Day-night differences in neural activation in histaminergic and serotonergic areas with putative projections to the cerebrospinal fluid in a diurnal brain

Abstract

In nocturnal rodents, brain areas that promote wakefulness have a circadian pattern of neural activation that mirrors the sleep/wake cycle, with more neural activation during the active phase than during the rest phase. To investigate whether differences in temporal patterns of neural activity in wake-promoting regions contribute to differences in daily patterns of wakefulness between nocturnal and diurnal species, we assessed Fos expression patterns in the tuberomammillary (TMM), supramammillary (SUM), and raphe nuclei of male grass rats maintained in a 12:12 h light-dark cycle. Day-night profiles of Fos expression were observed in the ventral and dorsal TMM, in the SUM, and in specific subpopulations of the raphe, including serotonergic cells, with higher Fos expression during the day than during the night. Next, to explore whether the cerebrospinal fluid is an avenue used by the TMM and raphe in the regulation of target areas, we injected the retrograde tracer cholera toxin subunit beta (CTB) into the ventricular system of male grass rats. While CTB labeling was scarce in the TMM and other hypothalamic areas including the suprachiasmatic nucleus, which contains the main circadian pacemaker, a dense cluster of CTB-positive neurons was evident in the caudal dorsal raphe, and the majority of these neurons appeared to be serotonergic. Since these findings are in agreement with reports for nocturnal rodents, our results suggest that the evolution of diurnality did not involve a change in the overall distribution of neuronal connections between systems that support wakefulness and their target areas, but produced a complete temporal reversal in the functioning of those systems.

Keywords: 3V; 4V; 5HT; CP; CSF; CTB; DR; DTM; Fos; HA; ICC; LSd; LV; ME; MR; NDS; NGS; PB; PBS; PLP; PVN; SCN; SUM; TBS; TMM; TX; VTM; ZT; cerebrospinal fluid; cholera toxin subunit beta; choroid plexus; circadian; dorsal raphe; dorsal tuberomammillary nucleus; fourth ventricle; grass rat; histamine; immunocytochemistry; lDR; lateral dorsal raphe; lateral septal nucleus, dorsal part; lateral ventricle; mDR3–5; medial dorsal raphe levels 3 through 5; median eminence; median raphe; normal donkey serum; normal goat serum; paraformaldehyde–lysine–sodium periodate; paraventricular nucleus of the hypothalamus; phosphate buffer; phosphate-buffered saline; serotonin; suprachiasmatic nucleus; supramammillary nucleus; third ventricle; tris-buffered saline; triton-X; tuberomammillary nucleus; vSPZ; ventral subparaventricular zone; ventral tuberomammillary nucleus; zeitgeber time.

Figure 1

Rostro-caudal illustrations and corresponding representative photomicrographs depicting the sampling regions used to quantify Fos expression in serotonergic and non-serotonergic cells of the lDR, MR, mDR3 (A, B), mDR4 (C, D), and mDR5 (E, F). See text for sampling box dimensions. Abbreviations: lDR, lateral dorsal raphe; mDR3-mDR5, medial dorsal raphe levels 3 through 5; MR, median raphe.

Figure 2

Fos expression (mean ± SEM) in serotonergic cells (A, C, and E) and non-serotonergic cells (B, D, and F) of the mDR4 (A and B), mDR5 (C and D), and MR (E and F) at six different zeitgeber times (white bars: light phase; grey bars: dark phase). Note that group means with different letters are significantly different from each other, here as well as in Figure 4. Abbreviations: 5HT-ir, serotonin like immunoreactive; mDR4-mDR5, medial dorsal raphe levels 4 and 5; MR, median raphe.

Figure 3

Photomicrographs of serotonergic cells expressing Fos in the mDR5 at ZT 10 (A) and ZT 22 (B). Arrowheads indicate some double-labeled cells. Fos: blue nuclear staining. 5HT: brown cell body staining. Abbreviations: 4V, fourth ventricle; mDR5, medial dorsal raphe level 5; ZT, zeitgeber time.

Figure 4

Fos expression (mean ± SEM) in the VTM (A), DTM (B), and SUM (C) at six different zeitgeber times (white bars: light phase; grey bars: dark phase). Abbreviations: DTM, dorsal tuberomammillary nucleus; Fos-ir, Fos like immunoreactive; SUM, supramammillary nucleus; VTM, ventral tuberomammillary nucleus.

Figure 5

Rostro-caudal photomicrographs of the CTB injection site in the 3V (A-E). Abbreviations: 3V, third ventricle; CTB, cholera toxin subunit beta; EC, ependymal cell layer; OX, optic chiasm; RCh, retrochiasmatic area.

Figure 6

Photomicrographs of areas that contained CTB-labeled cells after CTB injections to the ventricular system. Note the dense cell labeling in the mDR5 after injections to the ventricular system (A) in comparison to an injection that missed it (B). In addition, an injection restricted to the 3V produced scattered cell labeling in the SCN and vSPZ [C, the arrow indicates a CTB-positive bipolar neuron magnified in the inset (upper right corner)], Arc (D), PVN (E), and DTM (F). Arrowheads indicate some CTB-labeled cells. Abbreviations: 3V, third ventricle; 4V, fourth ventricle; Arc, arcuate nucleus; CTB, cholera toxin subunit beta; DTM, dorsal tuberomammillary nucleus; mDR5, medial dorsal raphe level 5; ME, median eminence; OX, optic chiasm; PVN, paraventricular nucleus of the hypothalamus; SCN, suprachiasmatic nucleus of the hypothalamus; vSPZ, ventral subparaventricular zone.

Figure 7

Photomicrographs depicting cells in the mDR5 after staining with Cy2 labeled 5HT (A, green fluorescence) and Cy3 labeled CTB (C, red fluorescence) in a representative animal that received a CTB injection directed to the 3V. Panel B is a composite image showing both labels. White asterisks indicate some double-labeled cells. Abbreviations: 4V, fourth ventricle; CTB, cholera toxin subunit beta; mDR5, medial dorsal raphe level 5.