ELSEVIER Brain Research 690 (1995) 254-258 BRAIN RESEARCH Short communication Non-photic manipulations induce expression of Fos protein in the suprachiasmatic nucleus and intergeniculate leaflet in the rat K. Edelstein *, S. Amir Center fi~r Studies in Behacioral Neurobiology, Department of Psychology, Concordia Uniuersity, Montreal, Canada H3G 1M8 Accepted 7 June 1995 Abstract Expression of Fos protein in the suprachiasmatic nucleus (SCN) and intergeniculate leaflet (IGL) is considered a cellular correlate of light-induced phase-shift of circadian rhythms in rodents. Non-photic stimuli also induce phase shifts, but their effects on Fos expression have not been established. We examined induction of Fos protein in SCN and IGL regions, in response to cage change, intraperitoneal saline injection, and restraint stress. Fos immunoreactivity was observed in SCN and IGL regions, with greater expression observed in IGL during the light phase of the light-dark cycle. Results suggest that cells in SCN and IGL respond to several types of non-photic manipulations and that expression of Fos in these regions is not light-specific. Kevwords: Circadian rhythm; Suprachiasmatic nucleus; Intergeniculate leaflet; Non-photic stimulus; Fos immunoreactivity Mammalian circadian rhythms are generated by a pace- maker located in the hypothalamic suprachiasmatic nu- cleus (SCN) [22]. Circadian rhythms are synchronized to the light-dark (LD) cycle via a direct retinal projection to the SCN, the retinohypothalamic tract (RHT), and an indirect projection originating in retinorecipient cells in the intergeniculate leaflet (IGL) of the lateral geniculate nu- cleus, the geniculohypothalamic tract (GHT) [17]. The RHT is necessary for photic entrainment of circadian rhythms. The GHT has been implicated in the circadian response to both photic [8,12,19] and non-photic cues (e.g. [ll]). Light pulses that induce phase shifts also induce Fos protein expression in SCN and IGL regions [14,20,21]. Little is known about induction of Fos protein in response to non-photic stimuli. Fos expression was not observed in the SCN of hamsters given subcutaneous saline injection or intraperitoneal (i.p.) triazolam injection, although these treatments induce phase shifts in circadian activity rhythms [16,25]. Exposure to a novel running wheel phase shifts circadian activity rhythms and has been associated with expression of Fos immunoreactive (Fos-IR) cells in ham- ster IGL [10]. * Corresponding author. Fax: (1) (514) 848-2817. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00736-9 We examined effects of cage change, i.p. saline injec- tion, and restraint stress during zeitgeber time (ZT) 4 (8 h before onset of dark, defined as ZT 12), and ZT 16 (4 h after onset of dark) on Fos expression in rat SCN and IGL. Activity-inducing stimuli induce phase shifts at ZT 4, and photic stimuli induce phase shifts and Fos expression in the SCN and IGL when given at ZT 16. Preliminary results of this work have been presented in abstract form [2]. Male Wistar rats (300-325 g) from Charles River Canada (St. Constant, Que.) were housed individually in plastic cages, in two light-tight, ventilated rooms under LD 12:12 cycles (lights on 08:00-20:00 or 21:00-09:00), with free access to rat chow and water for three days before testing. On the test day, rats were either restrained in plexiglass restrainers, removed from their home cages and placed into plastic buckets, or given an injection of 0.9% saline i.p. and returned to their home cages, at ZT 4 or at ZT 16. Rats treated at ZT 4 were exposed to ambient light (300 lux) while animals treated at ZT 16 were exposed to dim red light during treatment (< 5 lux). Additional rats housed under each LD schedule, untouched during treat- ment times, served as controls. All rats received an over- dose of sodium pentobarbital (100 mg/kg) 60 min after treatment. Anesthetized rats were perfused transcardially with 300 ml of cold 0.9% NaCI followed by 300 ml of cold 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.3).