GABA exerts opposite influence on warm and cold sensitive neurons in medial preoptic area in rats

The preoptic area regulates body temperature. GABA-ergic terminals and receptors are present in this area. Local microinjection studies have shown that GABA, its agonist, and its antagonist in this area may modulate body temperature. However, there are warm and cold sensitive neurons, and they are known to be affected by local and peripheral temperatures. In order to understand the mechanism of action of GABA in temperature regulation at the cellular level it was necessary to study the effect of GABA on individual thermosensitive neurons in in vivo preparations. Hence, in this study the responses of preoptic area thermosensitive and insensitive neurons to microiontophoretic application of picrotoxin, a GABA-A antagonist, were studied in anaesthetized rats. It was observed that a majority of both the thermosensitive and insensitive neurons were affected by microiontophoretic application of picrotoxin. Although almost an equal number of cold and warm sensitive neurons were affected, a majority of the cold sensitive neurons were excited, while a majority of the warm sensitive neurons were inhibited by picrotoxin. The results suggested that in normal conditions GABA acts through GABA-A receptor in modulating the spontaneous activity of thermosensitive neurons in the preoptic area. Furthermore, the results of the present study taken together with other reports suggest that normally GABA exerts a direct inhibitory action on the cold sensitive neurons, while it acts on presynaptic heteroreceptors, possibly on norepinephrinergic afferent input terminals on the warm sensitive neurons, for mediating its action.     

Interactions of reproductive steroids, osmotic pressure, and glucose on thermosensitive neurons in preoptic tissue slices

The preoptic area contains thermosensitive neurons, thought to be important in thermoregulation, and steroid-sensitive neurons, thought to be involved in reproduction. The preoptic area also contains osmosensitive neurons, considered important in water balance, and glucosensitive neurons, thought to function in the regulation of glucose. If these various neurons belong to separate populations, one might predict that most osmosensitive, glucosensitive, and steroid-sensitive neurons constitute the population of temperature-insensitive neurons rather than thermosensitive neurons. To test this hypothesis, single unit activity was recorded in preoptic tissue slices prepared from male rats. In addition to temperature changes, neuronal responses were examined with various perfusion media containing testosterone or estradiol (30 pg/mL), low glucose (1.0 mM), and increased osmotic pressure (309 mosmol/kg). It was found that the steroid-sensitive, osmosensitive, and glucosensitive neurons were not confined to the temperature-insensitive neurons; but that nearly half of the thermosensitive neurons responded to these nonthermal stimuli. This lack of specificity was also observed between osmosensitive and glucosensitive neurons; however, most of the steroid-sensitive neurons were highly specific for either estradiol or testosterone. Although these findings do not suggest a strong functional specificity for preoptic neurons, they do support studies emphasizing interactions between regulatory systems.     

Ultramicroscopic organization of the vascular plexus in the rabbit cerebral lateral ventricles and its changes under the effect of pyrogens

By means of electron microscopical methods organization of various components of the vascular plexus in the rabbit cerebral lateral ventricles--ependymal and supraependymal cells, as well as capillaries, making the base of the hemato-encephalic and hemato-liquor barriers of the plexus have been studied. Injection of bacterial pyrogen (pyrogenal) to the animals is accompanied with an increasing permeability in the barriers of the vascular plexus for certain blood cells (lymphocytes, monocytes), their activation and transformation into plasma cells and macrophages. Under pyrogenal effect in the ventricle cavities activation of the supraependymal cells and intensification of their interaction with the underlying ependyma take place. In the ependymal layer local dilatation of intercellular spaces and intensification of exocytic processes are observed. The changes revealed demonstrate an active reaction of some elements of the vascular plexus to fever, dependent on injection of the bacterial pyrogens into the organism.     

Effects of electrical stimulation of ventral septal area on firing rates of pyrogen-treated thermosensitive neurons in preoptic anterior hypothalamus from rabbits

Although there is considerable evidence supporting that fever evolved as a host defense response, it is important that the rise in body temperature would not be too high. Many endogenous cryogens or antipyretics that limit the rise in body temperature have been identified. Endogenous antipyretics attenuate fever by influencing the thermoregulatory neurons in the preoptic anterior hypothalamus (POAH) and in adjacent septal areas including ventral septal area (VSA). Our previous study showed that intracerebroventricular (I.C.V.) injection of interleukin-1beta (IL-1beta) affected electrophysiological activities of thermosensitive neurons in VSA regions, and electrical stimulation of POAH reversed the effect of IL-1beta. To further investigate the functional electrophysiological connection between POAH and VSA and its mechanisms in thermoregulation, the firing rates of thermosensitive neurons in POAH of forty-seven unit discharge were recorded by using extracellular microelectrode technique in New Zealand white rabbits. Our results show that the firing rates of the warm-sensitive neurons decreased significantly and those of the cold-sensitive neurons increased in POAH when the pyrogen (IL-1beta) was injected I.C.V. The effects of IL-1beta on firing rates in thermosensitive neurons of POAH were reversed by electrical stimulation of VSA. An arginine vasopressin (AVP) V1 antagonist abolished the regulatory effects of VSA on the firing rates in thermosensitive neurons of POAH evoked by IL-1beta. However, an AVP V2 antagonist had no effects. These data indicated that VSA regulates the activities of the thermosensitive neurons of POAH through AVP V1 but not AVP V2 receptor.     

The role of the medial preoptic area in the organization of paradoxical sleep

Influence of electrical stimulation of the medial preoptic area of cats on characteristics of paradoxical sleep and activity of medial preoptic neurons were studied in the course of sleep-waking cycle. Low-frequency stimulation of this structure in the state of slow-wave sleep evoked short-latency electrocortical desynchronization and induced transition to paradoxical sleep or paradocical sleep-like state. The same stimulation during the whole period of paradoxical sleep results in a reduction of its duration, practically complete disappearance of tonic stage, and increase in the density of rapid eye movements in phasic stage. The vast majority of meurons in the medial preoptic area decreased their firing rates during quiet waking and slow-wave sleep and dramatically increased their activity during paradoxical sleep. More than 50% of such neurons displayed activation 20-70 s prior to the appearance of electrocorticographic correlates of paradoxical sleep. Some neurons were selectively active during paradoxical sleep. Approximately 50% of cells increased their firing rates a few seconds prior to and/or during series of rapid eye movements. The results suggest that the medial preoptic area contains the units of the executive system (network) of paradoxical sleep and are involved in the mechanisms of neocortical desynchronization.     

The preoptic area of the domestic fowl

Summary The preoptic area of the domestic fowl (Gallus gallus) was studied by means of the Golgi technique. At least two regions can be recognized: (i) a medial and (ii) a lateral area, clearly distinguishable laterally from the adjacent telencephalic regions. The dendritic organization of the preoptic area is quite uniform. The neurons can be classified as isodendritic elements. The magnocellular elements are few and irregularly scattered mostly in the periventricular grey of the medial preoptic area. Of relevant interest is also the observation of some bipolar and horizontal neurons in the dorsal part of the medial preoptic area, near the anterior commissure.     

Ultrastructural analysis of estrogen receptor immunoreactive neurons in the medial preoptic area of the female rat brain

Neurons of the medial preoptic area were studied in the brain of the female rat by means of ultrastructural immunocytochemistry using a monoclonal antibody generated against purified estrogen receptor (ER), in order to delineate the morphological correlates of estrogen feedback mechanisms. In addition to the preoptic area, the bed nucleus of the stria terminalis, the arcuate and ventromedial nuclei of the hypothalamus exhibited an intense labelling for estrogen receptor. At the light microscopic level, the cell nuclei were immunoreactive. No major alterations were detected in the ER expression of medial preoptic neurons sampled during the estrous cycle, but proestrous rats did exhibit a slightly increased intensity of staining. At the ultrastructural level, the ER immunoreactivity was primarily confined to the nuclei and associated with the chromatin. Long term steroid deprivation elicited by either ovariectomy or ovariectomy plus adrenalectomy resulted in a marked intensity of nuclear labelling. This pattern was not influenced by acute estradiol replacement. These morphological data indicate that neurons of the medial preoptic area have the capacity to detect estrogens via receptor mechanisms and that changes in the level of the circulating ligand are manifested in an alteration in the staining for the estrogen receptor. The study also supports the revised concept of estrogen receptor action by demonstrating the presence of receptors in the nuclei of the cells, whether or not they are occupied by their ligand.     

Prostaglandin E2 attenuates preoptic expression of GABAA receptors via EP3 receptors

Prostaglandin E(2) (PGE(2)) has been shown to produce fever by acting on EP3 receptors within the preoptic area of the brain. However, there is little information about the molecular events downstream of EP3 activation in preoptic neurons. As a first step toward this issue, we examined PGE(2)-induced gene expression changes at single-cell resolution in preoptic neurons expressing EP3. Brain sections of the preoptic area from PGE(2)- or saline-injected rats were stained with an anti-EP3 antibody, and the cell bodies of EP3-positive neurons were dissected and subjected to RNA amplification procedures. Microarray analysis of the amplified products demonstrated the possibility that gene expression of gamma-aminobutyric acid type A (GABA(A)) receptor subunits is decreased upon PGE(2) injection. Indeed, we found that most EP3-positive neurons in the mouse preoptic area are positive for the alpha2 or gamma2 GABA(A) receptor subunit. Moreover, PGE(2) decreased the preoptic gene expression of these GABA(A) subunits via an EP3-dependent and pertussis toxin-sensitive pathway. PGE(2) also attenuated the preoptic protein expression of the alpha2 subunit in wild-type but not in EP3-deficient mice. These results indicate that PGE(2)-EP3 signaling elicits G(i/o) activation in preoptic thermocenter neurons, and we propose the possibility that a rapid decrease in preoptic GABA(A) expression may be involved in PGE(2)-induced fever.     

Melatonin decreases estrogen receptor expression in the medial preoptic area of inbred (LSH/SsLak) golden hamsters.

Daily late afternoon injections of melatonin (25 micrograms/day s.c.) were found to reduce the number of cells expressing estrogen receptor immunoreactivity in the medial preoptic area of ovariectomized inbred (LSH/SsLak) golden hamsters. Employing immunocytochemical analysis with the H222 monoclonal antibody to the human estrogen receptor, we examined the effects of melatonin on estrogen receptor expression in the hypothalamus, particularly the medial preoptic area, of ovariectomized virgin female hamsters. Analysis of the results showed that melatonin administration induced a 50-70% decrease in numbers of estrogen receptor-immunoreactive neurons in the medial preoptic area of ovariectomized female hamsters. Furthermore, an overall qualitative decrease in the intensity of estrogen receptor immunoreactivity was observed. In intact regularly cycling female hamsters used to monitor the efficacy of melatonin treatment, there were significant reductions in the serum levels of FSH, LH, and prolactin as measured by radioimmunoassay and in uterine and pituitary weights after 8 wk of melatonin treatment. These results suggest that melatonin may exert its anti-reproductive effects in hamsters by modulating estrogen receptor levels in medial preoptic area neurons, thus influencing steroid feedback mechanisms.     

Independent control of sexual and scent marking behaviors of male gerbils by cells in or near the medial preoptic area

This research studied the role of the medial preoptic area and adjacent cell populations in androgen control of scent marking and sexual behavior in male gerbils (Meriones unguiculatus). Experiment 1 replicated previous research showing that implants of testosterone propionate in or near the medial preoptic area reinstate marking behavior in castrates. Implant sites near the diagonal band of Broca or in the posterior part of the medial preoptic area, near the anterior hypothalamus, are more effective than other sites. Experiment 2 showed that medial preoptic area lesions permanently impair sexual behavior despite testosterone stimulation. Experiments 2–4 showed that lesions in or near the medial preoptic area can also disrupt scent marking; however, this behavior gradually recovered in many lesioned males, especially if they received testosterone. The data suggest that both scent marking and sexual behavior are controlled by androgens acting on cells in or near the medial preoptic area, but the cell populations involved in these two behaviors are probably not the same.     

Ultrastructural analysis of estrogen receptor immunoreactive neurons in the medial preoptic area of the female rat brain

Summary Neurons of the medial preoptic area were studied in the brain of the female rat by means of ultrastructural immunocytochemistry using a monoclonal antibody generated against purified estrogen receptor (ER), in order to delineate the morphological correlates of estrogen feedback mechanisms. In addition to the preoptic area, the bed nucleus of the stria terminalis, the arcuate and ventromedial nuclei of the hypothalamus exhibited an intense labelling for estrogen receptor. At the light microscopic level, the cell nuclei were immunoreactive. No major alterations were detected in the ER expression of medial preoptic neurons sampled during the estrous cycle, but proestrous rats did exhibit a slightly increased intensity of staining. At the ultrastructural level, the ER immunoreactivity was primarily confined to the nuclei and associated with the chromatin. Long term steroid deprivation elicited by either ovariectomy or ovariectomy plus adrenalectomy resulted in a marked intensity of nuclear labelling. This pattern was not influenced by acute estradiol replacement.These morphological data indicate that neurons of the medial preoptic area have the capacity to detect estrogens via receptor mechanisms and that changes in the level of the circulating ligand are manifested in an alteration in the staining for the estrogen receptor. The study also supports the revised concept of estrogen receptor action by demonstrating the presence of receptors in the nuclei of the cells, whether or not they are occupied by their ligand.Supported by grants from the IBRO/MacArthur Foundation Network Grant, the National Science Foundation (NSF INT 8703030), the Hungarian Academy of Sciences (OTKA 104), the National Institutes of Health (NS 19266), the National Foundation of Technical Development (OKKFT Tt 286/1986) and the Well-come Trust (14685/1.5)     

Coexistence of substance P and neurotensin-like peptides in single neurons of the rat hypothalamus

The coexistence of substance P with neurotensin-like immunoreactivity in certain neurons of the hypothalamus were demonstrated by the double immunofluorescence method. Substance P and neurotensin-like immunoreactivity coexisted within single neurons of some hypothalamic areas such as the medial preoptic area, perifornical area, anterior hypothalamic area, lateral hypothalamic area, periventricular nucleus and posterior hypothalamic nucleus, although they did not coexist in the majority of immunoreactive cells.     

Response of ERalpha-IR and ERbeta-IR cells in the forebrain of female rats to mating stimuli

Sexual behavior in female rats depends on the action of estradiol on estrogen receptors (ERs) found in particular brain regions. While hormonal regulation of female sexual behavior requires ERalpha, the possible functions of ERbeta remain to be clarified. Mating stimulation has several behavioral and physiological consequences and induces Fos expression in many brain areas involved in the regulation of reproductive behavior and physiology. In addition, some cells in which mating induces Fos expression coexpress ERalpha. To determine whether cells in which Fos is induced by a particular mating stimulus coexpress ERalpha, ERbeta, or both, we used a triple-label immunofluorescent technique to visualize ERalpha-, ERbeta-, and mating-induced Fos-immunoreactivity (Fos-ir) in neurons in which mating stimulation reliably increases Fos expression. Ovariectomized, hormone-primed rats were either unmated, received 15 mounts, or received 15 intromissions. In the rostral medial preoptic area, Fos-ir was induced by mounts alone primarily in cells coexpressing ERalpha-ir, while Fos-ir was induced by intromissions mainly in cells coexpressing both ERalpha-ir and ERbeta-ir (ERalpha/ERbeta-ir). In the dorsal part of the posterodorsal medial amygdala, Fos-ir was induced by intromissions in cells coexpressing ERalpha-ir and ERalpha/ERbeta-ir. However, in the ventral part of the posterodorsal medial amygdala, Fos-ir was induced by intromissions primarily in cells coexpressing only ERbeta-ir. These data suggest that qualitatively different sexual stimuli may be integrated through distinct ER-containing circuits in the rostral medial preoptic area and posterodorsal medial amygdala. The diversity in coexpression of type of ER in cells in different brain areas after various mating stimuli suggests a role for both ERalpha and ERbeta in the integration of hormonal information and information related to mating stimuli.     

A developmental study of the gonadotropin-releasing hormone neuronal system during sexual maturation in the male Djungarian hamster.

The number, morphology, and distribution of gonadotropin-releasing hormone cell bodies were studied in the brain of the male Djungarian hamster during sexual maturation. Males were reared in long days (16L:8D) and were killed at 15, 25, or 40 days of age, before (n = 5), during (n = 4), or after puberty (n = 4), respectively. Brain sections (60 microns) from the rostral olfactory tubercle to the medial basal hypothalamus were processed for GnRH immunocytochemistry. Unipolar and bipolar neurons were immunolabeled for GnRH; both subtypes had smooth cell contours. Analysis of every section from the olfactory tubercle to the arcuate nucleus indicated that at all ages more than 75% of all GnRH-immunoreactive cell bodies were distributed in the diagonal band of Broca, medial preoptic area, lateral preoptic area, and lateral hypothalamic area. GnRH-positive somata were also found in other brain regions, but in each of these areas they represented less than 6% of the total GnRH neuron number. In peripubertal 25-day-old males, during the rapid phase of testes growth, the number of unipolar, but not bipolar, GnRH-labeled cells nearly doubled in the diagonal band of Broca compared to soma numbers in this location in prepubertal 15-day-old males. The same number of unipolar GnRH-stained somata were found in this region in 40-day-old as in 25-day-old hamsters. In the medial preoptic area, a similar doubling of unipolar neuron numbers was observed at 25 days, but by 40 days the number of unipolar immunostained GnRH cells was secondarily reduced to a level comparable to that at 15 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diurnal levels of Fos immunoreactivity are elevated within hypocretin neurons in lactating mice

The hypocretins modulate arousal via actions across multiple terminal fields. Thus, alterations in hypocretin neurotransmission may contribute to altered sleep patterns observed during lactation. This study examined whether lactation is associated with alterations in the number of hypocretin neurons and in diurnal Fos-immunoreactivity within hypocretin neurons in female mice. Alterations in Fos-immunoreactivity were also examined within two hypocretin terminal regions; the medial preoptic area and the locus coeruleus. Fos-immunoreactivity was increased within hypocretin neurons and the medial preoptic area in lactating females. No differences were observed in the number of hypocretin neurons or in Fos-immunoreactivity within the locus coeruleus.     

Quantitative protein changes in the hypothalamic neurons of pubertal male rats, castrated neonatally.

The effect of neonatal castration of male rats on the sexual differentiation of the hypothalamus at puberty was studied. Male rats were castrated on days 1, 5 and 7 after birth. Their brains were processed for study on days 83-85. The neurons and cell nuclei of the preoptic area, mediobasal and ventromedial nuclei were assessed for changes in cell and nuclear sizes and dry weight (calculated using interferometric methods). Neonatal castration resulted in size as well as dry weight increase in the neurons of the anterior and mediobasal hypothalamus. The dry weight increased by 34% (P less than 0.001) in the medial preoptic area, by 25% (P less than 0.001) in the arcuate neurons and by 22% (P less than 0.001) in the ventromedial nucleus. The cell nuclei exhibited perceptible weight increase too--in the medial preoptic area 68% (P less than 0.001); 55% in the arcuate neurons (P less than 0.001), and 39% in the ventromedial region. The weight and size increases in neonatally castrated males were equal to those of females of the same age. In rats castrated on day 7, the cell sizes and dry weights of the ventromedial nucleus increased but the cell nuclei exhibited only little change. It is assumed that the changes in the dry weight may be the result of increased synthetic processes in these groups of neurons which are connected with the tonic and cyclic release of gonadotropins. These changes also point to the hypothalamic differentiation shifting to the female type in the absence of the inducing effect of androgens.     

杏仁内侧核注射AVP和AVPMcAb对家兔ET性发热效应的影响

目的和方法：在大脑杏仁内侧核微量注射精氨酸加压素（ＡＶＰ）和精氨酸加压素单克隆抗体（ＡＶＰＭｃＡｂ）,观察其对家兔内毒素（ＥＴ）性发热效应以及视前区一下丘脑前部（ＰＯＡＨ）温敏神经元放电活动的影响。结果：①杏仁内侧核微量注射ＡＶＰ能明显抑制家兔ＥＴ性发热效应,注射ＡＶＰＭｃＡｂ能明显易化家兔ＥＴ性发热效应;②杏仁外侧核分别注射ＡＶＰ和ＡＶＰＭｃＡｂ则对家兔ＥＴ性发热效应无明显影响;③杏仁内侧核分别注射ＡＶＰ和ＡＶＰＭｃＡｂ后ＰＯＡＨ热敏神经元和冷敏神经元放电活动均无明显变化。结论：家兔杏仁内侧核也是ＡＶＰ抗热效应的一个重要的作用部位,杏仁内侧核注射ＡＶＰ的抗热作用途径与隔区注射ＡＶＰ的抗热途径可能不同     

Sex difference in the neurotensin-immunoreactive cell populations of the preoptic area in quail (Coturnix japonica)

The distribution of neurotensin-immunoreactive cells and fibers was analyzed by immunocytochemistry in the forebrain of male and female Japanese quail (Coturnix japonica) by using an antibody directed against the C-terminal part of the molecule. Immunoreactive perikarya were located almost exclusively in the medial preoptic area with small populations also being present in the nucleus paraventricularis and in the tuberal region. Immunoreactive fibers were observed not only throughout the preoptic area-hypothalamus, but also in the septal region, nucleus intercollicularis, substantia grisea centralis and the classical catecholaminergic areas of the mesencephalon, such as the area ventralis of Tsai and the nucleus tegmenti pedunculo-pontinus, pars compacta. The preoptic neurotensin-immunoreactive cells were exclusively located within the boundaries of the sexually dimorphic medial preoptic nucleus. They were significantly more numerous in females than in males. In females, the number of neurotensin cells varied during the ovulatory cycle: fewer cells were observed in birds that were about to lay an egg (they had a calcified egg in the oviduct) than in those that had already laid or were not going to lay on that day. These data indicate major variations in the expression of neurotensin in response to neurochemical or neuroendocrine changes associated with ovulation.     

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.