Gamma‐protocadherins are enriched and transported in specialized vesicles associated with the secretory pathway in neurons

Gamma protocadherins (Pcdh‐γs) resemble classical cadherins and have the potential to engage in cell–cell interactions with homophilic properties. Emerging evidence suggests non‐conventional roles for some protocadherins in neural development. We sought to determine whether Pcdh‐γ trafficking in neurons is consistent with an intracellular role for these molecules. Here we show that, in contrast to the largely surface localization of classical cadherins, endogenous Pcdh‐γs are primarily intracellular in rat neurons in vivo and are equally distributed within organelles of subsynaptic dendritic and axonal compartments. A strikingly higher proportion of Pcdh‐γ‐containing organelles in synaptic compartments was observed at postnatal day 16. To determine the origin of Pcdh‐γ‐trafficking organelles, we isolated organelles with Pcdh‐γ antibody‐coupled magnetic beads from brain organelle suspensions. Vesicles with high levels of COPII and endoplasmic reticulum–Golgi intermediate compartment (ERGIC) components were isolated with the Pcdh‐γ antibody but not with the classical cadherin antibody. In cultured hippocampal neurons, Pcdh‐γ immunolabeling partially overlapped with calnexin‐ and COPII‐positive puncta in dendrites. Mobile Pcdh‐γ‐GFP profiles dynamically codistributed with a DsRed construct coupled to ER retention signals by live imaging. Pcdh‐γ expression correlated with accumulations of tubulovesicular and ER‐like organelles in dendrites. Our results are consistent with the possibility that Pcdh‐γs could have a unique function within the secretory pathway in addition to their documented surface roles.     

Adenosine receptors and muscarinic receptors cooperate in acetylcholine release modulation in the neuromuscular synapse

Adenosine receptors (ARs) are present in the motor terminals at the mouse neuromuscular junction. ARs and the presynaptic muscarinic acetylcholine receptors (mAChRs) share the functional control of the neuromuscular junction. We analysed their mutual interaction in transmitter release modulation. In electrophysiological experiments with unaltered synaptic transmission (muscles paralysed by blocking the voltage‐dependent sodium channel of the muscle cells with μ‐conotoxin GIIIB), we found that: (i) a collaborative action between different AR subtypes reduced synaptic depression at a moderate activity level (40 Hz); (ii) at high activity levels (100 Hz), endogenous adenosine production in the synaptic cleft was sufficient to reduce depression through A₁‐type receptors (A₁Rs) and A₂A‐type receptors (A_2ARs); (iii) when the non‐metabolizable 2‐chloroadenosine (CADO) agonist was used, both the quantal content and depression were reduced; (iv) the protective effect of CADO on depression was mediated by A₁Rs, whereas A_2ARs seemed to modulate A₁Rs; (v) ARs and mAChRs absolutely depended upon each other for the modulation of evoked and spontaneous acetylcholine release in basal conditions and in experimental conditions with CADO stimulation; (vi) the purinergic and muscarinic mechanisms cooperated in the control of depression by sharing a common pathway although the purinergic control was more powerful than the muscarinic control; and (vii) the imbalance of the ARs created by using subtype‐selective and non‐selective inhibitory and stimulatory agents uncoupled protein kinase C from evoked transmitter release. In summary, ARs (A₁Rs, A_2ARs) and mAChRs (M₁, M₂) cooperated in the control of activity‐dependent synaptic depression and may share a common protein kinase C pathway.     

Abnormal distribution of acetylcholinesterase activity in the hippocampal formation of the dreher mutant mouse

The distribution of acetylcholinesterase(AChE) in the hippocampal formation of the dreher mutant mouse was studied by comparing homozygous mutant (dr^sst-J/dr^sst-J) with littermate control (+ /? or + / +). In the control mice, AChE activity was most intense in the inner one-third of the stratum oriens and lacnosum of the hippocampus, and in the inner one-fifth of the molecular layer of the dentate gyrus. In contrast, in honozygous dreher mice, AChE activity in area CA3c of the hippocampus was not restricted to the stratum oriens, and extended upward into the infrapyramidal and suprapyramidal mossy fiber layers, the lower part of the stratum radiatum, the pyramidal cell layer, and downward toward the alveus. In addition, the distribution of AChE activity was modified by accompanying with ectopic pyramidal cells or with disruption of the pyramidal cell layer. AChE activity in the dentate gyrus of the dreher mouse was not confined to the inner one-fifth of the molecular layer. These findings indicated that the cholinergic input to the hippocampal formation is not normal in the dreher mutant mouse. Since the areas of AChE activity correspond to the presence of ectopic pyramidal cells in the dreher mouse, incoming cholinergic fibers may form synapses with these ectopic cells and with the dendrites of normal pyramidal cells that extend into the expanded area of AChE activity.     

Regulation by lipocalin-2 of neuronal cell death, migration, and morphology

A secreted protein, lipocalin-2 (LCN2), has been previously shown to regulate a variety of cellular phenotypes such as cell death, migration, and morphology. The role of LCN2, however, appears to be different depending on the cellular context. Here, we investigated how LCN2 influences neuronal phenotypes by using primary cortical neuronal cell cultures and neuroblastoma cell lines as a model. When exposed to LCN2 protein, neurons and neuroblastoma cells were sensitized to cell death evoked by nitric oxide, oxidative stress, and tumor necrosis factor-α (TNF-α). A forced expression of lcn2 in glia enhanced neuronal cell death in cocultures of glia and neurons, indicating that both exogenous protein addition and endogenous expression of lcn2 give rise to similar results. Iron and BCL2-interacting mediator of cell death (BIM) protein were involved in LCN2-induced cell death sensitization, based on the studies using iron donor, chelator, siderophore, and short hairpin RNA (shRNA)-mediated knockdown of bim expression. Furthermore, cell migration assay and immunofluorescence microscopic observation revealed that LCN2 accelerated neuronal motility and process extension, suggesting multiple roles for LCN2 in the regulation of neuronal cell death, migration, and morphology.     

Evidence for neuronal interactions by electrical field effects in the CA3 and dentate regions of rat hippocampal slices

During population spikes in slices of rat hippocampus, transmembrane differential recordings (intracellular minus extracellular) revealed electrical field-effect depolarizations in CA3 pyramidal and dentate granule neurons. The field-effect depolarizations were not seen in single-ended recordings, and were consistently shown to increase neuronal excitability. Therefore, as previously shown for the CA1 area, large population spikes from CA3 pyramidal and dentate granule cells are associated with transmembrane depolarizations that increase neuronal excitability.     

Selective suppression of horizontal propagation in rat visual cortex by norepinephrine

The release of norepinephrine in the cerebral cortex from axon terminals of locus coeruleus neurons was suggested to be involved in the control of attention. Accumulating data indicate that the responses of cortical neurons are varied when norepinephrine is applied iontophoretically in the vicinity of the cells being recorded. However, it is not known how the pattern of excitatory propagation is modified when norepinephrine is applied over a wide area in the visual cortex. By applying optical imaging to rat visuocortical slices, we found a new mode of norepinephrine action; a prominent suppression of the horizontal propagation in layers II/III. This action of norepinephrine was confirmed by the simultaneous recording of field potentials from multiple sites by use of a multi-electrode dish. Furthermore, our electrophysiological recordings showed that this norepinephrine action is exerted through suppression of excitatory neural transmission and enhancement of inhibitory transmission to the pyramidal neurons in these layers. Because the release of norepinephrine in the visual cortex is regulated by the level of attention, the neural basis of visual attention may relate partially to the suppression of the integration of visual information by norepinephrine resulting in a state-dependent restructuring of the receptive field.     

Short‐term synaptic plasticity at the main and vomeronasal olfactory receptor to mitral cell synapse in frog

Synaptic responses resulting from stimulation of the main olfactory and vomeronasal (VN) nerves were measured in main and accessory olfactory bulb (AOB) of frog, Rana pipiens, to test the hypothesis that properties of these synapses would reflect the distinct differences in the time course of odour delivery to each of these olfactory structures. Paired‐pulse depression dominated responses to repetitive stimulation of the main olfactory nerve for interstimulus intervals (ISI) up to several seconds. Inhibition of voltage‐gated Ca²⁺ channels by GABAb receptors contributes significantly to this inhibition of transmitter release, particularly for ISI > 0.5 s. In contrast, the monosynaptic connection between VN sensory neurons and mitral cells in the AOB showed enhancement with pairs or short trains of stimuli for ISI of 0.5 to > 10 s. A small inhibitory effect of GABAb receptors on presynaptic Ca²⁺ influx and release was only evident when a large proportion of the VN axons were stimulated simultaneously but even with inhibition present an overall enhancement of release was observed. Increasing the number of conditioning stimuli from one to five increased residual [Ca²⁺] and enhancement but a direct correlation between residual [Ca²⁺] and either the magnitude or the time course of enhancement was not observed. Enhanced transmitter release from VN afferent terminals results in effective integration of sustained low‐frequency activity, which may play a role in the detection of low‐intensity odourant stimuli by the VN system.     

Development and distribution of neuronal cilia in mouse neocortex

Neuronal primary cilia are not generally recognized, but they are considered to extend from most, if not all, neurons in the neocortex. However, when and how cilia develop in neurons are not known. This study used immunohistochemistry for adenylyl cyclase III (ACIII), a marker of primary cilia, and electron microscopic analysis to describe the development and maturation of cilia in mouse neocortical neurons. Our results indicate that ciliogenesis is initiated in late fetal stages after neuroblast migration, when the mother centriole docks with the plasma membrane, becomes a basal body, and grows a cilia bud that we call a procilium. This procilium consists of a membranous protrusion extending from the basal body but lacking axonemal structure and remains undifferentiated until development of the axoneme and cilia elongation starts at about postnatal day 4. Neuronal cilia elongation and final cilia length depend on layer position, and the process extends for a long time, lasting 8-12 weeks. We show that, in addition to pyramidal neurons, inhibitory interneurons also grow cilia of comparable length, suggesting that cilia are indeed present in all neocortical neuron subtypes. Furthermore, the study of mice with defective ciliogenesis suggested that failed elongation of cilia is not essential for proper neuronal migration and laminar organization or establishment of neuronal polarity. Thus, the function of this organelle in neocortical neurons remains elusive.     

The effect of nicotine on spiking activity and Ca2+ dynamics of dendritic spines in rat CA1 pyramidal neurons

Nicotinic acetylcholine receptors (nAChRs) of the hippocampus have been thought to contribute to cognitive enhancement by cigarette smoking. Although positive modulation on cognitive functions is linked to the smoked, low-dose nicotine, the cellular correlate behind this modulation is unknown. It has been accepted that cellular mechanisms underlying plastic effects on memory involve the association of backpropagating action potentials (bAPs) with synaptic activity in the hippocampus. Here, we show the effects of low-dose (1 microM) nicotine on bAP-evoked Ca2+ transients in basal dendrites and spines of pyramidal neurons in rat hippocampal slices. Although nicotine application failed to have any direct effect in low concentration, it could significantly enhance bAP-evoked Ca2+ transients through presynaptic nAChRs located on axon terminals innervating pyramidal cells. The activation of these receptors is known to release neurotransmitters and induce postsynaptic currents. High-dose (250-500 microM) nicotine could induce firing and Ca2+ accumulation in spines. Large amplitude currents were observed occasionally (8 out of 18 cells) in voltage clamp recordings in response to pressure application of high-dose nicotine. This may explain the relatively low incidence of nicotine-induced firing (7 out of 27 cells) under current clamp. These data indicate that (i) activation of presynaptic nAChRs can modulate backreporting in dendrites of pyramidal neurons and (ii) there is a group of pyramidal neurons with higher nicotine-sensitivity, producing firing at strong stimulations. Our data revealed a subcellular effect of nicotine through regulation of Ca2+ levels in the computational units of pyramidal neurons.     

Endoplasmic reticulum dynamics in hippocampal dendritic spines induced by agonists of type I metabotropic glutamate but not by muscarinic acetylcholine receptors

Neurons in the hippocampus exhibit subpopulations of dendritic spines that contain endoplasmic reticulum (ER). ER in spines is important for synaptic activity and its associated Ca(2+) signaling. The dynamic distribution of ER to spines is regulated by diacylglycerol and partly mediated by protein kinase C, metalloproteinases and γ-secretase. In this study, we explored whether pharmacological activation of type I metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChRs) known to activate phospholipase C would have any effect on spine ER content. We found that DHPG (100 μM) but not carbachol (10 μM) caused a reduction in the number of spines with ER. We further found that ER Ca(2+) depletion triggered by thapsigargin (200 nM) had no effect on ER localization in spines.     

Human cerebrospinal fluid promotes spontaneous gamma oscillations in the hippocampus in vitro

Gamma oscillations (30–80 Hz) are fast network activity patterns frequently linked to cognition. They are commonly studied in hippocampal brain slices in vitro, where they can be evoked via pharmacological activation of various receptor families. One limitation of this approach is that neuronal activity is studied in a highly artificial extracellular fluid environment, as provided by artificial cerebrospinal fluid (aCSF). Here, we examine the influence of human cerebrospinal fluid (hCSF) on kainate‐evoked and spontaneous gamma oscillations in mouse hippocampus. We show that hCSF, as compared to aCSF of matched electrolyte and glucose composition, increases the power of kainate‐evoked gamma oscillations and induces spontaneous gamma activity in areas CA3 and CA1 that is reversed by washout. Bath application of atropine entirely abolished hCSF‐induced gamma oscillations, indicating critical contribution from muscarinic acetylcholine receptor‐mediated signaling. In separate whole‐cell patch clamp recordings from rat hippocampus, hCSF increased theta resonance frequency and strength in pyramidal cells along with enhancement of h‐current (I_h) amplitude. We found no evidence of intrinsic gamma frequency resonance at baseline (aCSF) among fast‐spiking interneurons, and this was not altered by hCSF. However, hCSF increased the excitability of fast‐spiking interneurons, which likely contributed to gamma rhythmogenesis. Our findings show that hCSF promotes network gamma oscillations in the hippocampus in vitro and suggest that neuromodulators distributed in CSF could have significant influence on neuronal network activity in vivo.     

Differential distribution of release-related proteins in the hippocampal CA3 area as revealed by freeze-fracture replica labeling

Synaptic vesicle release occurs at a specialized membrane domain known as the presynaptic active zone (AZ). Several membrane proteins are involved in the vesicle release processes such as docking, priming, and exocytotic fusion. Cytomatrix at the active zone (CAZ) proteins are structural components of the AZ and are highly concentrated in it. Localization of other release-related proteins including target soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (t-SNARE) proteins, however, has not been well demonstrated in the AZ. Here, we used sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) to analyze quantitatively the distribution of CAZ and t-SNARE proteins in the hippocampal CA3 area. The AZ in replicated membrane was identified by immunolabeling for CAZ proteins (CAZ-associated structural protein [CAST] and Bassoon). Clusters of immunogold particles for these proteins were found on the P-face of presynaptic terminals of the mossy fiber and associational/commissural (A/C) fiber. Co-labeling with CAST revealed distribution of the t-SNARE proteins syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) in the AZ as well as in the extrasynaptic membrane surrounding the AZ (SZ). Quantitative analysis demonstrated that the density of immunoparticles for CAST in the AZ was more than 100 times higher than in the SZ, whereas that for syntaxin and SNAP-25 was not significantly different between the AZ and SZ in both the A/C and mossy fiber terminals. These results support the involvement of the t-SNARE proteins in exocytotic fusion in the AZ and the role of CAST in specialization of the membrane domain for the AZ.     

The genetic organization of neuron number in the pyramidal cell layer of hippocampal regio superior in house mice

This report concerns variations in neuron number within the pyramidal cell layer of hippocampal regio superior in 18 inbred strains of house mice. There is a genetically associated variability in the total number of neurons in this pyramidal layer. Systematic strain variations in the orientation of the pyramidal cell layer are also present. Relations between the numbers of neurons in variousexperimenter-defined sibdivisions of regio superior were examined following statistical corrections for the variations in orientation. This led to a preliminary delineation of 4genetically-defined subdivisions of the regio superior pyramidal cell layer.     

Intracortical distribution of axonal collaterals of pyramidal tract cells in the cat motor cortex

Slow and fast pyramidal tract cells (Pt cells) from the cat motor cortex were identified antidromically and injected with horseradish peroxidase (HRP). The axonal collaterals of these cells were mapped following HRP histochemistry with benzidine di-hydrochloride. All cells, slow or fast, show a similar arrangement of their collaterals. A proximal axonal network of 0.5–0.8 mm in diameter delimits a local field of action for collaterals in layers V and VI. The tangential expansion of this local field corresponds to that of the basal dendritic domain of Pt neurons. Much longer collaterals running for millimeters in the lower gray or white matter were observed in all cells. They form at a cortical level a distal field of action for Pt neurons. Many of these long branches were traced to other regions of area 4 or toward other cytoarchitectonic areas. In one case a collateral was seen entering and dividing in area 3a. Due to limitations of the HRP technique most of these long branches could not be followed to their terminals. On the basis of the laminar distribution of Pt cell collaterals (mostly in layers V and VI) synaptic sites where recurrent excitation and inhibition are produced on Pt neurons are discussed.     

Corticotropin-releasing factor-containing axon terminals synapse onto catecholamine dendrites and may presynaptically modulate other afferents in the rostral pole of the nucleus locus coeruleus in the rat brain

Physiological and immunohistochemical studies have suggested that corticotropin-releasing factor (CRF), the hypophysiotropic peptide that initiates endocrine responses to stress, may serve as a neurotransmitter to activate noradrenergic neurons in the nucleus locus coeruleus (LC). We combined immunoperoxidase labeling for CRF and immunogold-silver localization of the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in single sections through the rat LC to determine potential substrates for interactions between these two transmitters. Light microscopic analysis indicated that CRF processes are dense and highly varicose in the rostral LC region in the vicinity of noradrenergic dendrites. Electron microscopy of this rostral region revealed that immunoperoxidase labeling for CRF was mainly restricted to axons and axon terminals and was rarely seen in somata or dendrites. Axon terminals containing CRF immunoreactivity varied in size, content of synaptic vesicles, and formation of synaptic specializations. The postsynaptic targets of the CRF-labeled axon terminals consisted of both TH-labeled dendrites and dendrites lacking detectable TH-immunoreactivity. Of 113 CRF-immunoreactive axon terminals, approximately 70% were in direct contact with TH-labeled and unlabeled dendrites. Of the CRF-labeled axon terminals forming synapses with TH-labeled and unlabeled dendrites, they were either of the asymmetric (excitatory type; 19%) or symmetric (inhibitory type; 11%) variety or did not form identifiable contacts in the plane of section analyzed. Unlabeled axon terminals and glial processes were also commonly located adjacent to the plasma membranes of CRF-labeled axon terminals. These results provide the first direct ultrastructural evidence that axon terminals containing CRF-immunoreactivity 1) directly contact catecholamine-containing dendrites within the rostral pole of the LC, 2) may presynaptically modulate other afferents, and 3) are often enveloped by astrocytic processes. © 1996 Wiley-Liss, Inc.     

Nicotinic activity layer specifically modulates synaptic potentiation in the mouse insular cortex

Nicotinic acetylcholine receptors (nAChRs) in the insular cortex play an important role in nicotine addiction, but its cellular and synaptic mechanisms underlying nicotine addiction still remain unresolved. In layer 5 pyramidal neurons of the mouse insular cortex, activation of nAChRs suppresses synaptic potentiation through enhancing GABAergic synaptic transmission via activation of β2‐containing nAChRs in non‐fast‐spiking (non‐FS) interneurons. However, it has not been addressed whether and how activation of nAChRs modulates synaptic plasticity in layers 3 and 6 pyramidal neurons of the insular cortex. In this study, I demonstrate that activation of nAChRs oppositely modulates synaptic potentiation in layers 3 and 6 pyramidal neurons of the insular cortex. In layer 3 pyramidal neurons, activation of nAChRs depressed synaptic potentiation induced by combination of presynaptic stimulation with postsynaptic depolarization (paired training) through enhancing GABAergic synaptic transmission via activation of β2‐containing nAChRs in non‐FS interneurons. By contrast, in layer 6 pyramidal neurons, activation of nAChRs enhanced synaptic potentiation through activating postsynaptic β2‐containing nAChRs. These results indicate, in different layers of the mouse insular cortex, paired training‐induced synaptic potentiation is oppositely regulated by activation of nAChRs which are located on GABAergic interneurons (layer 3) and on pyramidal neurons (layer 6). Thus, layer‐specific modulation of synaptic potentiation may be involved in cellular and synaptic mechanisms of insular cortical changes in nicotine addiction.     

人参皂甙Rb_1、Rb_3、Rg_1对培养小鼠皮层细胞缺血损伤的保护作用及浓度—效应关系

目的:研究人参皂甙(Ginsenosides,Gs)的三种单体成分GSRb_1、GSRb_3、GSRg_1对培养小鼠皮层神经细胞缺血损伤的保护作用。方法:①将离体培养的ICR小鼠胎鼠神经细胞缺血培养以模仿缺血对中枢神经元的损伤,观察不同缺血时间神经元活性变化。②利用MTT比色法观察不同终浓度(0、20、40、60、80、100μmol/L)的GS Rb_1、Rb_3、Rg_1对缺血神经细胞的作用。③检测细胞外液LDH释放量以观察60μmol/L浓度的GSRb_1、Rb_3、Rg_1对缺血神经细胞存活的影响。结果:①培养的小鼠胎鼠皮层神经元随着缺血时间的延长,神经元活性逐渐降低,缺血时间越长,神经元活性下降越多、细胞受损越明显、死亡率增加。②在所研究GS浓度范围(20～60μmol/L)中,对神经细胞的保护作用呈现浓度依赖性,以60μmol/L终浓度的GS单体能明显提高缺血神经元的活性和生存能力,减轻细胞的形态学损伤,且这种保护作用以GSRb_2最明显,随着浓度的再进一步升高,保护作用减弱,100μmol/L的GS单体对缺血神经元无保护作用。结论:适宜浓度人参皂甙单体对缺血培养的小鼠胎鼠皮层神经细胞具保护作用。     

Influence of Ginkgo Biloba extract on beta-secretase in rat hippocampal neuronal cultures following chronic hypoxic and hypoglycemic conditions

BACKGROUND： Preparation of Ginkgo leaf has been widely used to improve cognitive deficits and dementia, in particular in Alzheirner＇s disease patients. However, the precise mechanism of action of Ginkgo leaf remains unclear. OBJECTIVE： To explore the effect of Ginkgo Biloba extract （Egb761）, Ginaton, on β -secretase expression in rat hippocampal neuronal cultures following chronic hypoxic and hypoglycemic conditions. DESIGN, TIME AND SETTNG： Completely by randomized, grouping study. The experiment was performed at the Laboratory of Molecular Imaging, Southeast University between August 2006 and August 2007. MATERIALS： A total of 128 Wistar rats aged 24 hours were selected, and hippocampal neurons were harvested for primary cultures. METHODS： On day 7, primary hippocampal neuronal cultures were treated with Egb761 （0, 25, 50, 100, 150, and 200μg/mL） under hypoxic/hypoglycemic or hypoglycemic culture conditions for 12, 24, and 36 hours, respectively. Hippocampal neurons cultured in primary culture medium served as control. MAIN OUTCOME MEASURES： Cell viability was assayed using 3-（4,5-Dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide （MTT）; fluorescence detection of β -secretase activity was performed; Western Blot was used to measure β -secretase expression. RESULTS： Cell viability under hypoxic/hypoglycemic or hypoglycemic culture conditions was significantly less than control cells （P 〈 0.05）. Under hypoxic/hypoglycemic or hypoglycemic culture conditions, treatment with 25 μg/mL Egb761 did not alter cell viability. However, 〉 25 μg/mL Egb761 induced greater cell viability （P 〈 0.05）. No differences were observed between hypoxic/hypoglycemic or hypoglycemic cells （P 〉 0.05）. α -secretase activity was increased after 12 hours in hypoxic/hypoglycemic culture （P 〈 0.01）. There were no significant differences between the 12-, 24-, or 36-hour Egb761 groups and the hypoxic/hypoglycemic groups （P 〉 0.05）. β -secretase activity was greater after