Ilia N. Karatsoreos

Phenotype Matters: Identification of Light-Responsive Cells in the Mouse Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus is the neural locus of the circadian clock. To explore the organization of the SCN, two strains of transgenic mice, each bearing a jellyfish green fluorescent protein (GFP) reporter, were used. In one, GFP was driven by the promoter region of the mouse Period1 gene (mPer1) (Per1::GFP mouse), whereas in the other, GFP was inserted in the promoter region of calbindin-D28K-bacterial artificial chromosome (CalB::GFP mouse). In the latter mouse, GFP-containing SCN cells are immunopositive for gastrin-releasing peptide. In both mouse lines, light-induced Per1 mRNA and Fos are localized to the SCN subregion containing gastrin-releasing peptide. Double-label immunohistochemistry reveals that most gastrin-releasing peptide cells (∼70%) contain Fos after a brief light pulse. To determine the properties of SCN cells in this light-responsive region, we examined the expression of rhythmic Period genes and proteins. Gastrin-releasing peptide-containing cells do not express detectable rhythms in these key components of the molecular circadian clock. The results support the view that the mammalian SCN is composed of functionally distinct cell groups, of which some are light induced and others are rhythmic with respect to clock gene expression. Furthermore, the findings suggest that gastrin-releasing peptide is a potential mediator of intercellular communication between light-induced and oscillator cells within the SCN.

Pubertal maturation and time of day differentially affect behavioral and neuroendocrine responses following an acute stressor

Puberty markedly influences stress responsiveness such that prepubertal animals show a more protracted corticosterone (CORT) and progesterone response following acute stress compared to adults. In both adult and juvenile rats, circadian time modulates adrenocortical steroids with basal CORT and progesterone levels rising prior to the onset of the dark phase of the light-dark cycle (i.e., active period). How time of day affects the pubertal difference in stress responsiveness and if the behaviors of prepubertal and adult animals are differentially affected by stress and time of testing remain unknown. Thus, we exposed group housed (3 per cage) prepubertal (28d) and adult (77d) male rats to 30 min of restraint in either the early portion of the behaviorally inactive, light (circadian nadir of CORT and progesterone) or behaviorally active, dark (circadian peak) phase of their light-dark cycle and measured ACTH, CORT, progesterone, and home cage behavior before and after the stressor. We found that the extended hormonal stress response demonstrated by prepubertal males occurred at both times of day. However, differences in post-stress behavior were dependent on time of testing. Specifically, although pre- and post-stress behaviors were similarly affected by the stressor in the light phase in prepubertal and adult males, during the dark phase, stress suppressed play behavior in the prepubertal males, and increased their time spent resting together (huddling), while these behaviors were unaffected by stress in the adults. These data indicate that pubertal development and time of day interact to modulate post-stress behavior and demonstrate a dissociation between post-stress hormonal and behavioral responses.

Gonadectomy reveals sex differences in circadian rhythms and suprachiasmatic nucleus androgen receptors in mice

In mammals, it is well established that circadian rhythms in physiology and behavior, including the rhythmic secretion of hormones, are regulated by a brain clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. While SCN regulation of gonadal hormone secretion has been amply studied, the mechanisms whereby steroid hormones affect circadian functions are less well known. This is surprising considering substantial evidence that sex hormones affect many aspects of circadian responses, and that there are significant sex differences in rhythmicity. Our previous finding that core and shell regions of the SCN differ in their expression of clock genes prompted us to examine the possibility that steroid receptors are localized to a specific compartment of the brain clock, with the discovery that the androgen receptor (AR) is concentrated in the SCN core in male mice. In the present study, we compare AR expression in female and male mice using Western blots and immunochemistry. Both of these methods indicate that ARs are more highly expressed in males than in females; gonadectomy eliminates and androgen treatment restores these sex differences. At the behavioral level, gonadectomy produces a dramatic loss of the evening activity onset bout in males, but has no such effect in females. Treatment with testosterone, or with the non-aromatizable androgen dihydrotestosterone, restores male locomotor activity and eliminates sex differences in the behavioral response. The results indicate that androgenic hormones regulate circadian responses, and suggest an SCN site of action.

Exploring spatiotemporal organization of SCN circuits

Suprachiasmatic nucleus (SCN) neuroanatomy has been a subject of intense interest since the discovery of the SCNs function as a brain clock and subsequent studies revealing substantial heterogeneity of its component neurons. Understanding the network organization of the SCN has become increasingly relevant in the context of studies showing that its functional circuitry, evident in the spatial and temporal expression of clock genes, can be reorganized by inputs from the internal and external environment. Although multiple mechanisms have been proposed for coupling among SCN neurons, relatively little is known of the precise pattern of SCN circuitry. To explore SCN networks, we examine responses of the SCN to various photic conditions, using in vivo and in vitro studies with associated mathematical modeling to study spatiotemporal changes in SCN activity. We find an orderly and reproducible spatiotemporal pattern of oscillatory gene expression in the SCN, which requires the presence of the ventrolateral core region. Without the SCN core region, behavioral rhythmicity is abolished in vivo, whereas low-amplitude rhythmicity can be detected in SCN slices in vitro, but with loss of normal topographic organization. These studies reveal SCN circuit properties required to signal daily time.

Diurnal regulation of the gastrin‐releasing peptide receptor in the mouse circadian clock

In mammals, circadian rhythms are generated by the suprachiasmatic nuclei (SCN) of the hypothalamus. SCN neurons are heterogeneous and can be classified according to their function, anatomical connections, morphology and/or peptidergic identity. We focus here on gastrin‐releasing peptide‐ (GRP) and on GRP receptor‐ (GRPr) expressing cells of the SCN. Pharmacological application of GRP in vivo or in vitro can shift the phase of circadian rhythms, and GRPr‐deficient mice show blunted photic phase shifting. Given the in vivo and in vitro effects of GRP on circadian behavior and on SCN neuronal activity, we investigated whether the GRPr might be under circadian and/or diurnal control. Using in situ hybridization and autoradiographic receptor binding, we localized the GRPr in the mouse SCN and determined that GRP binding varies with time of day in animals housed in a light–dark cycle but not in conditions of constant darkness. The latter results were confirmed with Western blots of SCN tissue. Together, the present findings reveal that changes in GRPr are light driven and not endogenously organized. Diurnal variation in GRPr activity probably underlies intra‐SCN signaling important for entrainment and phase shifting.

Androgens Modulate Structure and Function of the Suprachiasmatic Nucleus Brain Clock

Gonadal hormones can modulate circadian rhythms in rodents and humans, and androgen receptors are highly localized within the core region of the mouse suprachiasmatic nucleus (SCN) brain clock. Although androgens are known to modulate neural plasticity in other CNS compartments, the role of androgens and their receptors on plasticity in the SCN is unexplored. In the present study, we ask whether androgens influence the structure and function of the mouse SCN by examining the effects of gonadectomy (GDX) on the structure of the SCN circuit and its responses to light, including induction of clock genes and behavioral phase shifting. We found that after GDX, glial fibrillary acidic protein increased with concomitant decreases in the expression of the synaptic proteins synaptophysin and postsynaptic density 95. We also found that GDX exerts effects on the molecular and behavioral responses to light that are phase dependent. In late night [circadian time (CT)21], GDX increased light-induced mPer1 but not mPer2 expression compared with intact (INT) controls. In contrast, in early night (CT13.5), GDX decreased light induced mPer2 but had no effect on mPer1. At CT13.5, GDX animals also showed larger phase delays than did INT. Treatment of GDX animals with the nonaromatizable androgen dihydrotestosterone restored glial fibrillary acidic protein, postsynaptic density 95, and synaptophysin in the SCN and reinstated the INT pattern of molecular and behavioral responses to light. Together, the results reveal a role for androgens in regulating circuitry in the mouse SCN, with functional consequences for clock gene expression and behavioral responses to photic phase resetting stimuli.

Dose-Dependent Effects of Androgens on the Circadian Timing System and Its Response to Light

The hypothalamic suprachiasmatic nucleus (SCN) is the locus of a master clock that regulates circadian rhythms in physiology and behavior. Gonadectomy in male mice lengthens the period of circadian rhythms and increases the day-to-day variability of activity onset time. Both of these responses are rescued by the nonaromatizable androgen dihydrotestosterone. Androgen receptors (AR) are localized in SCN neurons that receive direct retinal input. To explore how androgens affect circadian clock function and its responsiveness to photic cues, we measured wheel-running behavior and SCN AR expression in intact, gonadectomized, and testosterone-replaced mice, held under various photic conditions. Gonadectomy lengthened circadian period in constant dim light but not in constant darkness. Increasing intensities of constant light parametrically increased circadian period, and this was potentiated at all intensities by gonadectomy. In contrast, gonadectomy did not alter light-induced pupil constriction, suggesting a nonretinal locus of hormone action. In hormone-replaced animals housed in constant darkness, T concentration was positively correlated with precision of activity onset and with SCN AR expression and negatively correlated with duration of activity. We infer the existence of two androgenic mechanisms: one modulates SCN responsiveness to light, and the second modulates SCN timekeeping and locomotor activity in a dose-dependent manner. Finally, the effects of androgens on period are a result of hormonal modulation of the SCNs response to photic input rather than to a change in the inherent period of oscillators in the absence of light.

Depression: What Is the Role of Physiological Dysregulation and Circadian Disruption?

ately lends itself to testing via a number of resultant hypotheses. Perhaps more importantly, this approach to depression opens the door a bit more to novel and exciting potential collaborations between previously (mostly) unrelated schools of thought—for example, psychodynamic theory and basic neuroscience. Such a wide-reaching collaboration promises many exciting and paradigm-shifting discoveries. REFERENCE