Abstract
Presynaptic terminal formation is a complex process that requires assembly of proteins responsible for synaptic transmission at sites of axo-dendritic contact. Accumulation of presynaptic proteins at developing terminals is facilitated by glutamate receptor activation. Glutamate is loaded into synaptic vesicles for release via the vesicular glutamate transporters VGLUT1 and VGLUT2. During postnatal development there is a switch from predominantly VGLUT2 expression to high VGLUT1 and low VGLUT2, raising the question of whether the developmental increase in VGLUT1 is important for presynaptic development. Here, we addressed this question using confocal microscopy and quantitative immunocytochemistry in primary cultures of rat neocortical neurons. First, in order to understand the extent to which the developmental switch from VGLUT2 to VGLUT1 occurs through an increase in VGLUT1 at individual presynaptic terminals or through addition of VGLUT1-positive presynaptic terminals, we examined the spatio-temporal dynamics of VGLUT1 and VGLUT2 expression. Between 5 and 12 days in culture, the percentage of presynaptic terminals that expressed VGLUT1 increased during synapse formation, as did expression of VGLUT1 at individual terminals. A subset of VGLUT1-positive terminals also expressed VGLUT2, which decreased at these terminals. At individual terminals, the increase in VGLUT1 correlated with greater accumulation of other synaptic vesicle proteins, such as synapsin and synaptophysin. When the developmental increase in VGLUT1 was prevented using VGLUT1-shRNA, the density of presynaptic terminals and accumulation of synapsin and synaptophysin at terminals were decreased. Since VGLUT1 knock-down was limited to a small number of neurons, the observed effects were cell-autonomous and independent of changes in overall network activity. These results demonstrate that up-regulation of VGLUT1 is important for development of presynaptic terminals in the cortex.
Highlights
The cortical maps responsible for perception and behavior are comprised of a complex network of synapses
To determine the extent to which these developmental changes occur through changes in the fraction of terminals expressing VGLUT1 and VGLUT2 or their expression levels at individual terminals of cortical neurons, we quantified the dynamics of the expression and co-localization of VGLUT1 and VGLUT2 at individual terminals during synapse formation
Neurons cultured from P0–P2 rat visual cortex were immunolabeled for VGLUT1, VGLUT2, and the integral synaptic vesicles (SV) protein synaptophysin at 5, 8–9 and 12DIV imaged using multi-channel fluorescence confocal microscopy
Summary
The cortical maps responsible for perception and behavior are comprised of a complex network of synapses Understanding how these synapses form has been a major focus in developmental neurobiology, and abnormal synapse development may be linked to a number of neurological disorders, including autism, epilepsy, amblyopia, depression and schizophrenia [1]. Because activity is known to play a major role in the establishment and refinement of cortical circuits and sensory maps [4,5,6], delineating the role of neurotransmitters in presynaptic terminal assembly is of particular interest [7,8,9]. Glutamate is loaded into SVs via vesicular glutamate transporters, of which there are three known isoforms: VGLUT1, VGLUT2 and VGLUT3 [10] They exhibit similar transport properties, each VGLUT isoform shows a unique spatio-temporal expression profile in the developing and adult brain. Others have suggested that VGLUT1 and VGLUT2 are targeted to separate terminals, even when expressed in the same neuron [16]
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