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

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

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

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

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

YFP expression in A11 neurons following Cre-dependent viral expression and retrograde tracing with FG from the spinal cord.

<p>Rostral region section of A11 from a TH-Cre mouse transduced with a Cre-dependent YFP AAV (green) and immunohistochemistry against FG (magenta) (A–D). Boxed inset in (B (b)) shows FG labeled cells in the motor cortex. Only cells in A11 were co-labeled with YFP and FG (C, D). Scale bars: 50 µm. D: Higher magnification of boxed area in A. Scale bar: 20 µm.</p

A11 TH positive neurons project to the spinal cord.

<p>Example of retrograde labelling in the A11 caudal area following FluoroGold (FG) injections into the lumbar spinal cord between lumbar vertebral segments L4 and L5. Representative double-fluorescent immunostaining of tyrosine hydroxylase (TH; green) and FluoroGold (FG; magenta). The white arrows point to double-labeled cells. Most of the TH positive cells also contain FG (arrows), one cell shows labelling for TH but not FG. Several cells that contain FG but not TH are also seen (not co-localizing). Scale bar: 50 µm.</p

A11 TH positive neurons are also expressing aromatic amino acid decarboxylase (AADC).

<p>Immunohistochemistry targeted against TH (green) and AADC (magenta) shown in a representative middle region section of A11 (A, B) and the locus coeruleus (C). Note the co-localization between TH and AADC positive neurons (arrows) in A11 (B). Locus coeruleus served as a positive control (C). Scale bar 50 µm. A and B are derived from data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109636#pone-0109636-g001" target="_blank">Figure 1</a>. B: Higher magnification of boxed area in A.</p

Characterization of tyrosine hydroxylase (TH) positive cells in A11.

<p>A: Diagram showing the middle region of the A11 area in the mouse. The red frame represents the area where representative micrographs were taken. The diagram was adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109636#pone.0109636-Paxinos1" target="_blank">[24]</a> with permission. B: TH immunohistochemistry (green) of the middle region of A11. Scale bar 100 µm. 3 V = third ventricle, PF = parafascicular thalamic nucleus, fr = fasciculus retroflexus, PH = posterior hypothalamic nucleus, mt = mammillothalamic tract. C: Diameter of TH positive cells in three regions - rostral (AP −2.0 mm), middle (AP −2.3 mm) and caudal region (AP −2.5 mm). The mean cell diameter was 16.7±0.3 µm and the cell diameters between sections were similar.</p