Serotoninergic innervation of the frog spinal cord and involvement of 5-HT5A receptors in the modulation of miniature glycinergic postsynaptic potentials of lumbar motoneurons

Abstract

The role of serotonin 5-HT5A receptors in the modulation of miniature inhibitory synaptic activity was studied using intracellular recording of miniature glycinergic inhibitory postsynaptic potentials (glymIPSPs) in the lumbar motoneurons of the isolated spinal cord of the frog Rana ridibunda. In a medium containing TTx, CNQX, DAP5, bicuculline, application of the serotonin receptor agonist 5-CT (10 µM) with high affinity for 5-HT5A led to a suppression of frequency by 86%, as well as the disappearance of high-amplitude glymIPSPs (200–500 µV) at preservation of rare potentials with an amplitude of about 100 μV. This effect indicates the possibility of pre- and postsynaptic action of 5-CT at such a concentration, not limited to its effect only on 5-HT5A receptors. The addition of methysergide, a blocker of 5-HT 1,2 receptors, to the medium reduced the average frequency of glymIPSPs by 67%, the frequency of high-amplitude events by 5 times and their average amplitude by 20%, which may indicate the participation of 5-HT5A receptors in pre- and postsynaptic modulation in glymIPSPs of motoneurons. Application of 1 μM 5-CT led to a decrease in the frequency of glymIPSPs by 49% without a noticeable change in the amplitude of glymIPSPs, and the subsequent introduction of SB-699551, a selective antagonist of 5-HT5A receptors, into the solution increased the frequency of events by 41%, which confirms the involvement of 5-HT5A receptors in presynaptic modulation of glymIPSPs. Immunofluorescence study showed that supra- and intraspinal 5-HT+ ir neurons produce abundant branching in the lumbar region with the possibility of forming axosomatic contacts with labeled motoneurons and axodendritic contacts on the proximal and distal portions of their dendrites. It is also possible to form contacts in the perimedullary plexus, penetrated by the distal dendrites of motoneurons and astrocytic processes. This represents the structural basis for post-, pre- and extrasynaptic modulation of motoneuron activity by serotonin. The possibility of postsynaptic modulation of motoneuron activity through 5-HT5A receptors is confirmed by the point-like fluorescence of the 5-HT5ARlike+ signal on the dendrites and bodies of labeled motoneurons, which is present in the neuropil but absent in the perimedullary plexus. Double labeling with antibodies to the 5-HT5A receptor and the Ca 2+ -binding protein, parvalbumin, revealed 5-HT5ARlike+ localization in the myelin sheath of dorsal and ventromedial funiculi fibers. In preparations after long-term stimulation of the ventral roots through suction electrodes when labeling motor neurons with biocytin, a bright 5-HT5ARlike+ signal was detected in the myelin of motor axons, dorsal root fibers entering the brain in the region of the dorsal horn and individual fibers of the ventromedial funiculus. The participation of extrasynaptic 5-HT5A receptors in the functioning of feedback circuits of lumbar motoneuron activity, with the possible participation of glial elements in these circuits, is discussed.