Figure 4: PVN CRH neurons project to the lateral hypothalamus.
From: Hypothalamic CRH neurons orchestrate complex behaviours after stress
(a) Schematic of experimental design. (b) c-Fos-positive cells in LH of CRHeYFP and CRHChR2 following photostimulation in PVN. (c) Summary data of c-Fos in LH (CRHeYFP: 100.5±21.1, n=4 versus CRHChR2: 318.8±60.4, n=5; P=0.0179; t-test). (d) In vivo photostimulation in LH (20hz, 5 min) increases grooming time (CRHeYFP: 5.4±3.0 s, n=3; versus CRHChR2: 36.9±7.3 s, n=7; P=0.0264; t-test). (e) Schematic map and experimental design of in vitro whole-cell recordings from LH neurons. (f) Biocytin filled recorded neurons in LH (red) surrounded by ChR2-eYFP-expressing fibres (green). (g) In voltage clamp (HP=−80 mV), blue light (2–5 ms) elicits fast inward currents (latency: 4.7±0.3 ms) that are abolished by TTX (baseline: 103.1±2.0 pA versus TTX: 4.7±0.7 pA, n=8; P<0.0001; repeated-measures one-way ANOVA) and partially restored by increasing light-pulse duration (7.5–10 ms) during application of 4-aminopyridine (4-AP)28,44 (40.1±9.0 pA; P<0.0001 versus baseline; P=0.0222 versus TTX, n=8; repeated-measures one-way ANOVA). (h) Sample traces show effects of optical stimulation on LH neuron firing. (i,j) oPSCs are unaffected by picrotoxin but are potently inhibited by DNQX (baseline: 90.7±12.6 pA, picro: 115.1±17.6 pA, DNQX: 11.3±3.5 pA, n=6; baseline versus DNQX, P=0.0007; repeated-measures one-way ANOVA). (k) Current clamp recordings of LH neurons reveal two distinct electrophysiological profiles. Cells depicted by grey square show no synaptic responses to blue light pulses; blue circle indicates cells with synaptic responses. (l) Action potential frequency–current relationship in responding/non-responding cells (n=16; P<0.0001; two-way ANOVA). (m) Differential hyperpolarization-induced ‘sag’ between groups (non-responder: 0.017±0.024, n=8; responder: 0.146±0.022, n=8; P=0.0016; t-test). Sag index calculation: (Vm max—Vm steady state)/Vm max, in response to −80 pA hyperpolarizing step). (n) Firing frequency (+60 pA step) versus sag index in responding and non-responding cells. Scale bars: (b) and (f), 50 μm; (g) and (i), 50 pA and 10 ms; (h) and (k), 50 mV and 50 ms; NS, not significant; *P<0.05; **P<0.01; ***P<0.0005; ****P<0.0001; Error bars,±s.e.m.
