Overexpression of striatal D2 receptors reduces motivation thereby decreasing food anticipatory activity.

Dopamine has been implicated in circadian timing underlying the food entrainable oscillator (FEO) circuitry and overexpression of the dopamine D2 receptor (D2R) in the striatum has been reported to reduce motivation to obtain food rewards in operant tasks. In the present study, we explored both of these mechanisms by examining food anticipatory activity (FAA) in dopamine D2 receptor-overexpressing (D2R-OE) mice under various durations of food availability. First, we noted that at baseline, there were no differences between D2R-OE mice and their littermates in activity level, food intake, and body weight or in circadian activity. Under conditions of very restricted food availability (4 or 6 hr), both genotypes displayed FAA. In contrast, under 8-hr food availability, control mice showed FAA, but D2R-OE mice did not. Normalization of D2R by administration of doxycycline, a tetracycline analogue, rescued FAA under 8-hr restricted food. We next tested for circadian regulation of FAA. When given ad libitum access to food, neither D2R-OE nor controls were active during the daytime. However, after an interval of food restriction, all mice showed elevated locomotor activity at the time of previous food availability in the day, indicating circadian timing of anticipatory activity. In summary, motivation is reduced in D2R-OE mice but circadian timing behavior is not affected. We conclude that an increase in striatal D2R reduces FAA by modulating motivation and not by acting on a clock mechanism.

Elevated zinc transporter ZnT3 in the dentate gyrus of mast cell-deficient mice.

Zinc is important in neurogenesis, but excessive levels can cause apoptosis and other pathologies leading to cognitive impairments. Mast cells are present in many brain regions including the hippocampus, an area rich in vesicular zinc. Mast cells contain zinc-rich granules and a well-developed mechanism for uptake of zinc ions; both features point to the potential for a role in zinc homeostasis. Prior work using the Timm stain supported this hypothesis, as increased labile zinc was detected in the hippocampus of mast cell-deficient mice compared to wild-type mice while no differences in total zinc were found between the two genotypes in the whole brain or other tissues. The current report further examines differences in zinc homeostasis between wild-type and mast cell-deficient mice by exploring the zinc transporter ZnT3, which transports labile zinc into synaptic vesicles. The first study used immunocytochemistry to localize ZnT3 within the mossy fibre layer of the hippocampus to determine whether there was differential expression of ZnT3 in wild-type versus mast cell-deficient mice. The second study used inductively coupled plasma mass spectrometry (ICP-MS) to determine total zinc content in the whole dentate gyrus of the two genotypes. The immunocytochemical results indicate that there are higher levels of ZnT3 localized to the mossy fibre layer of the dentate gyrus of mast cell-deficient mice than in wild-type mice. The ICP-MS data reveal no differences in total zinc in dentate gyrus as a whole. The results are consistent with the hypothesis that mast cells participate in zinc homeostasis at the level of synaptic vesicles.

Differential localization of PER1 and PER2 in the brain master circadian clock.

The hypothalamic suprachiasmatic nucleus (SCN), locus of the master circadian clock, bears many neuronal types. At the cellular-molecular level, the clock is comprised of feedback loops involving 'clock' genes including Period1 and Period2, and their protein products, PERIOD1 and PERIOD2 (PER1/2). In the canonical model of circadian oscillation, the PER1/2 proteins oscillate together. While their rhythmic expression in the SCN as a whole has been described, the possibility of regional differences remains unknown. To explore these clock proteins in distinct SCN regions, we assessed their expression through the rostro-caudal extent of the SCN in sagittal sections. We developed an automated method for tracking three fluorophores in digital images of sections triply labeled for PER1, PER2, and gastrin-releasing peptide (used to locate the core). In the SCN as a whole, neurons expressing high levels of PER2 were concentrated in the rostral, rostrodorsal, and caudal portions of the nucleus, and those expressing high levels of PER1 lay in a broad central area. Within these overall patterns, adjacent cells differed in expression levels of the two proteins. The results demonstrate spatially distinct localization of high PER1 vs. PER2 expression, raising the possibility that their distribution is functionally significant in encoding and communicating temporal information. The findings provoke the question of whether there are fundamental differences in PER1/2 levels among SCN neurons and/or whether topographical differences in protein expression are a product of SCN network organization rather than intrinsic differences among neurons.

Suprachiasmatic nucleus as the site of androgen action on circadian rhythms.

Androgens act widely in the body in both central and peripheral sites. Prior studies indicate that in the mouse, suprachiasmatic nucleus (SCN) cells bear androgen receptors (ARs). The SCN of the hypothalamus in mammals is the locus of a brain clock that regulates circadian rhythms in physiology and behavior. Gonadectomy results in reduced AR expression in the SCN and in marked lengthening of the period of free-running activity rhythms. Both responses are restored by systemic administration of androgens, but the site of action remains unknown. Our goal was to determine whether intracranial androgen implants targeted to the SCN are sufficient to restore the characteristic free-running period in gonadectomized male mice. The results indicate that hypothalamic implants of testosterone propionate in or very near the SCN produce both anatomical and behavioral effects, namely increased AR expression in the SCN and restored period of free-running locomotor activity. The effect of the implant on the period of the free-running locomotor rhythm is positively correlated with the amount of AR expression in the SCN. There is no such correlation of period change with amount of AR expression in other brain regions examined, namely the preoptic area, bed nucleus of the stria terminalis and premammillary nucleus. We conclude that the SCN is the site of action of androgen effects on the period of circadian activity rhythmicity.

Vasculature of the Suprachiasmatic Nucleus: Pathways for Diffusible Output Signals.

Transplant studies demonstrate unequivocally that the suprachiasmatic nucleus (SCN) produces diffusible signals that can sustain circadian locomotor rhythms. There is a vascular portal pathway between the SCN and the organum vasculosum of the lamina terminalis in mouse brain. Portal pathways enable low concentrations of neurosecretions to reach specialized local targets without dilution in the systemic circulation. To explore the SCN vasculature and the capillary vessels whereby SCN neurosecretions might reach portal vessels, we investigated the blood vessels (BVs) of the core and shell SCN. The arterial supply of the SCN differs among animals, and in some animals, there are differences between the 2 sides. The rostral SCN is supplied by branches from either the superior hypophyseal artery (SHpA) or the anterior cerebral artery or the anterior communicating artery. The caudal SCN is consistently supplied by the SHpA. The rostral SCN is drained by the preoptic vein, while the caudal is drained by the basal vein, with variations in laterality of draining vessels. In addition, several key features of the core and shell SCN regions differ: Median BV diameter is significantly smaller in the shell than the core based on confocal image measurements, and a similar trend occurs in iDISCO-cleared tissue. In the cleared tissue, whole BV length density and surface area density are significantly greater in the shell than the core. Finally, capillary length density is also greater in the shell than the core. The results suggest three hypotheses: First, the distinct arterial and venous systems of the rostral and caudal SCN may contribute to the in vivo variations of metabolic and neural activities observed in SCN networks. Second, the dense capillaries of the SCN shell are well positioned to transport blood-borne signals. Finally, variations in SCN vascular supply and drainage may contribute to inter-animal differences.

PPARγ Acetylation Orchestrates Adipose Plasticity and Metabolic Rhythms.

Systemic glucose metabolism and insulin activity oscillate in response to diurnal rhythms and nutrient availability with the necessary involvement of adipose tissue to maintain metabolic homeostasis. However, the adipose-intrinsic regulatory mechanism remains elusive. Here, the dynamics of PPARγ acetylation in adipose tissue are shown to orchestrate metabolic oscillation in daily rhythms. Acetylation of PPARγ displays a diurnal rhythm in young healthy mice, with the peak at zeitgeber time 0 (ZT0) and the trough at ZT18. This rhythmic pattern is deranged in pathological conditions such as obesity, aging, and circadian disruption. The adipocyte-specific acetylation-mimetic mutation of PPARγ K293Q (aKQ) restrains adipose plasticity during calorie restriction and diet-induced obesity, associated with proteolysis of a core circadian component BMAL1. Consistently, the rhythmicity in glucose tolerance and insulin sensitivity is altered in aKQ and the complementary PPARγ deacetylation-mimetic K268R/K293R (2KR) mouse models. Furthermore, the PPARγ acetylation-sensitive downstream target adipsin is revealed as a novel diurnal factor that destabilizes BMAL1 and mediates metabolic rhythms. These findings collectively signify that PPARγ acetylation is a hinge connecting adipose plasticity and metabolic rhythms, the two determinants of metabolic health.

Stomach ghrelin-secreting cells as food-entrainable circadian clocks.

Increases in arousal and activity in anticipation of a meal, termed "food anticipatory activity" (FAA), depend on circadian food-entrainable oscillators (FEOs), whose locations and output signals have long been sought. It is known that ghrelin is secreted in anticipation of a regularly scheduled mealtime. We show here that ghrelin administration increases locomotor activity in nondeprived animals in the absence of food. In mice lacking ghrelin receptors, FAA is significantly reduced. Impressively, the cumulative rise of activity before food presentation closely approximates a Gaussian function (r = 0.99) for both wild-type and ghrelin receptor knockout animals, with the latter having a smaller amplitude. For both groups, once an animal begins its daily anticipatory bout, it keeps running until the usual time of food availability, indicating that ghrelin affects response threshold. Oxyntic cells coexpress ghrelin and the circadian clock proteins PER1 and PER2. The expression of PER1, PER2, and ghrelin is rhythmic in light-dark cycles and in constant darkness with ad libitum food and after 48 h of food deprivation. In behaviorally arrhythmic-clock mutant mice, unlike control animals, there is no evidence of a premeal decrease in oxyntic cell ghrelin. Rhythmic ghrelin and PER expression are synchronized to prior feeding, and not to photic schedules. We conclude that oxyntic gland cells of the stomach contain FEOs, which produce a timed ghrelin output signal that acts widely at both brain and peripheral sites. It is likely that other FEOs also produce humoral signals that modulate FAA.

Light exposure induces short- and long-term changes in the excitability of retinorecipient neurons in suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the locus of a hypothalamic circadian clock that synchronizes physiological and behavioral responses to the daily light-dark cycle. The nucleus is composed of functionally and peptidergically diverse populations of cells for which distinct electrochemical properties are largely unstudied. SCN neurons containing gastrin-releasing peptide (GRP) receive direct retinal input via the retinohypothalamic tract. We targeted GRP neurons with a green fluorescent protein (GFP) marker for whole cell patch-clamping. In these neurons, we studied short (0.5-1.5 h)- and long-term (2-6 h) effects of a 1-h light pulse (LP) given 2 h after lights off [Zeitgeber time (ZT) 14:00-15:00] on membrane potential and spike firing. In brain slices taken from light-exposed animals, cells were depolarized, and spike firing rate increased between ZT 15:30 and 16:30. During a subsequent 4-h period beginning around ZT 17:00, GRP neurons from light-exposed animals were hyperpolarized by ∼15 mV. None of these effects was observed in GRP neurons from animals not exposed to light or in immediately adjacent non-GRP neurons whether or not exposed to light. Depolarization of GRP neurons was associated with a reduction in GABA(A)-dependent synaptic noise, whereas hyperpolarization was accompanied both by a loss of GABA(A) drive and suppression of a TTX-resistant leakage current carried primarily by Na. This suggests that, in the SCN, exposure to light may induce a short-term increase in GRP neuron excitability mediated by retinal neurotransmitters and neuropeptides, followed by long-term membrane hyperpolarization resulting from suppression of a leakage current, possibly resulting from genomic signals.

Targeted mutation of the calbindin D 28k gene selectively alters nonvisual photosensitivity.

Light intensity is an important determinant of diverse physiological and behavioral responses within the non-image-forming visual system. Thresholds differ among various photic responses, namely control of circadian rhythms, vigilance state, activity level and pupil constriction, but the mechanisms that regulate photosensitivity are not known. Calbindin D(28k) (CalB) is a calcium-binding protein associated with light processing in the mammalian circadian clock. Loss-of-function studies indicate that CalB-deficient mice (CalB(-/-)) have deficits in their ability to entrain to light-dark cycles. To explore the role of CalB in modulating photosensitivity, thresholds for three behaviors mediated by the non-image-forming visual system (entrainment, masking and pupillary light reflex; PLR) were compared in CalB(-/-) and wildtype mice, and the localization of CalB protein in these circuits was examined in adult and juvenile mice. The results reveal a divergence in how CalB affects thresholds to photic cues among these responses. Entrainment and masking were 40- to 60-fold less sensitive in CalB(-/-) than in wildtype mice. On the other hand, the PLR in CalB(-/-) mice was 80- to 200-fold more sensitive. Though CalB is expressed in the retina and in brain circuits regulating entrainment we found no CalB expression in any component of the PLR pathway, namely the olivary pretectal nucleus, Edinger-Westphal nucleus and ciliary ganglion. The behavioral and anatomical data together suggest that, in normal animals, the retinal response to light is blunted in the presence of CalB, but responsiveness of the higher order processes that transduce afferent retinal input is enhanced.

Characterization of orderly spatiotemporal patterns of clock gene activation in mammalian suprachiasmatic nucleus.

Because we can observe oscillation within individual cells and in the tissue as a whole, the suprachiasmatic nucleus (SCN) presents a unique system in the mammalian brain for the analysis of individual cells and the networks of which they are a part. While dispersed cells of the SCN sustain circadian oscillations in isolation, they are unstable oscillators that require network interactions for robust cycling. Using cluster analysis to assess bioluminescence in acute brain slices from PERIOD2::Luciferase (PER2::LUC) knockin mice, and immunochemistry of SCN from animals harvested at various circadian times, we assessed the spatiotemporal activation patterns of PER2 to explore the emergence of a coherent oscillation at the tissue level. The results indicate that circadian oscillation is characterized by a stable daily cycle of PER2 expression involving orderly serial activation of specific SCN subregions, followed by a silent interval, with substantial symmetry between the left and right side of the SCN. The biological significance of the clusters identified in living slices was confirmed by co-expression of LUC and PER2 in fixed, immunochemically stained brain sections, with the spatiotemporal pattern of LUC expression resembling that revealed in the cluster analysis of bioluminescent slices. We conclude that the precise timing of PER2 expression within individual neurons is dependent on their location within the nucleus, and that small groups of neurons within the SCN give rise to distinctive and identifiable subregions. We propose that serial activation of these subregions is the basis of robustness and resilience of the daily rhythm of the SCN.

Identification of the suprachiasmatic nucleus venous portal system in the mammalian brain.

There is only one known portal system in the mammalian brain - that of the pituitary gland, first identified in 1933 by Popa and Fielding. Here we describe a second portal pathway in the mouse linking the capillary vessels of the brain's clock suprachiasmatic nucleus (SCN) to those of the organum vasculosum of the lamina terminalis (OVLT), a circumventricular organ. The localized blood vessels of portal pathways enable small amounts of important secretions to reach their specialized targets in high concentrations without dilution in the general circulatory system. These brain clock portal vessels point to an entirely new route and targets for secreted SCN signals, and potentially restructures our understanding of brain communication pathways.

Arginine Vasopressin-Containing Neurons of the Suprachiasmatic Nucleus Project to CSF.

While it is well established that there are robust circadian rhythms of arginine vasopressin (AVP) in the cerebrospinal fluid (CSF), the route whereby the peptide reaches the CSF is not clear. A , AVP neurons constitute the largest fraction of the SCN neuronal population. Here, we show that processes of AVP-expressing SCN neurons cross the epithelium of the 3rd ventricular wall to reach the CSF (black arrows). Additionally, we report rostro-caudal differences in AVP neuron size and demonstrate that the localization of cells expressing the clock protein PER2 extend beyond the AVP population, thereby indicating that the size of this nucleus is somewhat larger than previously understood. B , Following lateral ventricle (LV) injection of cholera toxin ß subunit (CTß ; magenta) the retrograde tracer is seen in AVP neurons of the SCN, supporting the anatomical evidence that AVP neuronal processes directly contact the CSF.Arginine vasopressin (AVP) expressing neurons form the major population in the brain's circadian clock located in the hypothalamic suprachiasmatic nucleus (SCN). They participate in inter-neuronal coupling and provide an output signal for synchronizing daily rhythms. AVP is present at high concentrations in the cerebrospinal fluid (CSF) and fluctuates on a circadian timescale. While it is assumed that rhythms in CSF AVP are of SCN origin, a route of communication between these compartments has not been delineated. Using immunochemistry (ICC) and cell filling techniques, we determine the morphology and location of AVP neurons in mouse and delineate their axonal and dendritic processes. Cholera toxin ß subunit (CTß) tracer injected into the lateral ventricle tests whether AVP neurons communicate with CSF. Most importantly, the results indicate that AVP neurons lie in close proximity to the third ventricle, and their processes cross the ventricular wall into the CSF. We also report that contrary to widely held assumptions, AVP neurons do not fully delineate the SCN borders as PER2 expression extends beyond the AVP region. Also, AVP neurons form a rostral prong originating in the SCN medial-most and ventral-most aspect. AVP is lacking in the mid-dorsal shell but does occur at the base of the SCN just above the optic tract. Finally, neurons of the rostral SCN are smaller than those lying caudally. These findings extend our understanding of AVP signaling potential, demonstrate the heterogeneity of AVP neurons, and highlight limits in using this peptide to delineate the mouse SCN.

Phase Gradients and Anisotropy of the Suprachiasmatic Network: Discovery of Phaseoids.

Biological neural networks operate at several levels of granularity, from the individual neuron to local neural circuits to networks of thousands of cells. The daily oscillation of the brain's master clock in the suprachiasmatic nucleus (SCN) rests on a yet to be identified network of connectivity among its ∼20,000 neurons. The SCN provides an accessible model to explore neural organization at several levels of organization. To relate cellular to local and global network behaviors, we explore network topology by examining SCN slices in three orientations using immunochemistry, light and confocal microscopy, real-time imaging, and mathematical modeling. Importantly, the results reveal small local groupings of neurons that form intermediate structures, here termed "phaseoids," which can be identified through stable local phase differences of varying magnitude among neighboring cells. These local differences in phase are distinct from the global phase relationship, namely that between individual cells and the mean oscillation of the overall SCN. The magnitude of the phaseoids' local phase differences is associated with a global phase gradient observed in the SCN's rostral-caudal extent. Modeling results show that a gradient in connectivity strength can explain the observed gradient of phaseoid strength, an extremely parsimonious explanation for the heterogeneous oscillatory structure of the SCN.

Two forces for arousal: Pitting hunger versus circadian influences and identifying neurons responsible for changes in behavioral arousal.

The mechanisms underlying CNS arousal in response to homeostatic pressures are not known. In this study, we pitted two forces for CNS arousal against each other (circadian influences vs. restricted food availability) and measured the neuronal activation that occurs in a behaviorally defined group of animals that exhibited increased arousal in anticipation of feeding restricted to their normal sleeping time. The number of c-FOS+ neurons was significantly increased only in the ventromedial nucleus of the hypothalamus (VMH) in these mice, compared with control animals whose feeding was restricted to their normal active and feeding time (P < 0.01). Because the activation of VMH neurons coincides with the earliest signs of behavioral arousal preceding a change in meal time, we infer that VMH activation is involved in the increased arousal in anticipation of food.

Relationship of arousal to circadian anticipatory behavior: ventromedial hypothalamus: one node in a hunger-arousal network.

The mechanisms by which animals adapt to an ever-changing environment have long fascinated scientists. Different forces, conveying information regarding various aspects of the internal and external environment, interact with each other to modulate behavioral arousal. These forces can act in concert or, at times, in opposite directions. These signals eventually converge and are integrated to influence a common arousal pathway which, depending on all the information received from the environment, supports the activation of the most appropriate behavioral response. In this review we propose that the ventromedial hypothalamic nucleus (VMN) is part of the circuitry that controls food anticipation. It is the first nucleus activated when there is a change in the time of food availability, silencing of VMN ghrelin receptors decreases food-anticipatory activity (FAA) and, although lesions of the VMN do not abolish FAA, parts of the response are often altered. In proposing this model it is not our intention to exclude parallel, redundant and possibly interacting pathways that may ultimately communicate with, or work in concert with, the proposed network, but rather to describe the neuroanatomical requirements for this circuit and to illustrate how the VMN is strategically placed and connected to mediate this complex behavioral adaptation.

Musashi-2 and related stem cell proteins in the mouse suprachiasmatic nucleus and their potential role in circadian rhythms.

BACKGROUND: The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains the master circadian clock of the body and an unusually large number of cells expressing stem cell-related proteins. These seemingly undifferentiated cells may serve in entrainment of the SCN circadian clock to light cycles or allow it to undergo neural plasticity important for modifying its rhythmic output signals. These cells may also proliferate and differentiate into neurons or glia in response to episodic stimuli or developmental events requiring alterations in the SCN's control of physiology and behavior. PROBLEM: To characterize expression of stem cell related proteins in the SCN and the effects of stem-like cells on circadian rhythms. METHODS: Explant cultures of mouse SCN were maintained in medium designed to promote survival and growth of stem cells but not neuronal cells. Several stem cell marker proteins including SRY-box containing gene 2 (SOX2), nestin, vimentin, octamer-binding protein 4 (OCT4), and Musashi RNA-binding protein 2 (MSI2) were identified by immunocytochemistry in histological sections from adult mouse SCN and in cultures of microdissected SCN. A bioinformatics analysis located potential SCN targets of MSI2 and related RNA-binding proteins. RESULTS: Cells expressing stem cell markers proliferated in culture. Immunostained brain sections and bioinformatics supported the view that MSI2 regulates immature properties of SCN neurons, potentially providing flexibility in SCN neural circuits. Explant cultures had ongoing mitotic activity, indicated by proliferating-cell nuclear antigen, and extensive cell loss shown by propidium iodide staining. Cells positive for vasoactive intestinal polypeptide (VIP) that are highly enriched in the SCN were diminished in explant cultures. Despite neuronal cell loss, tissue remained viable for over 7 weeks in culture, as shown by bioluminescence imaging of explants prepared from SCN of Per1::luc transgenic mice. The circadian rhythm in SCN gene expression persisted in brain slice cultures in stem cell medium. Prominent, widespread expression of RNA-binding protein MSI2 supported the importance of posttranscriptional regulation in SCN functions and provided further evidence of stem-like cells. CONCLUSION: The results show that the SCN retains properties of immature neurons and these properties persist in culture conditions suitable for stem cells, where the SCN stem-like cells also proliferate. These properties may allow adaptive circadian rhythm adjustments. Further exploration should examine stem-like cells of the SCN in vivo, how they may affect circadian rhythms, and whether MSI2 serves as a master regulator of SCN stem-like properties.

Targeted mutation of the calbindin D28K gene disrupts circadian rhythmicity and entrainment.

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker in mammals. A salient feature of the SCN is that cells of a particular phenotype are topographically organized; this organization defines functionally distinct subregions that interact to generate coherent rhythmicity. In Syrian hamsters (Mesocricetus auratus), a dense population of directly retinorecipient calbindin D(28K) (CalB) neurons in the caudal SCN marks a subregion critical for circadian rhythmicity. In mouse SCN, a dense cluster of CalB neurons occurs during early postnatal development, but in the adult CalB neurons are dispersed through the SCN. In the adult retina CalB colocalizes with melanopsin-expressing ganglion cells. In the present study, we explored the role of CalB in modulating circadian function and photic entrainment by investigating mice with a targeted mutation of the CalB gene (CalB-/- mice). In constant darkness (DD), CalB-/- animals either become arrhythmic (40%) or exhibit low-amplitude locomotor rhythms with marked activity during subjective day (60%). Rhythmic clock gene expression is blunted in these latter animals. Importantly, CalB-/- mice exhibit anomalies in entrainment revealed following transfer from a light : dark cycle to DD. Paradoxically, responses to acute light pulses measured by behavioral phase shifts, SCN FOS protein and Period1 mRNA expression are normal. Together, the developmental pattern of CalB expression in mouse SCN, the presence of CalB in photoresponsive ganglion cells and the abnormalities seen in CalB-/- mice suggest an important role for CalB in mouse circadian function.

Circadian and homeostatic factors in arousal.

In the course of evolution, mechanisms have evolved to anticipate the timing of regularly occurring events. These mechanisms are encompassed in a circadian timing system that include a master clock localized to the suprachiasmatic nucleus of the hypothalamus and "slave" oscillators distributed throughout the body. This system serves multiple functions so as to ensure that various physiological processes occur at optimal and nonoverlapping times, to synchronize our activities to local environmental time, and to permit changes required to respond to new environmental circumstances. We suggest that a generalized concept of arousal (which includes alterations in responsiveness to homeostatic pressures, sensory stimuli and emotional reactivity, and to changes in motor activity) serves as a rubric in which to explore the multiple ways in which the circadian system modulates behavior.

Connectome of the Suprachiasmatic Nucleus: New Evidence of the Core-Shell Relationship.

A brain clock, constituted of ∼20,000 peptidergically heterogeneous neurons, is located in the hypothalamic suprachiasmatic nucleus (SCN). While many peptidergic cell types have been identified, little is known about the connections among these neurons in mice. We first sought to identify contacts among major peptidergic cell types in the SCN using triple-label fluorescent immunocytochemistry (ICC). To this end, contacts among vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), and calretinin (CALR) cells of the core, and arginine vasopressin (AVP) and met-enkephalin (ENK) cells of the shell were analyzed. Some core-to-shell and shell-to-core communications are specialized. We found that in wild-type (WT) mice, AVP fibers make extremely sparse contacts onto VIP neurons but contacts in the reverse direction are numerous. In contrast, AVP fibers make more contacts onto GRP neurons than conversely. For the other cell types tested, largely reciprocal connections are made. These results point to peptidergic cell type-specific communications between core and shell SCN neurons. To further understand the impact of VIP-to-AVP communication, we next explored the SCN in VIP-deficient mice (VIP-KO). In these animals, AVP expression is markedly reduced in the SCN, but it is not altered in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Surprisingly, in VIP-KO mice, the number of AVP appositions onto other peptidergic cell types is not different from controls. Colchicine administration, which blocks AVP transport, restored the numbers of AVP neurons in VIP-KO to that of WT littermates. The results indicate that VIP has an important role in modulating AVP expression levels in the SCN in this mouse.

Brain Activity during Methamphetamine Anticipation in a Non-Invasive Self-Administration Paradigm in Mice.

The ability to sense time and anticipate events is critical for survival. Learned responses that allow anticipation of the availability of food or water have been intensively studied. While anticipatory behaviors also occur prior to availability of regularly available rewards, there has been relatively little work on anticipation of drugs of abuse, specifically methamphetamine (MA). In the present study, we used a protocol that avoided possible CNS effects of stresses of handling or surgery by testing anticipation of MA availability in animals living in their home cages, with daily voluntary access to the drug at a fixed time of day. Anticipation was operationalized as the amount of wheel running prior to MA availability. Mice were divided into four groups given access to either nebulized MA or water, in early or late day. Animals with access to MA, but not water controls, showed anticipatory activity, with more anticipation in early compared to late day and significant interaction effects. Next, we explored the neural basis of the MA anticipation, using c-FOS expression, in animals euthanized at the usual time of nebulization access. In the dorsomedial hypothalamus (DMH) and orbitofrontal cortex (OFC), the pattern of c-FOS expression paralleled that of anticipatory behavior, with significant main and interaction effects of treatment and time of day. The results for the lateral septum (LS) were significant for main effects and marginally significant for interaction effects. These studies suggest that anticipation of MA is associated with activation of brain regions important in circadian timing, emotional regulation, and decision making.