Victoria M. Smith

Physiological responses of the circadian clock to acute light exposure at night

Circadian rhythms in physiological, endocrine and metabolic functioning are controlled by a neural clock located in the suprachiasmatic nucleus (SCN). This structure is endogenously rhythmic and the phase of this rhythm can be reset by light information from the eye. A key feature of the SCN is that while it is a small structure containing on the order of about 20,000 cells, it is amazingly heterogeneous. It is likely that anatomical heterogeneity reflects an underlying functional heterogeneity. In this review, we examine the physiological responses of cells in the SCN to light stimuli that reset the phase of the circadian clock, highlighting where possible the spatial pattern of such responses. Increases in intracellular calcium are an important signal in response to light, and this increase triggers many biochemical cascades that mediate responses to light. Furthermore, only some cells in the SCN are actually endogenously rhythmic, and these cells likely do not receive strong direct input from the retina. Therefore, this review also considers how light information is conveyed from the retinorecipient cells to the endogenously rhythmic cells that track circadian phase. A number of neuropeptides, including vasoactive intestinal polypeptide, gastrin-releasing peptide and substance P, may be particularly important in relaying such signals, but other neurochemicals such as GABA and nitric oxide may participate as well. A thorough understanding of the intracellular and intercellular responses to light, as well as the spatial arrangements of such responses may help identify important pharmacological targets for therapeutic interventions to treat sleep and circadian disorders.

Altered photic and non-photic phase shifts in 5-HT1A receptor knockout mice

The mammalian circadian clock located in the suprachiasmatic nucleus (SCN) is thought to be modulated by 5-HT. 5-HT is though to inhibit photic phase shifts by inhibiting the release of glutamate from retinal terminals, as well as by decreasing the responsiveness of retinorecipient cells in the SCN. Furthermore, there is also evidence that 5-HT may underlie, in part, non-photic phase shifts of the circadian system. Understanding the mechanism by which 5-HT accomplishes these goals is complicated by the wide variety of 5-HT receptors found in the SCN, the heterogeneous organization of both the circadian clock and the location of 5-HT receptors, and by a lack of sufficiently selective pharmacological agents for the 5-HT receptors of interest. Genetically modified animals engineered to lack a specific 5-HT receptor present an alternative avenue of investigation to understand how 5-HT regulates the circadian system. Here we examine behavioral and molecular responses to both photic and non-photic stimuli in mice lacking the 5-HT(1A) receptor. When compared with wild-type controls, these mice exhibit larger phase advances to a short late-night light pulse and larger delays to long 12 h light pulses that span the whole subjective night. Fos and mPer1 expression in the retinorecipient SCN is significantly attenuated following late-night light pulses in the 5-HT(1A) knockout animals. Finally, non-photic phase shifts to (+/-)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) are lost in the knockout animals, while attenuation of the phase shift to the long light pulse due to rebound activity following a wheel lock is unaffected. These findings suggest that the 5-HT(1A) receptor plays an inhibitory role in behavioral phase shifts, a facilitatory role in light-induced gene expression, a necessary role in phase shifts to 8-OH-DPAT, and is not necessary for activity-induced phase advances that oppose photic phase shifts to long light pulses.

A Single Generalized Seizure Alters the Amplitude, but Not Phase, of the Circadian Activity Rhythm of the Hamster

People with epilepsy exhibit high rates of sleep disturbances. In many cases, these sleep disruptions appear to be related to the occurrence of the seizures themselves. Changes in sleep structure may reflect underlying changes in the circadian clock, as circadian rhythms of locomotor activity, body temperature, and hormone release are disrupted following a seizure. The present study was designed to determine if a single generalized seizure could alter the phase and waveform of the circadian rhythm of wheel‐running behavior in the Syrian hamster. Animals were housed in constant darkness, and were administered either a sham treatment or a maximal electroconvulsive shock at one of three time‐points: 6 h before activity onset, 1 h after activity onset, or 6 h after activity onset. Seizures at all of these phases did not significantly affect the phase of the circadian activity rhythm. The circadian locomotor activity levels were significantly attenuated following seizures at all three phases. This attenuation was prominent over the 24 h following the seizure, and was also evident over the three post‐seizure days. These data suggest that while seizures do not affect phase, they may alter the amplitude of the circadian clock. Because the amplitude of the circadian clock affects sleep quality, these findings suggest one mechanism by which persistent seizures may decrease the quality of sleep in patients with epilepsy.

The cholinergic forebrain arousal system acts directly on the circadian pacemaker

Significance Sleep and wake states are regulated by a variety of mechanisms. One such important system is the circadian clock, which provides temporal structure to sleep and wake. Conversely, changes in behavioral state can influence the phase of the circadian clock. Here we demonstrate that the level of wakefulness is critical for arousal to reset circadian clock phase. We then show that treatments that produce arousal and reset the circadian clock activate the basal forebrain. Finally, we demonstrate that cholinergic input from the basal forebrain is both necessary and sufficient for eliciting this arousal-induced resetting of the circadian clock. These results establish a functional link between the major forebrain arousal center and the circadian system. Sleep and wake states are regulated by a variety of mechanisms. One such important system is the circadian clock, which provides temporal structure to sleep and wake. Conversely, changes in behavioral state, such as sleep deprivation (SD) or arousal, can phase shift the circadian clock. Here we demonstrate that the level of wakefulness is critical for this arousal resetting of the circadian clock. Specifically, drowsy animals with significant power in the 7- to 9-Hz band of their EEGs do not exhibit phase shifts in response to a mild SD procedure. We then show that treatments that both produce arousal and reset the phase of circadian clock activate (i.e., induce Fos expression in) the basal forebrain. Many of the activated cells are cholinergic. Using retrograde tract tracing, we demonstrate that cholinergic cells activated by these arousal procedures project to the circadian clock in the suprachiasmatic nuclei (SCN). We then demonstrate that arousal-induced phase shifts are blocked when animals are pretreated with atropine injections to the SCN, demonstrating that cholinergic activity at the SCN is necessary for arousal-induced phase shifting. Finally, we demonstrate that electrical stimulation of the substantia innominata of the basal forebrain phase shifts the circadian clock in a manner similar to that of our arousal procedures and that these shifts are also blocked by infusions of atropine to the SCN. These results establish a functional link between the major forebrain arousal center and the circadian system.

Serotonergic potentiation of photic phase shifts: examination of receptor contributions and early biochemical/molecular events.

The 5-HT mixed agonist/antagonist 1-(2-methoxyphenyl)4-[4-(phthalimido)butyl]-piperazine hydrobromide (NAN-190) has been shown to greatly potentiate photic phase shifts in hamsters. The mechanism of this potentiation has yet to be determined. NAN-190 is believed to act primarily through the 5-HT(1A) receptor, but also binds to several other receptors, making it uncertain as to which receptor underlies its potentiation of photic phase shifts. Also uncertain are the intracellular changes in the suprachiasmatic nucleus (SCN) which are associated with such enhanced phase shifting. Here we examine the role of the 5-HT(1A) receptor as well as the physiological underpinnings, in terms of both gene expression and biochemical activation, in the behavioral responses to photic stimuli following pretreatment with NAN-190. Administration of NAN-190 to wildtype mice significantly potentiated late subjective night photic phase shifts, while mice lacking the 5-HT(1A) receptor (knockouts) exhibited an attenuated behavioral response to light when pretreated with NAN-190. In wildtype mice, the protein product of the immediate-early gene c-fos, induced following photic stimulation, was found to be significantly decreased with NAN-190 pretreatment. Similarly, the levels of phosphorylated CREB protein, involved in a biochemical pathway leading to gene transcription, were also attenuated by NAN-190 in the wildtype mice. However, activation of the extracellular signal-regulated kinase I/II (ERK) pathway in wildtype mice, following the light pulse, was not affected by NAN-190 pretreatment, nor was the expression of the circadian clock components Period1 and Period2. These findings suggest that the 5-HT(1A) receptor plays a critical role in the potentiation effect observed with NAN-190, and that NAN-190 may potentiate photic phase shifts at least partly by down-regulating the activity of some (but not all) genes and biochemical pathways involved in coupling the light signal to the output of the circadian clock.

Non-photic modulation of phase shifts to long light pulses.

Circadian rhythms can be reset by both photic and non-photic stimuli. Recent studies have used long light exposure to produce photic phase shifts or to enhance non-photic phase shifts. The presence or absence of light can also influence the expression of locomotor rhythms through masking; light during the night attenuates locomotor activity, while darkness during the day induces locomotor activity in nocturnal animals. Given this dual role of light, the current study was designed to examine the relative contributions of photic and non-photic components present in a long light pulse paradigm. Mice entrained to a light/dark cycle were exposed to light pulses of various durations (0, 3, 6, 9, or 12 h) starting at the time of lights-off. After the light exposure, animals were placed in DD and were either left undisturbed in their home cages or had their wheels locked for the remainder of the subjective night and subsequent subjective day. Light treatments of 6, 9, and 12 h produced large phase delays. These treatments were associated with decreased activity during the nocturnal light and increased activity during the initial hours of darkness following light exposure. When the wheels were locked to prevent high-amplitude activity, the resulting phase delays to the light were significantly attenuated, suggesting that the activity following the light exposure may have contributed to the overall phase shift. In a second experiment, telemetry probes were used to assess what effect permanently locking the wheels had on the phase shift to the long light pulses. These animals had phase shifts fully as large as animals without any form of wheel lock, suggesting that while non-photic events can modulate photic phase shifts, they do not play a role in the full phase-shift response observed in animals exposed to long light pulses. This paradigm will facilitate investigations into non-photic responses of the mouse circadian system.

Effects of lighting condition on circadian behavior in 5-HT1A receptor knockout mice.

Serotonin (5-HT) is an important regulator of the mammalian circadian system, and has been implicated in modulating entrained and free-running rhythms, as well as photic and non-photic phase shifting. In general, 5-HT appears to oppose the actions of light on the circadian system of nocturnal rodents. As well, 5-HT mediates, at least in part, some non-photic responses. The 5-HT1A, 1B and 7 receptors regulate these acute responses to zeitgebers. 5-HT also regulates some entrained and free-running properties of the circadian clock. The receptors that contribute to these phenomena have not been fully examined. Here, we use 5-HT1A receptor knockout (KO) mice to examine the response of the mouse circadian system to a variety of lighting conditions, including a normal light-dark cycle (LD), T-cycles, phase advanced LD cycles, constant darkness (DD), constant light (LL) and a 6 hour dark pulse starting at CT5. Relative to wildtype mice, the 5-HT1A receptor KO mice have lower levels of activity during the first 8h of the night/subjective night in LD and LL, later activity onsets on transient days during re-entrainment, shorter free-running periods in LL when housed with wheels, and smaller phase shifts to dark pulses. No differences were noted in activity levels during DD, alpha under any light condition, free-running period in DD, or phase angle of entrainment in LD. While the 5-HT1A receptor plays an important role in regulating photic and non-photic phase shifting, its contribution to entrained and free-running properties of the circadian clock is relatively minor.

The serotonergic anxiolytic buspirone attenuates circadian responses to light

Serotonergic drugs modify circadian responses to light, with agonists attenuating and some partial agonists or antagonists potentiating photic phase shifts. The anxiolytic buspirone is a 5‐HT1A receptor partial agonist. Given that buspirone is used therapeutically to manage generalised anxiety disorder, it would be useful to understand if and how this drug may modify circadian responses to light, not only to help manage side effects, but also to examine its potential use as a chronobiotic. Here we examined behavioral and molecular responses to phase‐shifting light in mice and hamsters treated with buspirone. Phase advances to late subjective night light pulses in hamsters and wildtype mice were significantly attenuated by buspirone. 5‐HT1A receptor knockout mice exhibited potentiated photic phase shifts when pretreated with buspirone. In wildtype mice, the attenuated phase shifts were accompanied by increased cFos expression in the suprachiasmatic nucleus, whereas potentiated phase shifts in knockouts were accompanied by increased phosphorylation of extracellular signal‐regulated kinase (ERK) and cyclic AMP response element‐binding protein (CREB), and decreased cFos expression. Attenuated photic phase shifts in buspirone‐treated hamsters were accompanied by decreased phosphorylation of ERK and CREB. Chronic buspirone treatment decreased the amplitude of wheel‐running rhythms, lengthened the duration of the active phase and advanced the phase angle of entrainment. Buspirone administration at midday produced non‐photic phase advances in wildtype but not 5‐HT1A receptor knockout mice. These findings suggest that buspirone affected the circadian system in a manner similar to the 5‐HT1A/7 agonist (±)‐8‐Hydroxy‐2‐dipropylaminotetralin hydrobromide, primarily through the 5‐HT1A receptor, and suggest that therapeutic use of buspirone to manage anxiety may impact circadian function.

Serotonergic enhancement of circadian responses to light: Role of the raphe and intergeniculate leaflet

Light serves as the primary stimulus that synchronizes the circadian clock in the suprachiasmatic nucleus (SCN) to the external day/night cycle. Appropriately timed light exposure can reset the phase of the circadian clock. Some serotonergic drugs that bind to the serotonin 1A receptor can enhance phase shifts to light. The mechanism by which this potentiation occurs is not well understood. In this study, we examined where one of these drugs, 8‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl]‐8‐azaspiro[4.5]decane‐7,9‐dione dihydrochloride (BMY7378), might be working in the hamster brain. Systemic (5 mg/kg), intra‐dorsal raphe and intra‐median raphe (both 15.6 nmol in 0.5 μL), but not intra‐SCN (7.8 nmol or 15.6 nmol in 0.5 μL) injections of BMY7378 significantly potentiated phase shifts to light. Potentiation of photic shifts persisted when serotonergic innervation of the SCN was lesioned with infusions of the serotonin neurotoxin 5,7‐dihydroxytryptamine into the SCN. Light‐induced c‐Fos expression in the rostral and caudal intergeniculate leaflet (IGL) was attenuated with systemic BMY7378, suggesting that the IGL may be involved in this response. Both complete IGL lesions and depletion of serotonergic innervation of the IGL prevented systemic BMY7378 from potentiating photic phase shifts. Together, these findings suggest that the mechanism by which BMY7378 enhances photic responses is by changing the activity of the raphe nuclei to influence how the IGL responds to light, which subsequently influences the SCN as one of its downstream targets. Identification of the network that underlies this potentiation could lead to the development of useful therapeutic interventions for treating sleep and circadian disorders.

Neonatal medial frontal cortex lesions disrupt circadian activity patterns.

The medial frontal cortex (MFC) is involved in the temporal organization of behaviour. It receives timing information from the master circadian clock in the suprachiasmatic nucleus (SCN), and exhibits daily oscillations in gene expression itself. In this study, we evaluate various properties of circadian rhythms of locomotor activity following neonatal or adult MFC aspiration lesions. Mice with neonatal lesions were more active during the day than mice with adult lesions and less active during the early night than both mice with adult lesions and control mice. Compared to controls, mice with neonatal lesions exhibited smaller phase delays to an early-night light pulse and marginally larger phase advances to a late-night light pulse. Mice with adult lesions did not differ from controls on either measure. The results suggest that the timing of behaviour is determined by an interaction between the MFC and the SCN and that injury early in life has a significant effect on the ability of animals to organize such behaviours.