SNAP-25 is a component of the SNARE complex implicated in synaptic vesicle exocytosis. In this study, we demonstrate that hippocampal GABAergic synapses, both in culture and in brain, lack SNAP-25 and are resistant to the action of botulinum toxins type A and E, which cleave this SNARE protein. Relative to glutamatergic neurons, which express SNAP-25, GABAergic cells were characterized by a higher calcium responsiveness to depolarization. Exogenous expression of SNAP-25 in GABAergic interneurons lowered calcium responsiveness, and SNAP-25 silencing in glutamatergic neurons increased calcium elevations evoked by depolarization. Expression of SNAP-25(1-197) but not of SNAP-25(1-180) inhibited calcium responsiveness, pointing to the involvement of the 180-197 residues in the observed function. These data indicate that SNAP-25 is crucial for the regulation of intracellular calcium dynamics and, possibly, of network excitability. SNAP-25 is therefore a multifunctional protein that participates in exocytotic function both at the mechanistic and at the regulatory level.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a protein that participates in the regulation of synaptic vesicle exocytosis through the formation of the soluble NSF attachment protein receptor complex and modulates voltage-gated calcium channels activity. The Snap25 gene has been associated with schizophrenia, attention deficit hyperactivity disorder, and bipolar disorder, and lower levels of SNAP-25 have been described in patients with schizophrenia. We used SNAP-25 heterozygous (SNAP-25(+/-)) mice to investigate at which extent the reduction of the protein levels affects neuronal network function and mouse behavior. As interactions of genotype with the specific laboratory conditions may impact behavioral results, the study was performed through a multilaboratory study in which behavioral tests were replicated in at least 2 of 3 distinct European laboratories. Reductions of SNAP-25 levels were associated with a moderate hyperactivity, which disappeared in the adult animals, and with impaired associative learning and memory. Electroencephalographic recordings revealed the occurrence of frequent spikes, suggesting a diffuse network hyperexcitability. Consistently, SNAP-25(+/-) mice displayed higher susceptibility to kainate-induced seizures, paralleled by degeneration of hilar neurons. Notably, both EEG profile and cognitive defects were improved by antiepileptic drugs. These results indicate that reduction of SNAP-25 expression is associated to generation of epileptiform discharges and cognitive dysfunctions, which can be effectively treated by antiepileptic drugs.

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Summary Visual system development involves the formation of neuronal projections connecting the retina to the thalamic dorso-lateral geniculate nucleus (dLGN) and the thalamus to the visual cerebral cortex. Patients carrying mutations in the SOX2 transcription factor gene present severe visual defects, thought to be linked to SOX2 functions in the retina. We show that Sox2 is strongly expressed in mouse postmitotic thalamic projection neurons. Cre-mediated deletion of Sox2 in these neurons causes reduction of the dLGN, abnormal distribution of retino-thalamic and thalamo-cortical projections, and secondary defects in cortical patterning. Reduced expression, in mutants, of Sox2 target genes encoding ephrin-A5 and the serotonin transport molecules SERT and vMAT2 (important for establishment of thalamic connectivity) likely provides a molecular contribution to these defects. These findings unveil thalamic SOX2 function as a novel regulator of visual system development and a plausible additional cause of brain-linked genetic blindness in humans.

SUMMARYPurpose: Cortical dysplasia (CD) represents a wide range of histopathological abnormalities of the cortical mantle that are frequently associated with drug-resistant epilepsy. Recently, carmustine (1-3-bis-chloroethyl-nitrosurea [BCNU]), given to pregnant rats on embryonic day (E) 15, has been used to develop an experimental model mimicking human CD. The aim of this study was to characterize cytological and histological alterations in this model, and compare the results with those observed in human CD. Methods: Pregnant rats were given intraperitoneal injections of BCNU on E15. Sections of cerebral cortex from adult BCNU-treated rats were cytoarchitecturally and immunohistochemically analyzed using anti-SMI311, anticalbindin (CB), and antiparvalbumin (PV) antibodies. The density of the PVimmunoreactive (PV-ir) interneurons was quantitatively assessed by means of a two-dimensional cell-counting technique, and the spatial distribution of PV-ir neurons was evaluated by using the Voronoi tessellation. Results: The morphological features included reduced cortical size, laminar disorganization, and heterotopic clusters of neurons. We also identified large, disoriented SMI311-positive pyramidal neurons, and dysmorphic neurons intensely immunostained for neurofilaments, similar to those observed in human dysplastic cortex. An altered distribution of PV-immunoreactive cortical interneurons was also present. Conclusions: Although some of the cytoarchitectural abnormalities found in BCNU-exposed cortex are similar to those found in other CD models, we identified new alterations that recall the neuropathological description of type IIA (Taylor's type) CD. BCNU-treated rat could therefore be a useful additional model for investigating the pathogenic mechanisms involved in this CD.

Synaptosomal associated protein of 25 kDa (SNAP-25) is a SNARE component of the exocytotic apparatus involved in the release of neurotransmitter. We used multiple-labeling immunofluorescence, confocal microscopy, and ultrastructural immunocytochemistry to examine the expression of SNAP-25 in excitatory and inhibitory terminals from different rat and human brain areas. Glutamatergic and GABAergic terminals were identified by staining for the vesicular glutamate transporter (vGLUT1), glutamic acid decarboxylase (GAD67), or the vesicular GABA transporter (vGAT). In all examined areas GABAergic terminals did not display detectable levels of SNAP-25, whereas glutamatergic terminals expressed the protein to a variable extent. Codistribution analysis revealed a high colocalization between pixels detecting SNAP-25 labeling and pixels detecting vGLUT1 immunoreactivity. On the contrary, a low degree of pixel colocalization, comparable to that between two unrelated antigens, was detected between SNAP-25 and vGAT, thus suggesting a random overlap of immunofluorescence signals. Our immunofluorescence evidence was supported by ultrastructural data, which clearly confirmed that SNAP-25 was undetectable in GABAergic terminals identified by both their typical morphology and specific staining for GABA. Interestingly, our ultrastructural results confirmed that a subset of glutamatergic synapses do not contain detectable levels of SNAP-25. The present study extends our previous findings obtained in rodent hippocampus and provides evidence that SNAP-25 expression is highly variable between different axon terminals both in rat and human brain. The heterogeneous distribution of SNAP-25 may have important implications not only in relation to the function of the protein as a SNARE but also in the control of network excitability.

Our observation expands the spectrum of human GE anomalies, demonstrating that these may take place also without associated severe micro-lissencephalies.

SUMMARY:Ganglionic eminence is the main transitory proliferative structure of the ventral telencephalon in human fetal brain and it contributes for at least 35% to the population of cortical interneurons; however data on the human GE anomalies are scarce. We report 5 fetal MR imaging observations with bilateral symmetric cavitations in their GE regions resembling an inverted open C shape and separating the GE itself form the deeper parenchyma. Imaging, neuropathology, and follow-up features suggested a malformative origin. All cases had in common characteristics of lissencephaly with agenesis or severe hypoplasia of corpus callosum of probable different genetic basis. From our preliminary observation, it seems that GE cavitations are part of conditions which are also accompanied by severe cerebral structure derangement.ABBREVIATIONS: CC ϭ corpus callosum; GA ϭ gestational age; GE ϭ ganglionic eminence