Neurosecretory and neuropeptide phenotype of tdTomato cells in <i>Crh-IRES-Cre;Ai14</i> mice.

<p><b>A</b>) Retrograde transport of peripherally-delivered fluorogold in tdTomato neurons. A confocal image (40× magnification) shows tdTomato in the PVN (red) along with fluorogold immunoreactivity. Higher magnification (100×) image indicated by box is shown in lower left corner. Neuropeptide immunoreactivity for <b>B</b>) oxytocin (OT), <b>C</b>) vasopressin (VP), <b>D</b>) somatostatin (SST), or <b>E</b>) thyrotropin-releasing hormone (TRH; with 100× magnification inset) in <i>Crh-IRES-Cre;Ai14</i> mouse PVN, shown at 40× <b>F</b>) Bar graph showing the percent of tdTomato positive cells that coexpress each neuropeptide, each from n = 5 colchicine-treated mice. Graphed data are represented by mean ± SEM. Scale bars are 50 µm (A-E large), 20 µm (A, E inset).</p

Morphological and intrinsic membrane properties of <i>Crh-IRES-Cre;Ai14</i> tdTomato neurons.

<p><b>A</b>) Confocal Images (60× magnification), from 3 mouse PVN sections, showing morphology of single tdTomato neurons filled with Alexa488- and biocytin during whole-cell patch clamp recordings. <b>B</b>) Epifluorescence and differential interference contrast (DIC) images during whole-cell recording from a tdTomato positive PVN neuron. <b>C</b>) Left: current clamp trace of a tdTomato positive neuron. Membrane potential was −80 mV, current injection started at −20 pA with 10 pA increments. Scale bar: 0 mV/100 msec. Right,above: current-voltage plot for current injection (0±20 pA, Δ10 pA) in tdTomato neurons (n = 23 cells). Right,below: 50 pA current injection from −80 mV in a tdTomato negative neuron. Scale: 0 mV/100 msec <b>D</b>) Graph of action potential frequency for varied positive current steps from −80 mV in tdTomato+ (n = 23) and large soma tdTomato- (n = 15) neurons. <b>E</b>) Latency (in msec) to first action potential for varied current injection steps in tdTomato+/− neurons.</p

Anatomical distribution and CRH protein expression in tdTomato cells in the PVN of <i>Crh-IRES-Cre;Ai14</i> mice.

<p><b>A</b>) Confocal images (20× magnification) of the rostral-caudal extent of the PVN in a naïve tdTomato mouse. In lower left corner, Allen Brain Atlas level is indicated. <b>B</b>) confocal image (40× magnification) of a colchicine-treated <i>Crh-IRES-Cre;Ai14</i> mouse PVN. In green, immunostaining against corticotropin-releasing hormone (CRH) is shown.<b>C</b>) Higher magnification of the (100×) image of the box inset in (B). <b>D</b>) Colocalization of CRH immunoreactivity and tdTomato at the external zone of the median eminence, shown at 40× magnification. Scale bars are 100 µm (A), 50 µm (B, D), and 20 µm (C).</p

Fast glutamate and GABA transmission in Crh-IRES-Cre;Ai14 tdTomato neurons.

<p><b>A</b>) Left: Sample recording, in voltage-clamp mode, from a single tdTomato neuron in the presence of picrotoxin (100 µM) blocking GABAA receptors. Right: plots, obtained from analysis of 5 min recording showing a cumulative distribution of amplitudes (top) and inter event intervals (iei's) of spontaneous EPSCs during this period. <b>B</b>) Above: Averaged (black) and un-averaged (grey) evoked EPSCs. Paired-pulse interval is 50 msec. Below: Data from n = 12 tdTomato neurons showing paired-pulse ratio (PPR: evoke 2/evoke1). <b>C</b>) Left: Sample eEPSC traces recorded from an individual td+ cell at −80 mV (lower inward AMPAR-mediated current) and at +40 mV after addition of DNQX (10 µM; upper outward NMDAR current). Right: Ratio between inward AMPAR and outward NMDAR currents for n = 11 tdTomato neurons in the PVN. <b>D</b>) Left: spontaneous GABAAR-mediated inhibitory post-synaptic currents (IPSCs) recorded at −80 mV in a single td+ neuron with 10 µM DNQX. Right: cumulative distribution plots from this cell, of IPSC amplitudes and iei's. <b>E</b>) Above: evoked IPSCs with 50 msec interval. Averaged (black) and individual trials (grey) overlaid. Bottom: PPR data from n = 17 tdTomato neurons. <b>F</b>) Left: individual traces of evoked IPSCs (eIPSCs), in a td+ cell, recorded at varied holding potentials (−100 mV to −30 mV, 10 mV increment). Right: eIPSC current-voltage relationship showing eIPSC reversal potential in n = 6 td+ neurons. Data are represented by mean ± SEM. Scale bars 20 pA/50 msec in (<b>A,D</b>) and 50 pA/10 msec in (<b>B,C,E,F</b>).</p

Optogenetic activation of Crh-IRES-Cre PVN neurons by cre-dependent viral expression of channelrhodopsin.

<p><b>A</b>) DIC and epifluorescence images showing whole-cell recording from a YFP+ PVN neuron. <b>B</b>) example of a photocurrent recorded in voltage-clamp mode, elicited by 473 nm blue light exposure in the same YFP+ neuron. Light was delivered through a fibre optic cable end placed near the PVN <b>C</b>) examples of light-induced action potentials in a PVN neuron, and the variation of action potential waveform with varied light intensity. <b>D</b>) Trains of light pulses (2 msec pulse-width) at various frequencies and resulting action potentials. Scale bars are 500 pA/100 msec in (B), 10 mV/10 msec in (C), and 10 mV/500 msec in (D).</p

Induction of c-Fos protein in PVN tdTomato neurons following stress.

<p><b>A,B</b>) tdTomato expression in the PVN of naïve <i>Crh-IRES-Cre;Ai14</i> mice, and one exposed to a 15 min forced swim stress, 90 min prior to sacrifice. Confocal image at 15×. <b>C,D</b>) c-Fos immunoreactivity in these naïve or stress brain sections, showing an increased number of c-Fos containing nuclei following stress. <b>E,F</b>) Merged image showing high spatial colocalization of c-Fos and tdTomato in the stressed, but not naïve, mouse. <b>G</b>) Bar graph summarizing changes in the percentage of tdTomato neurons co-expressing c-Fos in naïve (n = 5) versus stressed (n = 6) mice. <b>H</b>) Higher magnification (100×), of c-Fos immunoreactivity in tdTomato cells from a stressed mouse. Data summarized in (H) are mean ± SEM. Scale bars are 200 µm (A–F) and 20 µm (H).</p