Voltage-gated sodium ion channel subtype 1.7 (NaV1.7) is a high interest target for the discovery of non-opioid analgesics. Compelling evidence from human genetic data, particularly the finding that persons lacking functional NaV1.7 are insensitive to pain, has spurred considerable effort to develop selective inhibitors of this Na+ ion channel target as analgesic medicines. Recent clinical setbacks and disappointing performance of preclinical compounds in animal pain models, however, have led to skepticism around the potential of selective NaV1.7 inhibitors as human therapeutics. In this Perspective, we discuss the attributes and limitations of recently disclosed investigational drugs targeting NaV1.7 and review evidence that, by better understanding the requirements for selectivity and target engagement, the opportunity to deliver effective analgesic medicines targeting NaV1.7 endures.

To understand visual functions mediated by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs), it is important to elucidate axonal projections from these cells into the brain. Initial studies reported that melanopsin is expressed only in retinal ganglion cells within the eye. However, recent studies in Opn4-Cre mice revealed Cre-mediated marker expression in multiple brain areas. These discoveries complicate the use of melanopsin-driven genetic labeling techniques to identify retinofugal projections specifically from mRGCs. To restrict labeling to mRGCs, we developed a recombinant adeno-associated virus (AAV) carrying a Cre-dependent reporter (human placental alkaline phosphatase) that was injected into the vitreous of Opn4-Cre mouse eyes. The labeling observed in the brain of these mice was necessarily restricted specifically to retinofugal projections from mRGCs in the injected eye. We found that mRGCs innervate multiple nuclei in the basal forebrain, hypothalamus, amygdala, thalamus and midbrain. Midline structures tended to be bilaterally innervated, whereas the lateral structures received mostly contralateral innervation. As validation of our approach, we found projection patterns largely corresponded with previously published results; however, we have also identified a few novel targets. Our discovery of projections to the central amygdala suggests a possible direct neural pathway for aversive responses to light in neonates. In addition, projections to the accessory optic system suggest that mRGCs play a direct role in visual tracking, responses that were previously attributed to other classes of retinal ganglion cells. Moreover, projections to the zona incerta raise the possibility that mRGCs could regulate visceral and sensory functions. However, additional studies are needed to investigate the actual photosensitivity of mRGCs that project to the different brain areas. Also, there is a concern of "overlabeling" with very sensitive reporters that uncover low levels of expression. Light-evoked signaling from these cells must be shown to be of sufficient sensitivity to elicit physiologically relevant responses.

Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are the only functional photoreceptive cells in the eye of newborn mice. Through postnatal day 9, in the absence of functional rods and cones, these ipRGCs mediate a robust avoidance behavior to a light source, termed negative phototaxis. To determine whether this behavior is associated with an aversive experience in neonatal mice, we characterized light-induced vocalizations and patterns of neuronal activation in regions of the brain involved in the processing of aversive and painful stimuli. Light evoked distinct melanopsin-dependent ultrasonic vocalizations identical to those emitted under stressful conditions, such as isolation from the litter. In contrast, light did not evoke the broad-spectrum calls elicited by acute mechanical pain. Using markers of neuronal activation, we found that light induced the immediate-early gene product Fos in the posterior thalamus, a brain region associated with the enhancement of responses to mechanical stimulation of the dura by light, and thought to be the basis for migrainous photophobia. Additionally, light induced the phosphorylation of extracellular-related kinase (pERK) in neurons of the central amygdala, an intracellular signal associated with the processing of the aversive aspects of pain. However, light did not activate Fos expression in the spinal trigeminal nucleus caudalis, the primary receptive field for painful stimulation to the head. We conclude that these light-evoked vocalizations and the distinct pattern of brain activation in neonatal mice are consistent with a melanopsin-dependent neural pathway involved in processing light as an aversive but not acutely painful stimulus.

Methylmercury (MeHg) is a ubiquitous toxicant that targets the developing fetal nervous system. MeHg interacts with the Notch signaling pathway, a highly-conserved intercellular signaling mechanism required for normal development. Notch signaling is conveyed by activation of the genes in the Enhancer of Split (E(spl)) locus in Drosophila. We have previously shown that acute high doses of MeHg upregulate several E(spl) genes in Drosophila neural-derived C6 cells. Furthermore, MeHg induction of E(spl) can occur independent of the Notch receptor itself. We now show that MeHg, unlike inorganic mercury (HgCl2), preferentially upregulates E(spl)mδ and E(spl)mγ in Drosophila C6 cells. This is distinct from Delta ligand-induced Notch signaling in which no induction of E(spl)mδ is seen. MeHg is also seen to specifically upregulate E(spl)mδ in Drosophila embryos where HgCl2 showed no such effect. Additionally, treatment of embryos with MeHg caused a consistent failure in axonal outgrowth of the intersegmental nerve (ISN). This ISN phenotype was partially replicated by genetic activation of the Notch pathway, but was not replicated by increasing expression of E(spl)mδ. These data suggest a role for Notch signaling and the E(spl)mδ target gene in MeHg toxicity, however, the site of action for E(spl)mδ in this system remains to be elucidated.

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A central mechanism in activation of the Notch signaling pathway is cleavage of the Notch receptor by ADAM metalloproteases. ADAMs also cleave Delta, the ligand for Notch, thereby downregulating Notch signals. Two ADAMs, Kuzbanian (Kuz) and TNF-α converting enzyme (TACE), are known to process both Delta and Notch, yet the role of these cleavages in signal propagation has remained controversial. Using an in-vitro model, we show that Kuz regulates Notch signaling primarily by activating the receptor and has little overall effect on signaling via disabling Delta. We confirm that Kuz-dependent activation of Notch requires stimulation of Notch by Delta. However, over-expression of Kuz gives ligand-independent Notch activation. In contrast, TACE, which is elevated in expression in developing Drosophila nervous system, can efficiently activate Notch in a ligand-independent manner. Altogether, these data demonstrate the potential for Kuz and TACE to participate in context-and mechanism-specific modes of Notch activation.

Melanopsin-expressing retinal ganglion cells (mRGCs) in the eye play an important role in many light-activated non-image-forming functions including neonatal photoaversion and the adult pupillary light reflex (PLR). MRGCs rely on glutamate and possibly PACAP (pituitary adenylate cyclase-activating polypeptide) to relay visual signals to the brain. However, the role of these neurotransmitters for individual non-image-forming responses remains poorly understood. To clarify the role of glutamatergic signaling from mRGCs in neonatal aversion to light and in adult PLR, we conditionally deleted vesicular glutamate transporter (VGLUT2) selectively from mRGCs in mice. We found that deletion of VGLUT2 in mRGCs abolished negative phototaxis and light-induced distress vocalizations in neonatal mice, underscoring a necessary role for glutamatergic signaling. In adult mice, loss of VGLUT2 in mRGCs resulted in a slow and an incomplete PLR. We conclude that glutamatergic neurotransmission from mRGCs is required for neonatal photoaversion but is complemented by another non-glutamatergic signaling mechanism for the pupillary light reflex in adult mice. We speculate that this complementary signaling might be due to PACAP neurotransmission from mRGCs.

A unique class of intrinsically photosensitive retinal ganglion cells in mammalian retinae has been recently discovered and characterized. These neurons can generate visual signals in the absence of inputs from rods and cones, the conventional photoreceptors in the visual system. These light sensitive ganglion cells (mRGCs) express the non-rod, non-cone photopigment melanopsin and play well documented roles in modulating pupil responses to light, photoentrainment of circadian rhythms, mood, sleep and other adaptive light functions. While most research efforts in mammals have focused on mRGCs in retina, recent studies reveal that melanopsin is expressed in non-retinal tissues. For example, light-evoked melanopsin activation in extra retinal tissue regulates pupil constriction in the iris and vasodilation in the vasculature of the heart and tail. As another example of nonretinal melanopsin expression we report here the previously unrecognized localization of this photopigment in nerve fibers within the cornea. Surprisingly, we were unable to detect light responses in the melanopsin-expressing corneal fibers in spite of our histological evidence based on genetically driven markers and antibody staining. We tested further for melanopsin localization in cell bodies of the trigeminal ganglia (TG), the principal nuclei of the peripheral nervous system that project sensory fibers to the cornea, and found expression of melanopsin mRNA ina subset of TG neurons. However, neither electrophysiological recordings nor calcium imaging revealed any light responsiveness in the melanopsin positive TG neurons. Given that we found no light-evoked activation of melanopsin-expressing fibers in cornea or in cell bodies in the TG, we propose that melanopsin protein might serve other sensory functions in the cornea. One justification for this idea is that melanopsin expressed in Drosophila photoreceptors can serve as a temperature sensor.