Spatial patterning of excitatory and inhibitory neuropil territories during spinal circuit development

Abstract

To generate rhythmic motor behaviors, both single neurons and neural circuits require a balance between excitatory inputs that trigger action potentials and inhibitory inputs that promote a stable resting potential (E/I balance). Previous studies have focused on individual neurons and have shown that, over a short spatial scale, excitatory and inhibitory (E/I) synapses tend to form structured territories with inhibitory inputs enriched on cell bodies and proximal dendrites and excitatory inputs on distal dendrites. However, systems-level E/I patterns, at spatial scales larger than single neurons, are largely uncharted. We used immunostaining for PSD-95 and gephyrin postsynaptic scaffolding proteins as proxies for excitatory and inhibitory synapses, respectively, to quantify the numbers and map the distributions of E/I synapses in zebrafish spinal cord at both an embryonic stage and a larval stage. At the embryonic stage, we found that PSD-95 puncta outnumber gephyrin puncta, with the number of gephyrin puncta increasing to match that of PSD-95 puncta at the larval stage. At both stages, PSD-95 puncta are enriched in the most lateral neuropil corresponding to distal dendrites while gephyrin puncta are enriched on neuronal somata and in the medial neuropil. Significantly, similar to synaptic puncta, neuronal processes also exhibit medial-lateral territories at both developmental stages with enrichment of glutamatergic (excitatory) processes laterally and glycinergic (inhibitory) processes medially. This establishment of neuropil excitatory-inhibitory structure largely precedes dendritic arborization of primary motor neurons, suggesting that the structured neuropil could provide a framework for the development of E/I balance at the cellular level. J. Comp. Neurol. 525:1649-1667, 2017. © 2016 Wiley Periodicals, Inc.

Defective Excitatory/Inhibitory Synaptic Balance and Increased Neuron Apoptosis in a Zebrafish Model of Dravet Syndrome

“…We recently showed that zebrafish gabra1 -/- and depdc5 -/- epileptic mutants displayed markedly fewer inhibitory synapses compared to age-matched control larvae, showing that these two mutants display brain development defects [21,22]. To determine whether Scn1Lab deficiency also induced unbalanced excitatory/inhibitory post-synaptic outputs, we analyzed and compared, by immunocytochemistry, the distribution of the two post-synaptic populations on brain sections of 5 dpf control and Scn1Lab-depleted larvae, using anti-PSD-95 and anti-gephyrin antibodies, which label excitatory and inhibitory post-synaptic terminals, respectively [23]. Quantification of the two post-synaptic populations showed a marked increase in PSD-95 labeling (Figure 2B,C; p < 0.01) combined with a clear decrease of that of gephyrin (Figure 1E,F; p < 0.01), in scn1Lab AUG morphants, compared to controls (Figure 2A,D).…”

“…We focused on excitatory and inhibitory synapses by studying two important proteins that are markers of synaptic integrity, PSD-95 and gephyrin [23]. PSD-95, by its PDZ domains, serves as a scaffolding protein with a docking site for signaling proteins clustering around NMDA- and AMPA- type glutamate receptors, which are crucial for excitatory neurotransmission and synaptic plasticity [34].…”

Restoring Shank3 in the rostral brainstem of shank3ab−/− zebrafish autism models rescues sensory deficits

“…Tissue blocks were then sectioned (30 µm) on a Leica cryostat, fixed in 4% formaldehyde (diluted from 16% paraformaldehyde Pierce Scientific) for 10 min and stained with synaptic antibodies as in ref. 38 . Anti-PSD-95 (1:500; Abcam; Cambridge, UK; ab-18258) and anti-Shank3ab (1:200; sc-30193, Santa Cruz Biotechnology, CA; this polyclonal has since been discontinued) were used as primary antibodies, with secondary antibodies conjugated to Alexa Fluor 568 (Abcam, ab175472) and Alexa Fluor 633 (Thermo Fisher Scientific, R21070), respectively.…”

Defective excitatory/inhibitory synaptic balance and increased neuron apoptosis in a zebrafish model of Dravet syndrome

“…We recently showed that zebrafish gabra1 -/-and depdc5 -/-epileptic mutants displayed markedly fewer inhibitory synapses compared to age-matched control larvae, showing that these two mutants display brain development defects [21], [22]. To determine whether Scn1Lab deficiency also induced unbalanced excitatory/inhibitory post-synaptic outputs, we analyzed and compared, by immunocytochemistry, the distribution of the two post-synaptic populations on brain sections of 5 dpf control and Scn1Lab-depleted larvae, using anti-PSD-95 and anti-gephyrin antibodies, which label excitatory and inhibitory post-synaptic terminals, respectively [23]. Quantification of the two post-synaptic populations showed a marked increase in PSD-95 labeling (Figure 2B, C; p < 0.01) combined with a clear decrease of that of gephyrin ( Figure 1E, F; p < 0.01), in scn1Lab AUG morphants, compared to controls (Figure 2A, 2D).…”