chicken anti mcherry  (AvesLabs)

Journal: bioRxiv

Article Title: Hypothalamic Orexin Input to the Medial Amygdala Links Vigilance to Arousal

doi: 10.64898/2026.03.09.710682

Figure Lengend Snippet: ( A ) Retrograde targeting strategy: Cre-dependent AAVrg-DIO-ChR2-mCherry was injected into the MeA of Orexin-Cre mice to label LHA Ox neurons projecting to the MeA, followed by implantation of optic fibers and EEG/EMG electrodes. ( B ) Representative images confirming viral expression in MeA and LHA; horizontal bars indicate individual fiber tip locations. Scale bars, 500 µm. ( C to D ) Optogenetic activation increased neuronal activity in the MeA, assessed by cFos immunostaining and quantification ( C , representative; D , summary). ( E ) Schematic of the isoflurane sedation arousal paradigm. ( F to H ) Under 0.75% isoflurane, 20 Hz stimulation of the LHA Ox →MeA pathway increased cortical activation and promoted behavioral arousal in ChR2 mice but not mCherry controls ( F , representative EEG/EMG and spectrogram; G , EEG power spectral density and band power analysis; H , arousal latency). ( I ) Schematic of the emergence paradigm and quantification of righting recovery: LHA Ox →MeA activation accelerated recovery of the righting reflex after deep anesthesia in ChR2 mice but not in controls. ( J ) Fiber photometry of orexin release in the MeA using the genetically encoded orexin sensor OxLight (OxL): OxL signal decreased during 2% isoflurane anesthesia and increased during emergence; AUC analysis summarizes state-dependent changes. ( K ) Strategy for simultaneous LHA Ox terminal stimulation and MeA orexin sensing (ChrimsonR in LHA; OxL in MeA) with representative expression images. ( L to M ) During isoflurane sedation, LHA Ox →MeA terminal stimulation increased MeA orexin release ( L ) and induced rapid arousal from light anesthesia ( M ). ( N to O ) During induction, pre-activation of LHA Ox →MeA terminals increased MeA orexin release ( N ) and delayed loss of the righting reflex ( O ). ( P to Q ) During emergence, terminal stimulation accelerated MeA orexin release ( P ) and shortened the time to recovery of the righting reflex ( Q ). Data are mean ± S.E.M.; statistical details and exact n values are provided in Supplementary Table 1. *p ≤ 0.05, **p ≤ 0.01, ****p < 0.0001.

Article Snippet: Primary and secondary antibodies (diluted in blocking solution) were as follows: mCherry: chicken anti-mCherry (1:400; Novus Biologicals, NBP2-25158); Cy3-conjugated goat anti-chicken IgY (1:400; Jackson ImmunoResearch, 103-165-155). cFos: rabbit anti-cFos (1:100; Cell Signaling Technology, 2250S); Alexa Fluor 488–conjugated donkey anti-rabbit IgG (1:400; Jackson ImmunoResearch, 711-546-152).

Techniques: Injection, Expressing, Activation Assay, Activity Assay, Immunostaining

Journal: bioRxiv

Article Title: Hypothalamic Orexin Input to the Medial Amygdala Links Vigilance to Arousal

doi: 10.64898/2026.03.09.710682

Figure Lengend Snippet: ( A ) Cre-dependent AAV5-FLEX-ChrimsonR-tdTomato was injected into the LHA of Orexin-Cre mice, and an optic fiber was implanted above the MeA. ( B ) Experimental timeline and schematic of the open field test (OFT). ( C to D ) Representative OFT trajectories ( C : mCherry, gray; ChrimsonR, red; laser-off and laser-on epochs; center zone outlined) and quantification of total distance traveled, percent time in the center, freezing time, and corner entropy ( D ). ( E ) Experimental timeline and schematic of real-time place preference (RTPP). ( F to G ) Representative RTPP heatmaps/trajectories ( F ) and quantification showing that LHA Ox →MeA terminal stimulation increased time spent and distance traveled in the laser-paired chamber in the ChrimsonR group but not in mCherry controls ( G ). ( H ) Experimental timeline and schematic of conditioned place preference (CPP). ( I to J ) Representative CPP heatmaps/trajectories from the same animal during baseline and test sessions ( I ) and quantification showing no change in time spent in the stimulation-paired chamber following conditioning ( J ). Data are mean ± S.E.M.; statistical details and exact n values are provided in Supplementary Table 1. *p ≤ 0.05, **p ≤ 0.01, ***p < 0.001.

Article Snippet: Primary and secondary antibodies (diluted in blocking solution) were as follows: mCherry: chicken anti-mCherry (1:400; Novus Biologicals, NBP2-25158); Cy3-conjugated goat anti-chicken IgY (1:400; Jackson ImmunoResearch, 103-165-155). cFos: rabbit anti-cFos (1:100; Cell Signaling Technology, 2250S); Alexa Fluor 488–conjugated donkey anti-rabbit IgG (1:400; Jackson ImmunoResearch, 711-546-152).

Techniques: Injection, Conditioned Place Preference

Journal: bioRxiv

Article Title: Hypothalamic Orexin Input to the Medial Amygdala Links Vigilance to Arousal

doi: 10.64898/2026.03.09.710682

Figure Lengend Snippet: ( A ) Strategy to activate MeA vGAT neurons (Flp-dependent ChrimsonR in MeA vGAT ) while monitoring prelimbic cortex (PrL) activity (AAV1-GCaMP6s in PrL) in vGAT-Flp mice; representative images confirm expression and fiber placement. ( B , D ) Fiber photometry showing PrL activity during MeA vGAT activation under isoflurane sedation ( B ) and 2% isoflurane anesthesia ( D ), with quantification. ( C ) MeA vGAT activation induces behavioral arousal from light anesthesia. ( E to F ) During induction, pre-activation of MeA vGAT neurons modulates PrL activity ( E ) and delays loss of the righting reflex (LoRR) ( F ). ( G to H ) During emergence, MeA vGAT activation increases PrL activity ( G ) and accelerates recovery of the righting reflex (RoRR) ( H ). ( I ) Strategy for behavioral assays: viral expression and optic fiber implantation above the MeA in vGAT-Flp mice. ( J to K ) Representative open-field trajectories ( J ) and quantification of exploration and defensive metrics ( K ). ( L to M ) Representative RTPP heatmaps/trajectories ( L ) and quantification showing increased time spent and distance traveled in the laser-paired chamber in ChrimsonR mice but not mCherry controls ( M ). ( N to O ) Representative CPP heatmaps/trajectories during baseline and test sessions ( N ) and quantification showing no change in time spent in the stimulation-paired chamber following conditioning ( O ). Data are mean ± S.E.M.; statistical details and exact n values are provided in Supplementary Table 1. *p ≤ 0.05, **p ≤ 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Primary and secondary antibodies (diluted in blocking solution) were as follows: mCherry: chicken anti-mCherry (1:400; Novus Biologicals, NBP2-25158); Cy3-conjugated goat anti-chicken IgY (1:400; Jackson ImmunoResearch, 103-165-155). cFos: rabbit anti-cFos (1:100; Cell Signaling Technology, 2250S); Alexa Fluor 488–conjugated donkey anti-rabbit IgG (1:400; Jackson ImmunoResearch, 711-546-152).

Techniques: Activity Assay, Expressing, Activation Assay

Journal: Cell reports

Article Title: RNA-programmable cell-type monitoring and manipulation in the human cortex with CellREADR

doi: 10.1016/j.celrep.2025.116037

Figure Lengend Snippet: Cell-type-specific optical manipulation of human neurons with CellREADR (A) CellREADR expression of an optogenetic effector, ChIEF, enables optogenetic control of AP firing in CALB2 - but not FOXP2 -targeted cells. CALB2- ChIEF neurons fired in response to 100 ms, 642 nm light pulses (left) or trains of 2 ms pulses (6/6 cells fired APs in response to both stimuli, with some failures in the train). Light stimuli depolarized FOXP2 -ChIEF but did not drive AP firing (0/10 cells fired in response to stimuli; gray traces, individual trials from a single cells; black traces, averages). Scale bars: 20 mV, 20 ms, 50 ms. (B) Representative targeting scheme for optical interrogation of CALB2 synaptic connectivity. Putative postsynaptic cells were labeled with a DLX2.0-YFP virus (top left, green). A binary CellREADR vector driving CALB2 -ChIEF-tdTomato was used to activate presynaptic cells (top right, red). During optical stimulation of the ChIEF-tdTomato + population, patch-clamp recordings were made from YFP + /ChIEF-tdTomato − cells; at the end of recordings, biocytin-filled patched cells were recovered (bottom right, purple, biocytin-streptavidin [SAV]). Scale bar: 50 μm. (C) Postsynaptic currents elicited by CALB2-ChIEF activation. Voltage-clamp recordings (average of 10 trials) made from a DLX2.0-YFP + /ChIEF-tdTomato − neurons during optical stimulation of the CALB2 -ChIEF population (left). Stimulation elicited optical postsynaptic currents (oPSCs) at the glutamate reversal potential (V rev(Glu) ) but not at the GABA reversal potential (V rev(GABA) ). Bicuculline was applied to verify that the observed oIPSCs were GABAergic. In DLX2.0-YFP + /ChIEF-tdTomato − cells, oPSCs were never observed at V rev(GABA) . Averaged traces were recorded from a hSyn-mCherry + pyramidal neuron during optical stimulation of the CALB2 -ChIEF population (right). In this example, oPSCs were detected at V rev(GABA) but not V rev(Glu) , indicating that the cell received glutamatergic inputs. These inputs were not blocked by bicuculline. No cell exhibited oPSCs at both V rev(GABA) and V rev(Glu) . Scale bars: 0.2 pA, 50 ms (left) and 2 pA, 50 ms (right). (D) Failure rate of optically triggered synaptic currents. To illustrate trial-to-trial variability in oPSC responses, 10 trials from a DLX2.0-YFP + /ChIEF-tdTomato − cell are shown at V rev(Glu). The first light pulse had a failure rate of 10% (1/10), while the fourth light pulse had a failure rate of 40% (4/10; averaged response, black). Scale bar: 2 pA, 50 ms. (E) Waveforms of optically evoked postsynaptic responses. Optical PSCs measured at V rev(Glu) from DLX2.0-YFP + /ChIEF-tdTomato − interneurons and hSyn-mCherry + pyramidal neurons were aligned to the light pulse and overlaid (left). All oPSCs were normalized to the peak current (right). The monophasic rise time and small jitter relative to the 2 ms light stimulus indicate that PSCs were monosynaptic events directly triggered by the optical pulse (averaged response, black). Scale bars: 2 pA, 10 ms (left) and 0.2 pA, 5 ms (right). (F) Quantification of postsynaptic responses to CALB2 -ChIEF activation. When recordings were made from DLX2.0-YFP + / CALB2 -ChIEF − interneurons, oIPSCs were observed in 6/16 cells (37.5%), and oEPSCs were not detected. Recordings were also made from hSyn-tdTomato + pyramidal neurons during CALB2 -ChIEF activation. oIPSCs were detected in 3/14 cells (21.4%), and oEPSCs were detected in 2/14 cells (14.3%).

Article Snippet: The following antibodies were used for immunohistochemical labeling of mNeon, mCherry, FoxP2, Calretinin and NeuN: mouse anti-mNeon Ab 1:500, Proteintech 32f6; chicken anti-mCherry Ab 1:500, Origene TA150127; rabbit anti-FoxP2 Ab 1:500, Abcam ab16046; rabbit anti-Calretinin Ab 1:500, Swant CR7697; mouse anti-NeuN Ab 1:500, Cell Signaling #94403S.

Techniques: Expressing, Control, Labeling, Virus, Plasmid Preparation, Patch Clamp, Activation Assay