Differential Localisation of GABAB Receptor Subtypes in the Brain, Their Functions in the Dentate Gyrus and Their Roles in the Control of Seizures.

GABAB receptors are G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. Functional GABAB receptors are formed by the assembly of GABAB1 and GABAB2 subunits. Genetic deletion of either subunit in mice abolished GABAB receptor-mediated inhibition resulting in epilepsy and shortened life-span. The two main isoforms of the GABAB1 subunit, GABAB1a and GABAB1b, form two receptor subtypes GABAB(1a, 2) and GABAB(1b, 2), and show differential synaptic localisation in several brain areas. In the amygdala, hippocampus, thalamus, and neocortex, the GABAB1a containing receptors have been demonstrated at presynaptic glutamatergic terminals, whereas, GABAB1b containing receptors were found at postsynaptic sites. In addition, both GABAB1 isoforms may function as autoreceptors on GABAergic terminals. This study aimed to further understand the roles of GABAB receptor subtypes in modulating synaptic inhibition and neuronal excitability. Receptor subtype expression was examined in the brain nuclei of GABAB1a-/- and GABAB1b-/- mice by immunohistochemistry using antibodies selective for the GABAB1 and the GABAB2 subunits. The modulation of synaptic inhibition by GABAB receptor subtypes was investigated in the dentate gyrus of the GABAB1a-/- and GABAB1b-/- mice by performing electrophysiological recordings in brain slices. The net effect of GABAB receptors on neuronal excitability was then compared by scoring seizure activities induced by a chemoconvulsant in GABAB1a -/- and GABAB1b-/- mice. GABAB1 staining was found to be widely reduced in both GABAB1a-/- and GABAB1b-/-brains. However, expression of GABAB2 was selectively reduced in the caudate putamen and amygdala in the GABAB1a-/- mice and in the thalamus of GABAB1b-/- mice, suggesting a predominant expression of the GABAB1a and GABAB1b subtypes in different brain nuclei. As the caudate putamen neurons were shown previously to have no intrinsic expression of GABAB2 transcripts, the GABAB1a containing subtypes are likely to be expressed on terminals of afferent projections to this region. The lack of GABAB1a may, therefore, also affect the interaction between different brain nuclei. The lack of GABAB1b subtypes, predominantly in the thalamus of GABAB1b-/- mice, may affect postsynaptic inhibition and result in increased neuronal excitability. In the hippocampal formation, glutamatergic pathways in the CA1 and CA3 are mainly modulated by the GABAB1a subtype. The dentate gyrus, a key region involved in seizures and epilepsy, receives excitatory inputs via the perforant pathway and the associational/commissural pathways and sends glutamateric outputs to the CA3 via mossy fibres. However, no GABAB heteroreceptor-mediated inhibition was found at the perforant pathway-dentate gyrus granule cell terminals. In agreement, staining in the dentate gyrus molecular layer associated with perforant pathway input to the granule cells was found not to differ between the genotypes. Staining of the glutamatergic associational/commissural pathway terminals showed GABAB1a containing receptors demonstrating a differential localisation for the receptors in the dentate gyrus. In contrast to the CA1 and CA3, activation of either the GABAB1a or GABAB1b receptors induced disinhibition via inhibition of GABAergic tramsission in the dentate gyrus. This excitatory effect was enhanced in the GABAB1b -/- mice and was likely due to loss of GABAB-mediated postsynaptic inhibition. Finally, acute seizure models were implemented to study the role of each receptor subtype in the control of excitability. GABAB1a-/- mice were found to display higher seizure activity invoked by the chemoconvulsant pentylenetetrazol, demonstrating that GABAB1a isoform exerts a greater overall inhibitory effect in the brain than the GABAB1b isoform. Pre-treatment with baclofen, a GABAB receptor agonist, decreased seizure activity in GABAB1a-/-, with little effect in GABAB1b-/- mice. Activation of the GABAB1a containing receptors causes excitation in the dentate gyrus, which is subsequently responsible for inhibition in the CA3 and CA1. In addition, the GABAB1a subtype is important for modulating excitatory communication between brain nuclei, their loss may promote spreading of seizure activity and reduction of seizure thresholds. Overall, the results of this study show differential expression of GABAB receptors subtypes in brain nuclei and between synaptic pathways. Collectively, it was found that the absence of GABAB(1a, 2) receptors increased the net excitability of the brain and enhanced chemoconvulsant-induced seizure activity. Genetic mutation and autoimmunity to the GABAB(1a, 2) may therefore underlie certain types of epilepsy and seizures observed clinically