did not demonstrate augmentation of GABA inhibition after I6 min of 5-HT application were designated as non-biphasic responders. Efects of serotonin on GABA -mediated during the initial phase

inhibitions

During the intial phase of the biphasic response, 81% (72/89) of the Purkinje cells showed a decrease in their response to GABA in the presence of 5-HT (1040 nA); 3% (3/89) of the cells showed an accentuated response, and 16% (14/89) did not change their response. For the 72 Purkinje cells whose GABA response was attenuated initially in the presence of 5-HT, GABA alone induced an average decrease in spontaneous activity of 21.9 + 1.0%; however, it elicited an average decrease of I I .3 + 0.8 (P < 0.0001) in the presence of S-HT, representing a 48.4% decrease in the GABA response. In these cells, continuous application of 5-HT caused an insignificant change in the spontaneous firing rate (average, 0.5 f 1.7% increase). This initial inhibitory component of 5-HT’s action began within 1 min, and lasted approximately 8 min. In the 3% of cells initially demonstrating an enhanced GABA response, control responses to GABA averaged 19.3 f 4.8% decrease in firing while in the presence of 5-HT. an average 51.3 & 6.7% inhibition was observed, representing a 215.5 k 124.3% increase in GABA effects. Again, the spontaneous rate was insignificantly affected by 5-HT. Thus, in the majority of cells, 5-HT initially decreased or blocked GABA-mediated inhibition in the absence of equivalent effects on the spontaneous firing rate. Eflticts of’ serotonin on GABA -mediated during the secondary phase

inhibitions

During the secondary phase, 6 1% (46/75) of Purkinje cells responded with an increase rather than a decrease in their sensitivity to pulsatile applications of GABA. Thirty two per cent (24/75) of the cells

Modulatory

role of serotonin

B

C 5-HT

30

nA

40

IO

5-HT

nA

on GABA

inhibition

I19

20 nA

nA

D

GABA

20 nA

1 min Fig. I. Effects of serotonin (5.HT) on GABA-induced inhibitions of the spontaneous activity of a Purkinje cell. (A) Control responses of a Purkinje cell to iontophoretic pulses of GABA (20 nA). (B). (C) Effects of continuous applications of 5-HT (IO, 20, 30 and 40 nA) on GABA-mediated inhibition. (D) Recovery responses of the same neuron to GABA after termination of 5-HT. In all illustrations. bars indicate the duration of the ejection period.

to show an attenuated response, while in 7% (5/75) of the cells the GABA response remained unaffected by serotonin. Fewer cells (75 vs 89) were tested in this secondary phase because recording instability prevented further tests of longer term applications of 5-HT. In the 46 cells in which 5-HT secondarily augmented GABA, GABA alone caused an average decrease of 20.8 + I .3%, whereas, in the presence of S-HT. GABA produced an average 47.8 k 2.8% decrease (P < 0.0001). The effects of continuous 5-HT on GABA-mediated inhibition represented an average increase of 172.4 f 26.9%, coincident with an average 23.7 k 3.5% decrease in the spontaneous firing rate. A representative tracing of 5-HT modulatory effects on GABA-induced inhibition appears in Fig. I. From this figure, it is apparent that 5-HT initially attentuated GABA inhibition, whereas 5-HT secondarily augmented GABA-elicited inhibition. This response pattern was typical irrespective of the current of 5-HT initially used. It appeared that the concentration of 5-HT was not critical once potenCation of GABA was present, although a strict quantitation of the concentration dependency of S-HT was not made. Serotonin also slightly augmented the duration of GABA-mediated inhibition in I7 of the 46 cells. The cells recovered from 5-HTmediated augmentation of GABA pulses within l-30 min after termination of 5-HT ejection (average time of 4.8 * I .O min). It is of interest that on occasions, 5-HT continued to potcntiate GABA-mediated inhibition for as long as 30 min after the current was terminated. Another characteristic of the augmentation was the nature of the cell’s response once it occurred. It was observed that when 5-HT augmented the GABA response. it occurred often with complete inhibition of simple continued

spike activity, although the cell continued to display complex discharges. In 24 instances (representing 32% of cells) in which 5-HT only attenuated GABA-mediated inhibition (during the secondary phase), GABA alone produced an average 21.9 + 1.7% decrease; whereas. in the presence of 5-HT (I040 nA), an average 9.1 + 0.9% decrease (P < 0.0001) to pulsatile applications of GABA was evident. This action of 5-HT represented an average 58.4% decrease in GABA effectiveness in the presence of long term 5-HT ejections which is similar to the effects of 5-HT on GABA responses observed during the initial phase. Of the 24 cells in which 5-HT attentuated GABA, an insignificant average increase in spontaneous firing rate of 2.4 + 4.4% was observed. The question arose as to which factors may have contributed to the differential response seen during the secondary phase of the biphasic response. To provide a possible explanation for these findings. a three-way comparison of the following factors was made: (a) the effects of 5-HT on GABA-mediated inhibitions during the secondary phase; (b) the tendency for direct effects of 5-HT; and (c) the initial spontaneous firing rates of the Purkinje cells. A significant difference (P < 0.0001) was found between the mean initial firing rates for the group in which 5-HT augmented GABA actions, and the group in which 5-HT attenuated GABA-mediated inhibition. The augmented group had an initial neuronal firing rate of 49.9 k 0.6 Hz. whereas the attenuated group had a lower initial rate of 32.6 f 2.6 Hz. Within the group in which 5-HT augmented GABA effects, 42 of the 46 cells displayed a tendency to be depressed by 5-HT. On the other hand. in the group in which 5-HT reduced GABA effects, 50% of the cells tended to be depressed and 50% showed a tendency to be excited

z

8

?

51

::

::

? 9

z I

=:

6 ::

SPONTANEOUS FIRING RATE (SPIKES I S)

Fig. 2. Correlation between the percentage of Purkinje cells in which 5-HT augmented and attenuated GABA-mediated inhibitions and the spontaneous firing rate. Unshaded area: attenuation of GABA inhibition; shaded area: augmentation of GABA inhibition. by 5-HT. In summary, the group of cells in which 5-HT augmented GABA-mediated inhibition displayed higher neuronal frequencies and tended to be depressed by SHT, whereas the attenuated group had slower neuronal firing rates and showed no predilection for 5-HT effects. In light of these findings, the total population of cells was divided into six groups on the basis of the drug-naive spontaneous firing rates (spikes/s): I O-l 9; 20-29; 30-39; 40-49; N-59; and W-89. The proportion of Purkinje cells in which 5-HT augmented and attenuated GABA-mediated inhibitions were calculated separately for each firing rate category. The results are shown in Fig. 2: with increasing tiring rates, the proportion of cells showing augmentation of GABA inhibition increased, and the proportion of cells displaying attenuation of GABA effects decreased (product moment correlation: r = 0.825. P < 0.05). Thus, in the 60-89 Hz group, 88% of the cells showed an augmented response to GABA in the presence of 5-HT. The percentages changed abruptly between the l(r-19 and 20-29 Hz groups, in that serotonin did not augment any of the cells in the lowest group. More subtle changes in the percentages were observed in the intermediate groups. Characterization of serotonergic influence on glycine mediated inhibitions To determine the specificity of the influence of 5-HT on GABA-mediated inhibition, ii-alanine and

glycine. two additional amino acids !hclt hypcrpolarize neurons by increasing prcsurnably (. I conductance were tested. Table 1 compares the effects of continuous 5-HT on the inhibition elicited by pulsatile applications of /I-alanine, glycinc and GABA. These studies were conducted identically to the GABA study, with the exception of the inhibitory agent chosen. As seen with GABA, 5-HT prcdominantly attenuated the glycine response during the initial phase in eight out of nine cells. Glyclnc alone caused an average decrease of 17.1 i_ I .7”1,: and in the presence of 5-HT, glycine induced an average 8.8 _t 2.9% decrease (P < 0.0001) in firing, reprcsenting a 48.5% attentuation of glycine-mediated inhibition. This is remarkably similar to the effects of S-HT on GABA responses. During exposure to continuous 5-HT, the spontaneous firing rates of the Purkinje cells decreased by 12.1 k 4.4%. Scrotonin continued to significantly attenuate glycine inhibition during the secondary phase of exposure to 5-HT (seven of the nine cells showing attenuation), rcsulting in an average 60.6 * 9.2% atlenuation (P < 0.002) of glycine-elicited inhibition, with a corresponding 3% decrease in the spontaneous firing rates. An example of the influence of S-HT on glycine-mediated inhibition is depicted In Fig. 3. Glycine (35 nA) induced a 6% decrease of Purkinje cell activity, whereas during the continuous application of 5-HT (15~-2.5 nA), glycine could no longer inhibit this cell even at currents of 5-HT that slowed the spontaneous activity from 59 to 28 spikes/s. Although

the inhibitory

actions

of glycine

in this example, GABA augmented during the continuous 5-HT. Two of the Purkinje cells showed a moderate augmentation 34% increase) of their response to presence of 5-HT. potentiated

Characterization of scrotonergic P-alanine-mediated inhibitions

were

not

was markedly application of in this group (approximately glycine in the

ir@4rncr

011

Consistent with the other inhibitory agents used, continuous 5-HT initially attenuated /j-aianinemediated inhibition of 24 of 26 (92%) Purkinje cells tested. /I-Alanine alone produced an average 14.5 +_ 1.0% inhibition, and in the presence of continuous 5-HT (IO-40 nA), fl-alanine elicited a 7.0 k 1.O% decrease (P < 0.0001) in cell firing, representing a 51.7% attenuation (which also is similar to the effects on GABA and glycine). Continuous sero-

Table I GABA Augmentation Attenuation No effect

3/89(215.3%) 72189 (48.4%) 14189

Initial phase /?-Alanine 2/26 (44.0%) 24126 (5 1.7%) O/26

Glycine 019 (-1 S/9 (48.5%) 119

GABA 46/75 (172.4%) 24175 (58.4%) 5175

Secondary p-Aianine 9/19(171.8%) 9/19 (55.7%) l/19

phase Glycine 2/9(34.0%) 7/9(60.6%) o/9

The expressed ratio represents the number of cells responding in the prescribed manner out of the total number of cells tested. The number in parentheses represents the quantitative assessment of the average magnitude of the response.

Modulatory

role of scrotonin

A

GLY __ 35 - no-

B

5-HT

15-25 “A GABA

o n.4

C GLY

Fig.

35

GABA -we-

nA

._ -

3. Ratemeter

o no

record

depicting the effects of 5-HT 35 nA) mediated illhibiti(~n. (A) (‘onrrol responses of a Purkmje cell to iontophorctic pulses of glycinc (35 nA). (B) Effect of continuous application of 5-HT (15 2.5 nA) on glycine-mediated and GABAmediated inhibitions. (C) Recovery responses of the same neuron to glycine and GABA.

(15 25nA) on glycine-(GLY.

a less than 2% increase in the sponfiring rates during the initial phase of the /f-alar-tine series. Two of the Purkinje cells in this group displayed augmentation of their response to /j-alanmc in the presence of 5-HT, which averaged 44.0 + ~.I%. During the secondary phase, 47% of the cells tested (nine of 19 cells) showed a significantly enhanced response to /I-alanine, which averaged 171.8 :t_ Sh.l% (P < 0.004). In contrast, 47% of the cells (nine of 19 cells) showed a significantly attenuatcd response to /I-alanine in the presence of 5-HT. which averaged 55.7 + 5.2% (P < 0.0002). The magnitudes of these responses were almost identical to S-HT’c effects on GABA-mediated inhibition. One of the 19 cells tested was not affected by 5-HT. Figure 4 depicts the efrects of 5-HT on /{-alanineelicited itlhibitions: 20 nA pulses of p-alanine induced average inhibitions of 15% in the spontaneous activity of this Purkinje cell. During the initial phase. the continuous administration of scrotonin (IO nA) caused the inhibitions elicited by /I-aianinc pulses to an average 71”/0 decrease from 7 10 2 spikes/s, attenuation. relative to a 7% suppression of background activity. During the secondary phase. [I-&nine-mediated inhibition changed from 7 to 15 spikcs’s. an average I t4”/o ~~ugmentation, relative to a 43% suppression of background activity. The predominant influence of 5-HT was to initially attenuate the inhibitory responses to each of the amino acids, whereas, during the secondary phase. GABAmediated inhibitions were predominantly augmented. tonin

produced

tancous

on GABA

121

inhibition

glycine effects were attenuated, and [1’-alaninc actions were augmented and attenuated in equal numbers by the presence of 5-HT. A direct comparison was made by testing a number of Purkinje cells with both GABA and either glycine or b-alaninc. Overall, the results were consistent with those reported when the agents were tested separately. In the glycine/GABA group. serotonin attenuated the glycine response during the initial and secondary phases, but initially attenuated and secondarily augmented the GABA responses in the two cells studied. Four cells were tested with /i-alaninc and GABA. Scrotonin initially attenuated the GABA response in three of the four cells, but augmented the GABA response of the remaining cell. However. during the secondary phase. GABA inhibition was augmented in all four cciis in the presence of S-HT. Consistent with the previous data. scrotonin initially attenuated /{-alanine responses in all four cells while attenuating /-alanine effects in two of the cells and augmenting @-alanine actions in the other two cells during the secondary phase.

With regard to the ability to modify GABA effects. a direct comparison was made between iontophorctic applications of norepinephrine (NE) and S-HT on six Purkinje cells. It is well documented that NE enhances GABA-mediated inl~ibition of cerebcllar Purkinje neurons. “M’ Intcrcstingly. both agents predominantly produced an augmentation of GABAmediated inhibition during the secondary phase 01 the response pattern. However. differences wcrc observed between the two agents during the initial phase of the response. Unlike 5-HT. NE augmcntcd G.i\BA-mediated inhibition during the initial phase in

A B-ALA

B

20 --- nA

10 nA

_--

--I

c

B’ -

5-HT

-

---

B-ALA

-20 nA--

1 min Fig. 4. Effect of 5-HT on p-atanirte-fB-ALA)induced inhibition of spontaneous activity of a Purkinjc cell. (A) Control responses to iontophoretic pulses of /i-alanine (20 nA). (B), (B’) Effects of 5-HT (IO nA) short-term. less than hmin (B) and long-term, greater than Xmin (B’) on ~~-alanine-mediated inhibition. (C) Recovery responses to /j-alamne.

50% of the cells (three of the six cells). A common finding was that the effectiveness of NE to augment GABA inhibition during the initial phase of the response appeared to correlate with the effect of NE on the spontaneous firing rate. In other words. cells whose spontaneous rates were depressed also displayed augmented responses to GABA; and those in which the spontaneous rates were increased by NE displayed attenuated GABA responses. The magnitude of the augmentation of GABA-mediated inhibition in the presence of NE was 20.7 & 7.2% (n = 3) with a corresponding decrease of 13.7 f 1.7% in the spontaneous firing rate. The remaining three cells showed an attenuation of GABA-mediated inhibition during the initial period, averaging 54.0) 10.3%, and a concomitant 8.3 t_ 5.8% increase in their spontaneous firing rate. During the secondary phase, five of the six cells (83% of cells tested) displayed a marked enhancement of GABAmediated inhibition in the presence of NE, averaging 87.0 k 30.5%, with a corresponding 15.2 f 6.7% decrease in spontaneous activity. The response to GABA of the sixth cell was attenuated by NE. In all six Purkinje cells, 5-HT decreased GABAmediated inhibition during the initial phase, while augmenting GABA inhibition during the secondary phase. During the initial phase. 5-HT decreased GABA-mediated inhibition by an average of 26.7 f 4.6%, with a corresponding 7.2 f 3.1% decrease in the spontaneous firing rate. Comparable to NE-induced augmentation during the secondary phase, 5-HT produced an average 90.5 & 11.8% increase in GABA-mediated inhibition, while decreasing the spontaneous rate by 17.3 i 8.3%.

Efect

oj’various

the serotonin-GABA

serotonin

untagonists

und sotalol

on

responses

In an attempt to characterize the receptors responsible for the influence of 5-HT on GABA-elicited inhibitions, various 5-HT antagonists were superimposed on the secondary phase of the serotoninGABA interactions. Continuous applications of the mixed 5-HT,, and 5-HT, antagonist spiperone (n = 8 cells), the 5-HT, antagonist ketanserin (n = 9 cells), and the mixed classical antagonist methysergide (n = 9 cells) failed to antagonize the effects of 5-HT on GABA inhibitions. In contrast, the actions of these compounds often appeared to be additive to the effects of 5-HT. To test the possibility that 5-HT may in some fashion utilize intrinsic noradrenergic neurotransmission to augment GABA-elicited inhibitions, the P-adrenergic antagonist sotalol was used. Sotalol (n = 6 cells), at iontophoretic ejection currents that markedly reduced NE-elicited enhancements of GABA effects, failed to affect 5-HT actions. Similar to the 5-HT antagonists, sotalol frequently augmented the actions of 5-HT. Therefore. noradrenergic transmission does not appear to be involved in the actions of 5-HT.

Charucteri~ation mediated

of serotonerSyk

inhibitions

of’ Purkinje

it$uew~~

oj G.-I BA

-

neurons

This study demonstrated that the influence of5-HT on GABA-mediated inhibition consisted of a biphasic response pattern in which 5-HT initially decreased GABA-mediated inhibition, and secondarily. 5-HT either continued to inhibit. or most frequently. elicited an augmentation of the GABA response. These data reinforce our previous suggestion that serotonin has an important modulatory role in the cerebellum. We previously showed that serotonin exerts a modulatory influence on amino acid-induced excitation, in that glutamate excitations w’ere antagonized by serotonin at currents that were ineffective in altering spontaneous activity.‘” Overall, our studies appear to indicate that the modulatory effects of serotonin may serve a biasing role to set the cell at a preferred firing rate, restricting or amplifying the magnitude of excitatory or inhibitory influences on Purkinje neurons. One possibility that could explain the initial 5-HT-elicited attenuation of GABA inhibition is an occlusion phenomenon due to sharing of a common ionic mechanism by GABA and 5-HT. The inhibitory action of GABA on cerebellar Purkinje neurons was shown to be related to an increase in K + conductance, as well as Cl conductance.2” It was also reported that inhibitory actions of 5-HT were mediated by increased K’ conductance in neurons of the dentate gyrus and CA1 regions of hippocampus; however. 5-HT mediated hyperpolarization of neurons in the CA3 region of the hippocampus was not related to increased K + conductance.‘T It was demonstrated electrophysiologically that a G-protein couples 5-HT and GABA, receptors to the same K + channels in the hippocampus.’ Furthermore, 5-HT-mediated inhibitions were antagonized by GABA antagonists within the cerebellar cortex.“.‘3 visual cortex and and hippocampus.‘” These studies hypothalamus.‘h indicate that there may be a common mechanism for inhibitory actions of 5-HT and GABA in some areas of the nervous system, which would lend support to the suggestion that occlusion of GABA and 5-HT inhibitions initially occurs within the cerebellum. During the secondary phase of the biphasic rcsponse, continuous 5-HT either markedly augmented GABA-mediated inhibition in two thirds of the cells tested, or attenuated the inhibitions in the remaining cells. The present data reveal that the effects of 5-HT may depend, in part, on the spontaneous firing rates of Purkinje cells. Serotonin would more likely augment GABA-mediated inhibitions in cells displaying higher initial frequencies. The same general correlation was established previously when the direct effects of 5-HT were compared with the initial spontaneous discharge rate of Purkinje cells.“ In that study, we showed that Purkinje neurons that rcsponded to 5-HT with an increase in firing rate had

123

Modulatory role of serotonin on GABA inhibition a significantly slower predrug firing frequency than those cells that were suppressed by 5-HT, implying that the direct effects of 5-HT in depressing faster firing cells may result, in part, from an augmentation of the intrinsic GABAergic system. It is not clear as to the exact mechanism responsible for the 5-HTiGABA interaction, although it has been reported that macroscopic conductance evoked by GABA varies with membrane potentialb.‘x such that more conductance was generated by GABA at depolarized potentials, presumably due to the opening of more channels for a longer time. Thus, any slight changes in membrane potential induced by 5-HT. changes that could be dependent upon the initial firing frequency, may produce marked alterations in GABA effectiveness. C‘harocterization a-&mine

of’

the

irzfiuence

and glycine-mediated

of’ serotonin

on

inhibitions

Continuous 5-HT initially attenuated glycinemediated inhibition as seen with GABA; however, during the secondary phase, 5-HT continued to attenuate glycine-mediated inhibitions, rather than augmenting the inhibition. These results would initially appear to suggest that the augmentation observed during the secondary phase may be selective for GABA-mediated inhibitions, whereas the initial phase may involve a mechanism common to both amino acids. A previous study using cultured spinal neurons suggested that GABA and glycine may share the same ion selectivity, implying that a single species of a multistate Cl channel exists as the effector coupled to both GABA and glycine receptors.‘” Our study shows that 5-HT interacts similarly with both glycine and GABA systems producing initial attenuated effects. These data could support the findings that glycine and GABA activate a similar ionic channel and attenuation of amino acid-elicited inhibition seen with 5-HT may be the result of an occlusion phenomenon. It is of interest that a distinct separation of effects was observed during the secondary phase of the response in which only the GABA response was augmented, implying that the augmentation of GABA in the presence of 5-HT was unique to the GABA system and involved another mechanism. On the other hand, 5-HT influenced p-alaninemediated inhibitions in a manner similar to those elicited by GABA. This finding was unexpected since Curtis and his colleagues’ described fi-alanine as a “glycinc-like” agent and, as is the case with glycine, /I-alanine effects are antagonized by strychnine. The similarity of action between GABA and /J-alanine during the secondary phase may possibly be explained by potcntiation of the intrinsic GABAergic system. based on the reported action of /I-alaninc in blocking GABA uptake.” Thus, the augmentation by serotonin of amino acid-elicited inhibition is more commonly seen with GABA. then /I-alanine. and

never with glycine. Alternatively, a-alanine affect glycine receptors on Purkinje cells.

of effects of

Comparison

on GABA-mediated

norepinephrine

may not

and serotonin

inhibitions

Similar effects of 5-HT on GABA-mediated inhibition of somatosensory cortical neurons were reported by Woodward et al.46 These investigators also demonstrated that norepinephrine and locus coeruleus stimulation augmented electrically evoked and GABA-mediated inhibitions in the cerebral cortexJY “+s and cerebellum.‘~‘y~4’ We found that 5-HT and NE produced marked augmentations of GABAmediated inhibitions during the secondary phase; whereas, during the initial phase. 5-HT showed a predominant decrease in GABA-mediated inhibition and NE displayed a mixed response. Analogous to the present study, Waterhouse and colleague? reported that NE facilitated GABA-mediated inhibition in 81% of the cells, antagonized GABA inhibition in 13% of cells, and failed to affect 6% of the cells; findings very similar to the present results. If we were to examine the overall response to GABA, irrespective of the duration of NE application. it could be concluded that NE-induced augmentation occurred sooner than did 5-HT-elicited augmentations. We do not believe that NE is involved in potentiation of GABA by 5-HT since sotalol, a beta-blocking agent shown to effectively block NE-induced augmentations of GABA.” failed to block 5-HT-elicited augmentations. In fact, this antagonist potentiated 5-HT-elicited augmentations of GABA inhibitions. Based on the present results using 5-HT antagonists, the 5-HT receptor rcsponsible for augmenting GABA-mediated inhibition does not appear to be of the 5-HT,, or 5-HT, subtype. Proposed

site

GABA-serotonin

und ,fimctionul

.signjfiwmv

for

the

interaction

A series of findings obtained in our laboratories indicate a close interaction between the GABAcrgic and serotonergic systems in the cerebellum. Our earlier studies indicated that the inhibitory cffccts of S-HT were consistently blocked by picrotoxin.” whereas the classical 5-HT antagonists were ineffective in antagonizing the 5-HT-elicited inhibition in the cerebellum.” Moreover, pentobarbital given systemically and iontophoretically potentiated the magnitude and duration of 5-HT- and GABAmediated inhibitions of Purkinje cells. and often reversed 5-HT excitation to that of inhibition.” Olsen” divided the GABA receptor/Cl ion ch:innel complex into various subunits, one of which is the picrotoxin;barbiturate modulatory site. Barbiturates modulate GABA. in part by prolonging the lifetime of GABA-activated Cl ion channels in the postsynaptic membrane.” yielding an increase in GABAmediated Cl flux. In addition. barbiturates wcrc

I24

J (‘.

Clxhttl t.YIX)Kt~ <‘Iiii.

reported to enhance the afhnity of membrane binding sites for GABA receptor agonists’,“,‘“,” by converting a low-affinity state to a high-affinity state.” This enhancement of GABA binding appears to relate to prolongation of the GABA-activated Cl channel lifetime. The GABA antagonist picrotoxin has been reported to act on the GABA receptor-linked Cl ion channels.“~” Based on these findings, serotonin may act on the same receptor-effecter complex as GABA, and thus share common pharmacological sensitivities for agents that act on the picrotoxin;barbituratc nlodulatory site for GABA. This suggestion could explain the ineffectiveness of the classical S-HT antagonists, on one hand. and the effectiveness of pentobarbital and picrotoxin, two GABAergic agents thought to act on ionophores associated with the GABAergic system. in altering S-H-T-mediated inhibitions on cerebellar Purkinje cells.

CONCLUSIONS

Our data do not directly address mechanisms involved in the effect of 5-HT on GAEA responses. Pertinent questions which remain unanswered concern which ionic currents are involved in the effects of S-HT and the possibility that different 5-HT receptor subtypes may mediate direct and modulatory effects of 5-HT. Classification of 5-HT receptors is rapidly evolving yet significant limitations are imposed by the lack of specificity of many 5-HT ligands. In an effort to uncover mechanisms underlying the complex effects of 5-WT on Purkinje cells,

we have begun current and voltage clamp rccordmgs of these neurons in the in z’itrn slice preparation Our present results raise some intriguing poss~bilities concerning the role of 5-I-IT as a modulator of cerebeiiar physiology and suggest that thz serotonergic innervation of the cerebellum may constitute part of a larger physiologic control system. Previously, we demonstrated that electrical stimulation of presumed serotonergic afferents in the raphe nuclei induced excitation and inhibition of Purkinje cells and fastigial nuclear cells.“‘.” Moreover, off-beam inhibition of Purkinje cells was markedly enhanced at stimulation strengths which elicited minimal effects on spontaneous activity.?’ We have also shown a facilitatory action of S-HT on complex spike-like bursts recorded from Purkinje celts.34 Given that the inferior olive also receives a significant serotonergic input,“4 our collective results would support a role of S-HT as a modulator of brainstem ccrebellar activity. Additional studies examining the neurophysiology of this system are definitely warranted; however, such studies will be difficult in view of recent reports demonstrating that cerebellar serotonergic afferents arise primarily from diffuse regions around and including nucleus gigantocellularis and paragigantocellularis3 and that a significant number of these afferents contain both 5-HT and GABA.lX Aeknowf~~~e~e~fs-This work was supported by NIH Grant NS 19296 to JCS and HKS. The authors thank Randal Pierce for computer programming and Etta Powell for assisting in the preparation of the manuscript. We extend our appreciation to Mead Johnson Pharmaceuticals and Sandoz Pha~a~uti~als for their generous supply of sotalol and methysergide, respectively.

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