Journal: Current Biology

Article Title: Ultra-sparse Connectivity within the Lateral Hypothalamus

doi: 10.1016/j.cub.2020.07.061

Figure Lengend Snippet: Optogenetic Connections within the LH Are Consistent with Ultra-sparse Synaptic Connectivity (A) Top: schematic of opsin and label delivery strategy to identify connections from VGAT neurons to orexin neurons. Middle: micrograph of an example coronal section with VGAT neurons expressing ChR2.mCherry and orexin neurons expressing GCaMP6s. f, fornix. Bottom: current-clamp recording shows a typical lack of synaptic response in orexin neurons while the simultaneously recorded VGAT neuron spikes in response to the light stimulus. (B) Examples of identified connections from the optogenetically activated VGLUT2 population to a GAD65-GFP neuron (top) and from the VGAT population to an orexin neuron (bottom). Amplitude scale bars in (A) and (B) only apply to black traces. Gray traces illustrate spiking in the channelrhodopsin-bearing population. (C) Summary data from all recordings showing that overall only 32% (43/136) of recorded neurons had a postsynaptic response when thousands of presynaptic neurons were activated. (D) Example voltage-clamp (−60 mV) recording of oPSCs and sPSCs from the GAD65-GFP neuron shown in (B), when VGLUT2 neurons were optogenetically tagged. Top: the trace is a segment of the recording showing a light-induced PSC and several sPSCs (asterisks). Bottom: from left, histogram of sPSC amplitudes and the average oPSC amplitude labeled, average oPSC across all light stimuli, average sPSC, average oPSC (detected and aligned with the same method as the sPSC for a reliable comparison), average of the largest 5% of sPSCs, and the average waveforms overlaid. (E) Another example recording of an n.m., GAD65-GFP, and VGLUT2-cre::DIO.ChR2 neuron simultaneously before (baseline, black) and after glutamate receptor blockade (CNQX, D-AP5, light blue). The VGLUT2 neuron shows runaway action currents indicating reliable spiking in response to light. (F) Summary data of average PSCs as labeled in (D) across all voltage-clamp data, showing that oPSCs were larger than sPSCs but not significantly different from the top 5% of sPSCs. ∗ p < 0.05; n.s., p > 0.05. The y axis in the amplitude plot is logarithmic. Error bars denote SD. (G) Histogram of the oPSC/sPSC amplitude ratio for all neurons in (F), showing the majority of optogenetic connections can be explained by 1 or 2 sPSCs.

Article Snippet: Chemicals for making solutions were purchased from Sigma-Aldrich, except synaptic blockers CNQX (50μM), D-AP5 (50μM) and gabazine (3μM) which were from Tocris.

Techniques: Expressing, Labeling, Comparison