Neural activity regulates the number and properties of GABAergic synapses in the brain, but the mechanisms underlying these changes are unclear. We found that blocking spike activity globally in developing hippocampal neurons from rats reduced the density of GABAergic terminals as well as the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Chronic inactivity later in development led to a reduction in the mIPSC amplitude, without any change in GABAergic synapse density. By contrast, hyperpolarizing or abolishing spike activity in single neurons did not alter GABAergic synaptic inputs. Suppressing activity in individual presynaptic GABAergic neurons also failed to decrease synaptic output. Our results indicate that GABAergic synapses are regulated by the level of activity in surrounding neurons. Notably, we found that the expression of GABAergic plasticity involves changes in the amount of neurotransmitter in individual vesicles.

Synaptic vesicles are recycled locally within presynaptic specializations. We examined how vesicles are reused after endocytosis, using transgenic mice expressing the genetically encoded fluorescent indicator synaptopHluorin in subsets of neurons. At both excitatory and inhibitory synapses in cultured hippocampal neurons, newly endocytosed vesicles did not preferentially enter the releasable pool of vesicles. Rather, they entered the reserve pool first and subsequently the readily releasable pool over a period of several minutes. All vesicles in the recycling pool could be accessed by spaced stimuli, arguing against preferential local reuse of the readily releasable vesicles. Interestingly, nearly half the vesicles at excitatory synapses, and a third at inhibitory synapses, could not be recruited for release even by sustained stimuli. We conclude that, at presynaptic terminals in the hippocampus, most vesicles vacate release sites after exocytosis and are replaced by existing vesicles from the reserve pool, placing constraints on kiss-and-run recycling.endocytosis ͉ exocytosis ͉ hippocampus ͉ kiss and run S ynaptic vesicles are recycled locally within the presynaptic terminal for reuse (1, 2). Several mechanisms of recycling have been proposed, including a conventional pathway involving clathrin coat formation (1, 2) and a clathrin-independent pathway involving kiss-and-run exocytosis (3-9) that might explain rapid endocytosis observed at some synapses (5, 10-12). Although recent attention has centered on modes of endocytosis, several key questions can also be raised regarding postendocytic traffic of synaptic vesicles. Where do vesicles go after endocytosis? Are recently recaptured vesicles preferentially reused? Rapid reuse of recently recaptured vesicles (13, 14) might be advantageous to synapses if it allows vesicles to become release-competent more rapidly than would be possible by recruitment from the reserve pool.Studies in hippocampal and cortical cultures generally have focused on glutamatergic synapses or assumed that the synapses under investigation are glutamatergic synapses because they are more abundant (15). It is unclear whether inhibitory presynaptic terminals have the same mechanisms of recycling as excitatory synapses. Some differences might be anticipated based on previous studies establishing differences in the molecular composition (16, 17) and short-term synaptic dynamics (16,18,19). Additionally, the loss of specific proteins, for example, synapsin I (20), appears to have different consequences in glutamatergic and GABAergic synapses. Therefore, it is important to understand the properties of and mechanisms in vesicle recycling at inhibitory synapses.Genetically encoded probes such as synaptopHluorin (spH), which is a fusion protein of the vesicle protein VAMP2 and a pH-sensitive EGFP (21), offer a method to target tracers to selected populations of neurons. We have generated lines of transgenic mice that express spH in subsets of neurons in the brain. We first show that exocytosis and en...

Exerting its actions pre-, post-and peri-synaptically, brain-derived neurotrophic factor (BDNF) is one of the most potent modulators of hippocampal synaptic function. Here, we examined the effects of BDNF on a rapidly recycling pool (RRP) of vesicles within excitatory synapses. First, we estimated vesicular release in hippocampal cultures by performing FM4-64 imaging in terminals impinging on enhanced green fluorescent protein (eGFP)-labelled dendritic spines -a hallmark of excitatory synapses. Consistent with a modulation of the RRP, BDNF increased the evoked destaining rate of FM4-64 only during the initial phase of field stimulation. Multiphoton microscopy in acute hippocampal slices confirmed these observations by selectively imaging the RRP, which was loaded with FM1-43 by hyperosmotic shock. Slices exposed to BDNF showed an increase in the evoked and spontaneous rates of FM1-43 destaining from terminals in CA1 stratum radiatum, mostly representing excitatory terminals of Schaffer collaterals. Variance-mean analysis of evoked EPSCs in CA1 pyramidal neurons further confirmed that release probability is increased in BDNF-treated slices, without changes in the number of independent release sites or average postsynaptic quantal amplitude. Because BDNF was absent during dye loading, imaging, destaining and whole-cell recordings, these results demonstrate that BDNF induces a long-lasting enhancement in the probability of transmitter release at hippocampal excitatory synapses by modulating the RRP. Since the endogenous BDNF scavenger TrkB-IgG prevented the enhancement of FM1-43 destaining rate caused by induction of long-term potentiation in acute hippocampal slices, the modulation of a rapidly recycling vesicle pool may underlie the role of BDNF in hippocampal long-term synaptic plasticity.

Synaptic depression is thought to underlie the loss of cortical responsiveness to an eye deprived of vision. Here, we establish a fundamental role for type 2 metabotropic glutamate receptors (mGluR2) in long-term depression (LTD) of synaptic transmission within primary visual cortex. Direct mGluR2 activation by (2S,2R,3R-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) persistently depressed layer 2͞3 field potentials in slices of mouse binocular zone when stimulated concomitantly. Chemical LTD was independent of N-methyl-D-aspartate (NMDA) receptors but occluded conventional LTD by low-frequency stimulation, indicating shared downstream events. Antagonists or targeted disruption of mGluR2 conversely prevented LTD induction by electrical lowfrequency stimulation to layer 4. In contrast, Schaeffer collateral synapses did not exhibit chemical LTD, revealing hippocampal area CA1, naturally devoid of mGluR2, to be an inappropriate model for neocortical plasticity. Moreover, monocular deprivation remained effective in mice lacking mGluR2, and receptor expression levels were unchanged during the critical period in wild-type mice, indicating that experience-dependent plasticity is independent of LTD induction in visual cortex. Short-term depression that was unaffected by mGluR2 deletion may better reflect circuit refinement in vivo.