GABA as an inhibitory transmitter in the pigeon isthmo-tectal pathway

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Neuroscience Letters 169 (1994) 212-214

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GABA as an inhibitory transmitter in the pigeon isthmo-tectal pathway Dominik Felix "'*, Gang-Yi Wu b, Shu-Rong Wang b "Division of Neurobiology, University of Berne, Erlachstrasse 9a, 3012 Berne, Switzerland ~Laboratory for VisualInJbrmation Processing, Institute of Biophysics, Academia Sinica, 100101 Beijing. China Received 22 October 1993; Revised version received 27 December 1993; Accepted 21 January 1994

Abstract

In the present investigation, the effects of inhibitory amino acids and their antagonists were tested on isthmo-tectal projection in the pigeon. The majority of superficial tectal cells are inhibited following stimulation of the parvocellular division of nucleus isthmi, A smaller portion of tectal cells showed excitatory responses followed by an inhibitory period. Both inhibitory mechanisms are blocked by the specific y-aminobutyric acid (GABA)-antagonist bicuculline but not by strychnine. Our results support the idea that GABA acts as an inhibitory neurotransmitter in the pigeon isthmo-tectal pathways. Key words." Pigeon; Nucleus isthmus; Isthmo-tectal pathway; Optic tectum; Microiontophoresis; 7-Aminobutyric acid; Bicuculline

There is electrophysiological and biochemical evidence that the inhibitory neurotransmitter GABA plays a prominent role within the pigeon optic tectum (for reviews, see [3,4]). Synaptosomal fraction from the optic tectum shows high-affinity uptake for GABA [6]. The GABA-synthesizing enzyme glutamate decarboxylase (GAD) is predominantly found within superficial layers of the tectum [5]. Microiontophoretic application of GABA inhibits large number of tectal neurones. Cells being more sensitive to GABA than to other inhibitory amino acids were found mostly in tectal layers llc and lid; the area in which most of the GAD is concentrated [6]. Electrical stimulation of the ipsi- or contralateral tectal surface results in inhibition of neurones in superficial layers [12]. The intra- as well as the intertectal inhibitions are antagonized by the specific GABA antagonist bicuculline [1]. Hunt and Kunzle [7] described three specific GABA systems in the avian tectum which were identified both from the uptake of tritiated GABA and from subsequent intracellular transport of the labeled substrate. GABA systems were either found in intrinsic tectal neurones while others implied extratectal connections. There is evidence from biochemical data that isthmo-tectal pathways uses glycine and GABA as inhibitory substances [9,11]. Using intracellular recording

* Corresponding author. 0304-3940194/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00081-K

techniques, Wang and Matsumoto [16] have shown that stimulation of the nucleus isthmi exerts inhibition on the majority of tectal cells. We have, therefore, attempted to test the action of inhibitory amino acids and their antagonists on the isthmo-tectal synaptic transmission with the use of electrophysiological and microiontophoretic techniques. All experiments were performed on adult homing pigeons Colurnba livia. The animals were anesthetized with urethane (1 ml/100 g body wt, 20% solution) and placed in a stereotaxic apparatus. The exposition of the surface of the optic tectum was done in conventional manner. Extracellular recording of action potentials were obtained from tectal neurones with a barrel of a 5-barrelled micropipette filled with 2 M NaCI and 100 mM cobalt chloride (4/~m diameter, 5-15 MI2 resistance). The other channels contained the following chemical compound to be ejected microiontophoretically by appropriate ionic currents: strychnine hydrochloride (Sigma, 2 mM in 165 mM NaCI), bicuculline methiodide (Sigma, 2 mM in 165 mM NaCI), ~,-aminobutyric acid (GABA, Fluka, 0.5 M, pH 3.0-3.5), glycine (Fluka, 0.5 M, pH 3,0-3.5). Visual stimuli comprised a 8° black or white disc which was moved manually against a diffuse grey background. Electrical stimuli to the parvocellular division of nucleus isthmi (Ipc) were applied through a concentric bipolar glass insulated tungsten electrode. Rectangular pulses of 100/~s and variable voltages (usually between

D. Felix et al./Neuroscience Letters 169 (1994) 212 214

I type

Co

(~

El

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type

Co

STR

BIC

BIC

Co

STR

Fig. 1. Effect of bicuculline (BIC) and strychnine (STR) on isthmo-tectal projection. A: inhibitory (I) type. Stimulation of nucleus isthmi inhibits tectal neurone (Co, control; three superimposed sweeps). Inhibitory period is blocked by bicucnlline (50 hA) but not by strychnine (50 hA). B: excitatory-inhibitory (El) type. Stimulation of nucleus isthmi excites tectal neurone (Co, upper trace, three superimposed sweeps) followed by an inhibitory period (Co, lower trace). Inhibition is blocked by bicuculline (BIC, 50 hA) but not by strychnine (STR, 50 nA). Scales: I A; 20 ms, 0.5 mV/cm, 1 B; Co, upper trace, 2 ms, 0.5 mV/cm; other traces, 10 ms, 0.5 mV/cm.

3 and 10 V) were given at a frequency of 0.2 Hz. The location of the stimulus electrode in Ipc was defined to AP-level 2.0-2.5 according to the atlas of Karten and Hodos [10]. At the end of each experiment, cobalt ions were ejected iontophoretically for histological verification of the recording points. Constant current pulses of 4 0 / t A for 60 s were applied through the stimulation electrode for the location of the stimulation area. Electrophysiology." Successful recordings were made from a total of 35 neurones. Only cells were taken on which all the antagonists could have been tested and recovery was observed. 15 cells were located in the superficial layers II-c-g according to the nomenclature of Cowan et al. [2]: 19 cells were found in layer III and the remaining cell was located in layer lI-j. Following stimulation of the parvocellular part of the nucleus isthmi (Ipc), 77% of tectal neurones tested were inhibited (I type, Fig. IA), 19% showed excitatory responses followed by an inhibitory period (EI type, Fig. 1B) and 4% were of inhibitory-excitatory type. In superficial area, the excitatory responses had latencies from 4.5-6.0 ms whereas neurones in layer IlI varied from 1.84.5 ms. By comparing equal stimulus strength, superficial neurones had longer duration period of inhibition (50-100 ms) than neurones in layer III (30-70 ms). The only cell found in layer II-i was of the excitatory-inhibitory type with 2.5-3.2 ms latencies followed by an inhibitory period of 60 ms. In a series of experiments, we tested the

effect of Ipc electrical stimulation to visual responses. Tectal neurones in superficial layers usually responded to the movement of the visual targets through their receptive fields and immediately stopped firing when the targets stopped moving. The visual responses were strongly inhibited by stimulation of Ipc (Fig. 2), lasting up to 250 ms. Pharmacology. In a second series of experiments, we tested the influence of the specific inhibitory amino acid antagonists strychnine and bicuculline on the Ipc-induced inhibitory mechanisms. The substances were usually ejected for 3-rain periods followed by an interval of 6 rain, thus, leaving enough time for the cell to recover. In case of negative results, the period of ejection as well as the doses were increased. A clear cut difference between the effect of bicuculline and strychnine could be observed on the majority of tectal inhibitory mechanisms. On all except one I-type cell, the inhibitory period was either reduced or blocked by bicuculline, ejected with currents of 50 100 nA (Fig. IA). Blockade of the inhibition usually started 1 2 min after onset of ejection and recovery occurred between 1 and 5 min after the offset of the ejection. In contrast, strychnine, applied with currents up to 150 nA and for longer periods never blocked the inhibition of I-type ceils. On few occasions, the inhibition was even prolonged. In a similar manner, we tested the two antagonists on the excitatory-inhibitory-type cells (Fig. 1B). Again, the inhibitory period

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D. Feli., el al./Neuroscience Let&,rs 169 (1994) 212 214

following the early excitation was blocked by bicuculline but not by strychnine. To rule out the possibility that the lacking effect of strychnine was due to inappropriate efflux from the electrode, we tested the alkaloid on glycine- and GABA-induced depression of spontaneous firing of tectal neurones. On all neurones tested, glycineinduced depression was blocked specifically by strychnine; the GABA-induced inhibition, however, was blocked by bicuculline only. The data obtained in our investigations agree with previous biochemical [6], enzymatic [5], autoradiographic [7] and electrophysiological [1] studies which suggested a prominent role for GABA as an inhibitory neurotransmitter in the pigeon optic tectum. There is, however, evidence that a glycine system is mediating inhibitory mechnisms to the tectum since glycine is taken up with high affinity by tectal synaptosomes [6] and exogenous glycine is released during electrical stimulation of an afferent pathway [11]. This is not in disagreement with present findings since most of the GABAergic system is confined to the rostral portion of Ipc whereas the glycine system lays in the more caudal part [8]. Our data suggest that the GABA system projects into layer IlI. Although our results rely on extracellular unit activity and are, therefore, somewhat indirect, our results supports earlier intracellular findings of a monosynaptic input from the nucleus isthmi into the tectum of the pigeon [17] and the bullfrog [16]. The inhibitory mechanisms found in the superficial layers of the tectum imply the activation of intrinsic GABAergic systems in these areas. From the work of Hunt and Kunzle [7], GABA systems were confined to layers II-c, having small cells with restricted superficially directed dendritic trees, layer II-d with horizontally organized structure and layer II-i with predominantly bipolar cells, with their axons passing into the layer I. Stimulation of the Ipc, therefore, could activate GABA-specific neurones which result in the inhibition of intratectal elements or the retinal input. Application of the specific GABA antagonist bicuculline interacts with this inhibitory mechanisms. It has been proposed that the nucleus isthmi may play a critical role on visual processing within the optic tecturn, possibly involving positive and negative loops which work together to make the animals orient to the most interesting object [13-15]. On the basis of our present findings, GABA may be the inhibitory transmitter in the negative isthmo-tectal negative feedback circuit. This work was supported by grants from the National Natural Science Foundation of China, the Chinese Academy of Sciences and from the Swiss National

Science Foundations. We wish also to thank S. Gygax for technical assistance and R. Schweizer for typing the manuscript. 1 Barth, R. and Felix, D., Influence of GABA and glycine and their antagonists on inhibitory mechanisms of pigeon's optic tectum, Brain Res., 80 (1974) 532-537. 2 Cowan, W.M., Adamson, L. and Powell, T.P.S., An experimental study of the avian visual system, J. Anat., 95 (1961) 545 563. 3 Cuenod, M. and Streit, P., Amino acid transmitters and local circuitry in optic rectum. In F.O. Schmitt and F.G. Worden (Eds.), The Neurosciences, Fourth Study Program, Volume II, MIT Press, Cambridge, MA, 1979, pp. 989od004. 4 Felix, D., Neurophysiological effects of neurotransmitters in the pigeon optic tectum, In G, Nistico and L. Bolis (Eds,), Progr. Nonmammalian Brain Res., Volume II, CRC Press, Boca Raton, FL, 1983, pp. 31-51. 5 Henke, H. and Fonnum, F., Topographical and subce|lular distribution of choline acetyltransferase and glutamate decarboxytase in pigeon optic tectum, J. Neurochem., 27 (1976) 387 491. 6 Henke, H., Sehenker, T.M. and Cuenod, M., Uptake of neurotransmitter candidates by pigeon optic tectum, J. Neurochem., 26 (1976) 125-130. 7 Hunt, S.P., Henke, H., Kunzle, H., Reubi, J.C., Schenker, T.M., Streit, P. Felix, D. and Cuenod, M., Biochemical neuroanatomy of the pigeon optic tectum, Exp. Brain Res. Suppl., 1 (1976) 521 525. 8 Hunt, S.P. and Kunzle, H., Observations on the projections and intrinsic organization of the pigeon optic tectum: an autoradiographic study based on anterograde and retrograde, axonal and dendritic flow, J. Comp. Neurol., 170 (1976) 173-190. 9 Hunt, S.E, Streit, E, Kunzle, H. and Cuenod, M., Characterization of the pigeon isthmo-tectal pathway by selective uptake and retrograde movement of radioactive compounds and by Golgi-like horseradish peroxidase labelling, Brain Res., 129 (1977) t97 212. 10 Karten, H.J. and Hodos, W., A stereotaxic atlas of the brain of the pigeon (Columbia livia), John Hopkins Press, Baltimore, MD, 1967. 1l Reubi, J.C. and Cuenod, M., Release of exogenous glycine in the pigeon optic tectum during stimulation ofa midbrain nucleus, Brain Res., 112 (1976) 347-361. 12 Robert, F. and Cuenod, M., Electrophysiology of the intertectal commissures in the pigeon. II. Inhibitory interaction, Exp. Brain Res., 9 (1969)123--136. 13 Sereno, M.I. and Ulinski, ES., Caudal topographic nucleus isthmi and the rostral nontopographic nucleus isthmi in the turtle, Pseuderays scripta, J. Comp. Neurol., 261 (1987) 319--346. 14 Wang, S.R., The nucleus isthmus is a visual center: neuroanatomy and electrophysiology. In D.T. Yew et al, (Eds.), Vision, Structure and Function, World Sci. Publ., Singapore, 1988, pp. 304-364. 15 Wang, Y.C. and Frost, B.J., Visual response characteristics of neurons in the nucleus isthmi magnocellularis and nucleus isthmi parvocellularis of pigeons, Exp. Brain Res., 87 (199I) 624-633. 16 Wang, S.R. and Matsumoto, N., Postsynaptic potentials and morphology of tectal ceils responding to electrical stimulation of the bullfrog nucleus isthmi, Vis. Neurosci., 5 (1990) 479~,88. 17 Wu, G.Y., Wang, S.R. and Yam K., Postsynaptic potentials and morphological features of tectal cells in homing pigeons, Sci. China, 36 (1993) 297-304,