Robust glycogen shunt activity in astrocytes: Effects of glutamatergic and adrenergic agents

The significance and functional roles of glycogen shunt activity in the brain are largely unknown. It represents the fraction of metabolized glucose that passes through glycogen molecules prior to entering the glycolytic pathway. The present study was aimed at elucidating this pathway in cultured astrocytes from mouse exposed to agents such as a high [K⁺], d-aspartate and norepinephrine (NE) known to affect energy metabolism in response to neurotransmission. Glycogen shunt activity was assessed employing [1,6-¹³C]glucose, and the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) to block glycogen degradation. The label intensity in lactate, reflecting glycolytic activity, was determined by mass spectrometry. In the presence of NE a substantial glycogen shunt activity was observed, accounting for almost 40% of overall glucose metabolism. Moreover, when no metabolic stimulant was applied, a compensatory increase in glycolytic activity was seen when the shunt was inhibited by DAB. Actually the labeling in lactate exceeded that obtained when glycolysis and glycogen shunt both were operational, i.e. supercompensation. A similar phenomenon was seen when astrocytes were exposed to d-aspartate. In addition to glycolysis, tricarboxylic acid (TCA) cycle activity was monitored, analyzing labeling by mass spectrometry in glutamate which equilibrates with α-ketoglutarate. Both an elevated [K⁺] and d-aspartate induced an increased TCA cycle activity, which was altered when glycogen degradation was inhibited. Thus, the present study provides evidence that manipulation of glycogen metabolism affects both glycolysis and TCA cycle metabolism. Altogether, the results reveal a highly complex interaction between glycogenolysis and glycolysis, with the glycogen shunt playing a significant role in astrocytic energy metabolism.     

Development of glutamatergic synaptic activity in cultured spinal neurons

The development of glutamatergic synapses involves a sequence of events that are still not well understood. We have studied the time course of the development of glutamatergic synapses in cultured spinal neurons by characterizing spontaneous synaptic currents recorded from cells maintained in vitro for different times. At short times in culture (2 days in vitro; DIV2), spontaneous synaptic activity consisted almost solely of N-methyl-D-aspartate (NMDA) receptor (NMDAR) openings. In contrast, older neurons (DIV5 to DIV8) displayed clear alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR)-mediated synaptic currents, while the NMDAR-mediated activity remained small. Between 8 and 14 days in vitro there was a large increase in the density of synaptically activated NMDARs, although there was no significant increase in the density of the NMDAR-mediated current activated by exogenous glutamate. The results indicate that there is a switch in NMDAR targeting from somatic to synaptic regions during the course of the second in vitro week. Finally, our results support the conclusion that the spontaneous synaptic activity displayed in culture depends on ongoing NMDAR-mediated activity, even when the expression of synaptic NMDARs is low.     

高糖对体外培养背根神经节神经元影响的研究进展

背根神经节神经元(DRGn)在糖尿病周围神经病变发展过程中起重要作用,近年来探讨高糖对体外培养背根神经节神经元(DRGn)的影响成为研究热点.高糖环境可致DRGn细胞凋亡;同时高糖环境下DRGn在氧化应激、膜受体mGluRs及TRPV1激活、DRGn/SCs共培养等方面均与正常培养基不同,具体机制仍需深入研究.     

High resolution in situ zymography reveals matrix metalloproteinase activity at glutamatergic synapses

Synaptic plasticity involves remodeling of extracellular matrix. This is mediated, in part, by enzymes of the matrix metalloproteinase (MMP) family, in particular by gelatinase MMP-9. Accordingly, there is a need of developing methods to visualize gelatinolytic activity at the level of individual synapses, especially in the context of neurotransmitters receptors. Here we present a high-resolution fluorescent in situ zymography (ISZ), performed in thin sections of the alcohol-fixed and polyester wax-embedded brain tissue of the rat (Rattus norvegicus), which is superior to the current ISZ protocols. The method allows visualization of structural details up to the resolution-limit of light microscopy, in conjunction with immunofluorescent labeling. We used this technique to visualize and quantify gelatinolytic activity at the synapses in control and seizure-affected rat brain. In particular, we demonstrated, for the first time, frequent colocalization of gelatinase(s) with synaptic N-methyl-D-aspartic acid (NMDA)- and AMPA-type glutamate receptors. We believe that our method represents a valuable tool to study extracellular proteolytic processes at the synapses, it could be used, as well, to investigate proteinase involvement in a range of physiological and pathological phenomena in the nervous system.     

Alterations in glutamatergic and gabaergic ion channel activity in hippocampal neurons following exposure to the abused inhalant toluene

Toluene, a representative member of the large class of abused inhalants, decreases neuronal activity and depresses behavior in both animals and humans. The sites of action of toluene are not completely known but recent studies suggest that ion channels that regulate neuronal excitability may be particularly sensitive. Previous studies with recombinant receptors showed that toluene decreases currents carried by N-methyl-D-aspartate (NMDA)-glutamate receptors without affecting those gated by non-NMDA receptors. In addition, toluene increases currents generated by GABA and glycine receptors. In the present study, primary cultures of rat hippocampal neurons were used to investigate the effects of acute and chronic toluene exposure on native excitatory and inhibitory ligand-gated ion channels. Toluene dose-dependently inhibited NMDA-mediated currents (IC50 1.5 mM) but had no effect on responses evoked by the non-NMDA agonist kainic acid. Prolonged treatment of neurons with toluene (1 mM; 4 days) increased whole-cell responses to exogenously applied NMDA, reduced those evoked by GABA but did not alter responses generated by kainic acid. Immunoblot analysis revealed that prolonged toluene exposure increased levels of NR2A and NR2B NMDA receptor subunits with no change in NR1. Immunohistochemical analysis with confocal imaging showed that toluene-treated neurons had significant increases in the density of NR1 subunits as compared with control neurons. Toluene exposure increased the amplitude of synaptic NMDA currents and decreased those activated by GABA. The results from this study suggest that toluene induces compensatory responses in the functional expression of ion channels that regulate neuronal excitability.     

Pharmacological and functional evidence for extracellular 3,4-dihydroxyphenylacetic acid as an index of metabolic activity of the adrenergic neurons: an in vivo voltammetry study in the rat rostral ventrolateral medulla

Catecholamine metabolism was studied in vivo in the C1 adrenergic area of the rostral ventrolateral medulla oblongata in rats, using differential normal pulse voltammetry coupled with an activated carbon fiber microelectrode. Pharmacological evidence indicates that 3,4-dihydroxyphenylacetic acid, the major dopamine metabolite, is responsible for the electrochemical signal appearance in the C1 group, and that it reflects the catecholamine synthesis rate, as previously reported in the locus coeruleus. Indeed, 3,4-dihydroxyphenylacetic acid was estimated to be formed from 77% of the intracellular dopamine, since its synthesis was increased by only 23%, after blockade of the dopamine-beta-hydroxylase activity. Neuronal activation by retrograde electrical stimulation increased the electrochemical signal, as well as hemorrhage and hypotension, suggesting that the level of extracellular 3,4-dihydroxyphenylacetic acid is a good biochemical index of the C1 adrenergic cellular activity in baseline conditions and during cellular activation.     

A role for primary cilia in glutamatergic synaptic integration of adult-born neurons

The sequential synaptic integration of adult-born neurons has been widely examined in rodents, but the mechanisms regulating the integration remain largely unknown. The primary cilium, a microtubule-based signaling center, is essential for vertebrate development, including the development of the CNS. We examined the assembly and function of the primary cilium in the synaptic integration of adult-born mouse hippocampal neurons. Primary cilia were absent in young adult-born neurons, but assembled precisely at the stage when newborn neurons approach their final destination, further extend dendrites and form synapses with entorhinal cortical projections. Conditional deletion of cilia from adult-born neurons induced severe defects in dendritic refinement and synapse formation. Deletion of primary cilia led to enhanced Wnt and β-catenin signaling, which may account for these developmental defects. Taken together, our findings identify the assembly of primary cilia as a critical regulatory event in the dendritic refinement and synaptic integration of adult-born neurons.     

Role of ionotropic glutamatergic and GABAergic inputs on the firing activity of neurons in the external pallidum in awake monkeys

The neurons in the external segment of the pallidum (GPe) in awake animals maintain a high level of firing activity. The level and pattern of the activity change with the development of basal ganglia disorders including parkinsonism and hemiballism. The GPe projects to most of the nuclei in the basal ganglia. Thus exploring the mechanisms controlling the firing activity is essential for understanding basal ganglia function in normal and pathological conditions. To explore the role of ionotropic glutamatergic and GABAergic inputs to the GPe, unit recordings combined with local injections of receptor antagonists were performed in awake monkeys. Observations on the effects of local application of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate antagonist 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide, the N-methyl-D-aspartic acid (NMDA) antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, and the GABAA antagonist gabazine as well as the effects of muscimol blockade of the subthalamic nucleus on the spontaneous firing rate, firing patterns, and cortical stimulation induced responses in the GPe suggested the following: sustained glutamatergic and GABAergic inputs control the level of the spontaneous firing of GPe neurons; both AMPA/kainate and NMDA receptors are activated by glutamatergic inputs; some GPe neurons receive glutamatergic inputs originating from areas other than the subthalamic nucleus; no GPe neurons became silent after a combined application of glutamate and GABA antagonists, suggesting that GPe neurons have intrinsic properties or nonionotropic glutamatergic tonic inputs that sustain a fast oscillatory firing or a combination of a fast and a slow oscillatory firing in GPe neurons.     

A high glucose environment improves survival of diabetic neurons in culture.

Dorsal root ganglia neurons from streptozotocin-induced diabetic and normal C57BL mice were cultured in serum-containing and serum-free media. The ratio of dead cells was higher in diabetic neurons than in controls in the early stages of culture. The effect of glucose concentration on survival in the culture medium was also measured for 1 week. Treatment with high glucose concentrations improved the survival of diabetic neurons, which was enhanced by duration of diabetes in the animal. These results indicate that exposure to hyperglycemia in vivo might adapt neurons to a high glucose environment in vitro.     

Endocannabinoids mediate synaptic plasticity at glutamatergic synapses on spiny neurons within a basal ganglia nucleus necessary for song learning

Activation of type 1 cannabinoid receptors (CB(1)R) in many central nervous system structures induces both short- and long-term changes in synaptic transmission. Within mammalian striatum, endocannabinoids (eCB) are one of several mechanisms that induce synaptic plasticity at glutamatergic terminals onto medium spiny neurons. Striatal synaptic plasticity may contribute a critical component of adaptive motor coordination and procedural learning. Songbirds are advantageous for studying the neural mechanisms of motor learning because they possess a neural pathway necessary for song learning and adult song plasticity that includes a striato-pallidal nucleus, area X (homologous to a portion of mammalian basal ganglia). Recent findings suggest that eCBs contribute to vocal development. For example, dense CB(1)R expression in song control nuclei peaks around the closure of the sensori-motor integration phase of song development. Also, systemic administration of a CB(1)R agonist during vocal development impairs song learning. Here we test whether activation of CB(1)R alters excitatory synaptic input on spiny neurons in area X of adult male zebra finches. Application of the CB(1)R agonist WIN55212-2 decreased excitatory postsynaptic current (EPSC) amplitude; that decrease was blocked by the CB(1)R antagonist AM251. Guided by eCB experiments in mammalian striatum, we tested and verified that at least two mechanisms indirectly activate CB(1)Rs through eCBs in area X. First, activation of group I metabotropic glutamate receptors with the agonist 3,5-dihydroxyphenylglycine (DHPG) induced a CB(1)R-mediated reduction in EPSC amplitude. Second, we observed that a 10 s postsynaptic depolarization induced a calcium-mediated, eCB-dependent decrease in synaptic strength that resisted rescue with late CB(1)R blockade. Together, these results show that eCB modulation occurs at inputs to area X spiny neurons and could influence motor learning and production.     

Evidence for a glutamatergic input to pontine preganglionic neurons of the superior salivatory nucleus in rat

Parasympathetic preganglionic neurons of the superior salivatory nucleus (SSN), which projects to the pterygopalatine ganglion (PPG), modulate salivation, lacrimation, and cerebrovascular tone. Our previous studies suggest that excitatory projections from the nucleus tractus solitarii modulate cerebrovascular tone by actions on SSN neurons. In this study we sought to test the hypothesis that N-methyl-D-aspartate (NMDA) type glutamate receptors and vesicular glutamate transporters (VGLUT) are present in the SSN and that SSN neurons receive glutamatergic input. In six rats we injected tetramethylrhodamine dextran (TRD), a fluorescent tracer, unilaterally into the PPG to label SSN neurons. Four days later, rats were perfused and brain stem sections containing the SSN were processed for fluorescent immunohistochemistry for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) and vesicular glutamate transporters (VGLUT1 and VGLUT2). Confocal laser scanning microscopy showed that 88+/-3% of TRD-labeled SSN neurons contained NMDAR1-immunoreactivity (IR). The surrounding neuropil contained numerous fibers labeled for VGLUT2-IR, but not VGLUT1-IR. Double fluorescent immunohistochemistry for NMDAR1 and VGLUT2 revealed that fibers containing VGLUT2-IR were often in close proximity to cell bodies or proximal dendrites of TRD-labeled SSN neurons that were positive for NMDAR1-IR. These studies support our hypothesis that NMDA receptors and VGLUT are present in the SSN. They further provide support for the suggestion that there are glutamatergic inputs to SSN neurons and would be consistent with an excitatory input that could regulate cerebrovascular tone.     

APPswe转基因小鼠海马结构中谷氨酸和γ-氨基丁酸能神经元的数量变化

目的:探讨谷氨酸和γ-氨基丁酸在阿尔茨海默病(AD)发生、发展中的作用.方法:利用硫磺素S染色技术检测APPswe转基因小鼠脑内沉积的老年斑;免疫荧光技术标记正常及APPswe转基因小鼠海马发育过程中谷氨酸、γ-氨基丁酸(GABA)阳性细胞. 结果: 模型组小鼠海马CA1、CA3区以及齿状回谷氨酸阳性细胞的数量明显少于正常对照组,而海马各区的GABA阳性细胞在两者之间变化不明显.结论: 谷氨酸与GABA能神经元数量的改变与Aβ诱导的神经元凋亡以及AD的病理发生有关.     

Posthypoxic disturbances in glutamatergic signal transduction in rat brain neurons: correction effect of preconditioning

Severe 3-h hypobaric hypoxia was followed by impairment of Ca²⁺-mediated glutamatergic signal transduction in the posthypoxic period (no less than 72 h). This impairment manifested in changes in the calcium response to glutamate application in slices of rat brain cortex. Moderate hypoxic preconditioning prevented these disturbances developed over the first day after sever hypoxia.     

Developmental timetables and gradients of neurogenesis in cerebellar Purkinje cells and deep glutamatergic neurons: A comparative study between the mouse and the rat

The aim of this report is to determine whether the times of neuron origin and neurogenetic gradients of PCs and Deep cerebellar nucli (DCN) glutamatergic neurons are different between mice and rats. Purkinje cells (PCs) were analyzed in each compartment of the cerebellar cortex (vermis, paravermis, medial, and lateral hemispheres), and deep glutamatergic neurons at the level of the medialis, interpositus, and lateralis nuclei. Tritiated thymidine ([³H]TdR) autoradiography was applied on sections. The experimental rodents were the offspring of pregnant dams injected with [³H]TdR on embryonic days (E) 11–12, E12–13, E13–14, E14–15, E15–16, and E16–17. Our results indicate that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar PCs and deep glutamatergic neurons between mice and rats. In mice, PCs and deep glutamatergic neurons neurogenesis extend from E10 to E14, with a predominance of neurogenesis on E12 for PCs, and on E12, E11, and E10 for the medialis, interpositus, and lateralis neurons, respectively. When neurogenesis in rats was considered, the data reveal that PCs and deep glutamatergic neurons production extends from E12 to E16, with a peak of production on E14 for PCs, and on E14, E13, and E12 for the medialis, interpositus, and lateralis neurons, respectively. Current data also indicate that, both in mice and rats, both types of macroneurons are generated according to a lateral-to-medial gradient. Thus, the lateral hemisphere and the lateralis nucleus present more early-generated neurons than the vermis and the medialis nucleus, which in their turn have more late-produced neurons.     

New candidates for GABAergic neurons in the rat cerebellum: an immunocytochemical study with anti-GABA antibody

Cerebellar cortical neurons immunoreactive to anti-gamma-aminobutyric acid (GABA) antibody were examined in the rat. In addition to the Purkinje, Golgi, basket and stellate cells, spindle-shaped cells lying just below the Purkinje cell layer were found to be strongly immunoreactive to the antibody. By the combination of immunofluorescence and hematoxylin stainings, these GABA-positive cells were shown to be the Lugaro cells. Unlike the immunopositive small Golgi cell, the pale cell was not immunoreactive to the antibody.     

Presynaptic long-term depression at a central glutamatergic synapse: a role for CaMKII

CaMKII is a calcium-activated kinase that is abundant in neurons and has been strongly implicated in memory and learning. Here we show that low-frequency stimulation of glutamatergic afferents in hippocampal slices from juvenile domestic chicks results in long-term depression of synaptic transmission. This reduction does not require activation of NMDA or metabotropic glutamate receptors and does not require a rise in postsynaptic calcium. However, buffering presynaptic calcium prevents the reduction of the excitatory postsynaptic potential or current that is induced by low-frequency stimulation. In addition, application of the calmodulin antagonist calmidazolium, or the specific CaMKII antagonist KN-93, completely blocks long-term depression. These findings demonstrate a newly discovered form of long-term synaptic depression in the avian hippocampus.     

Upper cervical inspiratory neurons in the rat: an electrophysiological and morphological study

Upper cervical inspiratory neurons form a distinct neuronal column located near the lateral edge of the intermediate grey matter in the rostral spinal segments. Previous studies conducted in cats have demonstrated synaptic inputs to these neurons from several respiratory related regions of the medulla, and long descending axonal projections mainly towards the motoneurons supplying the intercostal muscles. The aim of this study was to examine the electrophysiological and morphological properties of this propriospinal system in the rat. Extracellular recordings were made from 127 cervical inspiratory units, mainly in the C₁ and C₂ segments. Eighty-two percent could be antidromically activated from the C₇/C₈ border. No evidence of monosynaptic connection was obtained by cross-correlating the activity of some of these units with the discharge of the phrenic nerve. Intracellular recordings were made from seven neurons, three of which were labelled with biotinamide (neurobiotin). Long survival times after intracellular injections (up to 23 h) resulted in staining of axons for long distances, at least to the C₅ segment. Each of the three labelled axons issued only one short collateral which arborized in the region of the phrenic nucleus. These results demonstrate that upper cervical inspiratory neurons in the rat have features similar to those previously described in the cat, including only a limited projection to the phrenic nucleus. In addition, this study provides the first morphological identification of these neurons.