Fig. 3: The VTg nucleus sends monosynaptic inhibitory inputs to PMdCCK neurons. | Nature Communications

Fig. 3: The VTg nucleus sends monosynaptic inhibitory inputs to PMdCCK neurons.

From: Endocannabinoids disinhibit the ventral tegmental nucleus of Gudden to dorsal premammillary nucleus pathway to enhance escape behavior following learned threat experience

Fig. 3

a Scheme showing the approach to label VTg parvalbumin neurons projecting to the PMd. b A representative image showing the expression of both EGFP and mCherry in VTgPV neurons (b1). EGFP expression in VTgPV neurons (b2); Presynaptic PMd-projecting VTg neurons with mCherry (b3); Overlay (b4). Left, scale bar, 150 μm; right, scale bar, 200 μm. c Photograph showing EGFP+ synaptic terminals of VTgPV neurons and mCherry+ axon terminals of PMd-projecting VTg neurons in the PMd (c1). EGFP+ synaptic terminals of VTgPV neurons in the PMd (c2); mCherry+ axon terminals of PMd-projecting VTg neurons in the PMd (c3); Overlay (c4). Left, scale bar, 150 μm; right, scale bar, 200 μm. d Quantification of overlap between PMd-projecting and PV-Cre expressing neurons (N = 4 mice; n = 9 slices). e Scheme showing the strategy used to label VTgPV and PMdCCK neurons in the CCK-IRES-Cre::Pvalb-IRES-Cre mouse. f A representative image showing the expression of mCherry in VTgPV neurons. Scale bar, 100 μm. g Representative images showing the expression of both EGFP and mCherry in the PMd. EGFP expression in PMdCCK neurons (g1); Axon terminals of PMd-projecting VTg neurons with mCherry (g2); Overlay (g3). Scale bar, 200 μm. h 40x times magnified view of histology in (g). Scale bar, 50 μm. i PMd CCK neuron shown in (h1) showed an inhibitory response following blue light delivery to excite ChR2-expressing VTg PV axon terminals (h2) in an ex vivo slice preparation with kynurenic acid (h4). The response disappeared following the application of tetrodotoxin (TTX, 0.3 µM), and the response was rescued following the application of the potassium channel blocker (4-AP, 0.5 mM) in both single pulse (top) and pair pulse (bottom). j Quantification of the PMdCCK responses to stimulation of VTg PV terminals in (i). (N = 4 mice; n = 6 cells; One-way ANOVA followed by Tukey’s test, *p < 0.05, **p < 0.01). Scale bar, 0.5μm. Data are presented as means ± SEM. See also Supplementary Fig. 3. Source data are provided as a Source Data file. Aq, aqueduct; PV, parvalbumin; CCK, cholecystokinin; ACSF, the normal artificial cerebrospinal fluid; TTX, tetrodotoxin; 4-AP, 4-aminopyridine.

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