The daily variation in background light intensity (irradiance) can entrain the endogenous clock in the suprachiasmatic nucleus (SCN) to the external environment. The only source of this photic information in mammals is the eye, which is primarily a visual organ. It is therefore highly specialised to detect high frequency spatiotemporal modulations. This together with the adaptation which occurs within the retina could be present difficulties when encoding global irradiance. This raises the question of whether spatial patterns, which are present in our everyday viewing, might affect the ability of the SCN to receive 'true irradiance' signals and entrain to the external environment. My first approach was to determine whether individual SCN cells might receive a 'true irradiance' signal. To this end I mapped and characterised the receptive field properties of SCN neurons using in vivo electrophysiology. Indeed a handful of neurons (full field cells) responded to light anywhere in the visual scene and thus may act as 'irradiance detectors'. However, the vast majority of cells only sampled local radiance from a limited area of the visual scene. Having mapped the receptive field properties it became clear that cells which sampled from a limited area of the visual scene would be sensitive to spatial contrast (patterns). To examine the effect of spatiotemporal contrast on the SCN I examined two SCN outputs: locomotor activity and neuronal firing rates. Although spatiotemporal modulation in light intensity could induce large amplitude oscillations in neuronal activity; the time averaged firing rate and locomotor activity, which are believed to be determined by irradiance, were largely unaffected by spatial patterns. This led to the conclusion that the SCN can multiplex photic information into information regarding irradiance, and spatial information by encoding them under different timescales. Melanopsin has been heralded as the key photopigment for encoding irradiance and entraining the SCN. However such experiments have been only performed using diffuse light stimuli. Here I investigated the role of melanopsin under natural viewing conditions which incorporated spatial patterns. Under such stimuli the SCN response can be almost entirely accounted for by the melanopic irradiance of the stimuli.

In order to anticipate the predictable changes in the environment associated with the earth’s rotation, most organisms possess intrinsic biological clocks. To be useful, such clocks require a reliable signal of ‘time’ from the external world. In mammals, light provides the principle source of such information; conveyed to the suprachiasmatic nucleus circadian pacemaker (SCN) either directly from the retina or indirectly via other visual structures such as the thalamic intergeniculate leaflet (IGL). Nonetheless, while the basic pathways supplying sensory information to the clock are well understood, the sensory signals they convey or how these are processed within the circadian system are not. One established view is that circadian entrainment relies on measuring the total amount of environmental illumination. In line with that view, the dense bilateral retinal input to the SCN allows for the possibility that individual neurons could average signals from across the whole visual scene. Here I test this possibility by examining responses to monocular and binocular visual stimuli in the SCN of anaesthetised mice. In fact, these experiments reveal that SCN cells provide information about (at most) irradiance within just one visual hemisphere. As a result, overall light-evoked activity across the SCN is substantially greater when light is distributed evenly across the visual scene when the same amount of light is non-uniformly distributed. Surprisingly then, acute electrophysiological responses of the SCN population do not reflect the total amount of environmental illumination. Another untested suggestion has been that the circadian system might use changes in the spectral composition of light to estimate time of day. Hence, during ‘twilight’, there is a relative enrichment of shortwavelength light, which is detectable as a change in colour to the dichromatic visual system of most mammals. Here I used a ‘silent substitution’ approach to selectively manipulate mouse cone photoreception, revealing a subset of SCN neurons that exhibit spectrally-opponent (blue-yellow) visual responses and are capable of reliably tracking sun position across the day-night transition. I then confirm the importance of this colour discrimination mechanism for circadian entrainment by demonstrating a reliable change in mouse body temperature rhythms when exposed to simulated natural photoperiods with and without simultaneous changes in colour. This identification of chromatic influences on circadian entrainment then raises important new questions such as which SCN cell types process colour signals and do these properties originate in the retina or arise via input from other visual regions? Advances in mouse genetics now offer powerful ways to address these questions. Our original method for studying colour discrimination required transgenic mice with red-shifted cone sensitivity – presenting a barrier to applying this approach alongside other genetic tools. To circumvent this issue I validated a modified approach for manipulating wildtype cone photoreception. Using this approach alongside optogenetic cell-identification I then demonstrate that the thalamic inputs to the SCN are unlikely to provide a major source of chromatic information. To further probe IGL-contributions to SCN visual responses, I next used electrical microstimulation to show that the thalamus provides inhibitory input to both colour and brightness sensitive SCN cells. Using local pharmacological inhibition I then show that thalamic inputs supress specific features of the SCN light response originating with the contralateral retina, including colour discrimination. These data thus provide new insight into the ways that arousal signals reaching the visual thalamus could modulate sensory processing in the SCN. Together then, the work described in this thesis provides important new insight into sensory control of the circadian system and the underlying neural mechanisms.

Circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) regulates daily rhythms in behavior and physiology and is an important part of the mechanisms regulating mammalian homeostasis. SCN are synchronized with a 24hour cycle mainly by light, but they can also be regulated by a variety of nonphotic signals, such as growth factors, opioids, cytokines, or lipopolysaccharide (LPS), which act by inducing the JAK/STAT signaling pathway. STAT family proteins (i.e. signal transducers and activator of transcription) regulate many aspects of cellular physiology, from growth and differentiation to immune response. However, the JAK/STAT signaling pathway has not yet been studied in the SCN and the function of STAT proteins in the SCN has not yet been clarified. In the first part of the thesis, we focused on localization of STAT3 and STAT5 proteins in the rat SCN and determination of rhythm in proteins and mRNA. Our experiments showed the daily rhythm in the levels of STAT3 protein in SCN astrocytes of rat with low but significant amplitude and with maximum in the morning. In addition, we revealed strong but nonrhythmic expression of STAT5A protein in astrocytes and STAT5B protein in nonastrocytic cells of SCN. It was also found that Stat3 mRNA show, similarly to protein, circadian rhythm in...

The circadian system uses environmental cues to coordinate the plethora of physiological functions that occur with diurnal rhythmicity, with light being one of the strongest regulators of the circadian system. The suprachiasmatic nucleus (SCN) is positioned at the top of the circadian hierarchy, receiving photic input from the retina and using neuronal, humoral and endocrine signals to synchronize oscillatory patterns across virtually all organ systems. Though the circadian system is plastic to deviations from a normal light/dark (LD) cycle, there are biological limits as to the rate and degree to which the SCN can adapt to these deviations, with peripheral oscillators responding in a delayed manner to the master clock. Alzheimer’s disease (AD) has long been linked to disruptions in the circadian system, with circadian deficits generally portrayed as a consequence of disease development. Recent evidence, however, suggests that circadian disruptions may precede noticeable cognitive deficits associated with AD. Our study aims to determine whether inducible circadian disruption via exposure to our social jetlag protocol, taking advantage of manipulating light exposure to disrupt the circadian system, can exacerbate the rate and severity of AD pathology in both the AβPP/PS1 and APPNL-F/NL-F mouse models of AD. AβPP/PS1 and APPNL-F/NL-F subjected to a preliminary study at 2-4 months in which overall activity in normal LD conditions, re-entrainment to a maintained 8-hour phase advance of the LD cycle, and endogenous period in constant darkness were measured. Glucose Tolerance Test (GTT) was used to assess metabolic health. Following the preliminary study, wild type (WT; C57BL/6J) controls, AβPP/PS1 and APPNL-F/NL-F AD models were entrained to a control light/dark cycle starting at 6 weeks of age. Following 2-weeks of entrainment, half of the animals were maintained on the control schedule, and half were exposed to the social jetlag protocol, involving an 8-hour phase advance on day 5 and an 8-hour phase delay on day 7 of repeated 7-day sessions, effectively inducing chronic circadian disruption until the assigned 6 and 12-month endpoints. One month prior to the endpoint, activity measures and GTT were performed, following which the animals were all entrained to a normal 12:12 LD schedule for 3-5 weeks. Finally, animals underwent an 8-day Morris Water Maze (MWM) to assess effects of chronic disruption on AD-related cognitive decline. Animals were then sacrificed and tissues collected at Zeitgeber time (ZT) 12, time of lights off. At 2-4 months of age, preclinical stages in both AD models, APPNL-F/NL-F males displayed significantly higher fasting glucose levels and circadian period (day length). There were trending increases in overall activity levels under normal 12:12 LD conditions in both AD models over WT controls. 2-4 month females re-entrained to an 8-hour phase advance in significantly less time than males in all genotypes. AβPP/PS1 mice demonstrated hyperactivity as compared to age and sex-matched WT controls. Chronic circadian disruption dampened lights off activity in all cohorts. In all genotypes, female animals showed a higher degree of re-entrainment to the phase advancement of the lighting schedule going into the subjective weekend (day 6-7). Metabolic data as measured by glucose tolerance test on day 7 of the (social jet lag) SJL schedule indicated that disrupted animals were metabolically entrained to the day 1-5 schedule at the peak of rhythmic metabolic function, whereas control animals were at a low point in metabolic rhythms at the time of testing, indicating that circadian regulation of metabolic function was not able to adapt to the weekend phase shifts. Arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) expression in the SCN were significantly dampened as a result of chronic disruption in 12-month male AD mice. Amyloid plaque analysis indicated a severely worsened pathological phenotype in AβPP/PS1 mice as compared to age and sex-matched APPNL-F/NL-F mice. MWM data provides evidence for impaired spatial learning in both AD models that is significantly worsened by chronic jetlag exposure. Taken together, the data suggests that chronic exposure to the social jet lag schedule disrupts rhythmic behavior, metabolic function, and spatial learning significantly in both AD animal models.

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published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Os neurônios Kiss1 do núcleo anteroventral periventricular (AVPV/PeN) e núcleo arqueado (Arc) possuem o receptor de estrógeno alfa (ERα) e são essenciais no controle do ciclo ovulatório. Os neurônios Kiss1 do AVPV/PeN apresentam conexões com o núcleo supraquiasmático, através do qual a informação fótica poderia regular a ovulação. A vasopressina (VP) e o peptídeo intestinal vasoativo (VIP) são os principais peptídeos produzidos pelo NSQ, e estudos sugerem a participação deles na secreção de LH. Com isso, investigamos se fibras vasopressinérgicas estão em aposição com neurônios Kiss1 do AVPV/PeN e Arc; se os receptores de VP e VIP são expressos nestes núcleos; e se variações nos níveis circulantes de estradiol (E2) poderiam afetar a expressão destes receptores. Para isso, utilizamos camundongos divididos em: fêmeas em diestro; fêmeas que tiveram os ovários removidos (OVX); e fêmeas OVX que receberam reposição de E2 (OVX+E2). Observamos que neurônios Kiss1 do AVPV/PeN colocalizam com fibras vasopressinérgicas; e os níveis circulantes de estrógeno não afetou este contato e sim o número de neurônios Kiss1. No AVPV/PeN, as OVX tiveram aumento da expressão dos receptores VPAC1 e VPAC2; no Arc, a reposição com E2 induziu um aumento da expressão do Avpr1a. / The Kiss1 neurons in the periventricular anteroventral nucleus (AVPV/PeN) and arcuate nucleus (Arc) have the estrogen receptor alpha (ERα) and are essential in controlling the ovulatory cycle. The Kiss1 neurons of the AVPV/PeN have connections to the suprachiasmatic nucleus, through which the photic information could regulate ovulation. Vasopressin (VP) and vasoactive intestinal peptide (VIP) are the main peptides produced by the SCN, and studies suggest their participation in LH secretion. Thus, we investigated whether vasopressinergics fibers are in apposition with Kiss1 neurons in the AVPV/PeN and Arc; whether VP and VIP receptors are expressed in these nuclei; and if changes in circulating levels of estradiol (E2) could affect the expression of these receptors. We used mice divided into: females in estrus; females who had their ovaries removed (OVX); and OVX that receiving E2 replacement (OVX + E2). We observed that Kiss1 neurons of the AVPV/PeN in colocalization with vasopressinergics fibers; and circulating levels of estrogen did not affect this contact but the number of neurons Kiss1. In AVPV/PeN, the OVX had increased expression of VPAC1 and VPAC2 receptors; in the Arc, replacement with E2 induced an increased expression of Avpr1a.