Elsevier

Neuroscience Research

Volume 51, Issue 4, April 2005, Pages 475-492
Neuroscience Research

GABAergic neurons in inferior colliculus of the GAD67-GFP knock-in mouse: Electrophysiological and morphological properties

https://doi.org/10.1016/j.neures.2004.12.019Get rights and content

Abstract

Utilizing slice preparations of GAD67-GFP knock-in mouse, in which GABAergic neurons are specifically labeled with GFP fluorescence, we studied electrophysiological characteristics of GABAergic neurons of IC by whole-cell patch clamp-recording combined with biocytin-intracellular-staining techniques. GABAergic neurons of IC fell into two distinct firing types; (1) tonic type neurons and (2) transient (phasic) type neurons. Tonic type neurons showed regularly repetitive discharge pattern in response to a long depolarizing current pulse (200 ms), and transient type neurons showed spike discharges just at the onset of current pulse. Most of neurons of both types showed depolarizing sag in response to hyperpolarizing current pulse, which were blocked by 0.1 mM ZD7288 (Ih blocker). All two types of tonic neurons showed an AHP, which was blocked by Cd2+ (0.1 mM) and high concentration of apamin (2 μM). One of tonic type neurons (BP) revealed a long delay in spike onset or a longer first spike interval when they were stimulated from hyperpolarized potentials. The remaining tonic neurons (RS) did not show this property. Tonic type neurons were distributed in all region of IC. Morphologically, they were not identical; heterogeneous in somatic diameter, dendritic field size and its orientation. One of transient type neurons (Th−) revealed an AHP after the spike. The other transient type neurons (Th+) showed a depolarization hump after the spike, which were blocked by 0.1–0.2 mM Ni2+. Th+ type neurons were found only in the dorsolateral region of IC, having small dendritic field. Th+ type neurons are likely to be a distinct, homogenous group of GABAergic neuron in IC.

Introduction

Mammalian inferior colliculus (IC) is an integrative auditory processing center of the midbrain, receiving inputs from ascending, descending and intrinsic connections (Huffman and Henson, 1990, Saldana and Merchan, 1992, Casseday and Covey, 1996, Ehret, 1997, Oliver, 2000). IC is the richest source of γ-aminobutyric acid (GABAergic) neurons in the mammalian auditory tracts (gerbil, Roberts and Ribak, 1987; rat, Moore and Moore, 1987; cat, Oliver et al., 1994; bat, Winer et al., 1995), and about 20% of the central nucleus of IC neurons were reported to have GAD-immunoreactivity (Oliver et al., 1994, Winer et al., 1995). IC neurons receive GABAergic projections mainly from the dorsal nuclei of lateral lemniscus (DNLL) and intrinsic GABAergic neurons (Shneiderman et al., 1993, Oliver et al., 1994). Immunnohistochemical studies showed GABAergic axonal endings at dendrites or soma of IC neurons (Oliver and Beckius, 1992). IC neurons have a wide variety of response properties to sound stimulation (Casseday and Covey, 1996, Ehret, 1997). Previous studies have shown that GABAergic inhibitory inputs play an important role in shaping the response properties of IC neurons (Faingold et al., 1989, Faingold et al., 1991, Yang et al., 1992, Park and Pollak, 1993a, Park and Pollak, 1993b, Park and Pollak, 1994, Le-Beau et al., 1996). For example, iontophoretically applied bicuculline, a GABAA receptor blocker, broadened a receptive field of the tuning curve in IC of the bat (Yang et al., 1992, Jen et al., 2001). Electrophysiological studies in slice preparation also showed that a large populations of IC neurons have a synaptic response, which is sensitive to bicuculline (Moore et al., 1998, Reetz and Ehret, 1999). In spite of their functional significance, little is known about the firing characteristics of intrinsic IC GABAergic neurons. The difficulty in distinguishing GABAergic and non-GABAergic neurons in slice preapartion might have prevented an extensive electrophysiological study of the inhibitory neurons.

In our present study, we adopted glutamate decarboxylase-green fluorescent protein (GAD67-GFP) knock-in mouse (Yanagawa et al., 2001, Tamamaki et al., 2003) to elucidate the function of GABAergic neurons in IC. This knock-in mouse was made by the gene targeting of enhanced GFP (EGFP) to the locus of GAD67 gene, and the expression of EGFP is to be under the control of the endogenous GAD67 promoter. GABAergic neurons can be identified by the GFP-fluorescence in the knock-in mouse (Tamamaki et al., 2003). In this paper, we focused GFP-labeled neurons in IC to uncover the electrophysiological properties of the GABAergic neurons in it.

Section snippets

Knock-in mouse

Gene targeting of EGFP to the locus of GAD67 encoding lesion has been described elsewhere (Yanagawa et al., 2001, Tamamaki et al., 2003). Tamamaki et al. (2003) described two strains of transgenic mice, one is GAD67-GFP mouse, which retains a loxP-flanked neomycin-resistance cassette (PGK-Neo) and the other is GAD67-GFP mouse, which lacks the PGK-Neo cassette (Δneo). In this study we used the heterozygous transgenic mice, which were obtained by breeding the mice (PGK-Neo) with wild type ICR

GFP-positive neurons in IC

In this paper, we classified inferior colliculus (IC) into three parts, according to previous anatomical studies described in rats (Fay-Lund and Osen, 1985, Peruzzi et al., 2000). Three subdivisions are a dorsal cortex (DC), an external cortex (EC) and a central nucleus (CIC). We adopted this classification, because we observed almost same cytoarchitectonic areas and cortical structures in Nissl stained IC preparation in the mouse (Fig. 1A and B). Likely to rat (Fay-Lund and Osen, 1985), the

GABAergic neurons in IC

GABAergic neurons of the mouse IC can be roughly classified into two groups from their electrical properties: tonic and transient type neurons. Tonic type neurons showed regular and repetitive discharge pattern in response to a long depolarizing current pulse. And the firing frequency is almost proportional to the intensity of injected current before it reached to the maximum. Tonic type neurons are further divided into two subgroups of RS and BP neurons by the difference in firing pattern when

Acknowledgements

We appreciated Mr. M. Fukao for the technical assistance and Mr. K. Nakamura, Dr. T. Furuta, Professor. N. Tamamaki, Professor. H. Ohmori and Professor. T. Kaneko for helpful discussion. This work was supported by grants-in-aid from the ministry of education to K.K. (14580788), and Y.Y. (15500220, 16015315).

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