Sleep-inducing effects of adenosine microinjections into the medial preoptic area are blocked by flumazenil

Microinjection of a wide range of sedative agents, including triazolam, pentobarbital, ethanol and adenosine, into the medial preoptic area has been shown to increase sleep, suggesting that it is an important (though not necessarily the only) anatomic site mediating hypnotic effects of these compounds. The mechanism by which adenosine increases sleep at this site is not clear, but one possibility is that this is related to its effects on the GABA(A)-benzodiazepine receptor complex. In order to assess this possibility, this paper describes the administration of adenosine, alone and in combination with the benzodiazepine receptor blocker flumazenil, into the MPA. It was found that 12.5 and 25 nM of adenosine significantly reduced sleep latency and increased total sleep time. The sleep-inducing effect was blocked by flumazenil. Flumazenil caused a modest increase in total sleep, and prevented the increase in total sleep induced by the higher dose of adenosine. These data suggest that at least one aspect of the hypnotic properties of adenosine is mediated by a direct or indirect action on the GABA(A)-benzodiazepine receptor complex.     

Damage of the medial preoptic area impairs peripheral pilocarpine-induced salivary secretion

The existence of neural connections between the medial preoptic area (MPOA) and the salivary glands and the increase in salivation by thermal or electrical stimulation of the MPOA have suggested an important role of MPOA in the control of salivary gland function. Although direct cholinergic activation of the salivary glands induces salivation, recent studies have suggested that salivation produced by i.p. pilocarpine may also depend on the activation of central mechanisms. Therefore, in the present study, we investigated the effects of bilateral electrolytic lesions of the MPOA on the salivation induced by i.p. pilocarpine. Adult male Holtzman rats (n = 11-12/group) with bilateral sham or electrolytic lesions of the MPOA were used. One, five, and fifteen days after the brain surgery, under ketamine anesthesia, the salivation was induced by i.p. pilocarpine (1 mg/kg of body weight), and saliva was collected using pre-weighed small cotton balls inserted into the animal's mouth. Pilocarpine-induced salivation was reduced 1 and 5 days after MPOA lesion (341 +/- 41 and 310 +/- 35 mg/7 min, respectively, vs. sham lesions: 428 +/- 32 and 495 +/- 36 mg/7 min, respectively), but it was fully recovered at the 15th day post-lesion (561 +/- 49 vs. sham lesion: 618 +/- 27 mg/7 min). Lesions of the MPOA did not affect baseline non-stimulated salivary secretion. The results confirm the importance of MPOA in the control of salivation and suggest that its integrity is necessary for the full sialogogue effect of pilocarpine. However, alternative mechanisms probably involving other central nuclei can replace MPOA function in chronically lesioned rats allowing the complete recovery of the effects of pilocarpine.     

Induction of partial epileptic seizures by flumazenil

PURPOSE: This study addressed the efficacy of flumazenil (FMZ) to induce or activate interictal or ictal epileptic discharges in patients with medically intractable partial epilepsies. METHODS: Flumazenil, 1 mg, was injected intravenously in 67 patients undergoing presurgical monitoring for epilepsy surgery, 49 of whom had been treated with benzodiazepines (BZDs) before flumazenil was given. Continuous video electroencephalogram (EEG) monitoring with surface or intracranial electrodes was used to evaluate interictal EEG activity, ictal discharges, and the occurrence and semiology of clinically manifest epileptic seizures. RESULTS: Interictal epileptiform potentials did not change in frequency or distribution after FMZ. In patients not pretreated with BZDs, epileptic seizures could not be provoked. In eight of the 49 patients pretreated with BZDs, epileptic seizures occurred within 30 min of FMZ application. Seizure semiology and regional EEG onset were identical to seizures recorded without FMZ. Patients operated on according to seizure-onset localization with FMZ had a >75% reduction in seizure frequency or became seizure free. CONCLUSIONS: Seizure induction by FMZ seems to be a valid method for evaluating seizure semiology and localization of the seizure-onset zone during presurgical monitoring of patients with medically intractable localization-related epilepsies.     

Differential acute influence of medial and lateral preoptic areas on sleep-wakefulness in freely moving rats

The role of preoptic area (POA) in sleep-wakefulness and related EEG changes is well established. Anatomically the area is divided into medial (mPOA) and lateral (IPOA) portions having different physiological functions. Knowledge regarding the differential role, if any, of those two areas in sleep and wakefulness was lacking in the literature. Therefore, an attempt was made in this study, to investigate the same systematically. Experiments were conducted during day and night in freely moving rats. Electrophysiological parameters defining sleep and wakefulness were recorded before and after reversible inactivation of those two areas separately by microinjection of a local anaesthetic, marcain. The responses were opposite in nature depending upon the time, day or night, when the anaesthetic was applied. During the day, anaesthetization induced wakefulness while during the night, sleep was precipitated. However, anaesthetization of both the areas though induced similar qualitative response, the degree of the responses differed significantly. The results suggest that the mPOA is more effective in maintaining tonic sleep while the IPOA is more potent in the maintenance of tonic wakefulness in the normal rats. The finding supports and fits well with the existing knowledge.     

Efferents of the medial preoptic area in the guinea pig: An autoradiographic study

Medial preoptic axons were traced into the diagonal band of Broca and septum, particularly lateral septum. Other labeled fibers could be followed dorsally from medial preoptic area injections adjacent to the stria medullaris, and in the periventricular fiber system and the stria terminalis and its bed nucleus. The anterior and medial amygdaloid nuclei were labeled by fibers via the stria terminalis and others arching over the optic tract and through the substantia innominata. The lateral habenula was labeled. Labeled periventricular fibers reached the periventricular nucleus of the thalamus. Descending efferents were traced principally below the fornix and in the adjacent lateral hypothalamus to label the anterior hypothalamus, the tuberal nuclei, and median eminence. Axons of the medial preoptic area joined the medial part of the medial forebrain bundle and distributed to the reticular formation and the central gray of the midbrain and pons. A small amount of contralateral connections were described.     

Modulation of a viscerosomatic reflex by electrical and chemical stimulation of hypothalamic structures in the rat

Ventromedial forebrain structures were stimulated electrically with short (10-ms) trains of pulses to test for effects on a viscerosomatic reflex. Stimulation at many hypothalamic sites led to an attenuation or even a complete inhibition of reflex activity. The most sensitive sites, however (i.e. those requiring currents of 50 μA or less to inhibit the reflex), were located in the anterior hypothalamus/preoptic area (AH/POA) and rostrally in the diagonal band of Broca (DBB). At certain sites the effects of electrical stimulation were compared with those of microinjection of an excitatory amino acid (DL-homocysteic acid) which is known to excite neuronal cell bodies and not axons. The results of this part of the study indicated that activation of cell bodies located in the ventromedial AH/POA (from the level of the optic chiasma caudally to the level of DBB rostrally) mediate, at least in part, the inhibitory effects on visceral afferent processing. These data are discussed in relation to a possible role of AH/POA in the spinal processing of nociceptive information of visceral origin.     

Habituation and inhibitory avoidance training alter brain regional levels of benzodiazepine-like molecules and are affected by intracerebral flumazenil microinjection

The effects of habituation and inhibitory avoidance training on the rat brain regional levels of benzodiazepine (BZD)-like molecules and on central type BZD binding sites were examined. BZD-like immunoreactivity was decreased by 26-50% in the amygdala, cerebral cortex and septum of rats sacrificed immediately after stepping-down from the platform of an inhibitory avoidance apparatus (non-trained group) as compared to naive controls. Rats submitted to a second step-down session 20 h later (habituated group) have significantly lower BZD-like immunoreactivity in the septum (-60%) as compared to non-trained animals. Rats exposed to an inhibitory avoidance training, i.e. stepping-down and receiving a footshock (trained group), showed a significant reduction in the content of BZD-like molecules in cerebral cortex (-44%), amygdala (-68%), septum (-80%) and hippocampus (-82%) as compared to non-trained rats. In addition, the density of central type BZD binding sites was slightly increased in the hippocampus and septum of trained rats. No changes were observed in the apparent dissociation constant. No changes were observed in parallel measurements of [3H]-L-quinuclidinyl benzylate binding constants at cholinergic muscarinic binding sites. The immediate posttraining intrahippocampal bilateral injection of the central type BZD receptor antagonist flumazenil (10 nmol/hippocampus), enhanced the retention of habituation but not when injected in the amygdala or septum. In contrast, retention of the inhibitory avoidance task was significantly increased by flumazenil administered bilaterally into any of the 3 brain structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular serotonin variations during vigilance states in the preoptic area of rats: a microdialysis study

Numerous studies have shown that serotonergic transmission decreases from waking (W) to slow wave sleep (SWS) to paradoxical sleep (PS), suggesting an active role of serotonin (5-HT) in W but not in sleep. Conversely, the inhibition of 5-HT activity produces insomnia. This insomnia can be reversed by injections of 5-hydroxytryptophan in the preoptic area (POA), suggesting that 5-HT is necessary in this cerebral structure for sleep. Using microdialysis, we studied, 5-HT variations in the POA of rats in relation to vigilance states. 5-HT levels were higher during W than during during SWS and PS. 5-HT increased just before the rats fell asleep and then decreased during sleep. A decreased 5-HT transmission was also observed from SWS to PS. These data document a positive correlation between 5-HT levels in POA and wakefulness. Moreover, these observations are in favour of a permissive role of 5-HT in the POA during PS. A comparison between the POA and the prefrontal cortex in the sleep–wake cycle is discussed.     

Stimulation of the subcallosal fornix excites neurones in the cat preoptic region which project to the medial basal hypothalamus and in the medial forebrain bundle

The projection of neurones in the cat preoptic region driven by stimulation of the subcallosal fornix was systematically explored. We found 19% projected to the medial basal hypothalamus (MBH) and 10% projected in the medial forebrain bundle (MFB). Neurones projecting to 3he MBH were driven more often by stimulation of the lateral aspect of the fornix than the medial aspect (P = 0.006) and these neurones were thought to lie in the medial division of the preoptic nucleus (MPNm) since they were found significantly more often in the medial 0.6 mm of the preoptic region than more laterally (P = 0.028). A reverse projection from the preoptic region in the fornix is also suggested based on the finding of 24 antidromically activated neurones inthe preoptic region following stimulation of the fornix.     

Effects of lesions of a sexually dimorphic nucleus in the preoptic/anterior hypothalamic area on the expression of androgen- and estrogen-dependent sexual behaviors in male ferrets

The male nucleus of the preoptic/anterior hypothalamic area (MN-POA/AH) is a sexually dimorphic structure present in male, but not in female ferrets. Ovariectomized female ferrets given increasing dosages of estradiol benzoate (EB) normally run faster towards a stud male in an L-maze (i.e. become more proceptive). In two separate experiments, only gonadectomized males with bilateral damage to the MN-POA/AH following large or small electrolytic lesions approached stud males more quickly in response to EB. By contrast, males which received sham lesions, unilateral large POA/AH lesions, or bilateral lesions which missed the MN-POA/AH on at least one side failed to show EB-induced reductions in approach latencies in pre- or post-operative tests. Males with large POA/AH lesions also displayed significant post-operative decrements in masculine sexual behaviors during treatment with a high dose of testosterone propionate (TP). Less severe, but statistically significant deficits in masculine coital performance were also observed in males with small lesions which damaged the MN-POA/AH bilaterally; however, the ability of these males to achieve intromissions appeared normal. Together, these results suggest that the MN-POA/AH of the male ferret exerts an inhibitory influence on estrogen-dependent proceptive responsiveness, but play only a minor role in the control of masculine coital behavior.     

Sexual dimorphisms in the soma size of neurons in the brain of whiptail lizards (Cnemidophorus species)

Soma area was significantly larger in the anterior hypothalamus-preoptic area of male than female Cnemidophorus inornatus, and it was significantly larger in females than males in the ventromedial hypothalamus. These results parallel those on the volume of the brain areas in these animals, and therefore probably explain at least part of the dimorphisms seen in this sexually reproducing species. Soma size in parthenogenetic C. uniparens also parallels volume.     

Cytokines and Thermoregulation: Interleukin-9 Injected in preoptic area fails to evoke fever in rats

A number of the members of the family of cytokines including IL-1, IL-2, IL-6, and IL-11 act directly in the brain to induce a febrile response in the rat and other species. The purpose of this study was to examine the effect of interleukin-9 (IL9) when this cytokine is applied directly to the thermosensitive and pyrogen reactive region of the anterior hypothalamic, preoptic area (AH/POA). In male Sprague-Dawley rats, guide cannulae for microinjection into the AH/POA were implanted stereotaxically, and radio transmitters for monitoring body temperature (T_b) were placed intraperitoneally. Following postoperative recovery, recombinant murine macrophage inflammatory protein (MIP)-1β was microinjected in the AH/POA of each rat in a dose of 28 pg/1μl to identify pyrogen reactive sites in the AH/POA. Then recombinant human IL-9 was suspended in pyrogen-free CSF vehicle and microinjected in the same sites in concentrations of 2.4, 24, and 240 U/μl. In contrast to the pyrexic action of MIP-1β, IL-9 failed to elicit a significant alteration in the T_b of the rats at any of the doses tested. IL-9 was also without effect on the intakes of either water or food. These results demonstrate that IL-9 applied to the region of the diencephalon in which other cytokines act to evoke fever may not play a direct role in the thermogenic component underlying the acute phase response. However, as demonstrated in several different cell systems, IL-9 may require a cofactor related to pyrogen for a febrile response to develop.     

Microinjection of galanin-like peptide into the medial preoptic area stimulates food intake in adult male rats

Galanin-like peptide (GALP) is a neuropeptide implicated in the regulation of feeding behaviour, metabolism and reproduction. GALP is an endogenous ligand of the galanin receptors, which are widely expressed in the hypothalamus. GALP is predominantly expressed in arcuate nucleus (ARC) neurones, which project to the paraventricular nucleus (PVN) and medial preoptic area (mPOA). Intracerebroventricular or intraparaventricular (iPVN) injection of GALP acutely increases food intake in rats. The effect of GALP injection into the mPOA on feeding behaviour has not previously been studied. In the present study, intra-mPOA (imPOA) injection of GALP potently increased 0-1-h food intake in rats. The dose-response effect of imPOA GALP administration on food intake was similar to that previously observed following iPVN administration. The effects of GALP (1 nmol) or galanin (1 nmol) on food intake were then compared following injection into the PVN, mPOA, ARC, dorsal medial nucleus (DMN), lateral hypothalamus and rostral preoptic area (rPOA). GALP (1 nmol) increased food intake to a similar degree when injected into the imPOA or iPVN, but produced no significant effect when injected into the ARC, DMN, lateral hypothalamus or rPOA. Similarly, galanin (1 nmol) significantly increased food intake following injection imPOA and iPVN. However, the effect was significantly smaller than that following administration of GALP (1 nmol). Galanin also had no significant effect on food intake when administered into the ARC, DMN, lateral hypothalamus and rPOA. These data suggest that the mPOA and the PVN may have specific roles in mediating the orexigenic effect of GALP and galanin.     

Conditioned place preference produced by infusion of Met-enkephalin into the medial preoptic area

The conditioned place preference procedure was used to evaluate the rewarding properties ofd-Ala²-Met⁵-enkephalinamide (DALA) after bilateral infusion into the medial preoptic area. Doses of 60,250 and 1000 ng/cannula were used. It was found that all doses of DALA produced place preference. This suggests that the medial preoptic area is a structure where opioid reward is produced in doses as low as those required in already established reward systems. The significance of this in relation to sexual reward is discussed.     

The sex hormone-dependent development of opiate receptors in the rat medial preoptic area

The opiate receptor content of the sexually dimorphic medial preoptic area (MPOA) was examined in newborn and 5-day-old (D6) male and female rats. A significant increase of [³H]naloxone binding was observed in and around the sexually dimorphic nucleus of the preoptic area (SDN-POA) in D6 female rats, relative to newborn females. Opiate receptor labeling did not increase over this period in males, nor was labeling different between males and females at birth. This dramatic alteration of MPOA opiate receptor content was observed to occur in either sex in the absence of testosterone postnatally; that is, neonatally-castrated males exhibited the same increase of labeling by D6 as did normal females. Conversely, daily postnatal testosterone treatment of females from birth to D6 resulted in the development of male-like MPOA opiate receptor pattern. The sex hormone-dependence of MPOA opiate receptor development is discussed in relation to the sex hormone-dependent ontogeny of SDN-POA structure. The overlap of critical periods for the development of these structural and chemical sexual dimorphisms suggests a role for endogenous opioids in modulating MPOA development.     

Modifications in gonadotropin control and reproductive behavior in the female rat by hypothalamic and preoptic lesions

Effects of small hypothalamic and preoptic area lesions on vaginal cyclicity, ovulation and ovarian weights of female rats were examined. Animals were then gonadectomized and tested for lordosis behavior following injections of estrogen alone and estrogen plus progesterone. Male sex behavior was also measured during daily treatment with testosterone. Relative to sham operated rats, lesions in the dorsal preoptic area produced a significant increase in lordosis behavior, virtual elimination of male sex behavior, and only marginal effects on ovarian function. Animals with lesions in the ventral preoptic area showed constant vaginal cornification, lack of ovulation and significantly smaller ovaries than the other groups. These rats also tended to show more female sex behavior and less male sex behavior than sham operated rats. Animals with lesions in the anterior hypothalamus and dorsomedial hypothalamus showed normal ovarian function and levels of female and male sex behavior comparable to the ventral preoptic lesioned rats. Animals with lesions in the ventromedial hypothalamus tended to show lower levels of lordosis behavior than sham animals but displayed a dramatic and significant increase in male copulatory behavior relative to the other groups. These data indicate a clear dissociation between the neural control of cyclic gonadotropin activity and sex specific reproductive behavior.     

Sexually dimorphic opioid distribution in the preoptic area: manipulation by gonadal steroids

A striking sexual dimorphism has been found in the density of Met-enkephalin immunoreactive fibers in the periventricular region of the preoptic area in the rat: the enkephalinergic fiber system is much denser in females. The expression of this female-typical fiber plexus is regulated by the actions of gonadal steroids both during development and in adulthood. In light of abundant evidence demonstrating the ability of the opioid peptides to modulate various sexually differentiated neuroendocrine processes and behaviors, this dimorphic system may represent an important anatomical substrate underlying these functions.     

Partial reversal of ethanol-induced reductions in exploration by two benzodiazepine antagonists (flumazenil and ZK 93426)

A series of experiments was performed to examine the effects of two benzodiazepine antagonists, flumazenil and ZK 93426, on the behavioral changes caused by ethanol (2 g/kg) in a holeboard test. The interaction between ethanol and low doses of diazepam (0.2 and 0.5 mg/kg) was also investigated. Both flumazenil and ZK 93426 reversed the reduction in exploration caused by ethanol, but tended to increase exploration when administered alone. In contrast, diazepam, which also increased exploration alone, tended to enhance the reduction in exploration caused by ethanol. Diazepam reduced the motor stimulant action of ethanol. The partial reversal by the two antagonists of the reduction in exploration caused by ethanol was not due to alterations in blood ethanol concentrations but may have resulted from their intrinsic exploration-increasing effects or an antagonism of an endogenous ligand for central benzodiazepine receptors.     

Plasma LH levels in the long term ovariectomized rat after anterolateral deafferentation of the medial basal hypothalamus and lesions in the medial preoptic area

This study was designed to investigate the effect of anterolateral hypothalamic deafferentation (ALHD) and medial preoptic area (MPOA) lesions on plasma LH levels in the long term ovariectomized rat. The deafferentations were carried out with a Halasz-Pupp knife (radius of 1.5 mm and height of 2.0 mm) and the MPOA lesions with a platinum electrode. Sham treated and an intact group served as controls. Blood samples were obtained from the jugular vein under light ether anesthesia before and at 1, 2, 4 and 6 weeks after brain surgery. After the sixth week sample all rats were treated with 50 μg of estradiol benzoate (EB) and two days later blood samples were collected during the morning and afternoon. Hypothalamic deafferentation resulted in a more significant (p<0.01) drop in plasma LH levels in one half of the group (ALHD-1) than in the other half (p<0.05) (ALHD-2) when compared to the controls. Treatment of the controls with EB resulted in a significant (p<0.01) depression of LH levels in the morning and an LH surge during the afternoon. EB also resulted in a suppression (p<0.01) of LH levels during the morning in all of the ALHD rats; however, only the ALHD-1 group had an LH surge during the afternoon following EB. Plasma LH levels in the ALHD-2 remained suppressed during the afternoon after EB treatment. Lesions in the MPOA had no effect on plasma LH levels at 1 to 6 weeks when compared to controls. Treatment of the MPOA lesion group with EB resulted in a significant (p<0.01) drop in plasma LH levels during the morning as well as the afternoon. These data suggest that the fibers that are critical for the control of tonic and phasic LH secretion enter the medial basal hypothalamus laterally and that the deafferentations carried out here were selective in interrupting fibers involved with tonic LH secretion in some rats and those involved with the phasic secretion in others. These data also suggest that the MOPA components involved with tonic LH secretion are separate from those controlling phasic LH secretion.     

Fos expression induced by warming the preoptic area in rats

The preoptic area (POA) occupies a crucial position among the structures participating in thermoregulation, but we know little about its efferent projections for controlling various effector responses. In this study, we used an immunohistochemical analysis of Fos expression during local warming of the preoptic area. To avoid the effects of anesthesia or stress, which are known to elicit Fos induction in various brain regions, we used a novel thermode specifically designed for chronic warming of discrete brain structures in freely moving rats. At an ambient temperature of 22 degrees C, local POA warming increased Fos immunoreactivity in the supraoptic nucleus (SON) and the periaqueductal gray matter (PAG). Exposure of animals to an ambient temperature of 5 degrees C induced Fos immunoreactivity in the magnocellular paraventricular nucleus (mPVN) and the dorsomedial region of the hypothalamus (DMH). Concurrent warming of the POA suppressed Fos expression in these areas. These findings suggest that thermal information from the preoptic area sends excitatory signals to the SON and the PAG, and inhibitory signals to the mPVN and the DMH.