Differential distribution of GABA_A receptor subunits in soma and processes of cerebellar granule cells: Effects of maturation and a GABA agonist

Abstract

Quantitative analysis of the density of alpha 1 and beta 2/3 GABAA receptor subunits was performed at the electron microscope level after indirect pre-embedding immunogold labeling with subunit-specific antibodies of rat cerebellar granule cell cultures grown for 4 or 8 days and in the presence or absence of the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridin-3-ol (THIP). THIP (150 microM) induced a 2-fold increase in the number of alpha 1 and beta 2/3 subunits in both cell bodies and processes in 4-day-old cultures. Extending the culture period to 8 days led to a polarization of the receptor expression, since the increase in the number of subunits selectively was observed in the processes. Moreover, a general subcellular differentiation of the receptor population was observed in all culture conditions, since the ratio between the two subunits (beta 2/3; alpha 1) was four times higher in cell bodies compared to processes. A detailed analysis of the less mature (4-day-old) cultures revealed the existence of two populations of neurons exhibiting differences in the average number of receptors. During maturation neurons with few receptors developed into cells with a higher density of receptors resulting in a single population of the latter neurons, a process enhanced by exposure to THIP. This may indicate that receptor development is a discontinuous process with individual neurons following different temporal patterns. In double-labeling experiments, a spatially close association of the alpha 1 and beta 2/3 subunits could be seen, but the subunits were more frequently found separated from each other. In spite of the fact that exposure of the neurons to THIP increased the total number of receptor subunits, its presence apparently prevented formation of receptors with this subunit composition. Interestingly, receptor subunit clusters, consisting of alpha 1 alone, were more frequently observed than composite (alpha 1; beta 2/3) clusters. This substantiates the view that receptors not having alpha 1 and beta 2/3 subunits in the same complex may exist.

Normal formation of the postsynaptic elements of GABAergic synapses in the reeler cerebellum

“…These results suggest that developmental changes in subunit composition are independent of neuronal maturation, such as settling in the normal neuronal positions, maturation of excitatory networks, and formation of inhibitory synapses with basket and stellate cells. Therefore, as reported previously using culture granule cells [3,6,7,11,15,36,49,55], α1-expression may be initiated by GABAergic innervation from Golgi cells and neighboring Purkinje cells in the central cerebellar mass. In addition, the suppression of α2 and α3 subunit-expression might be related to the GABAergic innervation.…”

Modulatory actions of gamma aminobutyric acid (GABA) on GABA type a receptor subunit expression and function

“…The mechanism for this action of GABA involves activation of preexisting GABA, receptors (Belhage et al, 1990a,b) with subsequent stimulation of formation of GABA, receptor subunit mRNA and enhancement of protein synthesis (Belhage et al, 1990a;Kim et al, 1994). It appears to involve primarily mRNA for the subunits a 1 and p2 (Kim et al, 1994) in keeping with the immunocytochemical demonstration of a large increase in the expression of these subunits after exposure of neurons to the GABAA receptor specific agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) (Hansen et al, 1991;Elster et a]., 1995).…”

“…The mechanism for this action of GABA involves activation of preexisting GABA, receptors (Belhage et al, 1990a,b) with subsequent stimulation of formation of GABA, receptor subunit mRNA and enhancement of protein synthesis (Belhage et al, 1990a;Kim et al, 1994). It appears to involve primarily mRNA for the subunits a 1 and p2 (Kim et al, 1994) in keeping with the immunocytochemical demonstration of a large increase in the expression of these subunits after exposure of neurons to the GABAA receptor specific agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) (Hansen et al, 1991;Elster et a]., 1995).Although exposure of neurons to GABA or GABAA receptor agonists leads to an enhanced expression of GABAA receptors via stimulation of transcription and translation processes (Belhage et al, 1990a;Kim et al, 1994), the exact molecular mechanism remains to be established. That molecular events at the nuclear level may play an important role is underlined by the observation that polyamine biosynthesis and availability seem to be a determining factor (Abraham et al, 1994).…”

Morphological development and maturation of the GABAergic synapses in the mouse cerebellar granular layer

“…Moreover, the majority of both α1 and α6 subunit proteins accumulated adjacent to the Golgi cell axon terminals. These results demonstrated that (1) GAD-positive growth cones and varicosities probably undergo a change to GABAergic terminals [26,48], (2) GABAergic synapses were massively formed during the second postnatal week [2,28,31,37], and (3) GABAergic innervation might initiate the expression of the GABA A receptor α1 and α6 subunit genes, as well as trafficking and clustering of proteins to the synaptic site [1,8,12,13,16,17,20,24,46,48,61].…”