SYNAPSE 8:38-40 (1991)

GABA Depolarizes Neurons in the Rat Striatum: An In Vivo Study NICOLA BUG10 MERCURI, PAOLO CALABRESI, ALESSANDRO STEFANI, FRANCESCA STRATTA, AND GIORGIO BERNARD1 Clinica Neurologica, II Uniuersita’ di Roma, Dipartimento di Sanita’ Pubblica e Biologia Cellulare, 00173, Rome, Italy

KEY WORDS

Membrane depolarization, Intracellular recordings, In vivo preparation, -4nesthesia

ABSTRACT

GABA, applied by iontophoresis to striatal neurons of the rat in an in vivo preparation, depolarized the membrane potential and decreased the input resistance in a dose-dependent manner. The null potential of the GABA depolarization was about -50 mV. In addition, a fading of the GABA-induced response was observed for prolonged and relatively high amino acid application. We conclude that GABA has a depolarizing effect on striatal cells. This is in line with recent in vitro works describing a depolarizing effect of GABA in the rat neostriatum.

INTRODUCTIOK y-Aminobutyric acid (GABA)is abundant in the striatum where it exerts a depressant action on the neuronal activities. Electrophysiological studies in vivo (Bernardi et al., 1975,1976; Park et al., 1980; Herding, 1984; Mercuri et al., 1985) have shown that GABA hyper olarizes the cells and is responsible for an inhibitory yperpolarizing postsynaptic potential (IPSP) in the striatum. However, recent in vitro studies have roposed that GABA depolarizes striatal neurons, an it has been shown that endogenous GABA mediates short bicuculline-sensitive de olarizing potentials at rest. These potentials are mexiated by a GABA receptor-activated increase in chloride conductance (Misgeld et al., 1982; Lightall and Kitai, 1983; Calabresi et al., 1989,1990b). In addition, it has been recently shown in neostriatal neurons recorded in vivo that bicuculline- and pentylenetrazol-sensitive inhibitory potentials are depolarizing at resting membrane potentials and they reverse in a hyper olarizing direction when the cells are depolarized (Ca abresi et al., 1990a).

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resistance of 40-100 MR. Coordinates used for the striatum were 1.80 mm anterior and -1.20 mm posterior to bregma, lateral 1.813.8mm, and between 3.5 and 7 mm deep from the cortical surface. A WPI M 707 device was used as a biological amplifier. Data were stored on a magnetic tape for off-line analysis, displayed on a di .tal oscilloscope, and plotted on an x-y plotter. T e iontophoretic electrodes, lued to the shank of the recording electrode, were fi ed with GABA (Calbiochem, 0.5 M, pH 31, NaCl (1 M, pH 3, control current), and NaCl(2 M, balance barrel). A retaining current was routinely a plied between GABA a plications. The drug was ap Pied with automatic ba anced current. The intertip istance between the recording and the iontophoretic electrodes was about 50 Fm.

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RESULTS The resting membrane potential of unidentified striatal neurons ranged between -60 and -80 mV, and the spike amplitude ranged between 70 and 100 mV. The properties of these cells were similar to those already described for medium spiny striatal cells (Bishop et al., application 1982; Lightall and Kitai, 1983). GABA (15-150 nA) depolarized all the tested cells (n = 28) recorded with electrodes containing either preparation. K-acetate or K-citrate in rats anesthetized with thioMATERIALS AND METHODS ental. GABA also de olarized four neurons recorded Male Wistar rats, weighing 140400 ,were used for prom rats anesthetize (Pwith urethane. The depolarizathe ex eriments. The surgical metho has been de- tion ranged between 4 and 20 mV and was dependent on scribe in detail elsewhere (Mercuri et al., 1985). Gen- the amount of the iontophoretic current (Fig. 1A). The eral anesthesia was induced by i.p. injection of thiopen- GABA-induced depolarization was associated with a tal(50 mgkg) and then maintained by hourly injection marked decrease in input resistance and with cellular of the drug (10 mgkg). Some rats were also anesthetized inhibition (Figs. 1,2). with urethane (1.8-2 gkg, i. .). A cannula was placed in The decrease in membrane resistance was not only the trachea, and the anima s were paralyzed with suc- the result of changes in membrane potential-in fact, it cinylcholine and then artificial1 ventilated. The record- was already present when the cell membrane potential in electrodes (thin-wall ca il aries) were filled with ei&er K-acetate (2 M) or gcitrate (2 M) and had a Received July 5,1990; accepted in revised form October 24,1990

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0 1991 WILEY LISS, INC.

GABA IN THE STRIATUM

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Fig. 1. A. Upper trace: GABA, applied by iontophoresis for the period indicated by the bars, produces a depolarization of the membrane and a decrease in membrane resistance which depends on the dose applied. The downward deflections are hyperpolarizing voltage pulses (400ms duration). Membrane potential, -70 mV. Lower trace: Current. B: Left: GABA depolarizes another striatal cell. The same cell

is later exposed to GABA while the neuron is manually clamped to rest (-67 mV). Upper traces: voltage. Lower traces: current. Hyperpolarizing and depolarizing pulses are alternatively applied in the cell. Note the fading in GABA responses during the application ofthe amino acid. Voltage calibration in A is also valid for B.

was manually clamped at rest, by intracellular injection of current (Fig. 1B).A fading of the membrane depolarization and of the decrease in membrane resistance was often observed when relatively- high - doses of GABA were ap lied (Fig. 1). +he GABA-induced deDolarization decreased in amplitude b shiftin the membrane potential to less negative vafues an% had an equilibrium point about - 50 ? 3.5 mV, n = 5, mean 2 S.E. In three cases, the depolarizing shift of the membrane potential revealed a short lasting and slight (2-4 mV) hyperpolarization that preceded the depolarization (Fig. 2). DISCUSSION The major finding of this study is that GABA has a depolarizing effect on rat striatal cells intracellularly recorded in vivo. In previous in vivo studies, a hyperpolarizing effect of GABA had been described in the striatum (Bernardi et al., 1975, 1976; Park et al., 1980; Herrling, 1984; Mercuri et al., 1985). We are of the opinion that the difference in our results compared to earlier data may depend upon the higher resting potential of the cells recorded in this study. This is mainly due to the improvement of technical procedures used for obtaining in vivo intracellular recordings from rat striatal neurons.

In spite of the higher resting membrane otential obtained in this study, the mean null oint for tBe GABA response is similar to the value a ready reported in previous in vivo and in vitro ex eriments. The primary involvement of a chloride con uctance had been suggested by those works (Bernardi et al., 1975; Misgeld et al., 1982). The fading of the membrane depolarization observed during sustained and high application of GABA could be either due to a shift in ECLor to rece tor desensitization or to both. The observation that the ABA-induced drop in input resistance also fades during the application of GABA suggests a desensitization of the receptors. The short lasting hyperpolarization of the membrane, sometimes observed when the cells were depolarized, can result either from a different distribution of chloride in the intracellular compartments or from a resyna tic effect of GABA. It is unlikely that it coul depen on GABA B receptor-activated events, which have usually a slow onset and are long lasting (Lacey et al., 1988).In addition, no modification of the membrane otential is produced by the GABA B receptor agonist aclofen in striatal cells recorded in vitro (Calabresi et al., 1990~). Several cell ty es in the mammalian central nervous system respon to GABA with a depolarization that is dependent on the concentration of chloride within the

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Fig. 2. a: The depolarization produced by GABA decreases in amplitude by decreasing the membrane potential values. Steady intracellular current injection is used to bring the potential t o the values indicated. A transient membrane hyperpolarization roduced by GABA is observed when the cell is depolarized. The bottom trace is the current r e c o r l a t resting level. b: Plot of the GABA-induced depolarization versus the irnposed membrane potential.

neurons (Brown and Sholfield, 1979; Alger and Nicoll, 1982; Mis eld et al., 1982; Thompson et al., 1988). Our fin ings show that the striatal cells are readily depolarized and inhibited by GABA in vivo. Thus, the suggested difference in cellular response to GABA between in vivo and in vitro preparations can no longer be sustained.

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ACKNOWLEDGMENTS We are grateful to G. Gattoni and M. Tolu for their technical assistance and to R. Losacco for revising the English style. REFERENCES Alger, B.E., and Nicoll, R.A. (1982) Pharmacological evidence for two kinds of GABA receptors on rat hippocampal pyramidal cells studied in vitro. J. Physiol. (Lond.), 328:125-141. Bernardi, G., Marciani, M.G., Morocutti, C., and Giacomini, P. (1975) The action of GABA on rat caudate neurons rtxorded intracellularly. Brain Res., 92:511-515. Bernardi, G., Marciani, M.G., Morocutti, C., and Giacomini, P. (1976) The action of icrotoxin and bicuculline on rat caudate neurons inhibited by G h A . Brain Res., 102:379-384 Bishop, G.A., Chang, H.T., and Kitai, S.T. (1982) Morphological and hysiolo 'cal properties of neostriatal neurons: An intracellular orsera ish peroxidase study in the rat. Neuroscience, 7:179-191. Brown. D.A.. and Sholfield. C.N. (1979) Deoolarization of neurones in the isolated olfactory cortex ofthe guinea-'pig by y-aminobutyric acid. Br. J. Pharmacol., 65:339-345. Calabresi, P., Benedetti, M., Mercuri N.B., and Bernardi, G. (1989)

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Selective depression of synaptic transmission by tetanus toxin: A comparative study on hippocampal and neostriatal slices. Neuroscience, 30:663-670. Calabresi, P., Mercuri, N.B., Stefani, A,, and Bernardi, G. (1990a) Synaptic and intrinsic control of membrane excitability of neostriatal neurons. I. An in vivo analysis. J. Neurophysiol., 63:651-662. Calabresi, P., Mercuri N.B., and Bernardi, G. (1990b) Synaptic and intrinsic control of membrane excitability of neostriatal neurons. 11. An in vitro analysis. J. Neurophysiol., 63:663-675. Calabresi, P., Mercuri, N.B., De Murtas, M., and Bernardi, G. (1990~) Endogenous GABA mediates presynaptic inhibition of spontaneous and evoked excitatory synaptic potentials in the rat striatum. Neurosci Lett., 118:99-102. Herrling, P.L. (1984) Evidences for GABA as the transmitter for early cortically evoked inhibition of cat caudate neurons. Exp. Brain Res., 55:528-534. Lacey, M.G., Mercuri, N.B., and North, R.A. (1988) On the potassium conductance increase activated by GABA and dopamine D2 receptors in rat substantia nigra neurones. J Pkysiol., 401:437453. Lightall, J.W., and Kitai, S.T. (1983) A short duration GABAergic inhibition in identified neostriatal medium spiny neurons: In vitro slices study. Brain Res. Bull., 11:103-110. Mercuri, N.B., Bernardi, G., Calabresi, P., Cotugno, G., Levi, G., and Stanzione, P. (1985) Dopamine decrease cell excitability in rat striatal neurons by pre- and postsynaptic mechanisms. Brain Res., 358:llO-121. Misgeld, U., Wagner, A., and Ohno, T. (1982) Depolarizing IPSPs and depolarization by GABA of rat neostriatum cells in vitro. Exp. Brain Res., 45:108-124. Park, M.R., Lighthall, and Kitai, S.T. (1980) Recurrent inhibition in the rat neostriatum. Brain Res., 194:359-369. Thompson, S.M., Deisz, R.A., and Prince, D.A. (1988) Relative contributions of passive equilibrium and active transport to the distribution of chloride in mammalian cortical neurons. J. Neurophysiol., 60:105-124.