Probes for INS

Presynaptic cannabinoid-1 receptors (CB1-R) bind endogenous and exogenous cannabinoids to modulate neurotransmitter release. CB1-Rs are expressed throughout the basal ganglia, including striatum and substantia nigra, where they play a role in learning and control of motivated actions. However, the pattern of CB1-R expression across different striatal compartments, microcircuits and efferent targets, and the contribution of different CB1-R-expressing neurons to this pattern, are unclear. We use a combination of conventional techniques and novel genetic models to evaluate CB1-R expression in striosome (patch) and matrix compartments of the striatum, and in nigral targets of striatal medium spiny projection neurons (MSNs). CB1-R protein and mRNA follow a descending dorsolateral-to-ventromedial intensity gradient in the caudal striatum, with elevated expression in striosomes relative to the surrounding matrix. The lateral predominance of striosome CB1-Rs contrasts with that of the classical striosomal marker, the mu opioid receptor (MOR), which is expressed most prominently in rostromedial striosomes. The dorsolateral-to-ventromedial CB1-R gradient is similar to Drd2 dopamine receptor immunoreactivity and opposite to Substance P. This topology of CB1-R expression is maintained downstream in the globus pallidus and substantia nigra. Dense CB1-R-expressing striatonigral fibers extend dorsally within the substantia nigra pars reticulata, and colocalize with bundles of ventrally extending, striosome-targeted, dendrites of dopamine-containing neurons in the substantia nigra pars compacta (striosome-dendron bouquets). Within striatum, CB1-Rs colocalize with fluorescently labeled MSN collaterals within the striosomes. Cre recombinase-mediated deletion of CB1-Rs from cortical projection neurons or MSNs, and MSN-selective reintroduction of CB1-Rs in knockout mice, demonstrate that the principal source of CB1-Rs in dorsolateral striosomes is local MSN collaterals. These data suggest a role for CB1-Rs in caudal dorsolateral striosome collaterals and striosome-dendron bouquet projections to lateral substantia nigra, where they are anatomically poised to mediate presynaptic disinhibition of both striosomal MSNs and midbrain dopamine neurons in response to endocannabinoids and cannabinomimetics.

Abstract

BACKGROUND:

Global analyses of gene expression during development reveal specific transcription patterns associated with the emergence of various cell types, tissues, and organs. These heterogeneous patterns are instrumental to ensure the proper formation of the different parts of our body, as shown by the phenotypic effects generated by functional genetic approaches. However, variations at the cellular level can be observed within each structure or organ. In the developing mammalian limbs, expression of Hox genes from the HoxD cluster is differentially controlled in space and time, in cells that will pattern the digits and the forearms. While the Hoxd genes broadly share a common regulatory landscape and large-scale analyses have suggested a homogenous Hox gene transcriptional program, it has not previously been clear whether Hoxd genes are expressed together at the same levels in the same cells.

RESULTS:

We report a high degree of heterogeneity in the expression of the Hoxd11 and Hoxd13 genes. We analyzed single-limb bud cell transcriptomes and show that Hox genes are expressed in specific combinations that appear to match particular cell types. In cells giving rise to digits, we find that the expression of the five relevant Hoxd genes (Hoxd9 to Hoxd13) is unbalanced, despite their control by known global enhancers. We also report that specific combinatorial expression follows a pseudo-time sequence, which is established based on the transcriptional diversity of limb progenitors.

CONCLUSIONS:

Our observations reveal the existence of distinct combinations of Hoxd genes at the single-cell level during limb development. In addition, we document that the increasing combinatorial expression of Hoxd genes in this developing structure is associated with specific transcriptional signatures and that these signatures illustrate a temporal progression in the differentiation of these cells.

Detailed anatomical tracing and mapping of the viscerotopic organization of the vagal motor nuclei has provided insight into autonomic function in health and disease. To further define specific cellular identities, we paired information based on visceral connectivity with a cell-type specific marker of a subpopulation of neurons in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (nAmb) that express the autism-associated MET receptor tyrosine kinase. As gastrointestinal disturbances are common in children with autism spectrum disorder (ASD), we sought to define the relationship between MET-expressing (MET+) neurons in the DMV and nAmb, and the gastrointestinal tract. Using wholemount tissue staining and clearing, or retrograde tracing in a METEGFP transgenic mouse, we identify three novel subpopulations of EGFP+ vagal brainstem neurons: 1) EGFP+ neurons in the nAmb projecting to the esophagus or laryngeal muscles, 2) EGFP+ neurons in the medial DMV projecting to the stomach, and 3) EGFP+ neurons in the lateral DMV projecting to the cecum and/or proximal colon. Expression of the MET ligand, hepatocyte growth factor (HGF), by tissues innervated by vagal motor neurons during fetal development reveal potential sites of HGF-MET interaction. Furthermore, similar cellular expression patterns of MET in the brainstem of both the mouse and nonhuman primate suggest that MET expression at these sites is evolutionarily conserved. Together, the data suggest that MET+ neurons in the brainstem vagal motor nuclei are anatomically positioned to regulate distinct portions of the gastrointestinal tract, with implications for the pathophysiology of gastrointestinal comorbidities of ASD.

Abstract

BACKGROUND:

Levodopa-induced dyskinesias are an often debilitating side effect of levodopa therapy in Parkinson's disease. Although up to 90% of individuals with PD develop this side effect, uniformly effective and well-tolerated antidyskinetic treatment remains a significant unmet need. The pathognomonic loss of striatal dopamine in PD results in dysregulation and disinhibition of striatal CaV1.3 calcium channels, leading to synaptopathology that appears to be involved in levodopa-induced dyskinesias. Although there are clinically available drugs that can inhibit CaV1.3 channels, they are not adequately potent and have only partial and transient impact on levodopa-induced dyskinesias.

METHODS:

To provide unequivocal target validation, free of pharmacological limitations, we developed a CaV1.3 shRNA to provide high-potency, target-selective, mRNA-level silencing of striatal CaV1.3 channels and examined its ability to impact levodopa-induced dyskinesias in severely parkinsonian rats.

RESULTS:

We demonstrate that vector-mediated silencing of striatal CaV1.3 expression in severely parkinsonian rats prior to the introduction of levodopa can uniformly and completely prevent induction of levodopa-induced dyskinesias, and this antidyskinetic benefit persists long term and with high-dose levodopa. In addition, this approach is capable of ameliorating preexisting severe levodopa-induced dyskinesias. Importantly, motoric responses to low-dose levodopa remained intact in the presence of striatal CaV1.3 silencing, indicating preservation of levodopa benefit without dyskinesia liability.

DISCUSSION:

The current data provide some of the most profound antidyskinetic benefit reported to date and suggest that genetic silencing of striatal CaV1.3 channels has the potential to transform treatment of individuals with PD by allowing maintenance of motor benefit of levodopa in the absence of the debilitating levodopa-induced dyskinesia side effect.