Journal: Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

Article Title: Pituitary adenylate cyclase-activating polypeptide (PACAP) modulates dependence-induced alcohol drinking and anxiety-like behavior in male rats.

doi: 10.1038/s41386-020-00904-4

Figure Lengend Snippet: Fig. 2 Effect of chronic intermittent exposure to ethanol vapors on PAC1R-positive cell bodies in the BNST 8–10 h after vapor offset. No difference was found between groups (A). Drawing of a coronal rat brain slice representing the area analyzed (B). Representative pictures of PACAP immunohistochemistry in control, nondependent and in ethanol-dependent rats (C–D). STMA BNST medial-anterior, STLP BNST lateral- posterior, STLD BNST lateral-dorsal, STLJ BNST lateral-justacapsular. Scale bars represent 250 μm. Control and ethanol-dependent rats are depicted in orange and green, respectively. Data represent Mean ± SEM (n = 9–10/group).

Article Snippet: Sections were then incubated in either an anti-PACAP (1:4,000, T-4473, Bachem, Torrance, CA; RRID: AB_519166) [26, 54–61] or an anti-PAC1R primary antibody (1:250, sc-30018, Santa Cruz Biotechnology, Dallas, TX; RRID: AB_2305215) [62–64] in blocking solution for 24 h at 4 °C.

Techniques: Slice Preparation, Immunohistochemistry, Control

Journal: PLoS ONE

Article Title: Maxadilan Prevents Apoptosis in iPS Cells and Shows No Effects on the Pluripotent State or Karyotype

doi: 10.1371/journal.pone.0033953

Figure Lengend Snippet: List of primers.

Article Snippet: The membrane was incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-PAC1 antibody (1∶2000, Santa Cruz), rabbit polyclonal anti-OCT4 antibody (1∶1000, Cell Signaling), rabbit polyclonal anti-Nanog antibody (1∶1000, Cell Signaling), rabbit monoclonal anti-SOX2 antibody (1∶1000, Cell Signaling) and rabbit monoclonal anti-β-actin antibody (1∶3000, Cell Signaling).

Techniques:

Journal: PLoS ONE

Article Title: Maxadilan Prevents Apoptosis in iPS Cells and Shows No Effects on the Pluripotent State or Karyotype

doi: 10.1371/journal.pone.0033953

Figure Lengend Snippet: The expression of PAC1 mRNA in iPS cells by RT-PCR analysis (a). The expression of PAC1 protein in iPS cells by western blot analysis showed that there are three kinds of PAC1 isoforms (b). The molecular weights of these PAC1 isoforms (1, 2 and 3) vary from about 50 kDa to 80 kDa.

Article Snippet: The membrane was incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-PAC1 antibody (1∶2000, Santa Cruz), rabbit polyclonal anti-OCT4 antibody (1∶1000, Cell Signaling), rabbit polyclonal anti-Nanog antibody (1∶1000, Cell Signaling), rabbit monoclonal anti-SOX2 antibody (1∶1000, Cell Signaling) and rabbit monoclonal anti-β-actin antibody (1∶3000, Cell Signaling).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Maxadilan Prevents Apoptosis in iPS Cells and Shows No Effects on the Pluripotent State or Karyotype

doi: 10.1371/journal.pone.0033953

Figure Lengend Snippet: Immunofluorescence examining the protein expression of Nanog, OCT4, SOX2, SSEA-4 and TRA-1-60(s) in iPS cells of both the control group (a) and in iPS cells that had been treated with 100 nM maxadilan (b). iPS cells that were not treated with maxadilan served as the control. iPS cells treated with rabbit polyclonal anti-AQP1 antibody served as a negative control. Mouse anti-rabbit IgG secondary antibody, goat anti-mouse IgG secondary antibody and goat anti-mouse IgM secondary antibody were respectively used as secondary antibodies in this experiment. All scale bars are 100 µm.

Article Snippet: The membrane was incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-PAC1 antibody (1∶2000, Santa Cruz), rabbit polyclonal anti-OCT4 antibody (1∶1000, Cell Signaling), rabbit polyclonal anti-Nanog antibody (1∶1000, Cell Signaling), rabbit monoclonal anti-SOX2 antibody (1∶1000, Cell Signaling) and rabbit monoclonal anti-β-actin antibody (1∶3000, Cell Signaling).

Techniques: Immunofluorescence, Expressing, Control, Negative Control

Journal: Autism research : official journal of the International Society for Autism Research

Article Title: PAC1R genotype to phenotype correlations in Autism Spectrum Disorder

doi: 10.1002/aur.2051

Figure Lengend Snippet: Gene-level brain expression graphs were generated with data from the BrainSpan project (Kang et al., 2011) collected from post-mortem human brain samples. The x-axis shows periods of development with the dark vertical line signifying birth (developmental time points are defined in Table 1). Along the y-axis are values of median expression (Log2-Transformed Signal Intensity) for which values ≥ 6 signify high levels of expression in at least one post-mortem brain sample. A. Expression of PAC1R is observed in multiple brain regions starting at stage 1 of fetal development. B. Differential expression of PAC1R is observed between males and females in multiple brain regions with median values above 0.0 denoting higher expression in males and values below 0.0 denoting higher expression in females. C. Differential expression of PAC1R between males and females only in the amygdala. Abbreviations: AMY, amygdala; CBC, cerebellar cortex; HIP, hippocampus; MD, mediodorsal nucleus of the thalamus; NCX, neocortex; STR, striatum.

Article Snippet: PAC1R Interactome To investigate potential interactors of PAC1R protein with other proteins and a potential functional relationship with known ASD susceptibility genes, we generated a protein interactome utilizing Genomatix ( ).

Techniques: Expressing, Generated, Transformation Assay, Quantitative Proteomics

Journal: Autism research : official journal of the International Society for Autism Research

Article Title: PAC1R genotype to phenotype correlations in Autism Spectrum Disorder

doi: 10.1002/aur.2051

Figure Lengend Snippet: ISH analyses at E13.5 shows Pac1r mRNA expression in known amygdala progenitor zones (A, boxed regions). Higher magnification image shows numerous Pac1r+ cells (brown) in the emerging amygdala (B) and the VP (C, arrows), a source of progenitors destined to give rise to excitatory output neurons in the BLA. Pac1r+ cells (arrow) are also observed in the POA (D), a source of progenitors destined to give rise to inhibitory output neurons in the MeA. Abbreviations: AMY, Amygdala; BLA, Basolateral Amygdala; CeA, Central Amygdala; CTX, Cortex; HIP, Hippocampus; MeA, Medial Amygdala; LGE, Lateral Ganglionic Eminence; MGE, Medial Ganglionic Eminence; POA, Pre-Optic Area; VP, Ventral Pallium.

Article Snippet: PAC1R Interactome To investigate potential interactors of PAC1R protein with other proteins and a potential functional relationship with known ASD susceptibility genes, we generated a protein interactome utilizing Genomatix ( ).

Techniques: Expressing

Journal: Autism research : official journal of the International Society for Autism Research

Article Title: PAC1R genotype to phenotype correlations in Autism Spectrum Disorder

doi: 10.1002/aur.2051

Figure Lengend Snippet: ISH analyses at E18.5 shows high levels of Pac1r mRNA in developing amygdala nuclei (A, B). Higher magnification of the MeA (C) and BLA (D) show numerous Pac1r+ cells (arrows). (E) At post-natal day 7 many Pac1r+ cells are observed in multiple amygdala sub-nuclei. Abbreviations: BLA, basolateral amygdala; CeA, central nucleus of the amygdala; MeA, medial amygdala; HYP, hypothalamus; Pir, piriform cortex

Article Snippet: PAC1R Interactome To investigate potential interactors of PAC1R protein with other proteins and a potential functional relationship with known ASD susceptibility genes, we generated a protein interactome utilizing Genomatix ( ).

Techniques:

Journal: Autism research : official journal of the International Society for Autism Research

Article Title: PAC1R genotype to phenotype correlations in Autism Spectrum Disorder

doi: 10.1002/aur.2051

Figure Lengend Snippet: PAC1R is at a hub of different signaling cascades involved in stress, metabolism, and inflammatory responses, among others. Plot and legend adapted from Genomatix (v.s. 3.9).

Article Snippet: PAC1R Interactome To investigate potential interactors of PAC1R protein with other proteins and a potential functional relationship with known ASD susceptibility genes, we generated a protein interactome utilizing Genomatix ( ).

Techniques:

Journal: Autism research : official journal of the International Society for Autism Research

Article Title: PAC1R genotype to phenotype correlations in Autism Spectrum Disorder

doi: 10.1002/aur.2051

Figure Lengend Snippet: ADI behavioral scores by PAC1R SNP allele status. A significant correlation exists between Reciprocal Social scores and allele status. Bars show standard error, n = 129, p < 0.05.

Article Snippet: PAC1R Interactome To investigate potential interactors of PAC1R protein with other proteins and a potential functional relationship with known ASD susceptibility genes, we generated a protein interactome utilizing Genomatix ( ).

Techniques:

Journal: Addiction Biology

Article Title: PACAP Signalling Network in the Nucleus Accumbens Core Regulates Reinstatement Behaviour in Rat

doi: 10.1111/adb.70090

Figure Lengend Snippet: PACAP signalling in the NAcc. (A) Western blots of NAcc punch lysates confirming the presence of PACAP receptor (PAC1R), with specificity validated using a synthetic blocking peptide. (B) Quantification of endogenous PACAP levels in the NAcc extracellular lysate by microdialysis (176.70 ± 53.27 pM, mean ± SEM). (C) Diagram illustrating retrograde tracing, highlighting the neural pathway from the prefrontal cortex (PFC) to the NAcc. (D) Confocal image (10×) of the NAc injection site showing the spread of fluorescently conjugated cholera toxin subunit B (CTb 594) labelling (magenta) and cell nuclei stained with DAPI (blue), with the anterior commissure (aca) as an anatomical landmark. (E) Brain section highlighting prefrontal cortical regions with detailed anatomical labelling, including motor areas (M1 and M2), orbital cortices (MO, VO and LO) and agranular insular regions (AIV and AID). The inset (40 × composite) shows PACAP mRNA expression (green), CTb‐labelled projection neurons (magenta), nuclear DAPI staining (blue) and a merged image highlighting colocalization (yellow arrows). Abbreviations: OB (olfactory bulb), Cm (cerebellum), PrL (prelimbic cortex), Cg1 (cingulate cortex area 1), Fr3 (frontal cortex, Area 3).

Article Snippet: This is the first demonstration of PAC1R protein in the NAcc of Sprague–Dawley rats, which extends existing observations documenting the presence of PAC1R protein in the NAcc of Wistar rats [ , ].

Techniques: Western Blot, Blocking Assay, Retrograde Tracing, Injection, Staining, Expressing

Journal: bioRxiv

Article Title: Disruption of Pre-Bötzinger Complex neuropeptidergic tonality controls fear and metabolic response

doi: 10.64898/2026.01.01.697304

Figure Lengend Snippet: (A) Experimental design for assessing stress-induced activation of PAC1R neurons in the preBötC using the SEFL paradigm; Representative images of brain before and after tissue clearing, with corresponding 3D reconstructions of cFos immunolabeling in No SEFL and SEFL groups. B) Representative RNAscope FISH multiplexed with IHC images showing PAC1R mRNA (red), cFos protein (green) and DAPI (blue) expression in the preBötC of No-SEFL and SEFL mice. C) Quantification of cFos+ neurons in the preBötC in No-SEFL mice compared to SEFL mice (n=5–6; unpaired t-test, t=4.345, p=0.002); Correlation analysis showing a positive relationship between the number of cFos+ neurons in the preBötC and percent freezing behavior in SEFL mice (n=5–6; r² = 0.68, p=0.002). D) Pie chart illustrating the proportions of cFos+ and PAC1R+ neurons, and cFos+ and PAC1R neurons. E) Schematic of retrograde tracing strategy using CTB-Alexa 647 (CTB-A647) in ADCYAP1-GFP mice. F) Representative image of viral labeling of preBötC neurons by CTB-A647 in ADCYAP1-GFP mice. G) Representative image of the section containing a-NTS expressing PACAP (green) and CTB-A647 (red). H) Representative image of a coronal brain section containing the preBötC showing RNAscope labeling of PAC1R mRNA. I–J) Dual labeling of PAC1R mRNA with GAD2 (I) or Vglut2 (J) using RNAscope multiplexed with IHC. K) Quantification of PAC1R+/GAD2+ versus PAC1R+/Vglut2+ neurons in the preBötC (n=4, unpaired t-test, t=14.33, p<0.0001).

Article Snippet: A total of approximately 60 ten-week-old PAC1R floxed mice, and 10 ten-week-old ADCYAP-GFP mice, both males and females, were obtained from Jackson Laboratories.

Techniques: Activation Assay, Immunolabeling, RNAscope, Expressing, Retrograde Tracing, Labeling

Journal: bioRxiv

Article Title: Disruption of Pre-Bötzinger Complex neuropeptidergic tonality controls fear and metabolic response

doi: 10.64898/2026.01.01.697304

Figure Lengend Snippet: A) Experimental timeline of the loss-of-function study assessing fear behavior, anxiety-like phenotype, cardiorespiratory function, and metabolic adaptations. B) Representative images of PAC1R mRNA expression in mice receiving rAAV2-hSyn-GFP-Cre or rAAV2-hSyn-GFP injections (Ctrl) into the preBötC. C) Quantification of mean fluorescence intensity (MFI) of PAC1R mRNA in both Cre and Ctrl groups (n=4; unpaired t-test, t=5.216, p= 0.0022). D) Percent freezing measured on Day 2 of SEFL in male and female mice (n=12-20, two-way ANOVA, F(3,51)=17.46, p<0.0001). E) Shock reactivity assessed by measuring velocity in both male and female mice to evaluate their immediate response to the footshock stimulus on day 2 SEFL (n=6-10; two-way ANOVA, F(3,52)=0.6125, p=0.604). F) Percent freezing on Day 3 of SEFL in male and female mice (n=12-20, two-way ANOVA, F(3,52)=29, p<0.0001). G) Fear acquisition in the training context (Context A) was not affected by deletion (n=5; unpaired t-test, t=0.086, p=0.933). H) Example trace of deviation in heart rate (ΔHR) during 8s freezing bouts in Ctrl-SEFL and Cre-SEFL animals. I) Average ΔHR during the entire duration of freezing on Day 3 of the SEFL paradigm (n=12-13, one-way ANOVA, F(3,40)= 4.593, p=0.07). J) Example trace of breathing rate per minute (BRPM) measured in anesthetized Ctrl-SEFL and Cre-SEFL mice for 5 minutes. K) Average BRPM during 5 minutes of recording in Ctrl-SEFL and Cre-SEFL groups (n=6–8, unpaired t-test, t=2.625, p=0.02).

Article Snippet: A total of approximately 60 ten-week-old PAC1R floxed mice, and 10 ten-week-old ADCYAP-GFP mice, both males and females, were obtained from Jackson Laboratories.

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Disruption of Pre-Bötzinger Complex neuropeptidergic tonality controls fear and metabolic response

doi: 10.64898/2026.01.01.697304

Figure Lengend Snippet: (A) A) Schematic of the retrograde tracing strategy. B) Representative images showing RFP expression in the preBötC following retrograde labeling of PRV-RFP in the BAT. C) Representative images showing GFP expression in the preBötC following retrograde labeling of PRV-GFP in the larger lobe of the liver. D) Representative images showing GFP expression in the preBötC following retrograde labeling of PRV-GFP in the iWAT. E and F) Representative RNAscope FISH multiplexed with IHC images showing PAC1r mRNA (red) and PRV-GFP (green) expression in the preBötC retrograde tracing from BAT (E) and liver (F). G) Heatmap showing downregulated genes in Cre-SEFL compared to Ctrl-SEFL from BAT RNA-seq analysis. H) Downregulated pathway and gene network analysis from Cre-SELF BAT RNA-seq data. I) Representative images of TH staining in the BAT from Ctrl-SEFL and Cre-SEFL mice. G) Quantification of TH signal intensity normalized to tissue volume in Ctrl-SEFL and Cre-SEFL group (n=2, unpaired t-test, t=19.92, p<0.0001). K and L) Energy expenditure and oxygen consumption metabolic chamber data from indicated mouse groups with 60 hours of measurement (n=7-10, one-way ANOVA, p<0.05). M and N) Heatmap of DEGs from liver RNA-seq with rectangle highlighting genes that are upregulated in Cre-SEFL. O) Pathway analysis and transcription factor gene network that are upregulated in Cre-SEFL compared to Ctrl-SEFL. P) Blood glucose levels during GTT in Ctrl-SEFL and Cre-SEFL mice at multiple time points following glucose injection (n=7-9, two-way ANOVA, F(1,84)=5.86, p=0.017); Area under the curve (AUC) quantification of glucose response (n=7-9, unpaired t-test, t=2.235, p=0.0422) .

Article Snippet: A total of approximately 60 ten-week-old PAC1R floxed mice, and 10 ten-week-old ADCYAP-GFP mice, both males and females, were obtained from Jackson Laboratories.

Techniques: Retrograde Tracing, Expressing, Labeling, RNAscope, RNA Sequencing, Staining, Injection