Anterior paraventricular thalamus modulates light-induced phase shifts in circadian rhythmicity in rats

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1 Am J Physiol Regul Integr Comp Physiol 283: R897 R904, First published July 8, 2002; /ajpregu Anterior paraventricular thalamus modulates light-induced phase shifts in circadian rhythmicity in rats ALBERTO SALAZAR-JUÁREZ, 1 CAROLINA ESCOBAR, 2 AND RAÚL AGUILAR-ROBLERO 1 1 Departamento de Neurociencias, Instituto de Fisiología Celular and 2 Departamento de Anatomía, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F México Received 10 May 2002; accepted in final form 28 June 2002 Salazar-Juárez, Alberto, Carolina Escobar, and Raúl Aguilar-Roblero. Anterior paraventricular thalamus modulates light-induced phase shifts in circadian rhythmicity in rats. Am J Physiol Regul Integr Comp Physiol 283: R897 R904, First published July 8, 2002; /ajpregu The reciprocal connections between the paraventricular thalamic nucleus (PVT) and the suprachiasmatic nuclei suggest that PVT may participate in the regulation of circadian rhythms. We studied in rats the effect of lesions of the anterior and midposterior regions of the PVT on phase shifts of drinking circadian rhythm induced by light pulses at circadian times 6, 12, and 23, as well as the phase shifts produced by electrical or glutamatergic stimulation of the anterior PVT at the same circadian times. Lesion of the anterior PVT abolishes the advances induced by light during late subjective night, whereas midposterior PVT lesions did not affect the phase shifts. Electrical stimulation or glutamate injections in the anterior PVT mimic the phase-shifting effects of light pulses. These results indicate the participation of the anterior PVT as a modulator of entrainment of circadian rhythms to light. phase advances; paraventricular thalamic nucleus stimulation; paraventricular thalamic nucleus lesions; suprachiasmatic nucleus modulation; photic entrainment IN MAMMALS, THE SUPRACHIASMATIC nuclei (SCN) of the hypothalamus are the main pacemaker driving many behavioral, neuroendocrine, and autonomic circadian rhythms (20). Photic input to the SCN via the retinohypothalamic tract allows entrainment of circadian rhythmicity to the light-dark cycle (29). Other inputs to the SCN arising from the intergeniculate leaflet (17) and the mesencephalic raphe nuclei (30) modulate the entrainment of rhythmicity to light, either increasing or decreasing the response of the pacemaker to light, and seem to be involved in entrainment to nonphotic stimuli as well (29). Another input to the SCN arises from the anterior part of the paraventricular thalamic nucleus (PVT) (25, 27). This nucleus is part of the thalamic midline nuclei and has been architecturally divided into anterior and posterior subregions (5, 27). The PVT is a multisensorial structure and receives inputs from different nuclei from the brain stem, as well as the prefrontal cortex, Address for reprint requests and other correspondence: R. Aguilar- Roblero, Neurociencias, IFC/UNAM, Apartado Postal , México D. F , Mexico ( raguilar@ifisiol.unam.mx). amygdala, and many hypothalamic cell groups including the SCN (5, 16). Efferents from the SCN project to the posterior PVT (37, 38). The reciprocal connections of PVT with the SCN, along with the inputs to PVT from other components of the circadian system such as the subparaventricular zone and the intergeniculate leaflet, suggest that PVT might play a role in the regulation of circadian rhythms. However, to this moment, such a role has not been completely elucidated. Electrolitic lesions of the PVT in hamsters have no effect in either the expression of locomotor circadian rhythms in a 14:10-h photoperiod or in its ability to reentrain to an 8-h advance in the time of light onset (12). In blind rats, however, electrolitic lesion of the PVT produces lengthening of the free running period and concentrates the locomotor activity to the late subjective night (26). On the other hand, lesion of the posterior PVT increases food intake mainly during the light phase, which may be due to a disruption of information that regulates feeding where the PVT may serve as a convergence relay for the efferents from the circadian timing system, the hypothalamic nuclei involved in feeding and the limbic system (6). The phase response curve (PRC) is a valuable tool to study the dynamics and mechanisms of entrainment of circadian pacemakers to a discrete stimulus in subjects maintained under constant conditions and therefore expressing free running circadian rhythmicity. Typically, the PRC to light shows three characteristic regions; a nonresponsive interval during the subjective day (dead zone), while phase delays and advances are induced by light during the early and late subjective night, respectively (10). The use of the PRC has allowed for dissection among the different neurochemical and intracellular signaling pathways involved in lightinduced phase advances or delays (14, 15). In the present study, we used the PRC as the experimental tool to study the role of PVT in the process of entrainment to light of the circadian rhythm of drinking behavior in rats. First, we studied the effect of lesions of the anterior and midposterior regions of the PVT on the phase shifts induced by light pulses presented at the three characteristic regions of the PRC The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact /02 $5.00 Copyright 2002 the American Physiological Society R897

2 R898 [dead zone at circadian time (CT) 6, phase delays at CT12, and phase advances at CT23]. We also studied the phase shifts of circadian rhythmicity produced by electrical or chemical stimulation of the PVT at the same three circadian times. Present results indicate that 1) lesions of the anterior PVT alter the shape of the PRC to light by abolishing the advances induced by light during late subjective night and 2) electrical or chemical stimulation of PVT mimics the phase-shifting effects of light pulses. MATERIALS AND METHODS Adult male Wistar rats weighing g were used. Animals were housed individually in Plexiglas cages in a room with constant red light (DD; 50 lx). Drinking behavior was continuously monitored with a computerized system (1). Temperature in the room was kept between 20 and 22 C, and water and food were provided ad libitum. All experimental procedures were conducted according to the institutional guide for care and use of animal experimentation for the Unversidad Nacional Autónoma de México, which comply with the guiding principles for research from the American Physiological Society (3). Surgery All surgical procedures were performed in antiseptical conditions and under deep anesthesia with chloral hydrate (400 mg/kg body wt ip). For the first experiment, animals were randomly assigned to one of three groups; the first group received electrolytic lesions of the anterior PVT (AP 1.0 mm from bregma; L 0.0; V 6.0 from the skull surface), and the second group received lesions of the midposterior PVT (AP 2.6; L 0.0; V 5.5). The lesion was made by passing 1-mA direct current for 45 s through a stainless steel electrode (0000) insulated except for 0.5 mm at the tip. A third group of rats (sham lesioned) was treated exactly the same as rats receiving lesions but no current was passed through the electrode. Animals were allowed to recover for a week after surgery before starting the behavioral recording. For the second experiment, rats were implanted with a bipolar chrome-nickel electrode (300- m diameter) in the anterior PVT (AP 1.0; L 0.0; V 5.5); each electrode tip was welded to a miniature connector and the assemble was fixed to the skull with stainless steel screws and dental acrylic. This electrode was used to electrically stimulate the nucleus in the free moving animal. For the third experiment, animals were implanted with a stainless steel cannula (25 gauge) 3 mm above the anterior PVT, which was fixed to the skull with stainless steel screws and dental acrylic. This cannula was used as a guide to introduce an injector (27 gauge) into PVT to administer either vehicle or glutamate solutions to the free moving animal. Behavioral Recordings Behavioral recordings consisted of counting the number of touches to the water spout in 15-min bins. Data were stored in magnetic media and graphed as double-plot actograms for later inspection and further analysis. Estimation of activity onset (CT12) was done in a 10- to 15-day segment as follows: first, data were filtered to plot only those bins with values equal or above the mean for that segment, and the first bin of activity for each day of the segment was identified. Then a line was fitted by linear regression to adjust these activity onsets. Such line was plotted on a nonfiltered actogram for illustration purposes and indicates the estimated CT12 for each day of the segment. Other circadian times at which animals were manipulated, such as CT6 and CT23, were calculated from the estimated CT12. After the experimental manipulations (light pulses, electrical or chemical stimulation of PVT), CT12 was estimated during the next 10 consecutive cycles that showed a stable phase and a period similar to the one previous to the stimulus, and the line was projected to the day when the manipulation occurred. The transient cycles were counted from the day the light pulse was applied to the previous day when stable phase and period were found. Phase shifts induced by the experimental manipulations were measured from the difference of CT12 regression lines between the segments before and after the manipulation. Stimulation Procedures At the selected time of stimulation, each rat was transferred into a Plexiglas cage. Light stimulation consisted of 1-h light pulses of 400 lx (at the bottom of the cage). Electrical stimulation was provided by a Grass S-8800 electrical stimulator and consisted of a train of square direct current pulses (10 ms, 0.3 ma) at 0.5 Hz during 30 min. For the chemical stimulation, each rat received 1 l of glutamate (1.5 g/ l) in phosphate buffer saline (50 mm, ph 7.2) with a Hamilton microsyringe (7001) attached to a stainless steel injector (27 gauge) by tygon tubing. All procedures were carried out under red dim light (50 lx) with the exception of the light pulses that were applied inside the light-tight chamber provided with a 40-W white fluorescent tube. Control animals were handled as described but no stimulus was provided. For the chemical stimulation, control animals were injected with 1 l of vehicle. Histology At the end of the experiments, the animals were deeply anestethized with an overdose of pentobarbital sodium (200 mg/kg body wt ip) and transcardially perfused with physiological saline solution (NaCl, 0.9%) followed by 4% paraformaldehyde in phosphate buffer (0.1 M, ph 7.2). The brain was removed and cryoprotected in successive sucrose solutions (10, 20, and 30%). Four sets of serial coronal sections 40- m thick were obtained with a cryostat, from the anterior to the posterior commissures. One set of sections was stained for Nissl with cresyl violet, dehydrated with alcohol, cleared with xylene, and placed under a coverslip with permount. Serial reconstruction of the lesion placement or the position of either the electrode or the cannula was made from camera lucida drawings at 40. Experimental Design Experiment 1. This experiment was aimed to determine the effect of PVT lesions (PVTx) on light-induced phase shifts at three different circadian times. Three groups of nine rats each (sham lesions, anterior PVTx, and midposterior PVTx) were studied. Each animal received a light pulse (400 lx, 1 h) at CT6, CT12, or CT23 in a counterbalanced design; these time points were selected to explore the main regions of the PRC (dead zone, delays, and advances, respectively). After a baseline recording of 15 days, animals were stimulated at the selected time as previously described and then recorded for at least 15 additional days. The last 10 days of recording were used as the baseline for the next experimental segment. At the end of the experiment, all lesioned animals were pro-

3 R899 cessed for histology as described. The effects of the light stimulation on the magnitude and direction of the phase shifts and the number of transients were estimated as previously described. To determine the effect of the lesion on the endogenous period of rhythmicity, this parameter was estimated by the 2 periodogram (36), and the mean values were compared among the three groups by a one-way ANOVA followed by the Tukey post hoc test. In this and the remaining experiments, data were analyzed with the DiSPAC software developed and validated in our laboratory (1). Statistical comparisons were made using Statistica v 4.3 (Stat-Soft). Experiment 2. This experiment was aimed at determining the effect of electrical stimulation of the anterior PVT on the phase of activity onset at three different circadian times. One group of nine animals was implanted with a twisted bipolar electrode for electrical stimulation of PVT; at the three circadian times selected (CT6, CT12, and CT23), animals were stimulated or sham manipulated in a counterbalanced design as follows. After recovery from surgery, animals were recorded under DD for 10 days to establish CT12 (activity onset) and to determine the proper time of stimulation. After electrical stimulation, the recording continued for 15 days. The last 10 days of recording were used as the baseline for the next experimental segment. The parameters under study were the same as those of experiment 1. The effect of electrical stimulation and the time of manipulation on the direction and magnitude of the phase shifts were analyzed with a two-way ANOVA for repeated measurements followed by the Tukey post hoc test. At the end of the experiment, the animals were processed for histology to verify the position of the electrode in the brain. Experiment 3. To rule out the effect of electrical stimulation of fibers on passage or retrograde stimulation from terminals at the level of PVT, this experiment was aimed to determine the effect of glutamate stimulation at three different circadian times in the anterior PVT on the phase of activity onset. Twenty-seven animals were implanted with a stainless steel guide cannula to place an injector to chemically stimulate the nucleus with glutamate. These animals were randomly assigned to one of three groups (CT6, CT12, and CT23). The animals from each group (n 9) were injected at only one of the circadian times with glutamate and vehicle in a counterbalanced design. After recovery from surgery, animals were recorded under DD for 10 days to establish CT12 and to determine the time for next day injection with either glutamate or vehicle. The recording then continued for 15 days, the last 10 days of recording were used as the baseline for the next injection. Phase shifts were estimated as previously described. The effect of time of chemical stimulation on direction and magnitude of the shifts were analyzed with a one-way ANOVA for independent groups followed by the Tukey post hoc test. At the end of the experiment, animals were processed for histology to verify the position of the injector in the brain. RESULTS Experiment 1 Histological verification of the group with anterior PVTx showed that only five animals had complete destruction of the PVT (Fig. 1A), three animals showed varying degrees of partial damage of the dorsal aspect of the nucleus, and in one subject the lesion completely spared PVT. Complete lesions of the anterior PVT also involved surrounding thalamic nuclei such as the paratenial, centromedial, anteromedial, and stria medularis. With respect to the lesion of the midposterior PVT group, in six animals the lesion involved the middle and most of the midposterior parts of PVT and extended in varying degrees into the surrounding thalamic nuclei, such as the nucleus paratenialis, nucleus dorsal intermedius, lateral habenular nucleus, and middorsal nucleus of the thalamus (Fig. 1B). In the remaining three animals of this group, the lesion affected only partially the midposterior PVT. The results from animals with complete lesion of PVT (either anterior or midposterior) will be reported separate from those with incomplete lesions. According to the periodogram, the endogenous period of rhythmicity showed a nonsignificant decrease in animals with lesion of both anterior PVT ( min, means SE) and midposterior PVT ( min) with respect to sham-lesioned animals ( min). No evident effect of the lesions was found in the architecture of the rhythm. Light pulses in sham-lesioned animals induced phase shifts of rhythmicity similar to those found in intact animals (Fig. 2), at CT6 no evident shifts were found ( min, means SE), whereas at CT12 and CT23, light-induced phase delays ( min) or phase advances ( min) were present, respectively. Animals with lesion of the midposterior PVT (Fig. 2) showed a similar pattern of light-induced phase shifts at CT6 ( min) and CT12 ( min) and slightly larger advances induced at CT23 ( min) than sham-lesioned animals. In contrast, animals with lesion of the anterior PVT (Fig. 2) did not show phase advances at CT23 but rather phase delays ( min), although they showed similar effects of light at CT6 and CT12 ( and min, respectively) as their sham controls. These results are summarized in Fig. 3. Statistical analysis indicated significant differences in the phase shifts induced at CT23 in the group with anterior PVT lesion with respect to the other two groups [F(2,17) 132.1, P ; Tukey test P 0.01]. Animals with incomplete PVT lesion showed similar phase responses to those found in sham-lesioned controls. The shifts for anterior and posterior PVT incomplete lesions were as follows: at CT6, and ; at CT12, and ; and at CT23, and Experiment 2 The electrode tract was found within the boundaries of the anterior part of PVT in eight animals (Fig. 4, F). In the remaining rat, the electrode was found lateral to the nucleus and was discarded from the experiment (Fig. 4, *). The effects of the electrical stimulation of PVT and the sham manipulation at the three circadian times studied are summarized in Fig. 5. No effects on the phase of the free running rhythm were found after the sham manipulations at any of the circadian times nor after electrical stimulation of PVT at CT6. In contrast, electrical stimulation of PVT at CT12 and CT23 induced, respectively, significant phase delays

4 R900 Fig. 1. Schematic reconstruction of complete lesions (shadow area) in the anterior paraventricular thalamic nucleus (PVT; A) and midposterior PVT (B). PVT is indicated by *. The AP plane from bregma according to Paxinos and Watson (33) is indicated in each drawing. AM, anteromedial thalamic nucleus; AV, anteroventral thalamic nucleus; BST, bed nucleus of the stria terminalis; CM, centromedian thalamic nucleus; DG, dentate gyrus; fi, fimbria of the hippocampus; ic, internal capsule; ml, external medullary lamina; mt, mammillothalamic tract; Po, posterior thalamic nucleus; Re, nucleus reuniens; Rt, reticular thalamic nucleus; Sfi, septo fimbrial nucleus; SFO, subfornical organ; sm, stria medularis; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VPM, ventroposteromedial thalamic nucleus. ( min) and phase advances ( min), with a magnitude similar to those induced by light pulses in intact animals (see Fig. 3) in comparison to the sham manipulations at such times [F(1,7) 58.2, P ; Tukey P 0.05]. Experiment 3 The tip of the injection cannula was found within the anterior PVT in 23 of 27 implanted animals (Fig. 4, Œ) as follows: in seven animals from the group injected at CT6 and in eight animals from each of the groups studied at CT12 and CT23, respectively. In the remaining four animals, the tip of the cannula was found lateral or ventral to the PVT and was discarded from the experiment (Fig. 4, ). Also, one animal from each of the groups injected at CT12 and CT23 was randomly selected and discarded from the study to have an equal number of animals (n 7) in each group. The effect of glutamate or vehicle administration on the phase of the free running rhythm at the three circadian times under study is summarized in Fig. 6. No phase shifts were found after injection of vehicle at any of the circadian times or after the injection of glutamate at CT6. In contrast, injection of glutamate at CT12 induced phase delays ( min), whereas glutamate at CT23 induced phase advances ( min); such effects were statistically significant compared with the effects of vehicle administered at the same circadian times [F(2,18) 23.2, P ; Tukey P 0.05]. DISCUSSION Present data indicate that inputs to the SCN arising from the anterior portion of PVT play an important role in the shape of the PRC to light of drinking rhythm in rats kept under DD. Electrolytic lesion of anterior PVT

5 R901 Fig. 2. Lesion of the anterior PVT leads to phase delays instead of advances in response to a light pulse at circadian time (CT) 23. Double-plotted actograms of drinking behavior from animals of the sham-lesioned group (left), midposterior PVT lesion (middle), and anterior PVT lesion (right) are shown. The animals received a light pulse (1 h, 400 lx, triangle) at the time indicated on the left. A regression line was fitted to the activity onset (CT12) before and after the light pulse as indicated in MATERIALS AND METHODS. results in atypical phase delays induced by light pulses during late subjective night (CT23) instead of the phase advances found at this circadian time in shamlesioned animals. This effect was specific of lesions of the anterior PVT that project to the SCN, because neither lesions of the midposterior PVT nor lesions sparing most of the anterior PVT affected the phase shifts. Although the mechanism involved in such effect remains to be studied, we may speculate that PVT inputs to the SCN enable the mechanisms affected by light during late subjective night, which are involved in the phase advances in SCN neurons. In the present study, we did not find differences in the free running period in constant darkness among sham, anterior, and midposterior PVT-lesioned rats. A previous study with blinded rats indicated that lesions of anterior PVT lengthen the free running period of locomotor activity in constant darkness and shift the density of activity bouts toward the late subjective night (26). In contrast, electrolytic lesions of anterior PVT in hamsters do not alter the free running period of locomotor activity in constant darkness (12). Such differences were attributed either to the differences in organization of the circadian system between these species or to the absence of retinal inputs to the SCN. Present results support the notion that changes in period found in the previous study may be due to the lack of retinal inputs. In other rodents such as hamsters and mice, inputs to the SCN arising from the intergeniculate leaflet and the median raphe nucleus (MRN) participate with opposite effects in the entrainment of circadian rhythmicity by modulating the response of SCN neurons to light. Lesion of the intergeniculate leaflet decreases phase advances to light pulses, shortens the free running period, reduces the incidence of splitting in constant light, and prevents the lengthening of free running period induced by increasing illumination (17). On the other hand, lesion of serotoninergic neurons in the MRN increases phase delays induced by light pulses, increases the incidence of splitting in constant light, and lengthens the free running period in constant light (29, 30). Such studies indicate that inputs arising from intergeniculate leaflet increase the response of SCN to light, whereas inputs arising from the MRN decrease this response. Present findings indicate that PVT projections to the SCN are at least partially involved in setting the direction of the phase shift of the circadian pacemaker to light. Further studies are needed to characterize the effects of light at other

6 R902 Fig. 3. Atypical phase delays by light pulses at CT23 occur in animals with lesion of the anterior PVT but not in those with lesion of the midposterior PVT. Phase shifts (means SE) were induced by 1-h light pulse (400 lx) at 3 circadian times in sham-lesioned animals (open bar, n 6), midposterior PVT-lesioned animals (crosshatched bar, n 6), and anterior PVT-lesioned animals (black bar, n 5). *Significant differences with the remaining 2 groups at that circadian time (Tukey post hoc test, P 0.05). Fig. 5. Electrical stimulation of the anterior PVT induces phase shifts similar to those induced by light pulses. Phase shifts (means SE) were induced by a 30-min train (0.5 Hz) of square direct current pulses (10 ms, 0.3 ma) at 3 circadian times. The effect of the manipulation without turning on the stimulator (sham) is also shown. *Significant differences with the remaining 2 groups (n 9, Tukey post hoc test, P 0.05). circadian times to clarify whether phase advances occur at other times or are absent in PVT-lesioned animals. Electrical or chemical stimulation of the anterior PVT at the three characteristic circadian times of the PRC produced similar phase responses as those obtained by light. Because glutamate receptors are not present in fibers on passage, shifts found after glutamate stimulation of PVT indicate that these effects are due to activation of PVT neurons rather than to activation of fibers running through the stimulated area. Excitatory amino acid input to the SCN from PVT has previously been suggested from studies showing retrograde transport to PVT neurons of D-[ 3 H]aspartate injected into the SCN in hamsters (11) and rats (24). Thus it is possible that phase responses induced by PVT stimulation may be due to release of excitatory Fig. 4. Location of the electrodes (F, *) or injector (Œ, ) tips found, respectively, within or outside the anterior PVT (arrow). The AP plane from bregma according to Paxinos and Watson (33) is indicated in each drawing. Fig. 6. Phase shifts induced by stimulation of the anterior PVT depend on neuronal activation. Injection of glutamate (1.5 g/1 l, crosshatched bars) into the anterior PVT at 3 circadian times induces phase shifts (means SE) similar to those induced either by light pulses or by electrical stimulation of the PVT. Injection of the vehicle alone (open bars) had no effects. *Significant differences of glutamate with respect to vehicle injection (n 9 for each circadian time, Tukey post hoc test, P 0.05).

7 R903 amino acids in the SCN from fibers arising from the anterior PVT, because it is widely accepted that excitatory amino acids, most likely N-acetylaspartylglutamate, from retinal ganglion cells are neurotransmitters involved with the response of SCN neurons to light (29). Phase shifts in circadian rhythmicity induced by electrical or chemical stimulation of PVT differ from those shifts induced by stimulation of other SCN inputs. Electrical stimulation of the intergeniculate leaflet (35) or stimulation of the SCN with neuropeptide Y (2), which is released in the SCN from fibers arising from the intergeniculate leaflet, induces a nonphotic (or dark pulse) type of PRC. Electrical stimulation of the raphe (23) or stimulation of the SCN with agonists to serotonin, which is released from fibers originated from the MRN, induces a nonphotic type of PRC via serotonin receptors (7 9, 13, 34). In contrast, others found a PRC similar to that induced by light pulses but with systemic administration of quipazine, a nonselective agonist to serotonin receptors (19, 21, see Ref. 30 for a review). It is clear that activation of different receptors in SCN neurons has selective effects on the phase of the pacemaker, which depends not only of the chemical signal involved but also on the status of the pacemaker at the time at which the stimulus is presented. PVT has been implicated in visceral and autonomic functions (5, 16). In particular, the role in the control of some aspects of the sleep-wake states has been suggested from the dense innervation of this nucleus by the locus ceruleus and the raphe nuclei (4, 18, 22, 28) as well as the conspicuous expression of c-fos during wakefulness in both nocturnal and diurnal animals (31, 32). The present study clearly indicates the participation of the anterior PVT in the nonparametric entrainment of circadian rhythms to light. The results suggest that integrity of the anterior PVT is necessary for an adequate response of the pacemaker to light during late subjective night. It remains to be established whether PVT acts directly on SCN neurons or presynaptically on the terminals of the retinohypothalamic tract as well as the nature of the transmitter(s) involved. Furthermore, present data also suggest that anterior PVT inputs to the SCN may induce phase shifts in the pacemaker by acting through a similar neurochemical system as that involved in the response to light pulses. We thank J. L. Chavez for skillful technical assistance. This study was supported by grants from Consejo Nacional de Ciencia y Tecnología (33034-N), Dirección General de Asuntos del Personal Académico de la Universidad Nacional Autónoma de México (IN ), and Omnilife REFERENCES 1. Aguilar-Roblero R, Salazar-Juarez A, Rojas-Castañeda J, Escoba C, and Cintra L. Organization of circadian rhythmicity and suprachiasmatic nuclei in malnourished rats. Am J Physiol Regul Integr Comp Physiol 273: R1321 R1331, Albers HE, Ferris CF, Leeman SE, and Goldman BD. Avian pancreatic polypeptide phase shifts hamster circadian rhythms when microinjected into the suprachiasmatic region. Science 223: , American Physiological Society. Guiding principles for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 283: R282 R283, Azmitia EC and Segal M. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J Comp Neurol 179: , Bentivoglio M, Balercia G, and Krueger L. The specificity of the nonspecific thalamus: the midline nuclei. Prog Brain Res 7: 53 80, Bhatnagar S and Dallman MF. The paraventricular nucleus of the thalamus alters rhythms in core temperature and energy balance in a state-dependent manner. Brain Res 851: 66 75, Bobrzynska KJ, Godfrey MH, and Mrosovsky N. Serotonergic stimulation and nonphotic phase-shifting in hamsters. Physiol Behav 59: , Challet E, Scarbrough K, Penev PD, and Turek FW. Roles of suprachiasmatic nuclei and intergeniculate leaflets in mediating the phase-shifting effects of a serotonergic agononist and their photic modulation during subjective day. J Biol Rhythms 13: , Cutrera RA, Ouarour A, and Pevet P. Effects of the 5-HT 1a receptor agonist 8-OH-DPAT and other non-photic stimuli on te circadian rhythms of wheel-running activity in hamsters under different constant conditions. Neurosci Lett 19: 27 39, Daan S and Pittendrigh CS. A functional analysis of circadian pacemakers in nocturnal rodents. II. The variability of phase response curves. J Comp Physiol [A] 106: , De Vries MJ and Lakke EAJF. Retrograde labeling of retinal ganglion cells and brain neuronal subsets by [ 3 H]-D-aspartate injection in the Syrian master hypothalamus. Brain Res Bull 38: , Ebling F, Maywood E, Humby T, and Hastings M. Circadian and photoperiodic time measurment in male Syrian hamsters following lesions of the melatonin-binding sites of the paraventricular thalamus. J Biol Rhythms 7: , Edgar DM, Miller JD, Prosser RA, Dean RR, and Dement WC. Serotonin and the mammalian circadian system. II. Phase shifting rat behavioral rhythms with serotonergic agonist. J Biol Rhythms 8: 17 31, Gillette MU. Cellular and biochemical mechanisms underlying circadian rhythms in vertebrates. Curr Opin Neurobiol 7: , Golombek DA and Ralph MR. Let there be light: signal transduction in a mammalian circadian system. Braz J Med Biol Res 29: , Groenewegen HJ and Berendese HW. The specificity of the nonspecific midline and intralaminar thalamic nuclei. Trends Neurosci 17: 52 57, Harrington ME. The ventral lateral geniculate nucleus and the intergeniculate leaflet: interrelated structures in the visual and circadian systems. Neurosci Biobehav Rev 21: , Jones BE and Yang TZ. The efferent projections from the reticular formation and the locus coeruleus studied by anterograde and retrograde axonal transport in the rat. J Comp Neurol 242: 56 92, Kennaway DJ, Rowe SA, and Ferguson SA. Serotonin agonists mimic the phase shifting effect of light on the melatonin rhythms in rats. Brain Res 737: , Klein DC, Moore RY, and Reppert SM. Suprachiasmatic nucleus. In: The Mind s Clock. New York: Oxford Univ. Press, Kohler M, Kalkowski A, and Wollnik F. Serotonin agonist quazepine induces photic-like phase shifts of the circadian activity rhythm and c-fos expression in the rat suprachiasmatic nucleus. J Biol Rhythms 14: , Lindvall A, Björklund A, Nobin A, and Stenevi U. The adrenergic innervation of the rat thalamus as revealed by the glyoxylic acid fluorescence method. J Comp Neurol 154: , Meyer-Bernstein EL and Morin LP. Electrical stimulation of the median or dorsal raphe nuclei reduces light-induced fos

8 R904 protein in the suprachiasmatic nucleus and causes circadian activity rhythms phase shifts. Neuroscience 92: , Moga MM and Moore RY. Putative excitatory amino acid projections to the suprachiasmatic nucleus in the rat. Brain Res 743: , Moga MM and Moore RY. Organization of neural inputs to the suprachiasmatic nucleus in the rat. J Comp Neurol 389: , Moga MM and Moore RY. Paraventricular thalamus lesions alter circadian period and activity distribution in the blinded rat. Biol Rhythm Res 31: , Moga MM, Weis RP, and Moore RY. Efferent projections of the paraventricular thalamic nucleus in the rat. J Comp Neurol 359: , Moore RY, Halaris AE, and Jones BE. Serotonin neurons of the midbrain raphe: ascending projections. J Comp Neurol 180: , Morin L. The circadian visual system. Brain Res Rev 67: , Morin L. Serotonin and the regulation of mammalian circadian rhythmicity. Ann Med 31: 12 33, Novak CM and Nunez AA. Daily rhythms in Fos activity in the rat ventrolateral preoptic area and midline thalamic nuclei. Am J Physiol Regul Integr Comp Physiol 275: R1620 R1626, Novak CM, Smale L, and Nunez AA. Rhythms in Fos expression in brain areas related to the sleep-wake cycle in the diurnal Arvicanthis niloticus. Am J Physiol Regul Integr Comp Physiol 278: R1267 R1274, Paxinos G and Watson C. The Rat Brain in Stereotaxic Coordinates (2nd ed.). San Diego: Academic, Pickard GE and Rea M. Serotonergic innervation of the hypothalamic suprachiasmatic nucleus and photic regulation of circadian rhythms. Biol Cell 89: , Rusak B, Meijer JH, and Harrington ME. Hamster circadian rhythms are phase-shifted by electrical stimulation of the geniculo-hypothalamic tract. Brain Res 493: , Sokolove PG and Bushll WN. The chi square periodogram: its utility in the analysis of circadian rhythms. J Theor Biol 72: , Watts AG and Swanson LW. Efferent projections of the suprachiasmatic nucleus. II. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunohistochemistry in the rat. J Comp Neurol 258: , Watts AG, Swanson LW, and Sanchez-Watts G. Efferent projections of the suprachiasmatic nucleus. I. Studies using anterograde transport of phaseolus vulgaris leucoagglutinin in the rat. J Comp Neurol 258: , 1987.