Paola C. Yannielli

Ghrelin Effects on the Circadian System of Mice

The orexigenic peptide ghrelin stimulates both food intake and growth hormone release and is synthesized in the stomach and in hypothalamic areas involved in feeding control. The suprachiasmatic nuclei of the hypothalamus (SCN) control most circadian rhythms, although there is evidence that some oscillators, such as food-entrainable oscillators, can drive activity rhythms even after SCN ablation. Ghrelin levels exhibit a circadian rhythm and closely follow feeding schedules, making this peptide a putative candidate for food-related entraining signals. We examined the response of the SCN to ghrelin treatments in vitro, by means of electrophysiological and bioluminescence recordings, and in vivo, by assessing effects on the phase of locomotor activity rhythms. Ghrelin applied at circadian time 6 in vitro to cultured SCN slices induced an ∼3 h phase advance. In addition, ghrelin phase advanced the rhythm of PER2::LUC (Period2::Luciferase) expression in cultured SCN explants from mPer2Luc transgenic mice. In vivo, intraperitoneal administration of ghrelin or a synthetic analog, growth hormone-releasing protein-6 (GHRP-6), to ad libitum fed animals failed to alter circadian phase. When injected after 30 h of food deprivation, GHRP-6 induced a phase advance compared with saline-injected animals. These results indicate that ghrelin may play a role in the circadian system by exerting a direct action on the SCN and that the system as a whole may become sensitive to ghrelin and other feeding-related neuropeptides under conditions of food restriction.

Effects of neonatal clomipramine treatment on locomotor activity, anxiety-related behavior and serotonin turnover in Syrian hamsters.

Day-night differences in locomotor and anxiety-related behavior and brain serotonin metabolism were examined in adult Syrian hamsters that received clomipramine (15 mg/kg) or vehicle from day 8 to day 21 of life. Locomotor activity was significantly greater at the beginning of scotophase (20.00 h) than at noon (12.00 h) and it was highest in hamsters treated with clomipramine at both examined times. Significant day-night differences in anxiety-related behavior, as measured in a plus-maze paradigm, were found in saline-treated hamsters only, with higher values at night. Clomipramine-treated hamsters exhibited augmented 5-hydroxyindoleacetic acid/serotonin ratio in hypothalamus and midbrain raphe, while serotonin content decreased in frontal cortex and hypothalamic areas. The results indicate that neonatal clomipramine treatment produces a long-lasting change in locomotion and anxiety-related behavior, as well as reduces brain serotonin content in hamsters.

Daily rhythms in spontaneous and diazepam-induced anxiolysis in Syrian hamsters.

The diurnal variations in spontaneous and diazepam-induced anxiolysis and exploratory behavior were examined in Syrian hamsters in a plus-maze paradigm. The administration of diazepam or flunitrazepam augmented the percentage of time spent in the open arms, the percentage of entries to the open arms, and the number of crosses to both arms, whereas ethyl-beta-carboline injection decreased them. These three behavioral parameters showed significant daily variations, with the maxima being found at night (2400-0400 h). Flumazenil (5 mg/kg) injected at 0400 h decreased significantly the percentage of time spent in open arms and of entries to the open arms, without affecting significantly the total number of entries to both arms. Day-night differences in anxiety-related behavior persisted in hamsters kept under constant darkness for 3 days. Diazepam (0.5 mg/kg) increased the time spent in the open arms at 1600 and 2000 h only, and augmented the percent of entries to the open arms at 2000 h only. The total number of entries to both arms was augmented significantly by diazepam at all time intervals tested, except for 0400 h. The results indicate that Syrian hamsters exhibited significant diurnal changes in anxiolysis-related behavior in the plus-maze paradigm.

Daily variations in GABA receptor function in Syrian hamster cerebral cortex

The existence of diurnal changes in postsynaptic expression of gamma-aminobutyric acid (GABA) type A receptors was assessed in cerebral cortex of Syrian hamsters by measuring [3H]GABA binding and the influx of 36Cl- in synaptoneurosomes. A diurnal variation in dissociation constant of [3H]GABA binding to cerebral cortex membranes, and the absence of diurnal differences in maximal number of sites, were found. When the nycthemeral changes in muscimol-stimulated 36Cl- uptake by cortical synaptoneurosomes were assessed, a maximum occurred late at night (i.e. 0400 h). At 1600 h, micromolar concentrations of flunitrazepam potentiated significantly the influx of chloride induced by muscimol, while at 0400 h flunitrazepam did not exert any significant effect on 36Cl- uptake. The results indicate that postsynaptic type A GABAergic activity peaked at nocturnal hours in the cerebral cortex of Syrian hamsters.

Neurochemistry of Mammalian Entrainment: Signal Transduction Pathways in the Suprachiasmatic Nuclei

Neurochemical events leading to photic entrainment of circadian rhythms are reviewed. This entrainment pathway includes the retinohypothalamic tract and a glutamate-NMDA receptor (among others) interaction in the suprachiasmatic nuclei (SCN). The model we propose involves an increase in intracellular calcium levels and the activation of specific proteins in SCN neurons, including the Ca 2+ /calmodulin dependent protein kinase (CaM kinase) and phosphatase (calcineurin), other kinases (such as the cGMP-dependent protein kinase, PKG) and enzymes (nitric oxide synthase, NOS), which in turn activate specific transcription factors, in a cascade of events that is controlled both by light and by the circadian clock itself. Although the step at which the circadian gating of this process occurs is unknown, we propose it occurs downstream of glutamate binding, calcium entrance, and NOS activation. We conclude that a promising way of studying the function of the circadian pacemaker is to investigate the signal transduction pathway(s) leading to changes in the SCN, including the biochemical activity of its components.

Neonatal clomipramine treatment of Syrian hamsters: effect on the circadian system.

The circadian behavior of male Syrian hamsters injected with the serotonin/norepinephrine reuptake inhibitor clomipramine (15 mg/kg from postnatal days 8 to 21) was examined. Clomipramine treatment significantly augmented mean activity values of wheel running rhythm, as well as delayed its acrophase. After a 6-h phase advance of the light-dark cycle, reentrainment of clomipramine-treated hamsters took significantly longer than controls. Clomipramine-treated hamsters exhibited a shorter circadian period than controls in constant light conditions, but no differences were found in constant darkness. Light pulses applied at late subjective night to clomipramine-treated hamsters caused significantly reduced phase advances as compared to controls, while no differences were found in phase delay magnitudes when light pulses were applied during early subjective night. Administration of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) at circadian time 8 significantly advanced the onset of activity to a greater extent in clomipramine-treated hamsters than in controls. The results indicate that neonatal clomipramine treatment of hamsters causes long-lasting changes in the circadian system, by increasing activity levels and by partially inhibiting light-evoked responses. An enhancement of a non-photic, serotonergic-induced response was also unveiled.

Aging attenuates diurnal variation in hamster locomotion, anxiolysis and GABA turnover

A decrease in amplitude of wheel running circadian rhythms was found in old (18 month old) Syrian hamsters, as compared with young (3 month old.) animals. In a plus-maze paradigm, amplitude of variation of anxiety-related variables (2400 vs. 1600 h) was significantly impaired in aged hamsters. Cerebral cortex, hypothalamic and pineal gamma-aminobutyric acid (GABA) turnover was higher at night, amplitude of variation being significantly smaller in aged hamsters. The results further support the existence of impaired amplitude of circadian rhythms in aged Syrian hamsters.

Electrodermal Response in Nonglabrous Skin of Freely Moving Rats: Mediation by the Sympathetic Nervous System and Evaluation in an Animal Model of Depression

Electrodermal responses in the facial region of freely moving rats were recorded bilaterally. After a nociceptive stimulus (ammonia vapor exposure), the response (a transient negative potential followed by a longer-lasting positive potential) attained a similar amplitude on both sides. Surgical sympathetic denervation of facial skin by ipsilateral superior cervical ganglionectomy (SCGx) significantly decreased the electrodermal response. When an inferior cervical ganglionectomy was performed in addition to SCGx, a further decrease in electrodermal response was observed. Pretreatment of unilaterally SCGx rats with atropine blunted the electrical response in the control side to levels similar to those found in the SCGx side. Treatment with phenoxybenzamine or propranolol was ineffective. Skin potential responses were measured in adult rats administered with clomipramine from the 8th to the 21st day of life and exhibiting a long-lasting syndrome resembling human depression. Clomipramine-injected rats developed larger skin potential responses to sound stimulation than controls while responses to ammonia vapor were similar in both groups, as well as the habituation rate after repetitive exposure to ammonia vapor. The results indicate that some of the altered electrodermal responses found in depressed patients are detectable in the clomipramine animal model of endogenous depression.