Journal: Translational Psychiatry

Article Title: The ventral hippocampus to paraventricular thalamus circuit regulates context-dependent hyperlocomotion through PAC1 receptor signaling in the chronic stress-induced PTSD mouse model

doi: 10.1038/s41398-026-03963-1

Figure Lengend Snippet: (A) The immunofluorescent staining showed that CSDS treatment induced obvious increases in the expression of PAC1 receptor within PVT neurons (upper panel, amplification: 4 x; lower panel, amplification: 60 x). (B) The western blot revealed the apparent increases in the PAC1 receptor within PVT region and (C) the statistical analysis ( n = 5 mice for each group; Mann-Whitney unpaired two-tailed U test; U = 0, ** p = 0.0079). (D) The experimental strategy of cannula injection of PAC1 receptor antagonist and behavioral tests. (E) The context-dependent locomotion activity (One-way ANOVA with Tukey post-hoc test, F(2, 21.47) = 9.201, ** p = 0.0013; Ctrl vs. CSDS, * p = 0.0429; CSDS vs. CSDS + PA-915, *** p = 0.0006). (F) The time spent in open arm (One-way ANOVA with Tukey post-hoc test, F(2, 24.69) = 9.033, ** p = 0.0011; Ctrl vs. CSDS, ** p = 0.0041; CSDS vs. CSDS + PA-915, * p = 0.0163) and (G) number of open arm entries (One-way ANOVA with Tukey post-hoc test, F(2, 26.45) = 6.594, ** p = 0.0047; Ctrl vs. CSDS, * p = 0.0107; CSDS vs. CSDS + PA-915, * p = 0.0193) in the elevated plus maze test. (H) The sociability in the three-chamber test (One-way ANOVA with Tukey post-hoc test, F(2, 17.81) = 6.317, ** p = 0.0084; Ctrl vs. CSDS, * p = 0.0189; CSDS vs. CSDS + PA-915, * p = 0.0173) and (I) the innate motivation in the female encounter test (One-way ANOVA with Tukey post-hoc test, F(2, 22.87) = 7.469, ** p = 0.0032; Ctrl vs. CSDS, * p = 0.0122; CSDS vs. CSDS + PA-915, * p = 0.0102). (J) The arousal levels (One-way ANOVA with Tukey post-hoc test, F(2, 24.94) = 5.412, * p = 0.0112; Ctrl vs. CSDS, * p = 0.044; CSDS vs. CSDS + PA-915, * p = 0.0316). Data was represented as mean ± SD from three independent experiments. n = 10 mice per group per experiment.

Article Snippet: The diluted primary antibodies against PAC1R (CST, 1:1000) and GADPH (CST, 1:3000) were introduced, and the mixture was incubated for 12 h at 4 °C.

Techniques: Staining, Expressing, Amplification, Western Blot, MANN-WHITNEY, Two Tailed Test, Injection, Activity Assay

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

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: 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: Antibody specificity was confirmed by reprobing with and without a synthetic blocking PAC1R peptide (1:600, #BLP‐VR003, Alomone Labs, Jerusalem, Israel).

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