In Vivo Electrophysiology of Peptidergic Neurons in Deep Layers of the Lumbar Spinal Cord after Optogenetic Stimulation of Hypothalamic Paraventricular Oxytocin Neurons in Rats

Abstract

The spinal ejaculation generator (SEG) is located in the central gray (lamina X) of the rat lumbar spinal cord and plays a pivotal role in the ejaculatory reflex. We recently reported that SEG neurons express the oxytocin receptor and are activated by oxytocin projections from the paraventricular nucleus of hypothalamus (PVH). However, it is unknown whether the SEG responds to oxytocin in vivo. In this study, we analyzed the characteristics of the brain-spinal cord neural circuit that controls male sexual function using a newly developed in vivo electrophysiological technique. Optogenetic stimulation of the PVH of rats expressing channel rhodopsin under the oxytocin receptor promoter increased the spontaneous firing of most lamina X SEG neurons. This is the first demonstration of the in vivo electrical response from the deeper (lamina X) neurons in the spinal cord. Furthermore, we succeeded in the in vivo whole-cell recordings of lamina X neurons. In vivo whole-cell recordings may reveal the features of lamina X SEG neurons, including differences in neurotransmitters and response to stimulation. Taken together, these results suggest that in vivo electrophysiological stimulation can elucidate the neurophysiological response of a variety of spinal neurons during male sexual behavior.

Keywords: gastrin-releasing peptide neurons; in vivo extracellular recording; in vivo whole-cell patch-clamp recording; lamina X; optogenetics; oxytocin; spinal cord; spinal ejaculation generator.

Figure 1

In vivo electrophysiological recording from lamina X neurons in the rat spinal cord. (a) Schematic diagram of the electrophysiological recording and perfusion system. The lumber spinal cord was exposed by laminectomy and the surface of the cord was perfused continuously with warmed pre-oxygenated Krebs solution. Known concentrations of drugs were applied from the same line. The body temperature was monitored and kept at the normal range. (b) Schematic drawing of the transverse slice of lumbar cord and of the angle of a recording electrode (two-way arrow). Recordings were made from cells at a depth of 890–1330 μm (shown by two-way arrow) from the surface of spinal cord. (c) A representative image of the Nissl-stained spinal cord section after insertion of a tungsten electrode. The blocked area is enlarged. Scale bars, 100 µm in the low magnification, 50 µm in the high magnification.

Figure 2

In vivo extracellular recording from lamina X neurons in the rat spinal cord. Representative trace of spontaneous firing recorded from a lamina X neuron (upper panel). The spike shape of an action potential, indicated by an arrowhead, is enlarged on the right side of the trace. Summary showing the frequency of spontaneous firing (lower panel; left) and correlations between the frequency and recording depth in the spontaneous firing (lower panel; right).

Figure 3

In vivo patch-clamp recording from lamina X neurons in the rat spinal cord. (a) Representative trace of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from a lamina X neuron. (b) Summary showing the frequency (left) and the amplitude (right) of sEPSCs. (c) Under current clamp condition, representative trace of spontaneous excitatory postsynaptic potentials and action potentials recorded from a lamina X neuron. (d) Summary showing the frequency of spontaneous action potentials.

Figure 4

In vivo extracellular recordings from lamina X oxytocin-responsive neurons in the upper lumbar spinal cord (L3–L4 level) after superfusion of oxytocin or optogenetic stimulation of the paraventricular nucleus of the hypothalamus (PVH) of Oxtr-ChR2-EYFP transgenic rats. (a) Schematic drawing of the brain–spinal cord neural network controlling male sexual function and the location of optogenetic stimulation. The oxytocin neurons, expressing channel rhodopsin (ChR2; green) in the paraventricular nucleus of the hypothalamus project to the upper lumbar spinal cord (L3–L4 level) and act on the lamina X oxytocin-responsive neurons (magenta). (b) In vivo electrophysiology revealed that oxytocin-responsive neuronal firing was increased by oxytocin superfusion (1 μM) compared to control. (c) Quantification analysis showed that spontaneous firing was increased after oxytocin superfusion compared with control (the boxes show the 25th to 75th percentiles and the median, n = 9; paired t-test, * p < 0.05). (d) In vivo electrophysiology revealed that the frequency of oxytocin-responsive neuronal firing was significantly increased by the optogenetic stimulation of PVH when compared with control. (e) Quantification analysis showed that spontaneous firing was increased after optogenetic stimulation compared with control (the boxes show the 25th to 75th percentiles and the median, n = 7; paired t-test, ** p < 0.01). (b,d) were recorded from the same neuron. (f) Responsiveness of superfusion of oxytocin or optogenetic stimulation on spontaneous firing from lamina X neurons. (Left) Summary of recording depth on each neuron (plotted by numbers: 1–9). (Right) Summary showing effect of oxytocin or optogenetic stimulation on the relative frequency of firing in lamina X neurons.

Figure 5

Schematic drawing summarizing the brain–spinal cord neural network that controls the male sexual function. A group of oxytocin neurons located in the posterior part of the paraventricular nucleus of the hypothalamus (PVH) project to the lower spinal cord and control penile erection and ejaculation in male rats. The lamina X oxytocin-responsive neurons containing gastrin-releasing peptide (GRP) project axons to spinal centers of the lumbosacral cord that mediate penile reflexes and trigger ejaculation.