GABAergic dysfunction in mGlu7 receptor-deficient mice as reflected by decreased levels of glutamic acid decarboxylase 65 and 67 kDa and increased reelin proteins in the hippocampus

Abstract

Glutamate is the main excitatory neurotransmitter in the brain, while γ-aminobutyric acid (GABA) is a primary inhibitory neuromodulator. Both amino acids act through ionotropic and metabotropic receptors that are widely distributed in the central nervous system. There are at least eight subtypes of metabotropic glutamate receptors (mGlu), which have been divided into three groups (mGlu I, II, and III). The mGlu7 receptor subtype, which belongs to the mGlu III group, seems to play a special role, as it is abundant in brain structures that are known to be responsible for antidepressant and/or anxiolytic activity of drugs. In GABAergic neurons, GABA is synthesised from glutamate by the pyridoxal phosphate (PLP)-dependent enzyme glutamic acid decarboxylase (GAD). It is expressed as two major isoforms, GAD65 and GAD67, responsible for the synthesis of the vesicular and cytoplasmic pool of neurotransmitter, respectively. Moreover, GABAergic neurons express a variety of proteins such as reelin, involved in synaptic transmission and plasticity. The aim of our study was to investigate the regulation of GABA synthesis and the level of modulatory receptor for GABA in mice lacking mGlu7 receptor for glutamate. The levels of GAD mRNA, GADs, and reelin proteins in the hippocampi of mGlu7−/− and mGlu7−/+ mice were measured using in situ hybridisation, immunohistochemistry, and Western blotting (WB). GAD mRNAs in the CA and DG regions of the hippocampus were measured separately. The levels of GAD65, GAD67, and reelin proteins were determined in the homogenates using WB, and the number of stained neurons was estimated using a stereological method of counting. GABA_B receptor level was measured using a radioligand binding assay. Our results show that the mRNA and protein levels of both GADs were decreased in the hippocampi of animals lacking the mGlu7 receptor. Decreased levels of GAD67 mRNA were found in both the CA and DG regions, while the decrease in GAD65 mRNA was observed mainly in the CA region of the hippocampus. The protein levels of GAD65 was lowered in mGlu7−/− animals only, while GAD67 and GABA_B receptor number were decreased in both mGlu7+/− and mGlu7−/− mice when measured in the whole hippocampus. In contrast, reelin was shown to be increased both in mGlu7−/+ and mGlu7−/− mice. The results suggest that mGlu7 receptor is involved in the regulation of GABAergic system activity at the level of GABA synthesised enzymes, specific proteins expressed by GABAergic neurons and metabotropic receptor for GABA.

Introduction

Glutamate is the primary excitatory neurotransmitter in the central nervous system (CNS). Usually, it constitutes nearly 60% of the total amount of all neurotransmitters in the brain; the remaining 40% is GABA, the main inhibitory agent. All other monoamine or neuropeptide neurotransmitters make up only about 1% of the total. Under normal conditions, these two basic amino acids coexist in a physiological balance which ensures homeostasis in the brain. It is hypothesised that the disruption of this balance leads to a shift from health to disease, resulting in anxiety or depression when Glu activity increases, while hypofunction of both systems may lead to psychotomimetic symptoms (Palucha and Pilc, 2007, Wieronska and Pilc, 2009).

Glutamate acts through two types of receptors: ionotropic glutamate receptors (iGlu) and metabotropic glutamate receptors (mGlu). The latter are further divided into three groups according to sequence homology, pharmacology, and second messenger system activated (Pin and Duvoisin, 1995). Group III, which consists of mGlu4, mGlu6, mGlu7, and mGlu8 receptors, is the largest group of metabotropic Glu receptors (Pin and Duvoisin, 1995, Lavreysen and Dautzenberg, 2008). Among the subtypes of group III, mGlu7 receptors seem to have a very special role. They are the most highly conserved of all mGlu receptor subtypes across different mammalian species (Makoff et al., 1996, Flor et al., 1997) and are abundant in brain regions such as the hippocampus, amygdala, and locus coeruleus that are known to be critical in pathophysiology and that are important targets for the treatment of mood disorders (Kinoshita et al., 1998). Studies with mGlu7 knockout animals have shown that mGlu7 receptor ablation is associated with changes in behaviour predictive of antidepressant action, suggesting a crucial role of the receptor in the integration of stress response to aversive stimuli (Cryan et al., 2003, Palucha et al., 2007a, Stachowicz et al., 2008). As autoreceptors located in glutamatergic neurons, mGlu7 receptors regulate glutamate release, while as heteroreceptors located in GABAergic interneurons, they participate in the regulation of GABA activity (Sansig et al., 2001, Li et al., 2008, Somogyi et al., 2003).

GABA is synthesised in GABAergic neurons from glutamate by the pyridoxal phosphate (PLP)-dependent enzyme glutamic acid decarboxylase (GAD). As the enzyme responsible for mediation of this synthesis, GAD seems to be involved in the regulation of the balance between glutamate and GABA, giving it an important role in CNS function (Martin and Rimvall, 1993). GAD is expressed as two major isoforms, GAD65 and GAD67, which are the products of two independently regulated genes located in chromosomes 2 and 10, respectively (Laprade and Soghomonian, 1999). Each isoform is highly conserved among vertebrates; the amino acid sequences of the cat, rat, mouse, and human proteins share more than 95% identity (Kaufman et al., 1986, Erlander et al., 1991, Erlander and Tobin, 1991). Approximately 30–40% of GABAergic neurons express reelin, an extracellular matrix protein (ECM) critical for brain function that contributes to normal lamination in embryos and subserves synaptic plasticity in adult brain (Scotti and Herrmann, 2002; for review see Rice and Curran, 2001). Cells that can be double-labelled for reelin and the GABA synthesizing enzymes glutamic acid decarboxylase 65- and 67-kDa represent about 4% of the total neuron population and their density remains constant with age (Pesold et al., 1998). Since dysfunctions in GADs expression can directly influence the level of GABA, and since reelin plays a role in brain development and cell signalling, it can be speculated that these proteins strongly connected with the GABAergic system may be involved in the etiology of bipolar and/or mood disorders (Fatemi et al., 2000, Fatemi et al., 2001, 2002; Heckers et al., 2002).

The hippocampal formation is especially interesting as a brain structure involved in the pathology of depression, anxiety, and other psychiatric disorders. Therefore, in the current study, the role of mGlu7 receptors in the regulation of GABAergic system activity in the hippocampus was investigated using mGlu7 knockout mice. The levels of reelin and of GAD65- and 67-kDa proteins were examined because the normal production of these proteins reflects proper GABAergic cell function in the brain (Fatemi et al., 2005, Guidotti et al., 2005). The levels of mRNAs for both GADs were also investigated. Moreover, the expression of GABA_B receptor, the metabotropic GABA receptor frequently targeted in the pharmacotherapy of psychiatric disorders (Nowak et al., 2006, Pilc and Nowak, 2005, Cryan et al., 2005), was also studied.