Antonio A. Nunez

Mammalian Diurnality: Some Facts and Gaps

A major factor contributing to the evolution of mammals was their ability to be active during the night, a niche previously underused by terrestrial vertebrates. Diurnality subsequently reemerged multiple times in a variety of independent lineages. This paper reviews some recent data on circadian mechanisms in diurnal mammals and considers general themes that appear to be emerging from this work. Careful examination of behavioral studies suggests that although subtle differences may exist, the fundamental functions of the circadian system are the same, as seems to be the case with respect to the molecular mechanisms of the clock. This suggests that responses to signals originating in the clock must be different, either within the SCN or at its targets or downstream from them. Some features of the SCN vary from species to species, but none of these has been clearly associated with diurnality. The region immediately dorsal to the SCN, which receives substantial input from it, exhibits dramatically different rhythms in nocturnal lab rats and diurnal grass rats. This raises the possibility that it functions as a relay that transforms the signal emitted by the SCN and transmits different patterns to downstream targets in nocturnal and diurnal animals. Other direct targets of the SCN include neurons containing orexin and those containing gonadotropin-releasing hormone, and both of these populations of cells exhibit patterns of rhythmicity that are inverted in at least one diurnal compared to one nocturnal species. The patterns that emerge from the data on diurnality are discussed in terms of the implications they have for the evolution and neural substrates of a day-active way of life.

Female behavior is affected by male ultrasonic vocalizations in house mice.

The effects of male-mouse ultrasonic vocalizations on female preference behavior were investigated using Swiss-Webster mice. Intact females spent more time with an intact tethered male than with a devocalized tethered male during 3-min preference tests. Female preference behavior was similar during estrus and diestrus; but, a preference for the vocalizing male was absent after ovariectomy, and returned following replacement treatment with ovarian hormones. Similar effects on female behavior were obtained using synthetic 70 kHz ultrasounds presented behind one of two devocalized tethered males during preference tests. The results indicate that ultrasounds maintain the female close to the vocalizing male and may facilitate copulation.

Circadian rhythms in vasopressin deficient rats

Circadian rhythms in wheel running and drinking behavior were investigated using heterozygous an homozygous (diabetes insipidus) female Brattleboro rats Despite the lack of vasopressin in the suprachiasmatic nuclei of the diabetic rats, they showed coherent rhythms, both in cyclic light and in constant light. However, the periods of the free-running rhythms were longer for the diabetic rats, they were less active, and, of course, were severely polydipsic. Replacement treatment with systemic infusions of vasopressin reversed the polydipsia but did not affect the other measures.

Effects of bisphenol A on energy balance and accumulation in brown adipose tissue in rats.

Some environmental contaminants have the potential to affect humans or animals by mimicking the effects of hormones. Bisphenol A (BPA) is a weak estrogen agonist when tested using in vitro or in vivo bioassays. In addition to the well documented effects of estrogens on reproductive functions, ovarian hormones also have salient effects on mammalian energy balance and feeding behavior. In this study, we investigated the effects of BPA on body weight and food intake of ovariectomized adult female rats. Treatment with doses of 4 or 5 mg/day for 15 days resulted in a significant reduction of body weight gain with no reduction in food intake. A dose of 1 mg/day did not affect feeding or weight gain. BPA was detected in the blood, brain and adipose tissues of the BPA-treated animals but not in the vehicle control group. There was a preferential concentration of BPA in brown adipose tissue. These results indicate that BPA can affect energy balance and that brown adipose tissue may be a primary tissue into which BPA accumulates in mammals.

Retinofugal projections to the hypothalamus, anterior thalamus and basal forebrain in hamsters

In Part a of the study, the retinal inputs to the hypothalamus, anterior thalamus and basal forebrain of Syrian hamsters were studied using intraocular injections of horseradish peroxidase conjugated to cholera toxin (CT-HRP). In the hypothalamus, the heaviest retinal input was to the suprachiasmatic nucleus (SCN), however, many labeled fibers coursed through the SCN to reach more caudal, periventricular and lateral sites including the anterior and lateral hypothalamus, the paraventricular nucleus (PVN), the subparaventricular zone, the ventromedial nucleus and the pars compacta of the dorsomedial nucleus. Some of these fibers continued dorsally into the zona incerta (ZI). Other fibers emerged from the lateral optic chiasm and traveled either rostro-medially to end in the preoptic area (POA) or further laterally to reach the supraoptic nucleus. A subset of fibers extended laterally from the chiasm to form a well-defined tract which provided input to the pyriform cortex. The extrageniculate retinal input to the thalamus was to the anterior thalamic area (AT) via the stria terminalis. In Part b, injections of rhodamine-labeled latex microspheres were made in three brain areas that contained labeled fibers after intraocular injections of CT-HRP. Injections in the AT, PVN/ZI area and POA consistently produced a small number of labeled retinal ganglion cells, whereas control injections did not. Taken together, these results indicate that many regions of the brain involved in the control of reproductive and regulatory functions receive photic informations via direct retinal inputs. These retinal inputs may play a role in the photoperiodic modulation of physiology and behavior.

Differences in the suprachiasmatic nucleus and lower subparaventricular zone of diurnal and nocturnal rodents

Diurnal and nocturnal species are profoundly different in terms of the temporal organization of daily rhythms in physiology and behavior. The neural bases for these divergent patterns are at present unknown. Here we examine functional differences in the suprachiasmatic nucleus (SCN) and one of its primary targets in a diurnal rodent, the unstriped Nile grass rat (Arvicanthis niloticus) and in a nocturnal one, the laboratory rat (Rattus norvegicus). Grass rats and laboratory rats were housed in a 12:12 light:dark cycle, and killed at six time points. cFos-immunoreactive rhythms in the SCN of grass rats and laboratory rats were similar to those reported previously, with peaks early in the light phase and troughs in the dark phase. However, cFos-immunoreactivity in the lower subparaventricular zone (LSPV) of grass rats rose sharply 5 h into the dark phase, and remained high through the first hour after light onset, whereas in laboratory rats it peaked 1 h after light onset and was low at all other sampling times. Daily cFos rhythms in both the SCN and the LSPV persisted in grass rats, but not in laboratory rats, after extended periods in constant darkness. In grass rats, the endogenous cFos rhythm in the LSPV, but not the SCN, was present both in calbindin-positive and in calbindin-negative cells. Cells that expressed cFos at night in the region of the LSPV in grass rats were clearly outside of the boundaries of the SCN as delineated by Nissl stain and immunoreactivity for vasopressin and vasoactive intestinal peptide. The LSPV of the grass rat, a region that receives substantial input from the SCN, displays a daily rhythm in cFos expression that differs from that of laboratory rats with respect to its rising phase, the duration of the peak and its dependence on a light/dark cycle. These characteristics may reflect the existence of mechanisms in the LSPV that enable it to modulate efferent SCN signals differently in diurnal and nocturnal species.

Diurnal and nocturnal rodents show rhythms in orexinergic neurons.

Orexin (ORX) A and B (hypocretins) are excitatory neuropeptides produced by neurons of the lateral hypothalamus that have been implicated in the regulation of the sleep-wake cycle. In rats, Fos (the product of the cfos gene) expression shows daily rhythms in areas involved in sleep and wakefulness and orexinergic neurons show elevated Fos expression during the night. The present study directly compared the daily pattern of Fos expression in orexinergic neurons in diurnal (A. niloticus; grass rats) and nocturnal (R. norvegicus; lab rats) rodents. Animals kept on a 12:12 light-dark cycle were perfused at six different Zeitgeber times (ZT), with lights on at ZT 0: 1, 5, 13, 17, 20 and 23. In both nocturnal and diurnal rodents orexinergic neurons showed rhythms in Fos expression, with more Fos seen during the active phase of each species. In the diurnal species, Fos expression in cells of the lateral hypothalamus that do not produce ORX was elevated at ZT 20, a time when these animals sleep, and was low at ZT 13, a time of peak of activity. These results provide further evidence for a link between activity in orexinergic neurons and wakefulness and that in grass rats, other neurons of the lateral hypothalamus may work in an antagonistic fashion with respect to orexinergic neurons to regulate wakefulness in this diurnal species.

Daily rhythms in Fos activity in the rat ventrolateral preoptic area and midline thalamic nuclei

The present experiment investigated the expression of the nuclear phosphoprotein Fos over the 24-h light-dark cycle in regions of the rat brain related to sleep and vigilance, including the ventrolateral preoptic area (VLPO), the paraventricular thalamic nucleus (PVT), and the central medial thalamic nucleus (CMT). Immunocytochemistry for Fos, an immediate-early gene product used as an index of neuronal activity, was carried out on brain sections from rats perfused at zeitgeber time (ZT) 1, ZT 5, ZT 12.5, and ZT 17 (lights on ZT 0-ZT 12). The number of Fos-immunopositive (Fos+) cells in the VLPO was elevated at ZT 5 and 12.5 (i.e., during or just after the rest phase of the cycle). Fos+cell number increased at ZT 17 and ZT 1 in the PVT and CMT, 180° out of phase with the VLPO. A positive correlation was found between the numbers of Fos+ cells in the PVT and CMT, and Fos expression in each thalamic nucleus was negatively correlated with VLPO Fos+ cell number. The VLPO, PVT, and CMT may integrate circadian and homeostatic influences to regulate the sleep-wake cycle.

Daily rhythms of Fos expression in hypothalamic targets of the suprachiasmatic nucleus in diurnal and nocturnal rodents.

Little is known about the differences in the neural substrates of circadian rhythms that are responsible for the maintenance of differences between diurnal and nocturnal patterns of activity in mammals. In both groups of animals, the suprachiasmatic nucleus (SCN) functions as the principal circadian pacemaker, and surprisingly, several correlates of neuronal activity in the SCN show similar daily patterns in diurnal and nocturnal species. In this study, immunocytochemistry was used to monitor daily fluctuations in the expression of the nuclear phosphoprotein Fos in the SCN and in hypothalamic targets of the SCN axonal outputs in the nocturnal laboratory rat and in the diurnal murid rodent, Arvicanthis niloticus. The daily patterns of Fos expression in the SCN were very similar across the two species. However, clear species differences were seen in regions of the hypothalamus that receive inputs from the SCN including the subparaventricular zone. These results indicate that differences in the circadian system found downstream from the SCN contribute to the emergence of a diurnal or nocturnal profile in mammals.

Effects of medial preoptic lesions on male mouse ultrasonic vocalizations and copulatory behavior.

In male mice, lesions in the medial preoptic area (MPA) reduced the proportion of animals showing mounting behavior but failed to abolish ano-genital exploration of the female. The lesions did not effect the amount of 70 kHz ultrasound elicited by intact females or by soiled bedding obtained from female-occupied cages. These results suggest components of male sexual behavior are mediated by neural systems outside the MPA.