Somatostatin inhibits GABAergic transmission in the sensory thalamus via presynaptic receptors

Inhibition of GABAergic transmission by somatostatin

Pergamon PII: S0306-4522(00)00107-X

Neuroscience Vol. 98, No. 3, pp. 513–522, 2000 513 䉷 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

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SOMATOSTATIN INHIBITS GABAERGIC TRANSMISSION IN THE SENSORY THALAMUS VIA PRESYNAPTIC RECEPTORS N. LERESCHE,* E. ASPRODINI,†‡ Z. EMRI,†§ D. W. COPE† and V. CRUNELLI†k *Neurobiologie Cellulaire, Institut des Neurosciences, UMR CNRS 7624, Universite´ Pierre et Marie Curie, Paris, France †School of Biosciences, Cardiff University, Cardiff, U.K.

Abstract—The action of somatostatin on GABA-mediated transmission was investigated in cat and rat thalamocortical neurons of the dorsal lateral geniculate nucleus and ventrobasal thalamus in vitro. In the cat thalamus, somatostatin (10 mM) had no effect on the passive membrane properties of thalamocortical neurons and on the postsynaptic response elicited in these cells by bath or iontophoretic application of (^)baclofen (5–10 mM) or GABA, respectively. However, somatostatin (1–10 mM) decreased by a similar amount (45–55%) the amplitude of electrically evoked GABAA and GABAB inhibitory postsynaptic potentials in 71 and 50% of neurons in the lateral geniculate and ventrobasal nucleus, respectively. In addition, the neuropeptide abolished spontaneous bursts of GABAA inhibitory postsynaptic potentials in 85% of kitten lateral geniculate neurons, and decreased (40%) the amplitude of single spontaneous GABAA inhibitory postsynaptic potentials in 87% of neurons in the cat lateral geniculate nucleus. Similar results were obtained in the rat thalamus. Somatostatin (10 mM) had no effect on the passive membrane properties of thalamocortical neurons in this species, or on the outward current elicited by puff-application of (^)baclofen (5–10 mM). However, in 57 and 22% of neurons in the rat lateral geniculate and ventrobasal nuclei, respectively, somatostatin (1 mM) reduced the frequency, but not the amplitude, of miniature GABAA inhibitory postsynaptic currents by 31 and 37%, respectively. In addition, the neuropeptide (1 mM) decreased the amplitude of evoked GABAA inhibitory postsynaptic currents in 20 and 55% of rat ventrobasal neurons recorded in normal conditions and during enhanced excitability, respectively: this effect was stronger on bursts of inhibitory postsynaptic currents(100% decrease) than on single inhibitory postsynaptic currents (41% decrease). These results demonstrate that in the sensory thalamus somatostatin inhibits GABAA- and GABAB-mediated transmission via a presynaptic mechanism, and its action is more prominent on bursts of GABAergic synaptic currents/potentials. 䉷 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: sleep, epilepsy, baclofen, IPSC, IPSP.

Somatostatin is present in peripheral and central neurons and has been proposed to act as a neurotransmitter and a neuromodulator, 17,39 either via a direct action on postsynaptic neurones 9,23,28,36,51 or via presynaptic regulation of transmitter release. 7,18,19,26 Since in a number of brain areas, and in particular in the hippocampus, GABA and somatostatin are colocalized, 44 various studies have investigated the modulatory effects of somatostatin on GABAergic synaptic transmission. Although a reduction in both GABAA and GABAB inhibitory postsynaptic potentials (IPSPs) by somatostatin had been reported in earlier studies, 38,57 recent investigations analysing pharmacologically isolated synaptic currents have failed to reveal an effect on GABAA and GABAB IPSCs but indicated instead a clear presynaptic inhibition of glutamatergic synaptic currents. 6,48 In subicular neurons, somatostatin has been shown to decrease GABAA IPSPs indirectly (i.e. via a shunt of the postsynaptic membrane) and GABAB IPSPs via an action ascribed to a decreased synaptic conductance. 21 Furthermore,

in the dorsal septal nucleus, where co-localization of somatostatin and GABA has not been described, this neuropeptide inhibits GABAA and GABAB IPSPs through a postsynaptic mechanism. 52 Notwithstanding the fact that these different results may depend on the brain area under investigation, it remains unclear whether somatostatin is indeed capable of modulating GABAergic transmission by a presynaptic mechanism. In sensory thalamic nuclei, the main source of GABA is from either interneurons (located within a nucleus) and/or neurons of the nucleus reticularis thalami (NRT), a thin shell-like nucleus surrounding the lateral/anterior part of the main thalamic mass. 24 The role that these two types of GABAergic afferents play in the output of thalamocortical (TC) neurons spans from the processing of sensory and cortical information to the generation/modulation of behavioural state-dependent oscillatory activities. 24,45 The majority of the GABA-containing neurons in the cat NRT shows the presence of somatostatin-like immunoreactivity. 20,30 Autoradiographic studies have shown the presence of somatostatin receptors in the rat sensory thalamic nuclei and the NRT, 53 although Bodenant et al. 5 observed only a transient expression (up to postnatal day 21) in the dorsal lateral geniculate nucleus (LGN). Recent in situ hybridization experiments have shown a widespread presence of mRNA for somastostatin receptors-type 3 (SSTR-3) within the adult rat thalamus, including the NRT, 33,40,49 and with the use of a SSTR-5 antibody, strongly labelled cell bodies have been detected in the rat NRT with almost no labelling in the adjacent thalamic sensory nuclei. 47 In constrast, none of the thalamic nuclei appeared to be hybridized by a SSTR-1 probe and only a

kTo whom correspondence should be addressed. Tel.: ⫹ 44-1222-874091; fax: ⫹ 44-1222-874986. E-mail address: [email protected] (V. Crunelli). ‡Present address: Department of Pharmacology, University of Thessaly, Larisa, Greece §Present address: Department of Neuroanatomy, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary. Abbreviations: APV, dl-2-amino-5-phosphonovaleric acid; CNQX, 6cyano-7-nitroquinoxaline-2,3-dione; GYKI, 1-(4-aminophenyl)-4-methyl7,8-methylene-dioxy-5h-2,3-benzodiazepine; IPSC, inhibitory postsynaptic current; IPSP, inhibitory postsynaptic potential; LGN, dorsal lateral geniculate nucleus; MK-801, dizocilpine maleate; NRT, nucleus reticularis thalami; SSTR, somatostatin receptor; TC, thalamocortical; TEA, tetraethylammonium chloride; TTX, tetrodotoxin; VB, ventrobasal thalamus. 513

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few of the medial thalamic nuclei were moderately labelled by a SSTR-2 probe. 33,40,49 Notwithstanding this information on the thalamic distribution of somatostatin and its receptors, no electrophysiological study (but see Ref. 41) has examined the effect of somatostatin on membrane properties and synaptic potentials/ currents in thalamic sensory nuclei. This study, therefore, was undertaken to investigate the effect of somatostatin on GABAergic transmission in two thalamic sensory nuclei which differ for their GABAergic innervation, i.e. the rat/ cat LGN and cat ventrobasal thalamus (VB), where the GABAergic input is from both interneurons and NRT cells, and the rat VB which lacks interneurons. 24 Our results indicate that somatostatin decreases GABAA- and GABAB-mediated transmission in the sensory thalamus via a presynaptic mechanism, and that this effect is more pronounced on bursts of, than on single, IPSPs/IPSCs. A preliminary report of some of these results has appeared in abstract form. 2 EXPERIMENTAL PROCEDURES

All the procedures involving experimental animals were carried out in accordance with the U.K. Animals (Scientific Procedure) Act, 1986 and associated guidelines. All efforts were made to minimize animal suffering and the number of animals used. Microelectrode recordings The preparation and maintenance of LGN slices were similar to those described previously. 11 In brief, cats (six to 200 days) were anaesthetized (2:1 O2:NO2 plus 1.2% halothane), and a block of tissue containing the LGN was then separated from the rest of the brain by two cuts made parallel to the plane of the optic tract. Alternatively, a block of tissue containing the VB and NRT was cut in the horizontal plane. 50 The block of tissue was glued to the stage of a Vibroslice (Campden Instruments), bathed in a continously oxygenated (95% O2, 5% CO2) cold medium containing (in mM): NaCl 134, KCl 5, KH2PO4 1.25, CaCl2 2, MgSO4 5, NaHCO3 16, and glucose 10. Slices (400–500 mm thick) were subsequently transferred to a storage bath, where they remained submerged in the same medium at room temperature until they were transferred to the recording chamber. At least 1 h following the preparation of the slices, a single brain slice was transferred to an interface-type bath, where it was superfused with warmed (35 ^ 1⬚C) medium of similar composition to the one used for slice preparation, except for the following (in mM): CaCl2 4, MgSO4 0.5 and KCl 3.25 (when investigating spontaneous IPSPs in the kitten and cat LGN), or KCl 2 and MgSO4 2 (when investigating passive membrane properties and evoked IPSPs in the cat LGN and VB). Standard intracellular recordings under current clamp were made using glass microelectrodes (Clark Electromedical Instruments, U.K.) filled with 1 M potassium acetate or 1 M potassium chloride. Potentials were recorded with an Axoclamp-2A amplifier (Axon Instruments), and stored on a Bio-Logic DAT recorder (Intracell, Royston, Herts, U.K.). Data were subsequently analysed using pCLAMP (V 6.1, Axon Instruments). Synaptic potentials were elicited with the use of a tungsten bipolar electrode placed in the NRT (for recordings made in the VB), or within the LGN. Two-barrel iontophoretic pipettes were prepared as previously described 12,56 and contained GABA (1 M, pH 3.7) and NaCl (1 M) as the balancing barrel.

pulled from borosilicate glass using a Narishige micropipette puller, and coated with wax. When filled with the electrolyte, they had a resistance of 2–2.5 MV. Once a high-resistance seal (⬎1 GV) had been established, the holding potential was set to ⫺60 mV and the whole cell configuration was obtained. Values of access resistance (which were checked repeatedly during each experiment) ranged from 5–8 MV at the beginning of the recording to 10–12 MV at the end (usually 30–60 min later). At least 60% of these values were compensated. Patch clamp electrodes were connected to an Axopatch 200B amplifier (Axon Instruments). Membrane currents were filtered by a 4-pole Bessel filter set at a corner frequency of 5 kHz, digitalized on-line at a sampling rate of 5 kHz using a Labmaster interface and stored on disk or on a DAT Biologic tape recorder for later analysis. Data were not collected until ⱖ10 min after patch rupture to allow the internal and external solutions to equilibrate. Voltage protocols, acquisition and analysis of data were controlled by pClamp software, version 5.5 (Axon Instruments). The passive properties were examined by giving repetitive hyperpolarizing pulses of 10 mV. Spontaneous synaptic currents were detected and analysed off-line using a program written by Dr P. Vincent and described in detail elsewhere. 54 All neurons were recorded at a holding potential of ⫺60 mV, except when stated otherwise. Evoked postsynaptic currents were elicited by electrical stimulation using a glass pipette (tip diameter: 6–8 mm), positioned 40–60 mm away from the recorded neuron, and a remote 100 mm-thick platinium wire. Square pulses of 50–100 ms duration and 10–40 V amplitude were applied at a frequency of 0.1 Hz. Statistics and drugs Data are expressed as mean ^ S.E.M., and statistical analysis was carried out using the paired Student’s t-test. Comparison of cumulative amplitude distributions was performed using non-parametric Kolmogorov–Smirnov statistics. The number of data points in each control distribution corresponds to the number of mIPSCs used to build up the corresponding histogram, usually calculated from a 3 min epoch. The significance level for the Kolmogorov–Smirnov test was set at a value of P ⬍ 0.05. Somatostatin was always bath applied, and in order to exclude a possible desensitization of the somatostatin receptors, only one application per slice was generally performed, during both patch and microelectrode recordings. In few cases where a second application of somatostatin was tested in the same neuron, its effect was within 5– 10% of the one evoked by the first application. Baclofen was applied either in the perfusing solution or locally through a glass pipette (tip diameter: 100 mm) placed about 100 mm away from the recorded neuron. In the latter case, the duration of the puff was electronically controlled by the pClamp software. In the experiments on the effect of somatostatin on GABA IPSPs/ IPSCs, 29 (out of 70) rat and cat thalamocortical neurons exibited only small (⬍ 10%) changes in the investigated parameter(s) during application of the neuropeptide. In the majority (80%) of these neurons (i.e. 23 out of 29 neurons), washout of somatostatin (for up to 40 min and 2 h during patch and microelectrode recordings, respectively) failed to show a recovery of the putative effect. These 29 neurons, therefore, were classified as non-responders. Somatostatin, bicuculline methiodide, (^)baclofen and tetraethylammonium chloride (TEA) were purchased from Sigma Chemicals (France and U.K.); 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), dl-2-amino-5-phosphonovaleric acid (APV) and (5R,10S)-( ⫹ )-5methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate (MK 801) from Tocris Cookson (U.K.); tetrodotoxin (TTX) from Laxotan (France) and Alomone Labs (Israel); and 1-(4-aminophenyl)4-methyl-7,8-methylene-dioxy-5h-2,3-benzodiazepine (GYKI 52466) from RBI (U.K.).

Patch electrode recordings For whole-cell patch-clamp recordings, LGN or VB slices (250 mm thick) were prepared from 10–14-day-old male Wistar rats as described previously. 25 The extracellular medium was continously oxygenated (95% O2, 5% CO2) and contained (in mM): NaCl 125, KCl 2.5, NaH2PO4 1.25, CaCl2 2, MgCl2 1, NaHCO3 26, and glucose 25 (pH 7.3). The intracellular solution contained (in mM): CsCl 108 or KCl 115, CaCl2 1, MgCl2 5, EGTA 10, HEPES 10, Na-ATP 4, Na-GTP 0.4, phosphocreatine 15 and creatine phosphokinase 50 units/ml (pH 7.3). Membrane currents were recorded at room temperature from the soma of TC neurons under Nomarski optics using a Nikon microscope equipped with a 40 × water immersion lens. Recording pipettes were

RESULTS

Action of somatostatin on membrane properties and evoked inhibitory postsynaptic potentials in the cat thalamus Intracellular recordings with microelectrodes were obtained from TC neurons in the kitten LGN (n ˆ 16), and the cat LGN (n ˆ 15) and VB (n ˆ 16). The resting membrane potential and input resistance of cat LGN and VB neurons were indistinguishible (⫺67 ^ 6 mV and 95 ^ 12 MV,

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Fig. 1. Somatostatin decreases the amplitude of GABAA and GABAB IPSPs but has no effect on the passive membrane properties of cat TC neurons. (A1 –A2) Family of voltage responses to current steps shows the lack of action of 10 mM somatostatin on the membrane properties of an LGN TC neuron. Data from this neuron were used to construct the voltage–current plot depicted in A3. Action potentials in A1 and A2 have been truncated for clarity. (B) Bath application of 10 mM somatostatin reduces the amplitude of the GABAA and GABAB IPSPs elicited in a cat LGN TC neuron. Each trace is the average of five consecutive events. Superimposition of the traces obtained in control and during somatostatin perfusion is shown on the right-hand side. This experiment was carried out in the presence of CNQX (10 mM), GYKI 52466 (100 mM), APV (100 mM) and MK-801 (10 mM).

respectively) (n ˆ 31, pooled cat LGN and VB data). The resting membrane potential and input resistance of TC neurons in the kitten LGN were ⫺58 ^ 5 mV and 188 ^ 18 MV (n ˆ 16), respectively. 35 Application of somatostatin (1–10 mM) did not produce any change in the resting membrane potential or input resistance of TC neurons (Fig. 1A1 –A3), although in three neurones a small hyperpolarization (2–4 mV) accompanied by a small decrease (12 ^ 3%) in input resistance was observed in the presence of the neuropeptide. In order to study the effects of somatostatin on evoked IPSPs in the sensory thalamus, cat LGN and VB slices were perfused with a medium containing CNQX (10–20 mM), GYKI 52466 (100 mM), APV (50–100 mM) and MK-801 (10 mM), which fully abolished excitatory synaptic transmission. Under these conditions, synaptic responses evoked by focal electrical stimulation within the LGN produced a fast GABAA IPSP followed by a slow GABAB IPSP. 12,13 TC neurons in the VB responded to stimulation of the NRT with a similar biphasic response, consisting of a GABAA and a GABAB IPSP. 16 Somatostatin affected the evoked IPSPs in five out of seven (71%) and in four out of eight (50%) TC neurons in the LGN and VB, respectively. Superfusion of somatostatin (5– 10 mM) reversibly reduced the amplitude of the fast GABAA IPSP in the cat LGN from 14.4 ^ 2.2 mV to 7.6 ^ 2.4 mV (P ⬍ 0.01, n ˆ 5), and in the cat VB from 18.8 ^ 3.1 mV to 10.4 ^ 2.6 mV (P ⬍ 0.05, n ˆ 4) (Fig. 1B). The slow GABAB IPSP was also reversibly reduced in the LGN from 8.2 ^ 0.3 mV to 4.1 ^ 1.4 mV (P ⬍ 0.01, n ˆ 5), and in the

VB from 9.9 ^ 1.8 mV to 4.5 ^ 1.4 mV (P ⬍ 0.05, n ˆ 4) (Fig. 1B). This effect of somatostatin on GABAA and GABAB IPSPs was observed in the same TC neurons. Action of somatostatin on spontaneous inhibitory postsynaptic potentials in the cat thalamus In kitten LGN TC neurons recorded in conditions of enhanced excitability (i.e. 4 mM Ca 2⫹ and 0.5 mM Mg 2⫹), we have previously described the presence of spontaneous GABAA receptor-mediated IPSPs, 35 occurring in bursts (Fig. 2B), as single events or both (Fig. 2A1, A2, Control). An action of somatostatin (1–10 mM) on these IPSPs was observed in 12 out of 14 TC neurons (85%). In three (out of the 12) neurons, superfusion of the neuropeptide abolished the bursts of IPSPs, an effect that was fully reversible following washout of the neuropeptide (Fig. 2B). In the remaining neurons, somatostatin still abolished the long bursts of IPSPs, although few isolated IPSPs were still present during perfusion with the neuropeptide (Fig. 2A1, A2). As shown in the bottom histogram of Fig. 2A3, the suppression of the bursts of IPSPs was clearly indicated by the abolition of the smaller (⬍0.6 s) inter-IPSP intervals (representative of the intra-burst period) and by the marked reduction in the number of IPSPs within the 0.6–1.0 s range of inter-IPSP intervals, while only small changes were observed in the longer (⬎1.1 s) inter-IPSP intervals (representative of the inter-burst period). Identical analysis performed on the

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Fig. 2. The action of somatostatin on spontaneous bursts of GABAA IPSPs. (A1 –A2) In the absence of somatostatin, spontaneous GABAA IPSPs, occurring as single events and in bursts (each 2–10 s long) were recorded in a kitten LGN TC neuron. Application of somatostatin (10 mM) reversibly reduced the amplitude and frequency of the IPSPs and almost fully abolished the long bursts of IPSPs, so that the spontaneous activity of this TC neuron was now characterized by few single IPSPs. Examples of the spontaneous bursts marked by an asterisk in A1 are reproduced at a faster time base in A2. Membrane potential: ⫺53 mV. (A3) Amplitude distribution (top histogram) of the GABAA IPSPs shows the reversible reduction produced by somatostatin (data from the same neuron as in A1 – A2). Cut-off amplitude: 1 mV; bin size: 2 mV (analysis performed on 5 min long sections of data). Inter-IPSP interval distribution (bottom histogram) for the same neuron as in A1 –A2. The somatostatin-induced suppression of the bursts of IPSPs is indicated by the absence of inter-IPSP intervals ⬍0.61 s. Cut-off amplitude: 1 mV; bin size: 0.2 s (analysis performed on 5 min long sections of data). (B) Intracellular voltage records show application of 10 mM somatostatin to abolish reversibly the bursts of GABAA IPSPs in another LGN TC neuron. The effect was fully reversible following washout of the neuropeptide. Membrane potential: ⫺50 mV.

data obtained from another four TC neurons confirmed the results illustrated in Fig. 2A3 (bottom histogram): i.e. 100% block of inter-IPSP intervals ⬍0.6 s and no change in the number of inter-IPSP intervals ⬎1.1 s. The mean reduction in the amplitude of the IPSPs that were still present during

superfusion with somatostatin was 45 ^ 6% (n ˆ 5) (Fig. 2A3, top histogram). In seven out of eight TC neurons (87%) from the cat LGN, where we have previously shown that in conditions of increased excitability (i.e. 4 mM Ca 2⫹ and 0.5 mM Mg 2⫹)

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Fig. 3. Somatostatin decreases the frequency, but not the amplitude, of GABAA mIPSCs in the young rat LGN. (A) Three consecutive traces, recorded in the different experimental conditions indicated above each record. Traces were sampled at 10 kHz and filtered at 3 kHz. The histograms in (B) show the mIPSC amplitude distributions for 4 min recordings in the same conditions as the traces in A. Somatostatin (1 mM) reversibly reduced the frequency of the mIPSCs by 30%. (C) The lack of effect of somatostatin on the amplitude distribution is demonstrated by the cumulative distribution amplitude curves (P ⬎ 0.05), although in this neuron a small change was observed following washout of the neuropeptide. TTX (0.5 mM), CNQX (10 mM) and APV (50 mM) were present in the perfusion medium throughout the experiment.

spontaneous GABAA IPSPs occur only as single events, 43 superfusion of somatostatin significantly reduced (39 ^ 5%) their amplitude from 5.1 ^ 0.8 mV to 3.1 ^ 1.0 mV (n ˆ 7; P ⬍ 0.01) (not shown). Action of somatostatin on miniature GABAA inhibitory postsynaptic currents in the rat thalamus A total of 39 TC neurons (26 in the VB and 13 in the LGN) was recorded in young rats using CsCl-filled patch electrodes, and their input resistance, measured in the first minutes of whole cell recording, was 398 ^ 93 MV (n ˆ 20). We have previously shown that, in the presence of TTX (0.5 mM),

CNQX (10 mM) and APV (50 mM), GABAA miniature IPSCs (mIPSCs), originating from the activation of local interneurons and NRT neurons, or NRT neurons only, occur spontaneously in rat LGN or VB slices, respectively. 25 In LGN TC neurons, application of 1 mM somatostatin reversibly decreased the frequency of mIPSCs by 31 ^ 5% in four out of seven neurons (57%) without affecting the amplitude distribution (Fig. 3). In VB TC neurons, application of 1 mM somatostatin only affected 22% of the recorded neurons (i.e. two out of nine) by decreasing (37 and 38%) the frequency, but not the amplitude, of mIPSCs. Surprisingly, in one of these two neurons the mIPSCs occurred at a frequency of 15 Hz, a value much higher than the one recorded in the

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Fig. 4. Effect of somatostatin on evoked GABAA-IPSCs recorded in rat VB TC neurons. (A1) Five superimposed traces illustrating an atypical synaptic response composed of multiple components evoked by a single electrical shock in condition of normal excitability. (A2) Superimposition of the averages of the traces presented in A1. Note that somatostatin (1 mM) reversibly decreased the amplitude of the first component by 53% and abolished the subsequent components of the burst of IPSCs. (B) Somatostatin (1 mM) reversibly decreased the amplitude of the evoked IPSC by 50%. Each trace is the average of 10 IPSCs recorded in the presence of 1 mM TEA. (C) Three superimposed traces illustrating the multiple components of the synaptic response evoked following a single electrical shock in the presence of 1 mM TEA. Note that, as for the neuron illustrated in A, somatostatin (1 mM) reversibly decreased the amplitude of the first component by 32% and abolished the subsequent components of the burst. CNQX (10 mM) and APV (50 mM) were present in the perfusion medium throughout the experiments in A–C.

other eight neurons (1.55 ^ 0.83 Hz) and in any other neuron recorded in our previous study under identical experimental conditions. 25 Action of somatostatin on evoked GABAA inhibitory postsynaptic currents in the rat thalamus To investigate further why only a few rat VB neurons responded to somatostatin, we tested the effect of the neuropeptide on GABAA IPSCs evoked by electrical stimulation of the NRT. Application of 1 mM somatostatin, however, was again capable of reducing the amplitude of evoked GABAA IPSCs only in a small number of neurons (two out of eight, 25%) (Fig. 4A1) as it did for the mIPSCs. Interestingly,

the evoked GABAA synaptic response recorded in one of the two neurons that responded to somatostatin was rather atypical as it was characterized by the occurrence of multiple components following a single electrical shock. Somatostatin decreased the amplitude of the first component by 54% and abolished the late components (Fig. 4A1), an effect reminiscent of the one observed on spontaneous bursts of GABAA IPSPs in the kitten LGN (cf. Fig. 2A, B). Thus, two out of the four TC neurons from the rat VB, in which an effect of somatostatin had been observed, were characterized either by GABAA mIPSCs occurring at an unusually high frequency or by a burst of evoked GABAA IPSCs. In order to investigate this further, we enhanced excitability in VB slices either by increasing the extracellular Ca 2⫹ concentration from 2

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areas), 27,31 we investigated the action of somatostatin against the effect of exogenous application of baclofen in order to exclude a direct action of somatostatin on the postsynaptic GABAB receptor-effector system. Bath application of (^)baclofen (80 mM) to TC neurons in the cat LGN produced a marked hyperpolarization, which was associated with a decrease (32 ^ 11%, n ˆ 3) in input resistance, as previously shown for TC neurons of the rat and cat LGN. 12,32,55 In these three neurons, bath application of somatostatin (10 mM) had no effect on the baclofen-induced hyperpolarization (Fig. 5A). In the rat, six LGN neurons were recorded with KCl-filled patch electrodes (input resistance: 310 ^ 148 MV) in the presence of TTX (0.5 mM), CNQX (10 mM), APV (50 mM) and bicuculline (30 mM). In these neurons, puff application of baclofen (10 mM) elicited a long-lasting outward current that had a peak amplitude of 57 ^ 6 pA (n ˆ 6). The amplitude and duration of the baclofen-induced outward current were not affected by a concentration of either 1 mM (n ˆ 1), 6 mM (n ˆ 2) or 10 mM (n ˆ 3) somatostatin (Fig. 5C). Finally, somatostatin (10 mM) had no effect on the response elicited by activation of postsynaptic GABAA receptors in 4 LGN TC neurons (Fig. 5B), indicating that, as in the rat, the action of this peptide on GABAA IPSPs in the cat is mediated via a presynaptic route. Fig. 5. Action of somatostatin on the postsynaptic response of TC neurons to GABA and baclofen. (A) Intracellular voltage and current records (top and bottom trace, respectively) show the hyperpolarizing response of a TC LGN neuron to bath-applied (^)baclofen (80 mM) to be unaffected by 10 mM somatostatin. Break in the records is 15 min, and the shift in direct current was imposed at the peak of the baclofen response to compare the input resistance at the same membrane potential before and during the baclofen application. (B) Intracellular voltage and current records (top and bottom trace, respectively) show the depolarizing response of a TC LGN neurone to iontophoretically (arrows) applied GABA (145 nA) to be unaffected by a 17 min bath application of 10 mM somatostatin. As this neuron was recorded with a KCl electrode, the response to GABA is depolarizing, and it become smaller during steady depolarization of the neuron by d.c. injection. Upward deflections in the intracellular voltage records in A and B represent low threshold Ca 2⫹ potentials evoked by the preceding hyperpolarizing pulses (downward deflections). (C) Whole-cell currents elicited in a rat LGN TC neuron by a 60 ms puff of 10 mM (^)baclofen (arrow). Note the lack of effect of 10 mM somatostatin on both the amplitude and time course of the baclofen-elicited outward current. In this experiment, APV (50 mM), CNQX (10 mM), bicuculline (10 mM) and TTX (0.5 mM) were present in the perfusion medium, and TTX (0.5 mM) was also present in the local puff solution. Holding potential: ⫺50 mV.

to 4 mM (and simultaneously decreasing the Mg 2⫹ concentration from 1 to 0.5 mM, n ˆ 5) or by adding 1 mM TEA to the perfusion medium (n ˆ 4). In five out of these nine neurons (55%) the amplitude of the evoked GABAA IPSCs was reversibly decreased by 41 ^ 8% (n ˆ 4) from 892 ^ 306 pA to 498 ^ 136 pA (P ⬍ 0.05) in the presence of 1 mM somatostatin (Fig. 4B). In the fifth neuron, which responded with a burst of IPSCs following a single electrical shock, application of somatostatin decreased the amplitude of the first IPSC in the burst by 27% but abolished the subsequent components (Fig. 5C), again an effect reminiscent of the one observed on spontaneous bursts of GABAA IPSPs in the kitten LGN (cf. Fig. 2A, B). Effect of somatostatin on the postsynaptic action of GABA and baclofen As spontaneous/miniature GABAB IPSCs cannot be recorded in the thalamus (as well as in other brain

DISCUSSION

The main conclusions of this investigation are that in the sensory thalamus: (i) somatostatin inhibits GABAA-mediated transmission via an action presynaptic to TC neurons, (ii) somatostatin decreases the amplitude of GABAB IPSPs, most likely by a presynaptic mechanism, and (iii) activation of somatostatin receptors is particularly efficient in reducing GABAA synaptic responses that occur in bursts. Site and mechanism of action The conclusion that the inhibition of the GABAergic transmission in the rat sensory thalamus by somatostatin occurs via presynaptic receptors is strongly supported by the ability of this neuropeptide to decrease the frequency, but not the amplitude, of GABAA mIPSCs. The lack of action of somatostatin on the passive membrane properties of cat TC neurons and on their postsynaptic response to baclofen or GABA support our conclusion that in this species, as in the rat, the action of somatostatin on GABAergic transmission involves presynaptic receptors. To identify the precise location of this presynaptic action we need to take into account both the microcircuitry within and between the VB, LGN and NRT nuclei as well as the features of our VB and LGN slices. Whereas TC neurons of both cat/rat LGN and cat VB receive GABAergic afferents from local interneurons and NRT cells, TC neurons in the rat VB only receive GABAergic fibres originating in the NRT. 24 In our rat and cat LGN slices, neither the NRT nor the perigeniculate nucleus, the visual segment of the cat NRT, is present. 12,42 In the LGN slice, therefore, the site of the somatostatin-induced inhibition of mIPSCs can only be either on the interneuron axonal terminals and/or on the severed terminals of the NRT neurons: the present experiments cannot distinguish between these two possibilities. Although NRT cells are present in our VB slices, the ability of somatostatin to decrease mIPSPs in the rat VB (which does not

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N. Leresche et al. Table 1. Summary of the effect of somatostatin in thalamocortical neurons of the rat and cat ventrobasal thalamus and lateral geniculate nucleus

Species/nucleus

Type of IPSP/IPSC

Excitability

Responders*

Cat LGN Cat LGN

Spontaneous bursts Spontaneous single

Increased Increased

Cat LGN Cat VB

Evoked Evoked

Normal Normal

71% (5/7) 50% (4/8)

Rat LGN Rat VB

Miniatures Miniatures

Normal Normal

57% (4/7) 22% (2/9)

Rat VB

Evoked

Normal

25% (2/8)

Rat VB

Evoked

85% (12/14) 87% (7/8)

55% (5/9)

Decrease (%) in IPSP/IPSC amplitude† 100 (n ˆ 3)

45 ^ 6 (n ˆ 5) 39 ^ 5

GABAA: 47 ^ 5 GABAA: 45 ^ 7

GABAB: 53 ^ 7 GABAB: 55 ^ 8

1st component: 54

Other components: 100 31 (n ˆ 1) 1st component: 27 Other components: 100 41 ^ 8 (n ˆ 4)

Total (41/70) *In parentheses, number of neurons responding to somatostatin/number of neurons tested. †In parentheses, number of neurons producing the indicated response.

contain interneurons) leaves the NRT terminals within the VB itself as the only site for the presynaptic action of this neuropeptide. Our results indicate that somatostatin has a more consistent effect on GABAergic transmission in the cat/rat LGN than VB (with 71 vs 50% and 57 vs 22% of the evoked IPSPs and mIPSCs being affected, respectively) (Table 1). Whether this difference simply reflects the different GABAergic innervation within the two nuclei, or an additional direct action of the neuropeptide on the soma of the NRT cells present in the VB slice (only for evoked IPSPs), or is the result of a higher density and/or sensitivity of the somatostatin receptors in the LGN remains to be determined. Nevertheless, it is interesting that similar differences between the LGN and the VB as those reported in the present study have been described for the inhibitory modulation of EPSPs and IPSCs by presynaptic GABAB receptors. 16,25 To the best of our knowledge, this is the first report that provides conclusive evidence in support of an inhibitory action of somatostatin on GABAergic transmission via presynaptic receptors. In the subiculum and the dorsolateral septal nucleus, in fact, the effect of somatostatin on GABAmediated synaptic potentials has been shown to have a postsynaptic origin. 21,52 In the hippocampus, in contrast to earlier observations (see Introduction), recent investigations have shown a presynaptic action of somatostatin on excitatory transmission but no effect on GABAergic synaptic currents. 6,48 Interestingly, a stronger depression of the excitatory synaptic currents by somatostatin was observed in conditions of increased excitability in the hippocampus, 48 as we have shown here for the GABAergic transmission in both the rat and cat LGN and VB. Indeed, it is the strongly stimulated (i.e. by high veratridine or extracellular K ⫹ concentrations) GABA release that has been shown to be preferentially reduced by somatostatin in the caudatoputamen. 26 Physiological implications An interesting observation of the present study is that presynaptic somatostatin receptors seem to be particularly efficient in inhibiting bursts of GABAA IPSPs/IPSCs in cat and rat VB and LGN, occurring both in normal conditions and

during enhanced excitability. During periods of synchronized EEG activity in non-REM sleep, NRT neurons fire bursts of action potentials which are responsible for corresponding bursts of GABAA IPSPs in TC neurons. 1,10,14,15,45 Although the conditions required for the release of somatostatin from NRT neurons are unknown, evidence from other CNS regions indicates that peptides are released preferentially during bursts of action potentials which provide the adequate firing pattern for facilitation of the excitation–secretion coupling without the occurrence of secretory fatigue. 4,8 Thus, the conditions for an increased somatostatin release by thalamic GABAergic neurons (i.e. synchronized thalamic activity and burst firing) could be similar to those where a more pronounced modulation of GABAergic transmission by thalamic presynaptic somatostatin receptors occurs. This modulation might be responsible, at least in part, for the suppression of non-REM sleep that has been observed following exogenous administration of this neuropeptide or its analogues. 3,22,37 In addition, it cannot be excluded at present that there exists a tonic inhibition of GABAergic transmission resulting from a tonic activation of presynaptic somatostatin receptors in the sensory thalamus, as is the case for thalamic presynaptic GABAB receptors. 16,25,29 Within this scenario, it is not surprising that only a small and inconsistent action of somatostatin on the receptive field properties of LGN neurons in deeply anaesthetized preparations has been detected. 41 From a pathological perspective, bursts of GABAA IPSPs have been shown to characterize the activity of TC neurons during spike and wave discharges of absence seizures. 34,46 Although our data raise the possibility that the preferential reduction by somatostatin of bursts of GABAA IPSP might be deficient in absence epilepsy, a clear picture of the somatostatin involvement in this disease can only emerge when the action of this neuropeptide on thalamic GABAergic interneurons and NRT cells and on the excitatory transmission within the thalamocortical network is known. Acknowledgements—We wish to thank Bob Jones for photography. This work was funded by the Wellcome Trust (grant 37089/98) and the CNRS (UMR 7624). The support of Groupe Danone is also acknowledged.

Inhibition of GABAergic transmission by somatostatin

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