Enhanced glutamate release during REM sleep in the rostromedial medulla as measured by in vivo microdialysis

Anatomical studies and stimulation studies in the decerebrate animal have suggested that the muscle atonia of rapid eye movement (REM) sleep is mediated by a projection from cholinoceptive glutamatergic neurons in the pons to the nucleus magnocellularis (NMC) of medulla. This model suggests that glutamate release in NMC should be enhanced in REM sleep. In the present study, glutamate release across the sleep–wake cycle in NMC was measured by in vivo microdialysis. We found that glutamate release in NMC was significantly higher (p=0.0252) during REM sleep than during wakefulness (W). Glutamate release during REM sleep was not elevated either in nucleus paramedianus (NPM) or in the pontine inhibitory area (PIA) regions where cholinergic stimulation suppresses muscle tone. Acetylcholine (ACh) microinjection into PIA enhanced glutamate release in NMC. These results support the hypothesis that a glutamatergic pathway from PIA to NMC is responsible for the suppression of muscle tone in REM sleep.     

Differential distribution of GABAA receptor subunits on bulbospinal serotonergic and nonserotonergic neurons of the ventromedial medulla of the rat

Spinally projecting neurons of the ventromedial medulla (VMM) compose an important efferent pathway for the modulation of nociception. These neurons receive a substantial γ-aminobutyric acid (GABA)-ergic input, but the GABA receptor that mediates this input is unknown. This study examined the distribution of GABA_A receptor α1 and α3 subunits in serotonergic and nonserotonergic neurons of the VMM that project to the dorsal horn in the rat. A pledget of Gelfoam^TM soaked in Fluoro-Gold was placed at the thoracolumbar junction of the spinal cord to label spinally projecting neurons. Alternate sections of the medulla were then incubated with a mixture of antisera to either serotonin and the α1 subunit, or to serotonin and the α3 subunit of the GABA_A receptor. Nearly 30% of spinally projecting neurons in the VMM were immunoreactive for the α1 subunit. A similar percentage of spinally projecting neurons in the VMM were immunoreactive for the α3 subunit, although diffuse cellular labeling combined with intense staining of processes in the neuropil precluded a rigorous semi-quantitative estimation of this population. No α1-subunit-immunoreactive neurons colocalized serotonin. In contrast, serotonergic neurons were immunoreactive for the α3 subunit. However, these double-labeled neurons were a modest percentage of the serotonergic population. A small percentage of spinally projecting serotonergic neurons was immunoreactive for the α3 subunit. These results suggest that significant numbers of spinally projecting serotonergic and nonserotonergic neurons of the VMM possess GABA_A receptors that differ in their respective subunit compositions and that both classes of neurons may mediate the antinociception produced by the microinjection of GABA_A receptor antagonists in the VMM. J. Comp. Neurol. 384:337–348, 1997. © 1997 Wiley-Liss, Inc.     

Sleep-wake behavior and responses to sleep deprivation of mice lacking both interleukin-1 beta receptor 1 and tumor necrosis factor-alpha receptor 1

Data indicate that interleukin (IL)-1β and tumor necrosis factor-α (TNFα) are involved in the regulation of non-rapid eye movement sleep (NREMS). Previous studies demonstrate that mice lacking the IL-1β type 1 receptor spend less time in NREMS during the light period, whereas mice lacking the p55 (type 1) receptor for TNFα spend less time in NREMS during the dark period. To further investigate roles for IL-1β and TNFα in sleep regulation we phenotyped sleep and responses to sleep deprivation of mice lacking both the IL-1β receptor 1 and TNFα receptor 1 (IL-1R1/TNFR1 KO). Male adult mice (IL-1R1/TNFR1 KO, n = 14; B6129SF2/J, n = 14) were surgically instrumented with EEG electrodes and with a thermistor to measure brain temperature. After recovery and adaptation to the recording apparatus, 48 h of undisturbed baseline recordings were obtained. Mice were then subjected to 6 h sleep deprivation at light onset by gentle handling. IL-1R1/TNFR1 KO mice spent less time in NREMS during the last 6 h of the dark period and less time in rapid eye movement sleep (REMS) during the light period. There were no differences between strains in the diurnal timing of delta power during NREMS. However, there were strain differences in the relative power spectra of the NREMS EEG during both the light period and the dark period. In addition, during the light period relative power in the theta frequency band of the REMS EEG differed between strains. After sleep deprivation, control mice exhibited prolonged increases in NREMS and REMS, whereas the duration of the NREMS increase was shorter and there was no increase in REMS of IL-1R1/TNFR1 KO mice. Delta power during NREMS increased in both strains after sleep deprivation, but the increase in delta power during NREMS of IL-1R1/TNFR1 KO mice was of greater magnitude and of longer duration than that observed in control mice. These results provide additional evidence that the IL-1β and TNFα cytokine systems play a role in sleep regulation and in the alterations in sleep that follow prolonged wakefulness.     

Activating transcription factor 6α deficiency exacerbates oligodendrocyte death and myelin damage in immune‐mediated demyelinating diseases

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) play a critical role in immune‐mediated demyelinating diseases, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), by regulating the viability of oligodendrocytes. Our previous studies show that activation of the PERK branch of the UPR protects myelinating oligodendrocytes against ER stress in young, developing mice that express IFN‐γ, a key pro‐inflammatory cytokine in MS and EAE, in the CNS. Several studies also demonstrate that PERK activation preserves oligodendrocyte viability and function, protecting mice against EAE. While evidence suggests activation of the ATF6α branch of the UPR in oligodendrocytes under normal and disease conditions, the effects of ATF6α activation on oligodendrocytes in immune‐mediated demyelinating diseases remain unknown. Herein, we showed that ATF6α deficiency had no effect on oligodendrocytes under normal conditions. Interestingly, we showed that ATF6α deficiency exacerbated ER stressed‐induced myelinating oligodendrocyte death and subsequent myelin loss in the developing CNS of IFN‐γ‐expressing mice. Moreover, we found that ATF6α deficiency increased EAE severity and aggravated EAE‐induced oligodendrocyte loss and demyelination, without affecting inflammation. Thus, these data suggest the protective effects of ATF6α activation on oligodendrocytes in immune‐mediated demyelinating diseases.     

Corticotropin-releasing factor (CRF) produces analgesia in humans and rats

The analgesic activity of corticotropin releasing factor (CRF) was determined in a clinical model and in the rat hot plate test. Patients administered CRF reported significantly less postoperative pain than patients pretreated with placebo. In rats, injection of CRF resulted in a significant analgesia which was comparable in both intensity and duration to a 300 times greater molar dose of morphine. These findings suggest that endogenous CRF may play a physiologic role in modulating pain when released under conditions of stress.     

Cooperative regulation of nerve growth factor synthesis and secretion in fibroblasts and astrocytes by fibroblast growth factor and other cytokines

Acidic fibroblast growth factor (aFGF) enhances nerve growth factor (NGF) synthesis by astrocytes obtained from various brain regions. NGF secretion by fibrous-shaped astrocytes transformed by dibutyryl-cAMP (db-cAMP) pretreatment was less than that by untreated astrocytes. However, aFGF also enhanced NGF secretion by fibrous-shaped astrocytes. The effects of various kinds of intracellular signaling modulators on NGF synthesis were examined. None of the following second messenger effectors had an effect on NGF synthesis: protein kinase C (PKC) agonist (phorbol myristate acetate (PMA)) or antagonist (sphingosine (SP)). LiCl, and ionomycin (Iono). Further, increases of intracellular cAMP by forskolin (FK) or db-cAMP have no significant effect on NGF synthesis in astrocytes under a standard culture condition. However, NGF synthesis by astrocytes in the presence of aFGF was significantly enhanced by db-cAMP, but not by FK or sodium butyrate. These results indicate that an excessive amount of cAMP enhances the effect of aFGF on NGF synthesis in astrocytes. NGF synthesis in astrocytes was not affected by treatment with anti-aFGF or anti-bFGF neutralizing antibodies, indicating that FGFs are not involved in the autocrine regulation of NGF synthesis in astrocytes. Transforming growth factor-beta 1 (TGF-beta 1), which inhibits some effects of FGFs, increased NGF synthesis in concert with aFGF. Furthermore, the highest NGF synthesis was observed when astrocytes were stimulated by all of the following cytokines: aFGF, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) and TGF-beta 1. The mechanism regulating NGF synthesis in fibroblasts obtained from prenatal rat skin was also investigated. Acidic FGF, basic FGF (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha (TGF-alpha), TGF-beta 1, IL-1 beta, and TNF-alpha were found to be regulators of NGF synthesis in skin fibroblasts. Among these cytokines, aFGF is the most potent regulator of NGF synthesis in fibroblasts. NGF synthesis by skin fibroblasts, either in the presence or absence of aFGF, was not modified by any of the following: FK, PMA, SP, LiCl, and Iono. However, db-cAMP significantly enhanced NGF synthesis in both conditions. Sodium butyrate enhanced NGF synthesis in the presence of aFGF, but not in the absence of aFGF. These results suggest that an excessive amount of cAMP and butyrate moiety regulate NGF synthesis in skin fibroblasts in different ways.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of leukemia inhibitory factor in skeletal muscle regeneration

Although a number of cytokines have been implicated in tissue regeneration, it is unknown which ones actually function in vivo. Here, we use mice with a targeted mutation in the leukemia inhibitory factor (LIF) gene to examine the role of LIF in muscle regeneration. Using a muscle crush model, we show that muscle regeneration in LIF knockout mice is significantly reduced compared to control littermates. Further, targeted infusion of LIF in both normal and LIF knockout animals stimulated muscle regeneration, but the stimulation observed was much greater in the mutant animals than in controls. In contrast, interleukin-6 and transforming growth factor-α, which also stimulate myoblast proliferation in vitro, had no effect on regeneration. These findings demonstrate directly that LIF is involved in regeneration of injured muscle and points to the use of LIF as a therapeutic agent in the treatment of neuromuscular disease. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 815–822, 1997     

Different types of norepinephrinergic receptors are involved in preoptic area mediated independent modulation of sleep-wakefulness and body temperature

The preoptic area is known to regulate sleep-wakefulness and body temperature. It was suggested earlier that though sleep-wakefulness and body temperature may affect each other, the preoptic area mediated influence on those two physiological phenomena is likely to be independent of alteration in each other. Since intrapreoptic area norepinephrine could modulate both those functions, study of that system was undertaken. It was hypothesized that since the preoptic area has different types of norepinephrinergic receptors (viz. alpha 1, alpha 2 and beta), independent modulation of those two functions was probably due to activation or inactivation of separate receptors. Hence, the effects of different agonist and antagonist of those receptors individually as well as in combination into the preoptic area were studied on those two functions in freely moving rats. The results suggest that norepinephrine induced preoptic area mediated influence on the body temperature is primarily regulated by the alpha 1 receptors while the sleep and wakefulness are regulated by alpha 2 and beta receptors, respectively. The finding should help in explaining several poorly understood observations reported earlier and it suggests that similar phenomena may possibly exist in other system involving other neurotransmitters as well.     

Effects of REM sleep deprivation and desipramine on β-adrenergic binding sites in rat brain

Rats were subjected to REM sleep deprivation by the flower pot method for 7 days. Another group of rats received either desipramine (10 mg/kg, i.p.), a tricyclic antidepressant, or saline (i.p.) once daily for 7 days. beta-Adrenergic receptor sensitivity in rat brain cortices was assessed by determining the maximum number of [3H]dihydroalprenolol binding sites by Scatchard analysis. Administration of desipramine caused a 26% reduction in beta-adrenergic receptor density (P less than 0.01) while REM sleep deprivation reduced beta-adrenergic receptor density by 13%, which was not statistically significant.     

Effect of tumor necrosis factor α and β on human oligodendrocytes and neurons in culture

Cytokines produced by infiltrating hematogenous cells or by glial cells activated during the course of central nervous system disease or trauma are implicated as mediators of tissue injury. In this study, we have assessed the extent and mechanism of injury of human-derived CNS oligodendrocytes and neurons in vitro mediated by the cytokines tumor necrosis factor α and β and compared these with the tumor necrosis factor independent effects mediated by activated CD4⁺ T-cells. We found that activated CD4⁺ T-cells, but not tumor necrosis factor α or β, could induce significant release of lactate dehydrogenase, a measure of cell membrane lysis, from oligodendrocytes within 24 hr. Neither induced DNA fragmentation as measured using a fluorescence nick-end labelling technique. After a more prolonged time period (96 hr), tumor necrosis factor α did induce nuclear fragmentation changes in a significant proportion of oligodendrocytes without increased lactate dehydrogenase release. The extent of DNA fragmentation was comparable to that induced by serum deprivation. Tumor necrosis factor β effects were even more pronounced. In contrast to oligodendrocytes, the extent of DNA fragmentation, assessed by propidium iodide staining, induced in neurons by tumor necrosis factor α was less than that induced by serum deprivation. In-situ hybridization studies of human adult glial cells in culture indicated that astrocytes, as well as microglia, can express tumor necrosis factor α mRNA.     

Tumor necrosis factor α and transforming growth factor β upregulate astrocyte expression of monocyte chemoattractant protein-1

Astrocytes participate in the pathophysiology of central nervous system (CNS) inflammatory disease. Astrocyte expression of adhesion molecules, cytokines, and major histocompatibility complex antigens may contribute to these inflammatory processes. In addition, recent data suggested that astrocytes may be a source of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a member of the chemokine family of small cytokines and functions both as a chemoattractant as well as a stimulator of monocytes. To further characterize the role of astrocytes in CNS inflammation, we examined the effect of inflammatory cytokines on MCP-1 expression by astrocytes. Results of these studies demonstrate that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFa) upregulates MCP-1 message and protein expression. The pleiotropic cytokine transforming growth factor beta (TGFβ) also stimulated MCP-1 expression. When astrocytes were exposed to both cytokines simultaneously, an additive effect on MCP-1 message, but not MCP-1 protein expression, was observed. These data suggest that TNFa and TGFβ, each present during CNS inflammatory disease, may upregulate the expression of MCP-1 which, in turn, may function to both recruit monocytes to the site of inflammation as well as to activate those monocytes already present in an inflammatory lesion.     

Effects of β-endorphin and its fragments on inhibitory avoidance behavior in rats

The effects on retrieval of a one-trial learning inhibitory avoidance response of β-endorphin, α-endorphin, and γ-endorphin, given prior to test have been studied in rats. β-Endorphin (β-LPH_61–91) in a relatively low dose (1.5 μg sc. or 50 ng icv.) facilitated inhibitory avoidance behavior, while a higher dose (10 μg sc. or 100 ng icv.) caused bimodal changes (facilitation in 50% of the animals and attenuation in another 40%. Peripheral injection of γ-endorphin attenuated inhibitory avoidance behaviour in a dose-dependent manner. The C-terminus of β-endorphin (β-LPH_78–91) was ineffective. α-Endorphin facilitated inhibitory avoidance behavior in a dose-dependent manner. Naltrexone pretreatment antagonized the bimodal effect of β-endorphin: following pretreatment with the opiate antagonist the low latency component disappeared, but the facilitatory effect of the neuropeptide remained the same.It is suggested that β-endorphin carries more than one bit of behavioral information. Inherent activities either related or unrelated to naltrexone-sensitive opiate receptors as well as biotransformation into α- and γ-endorphin may contribute to the multiple behavioral effects of this neuropeptide.     

Combination of inflammatory cytokines increases nitrite and nitrate levels in the paraventricular nucleus of conscious rats

Inflammatory cytokines stimulate glial cells in vitro to produce nitric oxide (NO) from inducible NO synthase (iNOS). Whether the stimulation with cytokines produces NO derived from iNOS has not hitherto been demonstrated in the vivo brain. Nitrite and nitrate (NOx(-)) levels in the rat paraventricular nucleus (PVN) were measured before and after intraparenchymal microinjection of cytokines with a microdialysis technique. The cytokines, tumor necrosis factor (TNF)-alpha (10 ng), interleukin (IL)-1 beta (2 ng), and interferon (IFN)-gamma (2 ng) were microinjected. None of the cytokines alone had any effect on the NOx(-) levels for 8 h. But a combination of TNF-alpha and IFN-gamma gradually increased NOx(-) levels beginning at 140 min after the microinjection, and NOx(-) levels reached 1.8 times the basal level at 380 min. A combination of TNF-alpha and IL-1 beta increased NOx(-) beginning at 340 min, reaching 1.7 times the basal level at 440 min, whereas a combination of IL-1 beta and IFN-gamma had no effect. Microinjection of a mixture of all three cytokines increased NOx(-) levels beginning at 120 min, reaching 3.3 times the basal level at 400 min. Aminoguanidine, which is a selective inhibitor of iNOS, reduced NOx(-) levels induced by the mixed cytokine treatment. Semi-quantitative RT-PCR for iNOS mRNA was done. The intensity of the iNOS mRNA band for the cytokine-treated PVN was stronger than that for the vehicle-treated PVN. These results suggest that the increased NOx(-) after the treatment with mixed cytokines were dependent on iNOS activity. This is the first report to indicate that only cytokines induce NOS in vivo in the brain.     

Pregnenolone metabolism in rodent embryonic neurons and astrocytes

The rat CNS has been previously shown to synthesize pregnenolone (PREG) and to convert it into progesterone (PROG) and some of its 5α-reduced metabolites. However, the brain cell types involved in the metabolic conversions of PREG are poorly known. Selective conditions were used to obtain purified cultures of neurons and astrocytes from mouse or rat fetal striatum and cerebral cortex. Neurons converted PREG to only one identified metabolite, 20α-dihydro PREG, whereas astrocytes converted PREG also to PROG, 5α-dihydro PROG, and 3α (3β)-5α-tetrahydro PROG. Therefore, astrocytes can convert the neurosteroid PREG into the steroid hormone PROG and the neuromodulatory steroid 3α, 5α-tetrahydro PROG, whereas neurons lack the Δ5-3β-hydroxysteroid dehydrogenase isomerase activity (and cholesterol side-chain cleavage activity), necessary for the biosynthesis of PROG. Provision of steroid substrates is another example of cross-talk between glial cells and neurons.     

Antinociception produced by systemic R(+)-baclofen hydrochloride is attenuated by CGP 35348 administered to the spinal cord or ventromedial medulla of rats

This study examined the sites in the central nervous system at which subcutaneously-administered R(+) - baclofen hydrochloride (baclofen), the most active isomer of this prototypic γ-aminobutyric acid (GABAB receptor agonist, acts to produce antinociception in the rat. To determine whether baclofen acts in the spinal cord, either saline or the GABAB receptor antagonist CGP 35348 was injected intrathecally in rats pretreated 24 min earlier with 1 or 3 mg/kg s.c. baclofen. Intrathecal (i.t.) injection of 3 or 10 μg of CGP 35348 antagonized the increase in tail-flick and hot-plate latency produced by either dose of baclofen. To determine whether baclofen acts at sites in the ventromedial medulla (VMM), either saline or CGP 35348 was microinjected in the nucleus raphe magnus or nucleus reticularis gigantocellularis pars a of rats pretreated 24 min earlier with 1 or 3 mg/kg s.c. baclofen. Microinjection of 0.5 or 3 μg of CGP 35348 at sites in the VMM produced at best only a very modest attenuation of the antinociceptive effects of baclofen. These data suggest that systemically-administered baclofen acts at sites in both the spinal cord and the VMM, but that its antinociceptive effects are likely to be mediated to a greater extent by a spinal, rather than medullary site of action. However, a definitive comparison of the relative contribution of GABAB receptors in these two regions is precluded by differences in the diffusion and concentrations of the antagonist in the spinal cord and brainstem. Finally, microinjection of 0.5 or 3.0 μg of CGP 35348 in the nucleus raphe magnus or nucleus reticularis gigantocellularis pars a of saline-pretreated rats did not alter tail-flick or hot-plate latency. This finding suggests that, unlike GABA_A receptors, GABAB receptors do not mediate the tonic GABAergic input to neurons in these nuclei.     

Identification and topography of neuronal cell populations expressing; TNFα and IL-1α in response to hippocampal lesion

In previous studies, we have shown that a traumatic lesion to the hippocampus of adult mice induces the transitory expression of TNFα and IL-1α by neurons of different brain areas and also by glial cells at the site of injury. The aim of the present study was to establish whether the expression of TNFα and IL-1α is restricted to defined subpopulations, or else is common to most of the central neuronal populations. Using polyclonal anti-GAD 67, anti-TH and monoclonal anti-ChAT, and anti-5-HT antibodies in a double-labeling immunohistochemical procedure in combination with murine anti-TNFα and anti-IL-1α polyclonal antibodies, we show that most GABAergic, catecholaminergic, and serotoninergic neurons, and a subgroup of the cholinergic neurons, express these cytokines. Although not immunohistochemically characterized, neurons in some glutamatergic structures such as the hippocampus and the prefrontal cortex also express these cytokines. Thus, we conclude that the capacity of central neurons to express cytokines like TNFα and IL-1α in reaction to a brain Injury is not restricted to peculiar neuronal subtypes, but could include most of the neuronal populations of the brain. © 1996 Wiley-Liss, Inc.     

Sympathetically-mediated cardiovascular responses induced by neurochemical microinjection in the brainstem lateral tegmental field of cat

This study examines sympathetically-mediated cardiovascular responses arising from microinjections of AMPA and kynurenic acid in the medullary lateral tegmental field (LTF). The resulting behavior, which is predominately inhibitory, is nearly identical to that reported for the para-ambiguual area lying just lateral to the LTF, and suggests that the two regions may share other cardiovascular control functions.     

AbetaPP-overexpression and proteasome inhibition increase alphaB-crystallin in cultured human muscle: relevance to inclusion-body myositis

Amyloid-beta precursor protein (AbetaPP) and its fragment amyloid-beta (Abeta) are increased in s-IBM muscle fibers and appear to play an important role in the pathogenic cascade. alphaB-Crystallin (alphaBC) was shown immunohistochemically to be accumulated in s-IBM muscle fibers, but the stressor(s) influencing alphaBC accumulation was not identified. We now demonstrate, using our experimental IBM model based on genetic overexpression of AbetaPP into cultured normal human muscle fibers, that: (1) AbetaPP overexpression increased alphaBC 3.7-fold (p=0.025); (2) additional inhibition of proteasome with epoxomicin increased alphaBC 7-fold (p=0.002); and (3) alphaBC physically associated with AbetaPP and Abeta oligomers. We also show that in biopsied s-IBM muscle fibers, alphaBC was similarly increased 3-fold (p=0.025) and physically associated with AbetaPP and Abeta oligomers. We propose that increased AbetaPP is a stressor increasing alphaBC expression in s-IBM muscle fibers. Determining the consequences of alphaBC association with Abeta oligomers could have clinical therapeutic relevance.