SynapseJ: An Automated, Synapse Identification Macro for ImageJ

While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a pre-synaptic marker, bassoon, with two different post-synaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, post-synaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental nucleus (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation

Dopamine Receptor Dependence of Cocaine-Mediated Plasticity in Specific Excitatory Synapses Onto Midbrain Dopamine Neurons

Exposure to drugs of abuse, such as cocaine, has been known to cause synaptic plasticity, a process that involves changes in receptor expression and receptor-mediated transmission in response to overactivation of a synapse. Cocaine-induced synaptic plasticity has previously been demonstrated in various inputs onto neural pathways involved in motivation and reward, specifically dopaminergic pathways. Our research focused on characterizing cocaine-induced changes at one particular modulatory synapse onto the nigrostriatal dopaminergic pathway: that between the pedunculopontine tegmental nucleus (PPN) and substantia nigra pars compacta (SNc). Prior research has shown that, within the synapse between PPN glutamatergic neurons and SNc dopaminergic neurons, cocaine induces changes in glutamate receptor composition. Based on this finding and the role of cocaine in increasing extracellular dopamine, we wanted to determine whether PPN-SNc plasticity is dependent on one or both dopamine receptor subtypes: excitatory D1-like or inhibitory D2-like. As prior research had implicated D1-like receptors but not D2-like receptors in plasticity in a different dopaminergic pathway, we used a D1-like receptor antagonist to study its effect on cocaine-mediated plasticity at the PPN-SNc synapse. We utilized optogenetics to selectively excite PPN neurons and record current mediated by two ionotropic glutamate receptors: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) and N-methyl-D-aspartate receptors (NMDAR). While the project is still in progress, our data so far seem to diverge from the previously shown cocaine-induced decrease in the AMPA/NMDA ratio, but they suggest that blocking D1-like receptors affects AMPA receptors. These data bring up interesting considerations, such as the existence of SNc subpopulations, that could guide future studies