Cellular architecture of the mouse hippocampus: A quantitative aspect of chemically defined GABAergic neurons with stereology

Abstract

The structural organization of the central nervous system is an infrastructure of the modern neuroscience. Especially, anatomical research at the cellular level can serve as a fundamental resource to understand various novel findings from the molecular level to the system level. Of the decade, the importance of rigorous quantitative neuroanatomy is gradually realized, but earlier anatomical reports are usually presented in qualitative terms or quantified with biased methods. This article quantitatively describes the spatial distributions of chemically defined GABAergic neurons in the mouse hippocampus by using unbiased stereological techniques. We focus on the expression of nine major neurochemical markers: glutamic acid decarboxylase 67, three calcium binding proteins (parvalbumin, calretinin, calbindin D28K), four neuropeptides (somatostatin, neuropeptide Y, cholecystokinin, vasoactive intestinal protein) and neuronal nitric oxide synthase. Here we deal with their laminar distributions, numerical densities and the relative ratio to the total GABAergic neurons, with special reference to their differentiation along the dorsoventral axis of the mouse hippocampus. Furthermore, we estimate the absolute numbers of GABAergic neurons contained in a 300-μm-thick hypothetical slice. The present data outline the quantitative aspects of the cellular architecture of hippocampal GABAergic system and also give complementary information to the recent multidisciplinary analyses at the single cell level.

Introduction

Ever since Ramón y Cajal (1911) described the morphological difference of neurons, many studies reported the diversity of GABAergic neurons in the cortex from anatomical, neurochemical and electrophysiological viewpoints (DeFelipe, 1993, Cauli et al., 1997, Parra et al., 1998, Kubota and Kawaguchi, 2000; for review see, Freund and Buzsáki, 1996, Maccaferri and Lacaille, 2003, Markram et al., 2004, Somogyi and Klausberger, 2005). It is now well recognized that GABAergic neurons play various functions in the brain activity, including synchronized oscillation and synaptic plasticity (Whittington et al., 1995, Buzsáki et al., 1996, Blatow et al., 2003, Bacci and Huguenard, 2006; for review see, Mann et al., 2005). Furthermore, recent reports demonstrated that distinct subtypes of GABAergic neurons coordinated the activity of pyramidal neurons in a temporally different and brain-state-dependent manner (Klausberger et al., 2003, Klausberger et al., 2004). To date, characterization of GABAergic neuron subtypes is considered to be particularly important, and thus attracts much attention in the field of neuroscience.

Despite the above-mentioned extensive attention, GABAergic neuron research now confronts serious issues. One of the biggest problems may be the lack of agreement on the classifications of GABAergic neurons (Parra et al., 1998, Maccaferri and Lacaille, 2003). Conceivably, the most practically and widely used classification of GABAergic neurons is based upon the neurochemical markers. Previous studies reported the specific morphological characteristics and electrophysiological activities of chemically defined GABAergic neurons in the hippocampus and neocortex (Kawaguchi et al., 1987, Kawaguchi and Kubota, 1998, Wang et al., 2002), and thus the neurochemical markers were expected to work as a hub of the GABAergic neuron study (Fig. 1). But recent studies revealed the morphological and electrophysiological diversities of immunohistochemically defined subclasses of GABAergic neurons (Maccaferri et al., 2000, Pawelzik et al., 2002; for review see, Markram et al., 2004). In addition, some of the neurochemical markers were coexpressed in single GABAergic neurons (Kosaka et al., 1985a, Kosaka et al., 1985b, Sloviter and Nilaver, 1987, Dun et al., 1994, Jinno and Kosaka, 2000, Jinno and Kosaka, 2002, Jinno and Kosaka, 2004). Therefore, it is rather difficult to define distinct GABAergic neuron subtypes just by using the neurochemical markers. Even taking these short-comings into consideration, the analyses based on the neurochemical markers have some merits. One major advantage of the use of neurochemical markers is the compatibility with the quantitative analyses. Because immunostaining allows obtaining a mass of GABAergic neurons, it enables precise estimation of their numbers and/or densities in specific brain regions. In that reason, the neurochemical markers are quite helpful not only to investigate the anatomical organization of the brain, but also to estimate possible pathological changes in association with some diseases, such as ischemia (Arabadzisz and Freund, 1999) and epilepsy (Cossart et al., 2001, Cobos et al., 2005).

The latest development of molecular biology enabled the identification of individual GABAergic neurons by using multiplex reverse transcription polymerase chain reaction (mRT-PCR) at the single cell level (Markram et al., 2004). Some multidisciplinary studies reported that the specific gene expression profiles were able to predict the morphological and electrophysiological subclasses of GABAergic neurons in the neocortex (Wang et al., 2002, Toledo-Rodriguez et al., 2004, Sugino et al., 2006). Interestingly, Toledo-Rodriguez et al. (2005) demonstrated the strong relationship between specific combinations of three calcium binding proteins (parvalbumin, calretinin and calbindin D28K) and four neuropeptides (neuropeptide Y, somatostatin, cholecystokinin and vasoactive intestinal protein) gene expressions and morphological characteristics of neocortical GABAergic neurons. These modern research techniques have provided indispensable information about individual GABAergic neurons.

In this article, we quantitatively describe the spatial distributions of chemically defined hippocampal GABAergic neurons in mice. We focus on the patterns of expression of nine major neurochemical markers: glutamic acid decarboxylase 67 (GAD67), three calcium binding proteins [parvalbumin (PV), calretinin (CR) and calbindin D28K (CB)], four neuropeptides [neuropeptide Y (NPY), somatostatin (SOM), cholecystokinin (CCK) and vasoactive intestinal protein (VIP)] and neuronal nitric oxide synthase (NOS). The data shown here is mainly obtained from a series of our stereological analyses (Jinno et al., 1998, Jinno et al., 1999, Jinno and Kosaka, 2002, Jinno and Kosaka, 2003b). Furthermore, we newly estimated the absolute numbers of chemically defined GABAergic neurons contained in a 300-μm-thick hippocampal slice. Meanwhile, we have to accept that the neurochemical markers do not always represent distinct subclasses as described before, and thus they are not ideal for taxonomy of GABAergic neurons in the hippocampus. What is important is that they can outline the quantitative aspects of the cellular architecture of hippocampal GABAergic neurons. Considering that commonly used single cell analyses for multidisciplinary studies are not able to obtain unbiased quantitative results, the present data will also give complementary information. The aim of this review is to provide an infrastructure of the hippocampal research.