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

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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.

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N. M. Chmykhova

Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences

Author for correspondence.
Email: nchmykhova@gmail.com
Russian Federation, St. Petersburg

D. S. Vasilev

Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences

Email: nchmykhova@gmail.com
Russian Federation, St. Petersburg

N. P. Veselkin

Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences

Email: nchmykhova@gmail.com
Russian Federation, St. Petersburg

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2. Fig. 1. Parameters of GLIMTPS motor neuron, frequency and amplitude, with application of 10 µm 5-CT (experimental series I); (a) – examples of registration (complete records of 300 runs) of total spontaneous activity (sPSP), the isolated fraction of miniature glycine inhibitory postsynaptic potentials (GLIMTPS) and GLIMTPS after 10 minutes applications of 5-CT, an agonist of 5-HT receptors, (10 microns) in the lumbar motor neuron of the frog spinal cord; (b) fragments (4 seconds) of recordings of motor neuron activities: sPSP, glim TPSP, their suppression after 20 and 30 minutes of 5-CT application and restoration of spontaneous activity with the appearance of high-amplitude events (more than 1 mV) 20 minutes after the change of the experimental solution to the normal composition; (c) – histogram of the decrease in the number and amplitude of GLIMTPS in a motor neuron under the action of 5-CT (10 microns) (red bars) compared with the control (white bars).

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3. Fig. 2. The effect of 5-CT (10 microns) on glimTPSP with additional administration of an antagonist into a superfusing solution 5-HT1.2 methysergide (MS) receptors (10 microns) (experimental series II). (a) – registration of GLIMTPS in one of the five motor neurons. In a solution with 5-CT, there is a decrease in the number of GLIMTPS, which are blocked by subsequent administration of strychnine (Str) into the solution. Washing the drug with a solution of normal composition leads to spontaneous synaptic activity of the motor neuron. (b) – probability distribution of occurrence of various intervals of frequency and amplitude of the glimTPSP motor neuron before- (Control-black line) and under the action of 5-CT (10 microns) (red line) in the presence of methysergide in a superfusing solution. (c) – a comparison of the action of 5-CT (10 µm) in experiments without the presence of methysergide in solution and with methysergide shows a significant difference in the suppression of the frequency of GLIMTPS in lumbar motor neurons (p < 0.05, unpaired t –Student criterion).

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4. Fig. 3. The effect of SB‑699551, an antagonist of 5-HT5A receptors, on GLIMTPS motor neuron during superfusion with a solution without 5-CT containing 5-HT (10 microns) and methysergide (experimental series III). (a) – fragments of registrations in the motor neuron: 1, 2 - violent spontaneous glycine activity; 3, 4 – glimTPSP after addition to the TTx control solution; 5-8 – glimTPSP under the action of the SB‑699551 application – in the first 5 minutes and after 15 minutes. (b) – distributions of the probability of occurrence of various frequency and amplitude intervals of glimTPSP in the control (black line) and after 5 minutes of the action of the SB‑699551 antagonist (red line).

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5. Fig. 4. The effect of 5-CT and the 5-HT5A receptor antagonist SB‑699551na glimTPSP introduced into the solution (experimental series IV). (a) – 5-CT in low concentration (1mkM) also suppresses the frequency of glimTPSP with little effect on the amplitude of glimTPSP, and the antagonist of 5-HT5A receptors restores inhibitory activity (the two lower entries of glimTPSP). (b) – probability distribution of occurrence of different frequency and amplitude intervals of GLIMTPS in the control (black line) and after 10 minutes of injection into the solution, a 5-CT histogram (red line). (c) – a decrease in the frequency of glimTPSP under the action of a 5-CT serotonin receptor agonist at concentrations of 10 and 1 µm (n = 6 and 7, respectively ) with a significant difference in effects (p < 0.05, unpaired t–Student criterion).

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6. Fig. 5. Histograms representing the effect of serotonin receptor ligands on the frequency or MTPs (Control 1) of seven motor neurons. The agonist of 5-HT 5-CT receptors at a concentration (1 µm) reduces the frequency by an average of -48.6 + 8.0% (Control 2), the antagonist 5-HT5A receptors SB‑699551 restores the frequency of events by an average of 41.0 + 16.4% (m + SE).

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7. Fig. 6. Serotonin-immunoreactive (5-HT+ ir) neurons in the medulla oblongata and spinal cord of the frog Rana ridibunda: (a – c) – three horizontal slices of the medulla oblongata with double immunomarkation to 5-HT (red) and to PV (green), representing the location and comparative dimensions of 5-HT+ ir neurons and neurons of the reticular nucleus of parval- buminimmunoreactive (PV+ir) without serotonin immunoreactivity (5-HT – ir); (d) – diagram-an example of the location of intraspinal serotonin neurons (ISN) in one of the horizontal sections of the thoracic spinal cord (horizontal plan); (e) – sagittal section of the thoracic spinal cord with a chain of frequently repeated iSNS; (f) is a histogram of the average diameter of the ISN com in the thoracic and lumbar segments; (g) is the ventromedial section of the frontal section of the lumbar segment with ISN having branching dorsal and ventral dendrites. Plenty 5-HT+IR terminals surround the neuron. Like supraspinal serotonin neurons, ISN does not have an immu- nor signal

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8. Fig. 7. Distribution of serotonin-immunoreactive elements in lumbar segments and possible contacts of 5-HT+ ir terminals with motor neurons labeled intracellularly by injection of neurobiotin or application of markers to the venous root: (a) - frontal section of the lumbar segment IX, colored according to Nissl, for comparative orientation of sections of sections with 5-HT immunoreaction (b); (b) – frontal section of the X lumbar segment c5-HT+ir; (c) – histogram of intensities (in conventional units from 0 to 2.0) of the 5-HT+ir signal of sections (1-8) of the lumbar thickening of the spinal cord: 1– perimedullary plexus (marginal plexus); 2 – lateral cord; 3 – ventral commissura; 4 – dorsal horn; 5 – intermedial zone; 6 – motor nucleus; 7 – entrance area of dorsal root fibers; 8 – ventromedial cord; (d) is an image of a fragment of the lumbar segment IX with motor neurons produced by intracellular injection of neurobiotin and subsequent detection by a DAB reaction. Reconstruction in two ways

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9. Fig. 8. Detection of 5-HT5ARlike+ immunoreactivity signal in the lumbar thickening of the frog spinal cord (the slice areas are shown in the diagrams): (a) is the area of entry of dorsal root fibers into the spinal cord, where there are few terminals with 5-HT+ ir, but distinct profiles of PV+ ir afferent fibers are visible (green) and increased intensity of the 5-HT5ARlike+ signal (red). The arrows indicate the section of the axoplasm of one of the large afferents with PV+ ir (green), the section of its myelin sheath in the form of a ring (red) and the colocalization of two images with the insertion of an enlarged image of the axon profile; (b, c) - the composition of images of the medial region of the dorsal cord (b) and medial The areas of the ventral cord (c) also represent the presence of a 5-HT5ARlike+ signal in the myelin of PV+ ir fibers (indicated by arrows). The scale is 50 microns.

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10. Fig. 9. Retrograde biocytin-labeled motor neurons of the lumbar segments and 5–HT5ARlike+ immunoreactivity on frontal sections of the lumbar segment X: (a) - frontal section of the brain at low magnification of the lens with two fluorescence signals: a biocytin-labeled motor neuron (green) and 5-HT5ARlike+ signal (red) in the neuro- saw. A bright contrasting 5-HT5ARlike+ signal (arrows) is visible in the form of rings in the medial cord, in the form of spots, rings, stripes – in the area of the entrance of afferent fibers into the dorsal horn, in the ventral cord along the course of motor axons; (b) – separate images of the area with a fluorescent labeled motor neuron and 5-HT5ARlike+ fluorescence; (c) - the combined image shows the presence of 5–HT5ARlike+ signal in the form of grains on the soma and primary ventral dendrite of the motor neuron (arrow), in the ventral part of the gray matter with a bright strip of myelin sheath axon of a motor neuron; (d – i) – a composition of images of another slice with a selected area (g, h, i) to

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