Developmental and Adult Striatal Patterning of Nociceptin Ligand Marks Striosomal Population With Direct Dopamine Projections

Abstract

Circuit influences on the midbrain dopamine system are crucial to adaptive behavior and cognition. Recent developments in the study of neuropeptide systems have enabled high-resolution investigations of the intersection of neuromodulatory signals with basal ganglia circuitry, identifying the nociceptin/orphanin FQ (N/OFQ) endogenous opioid peptide system as a prospective regulator of striatal dopamine signaling. Using a prepronociceptin-Cre reporter mouse line, we characterized highly selective striosomal patterning of Pnoc mRNA expression in mouse dorsal striatum, reflecting the early developmental expression of Pnoc. In the ventral striatum, Pnoc expression in the nucleus accumbens core was grouped in clusters akin to the distribution found in striosomes. We found that Pnoc^tdTomato reporter cells largely comprise a population of dopamine receptor D1 (Drd1) expressing medium spiny projection neurons localized in dorsal striosomes, known to be unique among striatal projection neurons for their direct innervation of midbrain dopamine neurons. These findings provide a new understanding of the intersection of the N/OFQ system among basal ganglia circuits with particular implications for developmental regulation or wiring of striato-nigral circuits.

Keywords: dendron bouquet; dopamine; nociceptin; opioid; orphanin F/Q; striatum; striosome.

FIGURE 1

Pnoc^tdTomato reporter expression shows compartmental distribution across the striatum and corresponds with striosomes in the dorsal striatum. (a) Composite image showing Pnoc^tdTomato (magenta) and striosomal marker MOR1 (cyan) in the striatum. (b–d) Higher magnification images of the top boxed region from panel (a) with Pnoc^tdTomato (b, magenta) and MOR1 (c, cyan) shown separately. Merged image (d) shows strong overlap between Pnoc^tdTomato neurons and MOR1 expression. (e–g) same as (b–d) but for the lower boxed region in the ventral striatum from panel (a). (h) Quantification of Pnoc^tdTomato spatial distribution across the dorsal and ventral (nucleus accumbens core, medial shell, and lateral shell) striatum (bar graph, one‐way ANOVA with Bonferroni's multiple comparisons test, ****p < 0.0001, N = 12 mice, N = 19 sections, 1–2 sections/mouse. Data represented as mean ± SEM across mice). (i) Quantification of Pnoc^tdTomato spatial localization in striosomes vs. the matrix across the anterior to posterior axis in the dorsal striatum (two‐way ANOVA shows main effect of striatal compartment ***p < 0.001, no significant effect of anterior‐posterior axis p = 0.0851 bar graph, two‐way ANOVA with Bonferroni's multiple comparisons test, ***p < 0.001, ****p < 0.0001, N = 8 mice, 3 sections/mouse). Data represented as mean ± SEM across mice. ANT = anterior; DS = dorsal striatum; lNAcSh = lateral accumbens shell; MID = medial; mNAcSh = medial accumbens shell; NAc = nucleus accumbens; NAcC = nucleus accumbens core; POS = posterior.

FIGURE 2

Pnoc^tdTomato expressing striatal neurons cluster with Drd1 SPNs to near exclusion of Drd2. (a) Sagittal image of Pnoc^tdTomato cells (magenta) colocalizing with D1‐GFP‐expressing SPNs (green) in dorsal (b–e) and ventral (f–i) striatum. (b) Higher magnification image of top boxed region from panel (a) in the dorsal striatum. (c–e) Higher magnification images from inset in (b) of dorsal striatum. Arrowheads point to cells expressing markers for both Pnoc^tdTomato and D1‐GFP. (f) Higher magnification image of the bottom boxed region from panel (a) in the ventral striatum (nucleus accumbens). (g–i) Higher magnification images from inset in (f) of the ventral striatum. (j) Sagittal image of minimal overlap between Pnoc^tdTomato cells (magenta) and D2‐GFP expression (green) in dorsal (k–n) and ventral (o–r) striatum (same as [b–e] and [f–i] but for D2‐GFP expression). (s) Summary of IHC‐stained Pnoc^tdTomato reporter cell colocalization with in situ hybridization‐labeled Drd1 and Drd2 SPNs in dorsal striatum. (t) Quantification of Pnoc^tdTomato cells on Drd1 and Drd2 labeled SPNs across the anterior to posterior axis in the dorsal striatum (main effect of neuron type, two‐way ANOVA, ****p < 0.0001; bar graph, two‐way ANOVA with Bonferroni's multiple comparisons test, ****p < 0.0001, N = 6 mice, 3 brain sections/mouse). (u) Same as (s) for the nucleus accumbens. (v) Quantification in nucleus accumbens of Pnoc^tdTomato labeling in Drd1 and Drd2 SPNs (bar graph, one‐way ANOVA with Bonferroni's multiple comparisons test, ***p < 0.001, *p < 0.05, N = 6 mice, N = 8 sections, 1–2 sections/mouse). Data represented as mean ± SEM across mice. ANT = anterior; DS = dorsal striatum; MID = medial; NAc = nucleus accumbens; POS = posterior.

FIGURE 3

Pnoc^tdTomato reporter expression marks a population of NPY and SST co‐expressing interneurons. (a) Coronal image of Pnoc^tdTomato cells (magenta), somatostatin (white, SST), and neuropeptide Y (cyan, NPY) expression in the striatum. Higher magnification images of inset from panel (a) for (b) merged and (c–e) individual channels showing Pnoc^tdTomato (c) colocalization with a subset of striatal interneurons co‐expressing SST (d) and NPY (e). (f–j) Same organization as (a–e) for additional interneuron markers; Pnoc^tdTomato cells (magenta) show little colocalization with choline acetyltransferase (green, ChAT) or parvalbumin (blue, PV) expressing striatal interneurons. (k) Top: Summary of quantified Pnoc^tdTomato cells colocalized with different interneuron markers in the dorsal striatum. Bottom: Quantification of Pnoc^tdTomato cells colocalized with different interneuron markers across the anterior to posterior axis in the dorsal striatum (main effect of neuron type, two‐way ANOVA, ****p < 0.0001; bar graph, two‐way ANOVA with Bonferroni's multiple comparisons test, ****p < 0.0001, N = 6 mice, 3 sections/mouse). (l) Same as (k) but for nucleus accumbens (bar graph, one‐way ANOVA with Bonferroni's multiple comparisons test, ****p < 0.0001, **p < 0.01, *p < 0.05, N = 6 mice, N = 11 sections, 1–2 sections/mouse). Data represented as mean ± SEM across mice. ANT = anterior; lNAcSh = lateral accumbens shell; MID = medial; mNAcSh = medial accumbens shell; NAcC = nucleus accumbens core; POS = posterior.

FIGURE 4

Adult expression of striatal Pnoc mRNA is sparse relative to reporter‐labeled Pnoc^tdTomato cells. (a–d) Coronal image showing composite (a) of DAPI (blue, b), Pnoc mRNA expression evaluated by ISH (green, c), and reporter‐labeled Pnoc^tdTomato cells (magenta, d) in the adult striatum. Magnified insets from the dorsolateral striatum (e–g), dorsomedial striatum (h–j), nucleus accumbens core (k–m), and nucleus accumbens shell (n–p) show Pnoc^tdTomato reporter‐labeled cells that do not co‐label with adult Pnoc mRNA expression. (q) Quantification of mean Pnoc mRNA intensity in Pnoc^tdTomato cells (tdT⁺, green), nearby non‐reporter labeled cells (tdT⁻, yellow), and in Pnoc^tdTomato cells from adjacent control sections labeled with a negative control probe (NC, gray) in adult dorsal striatum. (Two‐way ANOVA with Bonferroni's multiple comparisons test, ***p < 0.001, **p < 0.01, ns = non‐significant, N = 4 mice, N = 7 sections, 1–2 sections/mouse). (r) Same as (q) but for adult ventral striatum (two‐way ANOVA with Bonferroni's multiple comparisons test, ****p < 0.0001, ***p < 0.001, ns = non‐significant, N = 4 mice, N = 7 sections, 1–2 sections/mouse). (s) Proportion of Pnoc^tdTomato reporter cells identified as Pnoc mRNA positive (Pnoc⁺/tdT⁺, green) or negative (Pnoc⁻/tdT⁺, magenta) in adult dorsal striatum. (t) Same as (s) but for adult ventral striatum. All data represented as mean ± SEM across mice. DLS = dorsolateral striatum; DMS = dorsomedial striatum; lNAcSh = lateral accumbens shell; mNAcSh = medial accumbens shell; NAcC = nucleus accumbens core.

FIGURE 5

Neonatal expression of striatal Pnoc mRNA recapitulates reporter‐labeled patterning in Pnoc^tdTomato cells. (a–d) Coronal image showing composite (a) of DAPI (blue, b), Pnoc mRNA expression evaluated by ISH (green, c), and reporter‐labeled Pnoc^tdTomato cells (magenta, d) in postnatal Day 1 (P1) brain sections. Magnified insets from the dorsolateral striatum (e–g), dorsomedial striatum (h–j), nucleus accumbens core (k–m), and nucleus accumbens shell (n–p) show Pnoc^tdTomato reporter‐labeled cells co‐labeled with neonatal Pnoc mRNA expression. (q) Quantification of mean Pnoc mRNA intensity in Pnoc^tdTomato cells (Pnoc⁻/tdT⁺, green), nearby non‐reporter labeled cells (Pnoc⁻/tdT⁻, yellow), and in Pnoc^tdTomato cells from adjacent control sections labeled with a negative control probe (NC, gray) in neonatal dorsal striatum. (Two‐way ANOVA with Bonferroni's multiple comparisons test, **p < 0.01, ns = non‐significant, N = 4 mice, N = 8 sections, 2 sections/mouse). (r) Same as (q) but for neonatal ventral striatum (two‐way ANOVA with Bonferroni's multiple comparisons test, ****p < 0.0001, ***p < 0.001, ns = non‐significant, N = 4 mice, N = 7 sections, 1–2 sections/mouse). (s) Proportion of Pnoc^tdTomato reporter cells identified as Pnoc mRNA positive (Pnoc⁺/tdT⁺, green) or negative (Pnoc⁻/tdT⁺, magenta) in neonatal dorsal striatum. (t) Same as (s) but for neonatal ventral striatum. (u) Proportion of Pnoc^tdTomato reporter cells identified as Pnoc mRNA positive in neonate (light purple) vs. adult (dark purple) tissue (two‐way ANOVA with Bonferroni's multiple comparisons test, ****p < 0.0001, N = 4 mice/group, N = 7 sections, 1–2 sections/mouse). (v) Fold‐change in mean Pnoc mRNA intensity in tdT⁺ cells relative to background (tdT⁻ cells) in neonate (light purple) vs. adult (dark purple) tissue (two‐way ANOVA with Bonferroni's multiple comparisons test, ****p < 0.0001, ***p < 0.001, **p < 0.01, ns = non‐significant, N = 4 mice/group, N = 7 sections, 1–2 sections/mouse). All data represented as mean ± SEM across mice. DLS = dorsolateral striatum; DMS = dorsomedial striatum; NAcC = nucleus accumbens core; NAcSh = nucleus accumbens shell.

FIGURE 6

Striosomal fibers from Pnoc^tdTomato targeted neurons project to the striosome‐dendron bouquet in SNc in both adults and neonates. In Pnoc^tdTomato reporter mice (a), tdTomato‐labeled fibers (magenta) project to TH‐labeled dopamine neurons (green) in adults (b–d) and neonates (e–g). (h) Schematic of mouse genetic strategy by viral labeling of striosomal cells in Pnoc‐Cre;loxP‐Stop‐loxP‐FlpO mice with a Flp‐dependent AAV expressing membrane‐GFP (mGFP). Virally targeted mGFP‐expressing PnocFlp neurons (i–j) exhibit projections to dopamine dendrons in SNc (k–m).