A comparative study of cytoarchitecture and serotonergic afferents in the suprachiasmatic nucleus of primates (Cebus apella and Callithrix jacchus) and rats (Wistar and Long Evans strains)

The suprachiasmatic nucleus, an essential diencephalic component of the circadian timing system, plays a role in the generation and modulation of behavioral and neuroendocrine rhythms in mammals. Its cytoarchitecture, neurochemical and hodological characteristics have been investigated in various mammalian species, particularly in rodents. In most species, two subdivisions, based on these aspects and considered to reflect functional specialization within the nucleus, can be recognized. Many studies reveal a typical dense innervation by serotonergic fibers in this nucleus, mainly in the ventromedial area, overlapping the retinal afferents. However, a different pattern occurs in certain animals, which lead us to investigate the distribution of serotonergic afferents in the suprachiasmatic nucleus of the Capuchin monkey, Cebus apella, compared to the marmoset, Callithrix jacchus, and two Rattus norvegicus lines (Long Evans and Wistar), and to reported findings for other mammalian species. Our morphometric data show the volume and length of the suprachiasmatic nucleus along the rostrocaudal axis to be greatest in C. apella > C. jacchus > Long Evans ≥ Wistar rats, in agreement with their body sizes. In C. apella, however, the serotonergic terminals occupy only some 10% of the nucleus' area, less than the 25% seen in the marmoset and rats. The distribution of the serotonergic fibers in C. apella does not follow the characteristic ventral organization pattern seen in the rodents. These findings raise questions concerning the intrinsic organization of the nucleus, as well as regarding the functional relationship between serotonergic input and retinal afferents in this diurnal species. © 2007 Elsevier B.V. All rights reserved.

Intrinsic organization of the suprachiasmatic nucleus in the capuchin monkey

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Exercise training normalizes an increased neuronal excitability of NTS-projecting neurons of the hypothalamic paraventricular nucleus in hypertensive rats

Stern JE, Sonner PM, Son SJ, Silva FC, Jackson K, Michelini LC. Exercise training normalizes an increased neuronal excitability of NTS-projecting neurons of the hypothalamic paraventricular nucleus in hypertensive rats. J Neurophysiol 107: 2912-2921, 2012. First published February 22, 2012; doi:10.1152/jn.00884.2011.-Elevated sympathetic outflow and altered autonomic reflexes, including impaired baroreflex function, are common findings observed in hypertensive disorders. Although a growing body of evidence supports a contribution of preautonomic neurons in the hypothalamic paraventricular nucleus (PVN) to altered autonomic control during hypertension, the precise underlying mechanisms remain unknown. Here, we aimed to determine whether the intrinsic excitability and repetitive firing properties of preautonomic PVN neurons that innervate the nucleus tractus solitarii (PVN-NTS neurons) were altered in spontaneously hypertensive rats (SHR). Moreover, given that exercise training is known to improve and/or correct autonomic deficits in hypertensive conditions, we evaluated whether exercise is an efficient behavioral approach to correct altered neuronal excitability in hypertensive rats. Patch-clamp recordings were obtained from retrogradely labeled PVN-NTS neurons in hypothalamic slices obtained from sedentary (S) and trained (T) Wistar-Kyoto (WKY) and SHR rats. Our results indicate an increased excitability of PVN-NTS neurons in SHR-S rats, reflected by an enhanced input-output function in response to depolarizing stimuli, a hyperpolarizing shift in Na+ spike threshold, and smaller hyperpolarizing afterpotentials. Importantly, we found exercise training in SHR rats to restore all these parameters back to those levels observed in WKY-S rats. In several cases, exercise evoked opposing effects in WKY-S rats compared with SHR-S rats, suggesting that exercise effects on PVN-NTS neurons are state dependent. Taken together, our results suggest that elevated preautonomic PVN-NTS neuronal excitability may contribute to altered autonomic control in SHR rats and that exercise training efficiently corrects these abnormalities.

Intrinsic organization of the suprachiasmatic nucleus in the capuchin monkey

The suprachiasmatic nucleus (SCN), which is the main circadian biological clock in mammals, is composed of multiple cells that function individually as independent oscillators to express the self-sustained mRNA and protein rhythms of the so-called clock genes. Knowledge regarding the presence and localization of the proteins and neuroactive substances of the SCN are essential for understanding this nucleus and for its successful manipulation. Although there have been advances in the investigation of the intrinsic organization of the SCN in rodents, little information is available in diurnal species, especially in primates. This study, which explores the pattern of expression and localization of PER2 protein in the SCN of capuchin monkey, evaluates aspects of the circadian system that are common to both primates and rodents. Here, we showed that PER2 protein immunoreactivity is higher during the light phase. Additionally, the complex organization of cells that express vasopressin, vasoactive intestinal polypeptide, neuron-specific nuclear protein, calbindin and calretinin in the SCN, as demonstrated by their immunoreactivity, reveals an intricate network that may be related to the similarities and differences reported between rodents and primates in the literature.

Purinergic and glutamatergic interactions in the hypothalamic paraventricular nucleus modulate sympathetic outflow

P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5′-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ± 13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflow

The cardiovascular actions of fractalkine/CX3CL1 in the hypothalamic paraventricular nucleus are attenuated in rats with heart failure

The paraventricular nucleus (PVN) of the hypothalamus plays an important role in the regulation of sympathetic nerve activity, which is significantly elevated in chronic heart failure (CHF). Fractalkine (FKN) and its cognate receptor, CX3CR1, are constitutively expressed in the central nervous system, but their role and physiological significance are not well known. The aims of the present study were to determine whether FKN plays a cardiovascular role within the PVN and to investigate how the actions of FKN might be altered in CHF. We show that both FKN and CX3CR1 are expressed on neurons in the PVN of rats, suggesting that they may have a physiological function in this brain nucleus. Unilateral microinjection of FKN directly into the PVN of anaesthetized rats elicited a significant dose-related decrease in blood pressure (1.0 nmol, -5 ± 3 mmHg; 2.5 nmol, -13 ± 2 mmHg; 5.0 nmol, -22 ± 3 mmHg; and 7.5 nmol, -32 ± 3 mmHg) and a concomitant increase in heart rate (1.0 nmol, 6 ± 3 beats min(-1); 2.5 nmol, 11 ± 3 beats min(-1); 5 nmol, 18 ± 4 beats min(-1); and 7.5 nmol, 27 ± 5 beats min(-1)) compared with control saline microinjections. In order to determine whether FKN signalling is altered in rats with CHF, we first performed quantitative RT-PCR and Western blot analysis and followed these experiments with functional studies in rats with CHF and sham-operated control rats. We found a significant increase in CX3CR1 mRNA and protein expression, as determined by quantitative RT-PCR and Western blot analysis, respectively, in the PVN of rats with CHF compared with sham-operated control rats. We also found that the blood pressure effects of FKN (2.5 nmol in 50 nl) were significantly attenuated in rats with CHF (change in mean arterial pressure, -6 ± 3 mmHg) compared with sham-operated control rats (change in mean arterial pressure, -16 ± 6 mmHg). These data suggest that FKN and its receptor, CX3CR1, modulate cardiovascular function at the level of the PVN and that the actions of FKN within this nucleus are altered in heart failure

The rat suprachiasmatic nucleus is a clock for all seasons.

Seasonal changes of daylength (photoperiod) affect the expression of hormonal and behavioral circadian rhythms in a variety of organisms. In mammals, such effects might reflect photoperiodic changes in the circadian pace-making system [located in the suprachiasmatic nucleus (SCN) of the hypothalamus] that governs these rhythms, but to date no functionally relevant, intrinsic property of the SCN has been shown to be photoperiod dependent. We have analyzed the temporal regulation of light-induced c-fos gene expression in the SCN of rats maintained in long or short photoperiods. Both in situ hybridization and immunohistochemical assays show that the endogenous circadian rhythm of light responsiveness in the SCN is altered by photoperiod, with the duration of the photosensitive subjective night under the short photoperiod 5-6 h longer than under the long photoperiod. Our results provide evidence that a functional property of the SCN is altered by photoperiod and suggest that the nucleus is involved in photoperiodic time measurement.

Two distinct oscillators in the rat suprachiasmatic nucleus in vitro.

In the rat suprachiasmatic nucleus slice culture, circadian rhythms in the release of arginine vasopressin and vasoactive intestinal polypeptide were measured simultaneously and longitudinally. The phase relationship between the two peptide rhythms was relatively constant in the culture without a treatment of antimitotic drugs but became diverse by an introduction of antimitotics, which is generally used to reduce the number of glial cells. By monitoring the two rhythms continuously for 6 days, different periods were detected in culture with the antimitotic treatment. Furthermore, N-methyl-D-aspartate shifted the phase of the two peptide rhythms in the same culture differently. These results indicate that the arginine vasopressin and vasoactive intestinal polypeptide release are under control of different circadian oscillators.

Hypothalamic paraventricular nucleus neurons regulate medullary catecholamine cell responses to restraint stress

Both physical and psychological stressors recruit catecholamine cells (CA) located in the ventrolateral medulla (VLM) and the nucleus of the solitary tract (NTS). In the case of physical stressors, this effect is initiated by signals that first access the central nervous system at or below the level of the medulla. For psychological stressors, however, CA cell recruitment depends on higher structures within the neuraxis. Indeed, we have recently provided evidence of a pivotal role for the medial amygdala (MeA) in this regard, although such a role must involve a relay, as MeA neurons do not project directly to the medulla. However, some of the MeA neurons that respond to psychological stress have been found to project to the hypothalamic paraventricular nucleus (PVN), a structure that provides significant input to the medulla. To determine whether the PVN might regulate medullary CA cell responses to psychological stress, animals were prepared with unilateral injections of the neurotoxin ibotenic acid into the PVN (Experiment 1), or with unilateral injections of the retrograde tracer wheat germ agglutinin-gold (WGA-Au) into the CA cell columns of the VLM or NTS (Experiment 2). Seven days later, animals were subjected to a psychological stressor (restraint; 15 minutes), and their brains were subsequently processed for Fos plus appropriate cytoplasmic markers (Experiment 1), or Fos plus WGA-Au (Experiment 2). PVN lesions significantly suppressed the stress-related induction of Fos in both VLM and NTS CA cells, whereas tracer deposits in the VLM or NTS retrogradely labeled substantial numbers of PVN cells that were also Fos-positive after stress. Considered in concert with previous results, these data suggest that the activation of medullary CA cells in response to psychological stress may involve a critical input from the PVN. (C) 2004 Wiley-Liss, Inc.

Dissociation of circadian and light inhibition of melatonin release through forced desynchronization in the rat

Pineal melatonin release exhibits a circadian rhythm with a tight nocturnal pattern. Melatonin synthesis is regulated by the master circadian clock within the hypothalamic suprachiasmatic nucleus (SCN) and is also directly inhibited by light. The SCN is necessary for both circadian regulation and light inhibition of melatonin synthesis and thus it has been difficult to isolate these two regulatory limbs to define the output pathways by which the SCN conveys circadian and light phase information to the pineal. A 22-h light-dark (LD) cycle forced desynchrony protocol leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral SCN (vlSCN) and the dorsomedial SCN (dmSCN). In the present study, we have used this protocol to assess the pattern of melatonin release under forced desynchronization of these SCN subregions. In light of our reported patterns of clock gene expression in the forced desynchronized rat, we propose that the vlSCN oscillator entrains to the 22-h LD cycle whereas the dmSCN shows relative coordination to the light-entrained vlSCN, and that this dual-oscillator configuration accounts for the pattern of melatonin release. We present a simple mathematical model in which the relative coordination of a single oscillator within the dmSCN to a single light-entrained oscillator within the vlSCN faithfully portrays the circadian phase, duration and amplitude of melatonin release under forced desynchronization. Our results underscore the importance of the SCN`s subregional organization to both photic input processing and rhythmic output control.

Calcium-binding proteins in the circadian centers of the common marmoset (Callithrix jacchus) and the rock cavy (Kerodon rupestris) brains

The hypothalamic suprachiasmatic nucleus (SCN) and the thalamic intergeniculate leaflet (IGL) are considered to be the main centers of the mammalian circadian timing system. In primates, the IGL is included as part of the pregeniculate nucleus (PGN), a cell group located mediodorsally to the dorsal lateral geniculate nucleus. This work was carried out to comparatively evaluate the immunohistochemical expression of the calcium-binding proteins calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR) into the circadian brain districts of the common marmoset and the rock cavy. In both species, although no fibers, terminals or perikarya showed PV-immunoreaction (IR) into the SCN, CB-IR perikarya labeling was detected throughout the SCN rostrocaudal extent, Seeming to delimit its cytoarchitectonic borders. CR-IR perikarya and neuropil were noticed into the ventral and dorsal portions of the SCN, lacking immunoreactivity in the central core of the marmoset and filling the entire nucleus in the rockcavy. The PGN of the marmoset presented a significant number of CB-, PV-, and CR-IR perikarya throughout the nucleus. The IGL of the rocky cavy exhibited a prominent CB- and CR-IR neuropil, showing similarity to the pattern found in other rodents. By comparing with literature data from other mammals, the results of the present study suggest that CB, PV, and CR are differentially distributed into the SCN and IGL among species. They may act either in concert or in a complementary manner in the SCN and IGL, so as to participate in specific aspects of the circadian regulation. (c) 2008 Elsevier Inc. All rights reserved.

Caracterização do perfil do ciclo sono-vigília em ratos sob dessincronização forçada

The circadian behavior associated with the 24 hours light-dark (LD) cycle (T24) is due to a circadian clock , which in mammals is located in the hypothalamic suprachiasmatic nucleus (SCN). Under experimental conditions in which rats are espoused to a symmetric LD 22h cycle (T22) the two SCN regions, ventrolateral (vl) and dorsomedial (dm), can be functionally isolated, suggesting that each region regulates distinct physiological and behavioral components. The vl region regulates the locomotor activity and slow wave sleep (SWS) rhythms, while the dm region assures the body temperature and paradoxical sleep (PS) rhythms regulation. This research aimed to deepen the knowledge on the functional properties of circadian rhythmicity, specifically about the internal desynchronization process, and its consequences to locomotor activity and body temperature rhythms as well as to the sleep-wake cycle pattern in rats. We applied infrared motion sensors, implanted body temperature sensors and a telemetry system to record electrocorticogram (ECoG) and electromyogram (EMG) in two rat groups. The control group under 24h period LD cycle (T24: 12hL-12hD) to the baseline record and the experimental group under 22h period LD cycle (T22: 11hL- 11hD), in which is known to occur the uncoupling process of the circadian locomotor activity rhythm where the animals show two distinct locomotor activity rhythms: one synchronized to the external LD cycle, and another expressed in free running course, with period greater than 24h. As a result of 22h cycles, characteristic locomotor activity moment appear, that are coincidence moments (T22C) and non coincidence moments (T22NC) which were the main focus or our study. Our results show an increase in locomotor activity, especially in coincidence moments, and the inversion of locomotor activity, body temperature, and sleep-wake cycle patterns in non coincidence moments. We can also observe the increase in SWS and decrease in PS, both in coincidence and non coincidence moments. Probably the increases in locomotor activity as a way to promote the coupling between circadian oscillators generate an increased homeostatic pressure and thus increase SWS, promoting the decreasing in PS

Serotonin and circadian rhythms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mechanisms involved in the pressor response to noradrenaline microinjection into the supraoptic nucleus of unanesthetized rats

We report on the cardiovascular effects of noradrenaline (NA) microinjection into the hypothalamic supraoptic nucleus (SON) as well as the central and peripheral mechanisms involved in their mediation. Microinjections of NA 1, 3, 10, 30 or 45 nmol/100 nL into the SON caused dose-related pressor and bradycardiac response in unanesthetized rats. The response to NA 10 nmol was blocked by SON pretreatment with 15 nmol of the alpha(2)-adrenoceptor antagonist RX821002 and not affected by pretreatment with equimolar dose of the selective alpha(1)-adrenoceptor antagonist WB4101, suggesting that local alpha(2)adrenoceptors mediate these responses. Pretreatment of the SON with the nonselective beta-adrenoceptor antagonist propranolol 15 nmol did not affect the pressor response to NA microinjection of into the SON. Moreover, the microinjection of the 100 nmol of the selective alpha(1)-adrenoceptor agonist methoxamine (MET) into the SON did not cause cardiovascular response while the microinjection of the selective alpha(2)adrenoceptor agonists BHT920 (BHT, 100 nmol) or clonidine (CLO, 5 nmol) caused pressor and bradycardiac responses, similar to that observed after the microinjection of NA. The pressor response to NA was potentiated by intravenous pretreatment with the ganglion blocker pentolinium and was blocked by intravenous pretreatment with the V(1)-vasopressin receptor antagonist dTyr(CH2)5(Me)AVP, suggesting an involvement of circulating vasopressin in this response. In conclusion, our results suggest that pressor responses caused by microinjections of NA into the SON involve activation of local alpha(2)-adrenoceptor receptors and are mediated by vasopressin release into circulation. (c) 2008 Published by Elsevier B.V.