Voluntary inhalation of methamphetamine: a novel strategy for studying intake non-invasively.

RATIONALE: The abuse of the psychostimulant methamphetamine (MA) is associated with substantial costs and limited treatment options. To understand the mechanisms that lead to abuse, animal models of voluntary drug intake are crucial. OBJECTIVES: We aimed to develop a protocol to study long-term non-invasive voluntary intake of MA in mice. METHODS: Mice were maintained in their home cages and allowed daily 1 h access to an attached tunnel leading to a test chamber in which nebulized MA was available. Restated, if they went to the nebulizing chamber, they self-administered MA by inhalation. This protocol was compared to injected and to imposed exposure to nebulized MA, in a series of seven experiments. RESULTS: We established a concentration of nebulized MA at which motor activity increases following voluntary intake resembled that following MA injection and imposed inhalation. We found that mice regulated their exposure to MA, self-administering for shorter durations when concentrations of nebulized MA were increased. Mice acquire the available MA by repeatedly running in and out of the nebulizing chamber for brief bouts of intake. Such exposure to nebulized MA elevated plasma MA levels. There was limited evidence of sensitization of locomotor activity. Finally, blocking access to the wheel did not affect time spent in the nebulizing chamber. CONCLUSIONS: We conclude that administration of MA by nebulization is an effective route of self-administration, and our new protocol represents a promising tool for examining the transitions from first intake to long-term use and its behavioral and neural consequences in a non-invasive protocol.

Differential contributions of intra-cellular and inter-cellular mechanisms to the spatial and temporal architecture of the suprachiasmatic nucleus circadian circuitry in wild-type, cryptochrome-null and vasoactive intestinal peptide receptor 2-null mutant mice.

To serve as a robust internal circadian clock, the cell-autonomous molecular and electrophysiological activities of the individual neurons of the mammalian suprachiasmatic nucleus (SCN) are coordinated in time and neuroanatomical space. Although the contributions of the chemical and electrical interconnections between neurons are essential to this circuit-level orchestration, the features upon which they operate to confer robustness to the ensemble signal are not known. To address this, we applied several methods to deconstruct the interactions between the spatial and temporal organisation of circadian oscillations in organotypic slices from mice with circadian abnormalities. We studied the SCN of mice lacking Cryptochrome genes (Cry1 and Cry2), which are essential for cell-autonomous oscillation, and the SCN of mice lacking the vasoactive intestinal peptide receptor 2 (VPAC2-null), which is necessary for circuit-level integration, in order to map biological mechanisms to the revealed oscillatory features. The SCN of wild-type mice showed a strong link between the temporal rhythm of the bioluminescence profiles of PER2::LUC and regularly repeated spatially organised oscillation. The Cry-null SCN had stable spatial organisation but lacked temporal organisation, whereas in VPAC2-null SCN some specimens exhibited temporal organisation in the absence of spatial organisation. The results indicated that spatial and temporal organisation were separable, that they may have different mechanistic origins (cell-autonomous vs. interneuronal signaling) and that both were necessary to maintain robust and organised circadian rhythms throughout the SCN. This study therefore provided evidence that the coherent emergent properties of the neuronal circuitry, revealed in the spatially organised clusters, were essential to the pacemaking function of the SCN.

Exploring spatiotemporal organization of SCN circuits.

Suprachiasmatic nucleus (SCN) neuroanatomy has been a subject of intense interest since the discovery of the SCN's 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.

Calbindin-D(28K) cells selectively contact intra-SCN neurons.

Calbindin-D(28K)-immunoreactive cells are tightly packed within a discrete region of the caudal aspect of the suprachiasmatic nuclei of hamsters. These cells receive direct retinal input and are Fos-positive in response to a light pulse. Knowledge of their afferent and efferent connections is necessary to understand suprachiasmatic nucleus organization. The first aim of the present study is to identify interconnections between calbindin and other peptidergic cells of the suprachiasmatic nuclei, using epi- and confocal microscopy and intra-suprachiasmatic nucleus tract tracing. The results indicate that essentially all calbindin cells receive numerous appositions from vasoactive intestinal polypeptide (VIP), neuropeptide Y and serotonin fibers and that most receive appositions from gastrin releasing peptide (GRP) and cholecystokinin (CCK) fibers. Reciprocal connections are seen from VIP, GRP and CCK cells but surprisingly, not from dorsomedial vasopressin cells. Injection of biotinylated dextran amine into the suprachiasmatic nucleus indicates that the ventrolateral suprachiasmatic nucleus projects to the entire nucleus, while the dorsal and medial regions of the suprachiasmatic nucleus project densely to most of the nucleus, except to the calbindin region. Analysis of colocalization of the peptides in the calbindin cell region shows that 91% of the substance P cells, 42% of the GRP cells and 60% of the VIP cells in the calbindin subnucleus coexpress calbindin-D(28K). Our results reveal a highly specialized topographical organization of connections among suprachiasmatic nucleus cells.

Diurnal and circadian variation of protein kinase C immunoreactivity in the rat retina.

We studied the dependence of the expression of protein kinase C immunoreactivity (PKC-IR) in the rat retina on the light:dark (LD) cycle and on circadian rhythmicity in complete darkness (DD). Two anti-PKC alpha antibodies were employed: One, which we call PKCalphabeta recognized the hinge region; the other, here termed PKCalpha, recognized the regulatory region of the molecule. Western blots showed that both anti-PKC antibodies stained an identical single band at approximately 80 kD. The retinal neurons showing PKC-IR were rod bipolar cells and a variety of amacrine neurons. After 3 weeks on an LD cycle, PKCalphabeta-IR in both rod bipolar and certain amacrine cells manifested a clear rhythm with a peak at zeitgeber time (ZT) of 06-10 hours and a minimum at ZT 18. No rhythm in total PKC-IR was observed when using the PKCalpha antibody, but, at ZT 06-10 hours, rod bipolar axon terminals showed increased immunostaining. After 48 hours in DD, with either antibody, rod bipolar cells showed increased PKC-IR. The PKCalpha antibody alone revealed that, after 48 hours, AII amacrine neurons, which lacked PKC-IR in an LD cycle, manifested marked PKC-IR, which became stronger after 72 hours. Light administered early in the dark period greatly increased PKCalphabeta-IR in rod bipolar and some amacrine neurons. Our data indicate that light and darkness exert a strong regulatory influence on PKC synthesis, activation, and transport in retinal neurons.

Expression of Period genes: rhythmic and nonrhythmic compartments of the suprachiasmatic nucleus pacemaker.

The mammalian circadian clock lying in the suprachiasmatic nucleus (SCN) controls daily rhythms and synchronizes the organism to its environment. In all organisms studied, circadian timekeeping is cell-autonomous, and rhythmicity is thought to be generated by a feedback loop involving clock proteins that inhibit transcription of their own genes. In the present study, we examined how these cellular properties are organized within the SCN tissue to produce rhythmicity and photic entrainment. The results show that the SCN has two compartments regulating Period genes Per1, Per2, and Per3 mRNA expression differentially. One compartment shows endogenous rhythmicity in Per1, Per2, and Per3 mRNA expression. The other compartment does not have rhythmic mRNA expression but has gated light-induced Per1 and Per2 and high levels of endogenous nonrhythmic Per3 mRNA expression. These results reveal the occurrence of differential regulation of clock genes in two distinct SCN regions and suggest a potential mechanism for producing functional differences in distinct SCN subregions.

Retinal innervation of calbindin-D28K cells in the hamster suprachiasmatic nucleus: ultrastructural characterization.

The authors have described a subregion of the hamster hypothalamic suprachiasmatic nucleus (SCN) containing cells that are immunopositive for the cytosolic calcium-binding protein, Calbindin-D28K (CaBP). Several lines of evidence indicate that this region may constitute the site of the pacemaker cells that are responsible for the regulation of circadian locomotor rhythms. First, 79% of the CaBP-immunoreactive (ir) neurons express Fos in response to photic stimulation, indicating that they are close to or part of the input pathway to pacemakers. Second, at the light microscopy level, retinal terminals innervate the CaBP subnucleus. Finally, destruction of this subnucleus renders animals arrhythmic in locomotor activity. In this study, the authors examined the ultrastructural relationship between cholera toxin (CTbeta) labeled retinal fibers and the CaBP-ir subregion within the hamster SCN. CTbeta-ir retinal terminals make primarily axo-somatic, symmetric, synaptic contacts with CaBP-ir perikarya. In addition, retinal terminals form synapses with CaBP processes as well as with unidentified profiles. There are also complex interactions between retinal terminals, CaBP perikarya, and unidentified profiles. Given that axo-somatic synaptic input has a more potent influence on a cell's electrical activity than does axo-dendritic synaptic input, cells of the CaBP subregion of the SCN are ideally suited to respond rapidly to photic stimulation to reset circadian pacemakers.

Calbindin expression in the hamster SCN is influenced by circadian genotype and by photic conditions.

Circadian rhythmicity in mammals, is controlled by the suprachiasmatic nuclei (SCN) of the hypothalamus. We previously described a discrete subnucleus in the core of the hamster SCN containing calbindin-D28k-positive cells which are fos-positive in response to a light pulse. Ablation of this subnucleus results in loss of circadian locomotor rhythmicity even when other parts of the SCN are spared. Here we show that Tau mutant hamsters have significantly more calbindin-D28k in the SCN than do wild type hamsters, and that SCN calbindin-immunoreactivity in the SCN increases in the dark. This is correlated with changes in magnitude of light mediated phase shifts in locomotion. The data are consistent with a role for calbindin cells in light mediated entrainment and phase shifting.

Multiple regulatory elements result in regional specificity in circadian rhythms of neuropeptide expression in mouse SCN.

It is well established that the mammalian circadian system consists of pacemaker cells in the suprachiasmatic nuclei (SCN). The mouse has become increasingly important in understanding the circadian timing system, due to the availability of mutant animals with abnormal circadian rhythms. In the present paper, we describe the organization of the mouse SCN, comparing the wild type and Clock mutant animal, with a special focus on those peptides bearing an upstream E-box element (vasopressin, vasoactive intestinal peptide, cholecystokinin and substance P). To this end, we describe the distribution of the foregoing SCN peptidergic cell types as well as gastrin-related peptide, calretinin, calbindin, somatostatin, neurotensin and retinal input to the SCN (determined by both tract tracing and fos-immunoreactivity in response to a light pulse). The Clock mutant mouse has decreased expression of vasopressin mRNA and protein in the SCN, with normal patterns of expression elsewhere in the brain. No other differences were detected between the Clock mutant and the wild type mouse. The results are consistent with the hypothesis that there are multiple regulatory elements of clock-controlled genes in the SCN.

Localization of a suprachiasmatic nucleus subregion regulating locomotor rhythmicity.

The bilaterally symmetrical suprachiasmatic nuclei (SCN) of the hypothalamus are the loci of the mammalian clock controlling circadian rhythms. Previous studies suggested that all regions of the SCN are equipotential as circadian rhythmicity is sustained after partial ablation, as long as approximately 25% of the nuclei are spared. In contrast to these results, we found that animals bearing partial lesions of the SCN that spared the subregion delimited by cells containing the calcium-binding protein calbindin-D28K (CaBP), sustained circadian locomotor rhythms. Furthermore, there was a correlation between the strength of the rhythm and the number of spared CaBP cells. Partial lesions that destroyed this region but spared other compartments of the SCN resulted in loss of rhythmicity. The next study indicates that transplants of half-SCN grafts that contain CaBP cells restore locomotor rhythms in SCN-lesioned host animals, whereas transplants containing SCN tissue but lacking cells of this subnucleus fail to restore rhythmicity. Finally, there was a correlation between the number of CaBP-positive cells in the graft and the strength of the restored rhythm. Taken together, the results indicate that pacemakers in the region of the CaBP subnucleus are necessary and sufficient for the control of locomotor rhythmicity and that the SCN is functionally heterogeneous.

Output signals of the SCN.

The suprachiasmatic nucleus (SCN) of the hypothalamus controls circadian rhythmicity in mammals (for reviews, see Refs. 33 and 59). Responses modulated by the SCN are numerous and include rhythms in sleep/wake cycles, locomotor, gnawing and general activity, temperature, ingestive behavior, and rhythms of hormonal and peptide secretions. Though a great deal is known about the neuroanatomical organization of the SCN, many elements of the structure-function relationships remain to be discovered. For example, it is not known which cellular components of the SCN function as driving pacemakers or which output signal(s) of these pacemakers are important for each of its functions. While some signals from pacemaker cells reach target regions by neural efferents, there is also evidence that rhythmic responses can be controlled by diffusible signals. This article reviews output signals from the SCN. The data available suggest that neural efferents are not necessary for the control of locomotor activity rhythms. Evidence that a diffusible signal is sufficient to restore activity rhythms in SCN-lesioned animals is described. Finally, possible physiological mechanisms for diffusible signals are suggested.

Fiber outgrowth from anterior hypothalamic and cortical xenografts in the third ventricle.

Fetal grafts of the anterior hypothalamus (SCN/AH) containing the suprachiasmatic nucleus (SCN) restore circadian rhythms to SCN-lesioned host hamsters and rats following implantation into the third ventricle. Previous studies suggest that intraventricular SCN/AH grafts are variable in their attachment sites, the extent of their outgrowth, and the precise targets innervated in the host brain. However, the use of different methods to analyze graft outgrowth in this model has previously led to inconsistent results. We have reevaluated the outgrowth of fetal rat SCN/AH grafts implanted in the third ventricle of hamsters by using two methods: the carbocyanine dye, 1,1'dioctadecyl-3,3'-tetramethylindocarbocyanine percholate (DiI), was placed directly onto grafted tissue; and a donor-specific neurofilament marker was used in conjunction with xenografts. We examined the specificity of outgrowth by comparing SCN/AH xenografts with that of control cortical (CTX) xenografts. To evaluate whether SCN/AH graft efferents arise from the donor SCN, we used micropunch grafts that contained minimal extra-SCN tissue. The results show that the use of a donor-specific neurofilament marker reveals more extensive SCN/AH graft outgrowth than DiI. SCN/AH graft efferents project into areas normally innervated by the intact SCN. However, this outgrowth is variable among graft recipients, is not specific to SCN/AH tissue, and does not necessarily derive from the donor SCN. The precise functional role of neural efferents arising from SCN/AH grafts in the restoration of circadian clock function and the extent of SCN-derived efferents remain to be determined.

Attachment site of grafted SCN influences precision of restored circadian rhythm.

Fetal hypothalamic grafts containing the suprachiasmatic nucleus (SCN) restore circadian locomotor rhythmicity when implanted into the third ventricle of SCN-lesioned hamsters. However, the quality of restored rhythms is variable, and the locomotor rhythms of grafted animals are generally less robust than those of intact animals. The present study explored whether anatomical features of the graft predict the quality of the recovered rhythm and whether such information might provide insight as to the target of the signal from the SCN that controls locomotor rhythmicity. The following graft parameters were assessed: distance between the attachment site of the graft and potential targets for the output signal from the SCN, number and overall size of SCN clusters, the size of the cluster closest to the SCN lesion site, and extent of vasoactive intestinal polypeptide (VIP) and vasopressin-associated neurophysin (NP) positive fiber outgrowth from the graft. The restored circadian activity rhythm was assessed by quantifying the precision of activity onset and the amount, period, and robustness of rhythmicity. The results indicate a significant positive correlation between the precision of activity onset and the proximity of the closest SCN cluster to the site of the lesioned host SCN. A more detailed analysis of the spatial location of the graft indicates that proximity of the graft in the dorsal and caudal directions, but not the rostral direction, is positively correlated with the precision of the recovered rhythm. This suggests two possibilities: the coupling signal may act on a site very near the SCN and travel preferentially in a rostro-caudal direction. Alternatively, the coupling signal may act on a site rostral to the SCN. That the site is not far rostral to the SCN was suggested by the lack of a correlation between the precision of the restored rhythm and the rostrally lying anterior medial preoptic nucleus. Finally, evaluation of NP- and VIP-ergic fibers in nuclei known to receive input from the SCN indicates that the extent of such innervation by graft efferents does not predict either the occurrence of recovery or the precision of the recovered rhythm. Overall, these results suggest that the target(s) of SCN pacemakers regulating locomotor rhythmicity lie in the hypothalamus, close to or rostral to the SCN.

A diffusible coupling signal from the transplanted suprachiasmatic nucleus controlling circadian locomotor rhythms.

The mammalian suprachiasmatic nuclei (SCN) transmit signals to the rest of the brain, organizing circadian rhythms throughout the body. Transplants of the SCN restore circadian activity rhythms to animals whose own SCN have been ablated. The nature of the coupling signal from the grafted SCN to the host brain is not known, although it has been presumed that functional recovery requires re-establishment of appropriate synaptic connections. We have isolated SCN tissue from hamsters within a semipermeable polymeric capsule before transplantation, thereby preventing neural outgrowth but allowing diffusion of humoral signals. Here we show that the transplanted SCN, like neural pacemakers of Drosophila and silkmoths, can sustain circadian activity rhythms by means of a diffusible signal.

Restoration of circadian rhythmicity by transplants of SCN "micropunches".

Although it is widely accepted that the suprachiasmatic nuclei (SCN) of the hypothalamus serve as biological pacemakers regulating circadian rhythmicity, a number of studies suggest that some circadian rhythms may be controlled by extra-SCN structures. Transplantation of fetal anterior hypothalamic tissue containing the SCN restores circadian locomotor rhythms in SCN-lesioned hosts. Such transplants, however, contain substantial extra-SCN hypothalamic tissue. In the present study, the authors examined the recovery of circadian locomotor rhythms in animals implanted with small grafts harvested by taking "micropunches" from vibratome-sectioned brain slices. Micropunches were taken from three areas of the hypothalamus known to receive retinal input: the SCN, the subparaventricular zone, and the supraoptic nucleus. The results indicate that transplants restricted to the SCN region are necessary and sufficient for restoration of circadian locomotor activity rhythms and that micropunches of tissues from other sources are ineffective.

Calbindin-D28K cells in the hamster SCN express light-induced Fos.

Although the suprachiasmatic nuclei (SCN) have been intensively analyzed, they contain a population of cells that has not yet been characterized. In this study, we examined the distribution of cells immunoreactive (ir) for calbindin-D28K (CaBP), calretinin (CR), parvalbumin, vasopressin-associated neurophysin (NP), substance P (SP), vasoactive intestinal peptide (VIP), and light-induced Fos-like protein. Previously unidentified cells in the core of the hamster SCN contained CaBP. Photic stimulation during the night induced Fos expression in about 75% of the CaBP-positive SCN cells, and about 50% of the Fos-positive cells in the core region expressed CaBP. These findings provide new information in the search for the cellular localization of pacemaker cells in the SCN, as photic input entrains the circadian system, and cells that receive photic input must be either part of the clock itself, or an upstream component of the clock.

How do fetal grafts of the suprachiasmatic nucleus communicate with the host brain?

Fetal grafts containing the hypothalamic suprachiasmatic nucleus (SCN), the site of an endogenous circadian pacemaker, can reinstate behavioral rhythms in lesioned recipients but the precise routes of communication between the graft and the host brain remain unknown. Grafts containing the SCN may convey temporal information to the host brain via neural efferents, diffusible factors, or a combination of both. We examined graft-host connections in anterior hypothalamic homografts (hamster-to hamster) and heterografts (rat-to hamster) implanted in the third ventricle by: (a) applying the carbocyanine dye, diI, directly onto homo- and heterografts in fixed tissue sections; and (b) using a donor-specific neurofilament (NF) antibody to immunocytochemically visualize heterograft efferents. DiI applied onto either homografts or heterografts labeled relatively few graft efferents which could be followed only short distances into the host brain. In contrast, NF-labeled heterograft efferents were both more numerous and extended for longer distances into the host brain than anticipated on the basis of diI tract tracing. The results suggest that anterior hypothalamic grafts implanted in the third ventricle provide substantial input to the adjacent host hypothalamus although it is not known whether these projections arise from SCN cells or from other extra-SCN hypothalamic tissue within these grafts. Nor is it known whether these projections are functional. To determine if neural efferents are required for the restoration of rhythmicity after grafting, we have encapsulated fetal anterior hypothalamus in a permselective polymer which prevents neurite outgrowth but allows diffusible signals to reach the host brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Host resets phase of grafted suprachiasmatic nucleus: a 2-DG study of time course of entrainment.

The object of the present experiment was to examine whether in an intact animal implanted with a hypothalamic graft, the phase of the host and grafted suprachiasmatic nucleus (SCN) would become synchronized. To this end, we first established the time at which daily fluctuations in local cerebral glucose utilization were maximal in the SCN in our population of adult hamsters. Next, we verified that rhythms of (14C)2-deoxyglucose uptake could be measured on the day after birth in pups that were to provide donor tissue. Host and donor animals were housed in opposite light:dark cycles. We then transplanted fetal SCN tissue into the third ventricle of intact hamsters, placed the grafted animals in constant darkness with access to running wheels and examined the phase of metabolic activity in host and donor SCN. For several days after grafting, there was no circadian fluctuation in the metabolic activity of either the host SCN or of the grafted SCN. During this time, the circadian locomotor rhythms were not disrupted, suggesting that pacemaker activity was not interrupted. By day 14 after transplantation, metabolic activity in the host SCN was elevated during subjective day and host and donor SCN were in synchrony, invariably with the phase of the host animal. We conclude that a signal from the host SCN resets the grafted SCN and not vice versa and that pacemaker cells communicate with each other rather than exerting independent effects on target sites.

Host resets phase of grafted SCN: influence of implant site, tissue specificity and pineal secretion.

The suprachiasmatic nuclei (SCN) have daily fluctuations in energy consumption with glucose utilization high in the day, and low at night. In a previous study, we used [14C]2-deoxyglucose (2-DG) uptake to index the phase of the SCN, and found that in intact hamsters bearing SCN grafts in the third ventricle (3V), the native and grafted SCN, which were initially 12 h out of phase, became synchronized to the phase of the host clock [32]. In the present study, adult males (host animals) and pregnant females (source of donor tissue) were housed in opposite light-dark cycles. Host animals were sacrificed 14 days after implantation with either (1) SCN grafts into the lateral ventricle (LV), or (2) cortical grafts into 3V, or (3) SCN grafts in 3V of pinealectomized hamster. The results indicate that rhythms of 2-DG uptake are not induced in cortical tissue grafts, that the host SCN does not entrain SCN grafts located in the LV, and that entrainment of the grafted SCN (located in 3V) by the host circadian system occurs in the absence of pineal secretions.

Heavy water lengthens the period of free-running rhythms in lesioned hamsters bearing SCN grafts.

Heavy water (D2O) lengthens the period of free-running circadian rhythms in most organisms. We compared the effect of D2O on free-running locomotor activity rhythms in intact and SCN-lesioned (SCN-X) hamsters that had recovered circadian rhythmicity following implantation of SCN grafts. The animals were housed individually in cages equipped with running wheels, and locomotor activity was monitored using a computer-based data acquisition system. At the end of the behavioral tests, animals were anesthetized and perfused. Brain sections were immunostained for vasoactive intestinal peptide (VIP) and vasopressin (VP) to evaluate the extent of the lesion and the presence of a functional graft. The D2O similarly lengthened the period of free-running activity without affecting amount of activity in both intact and in SCN-X grafted animals. The results indicate that D2O acts directly on the SCN to lengthen the free-running period, and suggest that coupling between pacemakers within the grafted SCN is as efficient as in the intact SCN.