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

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of PACAP and the PAC1 Receptor in the Basal Ganglia, Substantia Nigra and Centrally Projecting Edinger–Westphal Nucleus in the Rotenone model of Parkinson’s Disease

doi: 10.3390/ijms241411843

Figure Lengend Snippet: Adcyap1 and Adcyap1r1 mRNA expression, and PAC1R immunoreactivity in the caudate-putamen (CPu). Representative images show Adcyap1 mRNA transcripts (green in ( A – C ), white arrowheads), Adcyap1r1 mRNA signal puncta (white, ( E – G ), yellow arrowheads) and 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstaining (blue). Some cells are shown in higher magnification inserts within the same panels. Panels ( I – K ) show PAC1R-immunoreactive (ir) somata (red) in the CPu. Histogram ( D ) shows the number of Adcyap1 -expressing cells. Panel ( H ) illustrates the count of Adcyap1 transcripts per cell. Histogram ( L ) shows the number of PAC1R-ir cell counts. Histogram ( M ) shows the count of cells that contained Adcyap1r1. Panel ( N ) describes the magnitude of Adcyap1r1 expression. The count of Adcyap1 and Adcyap1r1 co-expressing cells is shown in histogram ( O ). The specific signal density (SSD) of PAC1R protein is shown in panel ( P ). Gray bars: oil- and saline-injected controls; red bars: rats treated with rotenone; pink bars: benserazide/levodopa (B/L)-treated parkinsonian rats. N = 6–8 * p < 0.05, ** p < 0.01, *** p < 0.001, according to Tukey’s post-hoc test. Bars: 50 µm.

Article Snippet: The specificity of PAC1R antibody (Cat. No.: AVR-003, RRID: AB_2756805, Alomone Laboratories) in the rat brain was tested by Western blot ( https://www.alomone.com/p/anti-pac1-receptor/AVR-003 accessed on 21 July 2023) and by preabsorption to the corresponding blocking peptide (Cat. No.: BLP-VR003, Alomone Laboratories).

Techniques: Expressing, Saline, Injection

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of PACAP and the PAC1 Receptor in the Basal Ganglia, Substantia Nigra and Centrally Projecting Edinger–Westphal Nucleus in the Rotenone model of Parkinson’s Disease

doi: 10.3390/ijms241411843

Figure Lengend Snippet: Adcyap1 and Adcyap1r1 mRNA expression in the globus pallidus (GP) and entopeduncular nucleus (EP). Representative images show Adcyap1 mRNA transcripts (green in ( A – C ), white arrowheads) and Adcyap1r1 mRNA signal puncta (white, ( E – G )) counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue) in the GP. Some cells were shown in higher magnification insets within the same panels. Histogram ( D ) shows the number of Adcyap1 -expressing cells in the GP. Panel ( H ) shows the count of cells that contained Adcyap1r1 in the GP. Histogram ( I ) illustrates the count of Adcyap1 transcripts per GP cell. Panel ( J ) describes the magnitude of Adcyap1r1 expression in the cells of GP. The count of PAC1R-immunoreactive (ir) cells in the GP is shown in ( K ). The specific signal density (SSD) of PAC1R protein in the GP is shown in panel ( L ). Histogram ( M ) shows the number of Adcyap1 -expressing cells in the EP. Histogram ( N ) illustrates the count of Adcyap1 transcripts per EP cell. Histogram ( O ) shows the number of Adcyap1r1 -expressing cells in the EP. The count of Adcyap1 and Adcyap1r1 co-expressing cells in the EP is shown in histogram ( P ). Panel ( Q ) shows the number of Adcyap1r1 transcripts in Adcyap1 -expressing EP cells. Histogram ( R ) indicates the number of Adcyap1r1 transcripts in Adcyap1 -negative EP cells. The count of PAC1R-ir cells in the EP is shown in ( S ). The SSD of PAC1R protein in the EP is shown in panel ( T ). Gray bars: oil- and saline-injected controls; red bars: rats treated with rotenone; pink bars: benserazide/levodopa (B/L)-treated parkinsonian rats. N = 6–8. * p < 0.05, ** p < 0.01, *** p < 0.001, t: trend ( p = 0.06), according to Tukey’s post-hoc test. Bars: 30 µm.

Article Snippet: The specificity of PAC1R antibody (Cat. No.: AVR-003, RRID: AB_2756805, Alomone Laboratories) in the rat brain was tested by Western blot ( https://www.alomone.com/p/anti-pac1-receptor/AVR-003 accessed on 21 July 2023) and by preabsorption to the corresponding blocking peptide (Cat. No.: BLP-VR003, Alomone Laboratories).

Techniques: Expressing, Saline, Injection

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of PACAP and the PAC1 Receptor in the Basal Ganglia, Substantia Nigra and Centrally Projecting Edinger–Westphal Nucleus in the Rotenone model of Parkinson’s Disease

doi: 10.3390/ijms241411843

Figure Lengend Snippet: Adcyap1 and Adcyap1r1 mRNA expression in the substantia nigra pars compacta (SNpc). Representative images show Adcyap1 mRNA transcripts (green in ( A – C ), white arrowheads) and Adcyap1r1 mRNA signal puncta (white, ( D – F )) in tyrosine hydroxylase (TH, red) immunoreactive (ir) and non-dopaminergic (white and yellow arrowheads) cells. Nuclear counterstaining: 4′,6-diamidino-2-phenylindole (DAPI, blue). Some cells are shown in higher magnification insets within the same panels. Histogram ( G ) shows the number of Adcyap1 -expressing TH-ir cells. Panel ( H ) shows the ratio of Adcyap1 -expressing TH-ir neurons. Histogram ( I ) illustrates the count of Adcyap1 transcripts in dopaminergic neurons. Panel ( J ) shows the number of dopaminergic cells that co-express Adcyap1 and Adcyap1r1. Graph ( K ) illustrates the count of TH-ir cells that contained Adcyap1r1 . Histogram ( L ) indicates the number of Adcyap1r1 transcripts in non-dopaminergic Adcyap1 -expressing cells. Panel ( M ) shows the count of Adcyap1 -expressing non-dopaminergic cells (see also the white arrowheads in ( A – C )). Histogram ( N ) shows the count of Adcyap1 and Adcyap1r1 co-expressing non-dopaminergic cells (see also yellow arrowheads in ( D – F )). Graph ( O ) shows the number of TH-immunonegative Adcyap1r1 -expressing cells. The number of Adcyap1r1 transcripts in non-dopaminergic SNpc cells is depicted in graph ( P ). Histogram ( Q ) illustrates the magnitude of Adcyap1r1 mRNA expression in non-dopaminergic cells of the SNpc. Gray bars: oil- and saline-injected controls; red bars: rats treated with rotenone; pink bars: benserazide/levodopa (B/L)-treated parkinsonian rats. N = 6–8. * p < 0.05, ** p < 0.01, *** p < 0.001, according to Tukey’s post-hoc test. Bars: 25 µm.

Article Snippet: The specificity of PAC1R antibody (Cat. No.: AVR-003, RRID: AB_2756805, Alomone Laboratories) in the rat brain was tested by Western blot ( https://www.alomone.com/p/anti-pac1-receptor/AVR-003 accessed on 21 July 2023) and by preabsorption to the corresponding blocking peptide (Cat. No.: BLP-VR003, Alomone Laboratories).

Techniques: Expressing, Saline, Injection

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of PACAP and the PAC1 Receptor in the Basal Ganglia, Substantia Nigra and Centrally Projecting Edinger–Westphal Nucleus in the Rotenone model of Parkinson’s Disease

doi: 10.3390/ijms241411843

Figure Lengend Snippet: PAC1R immunoreactivity in the substantia nigra pars compacta (SNpc). Representative images of tyrosine hydroxylase (TH, green, ( A – C )) and PAC1R (red, ( E – G )) double labeling and their overlay ( I – K ). Histogram ( D ) shows the count of TH immunoreactive (ir) cells that were also PAC1R immunopositive (see also white arrowheads in the images, and insets). Panel ( H ) illustrates the number of PAC1R-ir cells that were not dopaminergic (also pointed by blue arrowheads in the confocal photomicrographs and shown in insets). Graph ( L ) illustrates that most of the dopaminergic neurons were also positive for PAC1R. Yellow arrowheads in images indicate few TH-ir cells that were immunonegative for PAC1R. Graph ( M ) shows the proportion of dopaminergic neurons in the PAC1R-ir cell population if the SNpc. Histogram ( N ) illustrates the specific signal density (SSD) of PAC1R immunosignal in dopaminergic SNpc neurons. Panel ( O ) describes the SSD of PAC1R immunosignal in non-dopaminergic cells of the SNpc. Gray bars: oil- and saline-injected controls; red bars: rats treated with rotenone; pink bars: benserazide/levodopa (B/L)-treated parkinsonian rats. N = 6–8. * p < 0.05, ** p < 0.01, according to Tukey’s post-hoc test. Bars: 50 µm.

Article Snippet: The specificity of PAC1R antibody (Cat. No.: AVR-003, RRID: AB_2756805, Alomone Laboratories) in the rat brain was tested by Western blot ( https://www.alomone.com/p/anti-pac1-receptor/AVR-003 accessed on 21 July 2023) and by preabsorption to the corresponding blocking peptide (Cat. No.: BLP-VR003, Alomone Laboratories).

Techniques: Labeling, Saline, Injection

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of PACAP and the PAC1 Receptor in the Basal Ganglia, Substantia Nigra and Centrally Projecting Edinger–Westphal Nucleus in the Rotenone model of Parkinson’s Disease

doi: 10.3390/ijms241411843

Figure Lengend Snippet: Adcyap1 and Adcyap1r1 mRNA expression in the centrally projecting Edinger–Westphal nucleus (EWcp). Representative images show Adcyap1 mRNA transcripts (green in ( A – C ), white arrowheads) and Adcyap1r1 mRNA signal puncta (white, ( D – F )) in sections counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Some cells were shown in higher magnification insets within the same panels. Histogram ( G ) shows the number of peptidergic cells characterized by larger nuclei and pale, euchromatic karyoplasm (examples pointed out by arrowheads). Histogram H depicts the number of Adcyap1 -expressing peptidergic cells. Panel ( H ) illustrates the count of Adcyap1 transcripts in peptidergic neurons. Histogram ( I ) indicates the number of Adcyap1 mRNA transcripts in EWcp neurons. Graph ( J ) shows the count of peptidergic cells that contained Adcyap1r1 transcripts. Histogram ( K ) depicts the count of Adcyap1r1 transcripts in peptidergic EWcp cells. Bars in Figure ( L ) show the count of Adcyap1 and Adcyap1r1 co-expressing peptidergic EWcp cells. The number of PAC1R immunoreactive (ir) EWcp cells is shown in panel ( M ). The specific signal density (SSD) of PAC1R signal is shown in ( N ). Gray bars: oil- and saline-injected controls; red bars: rats treated with rotenone; pink bars: benserazide/levodopa (B/L)-treated parkinsonian rats. N = 6–8. * p < 0.05, ** p < 0.01, *** p < 0.001, according to Tukey’s post-hoc test. Bars: 20 µm.

Article Snippet: The specificity of PAC1R antibody (Cat. No.: AVR-003, RRID: AB_2756805, Alomone Laboratories) in the rat brain was tested by Western blot ( https://www.alomone.com/p/anti-pac1-receptor/AVR-003 accessed on 21 July 2023) and by preabsorption to the corresponding blocking peptide (Cat. No.: BLP-VR003, Alomone Laboratories).

Techniques: Expressing, Saline, Injection

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of PACAP and the PAC1 Receptor in the Basal Ganglia, Substantia Nigra and Centrally Projecting Edinger–Westphal Nucleus in the Rotenone model of Parkinson’s Disease

doi: 10.3390/ijms241411843

Figure Lengend Snippet: Adcyap1 and Adcyap1r1 mRNA expression in the insular cortex (CTX). Representative images show Adcyap1 mRNA transcripts (green in ( A – C ), white arrowheads) and Adcyap1r1 mRNA signal puncta (white, ( E – G )) in sections counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Some cells were shown in higher magnification insets within the same panels. Histogram ( D ) shows the number of cortical cells that contained Adcyap1 transcripts. The magnitude of Adcyap1 mRNA specific signal density (SSD) is shown in ( H ). Panel ( I ) describes the count of Adcyap1 and Adcyap1r1 co-expressing cortical cells. Graph ( J ) shows the count of Adcyap1r1 -expressing cells, while ( K ) illustrates the SSD of Adcyap1r1 in these neurons. The count of PAC1R-immunoreactive (ir) cells is shown in ( L ), and the SSD of PAC1R immunoreactivity is shown in ( M ). Gray bars: oil- and saline-injected controls; red bars: rats treated with rotenone; pink bars: benserazide/levodopa (B/L)-treated parkinsonian rats. N = 6–8. * p < 0.05, according to Tukey’s post-hoc test. Bars: 20 µm.

Article Snippet: The specificity of PAC1R antibody (Cat. No.: AVR-003, RRID: AB_2756805, Alomone Laboratories) in the rat brain was tested by Western blot ( https://www.alomone.com/p/anti-pac1-receptor/AVR-003 accessed on 21 July 2023) and by preabsorption to the corresponding blocking peptide (Cat. No.: BLP-VR003, Alomone Laboratories).

Techniques: Expressing, Saline, Injection