Neuroscience Letters, 119 (1990) 83-85

83

Elsevier Scientific Publishers Ireland Ltd. NSL 07258

Unusual features of G A B A responses in layers IV-V neurons of neocortex H. E1-Beheiry and E. Puil Department of Pharmacology and Therapeutics, and Department of Anaesthesiology, Faculty of Medicine, The University of British Columbia, Vancouver, B.C. (Canada) (Received 18 April 1990; Revised version received 30 June 1990; Accepted 6 July 1990)

Key words: Neocortex; Perikarya; GABA hyperpolarization; GABA depolarization; Dendrite; Epileptic Perikaryal application of GABA produced a hyperpolarization and increased the input conductance in neurons of layers IV-V of neocortex (guinea pig). This response faded during brief applications and had a long duration when the application period was increased from 4 s to > 10 s. The sensitivity of the first response to blockade by the selective antagonist, bicuculline, indicated a mediation by y-aminobutyric acid-A (GABA~ receptors. The longer duration response was mimicked to some extent by the GABAa agonist, baclofen. Dendritic application of GABA induced a depolarization and a conductance increase - a response which was not particularly sensitive to antagonism by bicuculline. The depolarizing response also did not have a clearly defined reversal potential and may be a consequence of complex changes in membrane conductance, possibly for C1 and Ca or Na. Fading in both types of responses may result from a concomitant postsynaptic activation of a C1 conductance with Na-dependent GABA uptake.

The original observations by Krnjevi6 and Schwartz [7] showed that extracellular applications of y-aminobutyrate (GABA) by iontophoresis hyperpolarized neocortical neurons in in vivo (cat) preparations. In hippocampal neurons, an application of GABA close to the perikaryon produces a Cl-mediated hyperpolarization [1, 2] whereas its application to the dendrites produces a depolarization in association with an increased membrane conductance. The depolarizing response to GABA, like that observed in olfactory cortical neurons [11], is attributable in a large measure, to an outward movement of CI- [1-4, 9, 12]. There have been no reports of GABA-evoked depolarizations in neocortical neurons. Long-lasting depolarizing potentials that may be mediated by dendritic GABAA receptors have been observed in human neocortical neurons [3]. Here, we investigated the effects of GABA administration at perikaryal and dendritic sites in neocortical neurons. Slices (500/tm thick) were prepared from sensorimotor cortex of guinea pig brain [6]. For intracellular recording, the slices were maintained under in vitro conditions at 34°C in a submersion type of chamber with oxygenated (95% 02-5% CO2) artificial cerebrospinal Correspondence: H. EI-Beheiry, Department of Pharmacology and Therapeutics, Faculty of Medicine, The University of British Columbia, 2176 Health Sciences Mall, Vancouver, B.C., V6T 1W5, Canada. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

fluid (ACSF) that contained (in mM): NaCI 124, KC1 3.75, KH2PO4 1.25, MgSO4 2, CaC12 2, NaHCO3 26, glucose 10. The microelectrodes were filled with 0.6 M K2SO4 (tip resistances, 70-90 MI2). Bridge balance techniques were used to determine input conductance [6]. GABA (0.5 M; pH 4.5), glutamate (0.5 M; pH 8.5), + baclofen (50 mM; pH 3, Ciba-Geigy) or NaCI (0.5 M; pH 4-4.5) were applied iontophoretically from a micropipette that was inserted into the slice ~ 150-200/~m from the recording electrode for dendritic applications of the amino acids. For perikaryal applications, the micropipette was glued to the recording electrode at an intertip distance of 30-50 pm. Tetrodotoxin (TTX) and bicuculline (Sigma) as well as baclofen were applied in the bath. The results were obtained from 33 pyramidal neurons in layers IV-V with mean input resistance of 46+2.3 MO and resting potential of - 7 6 _ 3.5 mV. The neurons fired spikes of >70 mV amplitude and < 2 ms in duration, in response to intracellular injections of depolarizing current pulses. As shown in Fig. 1, perikaryal application of GABA evoked a 5-9 mV hyperpolarization (9 out of 9 neurons) whereas its dendritic application evoked a 14-30 mV depolarization (24 out of 24 neurons). Neither effect could be mimicked by positive currents applied with similar magnitudes (50-100 nA) from an adjacent barrel containing NaCI [7]. The hyperpolarizing responses were associated with

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Fig. 1. Neuronal responses to GABA applied at the perikaryon (A,B) and dendrites (C-E). A: application of GABA (80 nA for 4s) evoked a hyperpolarizing response that was associated with an increased conductance which faded during the application. B: a long-lasting hyperpolarization was observed when the GABA application (80 nA) was 10 s. C: dendritic applications of GABA (100 nA for 5 s) evoked depolarizations. D: depolarization and conductance increase faded during application of GABA (I00 nA for 5 s). A plateau potential was maintained at the end of the GABA-current. Insets at right show resistance pulses near the peak (PI), after substantial fading (P2) and during the plateau (P 1) of the depolarization. Control (C) and recovery (R) pulses are also shown. Constant current pulses were 0.7 nA. E: continuous recording shows the tachyphylaxis that was apparent when GABA (70 nA) was applied repeatedly at short intervals. Note recovery in bottom trace. TTX (1-1.5 pM) was applied in A-D. Calibrations: voltage, 15 mV in (A) and (B), 20 mV in (C-E); time, 3 s in (E).

increases in input conductance (40-70%). In 4 neurons, the GABA-evoked conductance increase and hyperpolarization declined during the application period (Fig. 1A). When the duration of the application was increased from ~ 4 s to > 10 s a hyperpolarization without much change in conductance persisted for 30-90 s in the same 4 neurons (Fig. 1B). Bath application (7-10 min) of baclofen (50 /IM) induced small hyperpolarizations (2-3 mV) together with small increases in conductance (1015 %) in 3 neurons. The depolarizing effects of GABA also were accompanied by an increase in input conductance (40-70%). In 6 neurons, both the depolarization and conductance increase faded during the application to plateau levels lasting several seconds (Fig. 1D). In 18 neurons, the responses did not fade during the application period (Fig. 1C,E). Frequent administrations of GABA to 3 of these neurons revealed tachyphylaxis in the responses (Fig. IE). Spikes were often observed on the rising and the falling portions of the non-fading type of GABA depolarization (Fig. 1E). To determine if such responses also

could be inhibitory, glutamate was first applied to induce continuous spike firing and then GABA was additionally applied to the dendrites in 4 neurons. The spikes were suppressed completely during the GABA-evoked depolarizations. GABA was applied to the dendrites in 4 neurons where the resting potentials had been displaced by DC injection to levels between - 100 and - 30 mV, to determine the reversal potential for the depolarizing response (Fig. 2A). In the presence of TTX (1 #M) the GABA depolarization increased in amplitude at hyperpolarized potentials. The rate of rise of the GABA response decreased with imposed depolarization down to - 5 0 mV. The early and late parts of the GABA response were reversed to hyperpolarizations at imposed potentials that were depolarized to about - 40 mV or more. Bicuculline application (50 #M) for 4 min in 3 neurons blocked the hyperpolarizing type of GABA response (Fig. 2B); the depolarizing type was blocked only by prolonged applications ( > 15 min) of bicuculline in doses of 100-150 #M (Fig. 2C). Administration of baclofen to the dendrites of 6 neurons did not change their resting potentials, input conductances or glutamate-evoked discharges (Fig. 2D). The GABA-evoked depolarizations were unaffected by baclofen. The hyperpolarizing responses to GABA which were sensitive to bicuculline antagonism can be attributed to A

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Fig. 2. Properties of responses evoked by GABA. A: reversal potential for the depolarizing response could not be obtained in the range of - 100 to - 3 0 inV. TTX (1 ~M) was present throughout. B: bicuculline (BIC; 50 #M) blocked the hyperpolarizing response to GABA (50 nA) after ~ 4 rain (middle). Recovery was evident after 6 min (right). C: continuous trace shows that high doses of BIC (100 ~M) partially blocked the depolarizations evoked by applications of GABA (70 nA) and not glutamate (Glu; 100 nA). D: baclofen (100 nA) had no significant effects on the resting potential or spikes evoked by Glu (90 nA). GABA (100 nA) response is shown for comparison. Calibrations: voltage, 20 mV; time, 12s in A, B and D and 35 s in C.

85 interactions with G A B A A receptors on the perikarya [1, 7]. The longer-lasting hyperpolarizations that were m i m i c k e d to some extent by baclofen, m a y have resulted f r o m activation o f G A B A a receptors. The depolarizing responses to G A B A are less easily explained. As might be expected, they were relatively insensitive to a n t a g o n i s m by bicuculline and an exact reversal potential could not be found in the range o f - 100 to - 30 mV, partly because o f the n o n - u n i f o r m distribution o f the injected current in the dendrites relative to perikaryon. However, the multiphasic character o f this G A B A response at imposed depolarizations was suggestive o f complex or ' o v e r l a p p i n g ' changes in m e m b r a n e conductance, possibly for CI and C a or Na. The fading in the perikaryal and the dendritic types o f G A B A responses, could result f r o m a decreased ionic gradient (e.g. for Cl) or the activation o f C l - conductance together with a G A B A u p t a k e process [5]. Subsequent to the G A B A - i n d u c e d increase in conductance with the p r e s u m e d externally oriented gradient for Cl, the activation o f an u p t a k e process transporting G A B A and N a into the dendrites [5] could account for the G A B A - d e p o l a r i z a t i o n s , particularly those with maintained 'plateau' levels (cf. Fig. 1D). Despite the uncertainties a b o u t the exact ionic mechanism, the depolarizing actions o f G A B A were inhibitory and m a y mediate, as in olfactory cortex [11], inhibitory postsynaptic potentials in neocortex [cf. ref. 3]. The paradoxical excitatory p h e n o m e n o n (cf. Fig. 1E) observed when G A B A was applied to layers I V - V neurons during epileptiforrn afterdischarges in isolated slabs o f cat neocortex [10] m a y have been p r o d u c e d by a similar m e c h a n i s m with a c o m p r o m i s e d shunting effect o f the increased Cl-conductance, or activation o f C a - c o n d u c t a n c e [cf. 8].

The authors are grateful to the Medical Research Council o f C a n a d a for financial support (E.P.). 1 Alger, B.E. and Nicoll, R.A., Pharmacological evidence for two types of GABA receptors on rat hippocampal pyramidal neurons studied in vitro, J. Physiol., 328 (1982) 125-141. 2 Anderson, P., Dingledine, R., Gjerstad, L., Langmoen, I.A. and Mosfeldt-Laursen, A., Two different responses of hippocampal pyramidal cells to application of y-aminobutyric acid, J. Physiol., 305 (1980) 279-296. 3 Avoli, M. and Olivier, A., Electrophysiological properties and synaptic responses in the deep layers of the human epileptogenic neocortex in vitro, J. Neurophysiol., 61 (1989) 589-606. 4 Blaxter, T.J. and Carlen, P.L., GABA responses in rat dentate granule neurons are mediated by chloride, Can. J. Physiol. Pharmacol., 66 (1988) 637~42. 5 Constanti, A., Krnjevir, K. and Nistri, A., Interneuronal effects of inhibitory amino acids, Can. J. Physiol. Pharmacol., 58 (1980) 193204. 6 EI-Beheiry, H. and Puil, E., Postsynaptic depression induced by isoflurane and Althesin in neocortical neurons, Exp. Brain Res., 75 (1989) 361-368. 7 Krnjevi~, K. and Schwartz, S., The action of y-aminobutyric acid on cortical neurones, Exp. Brain Res., 3 (1967) 320-336. 8 Krnjevi~, K., The role of GABA receptors in the genesis of seizures. In U.Z. Littauer, Y. Dudai and T. Silman (Eds.), Neurotransmitters and their Receptors, Wiley, 1980, pp. 405-416. 9 Misgeld, U., Deisz, R.A., Dodt, H.V. and Lux, H.D., The role of chloride transport in post-synaptic inhibition of hippocampal neurons, Science, 232 (1986) 1413-1415. 10 Puil, E., Reiffenstein, R.J. and Triggle, C., Epileptiform afterdischarges and chemical responsiveness of cortical neurones, Electroencephalogr. Clin. Neurophysiol., 36 (1974) 265-273. 11 Scholfield, C.N., A depolarizing inhibitory potential in neurones of the olfactory cortex in vitro, J. Physiol., 275 (1978) 547-557. 12 Thalmann, R.H., Peck, E.J. and Ayala, G.F., Biphasic response of hippocampal pyramidal neurons to GABA, Neurosci. Lett., 21 (1981) 319-324.