Heterogeneous expression of SNAP‐25 in rat and human brain

Abstract

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.

VAMP-2, SNAP-25A/B and syntaxin-1 in glutamatergic and GABAergic synapses of the rat cerebellar cortex

“…These data agree with the assumption that the SNARE mechanisms largely characterize the excitatory synapses and that glutamate-containing vesicles release their content via SNARE-dependent mechanisms [12,15,33,34]. However, this study demonstrates subpopulations of glutamatergic synapses in the cerebellar cortex that do not contain the examined SNARE proteins.…”

“…(a) anti-GAD monoclonal antibody, raised in mouse against human GAD-65-GST fusion protein (Stressgen, Victoria, BC, Canada), and (b) anti-GAD polyclonal antibody, raised in rabbit against a synthetic peptide corresponding to the rat GAD-65 C-terminus residues 572-585 (Chemicon), which react to both GAD-65 and GAD-67 [63]; 4. (a) anti-VAMP monoclonal antibody, raised in mouse against crude synaptic immunoprecipitate (human) (Millipore), and (b) anti-VAMP polyclonal antibody, raised in rabbit against a synthetic peptide based on rat VAMP-2 (Stressgen), which react to VAMP-2 [48]; 5. anti-SNAP-25 monoclonal antibody, raised in against crude synaptic immunoprecipitate (human), which reacts with both SNAP-25A and SNAP-25B (Chemicon) [15]; 6. anti-syntaxin monoclonal antibody, raised in mouse against clone HCP-1, which reacts to syntaxin-1 (ABCAM, Cambridge, UK) [57]. …”

“…Finally, ATP hydrolysis by NSF leads to exocytosis and induces a conformational change in the complex that results in its disassembly [3-9]. Several biochemical and immunohistochemical studies suggest the existence of SNARE mechanisms virtually in all regions of the CNS [10-15]. …”

“…In particular, specific sets of SNARE proteins seem to be required to regulate trafficking of vesicles containing different types of neurotransmitters, possibly even one set per neurotransmitter [30,31]. According to this hypothesis, scholars have reported that SNARE proteins are strongly expressed in excitatory (glutamatergic) synapses and may be lacking in inhibitory (GABAergic) synapses [12,15,32-34]. …”

Endogenous SNAP-25 Regulates Native Voltage-gated Calcium Channels in Glutamatergic Neurons

“…Exogenous Expression of SNAP-25 in GABAergic Cells Negatively Regulates VGCC Properties-Because endogenous SNAP-25 expression is lower in GABAergic compared with glutamatergic neurons (13,26) we investigated whether SNAP-25-GFP overexpression in GABAergic neurons negatively modulates VGCC properties. Under these conditions, SNAP-25 is clearly detectable on the somatodendritic plasma membrane of GABAergic neurons (Fig.…”