Effects of microinjections of triazolam into the ventrolateral preoptic area on sleep in the rat

In view of interest in the ventrolateral preoptic area (VLPO), based on FOS protein accumulation during sleep as well as its output pathways to areas involved in sleep regulation, we have examined the effects of microinjections of triazolam into the VLPO. It was found that two doses of triazolam, noted previously to enhance sleep when injected into the medial preoptic area, had no significant effect on sleep or core temperature when administered into the VLPO. Although these data do not bear on the possibility that the VLPO is involved in physiological sleep regulation, they suggest that it is not a site of the pharmacologic action of hypnotic benzodiazepines.     

Melatonin microinjection into the medial preoptic area increases sleep in the rat

A wide variety of hypnotic compounds including triazolam, pentobarbital, ethanol and adenosine have been reported to enhance sleep when microinjected into the medial preoptic area (MPA) of the anterior hypothalamus of the rat. It is uncertain whether the pineal hormone melatonin, which may alter sleep/wake physiology in mammals, acts at this site. A previous report has indicated that a more widespread injection of melatonin into the hypothalamus of the cat induces sleep. In the present study we have examined the possibility that the MPA may mediate this effect. Nine adult rats were microinjected with melatonin 1 and 50 ug and vehicle into the MPA during the daytime in a repeated measures design study. It was found that melatonin increased total sleep time in a dose-dependent manner, primarily by increasing NREM sleep, and that wake time after sleep onset was significantly reduced. These data add melatonin to the growing list of compounds that increase total sleep after administration into the MPA, and suggest that the MPA may be a common site of action for such agents from a variety of pharmacologic classes. Based on previous studies, the possibility is raised that this sleep enhancement results from an alteration in function of the GABA(A)-benzodiazepine receptor complex.     

Hypnotic action of benzodiazepines: a possible mechanism

The objective of this investigation was to determine whether the effects of muscimol on benzodiazepine receptor binding relate to the hypnotic activity of nine benzodiazepines (clonazepam, triazolam, diazepam, flurazepam, nitrazepam, oxazepam, temazepam, clobazam, and chlordiazepoxide) and CL 218,872. There was no correlation between the basal receptor binding affinities of the drugs tested and their hypnotic potencies, whereas the benzodiazepine receptor agonists whose receptor bindings are strongly modulated by muscimol possess potent hypnotic activity. These results indicate that benzodiazepine receptors that couple to GABA receptors are involved in the hypnotic activity of the benzodiazepines.     

Noradrenergic afferents and receptors in the medial preoptic area: neuroanatomical and neurochemical links between the regulation of sleep and body temperature

Several studies have shown the importance of the medial preoptic area in the regulation of sleep-wakefulness and of body temperature. The medial preoptic area has a rich noradrenergic innervation, coming mostly from the lateral tegmental noradrenergic system. The accumulating evidences show that the noradrenergic afferents to the medial preoptic area are involved in the induction of sleep. This hypnogenic mechanism operates through the postsynaptic alpha1 and alpha2-adrenergic receptors. Noradrenergic afferents are also involved in the thermoregulatory mechanisms, and the activation of these fibers brings about a fall in body temperature. Though the body temperature changes are brought about by the same receptor subtypes as those involved in hypnogenesis, observations suggest the possibility of separate sets of noradrenergic afferents in the medial preoptic area for sleep regulation and thermoregulation. In this review, we present the compelling evidences, which showed that the noradrenergic afferents of the medial preoptic area bring about a fall in body temperature and other thermoregulatory behavioral alterations associated with sleep.     

Ghrelin microinjection into forebrain sites induces wakefulness and feeding in rats

Ghrelin, a gut-brain peptide, is best known for its role in the stimulation of feeding and growth hormone release. In the brain, orexin, neuropeptide Y (NPY), and ghrelin are parts of a food intake regulatory circuit. Orexin and NPY are also implicated in maintaining wakefulness. Previous experiments in our laboratory revealed that intracerebroventricular injections of ghrelin induce wakefulness in rats. To further elucidate the possible role of ghrelin in the regulation of arousal, we studied the effects of microinjections of ghrelin into hypothalamic sites, which are implicated in the regulation of feeding and sleep, such as the lateral hypothalamus (LH), medial preoptic area (MPA), and paraventricular nucleus (PVN) on sleep in rats. Sleep responses, motor activity, and food intake after central administration of 0.04, 0.2, or 1 mug (12, 60, or 300 pmol) ghrelin were recorded. Microinjections of ghrelin into the LH had strong wakefulness-promoting effects lasting for 2 h. Wakefulness was also stimulated by ghrelin injection into the MPA and PVN; the effects were confined to the first hour after the injection. Ghrelin's non-rapid-eye-movement sleep-suppressive effect was accompanied by attenuation in the electroencephalographic (EEG) slow-wave activity and changes in the EEG power spectrum. Food consumption was significantly stimulated after microinjections of ghrelin into each hypothalamic site. Together, these results are consistent with the hypothesis that forebrain ghrelinergic mechanisms play a role in the regulation of vigilance, possibly through activating the components of the food intake- and arousal-promoting network formed by orexin and NPY.     

Dose response curve for the phase-shifting effect of triazolam on the mammalian circadian clock

A dose response curve for the phase shifting effect of triazolam, a short-acting benzodiazepine commonly prescribed for the treatment of insomnia, on the circadian rhythm of locomotor activity was measured for the golden hamster. A single intraperitoneal injection of triazolam six hours before the onset of wheel-running activity induced a dose-dependent phase advance in the rhythm. A maximum phase advance, which averaged about 100 minutes, was observed in animals injected with 0.5 to 5.0 mg of triazolam. The use of drugs which promote sleep, and induce phase shifts in a central circadian clock, could be important in the treatment of sleep disorders associated with disrupted schedules and of mental and physical disorders associated with abnormal circadian rhythmicity.     

Microinjection of melanin concentrating hormone into the lateral preoptic area promotes non-REM sleep in the rat

The ventrolateral preoptic area (VLPO) has been recognized as one of the key structures responsible for the generation of non-REM (NREM) sleep. The melanin-concentrating hormone (MCH)-containing neurons, which are located in the lateral hypothalamus and incerto-hypothalamic area, project widely throughout the central nervous system and include projections to the VLPO. The MCH has been associated with the central regulation of feeding and energy homeostasis. In addition, recent findings strongly suggest that the MCHergic system promotes sleep. The aim of the present study was to determine if MCH generates sleep by regulating VLPO neuronal activity. To this purpose, we characterized the effect of unilateral and bilateral microinjections of MCH into the VLPO on sleep and wakefulness in the rat. Unilateral administration of MCH into the VLPO and adjacent dorsal preoptic area did not modify sleep. On the contrary, bilateral microinjections of MCH (100 ng) into these areas significantly increased light sleep (LS, 39.2 ± 4.8 vs. 21.6 ± 2.5 min, P < 0.05) and total NREM sleep (142.4 ± 23.2 vs. 86.5 ± 10.5 min, P < 0.05) compared to control (saline) microinjections. No effect was observed on REM sleep. We conclude that MCH administration into the VLPO and adjacent dorsal lateral preoptic area promotes the generation of NREM sleep.     

Body temperature and sleep: Are they controlled by the same mechanism?

Simultaneous changes in sleep and body temperature, produced either by lesion or by stimulation of the medial preoptic area (mPOA), have given reasons to suggest that thermoregulation and sleep regulation are controlled by the same set of neurons. The reasons for simultaneous changes in these parameters are discussed in the present paper with a view to explaining the relationship between thermoregulation and sleep regulation. Changes in body temperature and sleep on destruction of the preoptic area (POA) neurons and the sequence of these changes, suggest a separate control mechanism in the mPOA for regulation of sleep and body temperature. Evidence is put forward in the present paper to show that the mPOA is not involved in the downregulation or upregulation of changes in body temperature with alteration in the vigilance state. On the other hand, circadian modulation of body temperature is possibly involved in altering sleep propensity. A clear indication regarding separate control of sleep and body temperature came from the studies in which noradrenergic agents were applied into the mPOA of animals with and without lesion of the noradrenergic fibers projecting to the mPOA. Experiments in which sleep was analyzed after experimental manipulations of ambient temperature and body temperature, including peripheral, core and brain temperature, are presented here to show a close relationship between thermoregulation and sleep regulation. Various theories regarding the regulation of body temperature during slow wave sleep and rapid eye movement sleep are also discussed. The functional integrity of the mPOA may be essential not only for the regulation of body temperature and sleep-wakefulness but even for the homeostatic regulation of energy balance of the body in response to alterations in environmental temperature and sleep-wakefulness.

     

Microinjections of heat shock protein 70 kDa into the nucleus reticularis pontis oralis induce inhibition of rapid eye movement sleep in pigeons

Recently it was indicated that microinjections of heat shock proteins 70 kDa (Hsp70) into the third ventricle of brain in pigeons results in an increase in the duration of slow wave sleep and a decrease in somato-visceral indices. It is suggested that Hsp70 effect may be related to GABA(A) receptors activation in the preoptic area of the hypothalamus. However, what transmitter mechanisms of activation are related to the removal effect (in 2-3 hrs) of rapid eye movement sleep inhibition still remains poorly understood. To solve this problem in the present study, microinjections of Hsp70 into the Nucleus reticularis pontis oralis (NRPO) were done. It is well known that cholinergic neurons of the NRPO are crucial for rapid eye movement sleep generation. The data show that Hsp70 produces more early (for first two hrs) a decrease in number of episodes and total time of rapid eye movement sleep, a diminution of electroencephalogram (EEG) power spectra in the 9-14 Hz band, a decrease in contractile muscle activity and brain temperature. It is suggested that Hsp70 effects are realized due to activation of GABA(A) receptors in the NRPO and induced inhibition of cholinergic mechanisms of rapid eye movement sleep triggering. The microinjections of Hsp70 into the NRPO increase the slow wave sleep total time with long latency (for 8-12 hrs). This effect may be related to influence of Hsp70 on neurons population, which are responsible for slow wave sleep maintenance outside the NRPO.     

Central administration of muscimol phase-shifts the mammalian circadian clock

Summary The suprachiasmatic nucleus (SCN) of the hypothalamus contains a neural oscillatory system which regulates many circadian rhythms in mammals. Immunohistochemical evidence indicates that a relatively high density of GABAergic neurons exist in the suprachiasmatic region. Since intraperitoneal injections of the benzodiazepine, triazolam, have been shown to induce phase shifts in the free-running circadian rhythm of locomotor activity in the golden hamster, the extent to which microinjections of muscimol, a specific agonist for gamma-aminobutyric acid (GABA), may cause phase-shifts in hamster activity rhythms was investigated. Stereotaxically implanted guide cannulae aimed at the region of the SCN were used to deliver repeated microinjections in individual animals. A phase-response curve (PRC) generated from microinjections of muscimol revealed that the magnitude and direction of permanent phase-shifts in the activity rhythm were associated with the time of administration. The PRC generated for muscimol was characterized by maximal phase-advances induced 6 h before activity onset and by maximal phase-delays which occurred 6 h after activity onset. The PRC for muscimol had a shape similar to a PRC previously generated for the short-acting benzodiazepine, triazolam. Single microinjections of different doses of muscimol given 6 h before activity onset induced phase-advances in a dose-dependent fashion. Histological analysis revealed that phase shifts induced by the administration of muscimol were associated with the proximity of the injection site to the SCN area. These data indicate that a GABAergic system may exist within the suprachiasmatic region as part of a central biological clock responsible for the regulation of the circadian rhythm of locomotor activity in the golden hamster.Abbreviations CT circadian time - GABA gamma-aminobutyric acid - OC optic chiasm - PRC phase-response curve - SEM standard error of mean - SCN suprachiasmatic nuclei - T track - IIIV third ventricle     

Central neural pathways for angiotensin-induced thirst.

Evidence is reviewed implicating the preoptic region in angiotensin-induced thirst. The most responsive area according to results obtained with behavioral, electrophysiological, and autoradiographic mapping techniques is at the caudal border of the medial preoptic region and rostral border of the anterior hypothalamus. The neural pathway from this preoptic site for angiotensin-induced thirst extends along the medial forebrain bundle through the midlateral hypothalamus to the paramedial midbrain tegmentum and to an area ventrolateral to the central gray. Lesions of this pathway in the midlateral hypothalamus and rostral midbrain significantly attenuated drinking induced by microinjections of angiotensin II into the preoptic area but did not disrupt water intake induced by microinjections of angiotensin II into the subfornical organ or cerebral ventricles. Although the efferent pathways from angiotensin-receptive sites in the subfornical organ and cerebral ventricles are unknown, it appears from these observations that the medial forebrain bundle is not involved. Lesions of the medial forebrain bundle-lateral hypothalamus also do not disrupt drinking induced by microinjections of hypertonic saline into the preoptic region although lesions placed 1 mm further lateral do. Since fat lateral hypothalamic lesions are without effect on drinking induced by centrally administered angiotensin II, this suggests that intracellular and extracellular thirst signals are subserved by separate neural pathways in the hypothalamus.     

Evaluation of single-dose hypnotic treatment before elective operation.

A prospective randomised double-blind controlled trial was carried out to evaluate the place of a single dose of triazolam, flurazepam, and placebo on the evening before an elective operation in 96 patients. Features of sleep were recorded by patients and nurses on questionnaires. Onset of sleep was delayed and duration of sleep reduced in two-thirds of patients allocated placebo compared with their normal sleep pattern. Two-thirds of these patients also complained of waking more than twice during the night. Both hypnotics significantly improved the duration and time of onset of sleep and reduced the frequency of wakening when compared with the placebo. Patients who took triazolam, however, fell asleep faster and woke less often than those who took flurazepam. Furthermore, triazolam appeared to have advantages over flurazepam before surgery. Thus giving a single dose of a hypnotic on the night before an elective operation improves the patient''s sleep, and greater benefit was derived from triazolam than flurazepam.     

Cold-induced thermogenesis mediated by GABA in the preoptic area of anesthetized rats

Bilateral microinjections of GABA (300 mM, 100 nl) or the GABA(A) receptor agonist muscimol (100 microM, 100 nl) into the preoptic area (POA) of the hypothalamus increased the rate of whole body O(2) consumption (VO(2)) and the body core (colonic) temperature of urethane-chloralose-anesthetized, artificially ventilated rats. The most sensitive site was the dorsomedial POA at the level of the anterior commissure. The GABA-induced thermogenesis was accompanied by a tachycardic response and electromyographic (EMG) activity recorded from the femoral or neck muscles. Pretreatment with muscle relaxants (1 mg/kg pancuronium bromide + 4 mg/kg vecuronium bromide i.v.) prevented GABA-induced EMG activity but had no significant effect on GABA-induced thermogenesis. However, pretreatment with the beta-adrenoceptor propranolol (5 mg/kg i.v.) greatly attenuated the GABA-induced increase in VO(2) and tachycardic responses. Accordingly, the GABA-induced increase in VO(2) reflected mainly nonshivering thermogenesis. On the other hand, cooling of the shaved back of the rat by contact with a plastic bag containing 28 degrees C water also elicited thermogenic, tachycardic, and EMG responses. Bilateral microinjections of the GABA(A) receptor antagonist bicuculline (500 microM, 100 nl), but not the vehicle saline, into the POA blocked these skin cooling-induced responses. These results suggest that GABA and GABA(A) receptors in the POA mediate cold information arising from the skin for eliciting cold-induced thermogenesis.     

The hypnotic effect of propofol in the medial preoptic area of the rat

Recent introduction of the intravenous anesthetic propofol as an ICU sedative has allowed a deeply sedated state to be maintained for extended periods in the ICU without delays in emergence. Although such sedation has been advocated to promote physiologic sleep, little evidence exists to support such a strategy. To explore propofol's effect on sleep regulation, we administered propofol directly into the medial preoptic area (MPA) of the rat, an anatomic site where administration of other sedatives (triazolam and phenobarbital) also induce sleep. We performed three two-hour sleep studies in the daytime with the lights on following the administration of propofol (8 ng or 40 ng) or vehicle (intralipid). The higher dose of propofol significantly reduced sleep latency and increased nonREM and total sleep times when compared to vehicle. REM sleep times, intermittent waking times and number of transitions were not altered. Mean nonREM sleep bout length was increased significantly at the higher dose. These findings suggest that propofol may enhance sleep by acting at a hypothalamic site.     

Triazolam, an Anomalous Benzodiazepine Receptor Ligand: In Vitro Characterization of Alprazolam and Triazolam Binding

Both alprazolam and triazolam displaced clonazepam (but not Ro 5-4864) from rat brain membranes with high affinity, showing them to act at central but not peripheral benzodiazepine receptors. At 0 degrees C, 10 microM gamma-aminobutyric acid (GABA) increased the ability of alprazolam, but not of triazolam, to displace ethyl-beta-carboline-3-carboxylate (beta-CCE) and Ro 15-1788 from these receptors. At 37 degrees C, GABA increased the affinity of the receptors for both drugs, with a +GABA/-GABA ratio of 1.5 for each in promoting Ro 15-1788 binding displacement. As both triazolam and alprazolam act as anxiolytics in vivo, the results at 37 degrees C would be compatible with the hypothesis that GABA causes an increase in affinity of drugs that act in this way, but the results at 0 degrees C would not be compatible. At 37 degrees C, alprazolam had a higher IC50 for the benzodiazepine receptor than at 0 degrees C, whereas triazolam showed the reverse effect. The relative IC50 values in vitro at 37 degrees C correlated better with the potency in vivo than those obtained at 0 degrees C. At 0 degrees C, both drugs showed Hill plots with slopes of 0.9-1 with beta-CCE and Ro 15-1788. At 37 degrees C, the slopes with triazolam were much reduced, indicating that the drug may have a selective action on a subclass of central benzodiazepine receptors. In the studies reported here, alprazolam behaved like other benzodiazepines, whereas triazolam showed several anomalous properties. It would be of interest if these properties could be related either to the drug's use as a hypnotic or to the side effects it sometimes induces.     

Effects of acute microinjections of the thyroid hormone derivative 3-iodothyronamine to the preoptic region of adult male rats on sleep,thermoregulation and motor activity

The decarboxylated thyroid hormone derivative 3-iodothyronamine (T1AM) has been reported as having behavioral and physiological consequences distinct from those of thyroid hormones. Here, we investigate the effects of T1AM on EEG-defined sleep after acute administration to the preoptic region of adult male rats. Our laboratory recently demonstrated a decrease in EEG-defined sleep after administration of 3,3′,5-triiodo-l-thyronine (T3) to the same brain region. After injection of T1AM or vehicle solution, EEG, EMG, activity, and core body temperature were recorded for 24 h. Sleep parameters were determined from EEG and EMG data. Earlier investigations found contrasting systemic effects of T3 and T1AM, such as decreased heart rate and body temperature after intraperitoneal T1AM injection. However, nREM sleep was decreased in the present study after injections of 1 or 3 μg T1AM, but not after 0.3 or 10 μg, closely mimicking the previously reported effects of T3 administration to the preoptic region. The biphasic dose–response observed after either T1AM or T3 administration seems to indicate shared mechanisms and/or functions of sleep regulation in the preoptic region. Consistent with systemic administration of T1AM, however, microinjection of T1AM decreased body temperature. The current study is the first to show modulation of sleep by T1AM, and suggests that T1AM and T3 have both shared and independent effects in the adult mammalian brain.     

In Vivo High Intrinsic Efficacy of Triazolam: A Positron Emission Tomography Study in Nonhuman Primates

Abstract: The triazolobenzodiazepine triazolam is a central-type benzodiazepine receptor (BZR) ligand that is widely prescribed as a hypnotic agent. Triazolam produces its effects through potentiation of γ-aminobutyric acid-mediated neurotransmission. Findings reported from in vitro binding studies showed some discrepancies concerning the pharmacological characteristics of triazolam. The present study aims to characterize in vivo the biochemical properties of triazolam, i.e., cerebral pharmacokinetics, interaction with BZR, potency, and intrinsic efficacy. Triazolam was studied in living nonhuman primates using positron emission tomography. Two different studies were carried out: (a) a direct study using [¹¹C]triazolam and (b) an indirect competition study using the radiolabeled BZR antagonist [¹¹C]flumazenil. Results showed that, in the brain in vivo, triazolam binds specifically and competitively to the BZR. Its rapid cerebral kinetics is consistent with a hypnotic profile (maximal binding after 23 min, elimination half-life of 202 min). Triazolam is very potent in displacing [¹¹C]flumazenil (ID₅₀= 28 ± 6 μg/kg). Hill analysis of the displacement curve does not show obvious binding-site heterogeneity. Triazolam is 20 times more potent in displacing [¹¹C]flumazenil and 50 times more potent in inhibiting pentylenetetrazol-induced paroxysmal activity than the full benzodiazepine agonist diazepam. Interestingly, the simultaneous use of positron emission tomography and EEG recording allowed us to show that triazolam-positive intrinsic efficacy is slightly higher (20%) than that of diazepam. An attractive hypothesis proposes that the severity of side effects of BZR ligands is proportional to their intrinsic efficacy. Therefore, our study shows that triazolam side effects, as for other benzodiazepines, may be related to its high intrinsic efficacy in vivo.     

Studies on extrahypophyseal sites of estrogen action in the induction of ovulation in rats.

To discover possible extrahypophyseal sites of estrogen action in the induction of ovulation, the influence of a s.c. injection of estradiol benzoate (EB) on cell nuclear sizes in the limbic-medial preoptic continuum of progesterone-pretreated cyclic rats was evaluated. The ovulatory dose of 5 mug EB caused a significant increase of nuclear volumes in the medial preoptic nucleus and the anterior and posterior parts of the medial amygdaloid nucleus. Precocious ovulation was induced in prepuberal female rats by unilateral implantation of a molten EB: cholesterol mixture into the posterior part of the mediocortical amygdala (PMCA), but not by implantation into the anterior part of this region (AMCA) or the medial preoptic area (MPA). In adult females injected s.c. with 2.0 mg progesterone on the day post estrus, bilateral implantation of 0.1 or 0.2 mug crystalline EB on the following day did not abolish the delaying effect of progesterone on the preovulatory LH increase and ovulation, when the implants were located in the MPA, lateral septum (LS), bed nucleus of the stria terminalis (BST), AMCA, PMCA or dorsal hippocampus (DHPC), whereas intrapituitary implants were highly effective. However, the bilateral introduction of large tallow pellets containing 0.1 mug EB each, into the LS, BST, AMCA or PMCA advanced ovulation in rats with progesterone-induced 5-day cycles. Equal pellets did neither induced ovulation nor an LH increase after implantation into the MPA or the DHPC. The results suggest that the anterior pituitary, mediocortical amygdala, BST and LS, but not the MPA or DHPC, are sites of the stimulatory feedback of estrogen on gonadotropin secretion in female rats, and that the amygdaloid response to estrogen differs between prepuberal and cyclic females.     

Temperature dependence and GABA modulation of [3H]triazolam binding in the rat brain

The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Our major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of [3H]TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (Kd = 0.27 +/- 08 nM at 0 degree C; Kd = 1.96 +/- 0.85 nM at 37 degrees C) while the Bmax values remained unchanged (1220 +/- 176 fmoles/mg protein at 0 degree C and 1160 +/- 383 fmoles/mg protein at 37 degrees C). Saturation studies of [3H]TZ binding in the presence or absence of GABA (100 microM) showed a GABA-shift. At 0 degrees C the Kd values were (Kd = 0.24 +/- 0.03 nM/-GABA; Kd = 0.16 +/- 0.04/+GABA) and at 37 degrees C the Kd values were (Kd = 1.84 +/- 0.44 nM/-GABA; Kd = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, our findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists.