Leptin receptor signaling in the lateral parabrachial nucleus contributes to the control of food intake.

Pontine parabrachial nucleus (PBN) neurons integrate visceral, oral, and other sensory information, playing an integral role in the neural control of feeding. Current experiments probed whether lateral PBN (lPBN) leptin receptor (LepRb) signaling contributes to this function. Intra-lPBN leptin microinjection significantly reduced cumulative chow intake, average meal size, and body weight in rats, independent of effects on locomotor activity or gastric emptying. In contrast to the effects observed following LepRb activation in other nuclei, lPBN LepRb stimulation did not affect progressive ratio responding for sucrose reward or conditioned place preference for a palatable food. Collectively, results suggest that lPBN LepRb activation reduces food intake by modulating the neural processing of meal size/satiation signaling, and highlight the lPBN as a novel site of action for leptin-mediated food intake control.

Blockade of the cerebral aqueduct in rats provides evidence of antagonistic leptin responses in the forebrain and hindbrain.

Previously, we reported that low-dose leptin infusions into the fourth ventricle produced a small but significant increase in body fat. These data contrast with reports that injections of higher doses of leptin into the fourth ventricle inhibit food intake and weight gain. In this study, we tested whether exogenous leptin in the fourth ventricle opposed or contributed to weight loss caused by third ventricle leptin infusion by blocking diffusion of CSF from the third to the fourth ventricle. Male Sprague-Dawley rats received third ventricle infusions of PBS or 0.3 µg leptin/24 h from miniosmotic pumps. After 4 days, rats received a 3-µl cerebral aqueduct injection of saline or of thermogelling nanoparticles (hydrogel) that solidified at body temperature. Third ventricle leptin infusion inhibited food intake and caused weight loss. Blocking the aqueduct exaggerated the effect of leptin on food intake and weight loss but had no effect on the weight of PBS-infused rats. Leptin reduced both body fat and lean body mass but did not change energy expenditure. Blocking the aqueduct decreased expenditure of rats infused with PBS or leptin. Infusion of leptin into the third ventricle increased phosphorylated STAT3 in the VMHDM of the hypothalamus and the medial NTS in the hindbrain. Blocking the aqueduct did not change hypothalamic p-STAT3 but decreased p-STAT3 in the medial NTS. These results support previous observations that low-level activation of hindbrain leptin receptors has the potential to blunt the catabolic effects of leptin in the third ventricle.

Myosin IXa regulates epithelial differentiation and its deficiency results in hydrocephalus.

The ependymal multiciliated epithelium in the brain restricts the cerebrospinal fluid to the cerebral ventricles and regulates its flow. We report here that mice deficient for myosin IXa (Myo9a), an actin-dependent motor molecule with a Rho GTPase-activating (GAP) domain, develop severe hydrocephalus with stenosis and closure of the ventral caudal 3rd ventricle and the aqueduct. Myo9a is expressed in maturing ependymal epithelial cells, and its absence leads to impaired maturation of ependymal cells. The Myo9a deficiency further resulted in a distorted ependyma due to irregular epithelial cell morphology and altered organization of intercellular junctions. Ependymal cells occasionally delaminated, forming multilayered structures that bridged the CSF-filled ventricular space. Hydrocephalus formation could be significantly attenuated by the inhibition of the Rho-effector Rho-kinase (ROCK). Administration of ROCK-inhibitor restored maturation of ependymal cells, but not the morphological distortions of the ependyma. Similarly, down-regulation of Myo9a by siRNA in Caco-2 adenocarcinoma cells increased Rho-signaling and induced alterations in differentiation, cell morphology, junction assembly, junctional signaling, and gene expression. Our results demonstrate that Myo9a is a critical regulator of Rho-dependent and -independent signaling mechanisms that guide epithelial differentiation. Moreover, Rho-kinases may represent a new target for therapeutic intervention in some forms of hydrocephalus.

Selective blockade of the rat brain aqueduct with thermogelling hydrogel nanoparticle dispersion.

Experimental methods targeting molecules or drugs to specific neuronal tissue(s) can be important in determining function. In this study we focused on blockade of the small channel or aqueduct connecting the third and fourth ventricles of the rat brain. A cannula was placed into the aqueduct between the third and fourth ventricle. A second cannula was placed into the third or fourth ventricle. An aqueous dispersion of hydrogel nanoparticles, that maintains a liquid state at temperatures below 33 degrees C and solidifies near body temperature (35 degrees C), was infused into the aqueduct. Two interpenetrating polymer networks (IPN) of hydrogel nanoparticles with polymer concentrations at 2% by weight and 3% by weight were separately infused into the aqueduct to block cerebrospinal fluid (CSF) flow. Following infusion of hydrogel CSF was isolated to a particular ventricle as shown by the lack of dye movement between the ventricles. In addition, stress hormone, corticosterone, feeding behavior and blood glucose levels were measured. Results show upon reaching the aqueduct the hydrogel dispersion solidified and restricted the flow of CSF. A higher concentration of dispersion (3% wt.) was more effective in blocking the aqueduct and isolating the third from the fourth ventricle. Over the period of measurement, infusion of the dispersion had no measurable detrimental physiological effects on the animal. We conclude that isolation of ventricles in the brain can be completed for 48-h by using dispersions of hydrogel nanoparticles and the effects of drugs on certain brain tissues can be determined with this method.

Reduced nicotinic receptor-mediated antinociception following in vivo antisense knock-down in rat.

Pharmacological activation of neuronal nicotinic acetylcholine receptors can produce non-opioid antinociception in rodents. However, multiple nAChR subtypes exist, the most abundant of which contain alpha4 and beta2 subunits. The purpose of the present study was to investigate the role of alpha4-containing nAChRs in mediating nicotinic antinociception using an in vivo antisense strategy. Both i.c.v. infusion and repeated bolus injections into the cerebral aqueduct of an antisense oligonucleotide against the alpha4 subunit significantly attenuated the antinociceptive effects of the nAChR agonist A-85380 in the paw withdrawal test of acute thermal pain. Rats treated with a scrambled oligonucleotide displayed a full antinociceptive response to A-85380, while discontinuing antisense treatment restored the antinociceptive effects of the nicotinic agonist. Double immunohistochemical labeling revealed near-complete overlap of expression of the serotonin marker tryptophan hydroxylase and the alpha4 nAChR subunit in the dorsal raphe nucleus. The expression of alpha4-containing nAChRs by serotonergic neurons in the dorsal raphe offered a means to address nonspecific alpha4 knock-down, i.e., oligonucleotide-induced neurotoxicity. Immunohistochemical detection of alpha4 expression was reduced by nearly 50% in the dorsal raphe of antisense-treated rats as compared to either saline or missense-treated controls. In contrast, the expression of tryptophan hydroxylase, as well as, the alpha7 nAChR subunit in antisense-infused rats was similar to that observed in saline- and missense-treated controls. The results of these studies suggest that alpha4-containing nAChRs, possibly expressed by serotonergic neurons, are involved in nicotinic-mediated analgesia. However, these data do not eliminate the possibility that other nicotinic subunit combinations may also play a role in antinociception produced by nAChR activation.

[Serotoninergic nerve fibers in the area of the substantia grisea and the ependyma of the cerebral aqueduct]. / Serotoninergicheskie nervnye volokna v zone serogo veshchestva i épendimy vodoprovoda mozga.

An electron microscopic study of the midbrain gravy with the help of degeneration caused by serotonin-like neurotoxin 5,7-dihydroxytryptamine has shown that serotonergic terminals are responsible for the innervation of dendrites, neuron bodies and ependyma cells of the cerebral aqueduct. The detection of light, dark with prevailing fine transparent or large granular synaptic vesicles as well as vacuole degeneration evidences of the existence of several sources of serotonergic innervation of the central gray substance. Continuous damage of perivascular astrocytes of the central gray by neurotoxin allows a suggestion to be made that they are also the object of serotonergic innervation. Immunomorphological data on the presence of serotoninergic neurons in the cerebral gray are confirmed.

Microinjection of interleukin-1 into brain: separation of sleep and fever responses.

Interleukin-1 (IL1) and muramyl peptides are somnogenic, pyrogenic, immune response modifiers, Their central nervous system loci of action with respect to sleep and body temperature in rabbits were examined in this study using microinjection techniques. Unilateral microinjection of IL1 into various basal forebrain or brain stem sites resulted in elevated colonic temperatures (Tc), but the duration of slow-wave sleep (SWS) was unchanged compared to results obtained after control injections. Injection of IL1 into posterior hypothalamic areas failed to elicit either sleep or temperature responses. In contrast to these results, injection of either IL1 or muramyl dipeptide into the Aqueduct of Sylvius was followed by enhanced SWS and Tc. These results show that IL1-induced sleep and fever responses can be separated. Somnogenic sites of action for IL1 and muramyl peptides remain unknown.

Ventricular, paraventricular and circumventricular structures involved in peptide-induced satiety.

Cholecystokinin, bombesin or gastrin (2 microliter of 50 ng/microliter) was injected stereotaxically into the paraventricular nucleus of the hypothalamus, the arcuate/ventromedial area, the subfornical organ, the area postrema and the cerebral aqueduct of Sprague-Dawley rats and the effects of these injections on food and water intake were studied. While the injection of cholecystokinin reduced food intake when it was injected into both hypothalamic loci, food and water intake were most severely affected by the injection of this peptide into the cerebral aqueduct. Bombesin reduced food intake after its injection into all areas except the subfornical organ and reliable reductions in water intake were seen after injection of this peptide into all areas except the paraventricular nucleus. Minor reductions in food intake were seen following gastrin injection into the paraventricular nucleus while increased water consumption was observed after this peptide was injected into the paraventricular nucleus and cerebral aqueduct. In a second study 6-hydroxydopamine injections (2 microliter of 8 micrograms/microliter were made into the five areas studied 10 days before animals were injected with 100 micrograms/kg of cholecystokinin (i.p.). All 6-hydroxydopamine-injected animals reduced their food and water intake in response to the cholecystokinin challenge as did intact controls. These results indicate that while the changes in food and water intake produced by the central injection of cholecystokinin, bombesin or gastrin may involve central catecholamine systems, those occurring after its systemic administration do not. Therefore, if the release of gastrointestinal peptides during natural feeding is part of a homeostatic mechanism regulating hunger and satiety, this mechanism may operate without directly involving central catecholamine systems.

Analgesia, development of tolerance, and 5-hydroxytryptamine turnover in the rat after cerebral and systemic administration of morphine.

Morphine HCl (10 micrograms/0.5 microliter) was injected into the right striatum, the caudal aqueduct and the region of the nucleus raphe magnus of the rat. Turnover of 5-hydroxytryptamine (5-HT) in the brain was assessed by fluorimetric estimation of 5-hydroxyindol-3-ylacetic acid following the administration of probenecid. Injection into the right striatum (a region containing 5-HT terminals) increased 5-HT turnover in the right, but not in the left striatum or in the anterior medulla. The pain threshold was unaltered. Injection into the aqueduct accelerated 5-HT turnover in the anterior medulla, but the striata and spinal cord showed no such change. Analgesia was pronounced. Injection of morphine into the region of the nucleus raphe magnus analgesia and increased 5-HT turnover in the posterior medulla and the spinal cord. The action on the cord must have been the result of the stimulation of cells in the raphe. The effects of the local injections of morphine on 5-HT turnover were antagonized by systemic naloxone (1-2 mg/kg) in all the regions studied. When morphine was administered subcutaneously three times a day for five days, tolerance developed to the analgesic effect of morphine (7mg/kg). However, tolerance to its acceleration of 5-HT turnover was only seen in the spinal cord, not in striatum or anterior and posterior medulla. When morphine was withdrawn, its effects on analgesia and 5-HT turnover in the spinal cord recovered simultaneously. The results emphasize the likely part played by the descending serotoninergic pathway in the analgesic effect of morphine.

Antibodies to GM1 ganglioside inhibit morphine analgesia.

It has been shown that the injection of antiganglioside serum into the periaqueductal gray matter of rats blocks morphine induced analgesia. This result is due to the action of antibodies to GM1 ganglioside since the specific removal of these antibodies from the antiserum with pure GM1 ganglioside eliminates the blocking activity. Specificity for GM1 ganglioside was further shown by the blocking activity of choleragenoid, which binds specifically to GM1 sites. Antibodies to other brain constituents, namely S-100 protein and myelin, did not block the morphine analgesia.

The effects of serotonin antagonists on the inhibition of primate spinothalamic tract cells produced by stimulation in nucleus raphe magnus or periaqueductal gray.

The effects of serotonin antagonists were examined on the inhibition and excitation of nociceptive spinothalamic tract cells produced by brainstem stimulation with short (200 msec) or long (2 sec) stimulus trains. The inhibitory effects resulting from stimulation in either nucleus raphe magnus (NRM) or in the periaqueductal gray with short stimulus trains were significantly reduced after the administration of serotonin receptor blockers. Reductions in periaqueductal gray inhibition were also observed on inhibition produced by long duration trains, whereas the effects of NRM inhibition were dependent on stimulus duration, current strength and dose of antagonist. The rare excitatory effect of NRM stimulation was also found to be relatively reduced after the administration of a serotonin antagonist. The relatively weak effects of serotonin antagonists on NRM inhibition are discussed in relation to three hypotheses: 1) parallel pathways; 2) multiple receptors; or 3) corelease of serotonin and another transmitter from raphe-spinal neurons.

Lesions in nucleus reticularis gigantocellularis: effect on the antinociception produced by micro-injection of morphine and focal electrical stimulation in the periaqueductal gray matter.

The effect of bilateral electrolytic lesions in the nucleus reticularis giganto-cellularis (NGC) on the antinociceptive efficacy of morphine and electrical stimulation applied in the periaqueductal central gray matter (PAG) was investigated. Antinociception, evaluated by standard hot plate and tail-flick analgesiometric tests, was reliably produced by morphine (5 microgram) and focal electrical stimulation (40-200 micro A) administered in the PAG of rats via chronic indwelling cannula/electrode assemblies. Subsequent to the initial antinociceptive testing, bilateral electrolytic lesions were introduced in the NGC and the antinociceptive efficacy of morphine and stimulation in the PAG was again evaluated. Lesions in the NGC prevented the expression of the antinociception produced by the microinjection of morphine in the PAG whereas the antinociception resulting from electrical stimulation in the PAG was unaffected. Further, lesions in the NGC did not alter baseline (control) nociceptive thresholds in either analgesiometric test. These results provide additional support for involvement of the NGC in morphine-induced antinociception and, in addition, suggest that the NGC is not essential to a tonically-active inhibitory system or to the antinociception produced by focal electrical stimulation in the PAG.

Effects of ACTH, benzodiazepines and 5-HT antagonists on escape from periaqueductal grey stimulation in the rat.

1. The effects of drugs has been studied on escape behaviour of rats following electrical stimulation of the periaqueductal grey, in order to assess the feasibility of using the latency to escape as a measure of experimental anxiety. 2. Escape latencies were decreased by methysergide and cyproheptadine, whilst lorazepam and chlordiazepoxide (CDP) increased the latencies. 3. ACTH marginally decreased escape latencies, and did not alter the response to lorazepam. 4. Difficulties associated with using this paradigm to detect anxiolytic or anxiogenic effects of drugs are discussed.

Morphine and ACTH1-24: correlative behavioral excitations following micro-injections in rat periaqueductal gray.

Rats implanted with bilateral cannulas in the periaqueductal gray exhibited similar behavioral excitations following microinjections of morphine sulphate and ACTH1-24. Injections were more effective when the sites were located within rather than below the periaqueductal gray. Analgesia was observed following morphine but not ACTH microinjection. These results confirm that morphine exerts a dual action, inhibitory (i.e. analgesic) and excitatory, with ACTH mimicking only the latter action.

Periaqueductal gray neurons response to microiontophoretically injected morphine in naive and morphine-dependent rats.

The attempt of this study was to investigate the direct effects of increasing doses of morphine on the neuronal activity of the periaqueductal gray in morphine-naive and morphine-dependent rats. The microiontophoresis technique was used for this purpose. The four different responses induced by morphine exhibited dose-related patterns. Naloxone antagonized these responses in about 40% of the cases. Differences were found in the sensitivity of the neurons of morphine between naive and morphine-dependent rats. The phenomena of acute tolerance, chronic tolerance and dependence have been found. The results of this study indicate the presence of different neural populations in the periaqueductal gray in relation to their response to morphine, supporting the notion that subpopulations of opiate receptors exist within this brain area.

Selective synaptic antagonism by atropine and alpha-aminoadipate of pulvinar and cortical afferents to the suprasylvian visual area (Clare-Bishop area).

The synaptic excitations of cells of the Clare-Bishop cortical region produced by electrical stimulation of the pulvinar and ipsilateral cortex, have been shown to be differentially antagonized by iontophoretically applied antagonists. Atropine attenuated the responses evoked by pulvinar stimulation without having an appreciable effect against either iontophoretically applied aspartate or cortically evoked responses. alpha-Aminoadipate antagonized aspartate elicited excitations and those obtained with cortical stimulation while leaving unaffected acetylcholine and pulvinar evoked responses. The results are supportive of the view that acetylcholine and aspartate, or a similar excitatory amino acid, act as synaptic transmitters of some afferents from the pulvinar and ipsilateral cerebral cortex, respectively.

Effects of focal electrical stimulation and morphine microinjection in the periaqueductal gray of the rat mesencephalon on neuronal activity in the medullary reticular formation.

Neurons in the medullary reticular formation (MRF; nucleus reticularis gigantocellularis and nucleus reticularis paragigantocellularis) were evaluated for their involvement in the analgesia produced by focal electrical stimulation and microinjection of morphine into the periaqueductal gray region (PAG) of the rat mesencephalon. Analgesia-producing PAG stimulation altered the spontaneous activity of 80% of the neurons in the MRF (both excitation and inhibition were observed) and inhibited the noxious-evoked excitation of 75% of MRF neurons. Microinjection of morphine into the PAG also increased (50%) and decreased (17%) the spontaneous activity of MRF units and inhibited the noxious-evoked excitation of 47% of MRF neurons. These effects were specific for analgesia produced by the PAG manipulations and were partially reversed by naloxone. The role of the MRF in PAG-induced analgesias and the degree of overlap in neuronal systems influenced by intracranial morphine and electrical stimulation is discussed.

Inhibition of spinal cord interneurons by narcotic microinjection and focal electrical stimulation in the periaqueductal central gray matter.

Single cell evoked activity was recorded from spinal cord interneurons in rats prepared with microinjection cannulae or stimulating electrodes in the periaqueductal central gray matter (PAG). Morphine microinjections (4-16 microgram) inhibited the response evoked by a noxious stimulus in 55% of the wide dynamic range neurons tested. Microinjections of etorphine (0.25-0.5 microgram) inhibited 82% of the nociceptive neurons tested. Neither drug inhibited neurons which responded only to innocuous mechanical stimulation. The inhibition of wide dynamic range neurons produced by narcotic microinjection was antagonized by naloxone (1 mg/kg, i.p.) in 7 of 11 cases. Control experiments indicated that the effects obtained with microinjections could not be attributed to the drugs' diffusion to the spinal cord. Focal electrical stimulation of the PAG inhibited the responses to noxious stimuli of 60% of wide dynamic range neurons but was without effect on the responses of neurons that were activated only by innocuous stimuli. These experiments directly demonstrate that narcotic analgesics restricted to an intracerebral site of action activate a neural system which preferentially inhibits the responses of spinal cord wide dynamic range neurons to noxious stimuli. The system has a specificity for nociceptive input since non-nociceptive neurons were unaffected. Directly comparable results were produced by electrical stimulation of the PAG, supporting the concept that stimulation and narcotics modulate the transmission of nociceptive information by similar mechanisms.