Serotonergic Innervation of the Frog Spinal Cord and Involvement of 5-HT_5A Receptors in the Modulation of Miniature Glycinergic Postsynaptic Potentials of Lumbar Motoneurons

The role of serotonin 5-HT_5A receptorsin the modulation of miniature inhibitory synaptic activity was studiedusing intracellular recording of miniature glycinergic inhibitorypostsynaptic potentials (glymIPSPs) in the lumbar motoneurons ofthe isolated spinal cord of the frog Ranaridibunda. In a medium containing TTx, CNQX, D-AP5, bicuculline,application of the serotonin receptor agonist 5-CT (10 µM) withhigh affinity for 5-HT_5A led to a suppressionof frequency by 86%, as well as the disappearance of high-amplitudeglymIPSPs (200–500 µV) at preservation of rare potentials with an amplitudeof about 100 µV. This effect indicates the possibility of pre- andpostsynaptic action of 5-CT at such a concentration, not limitedto its effect only on 5-HT_5A receptors. Theaddition 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 averageamplitude by 20%, which may indicate the participation of 5-HT_5A receptorsin pre- and postsynaptic modulation in glymIPSPs of motoneurons.Application of 1 µM 5-CT led to a decrease in the frequency of glymIPSPsby 49% without a noticeable change in the amplitude of glymIPSPs,and the subsequent introduction of SB-699551, a selective antagonistof 5-HT_5A receptors, into the solution increasedthe frequency of events by 41%, which confirms the involvement of5-HT_5A receptors in presynaptic modulationof glymIPSPs. Immunofluorescence study showed that supra- and intraspinal5-HT+ ir neurons produce abundant branching in the lumbar regionwith the possibility of forming axosomatic contacts with labeled motoneuronsand axodendritic contacts on the proximal and distal portions oftheir dendrites. It is also possible to form contacts in the perimedullaryplexus, penetrated by the distal dendrites of motoneurons and astrocyticprocesses. This represents the structural basis for post-, pre-and extrasynaptic modulation of motoneuron activity by serotonin.The possibility of postsynaptic modulation of motoneuron activity through5-HT_5A receptors is confirmed by the point-likefluorescence of the 5-HT_5ARlike+ signal on thedendrites and bodies of labeled motoneurons, which is present inthe neuropil but absent in the perimedullary plexus. Double labelingwith antibodies to the 5-HT_5A receptor andthe Ca²⁺-binding protein, parvalbumin,revealed 5-HT_5ARlike+ localization in themyelin sheath of dorsal and ventromedial funiculi fibers. In preparationsafter long-term stimulation of the ventral roots through suctionelectrodes when labeling motor neurons with biocytin, a bright 5-HT_5ARlike+signal was detected in the myelin of motor axons, dorsal root fibersentering the brain in the region of the dorsal horn and individualfibers of the ventromedial funiculus. The participation of extrasynaptic5-HT_5A receptors in the functioning of feedbackcircuits of lumbar motoneuron activity, with the possible participationof glial elements in these circuits, is discussed.