Dorsal raphe nucleus serotonergic neurons innervate the rostral ventrolateral medulla in rat.

Stimulation of the dorsal raphe nucleus (DRN) alters arterial pressure, heart rate and cerebral blood flow, yet projections from the DRN to medullary autonomic nuclei have not been described. We examined whether serotonergic (5-HT) projections from the DRN terminate in the rostral ventrolateral medulla (RVL) and if so, whether the projection mediates cardiovascular responses to DRN stimulation. Studies were performed in adult male Sprague-Dawley rats. Horseradish peroxidase or choleratoxin B was injected unilaterally or bilaterally into the RVL. Levels of 5-HT, its precursors L-tryptophan and 5-hydroxytryptophan and the metabolite 5-hydroxyindole acetic acid were measured in the ventral medulla by HPLC three weeks following placement of electrolytic lesions in DRN. Serotonin transporter (3H-cyanoimipramine binding) was quantified by autoradiography in DRN-lesioned animals. Horseradish peroxidase or choleratoxin B injections into the medulla at the level of the RVL resulted in retrogradely labeled neurons bilaterally, with ipsilateral predominance, in the DRN. Labeled cells were preponderant in rostral ventrolateral portions of the DRN, but were also observed in the dorsal, lateral and interfascicular DRN subnuclei; fewer neurons were observed in caudal portions of the DRN. Three weeks following placement of electrolytic lesions in the DRN, the concentrations of 5-HT and 5-hydroxyindole acetic acid, but not L-tryptophan or 5-hydroxytryptophan, were reduced in the medulla by 45 and 48%, respectively, compared to sham-operated or unoperated controls. DRN lesions reduced binding to the 5-HT transporter in the RVL by approximately 30% compared to unlesioned controls. Unilateral lesions of the RVL reduced the evoked blood pressure response by 53+/-15%; bilateral RVL lesions reduced the response by 86+/-9%. The increase in cortical blood flow elicited by DRN stimulation was unchanged after unilateral or bilateral RVL lesions. These studies demonstrate that there is a descending serotonergic projection from the DRN to the RVL. This projection may mediate autonomic changes elicited by DRN stimulation.

Effect of chemical stimulation of the dorsal raphe nucleus on cerebral blood flow in rat.

We have previously shown that electrical stimulation of the dorsal raphe nucleus (DRN) can increase or decrease cerebral blood flow (CBF). We now sought to determine whether the CBF responses are mediated by DRN neurons or fibers of passage. In anesthetized rat, the DRN was stimulated chemically (L-glutamate, kainate) and CBF in parietal cortex was measured by laser-Doppler flowmetry. Microinjection of kainate (5 nmol), but not L-glutamate, into DRN increased CBF (28 +/- 22%; P < 0.05). Decreased CBF to DRN stimulation was not observed. We conclude that stimulation of DRN neurons increases CBF.

Computerized three-dimensional reconstruction reveals cerebrovascular regulatory subregions in rat brain stem.

Three-dimensional wireframe reconstructions were used to examine the relationship between the anatomical localization of electrode sites and the cerebrovascular response which was elicited by electrical stimulation of the dorsal raphe nucleus (DRN). Reconstructions of the rat brain and DRN were done from atlas plates and from Nissl-stained coronal sections (100-micron increments). Data points were entered and three-dimensional reconstructions were performed using commercially available software and a personal computer. Display of the entire brain yielded views which obscured visualization of the DRN. The data file was edited to reduce the number of contours without affecting the display resolution of the DRN. Selective display of the DRN and electronic rotation from the coronal to a sagittal view revealed a functional organization of the cerebral blood flow responses which was not apparent in two-dimensional coronal sections.

Regulation of cortical blood flow by the dorsal raphe nucleus: topographic organization of cerebrovascular regulatory regions.

We examined in rat: (1) the time-course and magnitude of change in cortical blood flow (CoBF) following electrical stimulation of the dorsal raphe nucleus (DRN) and (2) whether DRN lesions affect resting CoBF or the cerebrovascular response to CO2. Animals were anesthetized (chloralose), paralyzed, and artificially ventilated. The effect of stimulus frequency (1-200 Hz) and intensity (10-100 microA) on arterial pressure, heart rate, and CoBF was examined; lesions were made electrolytically. CoBF was measured using a laser-Doppler flowmeter with the probe placed extradurally over the parietal sensorimotor cortex. The DRN was computer reconstructed in three dimensions from Nissl stained coronal sections for localization of electrode placements. Brief stimuli (8 s; n = 6) elicited frequency and intensity-dependent increases in arterial pressure, heart rate, and CoBF. Sustained intermittent trains of stimuli of rostral DRN (200 Hz; 1 s on/1 s off; 70 microA) elicited a decrease (85 +/- 12% of baseline; n = 9) in CoBF (p less than 0.05) while stimulation in caudal DRN resulted in increased CBF (126 +/- 13% of baseline; n = 9). Phenylephrine infusion (0.1-1 microgram; i.v.; n = 8) increased arterial pressure and CoBF less than that elicited by brief DRN stimulation (p less than 0.05). DRN lesions did not affect resting CoBF (140 +/- 25 perfusion units (PU) before; 127 +/- 16 PU after DRN lesion; p greater than 0.05, n = 5) or mean arterial pressure (127 +/- 13 before; 120 +/- 11 after); nor did it affect the cerebrovascular response to change in arterial PCO2. Sustained intermittent stimulation of the DRN can evoke either increases or decreases in CoBF depending on the anatomical sublocalization. The DRN does not tonically maintain resting CoBF, nor participate in the cerebrovascular response to change in PCO2.

Effects of L-tryptophan and other amino acids on electroencephalographic sleep in the rat.

Electroencephalographic sleep was quantitated in adult male Sprague-Dawley rats following single injections of the methylesters of tryptophan, valine or alanine. The amino acids were administered at the onset of the daily light period (09.00 h); electrographic data were collected for the succeeding 6-h period. Saline served as the injection control, and fluoxetine, a serotonin-reuptake blocker, as a positive control. The injection of tryptophan methylester (125 mg/kg) caused a delay in rapid eye movement (REM) sleep onset, and significantly reduced the amount of REM sleep during the first 2 h postinjection. Tryptophan produced no effect on sleep onset, nor did it influence total sleep time. Fluoxetine (2.5 mg/kg) produced similar effects, as previously observed. The methylesters of valine and alanine were without effect on REM sleep, when injected at a molar dose equivalent to that for tryptophan. No consistent effects of any of the test substances were noted on non-REM (NREM) sleep or waking time, or on any of the other sleep indices quantitated. Together, the data indicate that tryptophan selectively reduces REM sleep; the effect is not due to a non-specific action of amino acids or their methylesters. The effect on REM sleep may be the consequence of a tryptophan-induced stimulation of 5-HT synthesis and release, since it is like that produced by fluoxetine, a drug that enhances transmission across serotonin synapses.

A microcomputer-based sleep system: data acquisition and system calibration programs.

A data acquisition program is described for the Apple II series of microcomputers that allows for continuous, direct monitoring of electrographic elements from cortical, hippocampal and muscle leads from rats. The program detects cortical delta waves and sigma activity, hippocampal theta activity and electromyographic activity. The detected elements are counted and stored in memory at 15 second intervals (bins). Every three hours, the data are transferred to disks for permanent storage and off-line analysis.