ABSTRACT
In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of GABAA and of benzodiazepine binding sites between membranes derived from this fraction (fraction G) and from a total cerebellar homogenate (fraction T) was studied. The benzodiazepine and GABA binding sites were measured by the binding of agonists [3H]flunitrazepam and [3H]muscimol, respectively. The results indicate that both binding sites are present, but only slightly enriched, in the glomerular synapses. We found a muscimol/flunitrazepam binding site ratio of two, which is consistent with the enrichment of muscimol binding sites in the granular layer shown by both autoradiographic with radioactive glutamatergic ligands and in situ hybridization experiments respectively.
Subject(s)
Cerebellum/metabolism , Flunitrazepam/metabolism , Muscimol/metabolism , Synapses/metabolism , Animals , Binding Sites , Cerebellum/cytology , Rats , Receptors, GABA-A/metabolismABSTRACT
Cerebellar glomeruli consist of large portions of the mossy fiber giant terminal, granule cell dendrites and Golgi neuron terminals. By modifying previously reported procedures we have developed a new method for bulk preparation of this polysynaptic complex from rat cerebellum. We obtained well preserved isolated glomeruli of satisfactory purity and homogeneity as indicated by electron microscopy and by determination of appropriate biochemical markers. The method is fast and simple, and it provides a glomerular fraction suitable for investigation of neurotransmitter receptors.
Subject(s)
Cerebellum/ultrastructure , Histological Techniques , Nerve Endings/ultrastructure , Nerve Fibers/ultrastructure , Animals , Astrocytes/chemistry , Biomarkers , Cerebellum/chemistry , Glutamates/metabolism , Glutamic Acid , Microscopy, Electron , Nerve Endings/chemistry , Nerve Fibers/chemistry , Neurons/chemistry , Rats , Rats, Inbred Strains , Subcellular Fractions/chemistry , Subcellular Fractions/ultrastructureABSTRACT
In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of the different types of NMDA and non-NMDA glutamate binding sites was studied in the membranes derived from this fraction (fraction G) as compared to that in the membranes prepared from a total cerebellar homogenate (fraction T). Cl-/Ca2+ independent [3H]glutamate binding sites were not abundant and could be reliably measured only in fraction G. Cl- dependent/Ca2+ activated [3H]glutamate binding sites were more abundant and exhibited a single KD in both fractions G and T. Quisqualate, NMDA, kainate, L-AP4 and trans-ACPD inhibited [3H]glutamate binding to different extents in the two membrane fractions. Quisqualate sensitive sites were predominant in all cases but more abundant in fraction T than in fraction G. An opposite distribution was observed for the NMDA sensitive binding sites while kainate sensitive binding sites were scarce everywhere. Trans-ACPD, a ligand presumed selective for metabotropic glutamate binding sites, displaced [3H]glutamate from fraction T but nor from fraction G, suggesting the absence of these sites from glomeruli. Similarly, no L-AP4 sensitive sites were present in fraction G while they were abundant in fraction T. Binding sites associated with ionotropic receptors of the quisqualate type were determined by measuring [3H]AMPA binding. The density of the high affinity [3H]AMPA binding sites in fraction T was twice as high as in fraction G, indicating that these sites are abundant in structures other than glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)
