State-dependent changes in glutamate, glycine, GABA, and dopamine levels in cat lumbar spinal cord

Abstract

Recent studies have indicated that the glycine receptor antagonist strychnine and the gamma-aminobutyric acid type A (GABA A) receptor antagonist bicuculline reduced the rapid-eye-movement (REM) sleep-specific inhibition of sensory inflow via the dorsal spinocerebellar tract (DSCT). These findings imply that the spinal release of glycine and GABA may be due directly to the REM sleep-specific activation of reticulospinal neurons and/or glutamate-activated last-order spinal interneurons. This study used in vivo microdialysis and high-performance liquid chromatography analysis techniques to provide evidence for these possibilities. Microdialysis probes were stereotaxically positioned in the L3 spinal cord gray matter corresponding to sites where maximal cerebellar-evoked field potentials or individual DSCT and nearby spinoreticular tract (SRT) neurons could be recorded. Glutamate, glycine, and GABA levels significantly increased during REM sleep by approximately 48, 48, and 14%, respectively, compared with the control state of wakefulness. In contrast, dopamine levels significantly decreased by about 28% during REM sleep compared with wakefulness. During the state of wakefulness, electrical stimulation of the nucleus reticularis gigantocellularis (NRGc) at intensities sufficient to inhibit DSCT neuron activity, also significantly increased glutamate and glycine levels by about 69 and 45%, respectively, but not GABA or dopamine levels. We suggest that the reciprocal changes in the release of glutamate, glycine, and GABA versus dopamine during REM sleep contribute to the reduction of sensory inflow to higher brain centers via the DSCT and nearby SRT during this behavioral state. The neural pathways involved in this process likely include reticulo- and diencephalospinal and spinal interneurons.

FIG. 1.

Experimental scheme illustrating procedures used for locating Clarke's column (CC) at the L₃ spinal cord segment (A), optimal site for conditioning the nucleus reticularis gigantocellularis (NRGc) in the medulla oblongata (B), and sampling spinal dialysates from CC (C). The location of CC was determined by monitoring maximal amplitude field potentials evoked by stimulation of the anterior cerebellar lobule (A). Conditioning stimuli were applied to the NRGc at intensities sufficient to suppress cerebellar-evoked field potentials via the reticulospinal pathway (RST) (B). The tip of the microdialysis probe placement in CC corresponded to stereotaxic coordinates 2,500 microns ventral to where maximal cerebellar field potentials were recorded. The dialysate in each collecting vial was stored at −80°C before high-performance liquid chromatography (HPLC) analyses (C; see methods for further details).

FIG. 2.

Examples of HPLC chromatograms of the amino acid neurotransmitters, glutamate (GLU), glycine (GLY), γ-aminobutyric acid (GABA), as well as dopamine (DA) during wakefulness and sleep. Behavioral states of wakefulness, nonrapid-eye-movement (NREM) sleep, rapid-eye-movement (REM) sleep, and awakening are indicated by 1.5-min continuous records of electroencephalographic (EEG), electrooculographic (EOG), pontogeniculooccipital (PGO), and electromyographic (EMG) activities (A). A relatively short 1.5-min time period is depicted to clearly illustrate the characteristic and marked differences in EEG, EOG, PGO, and EMG activities that were routinely used to score behavioral state when spinal dialysis samples for glutamate, glycine, GABA, and dopamine were collected. The chromatograms in B illustrate the change in glutamate, glycine, and GABA. There were marked increases in each of these neurotransmitters only during REM sleep. In contrast, there was a marked decrease in dopamine levels only during REM sleep compared with the other behavioral states (C).

FIG. 3.

State-dependent change in the levels of glutamate (A), glycine (B), GABA (C), and dopamine (D) in the L₃ spinal cord segment. Each histogram bar represents the group mean (±SE) of neurotransmitter level over 18 (A–C) and 16 (D) separate wake–sleep cycles comprised of consecutively occurring episodes of wakefulness (W), nonrapid-eye-movement sleep (NREM), rapid-eye-movement sleep (REM), and awakening from REM sleep (AW). Values obtained during W served as a control. Note that during REM sleep, glutamate, glycine, and GABA levels increased significantly by 47, 48, and 14%, respectively, whereas dopamine levels decreased by 28% (*P < 0.05, repeated-measures ANOVA, or Friedman repeated-measures ANOVA on ranks, Tukey test).

FIG. 4.

Example of HPLC chromatograms illustrating elution peaks for glutamate, glycine, and GABA during control periods of wakefulness, a test period of electrical stimulation of the NRGc, and recovery from stimulation. Each chromatogram represents an individual spinal dialysate sample collected over a 5-min period. Note that electrical stimulation of the NRGc (4 pulses, 0.8-ms duration, 2.5-ms interpulse interval, 120 μA, 1.0 Hz) resulted in enhanced magnitude of amino acid signals. The NRGc-induced increase in amino acid levels in this example was transient and occurred only during the presentation of electrical stimuli.

FIG. 5.

Overall action of NRGc stimulation (Stim) on neurotransmitter levels in the L₃ spinal cord segment. Each histogram bar in A and B represents the group mean (±SE) level of glutamate (A), glycine (B), GABA (C) (n = 14 experiments), and dopamine (D, n = 3 experiments) during the state of wakefulness before (Con), during NRGc stimulation, and at 5-min time intervals after NRGc stimulation. Note that during NRGc stimulation, glutamate and glycine levels increased significantly by about 69 and 45%, respectively (*P < 0.05, repeated-measures ANOVA or Friedman repeated-measures ANOVA on ranks, Tukey test).

FIG. 6.

A: histological serial section of the rostral L₃ spinal cord segment showing the tract (open arrow) and placement (filled arrow) of the tip of Eicom microdialysis probe. B: locations of microdialysis probes (black bars) implanted in the rostal, middle, and caudal regions of the Clarke's column nucleus (CC). Vertical lines represent the 1-mm length of dialysis membrane. The maximum field potential evoked by stimulation of the anterior cerebellar lobule corresponding to the location of Clarke's column was used as a guide for stereotaxic placement of the dialysis probes (see also Fig. 1A).