Calcium changes induced by presynaptic 5-hydroxytryptamine-3 serotonin receptors on isolated terminals from various regions of the rat brain

The serotonin 5-hydroxytryptamine-3 receptor is a ligand-gated ion channel that is distributed widely in the nervous system. Within the CNS, a signifi cant portion of the 5-hydroxytryptamine-3 receptors appears to be present o n presynaptic nerve terminals and, using an imaging approach, it was shown previously that presynaptic 5-hydroxytryptamine-3 receptors on individual i solated nerve terminals (synaptosomes) from rat corpus striatum display a d istinctive set of properties-slow onset, little desensitization and high ap parent permeability for Ca2+-when compared to those observed for 5-hydroxyt ryptamine-3 receptors localized at postsynaptic sites on neuronal cell bodi es. To consider whether their characteristic nature is a common feature of presynaptic 5-hydroxytryptamine-3 receptors across the brain, we used confo cal microscopy to measure changes in intracellular Ca2+ concentration resul ting from 5-hydroxytryptamine 3 agonist-induced responses in synaptosomes f rom representative rat brain regions, ranging in expression of overall leve ls of 5-hydroxytryptamine-3 receptors from relatively low (cerebellum) to i ntermediate (corpus striatum and hippocampus) to high (amygdala). Applicati on of 100 nM m-chlorophenyl biguanide, a specific 5-hydroxytryptamine-3 rec eptor agonist, induced changes in relative intracellular Ca2+ concentration in subsets of synaptosomes from the corpus striatum (similar to 6% of tota l), hippocampus (similar to 3% of total), amygdala (similar to 30% of total ) and cerebellum (similar to 32% of total). In order to assure the viabilit y of the synaptosomes that did not respond to 5-hydroxytryptamine-3 agonist stimulation, KCI (45 mM) was subsequently added to depolarize the same pop ulation of synaptosomes, and increases in intracellular Ca2+ concentration were then seen in 80-90% of the synaptosomes from all four regions. The kin etics of the intra synaptosomal Ca2+ changes produced by K+-evoked depolari zation were similar in all regions, showing a rapid rise to a peak followed by an apparent plateau phase. In contrast, the changes in intracellular Ca concentration evoked by m-chlorophenyl biguanide displayed substantially s lower kinetics, similar to previous findings, but which varied among respon ding synaptosomes from one region to another. In particular, in-chloropheny l biguanide-induced changes were notably slower in synaptosomes from the am ygdala (rise time constant, tau = 25 s), when compared to responses in syna ptosomes from other regions (striatum, tau = 12 s; hippocampus, tau = 9.6 s ; cerebellum, tau = 7 s). To independently demonstrate the presence of 5-hy droxytryptamine-3 receptors on nerve terminals in the various regions using a molecular approach, we double-immunostained the synaptosomes for the 5-h ydroxytryptamine-3 receptor and the synaptic vesicle protein synaptophysin, using, respectively, a polyclonal antibody raised against an N-terminal pe ptide of the 5-hydroxytryptamine-3 receptor and a monoclonal anti-synaptoph ysin antibody, and observed 5-hydroxytryptamine-3 receptors in varying subs ets of the synaptosomes from each region, providing direct support for the results obtained in our functional experiments. These results suggest that the distinctive properties of presynaptic 5-hydr oxytryptamine-3 receptors are found throughout the brain, with evident diff erences in the kinetics of the responses to agonist stimulation observed ac ross the brain regions studied. As expected, the proportion of the synaptos omal population that responded on application of 5-hydroxytryptamine-3 agon ist varied in preparations from one region to another; however, the presenc e of a relatively high proportion of presynaptic 5-hydroxytryptamine-3 rece ptors in the cerebellum contrasts with previous binding studies demonstrati ng a relatively low overall density of 5-hydroxytryptamine-3 receptors in t his region. We hypothesize that presynaptic 5-hydroxytryptamine-3 receptors present on nerve terminals regulate the functioning of select synapses thr oughout the rat brain. (C) 1999 IBRO. Published by Elsevier Science Ltd.