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Cardiac arrhythmias are a leading cause of cardiovascular death. It has long been accepted that life-threatening cardiac arrhythmias (ventricular tachycardia, ventricular fibrillation, and sudden cardiac death) are more likely to occur in the morning after waking. It is perhaps less well recognized that there is a circadian rhythm in cardiac pacemaking and other electrophysiological properties of the heart. In addition, there is a circadian rhythm in other arrhythmias, for example, bradyarrhythmias and supraventricular arrhythmias. Two mechanisms may underlie this finding: (1) a central circadian clock in the suprachiasmatic nucleus in the hypothalamus may directly affect the electrophysiology of the heart and arrhythmogenesis via various neurohumoral factors, particularly the autonomic nervous system; or (2) a local circadian clock in the heart itself (albeit under the control of the central clock) may drive a circadian rhythm in the expression of ion channels in the heart, which in turn varies arrhythmic substrate. This review summarizes the current understanding of the circadian rhythm in cardiac electrophysiology, arrhythmogenesis, and the underlying molecular mechanisms., (Copyright © 2018. Published by Elsevier Inc.)

Parkinson disease is one of the neurodegenerative diseases that benefited the most from the use of non-human models. Consequently, significant advances have been made in the symptomatic treatments of the motor aspects of the disease. Unfortunately, this translational success has been tempered by the recognition of the debilitating aspect of multiple non-motor symptoms of the illness. Alterations of the sleep/wakefulness behavior experienced as insomnia, excessive daytime sleepiness, sleep/wake cycle fragmentation and REM sleep behavior disorder are among the non-motor symptoms that predate motor alterations and inevitably worsen over disease progression. The absence of adequate humanized animal models with the perfect phenocopy of these sleep alterations contribute undoubtedly to the lack of efficient therapies for these non-motor complications. In the context of developing efficient translational therapies, we provide an overview of the strengths and limitations of the various currently available models to replicate sleep alterations of Parkinson's disease. Our investigation reveals that although these models replicate dopaminergic deficiency and related parkinsonism, they rarely display a combination of sleep fragmentation and excessive daytime sleepiness and never REM sleep behavior disorder. In this light, we critically discuss the construct, face and predictive validities of both rodent and non-human primate animals to model the main sleep abnormalities experienced by patients with PD. We conclude by highlighting the need of integrating a network-based perspective in our modeling approach of such complex syndrome in order to celebrate valid translational models., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The master circadian pacemaker in the suprachiasmatic nuclei (SCN) regulates the nocturnal secretion of the pineal hormone melatonin. Melatonin, in turn, has feedback effects on SCN neuronal activity rhythms via high affinity G protein-coupled receptors (MT(1) and MT(2) ). However, the precise effects of melatonin on the electrical properties of individual SCN neurones are unclear. In the present study, we investigated the acute effects of exogenous melatonin on SCN neurones using whole-cell patch-clamp recordings in brain slices prepared from Per1::d2EGFP-expressing transgenic mice. In current-clamp mode, bath applied melatonin, at near-physiological concentrations (1 nM), hyperpolarised the majority (63.7%) of SCN neurones tested at all times of the projected light/dark cycle. In addition, melatonin depolarised a small proportion of cells (11.0%). No differences were observed for the effects of melatonin between Per1::GFP or non-Per1::GFP SCN neurones. Melatonin-induced effects were blocked by the MT(1)/MT(2) antagonist, luzindole (1 μM) and the proportion of SCN neurones responsive to melatonin was greatly reduced in the presence of either tetrodotoxin (200 or 500 nM) or gabazine (20 μM). In voltage-clamp recordings, 1 nM melatonin increased the frequency of GABA-mediated currents. These findings indicate, for the first time, that exogenous melatonin can alter neuronal excitability in the majority of SCN neurones, regardless of whether or not they overtly express the core clock gene Per1. The results also suggest that melatonin acts mainly by modulating inhibitory GABAergic transmission within the SCN. This may explain why exogenous application of melatonin has heterogenous effects on individual SCN neurones., (© 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.)

The habenula complex is implicated in a range of cognitive, emotional and reproductive behaviors, and recently this epithalamic structure was suggested to be a component of the brain's circadian system. Circadian timekeeping is driven in cells by the cyclical activity of core clock genes and proteins such as per2/PER2. There are currently no reports of rhythmic clock gene/protein expression in the habenula and therefore the question of whether this structure has an intrinsic molecular clock remains unresolved. Here, using videomicroscopy imaging and photon-counting of a PER2::luciferase (LUC) fusion protein together with multiunit electrophysiological recordings, we tested the endogenous circadian properties of the mouse habenula in vitro. We show that a circadian oscillator is localized primarily to the medial portion of the lateral habenula. Rhythms in PER2:: LUC bioluminescence here are visualized in single cells and oscillations continue in the presence of the sodium channel blocker, tetrodotoxin, indicating that individual cells have intrinsic timekeeping properties. Ependymal cells lining the dorsal third ventricle also express circadian oscillations of PER2. These findings establish that neurons and non-neuronal cells in the epithalamus express rhythms in cellular and molecular activities, indicating a role for circadian oscillators in the temporal regulation of habenula controlled processes and behavior., ((c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Daily exercise promotes physical health as well as improvements in mental and neural functions. Studies in intact wild-type (WT) rodents have revealed that the brain's suprachiasmatic nuclei (SCN), site of the main circadian pacemaker, are also responsive to scheduled wheel running. It is unclear, however, if and how animals with a dysfunctional circadian pacemaker respond to exercise. Here, we tested whether scheduled voluntary exercise (SVE) in a running wheel for 6 hours per day could promote neural and behavioral rhythmicity in animals whose circadian competence is compromised through genetically targeted loss of vasoactive intestinal polypeptide (VIP(-/-) mice) or its VPAC(2) receptor (Vipr2(-/-) mice). We report that in constant dark (DD), rhythmic VIP(-/-) and Vipr2(-/-) mice show weak free-running rhythms with a period of <23 hours and all wild-type mice are strongly rhythmic with approximately 23.5-hour periodicity. VIP(-/-) and Vipr2(-/-) mice rapidly (<7 days) synchronize to daily SVE, while WT mice take much longer (>35 days). Following 21 to 50 days of SVE, WT mice show small changes in their rhythms, and most Vipr2(-/-) mice now sustain robust near 24-hour behavioral rhythms, whereas very few VIP(-/-) mice do. This study demonstrates that scheduled daily exercise can markedly improve circadian rhythms in behavioral activity and raises the possibility that this noninvasive approach may be useful as an intervention in clinical etiologies in which there are dysfunctions of circadian time keeping.

The coordinated activity of thousands of cellular oscillators in the suprachiasmatic nuclei (SCN) temporally regulates mammalian physiology to anticipate daily environmental changes across the seasons. The phasing of clock gene expression varies according to anatomical location in the SCN and is thought to encode photoperiodic information. However, it is unclear whether similar variations in phase occur in the electrical activity of SCN neurons, a measure of both intraSCN signaling and clock output. To address this, we recorded single-unit and multiunit activity (SUA/MUA) from dorsal and ventral subregions of the middle level of the rostrocaudal axis of the SCN in coronal brain slices prepared from mice housed under different photoperiods. We demonstrate that under a symmetrical (12 h light:12 h dark) photoperiod, cells in the dorsal SCN are less tightly synchronized than those in the ventral SCN. Comparison of recordings made from mice under short (8 h light:16 h dark) or long (16 h light:8 h dark) photoperiods shows that the phase distribution of ventral, but not dorsal, SCN neurons expands with increasing day length. Conversely, the duration that individual neurons are active increases in dorsal, but not ventral, SCN under long days. These data indicate that in the ventral SCN photoperiod is encoded at the network level, while this coding occurs at the level of individual cells in the dorsal SCN.

The study of neural arousal mechanisms has been greatly aided by the discovery of the orexin peptides (orexin A and orexin B), the subsequent identification of the neurons that synthesize these peptides, their projections in the brain, and the distribution of orexin receptors in the central nervous system. Orexin neuron activation is partly controlled by circadian signals generated in the brain's main circadian pacemaker, the suprachiasmatic nuclei (SCN). The SCN clock is in turn reset by arousal-promoting stimuli and, intriguingly, orexin fibers and receptor expression are detected in the SCN region. It is unclear, however, if orexin can alter SCN neuronal activity. Here using a coronal brain slice preparation, we found that orexin A and orexin B (0.1-1 microM) elicited significant changes in the extracellularly recorded firing rate and firing pattern in approximately 80% of rat SCN cells tested; the most common response was suppression of firing rate. Co-application of orexin A with a cocktail of ionotropic GABA and glutamate receptor antagonists did not alter the actions of this peptide on firing rate, but did change some its effects on firing pattern. We conclude that orexins can alter SCN neurophysiology and may influence the transmission of information through the SCN to other CNS regions.

Neuropeptide signaling plays key roles in coordinating cellular activity within the suprachiasmatic nuclei (SCN), site of the master circadian oscillator in mammals. The neuropeptide angiotensin II (ANGII) and its cognate receptor AT1, are both expressed by SCN cells, but unlike other SCN neurochemicals, very little is known about the cellular actions of ANGII within this circadian clock. We used multi-electrode, multiunit, extracellular electrophysiology, coupled with whole-cell voltage and current clamp techniques to investigate the actions of ANGII in mouse SCN slices. ANGII (0.001-10 microM) dose dependently stimulated and inhibited extracellularly recorded neuronal discharge in many SCN neurons ( approximately 60%). Both actions were blocked by pre-treatment with the AT1 receptor antagonist ZD7155 (0.03 microM), while suppressions but not activations were prevented by pre-treatment with the GABA A receptor antagonist bicuculline (20 microM). AT1 receptor blockade itself suppressed discharge in a subset ( approximately 30%) of SCN neurons, and this action was not blocked by bicuculline. In voltage-clamped SCN neurons (-70 mV), AT1 receptor activation dose-dependently enhanced the frequency of action potential-driven, GABA A receptor-mediated currents, but did not alter their responses to exogenously applied GABA. In current-clamped SCN neurons perfused with tetrodotoxin, ANGII induced a membrane depolarization with a concomitant decrease in input resistance. In conclusion we show that AT1 receptor activation by ANGII depolarizes SCN neurons and stimulates action potential firing, leading to increased GABA release in the mouse SCN. Additionally we provide the first evidence that endogenous AT1 receptor signaling tonically regulates the activities of some SCN neurons.

Vasoactive intestinal polypeptide and its receptor, VPAC2, play important roles in the functioning of the dominant circadian pacemaker, located in the hypothalamic suprachiasmatic nuclei (SCN). Mice lacking VPAC2 receptors (Vipr2-/-) show altered circadian rhythms and impaired synchronization to environmental lighting cues. However, light can increase phosphoprotein and immediate early gene expression in the Vipr2-/- SCN demonstrating that the circadian clock is readily responsive to light in these mice. It is not clear whether these neurochemical responses to light can be transduced to behavioral changes as seen in wild-type (WT) animals. In this study we investigated the diurnal and circadian wheel-running profile of WT (C57BL/6J) and Vipr2-/- mice under a 12-h light:12-h complete darkness (LD) lighting schedule and in constant darkness (DD) and used 1-h light pulses to shift the activity of mice in DD. Unlike WT mice, Vipr2-/- mice show grossly altered locomotor patterns making the analysis of behavioral responses to light problematic. However, analyses of both the onset and the offset of locomotor activity reveal that in a subset of these mice, light can reset the offset of behavioral rhythms during the subjective night. This suggests that the SCN clock of Vipr2-/- mice and the rhythms it generates are responsive to photic stimulation and that these responses can be integrated to whole animal behavioral changes.

In species where parents may commit infanticide, temporal kin recognition can help ensure parents kill unrelated young but care for their own offspring. This is not true recognition, but rather depends on accurate timing of the arrival of young and a behavioural switch from killing to caring for offspring. Mistakes have clear fitness consequences; how do species that use temporal kin recognition ensure accurate timing? We manipulated photic cues and show that the switch from infanticide to parental care in the burying beetle Nicrophorus vespilloides depends on day-length inputs. Extending the light period after carcass discovery influenced timing of both oviposition and the cessation of infanticide. Manipulation of the light : dark cycle after oviposition also influenced timing of the switch to parental care. The timing mechanism is therefore sensitive to photic cues and access to a carcass and is not triggered by oviposition. The behavioural switch is directly related to the timing mechanism rather than changes in reproductive physiology. Given the conserved nature and extensive homology of genetic influences on biological timing, we speculate that the molecular mechanisms regulating circadian behaviour may have been co-opted to allow beetles to determine how much time has passed after carcass discovery even though this is over 50 h.

Vasoactive intestinal polypeptide (VIP), acting via the VPAC(2) receptor, is a key signaling pathway in the suprachiasmatic nuclei (SCN), the master clock controlling daily rhythms in mammals. Most mice lacking functional VPAC(2) receptors are unable to sustain behavioral rhythms and lack detectable SCN electrical rhythms in vitro. Adult mice that do not produce VIP (VIP/PHI(-/-)) exhibit less severe alterations in wheel-running rhythms, but the effects of this deficiency on the amplitude, phasing, or periodicity of their SCN cellular rhythms are unknown. To investigate this, we used suction electrodes to extracellularly record multiple- and single-unit electrical activity in SCN brain slices from mice with varying degrees of VIP deficiency, ranging from wild-type (VIP/PHI(+/+)) to heterozygous (VIP/PHI(+/-)) and VIP/PHI(-/-) animals. We found decreasing proportions of rhythmic cells in SCN slices from VIP/PHI(+/+) ( approximately 91%, n = 23) through VIP/PHI(-/+) ( approximately 71%, n = 28) to VIP/PHI(-/-) mice (62%; n = 37) and a parallel trend toward decreasing amplitude in the remaining rhythmic cells. SCN neurons from VIP/PHI(-/-) mice exhibited a broad range in the period and phasing of electrical rhythms, concordant with the known alterations in their behavioral rhythms. Further, treatment of VIP/PHI(-/-) slices with a VPAC(2) receptor antagonist significantly reduced the proportion of oscillating neurons, suggesting that VPAC(2) receptors still become activated in the SCN of these mice. The results establish that VIP is important for appropriate periodicity and phasing of SCN neuronal rhythms and suggest that residual VPAC(2) receptor signaling promotes rhythmicity in adult VIP/PHI(-/-) mice.

The role of vasoactive intestinal polypeptide (VIP) receptors on excitable properties of neurones in slices acutely prepared from the suprachiasmatic nuclei (SCN) of wild-type (WT) and VPAC(2)-receptor-deficient (Vipr2 ( -/- )) mice was studied under voltage clamp with the use of patch-clamp recording in the whole-cell configuration. The resting membrane potential in Vipr2 ( -/- ) neurones was significantly hyperpolarised as compared to WT cells (-60+/-7 vs -72+/-6 mV, p<0.01). Bath application of 100 nM VIP or the VPAC(2) receptor agonist RO 25-1553 triggered a slow inward current in a subpopulation of WT SCN neurones; the VIP-induced current was not affected by slice incubation with 25 microM of bicuculline but disappeared completely when the cells were dialysed with CsCl-containing/K(+)-free solution. Application of VIP or RO 25-1553 to neurones from Vipr2 ( -/- ) mice did not induce currents in all cells tested. Incubation of WT slices with 100 nM VIP or RO 25-1553 resulted in inhibition of fast tetrodotoxin-sensitive sodium currents and delayed rectifier K(+) currents in most of the cells tested. This effect was completely absent in cells from Vipr2 ( -/- ) mice. We postulate that VIP receptors control excitability of SCN neurones at the postsynaptic level by direct modulation of membrane potential via inhibition of K(+) channels and by tonic inhibition of sodium and potassium voltage-gated currents.

Circadian rhythms of animals are reset by exposure to light as well as dark; however, although the parameters of photic entrainment are well characterized, the phase-shifting actions of dark pulses are poorly understood. Here, we determined the tonic and phasic effects of short (0.25 h), moderate (3 h), and long (6-9 h) duration dark pulses on the wheel-running rhythms of hamsters in constant light. Moderate- and long-duration dark pulses phase dependently reset behavioral rhythms, and the magnitude of these phase shifts increased as a function of the duration of the dark pulse. In contrast, the 0.25-h dark pulses failed to evoke consistent effects at any circadian phase tested. Interestingly, moderate- and long-dark pulses elevated locomotor activity (wheel-running) on the day of treatment. This induced wheel-running was highly correlated with phase shift magnitude when the pulse was given during the subjective day. This, together with the finding that animals pulsed during the subjective day are behaviorally active throughout the pulse, suggests that both locomotor activity and behavioral activation play an important role in the phase-resetting actions of dark pulses. We also found that the robustness of the wheel-running rhythm was weakened, and the amount of wheel-running decreased on the days after exposure to dark pulses; these effects were dependent on pulse duration. In summary, similarly to light, the resetting actions of dark pulses are dependent on both circadian phase and stimulus duration. However, dark pulses appear more complex stimuli, with both photic and nonphotic resetting properties.

Ageing alters fundamental aspects of circadian rhythmicity in mammals; the effects include reduced rhythm amplitude and alterations in period length and in entrainment to the light/dark cycle. Such changes may reflect disruptions in cellular function within the suprachiasmatic nucleus (SCN), the site of the predominant circadian pacemaker. In the SCN, vasoactive intestinal peptide (VIP)-synthesizing neurones receive various inputs, including retinohypothalamic projections containing pituitary adenylate cyclase activating peptide (PACAP). SCN VIP cells establish connections with local neurones and send efferents beyond the nucleus. Considerable evidence implicates VIP and PACAP in circadian rhythm maintenance and/or entrainment to photic Zeitgebers. These actions involve members of a distinct family of receptors; mRNAs for two such receptors, VPAC2 and PAC1, are present in the SCN. This study used isotopic in situ hybridization to examine the effects of ageing on expression of mRNAs for VIP, VPAC2 and PAC1 in the SCN of male rats under a 12 : 12 h light/dark cycle. Analysis of film autoradiographs from young adult (2-3 months) or aged (19-20 months) rats, at eight time points across the light/dark cycle, showed loss of diurnal rhythmicity and reduced levels for VIP mRNA in the aged group. A diurnal rhythm of VPAC2 receptor mRNA was present in both groups, but its levels were reduced in the aged rats. There were no differences between the two groups for PAC1 receptor mRNA expression. The present results indicate that ageing reduces VIP and VPAC2 receptor mRNA and eliminates diurnal expression of VIP mRNA within the SCN of aged male rats.

Biological oscillations with an endogenous period of near 24 h (circadian rhythms) are generated by the master circadian pacemaker or clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus. This clock is synchronised to recurring environmental signals conveyed by selective neural pathways. One of the main chemical constituents of SCN neurones is vasoactive intestinal polypeptide (VIP). Such neurones are retinorecipient and activated by light. Exogenous application of VIP resets the SCN circadian clock in a light-like manner, both in vivo and in vitro. These resetting actions appear to be mediated through the VPAC2 receptor (a type of receptor for VIP). Unexpectedly, genetically ablating expression of the VPAC2 receptor renders the circadian clock arrhythmic at the molecular, neurophysiological and behavioural levels. These findings indicate that this intrinsic neuropeptide acting through the VPAC2 receptor participates in both resetting to light and maintenance of ongoing rhythmicity of the SCN.

The suprachiasmatic nuclei (SCN) of the hypothalamus house the main circadian pacemaker in mammals. Vasoactive intestinal polypeptide (VIP) is the most abundant neuropeptide in the SCN and has been shown to phase-shift the electrical activity rhythm of SCN cells in vitro. However, the effects of VIP on the cellular activity of rat SCN neurones are unknown. In this study, we examined the acute effects of VIP on the extracellularly recorded spontaneous firing rate of SCN neurones in an in-vitro hypothalamic slice preparation. Furthermore, with the use of receptor-selective agonists and antagonists, we determined which receptors might mediate the effects of VIP in the SCN. Approximately 50% of cells responded to VIP; the main type of response was suppression in firing rate, although a few cells were activated. Suppression responses to VIP were mimicked by the VPAC(2) receptor agonist Ro 25-1553 and blocked by the selective VPAC(2) receptor antagonist PG 99-465. The PAC(1) receptor agonist maxadilan evoked responses from 40% of SCN cells, and activations to this agonist were not altered by PG 99-465. Responses to VIP were not blocked by antagonists to ionotropic glutamate receptors, but the duration of suppression was modulated by the GABA(A) receptor antagonist bicuculline. Our data indicate that VIP alters the electrical activity of rat SCN neurones in vitro, via both VPAC(2) and PAC(1) receptors.

The circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) receives photic information directly via the retinohypothalamic tract (RHT) and indirectly from retinally innervated cells in the thalamic intergeniculate leaflet (IGL) that project to the SCN. Using standard immunohistochemical methods, we examined the presence and distribution of substance P (SP) and the neurokinin-1 receptor (NK-1) in the SCN and IGL of rat and determined whether the patterns of immunostaining generalized to the SCN and IGL of Syrian hamster, Siberian hamster, and mouse. Terminals immunoreactive for SP were sparse within the SCN of Siberian and Syrian hamsters and mouse but were intense in the ventral, retinally innervated portion of the rat SCN. Immunostaining for the NK-1 receptor was mainly absent from the SCN of hamster and mouse. In contrast, a plexus of NK-1-ir cells and processes that was in close proximity to SP-ir terminals was found in the ventral SCN of the rat. Substance P-ir terminals were observed in the IGL of all four species, as were NK-1-ir cells and fibres. Double-labelled IGL sections of hamster or rat revealed SP-ir terminals in close apposition to NK-1-immunostained cells and/or fibres. These data indicate that SP could be a neurotransmitter of the RHT in rat, but not in hamster or in mouse, and they highlight potential species differences in the role of SP within the SCN circadian pacemaker. Such species differences do not appear to exist at the level of the IGL, where SP-ir and NK-1-ir were similar in all species studied., (Copyright 2001 Wiley-Liss, Inc.)

The orexins are recently discovered neuropeptides that reportedly play a role in energy homeostasis, in addition to various other physiological processes. The synthesis of orexin A undergoes diurnal variation in certain areas of the brain, while the mutation of the orexin receptor 2 gene has been implicated in canine narcolepsy. Since the circadian pacemaker in the suprachiasmatic nucleus modulates the sleep/wake cycle, there is a putative role for orexins in the mammalian circadian system. In this study, immunohistochemical techniques were used to determine the distribution of orexin A in the structures of the hypothalamus and thalamus of Syrian and Siberian hamsters. In both species, the pattern of immunoreactivity was similar. Cells immunoreactive for orexin A were noted in the lateral hypothalamic area. Immunoreactive varicose orexin A fibres were found throughout the hypothalamus. The suprachiasmatic nucleus possessed little or no immunoreactive orexin A fibres in its core, but had fibres at its periphery. The thalamus of both species contained comparatively few immunoreactive fibres, which were mainly localised around the midline. The thalamic intergeniculate leaflet contained a plexus of immunoreactive orexin A fibres throughout its rostro-caudal extent. Three areas of the brainstem, the dorsal and median raphe nuclei and the locus coeruleus, were also investigated owing to their relevance to the circadian system and all were found to contain immunoreactive orexin A fibres. The presence of orexin A-immunoreactive fibres in the neural architecture of the mammalian circadian system suggests an important role for orexin A in circadian timekeeping processes.

Pituitary adenylate cyclase activating polypeptide (PACAP38) is a putative neurochemical of the main retinal input to the mammalian circadian pacemaker housed in the suprachiasmatic nucleus (SCN). We assessed the phase-resetting effects of microinjection of PACAP38 into the SCN region on hamster wheel-running rhythms. When administered during the middle of the subjective day, PACAP38 evoked large but transient phase advances ( approximately 60 min), that were followed by small, steady-state phase delays. During the early subjective night, PACAP38 elicited small to moderate phase delays without any detectable concentration-dependence. Late in the subjective night, PACAP38 had no significant effects. Saline microinjection had no effect at any phase tested. These findings show that PACAP38 has small to moderate effects on the phase of the hamster SCN circadian pacemaker, including significant phase delays early in the subjective night.

In mammals, the principal circadian pacemaker is housed in the hypothalamic suprachiasmatic nuclei (SCN). The SCN exhibit high levels of vasoactive intestinal polypeptide (VIP) immunoreactivity and two of the three VIP receptors, VPAC(2) and PAC(1), are found in the rat SCN. However, the role of VIP in the SCN remains unclear. In this study, we examined the phase-resetting actions of VIP and selective VIP receptor agonists on the electrical activity rhythm of rat SCN neurons in vitro. Application of VIP during the subjective day did not shift the peak in the firing rate rhythm. However, VIP treatment during the early or late subjective night evoked a small phase delay or a large phase advance, respectively. The phase-advancing effect of VIP was reproduced by the novel VPAC(2) receptor agonist RO 25-1553, but not by pituitary adenylate cyclase-activating peptide (a potent PAC(1) receptor agonist), or by [K15,R16,L27]VIP(1-7)/GRF(8-27), a novel, selective VPAC(1) receptor agonist. These data show that VIP phase-dependently phase-resets the rodent SCN pacemaker in vitro, presumably via the VPAC(2) receptor. As the pattern of phase-shifting evoked by VIP and RO 25-1553 resembles the phase-resetting actions of light on rodent behavioural rhythms, these data support a role for VIP and the VPAC(2) receptor in photic entrainment of the rodent circadian pacemaker.

The neuropeptide neurotensin and two classes of its receptors, the neurotensin receptor-1 and 2, are present in the suprachiasmatic nucleus of the mammalian hypothalamus. The suprachiasmatic nucleus houses the mammalian central circadian pacemaker, but the effects of neurotensin on cellular activity in this circadian pacemaker are unknown. In this study, we examined the effects of neurotensin on the spontaneous discharge rate of rat SCN cells in an in vitro slice preparation. Neurotensin (1-10 microM) increased cell firing rate in approximately 50% of cells tested, while approximately 10% of suprachiasmatic cells tested showed a decrease in firing rate in response to neurotensin. These effects of neurotensin were not altered by the GABA receptor antagonist bicuculline (20 microM) or the glutamate receptor antagonists, D-aminophosphopentanoic acid (50 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). The neurotensin receptor selective antagonists SR48692 and SR142948a (10 microM) failed to antagonise neurotensin responses in the majority of cells examined. Compounds that function as agonists selective for the neurotensin-receptor subtypes 1 and 2, JMV-510 and JMV-431 respectively, elicited neurotensin-like responses in approximately 90% of cells tested. Six out of seven cells tested responded to both JMV-510 and JMV-431. Neuropeptide Y (100nM) treatment of suprachiasmatic nucleus slices was found to elicit profound suppression of neuronal firing rate. Co-application of neurotensin with neuropeptide Y significantly (P<0.05) reduced the duration of the response, as compared to that elicited with neuropeptide Y alone. Together, these results demonstrate for the first time the actions of neurotensin in the suprachiasmatic nucleus and raise the possibility that this neuropeptide may play a role in modulating circadian pacemaker function.

The existence of biological clocks was once the subject of considerable debate. However, research has confirmed that a 24 h (circadian) clock in our brain has a pronounced influence on when we sleep, eat, drink, etc. Disruptions in clock function compromise normal daily patterns of hormone release, sleep and body temperature, and can affect mental and physical well-being. The recent identification of clock genes and the neural pathways to the clock will facilitate the development of effective strategies to treat disorders of circadian function.

The main mammalian circadian pacemaker is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Gastrin-releasing peptide (GRP) and its receptor (BB(2)) are synthesized by rodent SCN neurons, but the role of GRP in circadian rhythm processes is unknown. In this study, we examined the phase-resetting actions of GRP on the electrical activity rhythms of hamster and rat SCN neurons in vitro. In both rat and hamster SCN slices, GRP treatment during the day did not alter the time of peak SCN firing. In contrast, GRP application early in the subjective night phase-delayed, whereas similar treatment later in the subjective night phase-advanced the firing rate rhythm in rat and hamster SCN slices. These phase shifts were completely blocked by the selective BB(2) receptor antagonist, [d-Phe(6), Des-Met(14)]-bombesin 6-14 ethylamide. We also investigated the temporal changes in the expression of genes for the BB(1) and BB(2) receptors in the rat SCN using a quantitative competitive RT-PCR protocol. The expression of the genes for both receptors was easily detected, but their expression did not vary over the diurnal cycle. These data show that GRP phase-dependently phase resets the rodent SCN circadian pacemaker in vitro apparently via the BB(2) receptor. Because this pattern of phase shifting resembles that of light on rodent behavioral rhythms, these results support the contention that GRP participates in the photic entrainment of the rodent SCN circadian pacemaker.

Melatonin acts on specific receptors in the suprachiasmatic nuclei (SCN) to phase-dependently regulate the phase of the circadian clock. How the gating of melatonin's effect is restricted to particular times of day is not known, but may be related to temporal differences in receptor availability. In the present study, we used a competitive reverse transcription-polymerase chain reaction (RT-PCR) method to determine if the expression of mt(1) melatonin receptor subtype mRNA in rat SCN varied across the 12:12 light-dark (LD) cycle. Measurement of core body temperature using radiotelemetry confirmed that the male Wistar rats used exhibited a robust diurnal rhythm. mt(1) receptor mRNA was readily detected in reduced SCN slices at all times of day. However, there was no significant variation in the amount of mt(1) mRNA with time of day. Expression of MT(2) melatonin receptor subtype mRNA in reduced SCN slices was confirmed by nested PCR. These results indicate that changes in the level of mt(1) mRNA do not underlie the diurnal and/or circadian variation in the response of the SCN circadian clock to the phase-resetting effects of melatonin.

A receptor for pituitary adenylate cyclase activating polypeptide (PACAP), denoted as PAC1, is expressed in the suprachiasmatic nuclei (SCN). Since the circadian clock demonstrates phase-dependent sensitivity to PACAP, we have used in situ hybridization histochemistry to examine whether PAC1 mRNA is differentially expressed in the rat SCN across the 24-h cycle. There was a significant variation in PAC1 mRNA within the SCN and supraoptic nuclei during the light-dark cycle and in constant darkness, with peaks at the middle of both the real and subjective day and night; no significant variation was observed in the cingulate cortex. The results suggest that the phase-dependent actions of PACAP on the clock may involve phase-specific changes in the availability of PAC1 receptors within the SCN., (Copyright 1998 Published by Elsevier Science B.V.)

Circadian functions of the suprachiasmatic nuclei (SCN) are influenced by cyclic AMP (cAMP). Adenylyl cyclase type II (AC-II) is a cAMP-generating enzyme which, in the context of activation by Gsalpha, is further stimulated by protein kinase C or G protein betagamma subunits. Using in situ hybridization we have found a biphasic variation in AC-II mRNA within the rat SCN during the light-dark cycle (peaks at Zeitgeber time 6 and 18) and also in constant darkness (peaks at circadian time 2 and 14). The cingulate cortex showed no such variation. These findings suggest that circadian changes in AC-II expression may be pertinent to the rhythmic functions of the SCN., (Copyright 1998 Elsevier Science B.V.)

We investigated the effects of neuropeptide Y and related analogues on the extracellularly recorded spontaneous firing rate activity of adult Syrian hamster suprachiasmatic nucleus neurones in vitro. Sixty-seven neurones were tested with neuropeptide Y: 45% were suppressed, 4% were activated, and the remaining 51% were unresponsive. These responses were not blocked by the GABA receptor antagonist bicuculline, indicating that neuropeptide Y-evoked responses did not appear to be dependent on GABA(A) receptor activation. We tested the effects of the neuropeptide Y Y1 receptor agonist [Leu31, Pro34]neuropeptide Y and the neuropeptide Y Y2 receptor agonist neuropeptide Y-(13-36) on nine cells suppressed by neuropeptide Y in order to determine the receptor subtype(s) mediating the effects of neuropeptide Y. Four of nine cells were suppressed by [Leu31, Pro34]neuropeptide Y only, one of nine was suppressed by neuropeptide Y-(13-36) only, two of nine were suppressed by both compounds, while the remaining two cells did not respond to either compound. These data suggest that neuropeptide Y can modulate suprachiasmatic nucleus function directly, without recruitment of GABA(A) interneurones. Further, our results indicate that neuropeptide Y may act on more than one receptor subtype within the adult hamster suprachiasmatic nucleus.

Previous studies have suggested a role for histamine in mediating effects of light on rodent circadian systems. We combined behavioural and neuroanatomical studies to analyze the potential role of histamine in circadian rhythm regulation of Syrian hamsters. The effects of histamine and the selective H2 receptor agonist dimaprit on free-running locomotor activity rhythms were monitored. In addition, histamine projections were mapped immunohistochemically. Histamine (0.5 microl of 1 mM) microinjected into the region of the suprachiasmatic nucleus induced very small delay phase-shifts, which were significantly greater than those elicited by vehicle at circadian time (CT) 18. No other significant differences were observed. Dimaprit (0.5 microl of 1 mM) microinjected into the region of the suprachiasmatic nucleus at CT 6 and CT 18, induced very small phase-shifts which were significantly different from the effects of vehicle, but did not resemble those of light. Numerous cell bodies immunopositive for histamine were found in the tuberomammillary nucleus of the posterior hypothalamus, while histamine immunoreactive fibres were seen in the periventricular nucleus and paraventricular nucleus of the anterior hypothalamus. The suprachiasmatic nuclei were largely devoid of immunostaining. Finally, preliminary electrophysiological studies demonstrated that ionophoretic application of histamine and the H2 receptor agonist dimaprit elicit inconsistent and weak responses in the hamster suprachiasmatic nucleus in vitro. The results presented here show that microinjections of HA or dimaprit into the SCN region do not mimic the effects of light on hamster circadian rhythms, and the SCN of the Syrian hamster is almost devoid of histaminergic fibres. These studies suggest that histamine does not play a prominent role in circadian rhythm regulation in this species.

The suprachiasmatic nuclei (SCN) in the hypothalamus function as the primary circadian pacemaker. A receptor for vasoactive intestinal peptide (VIP), denoted as VIP2, is abundantly expressed in the SCN. Since the rodent circadian clock demonstrates phase-dependent sensitivity to exogenous VIP, we investigated the possibility that VIP2 receptor mRNA is differentially expressed in the SCN across the 24 h cycle. To establish whether VIP2 receptor mRNA levels change across the 12:12 h light-dark (LD) cycle (lights on designated as Zeitgeber time (ZT)O), rats were killed at ZT 0, 2, 6, 10, 12, 14, 18 and 22. To determine if variation in this mRNA occurs in the absence of LD entrainment cues, lights were not turned on at the time of transition from dark to light (designated as CT O); the animals in this group were killed in constant darkness (DD) at CT 0, 2, 6, 10, 12, 14, 18 and 22. In situ hybridization histochemistry indicated no variations in VIP2 receptor mRNA in the cingulate cortex under either LD or DD conditions. There was, however, significant variation in the expression of VIP2 receptor mRNA within the SCN during the LD cycle, with one peak at ZT 6 and at ZT 22. A comparable biphasic pattern of mRNA expression was observed in DD animals with peaks at CT 10 and another at CT 22. The results suggest that the phase-dependent actions of VIP on the clock may involve phase-specific changes in the availability of VIP2 receptor within the SCN.

Glutamate is thought to mediate the effects of light on the circadian pacemaker contained in the suprachiasmatic nucleus. Glutamate can reset this pacemaker both in vivo and in vitro while glutamate antagonists can reduce photically induced phase shifts in activity rhythms and c-fos expression in the suprachiasmatic nucleus. Most behavioural and gene expression experiments investigating circadian rhythms use hamsters, but the majority of the anatomical data on the presence and distribution of selected glutamate receptor subunits in the suprachiasmatic nucleus has been collected from rat. In the present study, we examined the distribution of ionotropic glutamate receptor subunits in the hamster suprachiasmatic nucleus using mono- and polyclonal antibodies directed against these subunits. In addition, we examined the distribution of immunostaining for these subunits in a second structure of the mammalian circadian system, the intergeniculate leaflet of the thalamus since it also is thought to receive glutamatergic input from the retina and is important in the entrainment of circadian rhythms. The results indicated that all of the subunits investigated (GluR1, GluR2/3, GluR4, GluR5/6/7, and NMDAR1) were present in the suprachiasmatic nucleus and that all but GluR4 were present in the intergeniculate leaflet. Each of the subunits investigated had a unique pattern of distribution and intensity of staining. The distribution of immunoreactivity for these subunits in the hamster suprachiasmatic nucleus and intergeniculate leaflet differed from that reported in the rat. The presence of these subunits in the suprachiasmatic nucleus and intergeniculate leaflet implies the presence of functional NMDA and non-NMDA receptors in these structures that may have a role in photic entrainment of the circadian pacemaker.

The suprachiasmatic nucleus (SCN) receives a direct retinal projection, which in rats includes substance P (SP)-immunoreactive retinal ganglion cells. While SP has been shown to have neurophysiological effects on SCN cells in Syrian hamsters and rats, it is not known what effects SP in the SCN has on circadian rhythms in hamsters. We examined this question using male Syrian hamsters that were implanted with cannulas aimed at the SCN region and maintained in constant dim red lighting conditions. Hamsters received 0.5 microl microinjections of saline or SP (500 pmol in saline) at a variety of circadian times (CT). Saline injections had little or no phase-shifting effects at any phase tested. SP had no significant effects at CT4-8, 16-20, or 20-24, but did cause small phase delays of -23.7 +/- 7 min (mean +/- sem) at CT12-16. In order to examine the dose-response relations of this effect, hamsters were also microinjected with 50 and 2500 pmol of SP at CT12-16. Both the 50 and 2500 pmol doses induced very small phase delays (-14.2 +/- 7 min and -18.2 +/- 5 min, respectively), indicating no obvious dose dependence within this range. These results do not suggest that SP alone in the SCN mimics light effects on circadian rhythms or is a key neurotransmitter involved in photic entrainment. It remains to be determined whether SP interacts with other transmitters in the SCN to modulate their effects on rhythm phase.

Pituitary adenylate cyclase activating polypeptide (PACAP) is found in two forms of 27 and 38 amino acids (PACAP-27 and PACAP-38 respectively) in the mammalian central nervous system. Using antibodies to these two forms of PACAP, we examined the distribution of PACAP immunoreactivity in the rat hypothalamus and a number of extrahypothalamic areas. The patterns of immunostaining for PACAP-27 and PACAP-38 were similar: prominent terminal labelling was present in the retrochiasmatic area, median eminence, and posterior periventricular nucleus of the hypothalamus as well as the bed nucleus of the stria terminalis and amygdaloid complex. After colchicine treatment, immunopositive cell bodies were found in the preoptic region of the periventricular zone of the hypothalamus, the suprachiasmatic and paraventricular hypothalamic nuclei, neural structures adjacent to the median eminence (including the retrochiasmatic area, arcuate nucleus, ventromedial hypothalamus, and tuber cinereum), and the lateral mammillary and supramammillary nuclei. In all these areas, immunolabelling appeared specific since it was abolished by preabsorption of primary antisera with the appropriate PACAP peptide. However, the number of immunopositive cells in the suprachiasmatic nucleus was also reduced by preabsorption of PACAP-27/38 antisera with vasoactive intestinal polypeptide, suggesting that a subpopulation of cells in the suprachiasmatic nucleus express a peptide which has significant sequence homology with both PACAP-27/38 and vasoactive intestinal polypeptide. The distribution of PACAP immunoreactivity throughout the hypothalamus, bed nucleus of the stria terminalis, and amygdala suggests the involvement of PACAP in a number of processes including limbic, autonomic, and neuroendocrine functions as well as regulation of the circadian pacemaker.

We studied the influence on circadian rhythms of peptides that have been reported to be colocalized in suprachiasmatic nucleus (SCN) neurons. Gastrin-releasing peptide (GRP1-27), peptide histidine isoleucine (PHI), and vasoactive intestinal polypeptide (VIP) were microinjected into the suprachiasmatic nucleus (SCN) region of Syrian hamsters free running under three different constant lighting conditions. All peptide injections caused phase-dependent phase shifts of hamster locomotor activity rhythms which were unaffected by constant lighting conditions. GRP1-27 (150 pmol) caused large phase delays when injected at circadian times (CT) 12-16, modest phase advances when administered at CT20-24, and few shifts during the subjective day. Injections of saline vehicle at any of these phases caused only very small phase shifts. Phase delays induced by GRP1-27 at CT12-14 were dose dependent, unrelated to injection volume (at a constant dose), and attenuated by pretreatment with the BN/GRP-preferring receptor antagonist BIM 26226. VIP (150 pmol) caused moderate phase delays at CT12-14 and moderate phase advances at CT20-24. PHI (150 pmol) caused moderate phase delays at CT12-14 only. Coadministration of 150 pmol of GRP1-27, PHI, and VIP in an equimolar neuropeptide cocktail (50 pmol of each peptide) caused phase delays at CT12-14 and phase advances at CT20-24 which did not differ from those induced by 150 pmol of GRP1-27 alone at these phases. The shifts induced by 150 pmol of the peptide cocktail were smaller than the sum of the shifts induced by 50 pmol doses of each peptide administered separately at those phases. Since the phase-delaying effects of the cocktail were weaker than the summed effects of the component 50 pmol doses of the peptides, these data demonstrate a lack of synergism among the effects of these peptides. Since GRP1-27 (150 pmol) evoked shifts similar in magnitude to those of the cocktail, there is no evidence that these apparently colocalized neuropeptides must interact to exert maximal effects on the circadian pacemaker.

ionophoretic ejection of substance P (SP) activated 31% and suppressed 9% of hamster suprachiasmatic nucleus (SCN) cells in vitro. Hamster SCN cells did not demonstrate variation in sensitivity to SP across the circadian phases tested. SP modulated the response of 47% of hamster SCN cells to the excitatory amino acid (EAA) agonists glutamate and N-methyl-D-aspartate (NMDA). The results indicate that SP can alter both the spontaneous and EAA-evoked firing rate characteristics of hamster SCN neurons.

Ionophoresis of the smaller bombesin-like peptides (gastrin releasing peptide [GRP]-(18-27), neuromedin B, and bombesin) evoked responses from 30-60% of hamster suprachiasmatic nucleus cells recorded in a hypothalamic slice preparation, depending on the circadian phase. We also demonstrated for the first time that the putative bombesin-like peptide receptor antagonists [D-F5,D-Phe6,D-Ala11]bombesin-(6-13)methyl ester (BIM 26226) and [D-Phe6,Des-Met14]bombesin-(6-14)ethyl amide can be applied ionophoretically to block physiological responses to bombesin-like peptides. Together with earlier findings, these results show that bombesin-like peptides administered by several methods can potently alter the firing rates of hamster suprachiasmatic nucleus neurons in vitro. These results indicate that bombesin-like peptides affect suprachiasmatic nucleus cells and could play a role in modulating suprachiasmatic nucleus-mediated circadian rhythm entrainment.

The suprachiasmatic nuclei (SCN) of the rodent hypothalamus function as a light entrainable circadian pacemaker. The SCN contain moderate to high concentrations of a number of neuropeptides including peptides showing structural homology with the amphibian derived tetradecapeptide, bombesin (BN), called bombesin-like peptides (BNLPs). BNLPs include the 27 amino acid peptide, gastrin releasing peptide (GRP1-27), a smaller decapeptide (GRP18-27) and another decapeptide with less structural homology, neuromedin B (NmB). Immunoreactivity for BN and receptors for BNLPs have been demonstrated in the region of the rat SCN receiving photic input. We studied the effects of local pressure ejections of BNLPs dissolved in saline/1% bovine serum albumin (BSA) vehicle on the extracellularly recorded firing rates of Syrian hamster SCM neurons in a hypothalamic slice preparation. In one study, an ejecting electrode containing BN (10(-8) to 10(-4) M) was positioned 20 to 60 microm from a recording electrode. Of 74 cells tested with BN, 50 (67.6%) showed significant increases in firing rate, while 3 of 29 cells (15.8%) tested with vehicle ejections were activated. In a second study, a single electrode was used for both recordings and pressure ejections. Of 48 cells tested, BN (10(-6) to 10(-4) M) activated 30 (62.5%) and suppressed firing in 4 (8.3%). Of 208 cells tested with GRP1-27 (10(-9) to 10(-4) M), 105 (50.5%) were activated and 2 (1.0%) were suppressed. The percentage of cells responding increased with the concentration of GRP1027 used in the electrode. No circadian variation in responsiveness to GRP1-27 was detected. GRP18-27 (5 x 10(-5) to 10(-4) M) activated 10 out of 18 cells tested (55.6%), while NmB (10(-4) M) activated 2 out of 30 cells tested (6.7%) and vehicle ejections activated 1 out of 36 cells tested (2.8%). GRP1-27, GRP18-27 and BN, the BNLPs showing the greatest degree of structural homology, activate approximately 50% of SCN cells, apparently via the BN/GRP-preferring receptor subtype, and may play a role in photic entrainment.

Long-term consequences of neonatal blockade of bombesin (BN) receptors were examined in the present study. Rat pups were injected twice daily with [D-Phe6, phi Leu13-Cpa14]BN(6-14), a BN receptor antagonist, at either high (10 mg/kg; HD group) or low (5 mg/kg; LD group) doses from postnatal day 1 through 8. Their behavioral responses to a variety of conditions were compared to those of rats neonatally injected with saline (SAL group) or animals handled but not injected during infancy (UNT group). Adult HD rats entered and spent more time on the open arms of the elevated plus maze than LD, SAL, or UNT animals. Under the conditions of a water deprivation schedule, neither central nor peripheral injections of BN differentiated the neonatally pretreated groups as determined by measures of grooming, feeding, and drinking behaviors. These results indicate that at the dosage regimen employed, neonatal injections of [D-Phe6, phi Leu13-Cpa14]BN(6-14) had little effect on adult sensitivity to BN, but that such treatments could alter activity on the elevated plus maze through as yet unknown mechanisms.

Rhythms of spontaneous firing rate and of responsiveness to pressure ejection of melatonin were recorded from neurons in the Syrian hamster suprachiasmatic nuclei (SCN) in a slice preparation. In animals taken from light-dark cycles (LD 14:10), SCN cells had high firing rates during the projected day and lower rates during the projected night. The proportion of melatonin-suppressed cells (35% overall) was also high during the day and fell during the night, while melatonin activated approximately 23% of cells at all phases. To assess the source of the melatonin-responsiveness rhythm, hamsters were exposed for approximately 48 h to constant illumination (LL) to suppress melatonin secretion. LL exposure before slice preparation altered both firing-rate and melatonin-responsiveness rhythms. Firing rates failed to show a morning peak and remained at low levels, with no indication of daily rhythmicity. Melatonin responsiveness also failed to show the usual rhythm and even tended to rise at night. Overall melatonin responsiveness rose after LL exposure so that 50% of cells were suppressed and 21% activated. LL exposure also increased the proportions of cells which showed regular baseline firing rates. Control studies indicated that pressure artifacts did not account for either suppression or activation by melatonin, while the composition of the saline vehicle appeared to be responsible for the activations recorded. The results indicate that brief LL exposure alters SCN sensitivity to melatonin and SCN rhythmicity in Syrian hamsters, perhaps as a result of the loss of the daily melatonin secretion rhythm. Physiological melatonin patterns may have important effects on the rodent circadian pacemaker.

We review recent studies in our laboratory that have investigated the neural mechanisms underlying photic entrainment of the mammalian circadian system. The results from studies of extracellular single-unit recordings and of photic induction of Fos-like immunoreactivity (Fos-lir) indicate that excitatory amino acid (EAA) transmission, and particularly activation of the N-methyl-D-aspartate (NMDA) receptor subtype, is important for conveying photic information to suprachiasmatic nucleus (SCN) cells. We have also found that a subregion of the SCN still shows Fos-lir after blockade of EAA receptors, and we have evidence suggesting that these cells are innervated by a distinct subdivision of the retinal projection to the SCN. In addition, we have found that photic responses of cells in the intergeniculate leaflet (which projects to the SCN) and of SCN cells are modulated by serotonin (5-HT) via a receptor that resembles the 5-HT1A subtype.

This paper examined the grooming effects of central injections of the tetradecapeptide bombesin (BN) in the infant rat. Anaesthetized rat pups of 1-20 days of age (n = 7/dose/age) were injected intracerebroventricularly (i.c.v.) with either BN (0.01-1.0 micrograms) or saline (control condition) and placed in heated test cages. Following recovery from the anaesthetic, the subsequent behaviour displayed by the pups was videotaped for the next 60 min. These videotaped responses were then scored for a variety of grooming and other behaviours. Pups of all ages (1-20 days) groomed in response to BN with the 1-day-old pups the least and 20-day-olds the most sensitive to BN. In the 10-day-olds, scratching, in an immature, non-contact form, was elicited by BN. This was replaced by mature, contact type scratching in the sequence of grooming behaviour at 20 days of age. Scratching activities appeared to form a subsystem connected to but nonetheless separate from washing behaviours and this was particularly distinguishable at 20 days of age. Changes in BN-induced grooming appeared to reflect the maturation of the motor capabilities of the developing rat. In conclusion, these results indicate that BN binding sites in the developing rat central nervous system (CNS) are pharmacologically functional from an early stage in ontogeny when there is little or no measurable amount of the peptide itself present in the CNS.

Subcutaneous (sc) administration of the tetradecapeptide bombesin (BN) (1-10 mg/kg) elicited grooming in rat pups of 1-10 days of age and the magnitude of this response decreased as a function of age. The form of grooming induced was qualitatively different from that seen following central injection of BN to adult rats. Subchronic neonatal exposure to BN (5 or 10 mg/kg; sc, twice daily for the first 8 postnatal days) had no effect on subsequent adult behavior displayed under mildly stressful or novel conditions, in the open field or in an elevated plus maze. However, both saline and the high dose of BN (10 mg/kg) pretreatments increased adult sensitivity to central BN (0.1 micrograms; icv) as compared to noninjected but neonatally handled controls or those rats neonatally pretreated with the lower dose of BN (5 mg/kg). This was best demonstrated by increases in scratching activity at the 0.1-micrograms dose of icv BN. Neonatal pretreatments had no effect on later adult sensitivity to BN injected intraperitoneally (ip). These data indicate that BN receptors in the rat central nervous system are pharmacologically functional from an early stage in ontogeny. Systems utilizing BN-like peptides are, to a degree, plastic early in ontogeny and altered adult sensitivity to BN icv can be achieved via subchronic exposure to BN during infancy. Endogenous BN-based mechanisms did not appear to play a role in the development and/or expression of behavior(s) elicited under mildly stressful or novel conditions.

Central administration of bombesin elicits excessive grooming and locomotor activity in rats. This grooming activity is one characterised by vigorous scratching of the face, nape and body flanks. Pretreatment with the D1 receptor antagonist SCH 23390 inhibited the expression of bombesin-induced activity with grooming being more inhibited than locomotion. Blockade of D2 receptors with eticlopride significantly attenuated the behavioral responses to bombesin. When SCH 23390 and eticlopride were administered concurrently, it was apparent that D1 blockade had a greater effect on grooming and D2 blockade a larger effect on locomotion. Stimulation of D1 receptors by SKF 38393 elicited non-stereotyped locomotor activity and a form of grooming behavior characterised by vigorous washing of the face and ventral body surfaces. Co-administration of bombesin and SKF 38393 resulted in a form of grooming which resembled that elicited by SKF 38393 alone. The specific D2 agonist PPHT elicited a form of locomotion characterised by a downward oriented head posture and slow ambulatory activity around the cage perimeter. Co-administration of PPHT and bombesin resulted in a complete suppression of bombesin-induced behaviors and was largely indistinguishable from activity observed under PPHT alone conditions. These data implicate both D1 and D2 receptor based mechanisms in the modulation/mediation of the behavioral effects of bombesin. Part of the bombesin-induced behavioral effects may be explained by (indirect) activation of (a) dopamine system(s).

1. The objective of the current study was to assess the effect of concurrent D-1 and D-2 receptor blockade on bombesin (BN)-induced grooming and locomotion on the premise that dopamine could be mediating the behavioural effects of BN. 2. Rats implanted with ventricular cannulae were pretretreated systemically with SCH 23390 and/or eticlopride (ETIC) followed by BN (0.5 ug/3 ul; i.c.v.). Grooming and locomotory activity were assessed for 60 min. 3. Individually, SCH 23390 (0.033 mg/kg) and ETIC (0.05 mg/kg) significantly inhibited BN-elicited grooming but not locomotion. Combining ETIC (0.05-0.1 mg/kg) with the fixed dose of SCH 23390 (0.033 mg/kg) significantly inhibited both behaviours in a dose dependent manner with a greater effect on locomotion. Adding SCH 23390 (0.033-0.075 mg/kg) to ETIC (0.05 mg/kg) inhibited both behaviours in a dose dependent manner with a more pronounced effect on grooming. At the combination dosages utilized, SCH 23390 and/or ETIC failed to significantly alter baseline behaviours in control conditions. It is thus concluded that both dopamine receptor subtypes are involved in the expression of BN-induced behaviours with D-1 appearing to have a greater involvement with grooming and D-2 locomotion.