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. 2021 Aug 5;8(4):ENEURO.0093-21.2021. doi: 10.1523/ENEURO.0093-21.2021

Figure 15.

Figure 15.

Postsynaptic responses to optogenetic stimulation of Kiss1 AVPV/PeN fibers release GABA onto PVH neurons. A1, Low-power composite image of a coronal slice through AVPV. Injections of AAV-DIO:ChR2:mCherry into the AVPV/PeN labeled Kiss1AVPV/PeN neurons from rostral to caudal. A2, High-power composite image shows fluorescence projections are visible around the patched cells in the PVH. Low-power scale bar: 200 μm (A1); high-power scale bar: 40 μm (A2). B–C, Using a standard internal solution, whole-cell voltage clamp recordings were made in PVH neurons (Vhold = −10 mV). B, Fast outward currents were seen in response to blue light stimulation. Red trace is the averaged response. C, The GABAA antagonist picrotoxin (100 μm) effectively and reversibly blocked the optogenetically evoked outward currents to confirm GABAergic signaling. D, The optogenetically evoked currents were also blocked by TTX (1 μm), but recovered when the K+ blockers 4-AP (0.5 mm) and TEA (7.5 mm) were added to the bath when using high chloride internal solution (Vhold = −60 mV). This “rescue” of the response is physiological evidence of a direct synaptic connection. E, High-frequency optogenetic stimulation (20 Hz, 10 s) generated a slow IPSC in a PVH neuron using high chloride internal solution (Vhold = −60 mV; upper trace). In another cell, after blocking the LED-induced GABAA responses with picrotoxin at 100 μm, high-frequency optogenetic stimulation (20 Hz, 30 s) using normal internal solution still generated a slow IPSC that recovered in 10 min (middle trace). Furthermore, the GABAB antagonist CGP 55845 (1 μm) blocked the slow IPSC response in the presence of picrotoxin in another cell (bottom trace). Blue bar below or above the recordings indicate LED stimulus (B–E). F, Summary of the effects of CGP 55845 on the high-frequency optogenetic stimulation (20 Hz, 10 s)-induced slow IPSC in PVH neurons (un-paired t test, t(8) = 2.826, *p = 0.0223). Data points represent the mean ± SEM. Cell numbers are indicated. G, Representative gel images of scRT-PCR Pdyn and Vglut2 mRNA expression in eight responsive PVH neurons. MM, molecular markers; 1–3 and 1–5, the recorded PVH neurons; Ctrl, positive tissue control. H, A representative trace of a Pdyn neuron showing a “sag” (h-current) with hyperpolarizing current injection and rebound burst firing in current clamp. Two of three Pdyn neurons exhibited an h-current.