Association between vascular endothelial growth factor-mediated blood-brain barrier dysfunction and stress-induced depression.

Several lines of evidence suggest that stress induces the neurovascular dysfunction associated with increased blood-brain barrier (BBB) permeability, which could be an important pathology linking stress and psychiatric disorders, including major depressive disorder (MDD). However, the detailed mechanism resulting in BBB dysfunction associated in the pathophysiology of MDD still remains unclear. Herein, we demonstrate the role of vascular endothelial growth factor (VEGF), a key mediator of vascular angiogenesis and BBB permeability, in stress-induced BBB dysfunction and depressive-like behavior development. We implemented an animal model of depression, chronic restraint stress (RS) in BALB/c mice, and found that the BBB permeability was significantly increased in chronically stressed mice. Immunohistochemical and electron microscopic observations revealed that increased BBB permeability was associated with both paracellular and transcellular barrier alterations in the brain endothelial cells. Pharmacological inhibition of VEGF receptor 2 (VEGFR2) using a specific monoclonal antibody (DC101) prevented chronic RS-induced BBB permeability and anhedonic behavior. Considered together, these results indicate that VEGF/VEGFR2 plays a crucial role in the pathogenesis of depression by increasing the BBB permeability, and suggest that VEGFR2 inhibition could be a potential therapeutic strategy for the MDD subtype associated with BBB dysfunction.

Sirtuin 6 is a regulator of dendrite morphogenesis in rat hippocampal neurons.

Sirtuin 6 (SIRT6), a member of the Sirtuin family, acts as nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase, mono-adenosine diphosphate (ADP)-ribosyltransferase, and fatty acid deacylase, and plays critical roles in inflammation, aging, glycolysis, and DNA repair. Accumulating evidence has suggested that SIRT6 is involved in brain functions such as neuronal differentiation, neurogenesis, and learning and memory. However, the precise molecular roles of SIRT6 during neuronal circuit formation are not yet well understood. In this study, we tried to elucidate molecular roles of SIRT6 on neurite development by using primary-cultured hippocampal neurons. We observed that SIRT6 was abundantly localized in the nucleus, and its expression was markedly increased during neurite outgrowth and synaptogenesis. By using shRNA-mediated SIRT6-knockdown, we show that both dendritic length and the number of dendrite branches were significantly reduced in the SIRT6-knockdown neurons. Microarray and subsequent gene ontology analysis revealed that reducing SIRT6 caused the downregulation of immediate early genes (IEGs) and alteration of several biological processes including MAPK (ERK1/2) signaling. We found that nuclear accumulation of phosphorylated ERK1/2 was significantly reduced in SIRT6-knockdown neurons. Overexpression of SIRT6 promoted dendritic length and branching, but the mutants lacking deacetylase activity had no significant effect on the dendritic morphology. Collectively, the presented findings reveal a role of SIRT6 in dendrite morphogenesis, and suggest that SIRT6 may act as an important regulator of ERK1/2 signaling pathway that mediates IEG expression, which leads to dendritic development.

AUTS2 Regulation of Synapses for Proper Synaptic Inputs and Social Communication.

Impairments in synapse development are thought to cause numerous psychiatric disorders. Autism susceptibility candidate 2 (AUTS2) gene has been associated with various psychiatric disorders, such as autism and intellectual disabilities. Although roles for AUTS2 in neuronal migration and neuritogenesis have been reported, its involvement in synapse regulation remains unclear. In this study, we found that excitatory synapses were specifically increased in the Auts2-deficient primary cultured neurons as well as Auts2 mutant forebrains. Electrophysiological recordings and immunostaining showed increases in excitatory synaptic inputs as well as c-fos expression in Auts2 mutant brains, suggesting that an altered balance of excitatory and inhibitory inputs enhances brain excitability. Auts2 mutant mice exhibited autistic-like behaviors including impairments in social interaction and altered vocal communication. Together, these findings suggest that AUTS2 regulates excitatory synapse number to coordinate E/I balance in the brain, whose impairment may underlie the pathology of psychiatric disorders in individuals with AUTS2 mutations.

Comparison of physiological and behavioral responses to chronic restraint stress between C57BL/6J and BALB/c mice.

Rodent models of chronic restraint stress (CRS) are often used as simple models of depressive disorder. However, these models of stress have been mainly developed in rats, and the behavioral phenotypes of CRS models are still controversial. In this study, we compared the physiological and behavioral responses of C57BL/6J (B6) and BALB/c mice, which are commonly used in genetic and behavioral studies, to CRS. In addition to measuring physiological parameters and the levels of corticosterone (a stress hormone) in response to stress, we also examined changes in the levels of testosterone (an anti-stress hormone), which have rarely been studied in stressed mice. The mice were exposed to CRS for 6 h a day for 21 days. In both B6 and BALB/c mice, CRS elicited several physiological stress responses, including decreased body weight gain and changes in the tissue weights of stress-related organs. Accumulated corticosterone in the hair was measured, and BALB/c mice had significantly greater levels than control mice and B6 mice after CRS. On the other hand, in the case of accumulated testosterone in the hair, both B6 mice and BALB/c mice showed significantly higher concentrations than control mice, but the degree of change was not different between the two strains. In the sucrose preference test, BALB/c mice, but not B6 mice, showed anhedonia-like behavior after CRS. However, neither strain showed depressive-like behavior in the forced swim or tail suspension test. Our results show that the physiological and behavioral stress responses of BALB/c mice are greater than those of B6 mice, although anti-stress responses to CRS are similar in both strains. This suggests that BALB/c mice are likely to be advantageous for use as a CRS-induced depression model.

Interhemispherically dynamic representation of an eye movement-related activity in mouse frontal cortex.

Cortical plasticity is fundamental to motor recovery following cortical perturbation. However, it is still unclear how this plasticity is induced at a functional circuit level. Here, we investigated motor recovery and underlying neural plasticity upon optogenetic suppression of a cortical area for eye movement. Using a visually-guided eye movement task in mice, we suppressed a portion of the secondary motor cortex (MOs) that encodes contraversive eye movement. Optogenetic unilateral suppression severely impaired contraversive movement on the first day. However, on subsequent days the suppression became inefficient and capability for the movement was restored. Longitudinal two-photon calcium imaging revealed that the regained capability was accompanied by an increased number of neurons encoding for ipsiversive movement in the unsuppressed contralateral MOs. Additional suppression of the contralateral MOs impaired the recovered movement again, indicating a compensatory mechanism. Our findings demonstrate that repeated optogenetic suppression leads to functional recovery mediated by the contralateral hemisphere.

Construction of the common cortical space by spontaneous activity and its application in the mouse cortex.

Wide-field optical imaging of the animal brain is a useful technique for measuring brain dynamics, including spatial structure. However, quantitative inter-animal comparison is difficult due to lack of the common cortical space that can normalize individually imaged brains as done in human functional MRI studies. Here, by using wide-field functional Ca2+ imaging on anesthetized transgenic mice expressing G-CaMP7 in astrocytes and excitatory neutrons, we attempted to establish the common cortical space in mice, which can be useful as a standard of functional brain map. We initially reconstructed cortical areas embedded within spontaneous activity as the functional connectivity maps for the individual mice, then matched them in size, shape, and location across mice by geometric transformation. Finally, we assigned all the recorded signals into the transformed space, to make spatially normalized signals in the common cortical space. Using this method, we managed to extract activity patterns commonly observed across mice. These results suggest that the presented method is available to facilitate inter-animal comparison of brain dynamics, and has the potential to identify common brain activity across animals.

Streamlined sensory motor communication through cortical reciprocal connectivity in a visually guided eye movement task.

Cortical computation is distributed across multiple areas of the cortex by networks of reciprocal connectivity. However, how such connectivity contributes to the communication between the connected areas is not clear. In this study, we examine the communication between sensory and motor cortices. We develop an eye movement task in mice and combine it with optogenetic suppression and two-photon calcium imaging techniques. We identify a small region in the secondary motor cortex (MOs) that controls eye movements and reciprocally connects with a rostrolateral part of the higher visual areas (VRL/A/AL). These two regions encode both motor signals and visual information; however, the information flow between the regions depends on the direction of the connectivity: motor information is conveyed preferentially from the MOs to the VRL/A/AL, and sensory information is transferred primarily in the opposite direction. We propose that reciprocal connectivity streamlines information flow, enhancing the computational capacity of a distributed network.

Brief Novel Visual Experience Fundamentally Changes Synaptic Plasticity in the Mouse Visual Cortex.

LTP has been known to be a mechanism by which experience modifies synaptic responses in the neocortex. Visual deprivation in the form of dark exposure or dark rearing from birth enhances NMDAR-dependent LTP in layer 2/3 of visual cortex, a process often termed metaplasticity, which may involve changes in NMDAR subunit composition and function. However, the effects of reexposure to light after dark rearing from birth on LTP induction have not been explored. Here, we showed that the light exposure after dark rearing revealed a novel NMDAR independent form of LTP in the layer 2/3 pyramidal cells in visual cortex of mice of both sexes, which is dependent on mGluR5 activation and is associated with intracellular Ca2+ rise, CaMKII activity, PKC activity, and intact protein synthesis. Moreover, the capacity to induce mGluR-dependent LTP is transient: it only occurs when mice of both sexes reared in the dark from birth are exposed to light for 10-12 h, and it does not occur in vision-experienced, male mice, even after prolonged exposure to dark. Thus, the mGluR5-LTP unmasked by short visual experience can only be observed after dark rearing but not after dark exposure. These results suggested that, as in hippocampus, in layer 2/3 of visual cortex, there is coexistence of two distinct activity-dependent systems of synaptic plasticity, NMDAR-LTP, and mGluR5-LTP. The mGluR5-LTP unmasked by short visual experience may play a critical role in the faster establishment of normal receptive field properties.SIGNIFICANCE STATEMENT LTP has been known to be a mechanism by which experience modifies synaptic responses in the neocortex. Visual deprivation in the form of dark exposure or dark rearing from birth enhances NMDAR-dependent LTP in layer 2/3 of visual cortex, a process often termed metaplasticity. NMDAR-dependent form of LTP in visual cortex has been well characterized. Here, we report that an NMDAR-independent form of LTP can be promoted by novel visual experience on dark-reared mice, characterized as dependent on intracellular Ca2+ rise, PKC activity, and intact protein synthesis and also requires the activation of mGluR5. These findings suggest that, in layer 2/3 of visual cortex, as in hippocampus, there is coexistence of two distinct activity-dependent systems of synaptic plasticity.

Selective Suppression of Local Circuits during Movement Preparation in the Mouse Motor Cortex.

Prepared movements are more efficient than those that are not prepared for. Although changes in cortical activity have been observed prior to a forthcoming action, the circuits involved in motor preparation remain unclear. Here, we use in vivo two-photon calcium imaging to uncover changes in the motor cortex during variable waiting periods prior to a forepaw reaching task in mice. Consistent with previous reports, we observed a subset of neurons with increased activity during the waiting period; however, these neurons did not account for the degree of preparation as defined by reaction time (RT). Instead, the suppression of activity of distinct neurons in the same cortical area better accounts for RT. This suppression of neural activity resulted in a distinct and reproducible pattern when mice were well prepared. Thus, the selective suppression of network activity in the motor cortex may be a key feature of prepared movements.

3D clustering of GABAergic neurons enhances inhibitory actions on excitatory neurons in the mouse visual cortex.

Neocortical neurons with similar functional properties assemble into spatially coherent circuits, but it remains unclear how inhibitory interneurons are organized. We applied in vivo two-photon functional Ca(2+) imaging and whole-cell recording of synaptic currents to record visual responses of cortical neurons and analyzed their spatial arrangements. GABAergic interneurons were clustered in the 3D space of the mouse visual cortex, and excitatory neurons located within the clusters (insiders) had a lower amplitude and sharper orientation tuning of visual responses than outsiders. Inhibitory synaptic currents recorded from the insiders were larger than those of the outsiders. Single, isolated interneurons did not show such a location-tuning/amplitude relationship. The two principal subtypes of interneurons, parvalbumin- and somatostatin-expressing neurons, also formed clusters with only slightly overlapping each other and exhibited a different location-tuning relationship. These findings suggest that GABAergic interneurons and their subgroups form clusters to make their inhibitory function more effective than isolated interneurons.

Curtailing effect of awakening on visual responses of cortical neurons by cholinergic activation of inhibitory circuits.

Visual responsiveness of cortical neurons changes depending on the brain state. Neural circuit mechanism underlying this change is unclear. By applying the method of in vivo two-photon functional calcium imaging to transgenic rats in which GABAergic neurons express fluorescent protein, we analyzed changes in visual response properties of cortical neurons when animals became awakened from anesthesia. In the awake state, the magnitude and reliability of visual responses of GABAergic neurons increased whereas the decay of responses of excitatory neurons became faster. To test whether the basal forebrain (BF) cholinergic projection is involved in these changes, we analyzed effects of electrical and optogenetic activation of BF on visual responses of mouse cortical neurons with in vivo imaging and whole-cell recordings. Electrical BF stimulation in anesthetized animals induced the same direction of changes in visual responses of both groups of neurons as awakening. Optogenetic activation increased the frequency of visually evoked action potentials in GABAergic neurons but induced the delayed hyperpolarization that ceased the late generation of action potentials in excitatory neurons. Pharmacological analysis in slice preparations revealed that photoactivation-induced depolarization of layer 1 GABAergic neurons was blocked by a nicotinic receptor antagonist, whereas non-fast-spiking layer 2/3 GABAergic neurons was blocked only by the application of both nicotinic and muscarinic receptor antagonists. These results suggest that the effect of awakening is mediated mainly through nicotinic activation of layer 1 GABAergic neurons and mixed nicotinic/muscarinic activation of layer 2/3 non-fast-spiking GABAergic neurons, which together curtails the visual responses of excitatory neurons.

Reciprocal Homosynaptic and heterosynaptic long-term plasticity of corticogeniculate projection neurons in layer VI of the mouse visual cortex.

Most neurons in layer VI of the visual cortex project to the dorsal lateral geniculate nucleus (dLGN). These corticogeniculate projection neurons (CG cells) receive top-down synaptic inputs from upper layers (ULs) and bottom-up inputs from the underlying white matter (WM). Use-dependent plasticity of these synapses in layer VI of the cortex has received less attention than in other layers. In the present study, we used a retrograde tracer injected into dLGN to identify CG cells, and, by analyzing EPSPs evoked by electrical stimulation of the UL or WM site, examined whether these synapses show long-term synaptic plasticity. Theta-burst stimulation induced long-term potentiation (LTP) of activated synapses (hom-LTP) and long-term depression (LTD) of nonactivated synapses (het-LTD) in either pathway. The paired-pulse stimulation protocol and the analysis of coefficient variation of EPSPs suggested postsynaptic induction of these changes except UL-induced het-LTD, which may be presynaptic in origin. Intracellular injection of a Ca(2+)-chelator suggested an involvement of postsynaptic Ca(2+) rise in all types of long-term plasticity. Pharmacological analysis indicated that NMDA receptors and type-5 metabotropic glutamate receptors are involved in WM-induced and UL-induced plasticity, respectively. Analysis with inhibitors and/or in transgenic mice suggested an involvement of cannabinoid type 1 receptors and calcineurin in UL-induced and WM-induced het-LTD, respectively. These results suggest that hom-LTP and het-LTD may play a role in switching the top-down or bottom-up regulation of CG cell function and/or in maintaining stability of synaptic transmission efficacy through different molecular mechanisms.

Cell type-specific, presynaptic LTP of inhibitory synapses on fast-spiking GABAergic neurons in the mouse visual cortex.

Properties and plasticity of inhibitory synapses on fast-spiking (FS) GABAergic (FS-GABA) interneurons in layer II/III of the mouse visual cortex were examined in cortical slices by whole-cell recordings of IPSCs or IPSPs evoked by activation of presynaptic FS or non-FS GABAergic interneurons. Unitary IPSCs (uIPSCs) evoked by action potentials of FS-GABA neurons have shorter onset latency, faster rising slope, higher peak amplitude, and faster decay time than those evoked by action potentials of non-FS-GABA neurons. Tetanic activation of presynaptic FS-GABA neurons induced long-term potentiation (LTP) of uIPSCs, whereas that of presynaptic non-FS-GABA neurons did not induce LTP, indicating that long-term plasticity of inhibitory synapses on FS-GABA neurons is pathway specific. For further analysis of inhibitory synaptic plasticity, IPSPs evoked by electrical stimulation of an adjacent site in the cortex were recorded from FS-GABA neurons. Theta burst stimulation induced LTP of IPSPs in 12 of 14 FS-GABA neurons. The paired-pulse stimulation protocol and coefficient of variation analysis indicated that this form of LTP may be presynaptic in origin. Filling postsynaptic cells with a Ca(2+) chelator did not block the induction of LTP, suggesting no involvement of postsynaptic Ca(2+) rise. Also, this form of LTP was dependent neither on metabotropic glutamate receptors nor voltage-gated Ca(2+) channels of the L and T types. Further pharmacological analysis indicated that voltage-gated Ca(2+) channels other than the P/Q type, such as N and R types, were not involved in LTP, suggesting that P/Q-type channels are a candidate for factors inducing LTP of inhibitory synapses between FS-GABA neurons.

Selective control of inhibitory synapse development by Slitrk3-PTPδ trans-synaptic interaction.

Balanced development of excitatory and inhibitory synapses is required for normal brain function, and an imbalance in this development may underlie the pathogenesis of many neuropsychiatric disorders. Compared with the many identified trans-synaptic adhesion complexes that organize excitatory synapses, little is known about the organizers that are specific for inhibitory synapses. We found that Slit and NTRK-like family member 3 (Slitrk3) actS as a postsynaptic adhesion molecule that selectively regulates inhibitory synapse development via trans-interaction with axonal tyrosine phosphatase receptor PTPδ. When expressed in fibroblasts, Slitrk3 triggered only inhibitory presynaptic differentiation in contacting axons of co-cultured rat hippocampal neurons. Recombinant Slitrk3 preferentially localized to inhibitory postsynaptic sites. Slitrk3-deficient mice exhibited decreases in inhibitory, but not excitatory, synapse number and function in hippocampal CA1 neurons and exhibited increased seizure susceptibility and spontaneous epileptiform activity. Slitrk3 required trans-interaction with axonal PTPδ to induce inhibitory presynaptic differentiation. These results identify Slitrk3-PTPδ as an inhibitory-specific trans-synaptic organizing complex that is required for normal functional GABAergic synapse development.

Laminar-specific maturation of GABAergic transmission and susceptibility to visual deprivation are related to endocannabinoid sensitivity in mouse visual cortex.

The developmental period when neuronal responses are modified by visual experience is reported to start and end earlier in layer 4 than in layer 2/3 of the visual cortex, and the maturation of GABAergic inhibitory circuits is suggested to determine the timing of this period. Here, we examine whether the laminar difference in such timing corresponds to a difference in the time course of the functional maturation of GABAergic synaptic transmission to star pyramidal and pyramidal cells in layers 4 and 2/3, respectively, of the mouse visual cortex and whether the development of the strength of GABAergic transmission is affected by visual deprivation in a laminar-specific manner. Our analysis of developmental changes in inhibitory postsynaptic currents of star pyramidal and pyramidal cells evoked by electrical stimulation of afferents or action potentials of fast-spiking GABAergic neurons revealed that there was a sequential maturation of GABAergic function from layers 4 to 2/3. The maturation of inhibition in layer 4 occurred at postnatal week 3, which preceded by 1 week that of layer 2/3. Visual deprivation by dark rearing arrested the functional development of GABAergic transmission in layer 2/3, whereas dark rearing was not so effective in layer 4. GABAergic synapses in layer 2/3 were sensitive to an agonist for cannabinoid type 1 receptors and not normally matured in receptor knock-out mice, whereas those in layer 4 were not so. These results suggest laminar-specific maturation of inhibition and susceptibility to visual deprivation, which may be related to the laminar difference in sensitivity to endocannabinoids.

Ephrin-A5 and EphA5 interaction induces synaptogenesis during early hippocampal development.

BACKGROUND: Synaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca2+ fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents. CONCLUSIONS/SIGNIFICANCE: These studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation.

Difference in binocularity and ocular dominance plasticity between GABAergic and excitatory cortical neurons.

Neuronal circuits in the cerebral cortex consist mainly of glutamatergic/excitatory and GABAergic/inhibitory neurons. In the visual cortex, the binocular responsiveness of neurons is modified by monocular visual deprivation during the critical period of postnatal development. Although GABAergic neurons are considered to play a key role in the expression of the critical period, it is not known whether their binocular responsiveness and ocular dominance plasticity are different from those of excitatory neurons. Recently, the end of the critical period was found to be not strict so that cortical neurons in the adult still have some ocular dominance plasticity. It is not known, however, which type of neurons or both maintain such plasticity in adulthood. To address these issues, we applied in vivo two-photon functional Ca(2+) imaging to transgenic mice whose GABAergic neurons express a yellow fluorescent protein called Venus. We found that GABAergic neurons are more binocular than excitatory neurons in the normal visual cortex, and both types of neurons show the same degree of modifiability to monocular visual deprivation during the critical period, but the modifiability of GABAergic neurons is stronger than that of excitatory neurons after the end of the critical period.

Metabotropic glutamate receptor type 5-dependent long-term potentiation of excitatory synapses on fast-spiking GABAergic neurons in mouse visual cortex.

Long-term potentiation (LTP) of excitatory synapses on GABAergic neurons in layer II/III of visual cortical slices was examined in GAD67-GFP knock-in mice by whole-cell recordings of EPSPs evoked by layer IV stimulation. Theta burst stimulation (TBS) paired with postsynaptic depolarization induced LTP in 14 of 19 fast-spiking GABAergic (FS-GABA) neurons, whereas only in 6 of 17 non-FS GABAergic neurons. The mean magnitude of LTP in the former cell group was larger than that in the latter. The paired-pulse stimulation protocol and coefficient of variation analysis indicated that LTP of excitatory synapses on FS-GABA neurons may be postsynaptic in origin. Filling postsynaptic cells with a Ca2+-chelator blocked the induction of LTP, suggesting an involvement of postsynaptic Ca2+ rise. The developmental analysis of LTP indicated that almost the same magnitude of LTP was induced after postnatal day 17 to the young adulthood, suggesting no age dependence after eye opening. This form of LTP was dependent neither on NMDA receptors nor voltage-gated Ca2+ channels (L and T types). An antagonist for type 5 metabotropic glutamate receptors (mGluR5) blocked this form of LTP, whereas an antagonist for mGluR1 was not effective. An agonist for mGluR1/5 induced potentiation of EPSPs of FS-GABA neurons in concentration- and use-dependent manners. This potentiation and TBS-induced LTP occluded each other. Further pharmacological analyses suggested that this form of LTP at FS-GABA neurons is induced through an activation of mGluR5, which triggers Ca2+ release from internal stores via activations of phospholipase C and inositol triphosphate.

A local reduction in cortical GABAergic synapses after a loss of endogenous brain-derived neurotrophic factor, as revealed by single-cell gene knock-out method.

To address questions of whether brain-derived neurotrophic factor (BDNF) released from active excitatory neurons acts locally only on GABAergic presynaptic terminals contacting these neurons or generally also on GABAergic terminals contacting other inactive neurons, we developed a single-cell gene knock-out method in organotypic slice culture of visual cortex of floxed BDNF transgenic mice. A biolistic transfection of Cre recombinase with green fluorescence protein (GFP) plasmids to layer II/III of the cortex resulted in loss of BDNF in a single neuron or a small number of neurons, which expressed GFP at 13-14 d in vitro. Analysis with in situ hybridization and immunohistochemistry confirmed that neurons expressing GFP lacked BDNF mRNA and protein, respectively. Analysis with immunohistochemistry using antibody against GABA synthesizing enzyme showed that the number of GABAergic terminals on the soma of BDNF knock-out neurons was smaller than that of neighboring control neurons. Morphological analysis indicated that there was no significant difference in the soma size and branch points and length of dendrites between the BDNF knock-out and control neurons. Recordings of miniature IPSCs (mIPSCs) showed that the frequency of mIPSCs of BDNF knock-out neurons was lower than that of control neurons, although the amplitude was not significantly different, suggesting the smaller number of functional GABAergic synapses on whole the BDNF knock-out neuron. The present results suggest that BDNF released from postsynaptic target neurons promotes the formation or proliferation of GABAergic synapses through its local actions in layer II/III of visual cortex.