Haloperidol alters circadian clock gene product expression in the mouse brain

Abstract

Objectives. Circadian rhythms are patterns in behavioural and physiological measures that recur on a daily basis and are driven by an endogenous circadian timekeeping system whose molecular machinery consists of a number of clock genes. The typical anti-psychotic haloperidol has previously been shown to induce significant deficiencies in circadian timing in patients. In this study we examined the impact of haloperidol treatment on molecular components of the circadian clock in the mouse brain. Methods. We examined how haloperidol treatment, either acute (both at day and night) or chronically over 14 days, alters the expression of three clock gene protein products (PER1, PER2, BMAL1) across the mouse brain by means of immunohistochemistry. Results. Chronic haloperidol treatment significantly decreases the expression levels of PER1 in a number of brain areas, including the hippocampus, the prefrontal and cingulate cerebral cortex and the paraventricular nucleus of the hypothalamus. PER2 expression was only altered in the dentate gyrus and the CA3, and BMAL1 expression was only altered in the paraventricular nucleus of the hypothalamus. Conclusion. These data indicate that haloperidol has the potential to alter circadian rhythms via modulation of circadian clock gene expression.     

Recurring circadian disruption alters circadian clock sensitivity to resetting

A single phase advance of the light:dark (LD) cycle can temporarily disrupt synchrony of neural circadian rhythms within the suprachiasmatic nucleus (SCN) and between the SCN and peripheral tissues. Compounding this, modern life can involve repeated disruptive light conditions. To model chronic disruption to the circadian system, we exposed male mice to more than a month of a 20‐hr light cycle (LD10:10), which mice typically cannot entrain to. Control animals were housed under LD12:12. We measured locomotor activity and body temperature rhythms in vivo, and rhythms of PER2::LUC bioluminescence in SCN and peripheral tissues ex vivo. Unexpectedly, we discovered strong effects of the time of dissection on circadian phase of PER2::LUC bioluminescent rhythms, which varied across tissues. White adipose tissue was strongly reset by dissection, while thymus phase appeared independent of dissection timing. Prior light exposure impacted the SCN, resulting in strong resetting of SCN phase by dissection for mice housed under LD10:10, and weak phase shifts by time of dissection in SCN from control LD12:12 mice. These findings suggest that exposure to circadian disruption may desynchronize SCN neurons, increasing network sensitivity to perturbations. We propose that tissues with a weakened circadian network, such as the SCN under disruptive light conditions, or with little to no coupling, for example, some peripheral tissues, will show increased resetting effects. In particular, exposure to light at inconsistent circadian times on a recurring weekly basis disrupts circadian rhythms and alters sensitivity of the SCN neural pacemaker to dissection time.     

The axon-guidance roundabout gene alters the pace of the Drosophila circadian clock

Great efforts have been directed to the dissection of the cell-autonomous circadian oscillator in Drosophila . However, less information is available regarding how this oscillator controls rhythmic rest–activity cycles. We have identified a viable allele of roundabout , robo ^hy , where the period of locomotor activity is shortened. From its role in axon-pathfinding, we anticipated developmental defects in clock-relevant structures. However, robo ^hy produced minor defects in the architecture of the circuits essential for rhythmic behaviour. ROBO's presence within the circadian circuit strengthened the possibility of a novel role for ROBO at this postdevelopmental stage. Genetic interactions between pdf ⁰¹ and robo ^hy suggest that ROBO could alter the communication within different clusters of the circadian network, thus impinging on two basic properties, periodicity and/or rhythmicity. Early translocation of PERIOD to the nucleus in robo ^hy pacemaker cells indicated that shortened activity rhythms were derived from alterations in the molecular oscillator. Herein we present a mutation affecting clock function associated with a molecule involved in circuit assembly and maintenance.     

Serotonergic activation potentiates light resetting of the main circadian clock and alters clock gene expression in a diurnal rodent

The main circadian clock, localized in the suprachiasmatic nuclei (SCN) in mammals, can be synchronized by light and non-photic factors such as serotonergic cues. In nocturnal rodents, injections during the subjective day of the 5-HT1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) or its positive enantiomer, induce behavioral phase-advances in correlation with decreased expression of two clock genes, Per1/2. In addition, 8-OH-DPAT and the selective serotonin reuptake inhibitor fluoxetine reduce light-induced phase-shifts during the subjective night. Beside the chronobiotic effects of serotonin, changes of serotonergic activity in humans have been involved in mood disorders, that are often associated with alterations in circadian rhythmicity. To get insights into the circadian role of serotonin in diurnal species, we investigated its modulation of the SCN in Arvicanthis ansorgei housed in constant darkness. In striking contrast to nocturnal rodents, daily serotonin content in Arvicanthis SCN peaked during daytime while the sensitivity window of its SCN to (+)8-OH-DPAT occurred essentially during the subjective night. Moreover, fluoxetine produced behavioral phase-advances at circadian time (CT) 0 and CT12. Expression of Per1/2, Rev-erbalpha/beta and Roralpha/beta in the SCN was not modified after fluoxetine or (+)8-OH-DPAT injection. Furthermore, both treatments enhanced light-induced phase-advances and delays. Light responses of Per1 and Rorbeta expression at CT0 and those of Per2 and Rev-erbalpha at CT12 were markedly altered by serotonergic activation. The present findings demonstrate that the serotonergic modulation of the SCN clock appears to differ between nocturnal species and the diurnal Arvicanthis. The potentiating effects of fluoxetine on light resetting in a diurnal rodent may be clinically relevant.     

Activation of the maternal immune system alters cerebellar development in the offspring

A common pathological finding in autism is a localized deficit in Purkinje cells (PCs). Cerebellar abnormalities have also been reported in schizophrenia. Using a mouse model that exploits a known risk factor for these disorders, maternal infection, we asked if the offspring of pregnant mice given a mid-gestation respiratory infection have cerebellar pathology resembling that seen in these disorders. We also tested the effects of maternal immune activation in the absence of virus by injection of the synthetic dsRNA, poly(I:C). We infected pregnant mice with influenza on embryonic day 9.5 (E9.5), or injected poly(I:C) i.p. on E12.5, and assessed the linear density of PCs in the cerebellum of adult or postnatal day 11 (P11) offspring. To study granule cell migration, we also injected BrdU on P11. Adult offspring of influenza- or poly(I:C)-exposed mice display a localized deficit in PCs in lobule VII of the cerebellum, as do P11 offspring. Coincident with this are heterotopic PCs, as well as delayed migration of granule cells in lobules VI and VII. The cerebellar pathology observed in the offspring of influenza- or poly(I:C)-exposed mice is strikingly similar to that observed in autism. The poly(I:C) findings indicate that deficits are likely caused by the activation of the maternal immune system. Finally, our data suggest that cerebellar abnormalities occur during embryonic development, and may be an early deficit in autism and schizophrenia.     

Epidemiology of the human circadian clock

Humans show large inter-individual differences in organising their behaviour within the 24-h day-this is most obvious in their preferred timing of sleep and wakefulness. Sleep and wake times show a near-Gaussian distribution in a given population, with extreme early types waking up when extreme late types fall asleep. This distribution is predominantly based on differences in an individuals' circadian clock. The relationship between the circadian system and different "chronotypes" is formally and genetically well established in experimental studies in organisms ranging from unicells to mammals. To investigate the epidemiology of the human circadian clock, we developed a simple questionnaire (Munich ChronoType Questionnaire, MCTQ) to assess chronotype. So far, more than 55,000 people have completed the MCTQ, which has been validated with respect to the Horne-?stberg morningness-eveningness questionnaire (MEQ), objective measures of activity and rest (sleep-logs and actimetry), and physiological parameters. As a result of this large survey, we established an algorithm which optimises chronotype assessment by incorporating the information on timing of sleep and wakefulness for both work and free days. The timing and duration of sleep are generally independent. However, when the two are analysed separately for work and free days, sleep duration strongly depends on chronotype. In addition, chronotype is both age- and sex-dependent.     

MDMA alters the response of the circadian clock to a photic and non-photic stimulus

3,4-Methylenedioxymethamphetamine (MDMA or 'Ecstasy') is a widely used recreational drug that damages serotonin 5-HT neurons in animals and possibly humans. Published literature has shown that the serotonergic system is involved in photic and non-photic phase shifting of the circadian clock, which is located in the suprachiasmatic nuclei. Despite the dense innervation of the circadian system by 5-HT and the known selective neurotoxicity of MDMA, little is known about the effects of MDMA on the circadian oscillator. This study investigated whether repeated exposure to the serotonin neurotoxin MDMA alters the behavioural response of the Syrian hamster to phase shift to the serotonin 5-HT1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT). This agonist was administered under an Aschoff Type I (CT8) and Aschoff Type II (ZT8) paradigm (5 mg/kg) and was given before and after treatment with MDMA (10, 15 and 20 mg/kg administered on successive days). Pre-treatment with MDMA significantly attenuated phase shifts to 8-OH-DPAT. We also tested the ability of the clock to phase shift to a photic stimulus after treatment with MDMA. A 15-min light pulse (mean lux 125 at CT14 or ZT14) was administered before and after treatment with MDMA. Phase shifts to a photic stimulus were significantly attenuated by pre-treatment with MDMA. Our study demonstrates that repeated exposure to MDMA may alter the ability of the circadian clock to phase shift to a photic and non-photic stimulus in the hamster. Disruption of circadian function has been linked with a variety of clinical conditions such as sleep disorders, mood, concentration difficulties and depression, consequently outlining the potential dangers of long-term ecstasy use.     

Magnesium sulfate treatment alters fetal cerebellar gene expression responses to hypoxia

Prenatal perturbation of brain circulation and oxygenation is a leading cause of perinatal brain damage affecting about 0.3–0.9% of births. Hypoxia-ischemia (HI) in preterm human infants at gestational week 23–32 results in neurodevelopmental abnormalities in childhood, presenting as learning disability, seizure activity, motor impairment and in the most severe cases, death. Here, we examined the potential of MgSO₄ treatment, prior to foetal hypoxia, to attenuate hypoxia induced damage in a murine model of maternal hypoxia. We studied the time course of maternal hypoxia and MgSO₄ pre-treatment effects on cerebellar tissue by means of DNA microarray analyses. Mild hypoxia induced minor expression changes in most genes. However, there were 5 gene sets which were down-regulated by maternal hypoxia. MgSO₄ pre-treatment abrogated these decreases in gene. A cell cycle gene set which responded immediately (2 h) to hypoxia, showed a delayed response (24 h) when MgSO₄ pre-treatment was given. Similar proportions of cell death were observed in all groups before P7, where combined hypoxia and MgSO₄ treatment increased cell death in the internal granule layer. There were a higher number of BrdU positive cells at the end of hypoxic episodes and a down-regulation of Reelin signaling, compared to control. MgSO₄ pre-treatment prevented the enhancement of cell proliferation due to hypoxia and increased Reelin levels.     

The vascularization of the human cerebellar cortex

The blood vessels of the cerebellar cortex were studied by two methods of injection: india-ink and low viscosity resin (Mercox). The study is divided into two parts: (a) Pial vessels; few in number at the cerebellar surface, the pial vessels are quite dense, forming vascular laminae, within the sulci. Pial vessels do not frequently anastomose. (b) Intracortical vessels closely resemble those of the cerebral cortex. They may be divided into three categories: short, middle and long. There exist three vascular layers within the cerebellar cortex: superficial, middle and deep. The superficial vascular layer is situated within the molecular layer, the middle within the Purkinje cell layer, and the deep within the granular layer. It is therefore possible to establish a correlation between these vascular layers and the cortical cytoarchitecture. We accorded particular attention to the vascularization of the Purkinje cells. Arteries parallel to the cerebellar surface (parallel arteries) are in close relationship to the Purkinje cells whereas capillaries and veins are scarce. It is highly probable that the majority of Purkinje cells obtain nutritive elements by these parallel arteries. Possible implications in physiology and pathology are subsequently discussed.     

The microvasculature of the human cerebellar meninges

The vascular architecture of the human cerebellar meninges was investigated. The surface meninges were poor in vasculature. In the sulci, the meninges were highly vascular but had few capillaries. The venous blood vessels gave long side branches at right angles to the parent vessels in a cruciform pattern, running horizontally along the cerebellar sulci. They were situated at the origin of the secondary or tertiary sulci. Anastomoses between these horizontal branches gave a crosshatched appearance. Short branches often extended to the bases of the sulci, terminating in T-shaped bifurcations with numerous tiny branches, like the roots of a tree. The arteries ran perpendicular to venous branches which were parallel to each other exclusively along the sagittal plane. These arteries bifurcated to straddle the horizontally running veins at the origin of the secondary or tertiary sulci. They gave off many small branches like teeth of a fork from each artery in the secondary or tertiary sulci after they bifurcated to straddle the venous branches and penetrated the cerebellar cortex at the bases of sulci. These fork-like ramifications in the bases of the sulci were most likely responsible for the ready development of pronounced ischemic state. They might also play an important role in the occurrence of ischemic damage at the bases of sulci in cases of severe generalized ischemia.     

Developmental methylmercury administration alters cerebellar PSA-NCAM expression and Golgi sialyltransferase activity.

Brain dysmorphogenesis and persistent psychomotor disturbances are hallmarks of developmental methylmercury (MeHg) exposure, but the molecular mechanisms underlying these effects are poorly understood. Targets of developmental MeHg exposure include neural cell adhesion molecules (NCAMs), sialoglycoconjugate molecules whose proper temporal and spatial expression is important at all stages of neurodevelopment and especially during synaptic structuring. To investigate the effects of MeHg on the temporal expression of NCAM during development, rat pups were dosed with 7.0 mg/kg MeHgCl (s.c.) on alternate days from postnatal days (PNDs) 3-13 and killed on PNDs 15, 30 and 60. Brain MeHg concentrations were determined in a subset of litters injected with CH(3)203Hg. Expression of NCAM180 protein and of NCAM180 polysialylation was examined in whole cerebellum homogenates, cerebellar synaptosomes and isolated cerebellar growth cones by Western blotting and immunocytochemical staining. NCAM sialyltransferase activity was assayed in preparations of purified Golgi apparatus from the cerebelli of rats treated in vivo, or following in vitro incubation with 0, 1, 2.5, or 7.5 microM MeHg for 2 h. At PND15, no change in NCAM180 protein expression was observed in any cerebellar preparations, but decreased polysialylation of NCAM180 was observed in cerebellar whole homogenates, synaptosomes and isolated growth cones. At PND30, both NCAM180 protein expression and NCAM180 polysialylation were elevated in whole homogenate preparations but not in synaptosomes. NCAM180 expression in MeHg-treated rats was similar to controls at PND60, 47 days after the last methylmercury administration. In vivo studies of cerebellar Golgi sialyltransferase activity revealed significant reductions in PND15 MeHg-treated rats as compared to controls, but no changes in sialyltransferase activity in PND30 and PND60 animals. In vitro experiments revealed decreasing sensitivity of cerebellar sialyltransferases to MeHg as the developmental age of the rat increased. Toxic perturbation of the developmentally-regulated expression of polysialylated NCAM during brain formation may disturb the stereotypic formation of neuronal contacts and could contribute to the behavioral and morphological disturbances observed following MeHg poisoning.     

Acute ethanol alters the firing pattern and glutamate response of cerebellar Purkinje neurons in culture

Modified explant cultures derived from the cortical region of fetal rat cerebellum, and extracellular recording techniques were used to examine the sensitivity and response or cerebellar neurons, isolated from extracellular afferent input, to acute ethanol (EtOH) exposure. Recordings were made from Purkinje neurons (PNs) and granule cells maintained in culture for several weeks, with the emphasis on the PN. Both the PNs and granule cells exhibited spontaneous activity inculture, but, unlike the PNs, not all of the granule cells were spontaneously active. The majority of PNs studied exhibited a high frequency, regular simple spike firing pattern, previously shown to be endogenously generated by voltage-sensitive mechanisms intrinsic to the PN. The granule cells exhibited slow, irregular patterns of activity. EtOH at doses as low as 22 mM (100 mg%), a concentration that reflects blood levels during EtOH intoxication, altered the spontaneous activity of both neuronal types, demonstrating that EtOH has direct actions on cerebellar neurons. In the PNs, acute EtOH (20–80 mM) produced an increase in the regularity of the spontaneous activity and either a transient increase or no change in firing rate. Acute EtOH also significantly altered the response of PNs to the excitatory transmitter glutamate. In the granule cells, acute EtOH altered firing pattern with small and variable effects on firing rate. These data demonstrate that there are multiple sites of EtOH action in the cerebellum and that changes in PN activity with acute EtOH exposure may occur via direct actions on the PN and indirect actions via synaptically connected cerebellar neurons. The demonstration of EtOH-sensitive sites intrinsic to the cerebellum suggests that EtOH actions at these sites contribute to alterations in PN activity that occur in vivo after acute EhOH exposure.     

Eradication of cerebellar granular cells alters thedevelopmental expression of TRK receptors in the ratinferior olive

Granule cells which relay the mossy fibre afferent system to the cerebellar cortex aregenerated postnatally in mammals. In their absence, the climbing fibres, i.e. the second afferentsystem to the cerebellum originating in the inferior olivary nucleus, remain in an immature stage,and substantial elimination of redundant synapses they establish on the Purkinje cells does notoccur in the rat between day five (P5) and day fifteen (P15). It is generally assumed that synapseelimination is partly regulated by electrical activity which modulates the competition amongafferent fibres for the uptake of a limited amount of trophic factors released by the target. Theneurotrophins, whose expression is developmentally regulated in the cerebellum, especially ingranule cells, could be this retrograde signal. Using RT-PCR, we studied the expression of theirtrk receptors in the inferior olivary nucleus of developing and adult rats, and its alteration aftereradication of the granule cell precursors by X-irradiation on P5. From P0 to P90, the amount oftrkA mRNA is low and remains stable in control rats ; the high levels of trkB and C mRNAsdetected at P0 markedly decrease in parallel from P5 and reach their minimal values at P15,when the process of synapse elimination is completed in the cerebellum. X-irradiation of thecerebellum decreases the level of expression of the three trks, but a transient upregulation of trkCoccurs at P10. The downregulation of trkB and C expression in the inferior olivary nucleus,contemporary with the altered expression of neurotrophins in the cerebellum, suggest that NT-3and/or BDNF/NT-4/5 could be involved in the remodelling of olivocerebellar relationshipsduring development. In addition, the transient overexpression of trkC after granule cellseradication is consistent with a paracrin effect exerted on the olivary cells by granule cells releaseof NT-3, at the time when the climbing fibres invest the growing Purkinje cell dendrites in themolecular layer.     

Acute alcohol alters the excitability of cerebellar Purkinje neurons and hippocampal neurons in culture.

Acute exposure to ethanol at 22 and 44 mM concentrations altered several features of the current-evoked voltage responses of cerebellar Purkinje neurons and hippocampal neurons studied in culture model systems. Whole cell current clamp techniques were used. At 22 mM, ethanol depressed current-evoked spiking in the hippocampal neurons but enhanced the current-evoked spiking in the Purkinje neurons. In both neuronal types, 44 mM ethanol depressed spiking, the amplitude of the afterhyperpolarization generated at the termination of a current pulse and the amplitude of the off-response generated at the termination of a hyperpolarizing pulse. Ethanol had little or no effect on resting membrane potential or the passive membrane properties measured near resting level in either neuronal type. Some changes in the current-voltage curves were observed at more depolarized or hyperpolarized potentials in both neuronal types. In the Purkinje neurons, where spontaneous activity was a prominent feature of some recordings, exposure to ethanol reduced the frequency of the spontaneous events. These results indicate that acute exposure to ethanol at intoxicating doses alters the membrane excitability of these two CNS neuronal types. The ethanol induced changes in neuronal excitability presumably contribute to the changes in firing properties observed in extracellular recordings from these neuronal types in vivo and the behavioral effects observed during alcohol intoxication in animal models.     

Acute melatonin treatment alters dendritic morphology and circadian clock gene expression in the hippocampus of Siberian Hamsters

In the hippocampus of Siberian hamsters, dendritic length and dendritic complexity increase in the CA1 region whereas dendritic spine density decreases in the dentate gyrus region at night. However, the underlying mechanism of the diurnal rhythmicity in hippocampal neuronal remodeling is unknown. In mammals, most daily rhythms in physiology and behaviors are regulated by a network of circadian clocks. The central clock, located in the hypothalamus, controls melatonin secretion at night and melatonin modifies peripheral clocks by altering expression of circadian clock genes. In this study, we examined the effects of acute melatonin treatment on the circadian clock system as well as on morphological changes of hippocampal neurons. Male Siberian hamsters were injected with melatonin in the afternoon; 4 h later, mRNA levels of hypothalamic and hippocampal circadian clock genes and hippocampal neuron dendritic morphology were assessed. In the hypothalamus, melatonin treatment did not alter Period1 and Bmal1 expression. However, melatonin treatment increased both Period1 and Bmal1 expression in the hippocampus, suggesting that melatonin affected molecular oscillations in the hippocampus. Melatonin treatment also induced rapid remodeling of hippocampal neurons; melatonin increased apical dendritic length and dendritic complexity in the CA1 region and reduced the dendritic spine density in the dentate gyrus region. These data suggest that structural changes in hippocampal neurons are regulated by a circadian clock and that melatonin functions as a nighttime signal to coordinate the diurnal rhythm in neuronal remodeling. © 2014 Wiley Periodicals, Inc.     

Chronic exposure to alcohol during development alters the membrane properties of cerebellar Purkinje neurons in culture

The active and passive membrane properties of developing Purkinje neurons in control cultures and cultures chronically treated with 20 or 40 mM ethanol for 1 or 2 weeks were examined using whole-cell current-clamp techniques. The membrane properties were characterized by the features of the voltage responses evoked by intracellular current injection of a series of depolarizing and hyperpolarizing current pulses. Analysis of these responses and background spontaneous activity showed several differences between the control and ethanol-treated Purkinje neurons: (1) membrane input resistance was significantly larger in the ethanol-treated neurons; (2) the percentage of neurons exhibiting immature firing patterns was significantly higher in the ethanol-treated neurons; (3) the afterhyperpolarization following a current-evoked train of action potentials was significantly larger in the ethanol-treated neurons; (4) spontaneous activity (synaptic potentials and synaptically evoked spike events) was significantly reduced in neurons treated with 40 mM ethanol for 1 week; spontaneous activity in neurons treated with 20 mM ethanol for 1 or 2 weeks was similar to that observed in the control group. These differences indicate that ethanol exposure during development directly alters the physiological properties of this CNS neuronal type. These neuronal actions of ethanol may contribute to the behavioral deficits observed in animals models of fetal alcohol syndrome. Similar target sites of ethanol action are likely to be present in the human CNS neurons and may be involved in human fetal alcohol syndrome.     

Phase advancing the human circadian clock with blue-enriched polychromatic light

BackgroundPrevious studies have shown that the human circadian system is maximally sensitive to short-wavelength (blue) light. Whether this sensitivity can be utilized to increase the size of phase shifts using light boxes and protocols designed for practical settings is not known. We assessed whether bright polychromatic lamps enriched in the short-wavelength portion of the visible light spectrum could produce larger phase advances than standard bright white lamps.MethodsTwenty-two healthy young adults received either a bright white or bright blue-enriched 2-h phase advancing light pulse upon awakening on each of four treatment days. On the first treatment day the light pulse began 8 h after the dim light melatonin onset (DLMO), on average about 2 h before baseline wake time. On each subsequent day, light treatment began 1 h earlier than the previous day, and the sleep schedule was also advanced.ResultsPhase advances of the DLMO for the blue-enriched (92 ± 78 min, n = 12) and white groups (76 ± 45 min, n = 10) were not significantly different.ConclusionBright blue-enriched polychromatic light is no more effective than standard bright light therapy for phase advancing circadian rhythms at commonly used therapeutic light levels.