Embryonic markers of cone differentiation.

PURPOSE: Photoreceptor cells are born in two distinct phases of vertebrate retinogenesis. In the mouse retina, cones are born primarily during embryogenesis, while rod formation occurs later in embryogenesis and early postnatal ages. Despite this dichotomy in photoreceptor birthdates, the visual pigments and phototransduction machinery are not reactive to visual stimulus in either type of photoreceptor cell until the second postnatal week. Several markers of early cone formation have been identified, including Otx2, Crx, Blimp1, NeuroD, Trß2, Rorß, and Rxrγ, and all are thought to be involved in cellular determination. However, little is known about the expression of proteins involved in cone visual transduction during early retinogenesis. Therefore, we sought to characterize visual transduction proteins that are expressed specifically in photoreceptors during mouse embryogenesis. METHODS: Eye tissue was collected from control and phosducin-null mice at embryonic and early postnatal ages. Immunohistochemistry and quantitative reverse transcriptase-PCR (qPCR) were used to measure the spatial and temporal expression patterns of phosducin (Pdc) and cone transducin γ (Gngt2) proteins and transcripts in the embryonic and early postnatal mouse retina. RESULTS: We identified the embryonic expression of phosducin (Pdc) and cone transducin γ (Gngt2) that coincides temporally and spatially with the earliest stages of cone histogenesis. Using immunohistochemistry, the phosducin protein was first detected in the retina at embryonic day (E)12.5, and cone transducin γ was observed at E13.5. The phosducin and cone transducin γ proteins were seen only in the outer neuroblastic layer, consistent with their expression in photoreceptors. At the embryonic ages, phosducin was coexpressed with Rxrγ, a known cone marker, and with Otx2, a marker of photoreceptors. Pdc and Gngt2 mRNAs were detected as early as E10.5 with qPCR, although at low levels. CONCLUSIONS: Visual transduction proteins are expressed at the earliest stages in developing cones, well before the onset of opsin gene expression. Given the delay in opsin expression in rods and cones, we speculate on the embryonic function of these G-protein signaling components beyond their roles in the visual transduction cascade.

Rax is a selector gene for mediobasal hypothalamic cell types.

The brain plays a central role in controlling energy, glucose, and lipid homeostasis, with specialized neurons within nuclei of the mediobasal hypothalamus, namely the arcuate (ARC) and ventromedial (VMH), tasked with proper signal integration. Exactly how the exquisite cytoarchitecture and underlying circuitry becomes established within these nuclei remains largely unknown, in part because hypothalamic developmental programs are just beginning to be elucidated. Here, we demonstrate that the Retina and anterior neural fold homeobox (Rax) gene plays a key role in establishing ARC and VMH nuclei in mice. First, we show that Rax is expressed in ARC and VMH progenitors throughout development, consistent with genetic fate mapping studies demonstrating that Rax+ lineages give rise to VMH neurons. Second, the conditional ablation of Rax in a subset of VMH progenitors using a Shh::Cre driver leads to a fate switch from a VMH neuronal phenotype to a hypothalamic but non-VMH identity, suggesting that Rax is a selector gene for VMH cellular fates. Finally, the broader elimination of Rax throughout ARC/VMH progenitors using Six3::Cre leads to a severe loss of both VMH and ARC cellular phenotypes, demonstrating a role for Rax in both VMH and ARC fate specification. Combined, our study illustrates that Rax is required in ARC/VMH progenitors to specify neuronal phenotypes within this hypothalamic brain region. Rax thus provides a molecular entry point for further study of the ontology and establishment of hypothalamic feeding circuits.

Morphine Resistance in Spinal Cord Injury-Related Neuropathic Pain in Rats is Associated With Alterations in Dopamine and Dopamine-Related Metabolomics.

Opioids are not universally effective for treating neuropathic pain following spinal cord injury (SCI), a finding that we previously demonstrated in a rat model of SCI. The aim of this study was to determine analgesic response of morphine-responsive and nonresponsive SCI rats to adjunct treatment with dopamine modulators and to establish if the animal groups expressed distinct metabolomic profiles. Thermal thresholds were tested in female Long Evans rats (N = 45) prior to contusion SCI, after SCI and following injection of morphine, morphine combined with dopamine modulators, or dopamine modulators alone. Spinal cord and striatum samples were processed for metabolomics and targeted mass spectrometry. Morphine provided analgesia in 1 of 3 of SCI animals. All animals showed improved analgesia with morphine + pramipexole (D3 receptor agonist). Only morphine nonresponsive animals showed improved analgesia with the addition of SCH 39166 (D1 receptor antagonist). Metabolomic analysis identified 3 distinct clusters related to the tyrosine pathway that corresponded to uninjured, SCI morphine-responsive and SCI morphine-nonresponsive groups. Mass spectrometry showed matching differences in dopamine levels in striatum and spinal cord between these groups. The data suggest an overall benefit of the D3 receptor system in improving analgesia, and an association between morphine responsiveness and metabolomic changes in the tyrosine/dopamine pathways in striatum and spinal cord. PERSPECTIVE: Spinal cord injury (SCI) leads to opioid-resistant neuropathic pain that is associated with changes in dopamine metabolomics in the spinal cord and striatum of rats. We present evidence that adjuvant targeting of the dopamine system may be a novel pain treatment approach to overcome opioid desensitization and tolerance after SCI.

Dopamine D1 or D3 receptor modulators prevent morphine tolerance and reduce opioid withdrawal symptoms.

The long-term treatment of chronic pain by opioids is limited by tolerance and risk of addiction/dependence. Previously, we have shown that combination treatment of morphine with a dopamine D1 or D3 receptor modulator restored morphine analgesia in morphine-resistant neuropathic pain and decreased morphine's reward potential in an acute setting. Here, we investigated whether such adjunct therapy with a dopamine D1 receptor preferring antagonist (SCH 39166) or a dopamine D3 receptor preferring agonist (pramipexole) could prevent morphine tolerance and reduce withdrawal symptoms. Initially, tolerance to the combination of morphine + pramipexole was assessed in mice. Mice receiving intraperitoneal injections of morphine showed reduced thermal thresholds on Day 7 whereas those receiving morphine + pramipexole maintained analgesia at Day 7. Next, tolerance and withdrawal to both combinations were tested over 14 days in rats. Rats were assigned one of four drug conditions, (1) saline, 2) morphine, 3) morphine + SCH 39166, 4) morphine + pramipexole), for chronic administration via osmotic pumps. Chronic administration of morphine over 14 days resulted in a significant reduction of morphine analgesia. However, analgesia was maintained when morphine was administered with either the dopamine D1 receptor preferring antagonist or the D3 receptor preferring agonist. Withdrawal symptoms peaked at 48 h and were decreased in rats receiving either combination compared to morphine alone. The data suggests that adjunct therapy with dopamine D1 or D3 receptor preferring modulators prevents morphine tolerance and reduces the duration of morphine withdrawal symptoms, and thus this combination has potential for long-term pain management therapy.

Attenuated pain response of obese mice (B6.Cg-lep(ob)) is affected by aging and leptin but not sex.

Genetically obese mice (B6.Cg-lep(ob)) manifest decreased responses to noxious thermal stimuli (hotplate test) suggesting endogenous analgesia (Roy et al., 1981). To examine further the analgesic response of these mice, we conducted 4 experiments. Experiment 1 assessed the response of ob/ob mice to tail flick, another noxious thermal test. Tail-flick testing was performed on B6.Cg-lep(ob) mice (n=14) and B6.Cg-lep(OB/?) (n=12) across a range of temperatures. Ob/ob mice exhibited longer latencies than control mice at all temperatures tested. In Experiment 2, potential sex differences were examined. Tail-flick latencies in male and female ob/ob mice (n=6/group) did not differ. The final 2 experiments examined factors that could modulate endogenous analgesia. Experiment 3 assessed the effects of aging in ob/ob mice (n=10/group). Older mice displayed longer tail-flick latencies than did younger mice. Experiment 4 examined the effect of leptin administration in the leptin-deficient ob/ob mice. Two groups (n=10/group) of ob/ob mice received osmotic pump implants filled with either leptin or vehicle, and were tail-flick tested at days 7 and 14 post-implantation. Ob/ob mice receiving leptin showed shorter latencies than did vehicle-receiving ob/ob mice. Taken together, these results support earlier reports of heightened analgesia in ob/ob mice and suggest that aging further reduces the already impaired pain response. Furthermore, leptin deficiency partially contributes to decreased pain sensation of ob/ob mice.