Martínez, Schwartz, Smale & Nunez
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1 I. Artículos
2 Martínez, Schwartz, Smale & Nunez 12
3 Circadian regulation of daily rhythms in orexinergic neurons Circadian regulation of daily rhythms in orexinergic neurons in diurnal and nocturnal rodents Regulación circadiana de los ritmos diarios en neuronas orexinérgicas en roedores diurnos y nocturnos. Gladys S. Martínez Michael D. Schwartz Laura Smale Antonio A. Nunez Abstract Resumen 13
4 Martínez, Schwartz, Smale & Nunez Introduction 14
5 Circadian regulation of daily rhythms in orexinergic neurons General Method Subjects Experiment 1: Patterns of Fos expression in neurons expressing ORX in constant darkness Method Figure 1: Double plot actogram from a representative grass rat kept in constant darkness for 21 days. Grass rats are active at CTs 5 and 13, and less active (and apparently sleeping) at CT 22. Actograms for each animal were used to determine activity onset and time of perfusion. Each line represents a 48 hour period. 15
6 Martínez, Schwartz, Smale & Nunez Data Analysis Figure 2. (a) High magnification micro photographs of VIP fibers approaching ORX neurons in lab rats (left) and grass rats (right) (b) VP fibers approaching an ORX neuron in grass rats. Only those cases in which VIP or VP bouton-like structure was observed in contact with ORX neuron s and the bouton was a clear continuation of a fiber were scored as contacts. VIP vasoactive intestinal polypeptide; ORX, orexin; VP, arginine vasopressin 16
7 Circadian regulation of daily rhythms in orexinergic neurons Results Figure 3: ORX-immunoreactivity and Fos-immunoreactivity in grass rats as seen under high-power oil immersion lens (100X magnification). The image was edited with Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA). Double arrow, Orexin-ir neurons; arrowhead, Fos-ir nucleus; single arrow, Fosir orexin-ir neurons (b) Rectangle used to count cells in one section per animal. Cells were counted within and area defined using three landmarks: fornix (f), third ventricle (3V), and mammillothalamic tract (mt). Dark dots represent ORX-ir cells in the lateral hypothalamus of a grass rat. Distribution of ORX-ir cells 17
8 Martínez, Schwartz, Smale & Nunez Figure 4. (a) Mean (± S.E.M.) number of ORX-ir cells (both single and double labeled) at three different circadian times (CTs). (b) Mean ( ±S.E.M.) number of ORX-ir cells (both single and double labeled) in three different areas (medial, central, and lateral). Means with different letters (a, b, or c) are significantly different from each other. Fos Expression in ORX Neurons in Constant Darkness Figure 5: (a) Percentage (mean ±S.E.M.) of ORX-ir cells that contained Fos-ir nuclei at three different circadian times (CTs). (b) Percentage (mean ±S.E.M.) of ORX-ir cells that contained Fos-ir nuclei across areas. Means with different letters (a, b, or c) are significantly different. Fos expression in non-orx neurons Figure 6. (a) Percentage (mean ±S.E.M) cells that contained only Fos-ir nuclei at three different circadian times (CTs); (b) Percentage (mean ±S.E.M.) of cells containing Fos-ir nuclei and not labeled for ORX across areas. Means with different letters (a, b, or c) are significantly different from each other. 18
9 Circadian regulation of daily rhythms in orexinergic neurons Experiment 2: Distribution of VIP and VP fibers in the lateral hypothalamus in grass and lab rats Method VIP/ORX VP/ORX Results 19
10 Martínez, Schwartz, Smale & Nunez Figure 7: Distribution of VIP fibers in lab and grass rats. Similar to lab rats, VIP fibers in grass rats are dense in the spvz (a) and moderate in the PVT (c) but, in contrast to lab rats, only very few VIP fibers are present in the AHA of grass rats (b). VIP, vasoactive intestinal polypeptide; spvz, subparaventricular zone of the hypothalamus; PVT, paraventricular nucleus of the thalamus; AHA, anterior hypothalamic area. Figure 8: VP Fibers from SCN origin are fine caliber (a) and show irregularly spaced varicosities. In contrast, VP fibers from the SON and the PVN are large caliber and show few varicosities. VP, arginine vasopressin; SON, supraoptic nucleus; PVN, paraventricular hypothalamic nucleus. 20
11 Circadian regulation of daily rhythms in orexinergic neurons Figure 9: Top: VIP fibers and ORX neurons in the LHA in lab rats (left) and grass rats (right). Bottom: Percentage (mean ± S.E.M.) of ORX neurons in apparent contact with VIP fibers in lab and grass rats. Grass rats had proportionally more VIP contacts on ORX neurons compared to lab rats (U=0, p<0.05). General Discussion 21
12 Martínez, Schwartz, Smale & Nunez β β 22
13 Circadian regulation of daily rhythms in orexinergic neurons References 23
14 Martínez, Schwartz, Smale & Nunez 24
15 Circadian regulation of daily rhythms in orexinergic neurons 25
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