Sex differences in the daily rhythm of vasoactive intestinal polypeptide but not arginine vasopressin messenger ribonucleic acid in the suprachiasmatic nuclei

The timing of the preovulatory surge of LH in female rodents is tightly coupled to the environmental light/dark cycle. This coupling is mediated by the circadian pacemaker located in the suprachiasmatic nuclei (SCN). Studies indicate that vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP), which are synthesized in the SCN, transmit circadian information from the SCN to GnRH neurons, thereby regulating the timing of the LH surge. However, to date, the rhythmic expression of these two peptides in the SCN has only been examined in males. The pattern of VIP expression in males is difficult to reconcile with its role in the LH surge. The purpose of the present study was to assess the rhythm of VIP messenger RNA (mRNA) levels in the SCN of female rats under several endocrine conditions. We compared this rhythm to that in males and to AVP mRNA rhythms in all experimental groups. In all groups of females, VIP mRNA levels were rhythmic, with peak expression occurring during the light phase and a nadir occurring during the dark phase. The rhythm was approximately 12 h out of phase compared with that in males. The rhythmic expression of AVP mRNA in the SCN was virtually identical in all groups of animals. Based on these results, we conclude that 1) the rhythm of VIP seen in the SCN of females during the day may serve as a facilitory signal from the SCN to GnRH neurons; 2) the sex-specific pattern of VIP mRNA does not depend on estradiol; and 3) AVP gene expression within the SCN is not sexually differentiated or altered by estradiol.     

Requirement for Brn-3b in early differentiation of postmitotic retinal ganglion cell precursors

Postnatal development of the spinal cord serotonergic (5-HT) system and of swimming movements were studied in newborn Sprague-Dawley rats, in which the serotonin level in the central nervous system was lowered in the prenatal period. For this purpose, para-chlorophenylalanine (PCPA) (300 mg/kg) was administered intraperitoneally to pregnant mother rats on day 8 of gestation, followed by a daily injection of PCPA (80 mg/kg) from day 9 of gestation to delivery. The postnatal development of the 5-HT system in the spinal cord of the pups (PCPA-treated pups) born from the PCPA-administered mothers was markedly delayed during the period between PND 1 and PND 10 in comparison to that in the control pups born from healthy mothers. Postnatally, the control pups developed their swimming movements regularly through three distinct phases: forelimb dominant, forelimb and hindlimb well coordinated, hindlimb dominant. In contrast, in the PCPA-treated pups, swimming movements were disorganized during the period in which the development of 5-HT system was delayed. However, between PND 17 and 22 in which the 5-HT system developed to that extent observed in the control pups, the pups eventually developed swimming movements as observed in the control pups. These results suggest that the disorganized developmental process of swimming movements in the PCPA-treated pups is due to the possible failure in the prenatal and postnatal development of the 5-HT system and its target system in the brain stem and the spinal cord.     

Serotonin stimulates corticotropin-releasing factor gene expression in the hypothalamic paraventricular nucleus of conscious rats

To examine the direct effects of serotonin (5-HT) on the release and synthesis of corticotropin-releasing factor (CRF) in the hypothalamic paraventricular nucleus (PVN), 5-HT was microinjected just onto the bilateral PVN of conscious rats. Plasma adrenocorticotropic hormone (ACTH) levels peaked at 30 min and returned to the basal levels in 90 min. Northern blot analysis revealed that the CRF messenger RNA (mRNA) level in the PVN as well as the proopiomelanocortin mRNA level in the anterior pituitary significantly increased 120 min after the 5-HT injections (50-250 nmol/side). Pretreatment with intracerebroventricular (i.c.v.) injection of pindobind 5-HT1A (5 nmol) or LY-278584 (500 nmol) completely abolished the 5-HT-induced ACTH response, whereas LY-53857 (100 nmol) was without effect. These results suggest that 5-HT stimulates CRF release, which has interactions with 5-HT1A and 5-HT3 receptors on CRF neurons in the PVN, and activates CRF synthesis in conscious rats.     

Roles of suprachiasmatic nuclei and intergeniculate leaflets in mediating the phase-shifting effects of a serotonergic agonist and their photic modulation during subjective day

Serotonin (5-HT) has been implicated in the phase adjustment of the circadian system during the subjective day in response to nonphotic stimuli. Two components of the circadian system, the suprachiasmatic nucleus (SCN) (site of the circadian clock) and the intergeniculate leaflet (IGL), receive serotonergic projections from the median raphe nucleus and the dorsal raphe nucleus, respectively. Experiment 1, performed in golden hamsters housed in constant darkness, compared the effects of bilateral microinjections of the 5-HT1A/7 receptor agonist, 8-hydroxydipropylaminotetralin (8-OH-DPAT; 0.5 microgram in 0.2 microliter saline per side), into the IGL or the SCN during the mid-subjective day. Bilateral 8-OH-DPAT injections into either the SCN or the IGL led to significant phase advances of the circadian rhythm of wheel-running activity (p < .001). The phase advances following 8-OH-DPAT injections in the IGL were dose department (p < .001). Because a light pulse administered during the middle of the subjective day can attenuate the phase-resetting effect of a systemic injection of 8-OH-DPAT, Experiment 2 was designed to determine whether light could modulate 5-HT agonist activity at the level of the SCN and/or the IGL. Serotonergic receptor activation within the SCN, followed by a pulse of light (300 lux of white light lasting 30 min), still induced phase advances. In contrast, the effect of serotonergic stimulation within the IGL was blocked by a light pulse. These results indicate that the respective 5-HT projections to the SCN and IGL subserve different functions in the circadian responses to photic and nonphotic stimuli.     

Potency of CPR without artificial breathing

The effect of dorsal raphe nucleus (DR) stimulation on the unit discharge of suprachiasmatic nucleus (SCN) neurons was studied and analyzed pharmacologically in the Wistar rats. Experimental results showed that DR stimulation could significantly inhibit the light-induced discharge of SCN neurons. Pharmacologically, this inhibition could be enhanced by monoamine oxidase inhibitor pargyline, attenuated by 5-hydroxytryptamine (5-HT) synthesis inhibitor parachlorophenylalanine and blocked by 5-HT receptor antagonist cyproheptadine. It was suggested that the inhibitory effect of DR stimulation on the light-sensitive SCN neuron discharge might be mediated by 5-HT.     

Atherosclerosis in the human brachial artery

The hypothalamic suprachiasmatic nucleus (SCN) of the mammal is the circadian pacemaker responsible for generation of circadian rhythms. Several immediate-early genes are expressed in the SCN by light stimuli which induce phase shifts of animal activity rhythms. In the present study, we investigated whether Homer, a PDZ-like protein which is rapidly induced following synaptic activation, mRNA expression is regulated by light in rat SCN. Homer mRNA expression in the SCN of rat killed at 4 h after onset of the light and dark phases was very low. One hour light stimuli during the subjective night dramatically induced Homer mRNA expression in the ventrolateral portion of the SCN, whereas light stimuli during the subjective light phase did not. This finding implies that Homer may be involved in the photic entrainment of the circadian clock.     

The suprachiasmatic nucleus: cellular approach to clock functioning

The hypothalamic suprachiasmatic nucleus (SCN), the circadian clock in mammals, generates and maintains a variety of daily rhythms. The present review is an attempt to synthesis experimental data on the anatomical organisation and cellular activities within SCN. The clock exhibits an endogenous rhythmic activity and can also be entrained by environmental synchronisers such as the light/dark cycle. It can be also influenced by internal signals such as the rhythmic secretion of melatonin which is under control of SCN activity. This tiny structure contains a variety of peptides organised in a specific distribution. It receives three main inputs from the retina (glutamate), the intergeniculate leaflet (NPY) and the dorsal raphe (serotonin). VIP containing cells located in the ventral part of SCN receive all these afferences and innervate the whole structure. VIP, PHI and GRP are likely implicated in the entrainment of the clock. The vasopressin (VP) cells exhibiting an endogenous rhythmic synthesis are considered as an output of the clock. The specific induction of immediate early genes (c-fos, jun B) within SCN by light pulses during the subjective night suggests the participation of these genes in the process of cellular entrainment by the photic input. The demonstration of a rhythmic astrocytic activity within SCN suggests an active involvement of this cellular population in the functioning of the clock facilitating or not neuronal communication. Cellular disturbances such as a decrease in VIP or VP cell population, reduction in the amplitude of functional cellular rhythms, astrocytic proliferation could explain some pathologies observed with ageing.     

Light responsiveness of the suprachiasmatic nucleus: long-term multiunit and single-unit recordings in freely moving rats

The suprachiasmatic nuclei (SCN) of the hypothalamus contain a pacemaker that generates circadian rhythms in many functions. Light is the most important stimulus that synchronizes the circadian pacemaker to the environmental cycle. In this paper we have characterized the baseline neuronal firing patterns of the SCN as well as their response to light in freely moving rats. Multiunit and single-unit recordings showed that SCN neurons increase discharge during daytime and decrease discharge at night. Discharge levels of individual neurons that were followed throughout the circadian cycle appeared in phase with the population and were characterized by low discharge rates (often below 1 Hz), with a twofold increase during the day. The effect of light on the multiunit response was dependent on the duration of light exposure and on light intensity, with light thresholds of approximately 0.1 lux. The light response level showed a strong dependency on time of day, with large responsiveness at night and low responsiveness during day. At both phases of the circadian cycle, the response level could be raised by an increase in light intensity. Single-unit measurements revealed that the time-dependent light response of SCN neurons was present also at the level of single units. The results show that the basic light response characteristics that were observed at the multiunit level result from an integrated response of similarly behaving single units. Research at the single-unit level is therefore a useful approach for investigating the basic principles of photic entrainment.     

Photic responses of suprachiasmatic area neurons in diurnal degus (Octodon degus) and nocturnal rats (Rattus norvegicus)

Photic sensitivity of cells in the suprachiasmatic nuclei (SCN), the principal pacemaker of the mammalian circadian system, has been documented in several species. In nocturnal rodents, the majority of photically responsive SCN cells are activated by retinal illumination. One report identified mostly photic suppressions among SCN cells in a diurnal rodent, studied under somewhat different conditions. We examined photic sensitivity of SCN cells in a predominantly diurnal rodent, the degu, studied in vivo under identical conditions to rats, and found that a large majority of photic SCN cells were suppressed by light. In both rats and degus, SCN cells were more responsive to light during the subjective night than during the subjective day. Light-responsive cells did not show a daily rhythm in baseline firing rates in either species, but rat SCN cells that did not respond to light were more active spontaneously during the subjective day. Light-unresponsive SCN cells in degus did not show a similar pattern. There are substantial differences in the neurophysiological activity and photic responsiveness of SCN cells in diurnal degus and nocturnal rats.     

Role of serotonin in the amygdala in hypothalamo-pituitary-adrenocortical responses

In view of the fact that the amygdala (AMG) and hypothalamic serotonin (5-HT) have an excitatory effect on the hypothalamo-pituitary-adrenal (HPA) axis, we have investigated the role of 5-HT in the AMG on this response. In intact freely moving rats, a mildly stressing short photic stimulation caused depletion of median eminence CRH-41, due to its release into the portal circulation and a rise in serum ACTH and corticosterone levels. This effect was significantly inhibited in rats in which 5-HT was depleted in the AMG following local 5,7-dihydroxytryptamine administration, which did not affect hypothalamic 5-HT content. Also, local pretreatment with ketanserin (a 5-HT2 receptor antagonist) in the AMG had the same inhibitory effect on the HPA axis response. These results indicate that AMG 5-HT has an important role in the activation of the HPA axis following neural stimulation and that 5-HT2 receptors are involved.     

Serotonin depletion exacerbates changes in striatal gene expression following quinolinic acid injection

Controversy exists as to whether serotonin (5-HT) plays a neuroprotective role during brain injury. We sought to determine if prior 5-HT depletion alters gene expression patterns normally associated with NMDA receptor-mediated excitotoxicity of the rodent striatum. Adult male Sprague-Dawley rats were treated systemically with saline or p-chlorophenylalanine (pCPA, 350 mg/kg) to block 5-HT synthesis. After 3 days, these rats received unilateral injection (1 microliter) of quinolinic acid (QA, 40 micrograms in 0.1 M phosphate buffered saline, pH 7.4) or saline vehicle directly into the anterior striatum. All rats were sacrificed 6 or 48 h later. Striatal tissues containing the saline or QA injection site were subjected to Northern analysis of preprotachykinin (PPT), preproenkephalin (PPE), and zif/268 mRNAs, as well as HPLC-EC detection of monoamines. At the time of the intrastriatal injection, 5-HT levels were depleted greater than 95% by pCPA as compared to saline controls. At 48 h post-QA injection, PPT and PPE mRNAs were markedly reduced within the striatal lesion site of saline/QA and pCPA/QA groups with respect to their contralateral uninjected control sides. In the pCPA/QA group, striatal PPE and PPT mRNA levels were further reduced as compared to the saline/QA group with PPE mRNA reductions reaching statistical significance at 95% (ANOVA with Scheffe F-test). Exacerbation of the excitotoxic lesion in the 5-HT depleted rat was further exemplified by a larger increase in zif/268 mRNA measured at 6 h post-intrastriatal injection in the pCPA/QA group as compared to saline/QA animals (P < 0.05 by ANOVA with Scheffe F-test). These results suggest that 5-HT depletion may adversely affect neuronal survival following intrastriatal QA exposure and lend support to the hypothesis that increasing 5-HT levels during NMDA receptor-mediated excitotoxicity may spare neurons destined to degenerate.     

Role of tryptophan in the elevated serotonin-turnover in hepatic encephalopathy

The increase of the brain levels of 5-hydroxyindoleacetic acid (5-HIAA) in hepatic encephalopathy (HE) suggests an increased turnover of serotonin (5-HT). To study the role of tryptophan on the increased brain 5-HT metabolism in HE, we attempted to monitor brain levels of tryptophan in rats with thioacetamide-induced acute liver failure by intravenous infusion of branched-chain amino acids (BCAA). The effect of this treatment on 5-HT synthesis and metabolism was investigated in five brain areas. BCAA-infusions (1 and 2 gm/kg/24 h) increased the ratio BCAA/aromatic amino acids in plasma two- and fourfold, respectively, and lowered both plasma and brain levels of tryptophan. At the higher BCAA-dose all parameters suggesting an altered brain 5-HT metabolism (increased brain levels of 5-HT and 5-HIAA, increased 5-HIAA/5-HT ratio) were almost completely normalized. These results provide further evidence for the role of tryptophan in the elevation of brain 5-HT metabolism and for a potential role of BCAA in the treatment of HE.     

Analysis of the B7 costimulatory pathway in allograft rejection

Stimulus control induced by (-)-2,5-dimethoxy-4-methylamphetamine (DOM) is believed to be mediated by agonism at 5-HT2A receptors. We hypothesized that blockade of (-) DOM-induced stimulus control may thus prove useful in the pre-clinical characterization of novel antipsychotic agents by providing an in vivo index of antagonism at that receptor. A previous study (Fiorella et al., 1995a) observed no antagonism by typical agents such as haloperidol and thioridazine, partial antagonism by the atypical agent, clozapine, and full antagonism by risperidone, a second atypical antipsychotic. The present investigation extends these observations to include seven additional drugs: SCH 23390, sulpiride, amperozide, melperone, octoclothepin, tiospirone and ritanserin. Of the drugs tested in rats in which (-) DOM-induced stimulus control had reliably been established, only tiospirone and ritanserin produced complete antagonism of the (-) DOM stimulus. Intermediate levels of antagonism were observed following treatment with amperozide, melperone, and octoclothepin. Finally, SCH 23390 and sulpiride yielded no evidence of antagonistic activity in (-) DOM-trained animals. Because clozapine and risperidone are both classified as atypical antipsychotics yet yield different degrees of antagonism of (-) DOM-induced stimulus control, we tested the substitution of risperidone for clozapine in rats trained with clozapine as a discriminative stimulus. No significant substitution was observed. In conclusion it appears that complete or partial antagonism of the (-) DOM stimulus serves as an effective pre-clinical means of identifying antipsychotics with significant 5-HT2A antagonist properties. However, the failure of risperidone to substitute for clozapine in pigeons (Hoenicke et al., 1992) and in rats (present study) suggests that despite their shared 5-HT2A antagonist properties, clozapine and risperidone differ with respect to their stimulus effects.     

The role of the intergeniculate leaflet in entrainment of circadian rhythms to a skeleton photoperiod

Mammalian circadian rhythms are synchronized to environmental light/dark (LD) cycles via daily phase resetting of the circadian clock in the suprachiasmatic nucleus (SCN). Photic information is transmitted to the SCN directly from the retina via the retinohypothalamic tract (RHT) and indirectly from the retinorecipient intergeniculate leaflet (IGL) via the geniculohypothalamic tract (GHT). The RHT is thought to be both necessary and sufficient for photic entrainment to standard laboratory light/dark cycles. An obligatory role for the IGL-GHT in photic entrainment has not been demonstrated. Here we show that the IGL is necessary for entrainment of circadian rhythms to a skeleton photoperiod (SPP), an ecologically relevant lighting schedule congruous with light sampling behavior in nocturnal rodents. Rats with bilateral electrolytic IGL lesions entrained normally to lighting cycles consisting of 12 hr of light followed by 12 hr of darkness, but exhibited free-running rhythms when housed under an SPP consisting of two 1 hr light pulses given at times corresponding to dusk and dawn. Despite IGL lesions and other damage to the visual system, the SCN displayed normal sensitivity to the entraining light, as assessed by light-induced Fos immunoreactivity. In addition, all IGL-lesioned, free-running rats showed masking of the body temperature rhythm during the SPP light pulses. These results show that the integrity of the IGL is necessary for entrainment of circadian rhythms to a lighting schedule like that experienced by nocturnal rodents in the natural environment.     

Circadian expression of NMDA receptor mRNAs, epsilon 3 and zeta 1, in the suprachiasmatic nucleus of rat brain

The presence of the N-methyl-D-aspartate (NMDA) receptor channel subunit epsilon 3 and zeta 1 mRNAs in the rat suprachiasmatic nucleus (SCN) was detected by sensitive in situ hybridization. The daily fluctuations in the epsilon 3 and zeta 1 subunit mRNAs in their abundance were found in the SCN to be high during the day and lower during the night under 12 h light:12 h dark conditions (LD 12:12). Under constant darkness for 15 days, both the epsilon 3 and the zeta 1 mRNA levels in the SCN remained cyclic. Furthermore, after exposure of rats to light, the epsilon 3 and zeta 1 subunit mRNAs increased during the subjective night, but not during the subjective day. These results implicate the involvement of the epsilon 3 and zeta 1 subunits in neuronal signaling in the SCN and suggest that these subunits of the NMDA receptor channel are regulated by light and a circadian clock.     

Neuropeptidergic organization of the suprachiasmatic nucleus in the blind mole rat (Spalax ehrenbergi)

The blind mole rat, Spalax, is a subterranean rodent with atrophied, subcutaneous eyes. Whereas most of the visual system is highly degenerated, the retino-hypothalamic pathway in this species has remained intact. Although Spalax is considered to be visually blind, circadian locomotor rhythms are entrained by the light/dark cycle. In the present study we used anterograde tracing techniques to demonstrate retinal afferents to the suprachiasmatic nucleus (SCN) and immunohistochemistry to examine the distribution of neuropeptides that are known to be involved in the regulation or expression of circadian rhythmicity. Based on the localization of retinal afferents and neuropeptides, the SCN can be divided into two subdivisions. The ventral region, which receives retinal afferents, also contains vasoactive intestinal polypeptide (VIP)-containing neurons, and fibers that are immunopositive to neuropeptide Y (NPY) and serotonin (5-HT). The dorsal region contains vasopressinergic neurons, but this latter cell population is extremely sparse compared to that described in other rodents. The dorsal region is also characterized by numerous VIP-immunoreactive fibers. The presence of NPY and 5-HT fibers suggests that the SCN receives afferent projections from the intergeniculate leaflet and from the raphe nuclei, respectively. These neuroanatomical results, together with previous studies of behavior, visual tract tracing, and immediate early gene expression, confirm that an endogenous clock and the capacity for light entrainment of circadian rhythms are conserved in the blind mole rat.     

VIP neurons in the human SCN in relation to sex, age, and Alzheimer's disease

The brains of 46 control subjects and 21 Alzheimer's disease (AD) patients were studied to determine whether there are age-related or AD-related changes in the vasoactive intestinal polypeptide (VIP) neuron population of the human suprachiasmatic nucleus (SCN). The number of VIP expressing neurons in the SCN of females, ranging in age from 10-91 years, did not change during normal aging. In males, however, the number of VIP neurons in the SCN was highest in the young subjects (10-40 years of age), after which, a dramatic decrease occurred in middle-aged subjects. This resulted in an age-dependent sex difference in the VIP cell population of the SCN: young males had twice as many VIP expressing SCN neurons as young females, whereas in the middle-aged groups, the females had twice as many VIP SCN neurons as the males. A significant decrease in the number of VIP expressing neurons in the SCN was found in female presenile AD patients, i.e., those younger than 65 years.     

Serotonergic innervation of the hypothalamic suprachiasmatic nucleus and photic regulation of circadian rhythms

Converging lines of evidence have firmly established that the hypothalamic suprachiasmatic nucleus (SCN) is a light-entrainable circadian oscillator in mammals, critically important for the expression of behavioral and physiological circadian rhythms. Photic information essential for the daily phase resetting of the SCN circadian clock is conveyed directly to the SCN from retinal ganglion cells via the retinohypothalamic tract. The SCN also receives a dense serotonergic innervation arising from the mesencephalic raphe. The terminal fields of retinal and serotonergic afferents within the SCN are co-extensive, and serotonergic agonists can modify the response of the SCN circadian oscillator to light. However, the functional organization and subcellular localization of 5HT receptor subtypes in the SCN are just beginning to be clarified. This information is necessary to understand the role 5HT afferents play in modulating photic input to the SCN. In this paper, we review evidence suggesting that the serotonergic modulation of retinohypothalamic neurotransmission may be achieved via at least two different cellular mechanisms: 1) a postsynaptic mechanism mediated via 5HT1A or 5ht7 receptors located on SCN neurons; and 2) a presynaptic mechanism mediated via 5HT1B receptors located on retinal axon terminals in the SCN. Activation of either of these 5HT receptor mechanisms in the SCN by specific 5HT agonists inhibits the effects of light on circadian function. We hypothesize that 5HT modulation of photic input to the SCN may serve to set the gain of the SCN circadian system to light.     

Reduction of arthritis and pneumonitis in motheaten mice by soluble tumor necrosis factor receptor

A variety of observations from several rodent species suggest that a serotonin (5-HT) input to the suprachiasmatic nucleus (SCN) circadian pacemaker may play a role in resetting or entrainment of circadian rhythms by non-photic stimuli such as scheduled wheel running. If 5-HT activity within the SCN is necessary for activity-induced phase shifting, then it should be possible to block or attenuate these phase shifts by reducing 5-HT release or by blocking post-synaptic 5-HT receptors. Animals received one of four serotonergic drugs and were then locked in a novel wheel for 3 h during the mid-rest phase, when novelty-induced activity produces maximal phase advance shifts. Drugs tested at several doses were metergoline (5-HT1/2 antagonist; i.p.), (+)-WAY100135 (5-HT1A postsynaptic antagonist, which may also reduce 5-HT release by an agonist effect at 5-HT1A raphe autoreceptors; i.p.), NAN-190 (5-HT1A postsynaptic antagonist, which also reduces 5-HT release via an agonist effect at 5-HT1A raphe autoreceptors; i.p.) and ritanserin (5-HT2/7 antagonist; i.p. and i.c.v.). Mean and maximal phase shifts to running in novel wheels were not significantly affected by any drug at any dose. These results do not support a hypothesis that 5-HT release or activity at 5HT1, 2 and 7 receptors in the SCN is necessary for the production of activity-induced phase shifts in hamsters.