Brain Research, 552 (1991) 53-57 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116683A

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The effects of G A B A and benzodiazepines on neurones in the suprachiasmatic nucleus (SCN) of Syrian hamsters Robert Mason 1, Stephany M. Biello 2 and Mary E. Harrington 2 1Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham (U. K.) and 2Department of Psychology, Smith College, Northampton, MA 01063, (U.S.A.) (Accepted 8 January 1991) Key words: y-Aminobutyric acid; Benzodiazepine; Suprachiasmatic nucleus; Electrophysiology; Circadian rhythm

Administration of benzodiazepines at appropriate times in the circadian cycle induce phase-shifts in circadian locomotor activity. The possibility that benzodiazepine-induced shifts are mediated at the level of the suprachiasmatic nuclei (SCN), identified as the circadian pacemaker in mammals, was examined electrophysiologically. Extracellular recordings were made from Syrian hamster (Mesocricetus auratus) hypothalamic SCN neurones in vitro to assess (1) the effects of y-aminobutyric acid (GABA) on SCN neuronal activity and (2) the effects of benzodiazepines (chlordiazepoxide and flurazepam) on GABA-evoked responses. Of 93 SCN cells tested, 86 were suppressed by iontophoresed GABA (20 mM) in a current(dose)-dependent manner, while 6 were unaffected; suppression was found during both the projected light and dark phases of the circadian cycle. Application of bicuculline methiodide alone elevated mean discharge activity, while GABA-evoked suppressions were blocked by bicucuUine (n = 9/11 cells). Iontophoresis of chlordiazepoxide or flurazepam (20 mM; 1-10 nA) alone produced a current(dose)-dependent prolonged suppression of cell firing which was antagonised by bicuculline. These results indicate that benzodiazepine/GABA-evoked responses are at least partially mediated by GABAA receptors within the SCN and suggest the SCN may be a possible locus for the action of benzodiazepines in their induction of phase-shifts in circadian function.

INTRODUCTION The hypothalamic suprachiasmatic nuclei (SCN), identified as the circadian pacemaker in mammals 21'3°, regulates a range of physiological and behaviourial rhythms. Administration of benzodiazepines at appropriate times in the circadian cycle induces phase-shifts (either phase advances or phase delays) in circadian locomotor behaviour 16,27,39. These phase-shifts are blocked by pretreatment with benzodiazepine antagonists (Flumazenil, R o 15-1788) or G A B A A receptor antagonists (bicuculline) 16'26'27'39. Benzodiazepines potentiate G A B A (~,-aminobutyric acid) neurotransmission at GABAA-benzodiazepine receptors 7'23'31 and so implicate the neurotransmitter G A B A in the modulation of circadian rhythmicity 27. The G A B A synthetic enzyme, glutamic acid decarboxylase ( G A D ) , and G A B A itself are present in the SCN 25'41. There is a high density of G A D and G A B A - i m m u n o r e a c t i v e terminals within the SCN 4, 5,22,25,40,41 and G A B A e r g i c cell bodies have been identified in the SCN 22'40"41. This intrinsic SCN G A B A e r g i c system may be involved in interneuronal local circuit

communication within the SCN and as such a target for drugs acting on G A B A neurotransmission. The G A B A A receptor is a component of a supermolecular receptor complex with binding sites for G A B A , benzodiazepines, barbiturates, picrotoxin and steroid anaesthetics 3"23'31. G A B A at these receptors gates the conductance of chloride ions through a chloride channel associated with the complex 3'23,31. A second type of G A B A receptor, the G A B A B receptor, is not associated with either a chloride channel or benzodiazepine binding sites 23 and is also found in the SCN where they appear to modulate the release of 5-HT from raphe terminals 4'6. The possibility that the benzodiazepine-induced shifts are mediated at the level of the SCN is addressed electrophysiologically in this paper by examination of the effects of locally applied G A B A and water-soluble benzodiazepines (chlordiazepoxide and flurazepam). These benzodiazepines cause phase-shifts in activity rhythms 2'16 similar to those obtained with triazolam, a short-acting but water-insoluble benzodiazepine 39. A preliminary report has appeared elsewhere in abstract form 17.

Correspondence: R. Mason, Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K.

54 responsive only if they showed a consistent incrcasc or decrease in firing rate on repeated tests.

MATERIALS AND METHODS Male Syrian hamsters (Mesocricetus auratus) were housed in a 12-h light-12-h dark cycle (LD12:12, n = 32); lights-on corresponds to 00.00 h. On the experimental day, at different times during the LD cycle, animals were anaesthetized with halothane and decapitated. The brain was trimmed to a block containing the hypothalamus and coronal hypothalamic slices (450-500/~m) containing the SCN were cut. Slices were transferred to a brain slice chamber ~8"19 and maintained at 35° + 0.5 °C at the gas/fluid interface between warm humidified O2-CO2 (95%:5%) and artificial cerebrospinal fluid (aCSF, in mM: NaCI 124, KCI 3.3, KH2P04 1.2, CaCI 2 2.5, MgSO 4 1, NaHCO 3 2.5, glucose 10) at pH 7.4. ExtraceUular recordings were obtained with single-barrel or 5-barrel micropipettes, the recording barrel containing 2% Pontamine sky blue in 0.5 M NaCI. The side-barrels were each filled with one of the following: 5-hydroxytryptamine creatinine sulphate (5-HT: 2 mM in physiological saline [165 mM NaCI], Sigma), GABA (20 mM, pH 4, Sigma), bicuculline methiodide (20 mM, pH 3.5, Sigma), chlordiazepoxide-HCl (20 mM; pH 5, Roche) or flurazepam-HCl (20 mM; pH 5, Roche). The competitive benzodiazepine antagonist Flumazenil (Ro 15-1788, 10-5 M in DMSO) was administered via the perfusion line. In some experiments independently manipulated recording and microiontophoretic pipettes were used; the drug-containing 5-barrel pipette was positioned 20-100/am away from the recording pipette and then lowered into the slice until the shortest latency to the onset of the response was obtained. Micropipettes were guided to the SCN under visual control with the aid of a trinocular stereo-microscope, the SCN being clearly seen as paired opaque ovoid structures situated in the mid-line and dorsal to the optic chiasma. Electrophysiological recording, display and processing of signals was conventional~5. Discriminated action potential spikes were counted in successive 5-s epochs by an electronic rate meter with an output to a chart recorder 15. Drugs were delivered iontophoretically using a laboratory-built solid-state microiontophoresis instrument. Retaining currents (-10 nA) were used routinely with continuous automatic current balancing between ejections via a side-barrel containing 2% Pontamine sky blue in 0.5 M NaCI. Ejection currents ranged between 0-100 nA for examining current (dose-)response relationships. The effects of drugs on the spontaneous discharge of SCN neurones were assessed at all daily phases of the circadian cycle. Cells were considered SCNvl

RESULTS

Effects of GABA and bicuculline T h e s p o n t a n e o u s discharge activity in 86/93 SCN n e u r o n e s tested was s u p p r e s s e d b y i o n t o p h o r e s e d G A B A in a c u r r e n t ( d o s e ) - d e p e n d e n t m a n n e r (Fig. 1); the rem a i n i n g 6 cells were u n a f f e c t e d . This G A B A - e v o k e d s u p p r e s s i o n was o b s e r v e d d u r i n g b o t h the p r o j e c t e d light (n = 51/54) a n d d a r k phases (n = 35/39) of the circadian cycle. T h e r e was n o e v i d e n c e of S C N n e u r o n e s exhibiting a circadian v a r i a t i o n in sensitivity to i o n t o p h o r e s e d G A B A , s u p p o r t i n g o b s e r v a t i o n s in vivo t5 a n d in vitro 13 in the rat. G A B A - e v o k e d r e s p o n s e s were f o u n d t h r o u g h o u t the SCN, i n c l u d i n g the r e t i n o - h y p o t h a l a m i c termin a t i o n r e g i o n a n d n o n - v i s u a l r e c i p i e n t zones of the SCN. T h e firing p a t t e r n s of s p o n t a n e o u s l y discharging SCN n e u r o n e s have b e e n c h a r a c t e r i s e d as r e g u l a r (type I), irregular (type II) a n d burst firing (type 11I) 19'32'38. D u r i n g this study cells with all firing p a t t e r n types were f o u n d to be i n h i b i t e d b y i o n t o p h o r e s e d G A B A . I o n t o p h o r e s i s of the G A B A A a n t a g o n i s t bicuculline m e t h i o d i d e a l o n e e l e v a t e d the m e a n discharge activity in the m a j o r i t y of cells (n = 21/24) e x a m i n e d (Fig. 1). This i n c r e a s e d firing suggests the tonic release of G A B A from intrinsic G A B A e r g i c n e u r o n e s w i t h i n the SCN. T h e G A B A - e v o k e d suppressions w e r e b l o c k e d by c o n c u r r e n t e j e c t i o n of bicuculline (n = 15/19 cells; Fig. 1). Similar o b s e r v a t i o n s have b e e n r e p o r t e d in the rat SCN 13'38. Flurozepom

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Fig. 2. Upper panel: integrated firing rate histogram for a SCN neurone illustrating the current(dose)-dependent nature of the inhibitory response to iontophoresis of benzodiazepines. Note the prolonged suppression at the higher (20 nA) ejection current. The smaller flurazepam-evoked suppression of firing obtained with a lower ejection current (5 hA) is blocked by concurrent ejection of bicuculline methiodide (10 nA). Lower panel: the current(dose)-dependent nature of the flura,lepam-evokedsuppression of spontaneous activity with respect to both% suppression and duration of suppression.

Effects of benzodiazepines

DISCUSSION

Iontophoretically applied chlordiazepoxide and flurazepam, at response subthreshold ejection currents which failed to alter firing, enhanced GABA-evoked suppression of SCN neuronal discharge activity (Fig. 1). At higher (supra-threshold) iontophoretic currents, chlordiazepoxide or flurazepam ejection alone produced a prolonged suppression of spontaneous firing in SCN neurones (n = 27/35 cells; Fig. 2). Both the magnitude of the benzodiazepine-evoked suppression and its time course to recovery were dependent on the ejection current (Fig. 2, lower panel). This effect was specific to GABA as inhibitions induced by iontophoresed 5-HT were not affected by simultaneous ejection of flurazepam or chlordiazepoxide (n = 6 cells). Addition of the competitive benzodiazepine antagonist Flumazenil (Ro 15-1788; 10-5 M) or concurrent ejection of bicuculline blocked the suppression in firing evoked by iontophoresed flurazepam (n = 5 cells; data not shown). The benzodiazepine enhancement of the GABAevoked responses in the SCN was observed during both the light and dark phases of the circadian cycle. No quantitative evaluation of a possible circadian variation in the efficacy of the benzodiazepine potentiation of GABA-evoked responses was attempted during the present investigation.

The present results provide evidence for the SCN, identified as a circadian pacemaker, as a possible locus for the action of benzodiazepines in their induction of phase-shitts in circadian behaviour 16'26'35'39. The majority of SCN cells tested were suppressed by iontophoresed G A B A in a current(dose)-dependent manner; suppression was found during both the projected light and dark phases of 1he circadian cycle. Application of the G A B A A antagonist bicuculline methiodide elevated mean discharge activity and blocked GABA-evoked suppressions. Iontophotesis of the benzodiazepines chlordiazepoxide or flurazopam produced a current(dose)-dependent prolonged suppression of cell firing which was also antagonised by bicuculline, indicating that the benzodiazepineevoked responses are at least partially mediated via GABA~, receptors within the SCN. Similar effects of GABA aid benzodiazepines applied by bath superfusion have recontly been reported in the rat SCN in vitro 13. There '.are at least three possible sites at which benzodia~epines could mediate their effects on phaseshifting circadian activity rhythms; the SCN, the intergeniculat(t leaflet of the thalamus (IGL) and/or the raphe nuclei. The SCN receives a terminal innervation from NPY-conqaining IGL neurones 5'9 and an overlapping i

56 serotonergic innervation from the raphe nuclei 5. The I G L itself also receives a serotonergic innervation from the raphe 24. There is reported to be a convergent innervation of G A B A and 5-HT terminals on VIP-containing neurones within the SCN 4, further N P Y axon terminals also make synaptic contacts with VIP-immunoreactive SCN neurones 1°, some target neurones receiving both NPY and 5-HT synaptic terminals 8"12. These immunocytochemical identified synaptic junctions were observed to make axo-axonal appositions 8 and/or contact with dendrites or perikarya 8'1°'12 in the SCN. This provides an anatomical substrate for multiple synaptic interactions between these different SCN neurotransmitter systems. The suprachiasmatic pacemaker itself has been examined in the present electrophysiological study which has demonstrated that many SCN neurones in the Syrian hamster are responsive to exogenously applied G A B A and support similar observations made in vitro and in vivo in the rat SCN 15'32'38. In addition benzodiazepines augment G A B A - e v o k e d suppression of SCN firing and reduce SCN firing. The elevation in spontaneous neuronal discharge activity in the SCN mediated by bicuculline suggests an ongoing tonic release of G A B A from G A B A e r g i c neurones within the SCN. This interpretation would also explain the suppression in SCN neuronal discharge produced by benzodiazepine ejection alone (Fig. 2). These direct inhibitory effects of benzodiazepines are not seen in recordings from serotonergic raphe neurones 7'42 (also Mason and Garratt, unpublished observations). This latter observation suggests that benzodiazepine-induced phase-shifts at the behavioural level are unlikely to be mediated via raphe projections to the SCN. Further, G A B A modulation of 5-HT release from raphe terminals in the SCN is mediated via G A B A B receptors, not by a GABAA-benzodiazepine receptor 4'6. While L G N v and I G L neurones have both functional G A B A A and G A B A B receptors, optic nerve stimulation evokes specifically G A B A A - m e d i a t e d IPSPs in these cells 36. I G L / L G N v neurones recorded in vitro exhibit a

low spontaneous discharge which was elevated in the presence of bicuculline (Mason, unpublished observations), further indicating G A B A A receptors at this site. The effect of benzodiazepines on I G L neurones requires examination, although autoradiographic studies indicate little or no benzodiazepine binding in the I G L TM. There may be differential effects of benzodiazepines on the SCN and IGL. Regional differences in the sensitivity of neurones to G A B A following benzodiazepine treatment have been documented t4"42, perhaps reflecting molecular heterogeneity in GABA-benzodiazepine receptor subunit composition and associated variations in affinity for ligands 3~3J" In preliminary studies we have observed a shift in the timing of the peak in the circadian variation in SCN neuronal firing rate recorded in vitro ~9. induced by benzodiazepine administration in vivo 1-3 days prior to recording 17. This shift may be a consequence of benzodiazepines acting at the I G L and/or the SCN in vivo prior to SCN recording. Lesions of the I G L / L G N v prevent the behavioural phase-shifts induced by benzodiazepines 2"~. This apparent loss of sensitivity to benzodiazepine treatment could result from the destruction of target neurones in the I G L and/or a subsequent change in the modulation of NPY or enkephalin neurotransmission from I G L afferents in the SCN 9'19'37. The SCN exhibits a circadian variation in its sensitivity to stimulation of the I G L and N P Y both behaviourally 1'28 and electrophysiologically ~9" 33, a property which may underlie the variation in magnitude and direction of phase-shifts induced by benzodiazepines administered at different circadian times. Further experiments are required to investigate if the sensitivity of SCN neurones to G A B A and/or benzodiazepines is altered following I G L lesions and if NPY modulates GABA/benzodiazepine responses in a time-dependent fashion. Acknowledgements. This work was supported in part by grants from the Royal Society (U.K.) and Smith Kline (1982) Foundation.

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The effects of GABA and benzodiazepines on neurones in the suprachiasmatic nucleus (SCN) of Syrian hamsters.

Administration of benzodiazepines at appropriate times in the circadian cycle induce phase-shifts in circadian locomotor activity. The possibility tha...
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