Astrocyte-derived adenosine excites sleep-promoting neurons in the ventrolateral preoptic nucleus: Astrocyte-neuron interactions in the regulation of sleep.

Although ATP and/or adenosine derived from astrocytes are known to regulate sleep, the precise mechanisms underlying the somnogenic effects of ATP and adenosine remain unclear. We selectively expressed channelrhodopsin-2 (ChR2), a light-sensitive ion channel, in astrocytes within the ventrolateral preoptic nucleus (VLPO), which is an essential brain nucleus involved in sleep promotion. We then examined the effects of photostimulation of astrocytic ChR2 on neuronal excitability using whole-cell patch-clamp recordings in two functionally distinct types of VLPO neurons: sleep-promoting GABAergic projection neurons and non-sleep-promoting local GABAergic neurons. Optogenetic stimulation of VLPO astrocytes demonstrated opposite outcomes in the two types of VLPO neurons. It led to the inhibition of non-sleep-promoting neurons and excitation of sleep-promoting neurons. These responses were attenuated by blocking of either adenosine A1 receptors or tissue-nonspecific alkaline phosphatase (TNAP). In contrast, exogenous adenosine decreased the excitability of both VLPO neuron populations. Moreover, TNAP was expressed in galanin-negative VLPO neurons, but not in galanin-positive sleep-promoting projection neurons. Taken together, these results suggest that astrocyte-derived ATP is converted into adenosine by TNAP in non-sleep-promoting neurons. In turn, adenosine decreases the excitability of local GABAergic neurons, thereby increasing the excitability of sleep-promoting GABAergic projection neurons. We propose a novel mechanism involving astrocyte-neuron interactions in sleep regulation, wherein endogenous adenosine derived from astrocytes excites sleep-promoting VLPO neurons, and thus decreases neuronal excitability in arousal-related areas of the brain.

Astrocytes in the Ventrolateral Preoptic Area Promote Sleep.

Although ventrolateral preoptic (VLPO) nucleus is regarded as a center for sleep promotion, the exact mechanisms underlying the sleep regulation are unknown. Here, we used optogenetic tools to identify the key roles of VLPO astrocytes in sleep promotion. Optogenetic stimulation of VLPO astrocytes increased sleep duration in the active phase in naturally sleep-waking adult male rats (n = 6); it also increased the extracellular ATP concentration (n = 3) and c-Fos expression (n = 3-4) in neurons within the VLPO. In vivo microdialysis analyses revealed an increase in the activity of VLPO astrocytes and ATP levels during sleep states (n = 4). Moreover, metabolic inhibition of VLPO astrocytes reduced ATP levels (n = 4) and diminished sleep duration (n = 4). We further show that tissue-nonspecific alkaline phosphatase (TNAP), an ATP-degrading enzyme, plays a key role in mediating the somnogenic effects of ATP released from astrocytes (n = 5). An appropriate sample size for all experiments was based on statistical power calculations. Our results, taken together, indicate that astrocyte-derived ATP may be hydrolyzed into adenosine by TNAP, which may in turn act on VLPO neurons to promote sleep.SIGNIFICANCE STATEMENT Glia have recently been at the forefront of neuroscience research. Emerging evidence illustrates that astrocytes, the most abundant glial cell type, are the functional determinants for fates of neurons and other glial cells in the central nervous system. In this study, we newly identified the pivotal role of hypothalamic ventrolateral preoptic (VLPO) astrocytes in the sleep regulation, and provide novel insights into the mechanisms underlying the astrocyte-mediated sleep regulation.

Loss-of-function of EBP50 is a new cause of hereditary peripheral neuropathy: EBP50 functions in peripheral nerve system.

Finding causative genetic mutations is important in the diagnosis and treatment of hereditary peripheral neuropathies. This study was conducted to find new genes involved in the pathophysiology of hereditary peripheral neuropathy. We identified a new mutation in the EBP50 gene, which is co-segregated with neuropathic phenotypes, including motor and sensory deficit in a family with Charcot-Marie-Tooth disease. EBP50 is known to be important for the formation of microvilli in epithelial cells, and the discovery of this gene mutation allowed us to study the function of EBP50 in the nervous system. EBP50 was strongly expressed in the nodal and paranodal regions of sciatic nerve fibers, where Schwann cell microvilli contact the axolemma, and at the growth tips of primary Schwann cells. In addition, EBP50 expression was decreased in mouse models of peripheral neuropathy. Knockout mice were used to study EBP50 function in the peripheral nervous system. Interestingly motor function deficit and abnormal histology of nerve fibers were observed in EBP50+/- heterozygous mice at 12 months of age, but not 3 months. in vitro studies using Schwann cells showed that NRG1-induced AKT activation and migration were significantly reduced in cells overexpressing the I325V mutant of EBP50 or cells with knocked-down EBP50 expression. In conclusion, we show for the first time that loss of function due to EBP50 gene deficiency or mutation can cause peripheral neuropathy.

Effect of carbamazepine on tetrodotoxin-resistant Na+ channels in trigeminal ganglion neurons innervating to the dura.

Migraine is a neurological disorder characterized by recurrent and disabling severe headaches. Although several anticonvulsant drugs that block voltage-dependent Na+ channels are widely used for migraine, far less is known about the therapeutic actions of carbamazepine on migraine. In the present study, therefore, we characterized the effects of carbamazepine on tetrodotoxin-resistant (TTX-R) Na+ channels in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na+ currents were measured in medium-sized DiIpositive neurons using the whole-cell patch clamp technique in the voltage-clamp mode. While carbamazepine had little effect on the peak amplitude of transient Na+ currents, it strongly inhibited steady-state currents of transient as well as persistent Na+ currents in a concentration-dependent manner. Carbamazepine had only minor effects on the voltage-activation relationship, the voltage-inactivation relationship, and the use-dependent inhibition of TTX-R Na+ channels. However, carbamazepine changed the inactivation kinetics of TTX-R Na+ channels, significantly accelerating the development of inactivation and delaying the recovery from inactivation. In the current-clamp mode, carbamazepine decreased the number of action potentials without changing the action potential threshold. Given that the sensitization of dural afferent neurons by inflammatory mediators triggers acute migraine headaches and that inflammatory mediators potentiate TTX-R Na+ currents, the present results suggest that carbamazepine may be useful for the treatment of migraine headaches.

Pathogenic Upregulation of Glial Lipocalin-2 in the Parkinsonian Dopaminergic System.

UNLABELLED: Lipocalin-2 (LCN2) is a member of the highly heterogeneous secretory protein family of lipocalins and increases in its levels can contribute to neurodegeneration in the adult brain. However, there are no reports on the role of LCN2 in Parkinson's disease (PD). Here, we report for the first time that LCN2 expression is increased in the substantia nigra (SN) of patients with PD. In mouse brains, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment for a neurotoxin model of PD significantly upregulated LCN2 expression, mainly in reactive astrocytes in both the SN and striatum. The increased LCN2 levels contributed to neurotoxicity and neuroinflammation, resulting in disruption of the nigrostriatal dopaminergic (DA) projection and abnormal locomotor behaviors, which were ameliorated in LCN2-deficient mice. Similar to the effects of MPTP treatment, LCN2-induced neurotoxicity was also observed in the 6-hydroxydopamine (6-OHDA)-treated animal model of PD. Moreover, treatment with the iron donor ferric citrate (FC) and the iron chelator deferoxamine mesylate (DFO) increased and decreased, respectively, the LCN2-induced neurotoxicity in vivo In addition to the in vivo results, 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in cocultures of mesencephalic neurons and astrocytes was reduced by LCN2 gene deficiency in the astrocytes and conditioned media derived from MPP(+)-treated SH-SY5Y neuronal enhanced glial expression of LCN2 in vitro Therefore, our results demonstrate that astrocytic LCN2 upregulation in the lesioned DA system may play a role as a potential pathogenic factor in PD and suggest that inhibition of LCN2 expression or activity may be useful in protecting the nigrostriatal DA system in the adult brain. SIGNIFICANCE STATEMENT: Lipocalin-2 (LCN2), a member of the highly heterogeneous secretory protein family of lipocalins, may contribute to neuroinflammation and neurotoxicity in the brain. However, LCN2 expression and its role in Parkinson's disease (PD) are largely unknown. Here, we report that LCN2 is upregulated in the substantia nigra of patients with PD and neurotoxin-treated animal models of PD. Our results suggest that LCN2 upregulation might be a potential pathogenic mechanism of PD, which would result in disruption of the nigrostriatal dopaminergic system through neurotoxic iron accumulation and neuroinflammation. Therefore, inhibition of LCN2 expression or activity may be useful in protecting the nigrostriatal dopaminergic projection in PD.

Pyruvate Dehydrogenase Kinase-mediated Glycolytic Metabolic Shift in the Dorsal Root Ganglion Drives Painful Diabetic Neuropathy.

The dorsal root ganglion (DRG) is a highly vulnerable site in diabetic neuropathy. Under diabetic conditions, the DRG is subjected to tissue ischemia or lower ambient oxygen tension that leads to aberrant metabolic functions. Metabolic dysfunctions have been documented to play a crucial role in the pathogenesis of diverse pain hypersensitivities. However, the contribution of diabetes-induced metabolic dysfunctions in the DRG to the pathogenesis of painful diabetic neuropathy remains ill-explored. In this study, we report that pyruvate dehydrogenase kinases (PDK2 and PDK4), key regulatory enzymes in glucose metabolism, mediate glycolytic metabolic shift in the DRG leading to painful diabetic neuropathy. Streptozotocin-induced diabetes substantially enhanced the expression and activity of the PDKs in the DRG, and the genetic ablation of Pdk2 and Pdk4 attenuated the hyperglycemia-induced pain hypersensitivity. Mechanistically, Pdk2/4 deficiency inhibited the diabetes-induced lactate surge, expression of pain-related ion channels, activation of satellite glial cells, and infiltration of macrophages in the DRG, in addition to reducing central sensitization and neuroinflammation hallmarks in the spinal cord, which probably accounts for the attenuated pain hypersensitivity. Pdk2/4-deficient mice were partly resistant to the diabetes-induced loss of peripheral nerve structure and function. Furthermore, in the experiments using DRG neuron cultures, lactic acid treatment enhanced the expression of the ion channels and compromised cell viability. Finally, the pharmacological inhibition of DRG PDKs or lactic acid production substantially attenuated diabetes-induced pain hypersensitivity. Taken together, PDK2/4 induction and the subsequent lactate surge induce the metabolic shift in the diabetic DRG, thereby contributing to the pathogenesis of painful diabetic neuropathy.

Astrocyte-derived lipocalin-2 mediates hippocampal damage and cognitive deficits in experimental models of vascular dementia.

Lipocalin-2 (LCN2) has diverse functions in multiple pathophysiological conditions; however, its pathogenic role in vascular dementia (VaD) is unknown. Here, we investigated the role of LCN2 in VaD using rodent models of global cerebral ischemia and hypoperfusion with cognitive impairment and neuroinflammation. Mice subjected to transient bilateral common carotid artery occlusion (tBCCAo) for 50 min showed neuronal death and gliosis in the hippocampus at 7 days post-tBCCAo. LCN2 expression was observed predominantly in the hippocampal astrocytes, whereas its receptor was mainly detected in neurons, microglia, and astrocytes. Furthermore, Lcn2-deficient mice, compared with wild-type animals, showed significantly weaker CA1 neuronal loss, cognitive decline, white matter damage, blood-brain barrier permeability, glial activation, and proinflammatory cytokine production in the hippocampus after tBCCAo. Lcn2 deficiency also attenuated hippocampal neuronal death and cognitive decline at 30 days after unilateral common carotid artery occlusion (UCCAo). Furthermore, intracerebroventricular (i.c.v) injection of recombinant LCN2 protein elicited CA1-neuronal death and a cognitive deficit. Our studies using cultured glia and hippocampal neurons supported the decisive role of LCN2 in hippocampal neurotoxicity and microglial activation, and the role of the HIF-1α-LCN2-VEGFA axis of astrocytes in vascular injury. Additionally, plasma levels of LCN2 were significantly higher in patients with VaD than in the healthy control subjects. These results indicate that hippocampal damage and cognitive impairment are mediated by LCN2 secreted from reactive astrocytes in VaD.

Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis.

Pyruvate dehydrogenase kinases (PDK1-4) are mitochondrial metabolic regulators that serve as decision makers via modulation of pyruvate dehydrogenase (PDH) activity to convert pyruvate either aerobically to acetyl-CoA or anaerobically to lactate. Metabolic dysregulation and inflammatory processes are two sides of the same coin in several pathophysiological conditions. The lactic acid surge associated with the metabolic shift has been implicated in diverse painful states. In this study, we investigated the role of PDK-PDH-lactic acid axis in the pathogenesis of chronic inflammatory pain. Deficiency of Pdk2 and/or Pdk4 in mice attenuated complete Freund's adjuvant (CFA)-induced pain hypersensitivities. Likewise, Pdk2/4 deficiency attenuated the localized lactic acid surge along with hallmarks of peripheral and central inflammation following intraplantar administration of CFA. In vitro studies supported the role of PDK2/4 as promoters of classical proinflammatory activation of macrophages. Moreover, the pharmacological inhibition of PDKs or lactic acid production diminished CFA-induced inflammation and pain hypersensitivities. Thus, a PDK-PDH-lactic acid axis seems to mediate inflammation-driven chronic pain, establishing a connection between metabolism and inflammatory pain. SIGNIFICANCE STATEMENT: The mitochondrial pyruvate dehydrogenase (PDH) kinases (PDKs) and their substrate PDH orchestrate the conversion of pyruvate either aerobically to acetyl-CoA or anaerobically to lactate. Lactate, the predominant end product of glycolysis, has recently been identified as a signaling molecule for neuron-glia interactions and neuronal plasticity. Pathological metabolic shift and subsequent lactic acid production are thought to play an important role in diverse painful states; however, their contribution to inflammation-driven pain is still to be comprehended. Here, we report that the PDK-PDH-lactic acid axis constitutes a key component of inflammatory pain pathogenesis. Our findings establish an unanticipated link between metabolism and inflammatory pain. This study unlocks a previously ill-explored research avenue for the metabolic control of inflammatory pain pathogenesis.

Phenotypic polarization of activated astrocytes: the critical role of lipocalin-2 in the classical inflammatory activation of astrocytes.

Astrocytes provide structural and functional support for neurons, as well as display neurotoxic or neuroprotective phenotypes depending upon the presence of an immune or inflammatory microenvironment. This study was undertaken to characterize multiple phenotypes of activated astrocytes and to investigate the regulatory mechanisms involved. We report that activated astrocytes in culture exhibit two functional phenotypes with respect to pro- or anti-inflammatory gene expression, glial fibrillary acidic protein expression, and neurotoxic or neuroprotective activities. The two distinct functional phenotypes of astrocytes were also demonstrated in a mouse neuroinflammation model, which showed pro- or anti-inflammatory gene expression in astrocytes following challenge with classical or alternative activation stimuli; similar results were obtained in the absence of microglia. Subsequent studies involving recombinant lipocalin-2 (LCN2) protein treatment or Lcn2-deficient mice indicated that the pro- or anti-inflammatory functionally polarized phenotypes of astrocytes and their intracellular signaling pathway were critically regulated by LCN2 under in vitro and in vivo conditions. Astrocyte-derived LCN2 promoted classical proinflammatory activation of astrocytes but inhibited IL-4-STAT6 signaling, a canonical pathway involved in alternative anti-inflammatory activation. Our results suggest that the secreted protein LCN2 is an autocrine modulator of the functional polarization of astrocytes in the presence of immune or inflammatory stimuli and that LCN2 could be targeted therapeutically to dampen proinflammatory astrocytic activation and related pathologies in the CNS.

Chronic sleep deprivation-induced proteome changes in astrocytes of the rat hypothalamus.

Sleep deprivation (SD) can influence cognition, memory, and sleep/wake homeostasis and can cause impairments in many physiological processes. Because the homeostatic control of the sleep/wake cycle is closely associated with the hypothalamus, the current study was undertaken to examine proteomic changes occurring in hypothalamic astrocytes following chronic partial SD. After chronic partial SD for 7 days, astrocytes were prepared from rat hypothalamus using a Percoll gradient method, and their proteome profiles were determined by LC-MS/MS. Comparisons of the proteome profiles of hypothalamic astrocytes revealed that chronic partial SD increased (≥1.5-fold) 89 proteins and decreased (≤0.7-fold) 50 proteins; these changes in protein expression were validated by western blot or immunohistochemistry. DAVID and IPA analyses of these proteins suggested that SD may influence gliotransmission and astrocyte activation. PPP2R1A, RTN4, VAMP-2, LGI-1, and SLC17A7 were identified and validated as the main targets of SD in astrocytes. Our results suggest that SD may modulate gliotransmission in the hypothalamus, thereby disturbing sleep/wake homeostasis and increasing susceptibility to neurological disease; however, further studies are required to confirm whether the proteome changes are specific to SD.

Push-in Head Restraining Apparatus for Intracranial Self Stimulation Tasks in Rats.

Head restraining is an experimental technique that firmly secures the animal's head to a fixation apparatus for the precise control and sensing of behaviors. However, procedural and surgical difficulties and limitations have been obstructing the use of the technique in neurophysiological and behavioral experiments. Here, we propose a novel design of the head-restraining apparatus which is easy to develop and convenient for practical use. Head restraining procedure can be completed by sliding the head mounter, which is molded by dental cement during implantation surgery, into the port, which serves as matching guide rails for the mounter, of the fixation bar. So neither skull-attached plates nor screws for fixation are needed. We performed intracranial self stimulation experiment in rats using the newly designed device. Rats were habituated to acclimatize the head-restraint environment and trained to discriminate two spatially distinguished cues using a customized push-pull lever as an operandum. Direct electrical stimulation into the medial forebrain bundle served as reward. We confirmed that head restraining was stable throughout experiments and rats were able to learn to manipulate the lever after successful habituation. Our experimental framework might help precise control or sensing of behavior under head fixed rats using direct electrical brain stimulation as a reward.

α(2A) adrenoceptor-mediated presynaptic inhibition of GABAergic transmission in rat tuberomammillary nucleus neurons.

Histaminergic neurons within the tuberomammillary nucleus (TMN) play an important role in the regulation of sleep-wakefulness. Here, we report the adrenergic modulation of GABAergic transmission in rat TMN histaminergic neurons using a conventional whole-cell patch clamp technique. Norepinephrine (NE) reversibly decreased the amplitude of action potential-dependent GABAergic inhibitory post-synaptic currents (IPSCs) and increased the paired pulse ratio. The NE-induced inhibition of GABAergic IPSCs was mimicked by clonidine, a selective α2 adrenoceptor agonist. However, cirazoline and isoproterenol, nonselective α1 and ß adrenoceptor agonists, respectively, had no effect on GABAergic IPSCs. The NE-induced inhibition of GABAergic IPSCs was significantly blocked by BRL44408, a selective α2A adrenoceptor antagonist, but not imiloxan or JP1302, a selective α2B and α2C adrenoceptor antagonists. The extent of NE-induced inhibition of GABAergic IPSCs was inversely proportional to the extracellular Ca(2+) concentration. Pharmacological agents affecting the activities of adenylyl cyclase or G-protein-coupled inwardly rectifying K(+) channels did not affect the NE-induced inhibition of GABAergic IPSCs. However, NE had no effect on the frequency and amplitude of GABAergic miniature IPSCs. These results suggest that NE acts on presynaptic α2A adrenoceptor to inhibit action potential-dependent GABA release via the inhibition of Ca(2+) influx from the extracellular space to GABAergic nerve terminals, and that this α2A adrenoceptor-mediated modulation of GABAergic transmission may be involved in regulating the excitability of TMN histaminergic neurons.

Natural flavone jaceosidin is a neuroinflammation inhibitor.

Jaceosidin is a naturally occurring flavone with pharmacological activity. Jaceosidin, as one of the major constituents of the medicinal herbs of the genus Artemisia, has been shown to exert anticancer, anti-oxidative, anti-inflammatory, and immunosuppressive effects. This study was undertaken to determine the effect of jaceosidin on microglia and neuroinflammation. Microglia are the innate immune cells in the central nervous system, and they play a central role in the initiation and maintenance of neuroinflammation. We report that jaceosidin inhibits inflammatory activation of microglia, reducing nitric oxide (NO) production and proinflammatory cytokine expression. IC50 for NO inhibition was 27 ± 0.4 µM. The flavone also attenuated microglial neurotoxicity in the microglia/neuroblastoma co-culture. Systemic injection of jaceosidin ameliorated neuroinflammation in the mouse model of experimental allergic encephalomyelitis. These results indicate that plant flavone jaceosidin is a microglial inhibitor with anti-neuroinflammation activity.

Melanin-concentrating hormone neurons discharge in a reciprocal manner to orexin neurons across the sleep-wake cycle.

Neurons containing melanin-concentrating hormone (MCH) are codistributed with neurons containing orexin (Orx or hypocretin) in the lateral hypothalamus, a peptide and region known to be critical for maintaining wakefulness. Evidence from knockout and c-Fos studies suggests, however, that the MCH neurons might play a different role than Orx neurons in regulating activity and sleep-wake states. To examine this possibility, neurons were recorded across natural sleep-wake states in head-fixed rats and labeled by using the juxtacellular technique for subsequent immunohistochemical identification. Neurons identified as MCH+ did not fire during wake (W); they fired selectively during sleep, occasionally during slow wave sleep (SWS) and maximally during paradoxical sleep (PS). As W-Off/Sleep-On, the MCH neurons discharged in a reciprocal manner to the W-On/Sleep-Off Orx neurons and could accordingly play a complementary role to Orx neurons in sleep-wake state regulation and contribute to the pathophysiology of certain sleep disorders, such as narcolepsy with cataplexy.

Long-termin-vivorecording performance of flexible penetrating microelectrode arrays.

Objective. Neural interfaces are an essential tool to enable the human body to directly communicate with machines such as computers or prosthetic robotic arms. Since invasive electrodes can be located closer to target neurons, they have advantages such as precision in stimulation and high signal-to-noise ratio (SNR) in recording, while they often exhibit unstable performance in long-termin-vivoimplantation because of the tissue damage caused by the electrodes insertion. In the present study, we investigated the electrical functionality of flexible penetrating microelectrode arrays (FPMAs) up to 3 months inin-vivoconditions.Approach. Thein-vivoexperiment was performed by implanting FPMAs in five rats. Thein-vivoimpedance as well as the action potential (AP) amplitude and SNR were analyzed over weeks. Additionally, APs were tracked over time to investigate the possibility of single neuron recording.Main results. It was observed that the FPMAs exhibited dramatic increases in impedance for the first 4 weeks after implantation, accompanied by decreases in AP amplitude. However, the increase/decrease in AP amplitude was always accompanied by the increase/decrease in background noise, resulting in quite consistently maintained SNRs. After 4 weeks of implantation, we observed two distinctive issues regarding long-term implantation, each caused by chronic tissue responses or by the delamination of insulation layer. The results demonstrate that the FPMAs successfully recorded neuronal signals up to 12 weeks, with very stably maintained SNRs, reduced by only 16.1% on average compared to the first recordings, although biological tissue reactions or physical degradation of the FPMA were present.Significance. The fabricated FPMAs successfully recorded intracortical signals for 3 months. The SNR was maintained up to 3 months and the chronic function of FPMA was comparable with other silicon based implantable electrodes.

Discharge profiles of identified GABAergic in comparison to cholinergic and putative glutamatergic basal forebrain neurons across the sleep-wake cycle.

Whereas basal forebrain (BF) cholinergic neurons are known to participate in processes of cortical activation during wake (W) and paradoxical sleep (PS or P, also called REM sleep), codistributed GABAergic neurons have been thought to participate in processes of cortical deactivation and slow-wave sleep (SWS or S). To learn the roles the GABAergic neurons might play, in relation to cholinergic and glutamatergic neurons, we juxtacellularly recorded and labeled neurons during natural sleep-wake states in head-fixed rats. Neurobiotin (Nb)-labeled cells were identified immunohistochemically as choline acetyltransferase (ChAT)+, glutamic acid decarboxylase (GAD)+, or ChAT-/GAD-. Of the latter, some were identified as glutamatergic by immunostaining of their terminals with the vesicular glutamate transporter (VGluT2). In contrast to ChAT+ neurons, which all discharged maximally during W and PS, GAD+ neurons comprised multiple sleep-wake subgroups. Some GABAergic neurons discharged maximally during W and PS, as WP-max active cells (36%), and in positive correlation with gamma electroencephalographic (EEG) activity. Some discharged maximally during SWS, as S-max active cells (28%), and in positive correlation with delta EEG activity. Others increased their discharge progressively during sleep to discharge maximally during PS, as P-max active cells (36%), and in negative association with electromyographic (EMG) activity. ChAT-/GAD- cells comprised WP-max (46%), S-max (17%), P-max (17%), and W-max active cells (14%), whose discharge was positively correlated with EMG activity. GABAergic neurons would thus play similar or reciprocal roles to other cholinergic and glutamatergic BF neurons in regulating cortical activity and muscle tone along with behavior across sleep-wake states.

Compound K, a metabolite of ginsenosides, facilitates spontaneous GABA release onto CA3 pyramidal neurons.

Ginsenoside Rb1, a major ingredient of ginseng saponins, can affect various brain functions, including learning and memory. When ingested orally, ginsenoside Rb1 is not found in plasma as well as urine, but its metabolite compound K (ComK) reaches the systemic circulation in animals and human. Nevertheless, the pharmacological actions of ComK are still poorly known. In the present study, we investigated the effect of ComK on GABAergic spontaneous miniature inhibitory post-synaptic currents (mIPSCs) in acutely isolated rat hippocampal CA3 pyramidal neurons using a conventional whole-cell patch-clamp technique. While ComK significantly increased mIPSC frequency in a concentration-dependent manner, it had no effect on the current amplitude, suggesting that ComK acts pre-synaptically to increase the probability of spontaneous GABA release. ComK still increased mIPSC frequency even in a Ca(2+) -free external solution, suggesting that the ComK-induced increase spontaneous GABA release is not related to Ca(2+) influx from the extracellular space. However, the ComK-induced increase mIPSC frequency was significantly decreased after the blockade of either sarcoplasmic/endoplasmic reticulum Ca(2+) -ATPase or Ca(2+) release channels. These results strongly suggest that ComK enhances spontaneous GABA release by increasing intraterminal Ca(2+) concentration via Ca(2+) release from pre-synaptic Ca(2+) stores. The ComK-induced modulation of inhibitory transmission onto CA3 pyramidal neurons could have a broad impact on the excitability of CA3 pyramidal neurons and affect the physiological functions mediated by the hippocampus.

GABAergic neurons intermingled with orexin and MCH neurons in the lateral hypothalamus discharge maximally during sleep.

The lateral hypothalamus (LH), where wake-active orexin (Orx)-containing neurons are located, has been considered a waking center. Yet, melanin-concentrating hormone (MCH)-containing neurons are codistributed therein with Orx neurons and, in contrast to them, are active during sleep, not waking. In the present study employing juxtacellular recording and labeling of neurons with Neurobiotin (Nb) in naturally sleeping-waking head-fixed rats, we identified another population of intermingled sleep-active cells, which do not contain MCH (or Orx), but utilize gamma-aminobutyric acid (GABA) as a neurotransmitter. The 'sleep-max' active neurons represented 53% of Nb-labeled MCH-(and Orx) immunonegative (-) cells recorded in the LH. For identification of their neurotransmitter, Nb-labeled varicosities of the Nb-labeled/MCH- neurons were sought within sections adjacent to the Nb-labeled soma and immunostained for the vesicular transporter for GABA (VGAT) or for glutamate. A small proportion of sleep-max Nb+/MCH- neurons (19%) discharged maximally during slow-wave sleep (called 'S-max') in positive correlation with delta electroencephalogram activity, and from VGAT staining of Nb-labeled varicosities appeared to be GABAergic. The vast proportion of sleep-max Nb+/MCH- neurons (81%) discharged maximally during paradoxical sleep (PS, called 'P-max') in negative correlation with electromyogram amplitude, and from Nb-labeled varicosities also appeared to be predominantly GABAergic. Given their discharge profiles across the sleep-wake cycle, P-max together with S-max GABAergic neurons could thus serve to inhibit other neurons of the arousal systems, including local Orx neurons in the LH. They could accordingly dampen arousal with muscle tone and promote sleep, including PS with muscle atonia.

Presynaptic glycine receptors facilitate spontaneous glutamate release onto hilar neurons in the rat hippocampus.

Although glycine receptors are found in most areas of the brain, including the hippocampus, their functional significance remains largely unknown. In the present study, we have investigated the role of presynaptic glycine receptors on excitatory nerve terminals in spontaneous glutamatergic transmission. Spontaneous EPSCs (sEPSCs) were recorded in mechanically dissociated rat dentate hilar neurons attached with native presynaptic nerve terminals using a conventional whole-cell patch recording technique under voltage-clamp conditions. Exogenously applied glycine or taurine significantly increased the frequency of sEPSCs in a concentration-dependent manner. This facilitatory effect of glycine was blocked by 1 microM strychnine, a specific glycine receptor antagonist, but was not affected by 30 microM picrotoxin. In addition, Zn(2+) (10 microM) potentiated the glycine action on sEPSC frequency. Pharmacological data suggested that the activation of presynaptic glycine receptors directly depolarizes glutamatergic terminals resulting in the facilitation of spontaneous glutamate release. Bumetanide (10 microM), a specific Na-K-2C co-transporter blocker, gradually attenuated the glycine-induced sEPSC facilitation, suggesting that the depolarizing action of presynaptic glycine receptors was due to a higher intraterminal Cl(-) concentration. The present results suggest that presynaptic glycine receptors on excitatory nerve terminals might play an important role in the excitability of the dentate gyrus-hilus-CA3 network in physiological and/or pathological conditions.

Differential pharmacological properties of GABAA receptors in axon terminals and soma of dentate gyrus granule cells.

Although it has been well established that GABA(A) receptors are molecular targets of a variety of allosteric modulators, such as benzodiazepines, the pharmacological properties of presynaptic GABA(A) receptors are poorly understood. In this study, the effects of diazepam and Zn(2+) on presynaptic GABA(A) receptors have been investigated by measuring the GABA(A) receptor-mediated facilitation of spontaneous glutamate release in mechanically dissociated rat CA3 pyramidal neurons. Diazepam significantly enhanced the muscimol-induced facilitation (particularly at submicromolar concentrations) of spontaneous glutamate release and shifted the concentration-response relationship for muscimol toward the left, whereas Zn(2+) (