Charles A. Fuller

Effect of SCN lesions on sleep in squirrel monkeys: evidence for opponent processes in sleep-wake regulation

Sleep and wakefulness are governed by both the suprachiasmatic nuclei of the hypothalamus (SCN), and a sleep homeostatic process; however, the interaction of these control systems is not well understood. From rodent studies it has been assumed that the SCN promote neither wake nor sleep but gate the homeostatic sleep-promoting process. Yet in humans sleep tendency is lowest during the later waking hours of the day, and sleep duration can be predicted because of the precise circadian timing of waking. Thus in primates, the SCN could assure sleep-wake cycle consolidation by actively promoting or facilitating wakefulness. To evaluate this hypothesis, we examined the sleep-wake and sleep-stage patterns of intact and SCN-lesioned (SCNx) squirrel monkeys maintained in constant light. This diurnal primate has consolidated sleep and wake patterns more similar to man than rodents. Sleep-wake, sleep stages, brain temperature, and drinking circadian rhythms were eliminated, and total sleep time was significantly increased (4.0 hr, P < 0.01) in SCNx monkeys. However, total times in deeper stages of non-rapid eye movement (non-REM; e.g., delta sleep) and REM sleep were not significantly affected by SCN lesions. Increased total sleep time was associated with a reduction in subjective day wake consolidation, as evidenced by substantially shorter wake bout lengths in SCNx monkeys (15 +/- 6 min) as compared to intact monkeys (223 +/- 10 min; P < 0.0001, ANOVA). These findings show that the SCN influence the regulation of daily total wake and sleep times, and implicate an alternative sleep-wake regulatory model in which an SCN-dependent process actively facilitates the initiation and maintenance of wakefulness and opposes homeostatic sleep tendency during the subjective day in diurnal primates.

Neurovestibular modulation of circadian and homeostatic regulation: vestibulohypothalamic connection?

Chronic exposure to increased force environments (+G) has pronounced effects on the circadian and homeostatic regulation of body temperature (Tb), ambulatory activity (Act), heart rate, feeding, and adiposity. By using the Brn 3.1 knockout mouse, which lacks vestibular hair cells, we recently described a major role of the vestibular system in mediating some of these adaptive responses. The present study used the C57BL/6JEi-het mouse strain (het), which lacks macular otoconia, to elucidate the contribution of specific vestibular receptors. In this study, eight het and eight WT mice were exposed to 2G for 8 weeks by means of chronic centrifugation. In addition, eight het and eight WT mice were maintained as 1G controls in similar conditions. Upon 2G exposure, the WT exhibited a decrease in Tb and an attenuated Tb circadian rhythm. Act means and rhythms also were attenuated. Body mass and food intake were significantly lower than the 1G controls. After 8 weeks, percent body fat was significantly lower in the WT mice (P < 0.0001). In contrast, the het mice did not exhibit a decrease in mean Tb and only a slight decrease in Tb circadian amplitude. het Act levels were attenuated similarly to the WT mice. Body mass and food intake were only slightly attenuated in the het mice, and percent body fat, after 8 weeks, was not different in the 2G het group. These results link the vestibular macular receptors with specific alterations in homeostatic and circadian regulation.

The response of suprachiasmatic neurons of the rat hypothalamus to photic and serotonergic stimulation.

Single-unit activity of the rat suprachiasmatic nucleus and surrounding regions was recorded in vivo. Photically responsive neurons were typically inhibited by low intravenous doses of the serotonin (5-HT) agonist quipazine. The 5-HT antagonist, metergoline, administered in a low intravenous dose, reversed the effects of quipazine. The subsequent response to photic stimulation was essentially identical to the initial photic response. Thus the photic response of neurons in the suprachiasmatic nucleus (SCN) and adjacent regions is relatively uninfluenced by 5-HT blockade.

The retinohypothalamic tract in the cat: retinal ganglion cell morphology and pattern of projection

The pattern of retinal projection to the hypothalamus and the morphological properties of the retinal ganglion cells that comprise the retinohypothalamic tract have been examined in the cat. Intraocular injections of horseradish peroxidase revealed a dense retinal projection to the ventral suprachiasmatic nucleus; however, lighter projections were seen in the dorsal suprachiasmatic nucleus, and in hypothalamic regions both dorsal and lateral to the suprachiasmatic nucleus. Intrasuprachiasmatic nucleus injections of horseradish peroxidase retrogradely labelled retinal ganglion cells that were small to medium in soma size. The labelled ganglion cells exhibited long thin dendrites that were sparsely branched. The labelled retinal ganglion cells exhibited a significant change in soma size associated with retinal eccentricity. The morphological characteristics of the ganglion cells that project to the suprachiasmatic nucleus are similar to those of gamma cells.

Diurnal modulation of long-term potentiation in the hamster hippocampal slice.

Long-term potentiation (LTP) was examined in hippocampal slices from Syrian hamsters entrained to a LD 14:10 cycle. Population spike (PS) amplitudes from CA1 pyramidal cells were measured before (control) and after tetanizing the Schaffer/collateral commissural pathway. Slices from animals sacrificed during the day, between zeitgeber time (ZT) 0430 and 0530, were incubated, and then tetanized between ZT 1340 and 1930, where ZT=0 denotes lights on. Slices from animals sacrificed during the night, between ZT 1830 and 1930, were incubated, and tetanized between ZT 0030 and 0410. LTP, a sustained increase in PS amplitude following tetanus, was evoked in both groups. PS amplitude increased by 102.7+/-20.3% in animals sacrificed during the day and by 48.0+/-7.5% in animals sacrificed during the night (p<0.05). Thus hamster slices prepared during the day show more robust LTP (a doubling of PS amplitude), a difference persisting in slices incubated for several hours.

Evidence for vestibular regulation of autonomic functions in a mouse genetic model

Physiological responses to changes in the gravitational field and body position, as well as symptoms of patients with anxiety-related disorders, have indicated an interrelationship between vestibular function and stress responses. However, the relative significance of cochlear and vestibular information in autonomic regulation remains unresolved because of the difficulties in distinguishing the relative contributions of other proprioceptive and interoceptive inputs, including vagal and somatic information. To investigate the role of cochlear and vestibular function in central and physiological responses, we have examined the effects of increased gravity in wild-type mice and mice lacking the POU homeodomain transcription factor Brn-3.1 (Brn-3b/Pou4f3). The only known phenotype of the Brn-3.1−/− mouse is related to hearing and balance functions, owing to the failure of cochlear and vestibular hair cells to differentiate properly. Here, we show that normal physiological responses to increased gravity (2G exposure), such as a dramatic drop in body temperature and concomitant circadian adjustment, were completely absent in Brn-3.1−/− mice. In line with the lack of autonomic responses, the massive increase in neuronal activity after 2G exposure normally detected in wild-type mice was virtually abolished in Brn-3.1−/− mice. Our results suggest that cochlear and vestibular hair cells are the primary regulators of autonomic responses to altered gravity and provide genetic evidence that these cells are sufficient to alter neural activity in regions involved in autonomic and neuroendocrine control.

Alterations in endogenous circadian rhythm of core temperature in senescent Fischer 344 rats

We assessed whether alterations in endogenous circadian rhythm of core temperature (CRT) in aging rats are associated with chronological time or with a biological marker of senescence, i.e., spontaneous rapid body weight loss. CRT was measured in male Fischer 344 (F344) rats beginning at age 689 days and then continuously until death. Young rats were also monitored. The rats were housed under constant dim red light at 24-26°C, and core temperature was recorded every 10 min via biotelemetry. The CRT amplitude of the body weight-stable (presenescent) old rats was significantly less than that of young rats at all analysis periods. At the onset of spontaneous rapid weight loss (senescence), all measures of endogenous CRT differed significantly from those in the presenescent period. The suprachiasmatic nucleus (a circadian pacemaker) of the senescent rats maintained its light responsiveness as determined by an increase in c- fos expression after a brief light exposure. These data demonstrate that some characteristics of the CRT are altered slowly with chronological aging, whereas others occur rapidly with the onset of senescence.

Endogenous thermoregulatory rhythms of squirrel monkeys in thermoneutrality and cold

Whole body heat production (HP) and heat loss (HL) were examined to determine if the free-running circadian rhythm in body temperature (Tb) results from coordinated changes in HP and HL rhythms in thermoneutrality (27°C) as well as mild cold (17°C). Squirrel monkey metabolism ( n = 6) was monitored by both indirect and direct calorimetry, with telemetered measurement of Tb and activity. Feeding was also measured. Rhythms of HP, HL, and conductance were tightly coupled with the circadian Tb rhythm at both ambient temperatures (TA). At 17°C, increased HP compensated for higher HL at all phases of the Tb rhythm, resulting in only minor changes to Tb. Parallel compensatory changes of HP and HL were seen at all rhythm phases at both TA. Similar time courses of Tb, HP, and HL in their respective rhythms and the relative stability of Tb during both active and rest periods suggest action of the circadian timing system on Tb set point.

Evidence for macular gravity receptor modulation of hypothalamic, limbic and autonomic nuclei

Mice lacking normal vestibular gravity reception show altered homeostatic, circadian and autonomic responses to hypergravity (+G) exposure. Using c-Fos as a marker of neuronal activation, the current study identifies CNS nuclei that may be critical for initiating and integrating such responses to changes in vestibular signaling. This experiment utilized the mutant C57BL/6JEi-het mouse (het), which lacks macular otoconia and thus gravity receptor function. Following 2 h of 2G (2x Earths gravity) exposure (via centrifugation) the neuronal responses of the het mice were compared with wildtype mice similarly exposed to 2G, as well as het and wildtype 1G controls. Wildtype mice exposed to 2G demonstrated robust c-Fos expression in multiple autonomic, hypothalamic and limbic nuclei, including: the lateral septum, bed nucleus of the stria terminalis, amygdala, paraventricular hypothalamus, dorsomedial hypothalamus, arcuate, suprachiasmatic hypothalamus, intergeniculate leaflet, dorsal raphe, parabrachial and locus coeruleus. The het mice exposed to 2G demonstrated little to null c-Fos expression in these nuclei with a few exceptions and, in general, a similar pattern of c-Fos to 1G controls. Data from this study further support the existence of a complex and extensive influence of the neurovestibular system on homeostatic, circadian and possibly autonomic regulatory systems.

Immunotoxin-induced ablation of melanopsin retinal ganglion cells in a non-murine mammalian model.

In mammals, non‐image‐forming visual functions, including circadian photoentrainment and the pupillary light reflex, are thought to be mediated by the combination of rods, cones, and the melanopsin‐expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). Although several genetic models have been developed to clarify the individual roles of the rod, cone, and ipRGC systems in mediating non‐image visual function, assessing the in vivo role(s) of the ipRGCs has been complicated by the possibility of ontogenetic issues in these genetically modified animal models. In the present study, we describe the development and validation of an immunotoxin that specifically targets the ipRGC population in the mature mammalian retina. This ipRGC immunotoxin, consisting of saporin conjugated to a melanopsin polyclonal antibody, was evaluated with respect to its effectiveness and specificity in depleting the ipRGC population in the fully developed rat retina. The results showed that the ipRGC toxin rapidly and permanently depleted ∼70% of the ipRGC population, without inducing appreciable changes in the cell number or morphology of any of the non‐melanopsin‐containing retinal cell populations investigated. These findings suggest that the newly developed ipRGC immunotoxin provides a potent method for achieving relatively rapid, permanent, and selective depletion of the ipRGC population in a non‐murine model system. The development of this ipRGC‐ablation method is the next step in elucidating the role of ipRGCs in mediating non‐visual and circadian light‐resetting responses in a wide range of non‐murine mammalian models. J. Comp. Neurol. 516:125–140, 2009.