Antidepressant-like and anorectic effects of the cannabinoid CB1 receptor inverse agonist AM251 in mice.

Psychopathological disorders, and depression in particular, are strongly linked to eating attitude in obese patients. The identification of cannabinoid CB1 receptors (CB1Rs) in areas of the central nervous system (CNS) that have been implicated in regulation of mood and food intake suggests that these receptors may mediate such a behavioral link. The goal of this study was to evaluate CB1R modulation of antidepressant-like effects and food intake. For this purpose, 129/SVE and C57BL/6 male mice were acutely dosed intraperitoneally (i.p.) with the CB1R inverse agonist AM251 (3-30 mg/kg) and tested, respectively, in the tail-suspension test (TST) and in the forced-swim test (FST), which have been used widely as tests sensitive to antidepressant compounds. Like the antidepressant desipramine (DMI, 16 mg/kg), AM251 significantly reduced immobility at 10 mg/kg in the TST and at 1 and 10 mg/kg in the FST. Such a decrease of immobility was not accompanied by an increase in motor activity in the open field, suggesting that occupancy of CB1R by AM251 induced antidepressant-like effects. This was supported by two additional experiments. First, the co-administration of the CB1R agonist CP55940, at a dose that did not induce motor impairment or profound hypothermia (0.01 mg/kg), reversed effects of AM251 in the TST. Secondly, effects of AM251 in the FST were absent in CB1R knockout (KO) mice. In addition to an antidepressant-like effect, AM251 reduced fasting-induced hyperphagia over a comparable dose range. Taken together, these data suggest that regulation of mood and food intake might be obtained through inverse agonism of CB1R.

Plasma agouti-related protein level: a possible correlation with fasted and fed states in humans and rats.

We measured plasma concentrations of agouti-related protein (AGRP) in humans and rats and determined whether these were affected by ingestion of a meal after fasting. In 17 healthy human subjects, the mean plasma concentration of AGRP was lower in the fed state than in the fasted state. Two hours after a breakfast meal, AGRP levels dropped by 39%. By contrast, a continued fast for 2 h increased the average AGRP concentration by 73%. In rats with diet-induced obesity, refeeding resulted in a 50% decrease in plasma AGRP concentrations following a fasting-refeeding protocol. Our results support the notion that plasma AGRP may serve as a biomarker for the transition from a fasted to the satiated state.

Distinct pharmacological mechanisms leading to c-fos gene expression in the fetal suprachiasmatic nucleus.

Maternal treatment with cocaine or a D1-dopamine receptor agonist induces c-fos gene expression in the fetal suprachiasmatic nuclei (SCN). Other treatments that induce c-fos expression in the fetal SCN include caffeine and nicotine. In the current article, the authors assessed whether these different pharmacological treatments activate c-fos expression by a common neurochemical mechanism. The results indicate the presence of at least two distinct pharmacological pathways to c-fos expression in the fetal rat SCN. Previous studies demonstrate that prenatal activation of dopamine receptors affects the developing circadian system. The present work shows that stimulant drugs influence the fetal brain through multiple transmitter systems and further suggests that there may be multiple pathways leading to entrainment of the fetal biological clock.

Targeted disruption of the mPer3 gene: subtle effects on circadian clock function.

Neurons in the mammalian suprachiasmatic nucleus (SCN) contain a cell-autonomous circadian clock that is based on a transcriptional-translational feedback loop. The basic helix-loop-helix-PAS proteins CLOCK and BMAL1 are positive regulators and drive the expression of the negative regulators CRY1 and CRY2, as well as PER1, PER2, and PER3. To assess the role of mouse PER3 (mPER3) in the circadian timing system, we generated mice with a targeted disruption of the mPer3 gene. Western blot analysis confirmed the absence of mPER3-immunoreactive proteins in mice homozygous for the targeted allele. mPer1, mPer2, mCry1, and Bmal1 RNA rhythms in the SCN did not differ between mPER3-deficient and wild-type mice. Rhythmic expression of mPer1 and mPer2 RNAs in skeletal muscle also did not differ between mPER3-deficient and wild-type mice. mPer3 transcripts were rhythmically expressed in the SCN and skeletal muscle of mice homozygous for the targeted allele, but the level of expression of the mutant transcript was lower than that in wild-type controls. Locomotor activity rhythms in mPER3-deficient mice were grossly normal, but the circadian cycle length was significantly (0.5 h) shorter than that in controls. The results demonstrate that mPer3 is not necessary for circadian rhythms in mice.

Interacting molecular loops in the mammalian circadian clock.

We show that, in the mouse, the core mechanism for the master circadian clock consists of interacting positive and negative transcription and translation feedback loops. Analysis of Clock/Clock mutant mice, homozygous Period2(Brdm1) mutants, and Cryptochrome-deficient mice reveals substantially altered Bmal1 rhythms, consistent with a dominant role of PERIOD2 in the positive regulation of the Bmal1 loop. In vitro analysis of CRYPTOCHROME inhibition of CLOCK: BMAL1-mediated transcription shows that the inhibition is through direct protein:protein interactions, independent of the PERIOD and TIMELESS proteins. PERIOD2 is a positive regulator of the Bmal1 loop, and CRYPTOCHROMES are the negative regulators of the Period and Cryptochrome cycles.

CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP.

DBP, the founding member of the PAR leucine zipper transcription factor family, is expressed according to a robust daily rhythm in the suprachiasmatic nucleus and several peripheral tissues. Previous studies with mice deleted for the Dbp gene have established that DBP participates in the regulation of several clock outputs, including locomotor activity, sleep distribution, and liver gene expression. Here we present evidence that circadian Dbp transcription requires the basic helix-loop-helix-PAS protein CLOCK, an essential component of the negative-feedback circuitry generating circadian oscillations in mammals and fruit flies. Genetic and biochemical experiments suggest that CLOCK regulates Dbp expression by binding to E-box motifs within putative enhancer regions located in the first and second introns. Similar E-box motifs have been found previously in the promoter sequence of the murine clock gene mPeriod1. Hence, the same molecular mechanisms generating circadian oscillations in the expression of clock genes may directly control the rhythmic transcription of clock output regulators such as Dbp.

Cocaine up-regulates norepinephrine transporter binding in the rat placenta.

We investigated the influence of 3 days of continuous cocaine exposure on norepinephrine transporter binding in the rat placenta. On gestational day 17, pregnant rats were implanted subcutaneously with two cocaine-containing Silastic capsules. There were two control groups, one that received capsules with vehicle only and was pair-fed to the cocaine-treated females, and a second group that was untreated and fed ad libitum. Placentas and fetal brains were harvested and frozen on gestational day 20, and subsequently subjected to saturation analyses for norepinephrine transporter binding using the selective ligand [3H]nisoxetine. There was a marked increase in the density (B(max)) of norepinephrine transporter binding sites in the placentas of the cocaine-treated animals compared to both control groups, but no change in the fetal brain. The mechanism underlying this up-regulation of the placental norepinephrine transporter is not yet known, but it could involve a beta-adrenoceptor- and cAMP-mediated induction of transporter gene expression.

mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop.

We determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1 -mediated transcription. This suggested cryptochrome involvement in the negative limb of the feedback loop. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially.

Photic induction of Period gene expression is reduced in Clock mutant mice.

The Clock mutation leads to abnormal circadian behavior and defective transcriptional activity of CLOCK, a basic helix-loop-helix (bHLH)/PAS protein. In situ hybridization was used to assess whether the Clock mutation affects the photic induction of mPer1, mPer2, and c-fos in the mouse suprachiasmatic nucleus (SCN). Exposure of wild-type mice to a 15 min light pulse at night rapidly induced expression of c-fos mRNA, with mPer1 and mPer2 mRNAs peaking later. Light exposure also increased c-fos, mPer1 and mPer2 mRNA levels in the SCN of homozygous Clock mutant mice, but the amplitude of the response to light was significantly reduced. Clock appears to play a role in circadian photoreception that is distinct from its role in the circadian oscillatory mechanism.

Expression of basic helix-loop-helix/PAS genes in the mouse suprachiasmatic nucleus.

The suprachiasmatic nuclei contain a circadian clock that drives rhythmicity in physiology and behavior. In mice, mutation of the Clock gene produces abnormal circadian behavior [Vitaterna M. H. et al. (1994) Science 264, 715-725]. The Clock gene encodes a protein containing basic helix-loop-helix and PAS (PER-ARNT-SIM) domains [King D. P. et al. (1997) Cell 89, 641-653]. The PAS domain may be an important structural feature of a subset of genes involved in photoreception and circadian rhythmicity. The expression and regulation of messenger RNAs encoding eight members of the basic helix-loop-helix/PAS protein superfamily were examined by in situ hybridization. Six of the genes studied (aryl hydrocarbon receptor nuclear transporter, aryl hydrocarbon receptor nuclear transporter-2, Clock, endothelial PAS-containing protein, hypoxia-inducible factor-1alpha and steroid receptor coactivator-1) were expressed in the suprachiasmatic nucleus of adult and neonatal mice. No evidence for rhythmicity of expression was observed when comparing brains collected early in the subjective day (circadian time 3) with those collected early in subjective night (circadian time 15). Neuronal PAS-containing protein-1 messenger RNA was expressed in the suprachiasmatic nucleus of adult (but not neonatal) mice, and a low-amplitude rhythm of neuronal PAS-containing protein-1 gene expression was detected in the suprachiasmatic nucleus. Neuronal PAS-containing protein-2 messenger RNA was not detected in adult or neonatal suprachiasmatic nucleus. Exposure to light at night (30 or 180 min of light, beginning at circadian time 15) did not alter the expression of any of the genes studied. The expression of multiple members of the basic helix-loop-helix/PAS family in the suprachiasmatic nucleus suggests a rich array of potential interactions relevant to the regulation of the suprachiasmatic circadian clock.

A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock.

We examined the transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN). A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. We show that the RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm.

Molecular analysis of mammalian timeless.

We cloned the mouse cDNA of a mammalian homolog of the Drosophila timeless (tim) gene and designated it mTim. The mTim protein shows five homologous regions with Drosophila TIM. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays revealed an array of interactions between the various mPER proteins but no mPER-mTIM interactions. The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock.

Relationship between [125I]RTI-55-labeled cocaine binding sites and the serotonin transporter in rat placenta.

We investigated the characteristics of cocainelike binding sites in rat placenta using [125I]RTI-55. [3H]paroxetine binding and immunocytochemical staining for serotonin [5-hydroxytryptamine (5-HT)] and for the 5-HT transporter were also used to obtain evidence for rat placental 5-HT uptake. [125I]RTI-55 saturation analyses with membranes from normal gestational day 20 placentas yielded curvilinear Scatchard plots that were resolved into high- and low-affinity components (mean dissociation constants of 0.29 and 7.9 nM, respectively). Drug competition studies with various monoamine uptake inhibitors gave rise to complex multiphasic displacement curves, although the results obtained with the selective 5-HT uptake inhibitor citalopram suggest that the 5-HT transporter is an important component of placental high-affinity [125I]RTI-55 binding. The presence of a rat placental 5-HT uptake system was additionally supported by the [3H]paroxetine binding experiments and by the presence throughout the placenta of immunoreactivity for 5-HT and the 5-HT transporter. Immunostaining with both antibodies was most intense in the junctional zone, whereas the density of [125I]RTI-55 binding sites was greater in the placental labyrinth. This discrepancy may be due to the fact that [125I]RTI-55 appears to be labeling additional cellular components besides the 5-HT transporter. The presence of cocaine- and antidepressant-sensitive 5-HT transporters in the placenta has important implications for the possible effects of these compounds on pregnancy and fetal development.

Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain.

We have cloned and characterized the mouse cDNA of a third mammalian homolog of the Drosophila period gene and designated it mPer3. The mPER3 protein shows approximately 37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. In the SCN, mPer3 RNA levels are not acutely altered by light exposure at different times during subjective night. This contrasts with the acute induction by light of mPer1 and mPer2 RNA levels during early and late subjective night. mPer3 is widely expressed in tissues outside of brain. In liver, skeletal muscle, and testis, mPer RNAs exhibit prominent, synchronous circadian oscillations. The results highlight the differential light responses among the three mammalian Per genes in the SCN and raise the possibility of circadian oscillators in mammals outside of brain and retina.

Norepinephrine transporters in rat placenta labeled with [3H]nisoxetine.

Previous research has identified a norepinephrine (NE) transporter in brush-border membranes from human placental syncytiotrophoblastic cells. In the present study, we used the selective ligand [3H]nisoxetine to demonstrate the presence of an NE transporter in rat placental membranes, determine the binding characteristics of the transporter and ascertain its localization by means of in vitro film and dry-emulsion autoradiography. Additional membrane binding studies were performed with [3H]GBR 12935 to determine whether a dopamine transporter also was present in rat placenta. Saturation analyses carried out on washed membrane fractions from whole rat placentas at gestational day 20 showed saturable [3H]nisoxetine binding (mean Kd = 1.00 nM, Bmax = 1.24 pmol/mg of protein) but no saturable binding of [3H]GBR 12935. When various monoamine uptake inhibitors were tested for their potency to inhibit placental [3H]nisoxetine binding, the results supported the conclusion that the radioligand was labeling an NE transporter. Autoradiographic studies showed the presence of [3H]nisoxetine binding in all three cellular zones of the rat placenta: the decidua, junctional zone and labyrinth. Binding was greatest in the junctional zone, particularly in the giant trophoblastic cells. These findings indicate the presence of a high density of NE transporters in the late-gestation rat placenta. Catecholamine uptake probably has a multifunctional role in placental physiology, and blockade of the NE transporter by certain drugs such as cocaine may therefore contribute to the adverse effects of such compounds on pregnancy outcome and offspring development.

Widespread expression of functional D1-dopamine receptors in fetal rat brain.

Maternal treatment with cocaine or the D1-dopamine receptor agonist, SKF 38393, induces expression of the immediate-early gene, c-fos, in fetal rodent brain. Our previous studies have focused on the suprachiasmatic nucleus late in gestation. In the present report, we examined the anatomical distribution of functional D1-dopamine receptors throughout fetal rat brain. Functional D1 receptors were defined using three complementary methods: in situ hybridization to detect D1 receptor mRNA, autoradiographic detection of 125I-SCH 23982 binding, and in situ hybridization to detect c-fos gene expression induced by maternal treatment with SKF 38393. D1-dopamine receptor binding, receptor mRNA, and SKF 38393-induced c-fos gene expression are widespread in fetal brain by late gestation. These data indicate that the fetal brain is sensitive to dopamine receptor activation, and suggest that gestational exposure to drugs of abuse acting via dopaminergic mechanisms may influence fetal brain function.

Haloperidol regulates neurotensin gene expression in striatum of c-fos-deficient mice.

The immediate-early gene c-fos has been proposed to play a role in induction of neurotensin/neuromedin N (NT/N) gene expression in the striatum following acute haloperidol (HAL) treatment. We utilized mice with targeted disruption of the c-fos gene to directly test this hypothesis. A robust increase in NT/N gene expression was observed in the dorsolateral striatum (DLSt) in both wild-type (WT) and c-fos-deficient mice 4-6 h after a single injection of HAL (1 or 4 mg/kg) indicating that products of the c-fos gene are not absolutely required for induction of NT/N mRNA. The basal expression of preprotachykinin, preproenkephalin and preprocholecystokinin mRNAs did not differ between WT and c-fos knockout mice. HAL treatment first increased striatal NT/N mRNA on postnatal day (PD) 10. HAL-induced NT/N mRNA levels were significantly lower in c-fos knockout mice than in WT mice on PD 10 and 15. These findings indicate that reliance on c-fos may be greater earlier in development and that redundant molecular pathways can lead to induction of NT/N mRNA in mouse striatum.

Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock.

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

[125I]4-aminobenzyl-5'-N-methylcarboxamidoadenosine (125I)AB-MECA) labels multiple adenosine receptor subtypes in rat brain.

Adenosine modulates neuronal activity and neurotransmitter release through interaction with cell surface receptors. Four adenosine receptor subtypes, A1, A2A, A2B, and A3 receptors, have been cloned and characterized. The agonist ligand, [125I]AB-MECA ([125I]4-aminobenzyl-5'N-methylcarboxamidoadenosine) has high affinity for recombinant A1 and A3 receptors [Olah et al., Mol. Pharmacol, 45 (1994) 978-982]. Rodent A3 receptors are relatively insensitive to xanthines; inhibition of A1 receptors with xanthines allows selective detection of A3 receptors despite the lack of selectivity of the ligand. We studied whether [125I]AB-MECA is useful for localization and characterization of A3 receptors in rat brain. The autoradiographic distribution of total [125I]AB-MECA (400 pM) binding closely resembled the pattern of A1 receptor binding, with highest levels in cerebellum, hippocampus, and thalamus, and moderate levels in cortex and striatum. Drug competition studies confirmed that almost all [125I]AB-MECA binding could be attributed to labeling of A1 receptors. Xanthine amine congener (1 microM) reduced specific [125I]AB-MECA binding by > 95%, indicating that xanthine-resistant A3 receptors represent a quantitatively minor subtype. Despite the use of a radioligand with high affinity and high specific activity, the low density of A3 receptors in rat brain appears insufficient to allow localization, or even consistent detection, of this receptor subtype. In the presence of DPCPX (50 nM, to block A1 receptors), residual [125I]AB-MECA binding to A2A receptors was observed in the striatum. Thus [125I]AB-MECA labels primarily A1 and A2A adenosine receptors in rat brain.

Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei.

We have characterized a mammalian homolog of the Drosophila period gene and designated it Per2. The PER2 protein shows >40% amino acid identity to the protein of another mammalian per homolog (designated Per1) that was recently cloned and characterized. Both PER1 and PER2 proteins share several regions of homology with the Drosophila PER protein, including the protein dimerization PAS domain. Phylogenetic analysis supports the existence of a family of mammalian per genes. In the mouse, Per1 and Per2 RNA levels exhibit circadian rhythms in the SCN and eyes, sites of circadian clocks. Both Per1 and Per2 RNAs in the SCN are increased by light exposure during subjective night but not during subjective day. The results advance our knowledge of candidate clock elements in mammals.