E-cadherin is required at GABAergic synapses in cultured cortical neurons

Classical cadherins are cell adhesion molecules that are thought to contribute to the control of synapse formation, synaptic transmission, and synaptic plasticity. This is largely based on studies investigating the functions of N-cadherin at glutamatergic synapses, whereas other classical cadherins have hardly been examined at central synapses. We have now used a conditional knockout approach in cultured cortical neurons to address the role of E-cadherin mainly at inhibitory, GABAergic synapses. Cortical neurons were cultured from mouse fetuses carrying floxed E-cadherin alleles in homozygous configuration. E-cadherin knockout was induced in individual neurons by expression of an EGFP-Cre fusion protein. Immunocytochemical stainings for the vesicular GABA (VGAT) and glutamate (VGLUT1) transporters revealed a reduced density of dendritic GABAergic synapses in E-cadherin knockout neurons, whereas glutamatergic synapses were unaffected. Electrophysiological recordings of miniature and action potential-evoked, GABA_A receptor-mediated postsynaptic currents confirmed an impairment of GABAergic synapses at the functional level. In summary, our immunocytochemical and electrophysiological analysis of E-cadherin knockout neurons suggested that E-cadherin signaling importantly contributes to the regulation of GABAergic synapses in cortical neurons.     

Cyclic AMP controls BDNF-induced TrkB phosphorylation and dendritic spine formation in mature hippocampal neurons

Synaptic actions of brain-derived neurotrophic factor (BDNF) are 'gated' by cyclic AMP (cAMP), but the underlying molecular mechanisms remain unclear. Here we report that cAMP regulates BDNF function in mature hippocampal neurons by modulating the signaling and trafficking of its receptor TrkB. cAMP gated the TrkB tyrosine kinase with three characteristic features: BDNF-induced TrkB phosphorylation was attenuated by inhibitors of cAMP signaling, it was potentiated by cAMP analogs, and activation of the cAMP pathway alone had no effect. In addition, cAMP facilitated trafficking of TrkB to dendritic spines, possibly by promoting its interaction with synaptic scaffolding protein PSD-95. Norepinephrinergic and dopaminergic agonists, which elevate intracellular cAMP concentration, also enhanced TrkB phosphorylation and its translocation to spines. cAMP gated long-term modulation by BDNF of spine density, but not the number of primary dendrites. These results reveal a specific role of cAMP in controlling BDNF actions in the brain, and provide new insights into the molecular mechanism underlying cAMP gating.     

Binocular suppression in cortical neurons

Summary Dichoptic presentation of patterns similar in shape but of very different contrast results in the perception of only the high contrast pattern (binocular suppression). When recording from binocular neurons of the cat visual cortex, we have found an effect which is strikingly similar to this perceptual phenomenon. If a high and a low contrast grating are presented simultaneously, one to each eye, the cell's response to the low contrast stimulus is suppressed.     

miRNA慢病毒表达载体的构建及其在原代培养的大鼠胚胎大脑皮层神经细胞中的稳定表达

目的:构建miRNA的慢病毒表达载体,使其在原代培养的大鼠胚胎大脑皮层神经细胞中稳定表达。方法:将该干扰片段构建入慢病毒载体(pL KD-Ubc-eG FP-U6-shRNA)的U6启动子下游,该载体可以实现在干扰小片段RNA的同时表达绿色荧光蛋白EGFP基因,便于观察载体工作状态。通过脂质体转染法转染原代培养的大鼠胚胎大脑皮层神经细胞,观察转染的原代培养的大鼠胚胎大脑皮层神经细胞的形态及表达情况。结果:针对筛选的miRNA构建的慢病毒载体,经过DNA测序结果和琼脂糖凝胶电泳鉴定成功构建了筛选的miRNA的慢病毒表达载体,重组质粒转染原代培养的大鼠胚胎大脑皮层神经细胞,经24 h后在荧光倒置显微镜下可以观察到部分细胞带有绿色荧光,筛选的miRNA构建的慢病毒载体在原代培养的大鼠胚胎大脑皮层神经细胞中稳定表达。结论:筛选的miRNA的慢病毒表达载体构建成功,并稳定的转染到原代培养的大鼠胚胎大脑皮层神经细胞中,为miRNA对神经细胞存活功能的研究提供了平台。     

NR2B-containing NMDA autoreceptors at synapses on entorhinal cortical neurons

We have previously shown that presynaptic N-methyl-D-aspartate receptors (NMDARs) can facilitate glutamate release onto principal neurons in the entorhinal cortex (EC). In the present study, we have investigated the subunit composition of these presynaptic NMDARs. We recorded miniature alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (mEPSCs), from visually identified neurons in layers II and V of the EC in vitro. In both layers, bath application of the NR2A/B subunit-selective agonist, homoquinolinic acid (HQA), resulted in a marked facilitation of mEPSC frequency. Blockade of presynaptic Ca(2+) entry through either NMDARs or voltage-gated Ca(2+) channels with Co(2+) prevented the effects of HQA, confirming that Ca(2+) entry to the terminal was required for facilitation. When the NR2B-selective antagonist, ifenprodil, was applied prior to HQA, the increase in mEPSC frequency was greatly reduced. In addition, we found that an NMDAR antagonist blocked frequency-dependent facilitation of evoked release and reduced mEPSC frequency in layer V. Thus we have demonstrated that NMDA autoreceptors in layer V of the EC bear the NR2B subunit, and that NMDARs are also present at terminals onto superficial neurons.     

Activity- and BDNF-induced plasticity of miniature synaptic currents in ES cell-derived neurons integrated in a neocortical network

In vitro differentiated embryonic stem (ES) cells have been proposed as potential donor cells for cell replacement therapies of neurodegenerative diseases. The functional synaptic integration of such cells appears conceivable because ES cell-derived neurons are well known to establish excitatory and inhibitory synapses. However, long-term synaptic plasticity, a prerequisite of memory formation, has not yet been demonstrated at these synapses. After in vitro differentiation and purification by immunoisolation, we co-cultured ES cell-derived neurons with neocortical explants, which strongly innervated the ES cell-derived target neurons. ES cell-derived neurons exhibited action potential firing similar to primary cultured neocortical neurons. The formation of glutamatergic synapses was indicated by AMPA receptor-mediated miniature excitatory postsynaptic currents (AMPA mEPSCs). In addition, a N-methyl-D-aspartate receptor-mediated, D-2-amino-5-phosphonopentanoic acid-sensitive mEPSC component was observed. We first studied activity-dependent homeostatic plasticity (synaptic scaling) of mEPSCs at glutamatergic synapses. Chronic blockade of action potential activity by TTX resulted in an increase in the amplitudes of AMPA mEPSCs. This indicates that ES cell-derived neurons are capable of a homeostatic regulation of postsynaptic AMPA receptors. In addition, we investigated neurotrophin-induced synaptic plasticity of mEPSCs at glutamatergic synapses. Chronic addition of brain-derived neurotrophic factor (BDNF; 100 ng/ml) to the culture medium resulted in an increase in both the frequency and the amplitudes of AMPA mEPSCs. These results suggest that BDNF induces the formation and/or the functional maturation of presynaptic release sites in parallel with an upregulation of postsynaptic AMPA receptors. Thus BDNF represents a potential co-factor that could improve functional synaptic integration of ES cell-derived neurons into neocortical networks.     

BDNF-induced facilitation of afferent-evoked responses in lamina II neurons is reduced after neonatal spinal cord contusion injury

We previously reported that brain-derived neurotrophic factor (BDNF), a pronociceptive neurotransmitter, induces synaptic facilitation of excitatory postsynaptic current (EPSC) in lamina II neurons of neonatal rats up to P14 in a N-methyl-d-aspartate (NMDA) receptor-dependent manner. Here we used the patch-clamp technique to study synaptic and NMDA-evoked responses in transverse spinal slices in the lumbar enlargement as well as the ability of BDNF to modify these responses from 1 day to 6 wk after neonatal contusion. In older uninjured animals (>P14), BDNF continued to evoke synaptic facilitation although superfusion of NMDA (in TTX) induced inward current of significantly smaller amplitude than that observed in younger rats. After contusion injury, BDNF was unable to facilitate dorsal root-evoked EPSCs in lamina II neurons despite the finding that NMDA-evoked currents were only slightly smaller than those observed in age-matched uninjured animals. These findings suggest that although BDNF-induced facilitation of the AMPA/kainate receptor-mediated response to dorsal root stimulation is maintained in the mature dorsal horn from intact rats, BDNF may no longer elicit these pronociceptive actions after neonatal contusion injury. The lack of change in NMDA-evoked currents in contused cords suggests that diminished NMDA receptor function is not the major cause of the decline in BDNF action after contusion. It seems more likely that diminished trkB expression and enhanced expression of truncated trkB receptors in the contused cord play a significant role in determining the reduced effect of BDNF under these conditions.     

Visual cortical organization at the single axon level: a beginning

Single axon analysis of visual cortical connections is an important extension of previous anterograde studies using 3H-amino acids or wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). The higher resolution tracers-Phaseolus vulgaris-leucoagglutinin (PHA-L), biocytin, biotinylated dextran amine (BDA) and dextran-conjugates-have already produced new results, simply by providing improved visualization, concerning laminar definition and possible subtypes of connections, as well as the beginning of a database of morphometrics and microstructure. The comparative approach, comparing geniculocortical terminations and cortical connections across several areas, has suggested both specific structural-functional correlations (for example, in extrastriate area MT/V5) and more subtle, possibly gradient-wise variations. Likely future directions for this line of research include more direct correlations of axon geometry with functional architectures, investigations of microcircuitry at the level of electron or confocal microscopy, anatomical and functional investigations of connectional convergence and interactions, and, not least, a more comprehensive database.     

Functionally distinct inhibitory neurons at the first stage of visual cortical processing

Here we explore inhibitory circuits at the thalamocortical stage of processing in layer 4 of the cat's visual cortex, focusing on the anatomy and physiology of the interneurons themselves. Our immediate aim was to explore the inhibitory mechanisms that contribute to orientation selectivity, perhaps the most dramatic response property to emerge across the thalamocortical synapse. The broader goal was to understand how inhibitory circuits operate. Using whole-cell recording in cats in vivo, we found that layer 4 contains two populations of inhibitory cells defined by receptive field class--simple and complex. The simple cells were selective for stimulus orientation, whereas the complex cells were not. Our observations help to explain how neurons become sensitive to stimulus orientation and maintain that selectivity as stimulus contrast changes. Overall, the work suggests that different sources of inhibition, either selective for specific features or broadly tuned, interact to provide appropriate representations of elements within the environment.     

Prolonged synaptic integration in perirhinal cortical neurons

Layer II/III of rat perirhinal cortex (PR) contains numerous late-spiking (LS) pyramidal neurons. When injected with a depolarizing current step, these LS cells typically delay spiking for one or more seconds from the onset of the current step and then sustain firing for the duration of the step. This pattern of delayed and sustained firing suggested a specific computational role for LS cells in temporal learning. This hypothesis predicts and requires that some layer II/III neurons should also exhibit delayed and sustained spiking in response to a train of excitatory synaptic inputs. Here we tested this prediction using visually guided, whole cell recordings from rat PR brain slices. Most LS cells (19 of 26) exhibited delayed spiking to synaptic stimulation (>1 s latency from the train onset), and the majority of these cells (13 of 19) also showed sustained firing that persisted for the duration of the synaptic train (5-10 s duration). Delayed and sustained firing in response to long synaptic trains has not been previously reported in vertebrate neurons. The data are consistent with our model that a circuit containing late spiking neurons can be used for encoding long time intervals during associative learning.     

Synaptic interactions between thalamic and cortical inputs onto cortical neurons in vivo

To study the interactions between thalamic and cortical inputs onto neocortical neurons, we used paired-pulse stimulation (PPS) of thalamic and cortical inputs as well as PPS of two cortical or two thalamic inputs that converged, at different time intervals, onto intracellularly recorded cortical and thalamocortical neurons in anesthetized cats. PPS of homosynaptic cortico-cortical pathways produced facilitation, depression, or no significant effects in cortical pathways, whereas cortical responses to thalamocortical inputs were mostly facilitated at both short and long intervals. By contrast, heterosynaptic interactions between either cortical and thalamic, or thalamic and cortical, inputs generally produced decreases in the peak amplitudes and depolarization area of evoked excitatory postsynaptic potentials (EPSPs), with maximal effect at approximately 10 ms and lasting from 60 to 100 ms. All neurons tested with thalamic followed by cortical stimuli showed a decrease in the apparent input resistance (R(in)), the time course of which paralleled that of decreased responses, suggesting that shunting is the factor accounting for EPSP's decrease. Only half of neurons tested with cortical followed by thalamic stimuli displayed changes in R(in). Spike shunting in the thalamus may account for those cases in which decreased synaptic responsiveness of cortical neurons was not associated with decreased R(in) because thalamocortical neurons showed decreased firing probability during cortical stimulation. These results suggest a short-lasting but strong shunting between thalamocortical and cortical inputs onto cortical neurons.     

Presenilin-1 deficiency impairs glutamate-evoked intracellular calcium responses in neurons

Presenilin 1 (PS1) plays a critical role in cleaving amyloid precursor protein (APP) to produce amyloid-beta (Abeta), the primary proteinaceous component of the senile plaques associated with Alzheimer's disease. In addition to mediating the cleavage of APP and a number of other proteins, a growing body of evidence suggests that PS1 also regulates intracellular endoplasmic reticulum calcium levels. Such findings suggest that PS1 activity may modulate neuronal excitability, as well. To address this issue we examined cytosolic intracellular calcium responses in PS1-deficient neurons stimulated by the excitatory amino acid neurotransmitter, glutamate. We found that glutamate-induced intracellular calcium levels were markedly reduced in neurons lacking PS1 (-/-) compared with heterozygous (+/-) and wild-type (+/+) neurons. To prove that PS1 was sufficient to mediate normal glutamate-induced calcium responses, we used a Semliki-forest virus (SFV) vector to express wild-type PS1 in PS1 knock-out neurons. We found that heterologous PS1 expression restored glutamate-evoked calcium responses in PS1-deficient neurons to levels matching non-infected wild-type cells. PS1-deficient neurons infected with SFV directing expression of beta-galactosidase failed to rescue the wild-type phenotype. These results support the idea that normal PS1 activity regulates neuronal responses to neurotransmitter stimulation.     

Spread of electrical activity at cortical level after repetitive magnetic stimulation in normal subjects

In normal subjects, focal repetitive transcranial magnetic stimulation (rTMS) of the hand motor area evokes muscle potentials (MEPs) from muscles in the hand (target muscles) and the arm (non-target muscles). In this study we investigated the mechanisms underlying the spread of MEPs induced by focal rTMS in non-target muscles. rTMS was delivered with a Magstim stimulator and a figure-of-eight coil placed over the first dorsal interosseus (FDI) motor area of the left hemisphere. Trains of 10 stimuli were given at a suprathreshold intensity (120% of motor threshold) and at frequencies of 5, 10 and 20 Hz at rest. Electromyographic (EMG) activity was recorded simultaneously from the FDI (target muscle) and the contralateral biceps muscle and from the FDI muscle ipsilateral to the side of stimulation (non-target muscle). rTMS delivered in trains to the FDI motor area of the left hemisphere elicited MEPs in the contralateral FDI (target muscle) that gradually increased in amplitude over the course of the train. Focal rTMS trains also induced MEPs in the contralateral biceps (non-target muscle) but did so only after the second or third stimulus; like target-muscle MEPs, in non-target muscle MEPs progressively increased in amplitude during the train. At no frequency did rTMS elicit MEPs in the FDI muscle ipsilateral to the site of stimulation. rTMS left the latency of EMG responses in the FDI and biceps muscles unchanged during the trains of stimuli. The latency of biceps MEPs was longer after rTMS than after a single TMS pulse. In conditioning-test experiments designed to investigate the cortical origin of the spread, a single TMS pulse delivered over the left hemisphere at an interstimulus interval (ISI) of 50, 100 and 150 ms reduced the amplitude of the test MEP evoked by a single TMS pulse delivered over the right hemisphere; and a conditioning rTMS train delivered over the left hemisphere increased the amplitude of the test MEP evoked by a single TMS pulse over the right hemisphere. A conditioning rTMS train delivered over the left hemisphere and paired magnetic shocks (test stimulus) at 3 and 13 ms ISIs over the right hemisphere reduced MEP inhibition at the 3-ms ISI but left the MEP facilitation at 13 ms unchanged. Using a control MEP size matched with that observed after a conditioning contralateral rTMS, we found that paired-pulse inhibition remained unchanged. Yet a single TMS conditioning pulse sufficiently strong to evoke a MEP in the contralateral FDI and biceps muscles simultaneously (as rTMS did) left paired-pulse inhibition unchanged. We conclude that the spread of EMG activity to non-target muscles depends on cortical mechanisms, mainly including changes in the excitability of the interneurones mediating intracortical inhibition. Electronic Publication     

RNA expression profiling at the single molecule level

We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration--corresponding to only 39,000 molecules in the sample solution--and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 10(4) cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.