LKB1表达下调对培养海马神经元突触mEPSC的影响

目的研究离体培养的海马神经元LKB1表达下调对于神经元微小兴奋性突触后电流(mEP-SC)的影响。方法选用17d的胚胎大鼠培养海马神经元,分别用电穿孔的方法转染CAG-RE质粒和LKB1RNAi质粒,培养10~12d后进行电生理记录,选用全细胞膜片钳方式及自由记录模式,细胞外液加TTX阻断动作电位,加Bicucullin抑制GABA电流,记录神经元的mEPSC,比较2组神经元的mEPSC频率和幅度的差别。结果转染CAG-RE的神经元mEPSC幅度平均为25.6pA,频率平均为(5.21±0.25)Hz,是基线的99.8%;转染LKB1RNAi的神经元mEPSC幅度平均为35.1pA,频率平均为(5.79±0.27)Hz,是基线的127.1%;比较2组间频率、幅度变化,差别有显著性意义(P<0.05)。结论LKB1基因表达下调增强了培养海马神经元突触传递的效率。     

Compensation by reduced L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor responses in a mouse model with reduced gamma-aminobutyric acid type A receptor-mediated synaptic inhibition

L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists increase the threshold for electroshock-induced convulsions. Here, we show that a transgenic mouse line overexpressing cerebellum-restricted gamma-aminobutyric acid type A (GABA(A)) receptor alpha6 subunit in the hippocampal CA1 pyramidal cells (Thy1alpha6 mouse line) exhibits about a 20% increase in the electroshock current intensity inducing tonic hindlimb extension convulsion in 50% of the mice compared with that of their wild-type controls. AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in patch clamp recordings of CA1 pyramidal neurons in hippocampal slices had decreased amplitudes (8.4 +/- 2.2 pA) in the transgenics compared with the wild types (10.3 +/- 2.5 pA) but showed no change in current decay or frequency. Our results suggest that decreased AMPA-mediated neurotransmission might explain the increased threshold for electroconvulsions and warrant further studies on the regulation between various components of inhibition and excitation in neurons.     

Spontaneous and synaptic input from granule cells and the perforant path to dentate basket cells in the rat hippocampus

To characterize excitatory inputs to dentate basket cells from dentate granule cells and the perforant path, the whole-cell recording technique was used in neonatal rat hippocampal slices. Spontaneous excitatory input to basket cells was also examined and compared to that of other interneurons in the dentate gyrus. Basket cells were separable from other neurons in the dentate gyrus based on morphology and location, as determined by biocytin staining following recording, and by resting membrane potential, propensity to fire action potentials spontaneously, and miniature excitatory postsynaptic current (EPSC) characteristics. Minimal electrical stimulation of the granule cell layer evoked in basket cells short latency EPSCs that were composed of both N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) components as judged by their time course, voltage dependence, and blockade by selective antagonists. Perforant path EPSCs exhibited slower kinetics than EPSCs evoked by granule cell stimulation. Like granule cell evoked EPSCs, however, perforant path EPSCs were composed of both NMDA and AMPA components. Minimal electrical stimulation of the granule cell layer and perforant path evoked monosynaptic EPSCs in only 67% and 62% of the trials, respectively, suggesting that these inputs are as unreliable as previously determined inputs from CA3 pyramidal cells (48%). Tetrodotoxin-insensitive spontaneous miniature EPSCs were frequent in basket cells and non-basket interneurons residing either at the border between the granule cell layer and the hilus or deep within the hilus. Miniature EPSCs recorded from all cells held at ?70 mV were blocked completely by 3 μSM 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). Though a component of the miniature EPSCs recorded from border and deep hilar interneurons at +40 mV was blocked by the NMDA receptor antagonist D -2-amino-phosphonovaleric acid (D-APV), miniature EPSCs in basket cells were insensitive to D-APV. We conclude that input from granule cells and the perforant path results in activation of basket cells via glutamatergic synapses that employ both NMDA and AMPA receptors. These inputs to basket cells likely contribute to feedback and feedforward inhibition of granule cells. The absence of an NMDA receptor component in spontaneous miniature EPSCs of dentate basket cells implies a difference in organization of excitatory synapses made onto basket cells compared with other hilar interneurons. © 1995 Wiley-Liss, Inc.     

Influence of the NR3A subunit on NMDA receptor functions

Various combinations of subunits assemble to form the NMDA-type glutamate receptor (NMDAR), generating diversity in its functions. Here we review roles of the unique NMDAR subunit, NR3A, which acts in a dominant-negative manner to suppress receptor activity. NR3A-containing NMDARs display striking regional and temporal expression specificity, and, unlike most other NMDAR subtypes, they have a low conductance, are only modestly permeable to Ca²⁺, and pass current at hyperpolarized potentials in the presence of magnesium. While glutamate activates triheteromeric NMDARs composed of NR1/NR2/NR3A subunits, glycine is sufficient to activate diheteromeric NR1/NR3A-containing receptors. NR3A dysfunction may contribute to neurological disorders involving NMDARs, and the subunit offers an attractive therapeutic target given its distinct pharmacological and structural properties.     

Ivermectin potentiates ATP-induced ion currents in cortical neurones: Evidence for functional expression of P2X4 receptors?

We examined the effects of an antiparasitic agent ivermectin on ATP- and alpha,beta-methylene-ATP (alpha,beta-me-ATP)-activated currents in neurones acutely isolated from slices of somato-sensory cortex of 17-22 days old CBL57 mice. Membrane currents were monitored using whole-cell patch clamp combined with a "concentration-clamp" fast agonist application system. Ivermectin potentiated membrane currents induced by both ATP and alpha,beta-me-ATP applied alone or in the presence of broad P2X antagonist PPADS. Ivermectin also significantly increased amplitude and frequency of spontaneous P2X-mediated EPSCs recorded from cortical slices incubated with mixture of glutamate (CNQX, D-APV, SYM2081), GABA (picrotoxin), nicotinic cholinoreceptor (hexamethonium) antagonists and sodium channel blocker tetrodotoxin. These results indicate functional expression of P2X4 receptors in cortical neurones and their involvement in purinergic synaptic transmission in cortex.     

Miniature release events of glutamate from hippocampal neurons are influenced by the dystonia-associated protein torsinA

TorsinA is an evolutionarily conserved AAA+ ATPase, and human patients with an in‐frame deletion of a single glutamate (ΔE) codon from the encoding gene suffer from autosomal‐dominant, early‐onset generalized DYT1 dystonia. Although only 30–40% of carriers of the mutation show overt motor symptoms, most experience enhanced excitability of the central nervous system. The cellular mechanism responsible for this change in excitability is not well understood. Here we show the effects of the ΔE‐torsinA mutation on miniature neurotransmitter release from neurons. Neurotransmitter release was characterized in cultured hippocampal neurons obtained from wild‐type, heterozygous, and homozygous ΔE‐torsinA knock‐in mice using two approaches. In the first approach, patch‐clamp electrophysiology was used to record glutamate‐mediated miniature excitatory postsynaptic currents (mEPSCs) in the presence of the Na⁺ channel blocker tetrodotoxin (TTX) and absence of GABA_A receptor antagonists. The intervals between mEPSC events were significantly shorter in neurons obtained from the mutant mice than in those obtained from wild‐type mice. In the second approach, the miniature exocytosis of synaptic vesicles was detected by imaging the unstimulated release of FM dye from the nerve terminals in the presence of TTX. Cumulative FM dye release was higher in neurons obtained from the mutant mice than in those obtained from wild‐type mice. The number of glutamatergic nerve terminals was also assessed, and we found that this number was unchanged in heterozygous relative to wild‐type neurons, but slightly increased in homozygous neurons. Notably, in both heterozygous and homozygous neurons, the unitary synaptic charge during each mEPSC event was unchanged. Overall, our results suggest more frequent miniature glutamate release in neurons with ΔE‐torsinA mutations. This change may be one of the underlying mechanisms by which the excitability of the central nervous system is enhanced in the context of DYT1 dystonia. Moreover, qualitative differences between heterozygous and homozygous neurons with respect to certain synaptic properties indicate that the abnormalities observed in homozygotes may reflect more than a simple gene dosage effect. Synapse 66:807–822, 2012. © 2012 Wiley Periodicals, Inc.     

Control of synapse development and plasticity by Rho GTPase regulatory proteins

Synapses are specialized cell–cell contacts that mediate communication between neurons. Most excitatory synapses in the brain are housed on dendritic spines, small actin-rich protrusions extending from dendrites. During development and in response to environmental stimuli, spines undergo marked changes in shape and number thought to underlie processes like learning and memory. Improper spine development, in contrast, likely impedes information processing in the brain, since spine abnormalities are associated with numerous brain disorders. Elucidating the mechanisms that regulate the formation and plasticity of spines and their resident synapses is therefore crucial to our understanding of cognition and disease. Rho-family GTPases, key regulators of the actin cytoskeleton, play essential roles in orchestrating the development and remodeling of spines and synapses. Precise spatio-temporal regulation of Rho GTPase activity is critical for their function, since aberrant Rho GTPase signaling can cause spine and synapse defects as well as cognitive impairments. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and inhibited by GTPase-activating proteins (GAPs). We propose that Rho-family GEFs and GAPs provide the spatiotemporal regulation and signaling specificity necessary for proper Rho GTPase function based on the following features they possess: (i) existence of multiple GEFs and GAPs per Rho GTPase, (ii) developmentally regulated expression, (iii) discrete localization, (iv) ability to bind to and organize specific signaling networks, and (v) tightly regulated activity, perhaps involving GEF/GAP interactions. Recent studies describe several Rho-family GEFs and GAPs that uniquely contribute to spinogenesis and synaptogenesis. Here, we highlight several of these proteins and discuss how they occupy distinct biochemical niches critical for synaptic development.     

Chronically saturating levels of endogenous glycine disrupt glutamatergic neurotransmission and enhance synaptogenesis in the CA1 region of mouse hippocampus

Glycine serves a dual role in neurotransmission. It is the primary inhibitory neurotransmitter in the spinal cord and brain stem and is also an obligatory coagonist at the excitatory glutamate, N-methyl-D-aspartate receptor (NMDAR). Therefore, the postsynaptic action of glycine should be strongly regulated to maintain a balance between its inhibitory and excitatory inputs. The glycine concentration at the synapse is tightly regulated by two types of glycine transporters, GlyT1 and GlyT2, located on nerve terminals or astrocytes. Genetic studies demonstrated that homozygous (GlyT1-/-) newborn mice display severe sensorimotor deficits characterized by lethargy, hypotonia, and hyporesponsivity to tactile stimuli and ultimately die in their first postnatal day. These symptoms are similar to those associated with the human disease glycine encephalopathy in which there is a high level of glycine in cerebrospinal fluid of affected individuals. The purpose of this investigation is to determine the impact of chronically high concentrations of endogenous glycine on glutamatergic neurotransmission during postnatal development using an in vivo mouse model (GlyT1+/-). The results of our study indicate the following; that compared with wild-type mice, CA1 pyramidal neurons from mutants display significant disruptions in hippocampal glutamatergic neurotransmission, as suggested by a faster kinetic of NMDAR excitatory postsynaptic currents, a lower reduction of the amplitude of NMDAR excitatory postsynaptic currents by ifenprodil, no difference in protein expression for NR2A and NR2B but a higher protein expression for PSD-95, an increase in their number of synapses and finally, enhanced neuronal excitability.     

突触内NMDAR通道电流在培养神经元发育中的变化

 [目的]观察培养大鼠海马神经元突触内NMDA受体（NMDAR）通道电流在发育中的变化。[方法]取新生1dSD大鼠海马制成细胞悬液,接种在培养板上进行培养。培养到1周和2周时,采用膜片钳全细胞模式记录神经元突触内自发的微小兴奋性突触后电流（mEPSC）。[结果]培养2周海马神经元突触内NMDA受体介导的mEPSC（mEPSCNMDA）幅度比培养1周神经元小,对NR2B的特异拮抗剂ifenprodil的敏感性降低。Ifenprodil对培养1周神经元mEPSCNMDA的抑制作用达到（80.47±6.12）%,却只抑制（12.27±2.02）%培养2周神经元的mEPSCNMDA。[结论]培养海马神经元突触内NMDA受体通道电流有发育变化,提示培养1周神经元突触内NMDA受体NR2亚单位主要为NR2B;而神经元培养到2周时,突触内NR2B亚单位逐渐被NR2A取代。