Cyclic GMP alters the firing rate and thermosensitivity of hypothalamic neurons

The rostral hypothalamus, especially the preoptic-anterior hypothalamus (POAH), contains temperature-sensitive and -insensitive neurons that form synaptic networks to control thermoregulatory responses. Previous studies suggest that the cyclic nucleotide cGMP is an important mediator in this neuronal network, since hypothalamic microinjections of cGMP analogs produce hypothermia in several species. In the present study, immunohistochemisty showed that rostral hypothalamic neurons contain cGMP, guanylate cyclase (necessary for cGMP synthesis), and CNG A2 (an important cyclic nucleotide-gated channel). Extracellular electrophysiological activity was recorded from different types of neurons in rat hypothalamic tissue slices. Each recorded neuron was classified according to its thermosensitivity as well as its firing rate response to 2-100 microM 8-bromo-cGMP (a membrane-permeable cGMP analog). cGMP has specific effects on different neurons in the rostral hypothalamus. In the POAH, the cGMP analog decreased the spontaneous firing rate in 45% of temperature-sensitive and -insensitive neurons, an effect that is likely due to cGMP-enhanced hyperpolarizing K(+) currents. This decreased POAH activity could attenuate thermoregulatory responses and produce hypothermia during exposures to cool or neutral ambient temperatures. Although 8-bromo-cGMP did not affect the thermosensitivity of most POAH neurons, it did increase the warm sensitivity of neurons in other hypothalamic regions located dorsal, lateral, and posterior to the POAH. This increased thermosensitivity may be due to pacemaker currents that are facilitated by cyclic nucleotides. If some of these non-POAH thermosensitive neurons promote heat loss or inhibit heat production, then their increased thermosensitivity could contribute to cGMP-induced decreases in body temperature.     

An autoradiographic analysis of hypothalamic magnocellular neurons

Summary A light and electron microscopic autoradiographic analysis revealed that H³-valine infused into the lateral ventricle of normal and acutely dehydrated cats is preferentially taken up by the supraoptic (SON) and paraventricular nucleus (PVN) of the hypothalamus. Grain counts for these magnocellular neurons in normal unstressed cats were highest at one hour post infusion with a significant fall off by three hours. Uptake by the SON and PVN at one hour exceeded neighboring nuclear groups by a factor of 7 and 4 fold respectively. Electron microscopic autoradiographs from acutely dehydrated cats revealed the presence of emission grains in association with rough endoplasmic reticulum and large osmiophilic neurosecretory vesicles. In view of statistically significant uptake values and rapid turnover of H³-valine by SON and PVN in normal animals, coupled with emission tracks in direct association with underlying neurosecretory product in acutely dehydrated ones, it is speculated that valine may be an amino acid component of one or both of the neurophysins to which neurohypophyseal hormones are non-covalently linked.Supported by U.S.P.H.S. Grant NS 08171.U.S.P.H.S. Career Development Awardee K04 GM70001.The authors are deeply indebted to Dr. Finley P. Gibbs and Dr. Sandy Sorrentino, Jr. for their advice and assistance in statistically quantifying autoradiographic data.     

Glucagon-like immunoreactivity in hypothalamic neurons of the rat

Summary Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.     

CNS thermosensitivity and control of latent heat dissipation in the pigeon

Thermoregulatory responses to heat exposure were studied in 12 hand-reared, acclimated pigeons (Columbia livia). Measurements of body temperature (Tcl), brain temperature (Tbr), cutaneous water evaporation (CWE) and respiratory frequency (fr) were carried out in intact conscious heat exposed birds. In a second group of lightly restrained birds, fr and CWE were taken when temperatures of the trunk, brain and air (Ta) were independently changed. Increasing Tbr to 43.5–43.8°C induced a pronounced polypnea (deep and fast, (300 breaths min⁻¹) when Tcl regulated at 42.4°C. Moreover, when hyperthermia (Tcl = 43.0°C) was combined with increased Tbr (43.0–43.8°C) shallow and fast panting (>500 breaths min⁻¹) was evoked. CWE was probably elicited by inputs generated by the skin warm receptors as a result of increased Ta. Moreover it was demonstrated that warming the brain to 42.5°C elicits cutaneous water evaporation in birds exposed to 26°C. When a high Ta (60°C) is accompanied by a high relative humidity (17%), the combined effect generates inputs eliciting intensive panting. The integration of the present and earlier data allows us to generate a model demonstrating the distinguished significance of the trunk, skin and brain thermosensors in the regulation of both respiratory and cutaneous latent heat dissipation. The present model also emphasizes the fact that the highly thermosensitive pigeon brain responds in a similar pattern to that found in mammals     

Effects of cold adaptation and noradrenaline on thermosensitivity of rat hypothalamic neurons studiedin vitro

The experiments performed on rat brain slices have shown that cold adaptation of an animal influences the thermosensitivity of hypothalamic medial preoptical neurons. The adaptation is followed by an increase in the proportion of 38–41°C-thermoresponsive neurons and by a decrease in the proportion of 35–38°C-thermoresponsive units. In control animals, noradrenaline (NA) increased the responses of hypothalamic neurons to the action of 35–38°C temperature and decreased them to the action of 38–41°C temperature. Cold adaptation prevented the effects of NA on neuronal thermosensitivity, which suggests that their NA sensitivity is modified by cold adaptation.Neirofiziologiya/Neurophysiology, Vol. 26, No. 3, pp. 171–176, May–June, 1994.     

In vitro differentiation of hypothalamic magnocellular neurons of guinea pigs

Summary The development of neurophysin-oxytocin and neurophysinvasopressin containing neurons of the guinea pig was studied in vitro. Supraoptic (SO) and paraventricular (PV) nuclei were explanted from guinea pig foetuses at the 40th day of gestation and cultured in Maximov slides for nearly fifty days. The cultures were observed daily under a phase-contrast microscope. Explants were fixed every five days for observation with the electron microscope. At the time of explantation, magnocellular neurons were still immature. They acquired the morphologic characteristics of mature neurons, with axosomatic synapses, after about 10 days in vitro. After 15–20 days in vitro, they contained in addition neurosecretory granules (NSG), first in the Golgi region, then also dispersed in the cytoplasm. In the oldest culture (45–50 days), signs of granulolysis were regularly found. It appears that magnocellular neurosecretory neurons are able to differentiate in vitro from a primitive state in the absence of specific stimulation.The authors are indebted to M.J. Drian for help with tissue culture, and to D. Le Cren for photographic work     

Melanin-concentrating hormone producing neurons: Activities and modulations

Regulation of energy homeostasis in animals involves adaptation of energy intake to its loss, through a perfect regulation of feeding behavior and energy storage/expenditure. Factors from the periphery modulate brain activity in order to adjust food intake as needed. Particularly, “first order” neurons from arcuate nucleus are able to detect modifications in homeostatic parameters and to transmit information to “second order” neurons, partly located in the lateral hypothalamic area. These “second order” neurons have widespread projections throughout the brain and their proper activation leads them to a coordinated response associated to an adapted behavior. Among these neurons, melanin-concentrating hormone (MCH) expressing neurons play an integrative role of the various factors arising from periphery, first order neurons and extra-hypothalamic arousal systems neurons and modulate regulation of feeding, drinking and seeking behaviors. As regulation of MCH release is correlated to regulation of MCH neuronal activity, we focused this review on the electrophysiological properties of MCH neurons from the lateral hypothalamic area. We first reviewed the knowledge on the endogenous electrical properties of MCH neurons identified according to various criteria which are described. Then, we dealt with the modulations of the electrical activity of MCH neurons by different factors such as glucose, glutamate and GABA, peptides and hormones regulating feeding and transmitters of extra-hypothalamic arousal systems. Finally, we described the current knowledge on the modulation of MCH neuronal activity by cytokines and chemokines. Because of such regulation, MCH neurons are some of the best candidate to account for infection-induced anorexia, but also obesity.     

Modulate the phase transition temperature of hydrogels with both thermosensitivity and biodegradability

The synthesis and characterization of thermoresponsive hydrogels on the basis of N-isoproplyarylamide (NIPAAm) and acrylamide (AAm) copolymers crosslinked with a novel biodegradable crosslinker (PEG-co-PLA) were carried out in this study. Swelling measurement results demonstrated that four gels of PNAM5, PNAM10, PNAM12 and PNAM15 are thermoresponsive. The equilibrium swelling ratio and degradation of the hydrogels strongly depend on hydrogels composition. The morphology of the hydrogels was observed by scanning electron microscopy (SEM), and their thermal property was characterized by differential scanning calorimetry (DSC). The results show that the proportion of AAm in the copolymer has notable effect on the low critical solution temperature (LCST) of the hydrogel. When the molar ratio of AAm to NIPAAm was increased from 1:10 to 3:10 the LCST of the copolymer increased from 39.7 to 64.2 °C. The compression modulus of PNAM15 is of the highest among other hydrogels, because PNAM15 hydrogel has a more compact structure.     

Induction and decay of thermosensitivity in the flesh fly,Sarcophaga crassipalpis

When pharate adults of the flesh fly Sarcophaga crassipalpis are exposed to 40°C for 4 h they become more tolerant of high temperatures that are normally lethal (thermotolerance). In contrast, a 1-h exposure to 45°C decreases tolerance to a subsequent high temperature challenge (thermosensitivity). While control flies experience little mortality when held at 35°C for 24–48 h the thermosensitized flies die when exposed to 35°C. Sensitivity to a second thermal challenge slowly decays over a 72-h period. The acquisition of thermotolerance prevents the development of thermosensitivity. Brains from thermosensitized flies cultured at 43°C express the 72-kDa heat-shock protein and normal protein synthesis is inhibited. This implies that development of thermosensitivity is not associated with a loss in the capacity to express the 72-kDa heat-shock protein.Abbreviations ICN ICN Biomedicals, Inc. PO Box 19536, Irvine, CA 92713-9921 - LD light dark cycle - LT₅₀ time required to kill 50% of the test animals - SDS sodium dodecyl sulfate - TRIS Tris(hydroxymethyl)aminomethane     

Central,but not peripheral application of motilin increases c-Fos expression in hypothalamic nuclei in the rat brain

Previous immunocytochemical studies have shown the presence of motilin-immunoreactive neurons in specific brain areas of rats and autoradiographic studies in rabbits demonstrated motilin-binding sites in the central nervous system as well. Therefore, the aim of this study was to determine the anatomical localisation and neurochemical features of neurons activated by central administration of motilin (Mo) in rats. One week after cannulation, an intracerebroventricular injection of Mo (ICV, 3 g/6 l 0.9% saline) was given. For comparative purposes, a group of animals received an intravenous injection of motilin (IV, 9 g/300 l 0.9% saline) or an equal volume of saline. Neuronal excitation was assessed by c-Fos immunocytochemistry and combined with immunostaining for neurotransmitter markers. In contrast to the IV motilin-treated animals, the ICV motilin-treated animals displayed a significant increase in c-Fos expression in the supraoptic nuclei (SO) and paraventricular nuclei of the hypothalamus (PVH). At the level of the dorsomedial, ventromedial and lateral hypothalamic nuclei, ICV administration of motilin did not induce changes in c-Fos expression. In addition, the cerebellum did not show c-Fos expression after ICV motilin administration either. These findings might suggest distinct pathways and actions of centrally released and systemic motilin, but, particularly in rodents, do not rule out the possibility that the effects seen in the SO and PVH after ICV application are aspecific in nature. At present, we cannot exclude the fact that the results observed with motilin in rodents are due to cross-interaction with other related (e.g. ghrelin) or not yet identified receptors.     

Nematosomes or nucleolus-like bodies in hypothalamic neurons,the subfornical organ and adenohypophysial cells of the rat

Summary Fibrillar intracytoplasmic bodies, generally referred to as nematosomes or nucleolar like bodies (NLBs), are not only observed in various types of neurons in the hypothalamus and subfornical organ but also in the glandular cells of the pars tuberalis and the pars intermedia hypophyses. According to their cytochemical properties the NLBs are probably of ribonucleoprotein nature. Within the neurons NLBs occur within perikarya and processes. Their presence within the neurosecretory nerve fibers of the neural lobe proves their ability to migrate within the axon. Morphologic modifications of NLBs are observed in stimulated neurons and after colchicine treatment. Colchicine causes a characteristic dense texture of NLBs and a peripheral agglomeration of mitochondria very similar to the rosette arrangement observed in oocytes. Our findings suggest a structural and functional similarity of NLBs in neurons and oocytes, in which their nucleolar origin appears obvious and where they seem to represent preribosomal material. It is very likely that the axonal migration of the NLBs reflects transport of ribosomal RNA for delayed utilization (as in oocytes).This paper is dedicated to Prof. F. Stutinsky for his 65th birthday.     

Intracellular regions of potassium channels: Kv2.1 and heag

Intracellular regions of voltage-gated potassium channels often comprise the largest part of the channel protein, and yet the functional role of these regions is not fully understood. For the Kv2.1 channel, although there are differences in activation kinetics between rat and human channels, there are, for instance, no differences in movement of the S4 region between the two channels, and indeed our mutagenesis studies have identified interacting residues in both the N- and C -terminal intracellular regions that are responsible for these functional effects. Furthermore, using FRET with fluorescent-tagged Kv2.1 channels, we have shown movement of the C-termini relative to the N-termini during activation. Such interactions and movements of the intracellular regions of the channel appear to form part of the channel gating machinery. Heag1 and heag2 channels also display differing activation properties, despite their considerable homology. By a chimeric approach, we have shown that these differences in activation kinetics are determined by multiple interacting regions in the N-terminus and membrane-spanning regions. Furthermore, alanine mutations of many residues in the C-terminal cyclic nucleotide binding domain affect activation kinetics. The data again suggest interacting regions between N- and C- termini that participate in the conformational changes during channel activation. Using a mass-spectrometry approach, we have identified α-tubulin and a heat shock protein as binding to the C-terminus of the heag2 channel, and α-tubulin itself has functional effects on channel activation kinetics. Clearly, the intracellular regions of these ion channels (and most likely many other ion channels too) are important regions in determining channel function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

Globular adiponectin inhibits GnRH secretion from GT1-7 hypothalamic GnRH neurons by induction of hyperpolarization of membrane potential

Reproduction is accurately regulated by metabolic states in mammals. Adiponectin regulates luteinizing hormone (LH) secretion in the pituitary and energy homeostasis in the hypothalamus. We further investigated the gonadotropin-releasing hormone (GnRH) secretion regulation by adiponectin and its related molecular and electrophysiological mechanisms. The results showed that adiponectin receptors (AdipR1 and 2) were expressed in GT1-7 cells derived from hypothalamus neurons. GnRH secretion was inhibited via activation of AMP-activated protein kinase (AMPK). Moreover, we revealed that hyperpolarization of plasma membrane potentials and reduction of calcium influx was also caused by adiponectin.     

Toward an integrative theory of sleep and dreaming

Non-rapid-eye-movement sleep (NREMS) is triggered by the accumulation of adenosine, as a result of the perceptual overload of the brain cortex. NREMS starts in the most burdened regions of the cortex first and then eventually, after the released adenosine has reached the ventrolateral pre-optic nucleus area of the hypothalamus, triggers the "general NREMS pattern". This is accompanied by the usual familiar changes in the thalamocortical system. When NREMS reaches the slow-wave sleep (SWS) phase, with its predominant delta activity, brain metabolism drops significantly with the brain temperature, and this is recognized by the alarm system in the pre-optic anterior hypothalamus and/or the other thermostat circuit in the brainstem as a life-threatening situation. This alarm system triggers a reaction similar to abortive or partial awakening called rapid-eye-movement sleep (REMS), which is aimed at restoring the optimal body-core temperature. As soon as this restoration is accomplished by the activation of the brainstem-to-cortex ascending pathways, NREMS may continue, as may the interchange of the two sleep phases during the entire sleep period. During both NREMS and REMS, the same essential pattern occurs in the cortex: the loops "used" during the previous waking period, now deprived of external input, replay their waking activity at a lower frequency, one which enables them to restore the membrane's potential (possibly by means of LTD). During REMS, however, the cholinergic flood originating in the LTD/PPT nuclei of the pons tegmentum, increases in the basal forebrain and, provoking theta activity in the medial septum is extended to the hippocampus, causing the circuits that are active at that particular moment in the cortex, to store the information they carry as memory. This is the explanation of both the memory improvement known to be related to REMS and of dreams. Both phenomena are clearly side effects of REMS.     

Threshold temperature responses of thermosensitive neurons in guinea-pig hypothalamic slices

1. 1.|Neuronal activity in slices of the preoptic and anterior hypothalamic area of guinea-pigs during slow low-amplitude temperature changes analogous to temperature changes in the brain of endothermic animals, was extracellularly recorded.

2. 2.|42% of neurons showed threshold temperature responses. The threshold of response averaged 37.4°C for warm-sensitive neurons during warming and 37.0°C for cold-sensitive neurons during cooling.

3. 3.|The thresholds differed, on average, by 0.1°C in the same neuron at repeated temperature changes.

4. 4.|With temperatures 0.8°C above threshold on average (0.2°C in some units) neuronal activity reached a new high level that did not change either during a further exceeding of the threshold or prolonged maintenance of suprathreshold temperature.

5. 5.|The characteristics of the threshold temperature response of a hypothalamic neuron meet the criteria of thermoinduced structural rearrangements of cell membranes, caused by phase transitions of lipids, changes in protein conformation and cytoskeletal activity.

Effects of hypothalamic microinjection of PGE2 on body temperature and sympathetic nervous activities in the rabbit

The febrile response and sympathetic nervous response to hypothalamic microinjections of prostaglandin E₂ (PGE₂) were investigated in anesthetized rabbits. Microninjection of PGE₂ (500–1000 ng) caused an increase in rectal temperature of more than 0.3°C in 13 of 50 loci in the preoptic and anterior hypothalamic area (PO/AH). At 8 of these 13 loci, PGE₂ elicited response patterns in the sympathetic nervous system, such as an increase in cutaneous sympathetic nervous activity and decrease in renal sympathetic nervous activity. This pattern of sympathetic nervous responses was induced with a simultaneous increase in rectal temperature of more than 0.5°C. The 8 loci were distributed in the preoptic area, especially in the vicinity of the supraoptic nucleus. Electrolytic lesions of this region were made bilaterally, and intracerebroventricular injection of PGE₂ (8 µg/kg) was found to inhibit fever and sympathetic activity. The results demonstrate that the action of PGE₂ is responsible for the response patterns of sympathetic twigs during fever. The preoptic area, especially in the vicinity of the supraoptic nucleus, is most sensitive to PGE₂ for the patternized response of sympathetic neurons and fever.     

NO调控下丘脑钙激活钾通道的机制研究

目的 :研究NO对下丘脑神经元钙激活钾通道 (KCa)的作用及其机制。方法 :采用膜片钳技术内面向外式及细胞贴附式。结果 :NO可直接或通过升高cGMP来提高KCa通道的开放概率 (Po) ,这种增强作用是因为通道开放时间延长及开放频率增加。结论 :下丘脑神经元中NO可通过不同机制激活KCa。     

Morphological analysis of the neurons in the area of the hypothalamic magnocellular dorsal nucleus of the guinea pig

Summary In the guinea-pig hypothalamus, a group of enkephalinergic cells forms a well-circumscribed nuclear area called the magnocellular dorsal nucleus (MDN). This nucleus gives rise to a prominent projection to the lateral spetum: the hypothalamo-septal enkephalinergic pathway. In the present study, MDN neurons visualized by Golgi impregnation were subjected to morphological analysis in order to define the potential segregation of cellular types within the MDN. This study was complemented by additional observations of MDN neurons intracellularly injected by Lucifer yellow (LY) or horseradish peroxidase (HRP) during the in vitro incubation of hypothalamic slices. The following results were obtained from the analysis of 200 neurons: 163 Golgi-impregnated cells plus 37 injected cells (LY=14; HRP=23). Thirteen HRP-injected cells were precisely located in the MDN and 10 were located in the perifornical area surrounding the MDN. Four different cellular types were identified. Type-I neurons (41%) displayed a globular perikaryon, a variable number of primary dendrites that were poorly ramified, no preferential orientation, and an axon emerging from the perikaryon. Type-II neurons (30.5%) had a triangular perikaryon, three well-ramified primary dendrites, an orientation perpendicular to the third ventricle, and an axon emerging from the perikaryon. Type-III neurons (22%) exhibited a spindle-shaped perikaryon, two opposed well-ramified primary dendrites, an orientation perpendicular to the third ventricle, and an axon emerging from a primary dendrite. Type-IV neurons (6.5%), showed a globular perikaryon, a variable number of primary dendrites, poorly ramified dendrites, an orientation parallel to the third ventricle, and an axon whose orientation could not be identified. Neurons labeled after intracellular injection belonged to the first three cellular types.     

The role of prostaglandins and the hypothalamus in thermoregulation in the lizard, Phrynocephalus przewalskii (Agamidae)

Typically, small lizards rely heavily on behavioral thermoregulation rather than physiological mechanisms to control their rates of warming and cooling. We tested the hypothesis that prostaglandins participate in mediating the cardiovascular response to heating and cooling and temperature regulating neurons in the hypothalamus of the small lizard Phrynocephalus przewalskii. In vivo and in vitro treatments, heart rates (HRs) were all found to be higher during heating than during cooling, hysteresis was distinct below 30 and 26°C, respectively. In vivo, as administration of COX inhibitor, there were no differences in HR between heating and cooling at any body temperature and administration of agonist prostaglandins only produced a significant effect on HR below 25°C. Single-unit activity was recorded extracellularly in vitro with microelectrodes, found the firing rate of the continuous unit increased 23% when the temperature of the artificial cerebrospinal fluid dropped from 30–20°C. We conclude that prostaglandins appear to play only a limited role in modulating heart activity in Phrynocephalus przewalskii and suggest that cold-sensitive neurons in the preoptic and anterior hypothalamus (PO/AH) are involved in thermoregulatory control during heating or cooling.     

The Notch effector gene Hes1 regulates migration of hypothalamic neurons, neuropeptide content and axon targeting to the pituitary

Proper development of the hypothalamic-pituitary axis requires precise neuronal signaling to establish a network that regulates homeostasis. The developing hypothalamus and pituitary utilize similar signaling pathways for differentiation in embryonic development. The Notch signaling effector gene Hes1 is present in the developing hypothalamus and pituitary and is required for proper formation of the pituitary, which contains axons of arginine vasopressin (AVP) neurons from the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). We hypothesized that Hes1 is necessary for the generation, placement and projection of AVP neurons. We found that Hes1 null mice show no significant difference in cell proliferation or death in the developing diencephalon at embryonic day 10.5 (e10.5) or e11.5. By e16.5, AVP cell bodies are formed in the SON and PVN, but are abnormally placed, suggesting that Hes1 may be necessary for the migration of AVP neurons. GAD67 immunoreactivity is ectopically expressed in Hes1 null mice, which may contribute to cell body misplacement. Additionally, at e18.5 Hes1 null mice show continued misplacement of AVP cell bodies in the PVN and SON and additionally exhibit abnormal axonal projection. Using mass spectrometry to characterize peptide content, we found that Hes1 null pituitaries have aberrant somatostatin (SS) peptide, which correlates with abnormal SS cells in the pituitary and misplaced SS axon tracts at e18.5. Our results indicate that Notch signaling facilitates the migration and guidance of hypothalamic neurons, as well as neuropeptide content.