Diane lipscombe

Neurons have the unique capacity to adapt output in response to changes in their environment. Within seconds, sensory nerve endings can become hypersensitive to stimuli in response to potentially damaging events. The underlying behavioral response is well studied, but several of the key signaling molecules that mediate sensory hypersensitivity remain unknown. We previously discovered that peripheral voltage-gated CaV 2.2 channels in nerve endings in skin are essential for the rapid, transient increase in sensitivity to heat, but not to mechanical stimuli, that accompanies intradermal capsaicin...

Neurons have the unique capacity to adapt output in response to changes in their environment. Within seconds, sensory nerve endings can become hypersensitive to stimuli in response to potentially damaging events. The underlying behavioral response is well studied, but several of the key signaling molecules that mediate sensory hypersensitivity remain unknown. We previously discovered that peripheral voltage-gated Ca V 2.2 channels in nerve endings in skin are essential for the rapid, transient increase in sensitivity to heat, but not to mechanical stimuli, that accompanies intradermal capsaicin...

SIGNIFICANCE: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. AIM: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs...

SIGNIFICANCE: Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). AIM: To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. APPROACH: We developed novel luciferases optimized for Förster resonance energy transfer when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1)...

SIGNIFICANCE: Pain is comprised of a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms...

SIGNIFICANCE: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. AIM: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs...

OBJECTIVE: Incorporating diversity, equity, inclusion, and anti-racism principles into clinical and didactic education is essential because each influence cognitive and affective attitudes in pharmacy practice. Educators must learn from the past to enlighten the future. For example, race is a social construct, not a biological construct. However, it persistently acts as a surrogate for determining medical diagnoses and treatment. FINDINGS: Precision medicine and pharmacogenomics can serve as a basis for deconstructing social constructs surrounding race and other social determinants of health...

Cell-specific alternative splicing of Cacna1b pre-mRNA generates functionally distinct voltage-gated Ca V 2.2 channels. Ca V 2.2 channels mediate the release of glutamate from nociceptor termini in the dorsal horn spinal cord and they are implicated in chronic pain. One alternatively spliced exon in Cacna1b , e37a, is highly expressed in dorsal root ganglia, relative to other regions of the nervous system, and it is particularly important in inflammatory hyperalgesia. Here we studied the effects of two ω-phonetoxins, PnTx3-4 and Phα1β, derived from the spider Phoneutria nigriventer on Ca V 2...

We developed a platform that utilizes a calcium-dependent luciferase to convert neuronal activity into activation of light sensing domains within the same cell. The platform is based on a Gaussia luciferase variant with high light emission split by calmodulin-M13 sequences that depends on influx of calcium ions (Ca 2+ ) for functional reconstitution. In the presence of its luciferin, coelenterazine (CTZ), Ca 2+ influx results in light emission that drives activation of photoreceptors, including optogenetic channels and LOV domains...

The ability to manipulate neuronal activity both opto-and chemogenetically with a single actuator molecule presents unique and flexible means to study neural circuit function. We previously developed methodology to enable such bimodal control using fusion molecules called luminopsins (LMOs), where a channelrhodopsin actuator can be activated using either physical (LED driven) or biological (bioluminescent) light. While activation of LMOs using bioluminescence has previously allowed manipulation of circuits and behavior in mice, further improvement would advance the utility of this technique...

Ca 2+ plays many critical roles in cell physiology and biochemistry, leading researchers to develop a number of fluorescent small molecule dyes and genetically encodable probes that optically report changes in Ca 2+ concentrations in living cells. Though such fluorescence-based genetically encoded Ca 2+ indicators (GECIs) have become a mainstay of modern Ca 2+ sensing and imaging, bioluminescence-based GECIs-probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein-have several distinct advantages over their fluorescent counterparts...

UNLABELLED: Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder affecting brain and spinal cord motor neurons. Mutations in the copper/zinc superoxide dismutase gene ( SOD1 ) are associated with ∼20% of inherited and 1-2% of sporadic ALS cases. Much has been learned from mice expressing transgenic copies of mutant SOD1, which typically involve high-level transgene expression, thereby differing from ALS patients expressing one mutant gene copy. To generate a model that more closely represents patient gene expression, we created a knock-in point mutation (G85R, a human ALS-causing mutation) in the endogenous mouse Sod1 gene, leading to mutant SOD1 G85R protein expression...

Understanding percepts, engrams and actions requires methods for selectively modulating synaptic communication between specific subsets of interconnected cells. Here, we develop an approach to control synaptically connected elements using bioluminescent light: Luciferase-generated light, originating from a presynaptic axon terminal, modulates an opsin in its postsynaptic target. Vesicular-localized luciferase is released into the synaptic cleft in response to presynaptic activity, creating a real-time Optical Synapse...

CACNA1I is implicated in the susceptibility to schizophrenia by large-scale genetic association studies of single nucleotide polymorphisms. However, the channelopathy of CACNA1I in schizophrenia is unknown. CACNA1I encodes CaV3.3, a neuronal voltage-gated calcium channel that underlies a subtype of T-type current that is important for neuronal excitability in the thalamic reticular nucleus (TRN) and other regions of the brain. Here, we present an extensive functional characterization of 57 naturally occurring rare and common missense variants of CACNA1I derived from a Swedish schizophrenia cohort of more than 10,000 individuals...

Voltage-gated CaV 2.2 calcium channels are expressed in nociceptors at presynaptic terminals, soma, and axons. CaV 2.2 channel inhibitors applied to the spinal cord relieve pain in humans and rodents, especially during pathological pain, but a biological function of nociceptor CaV 2.2 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is underappreciated. Here, we demonstrate that functional CaV 2.2 channels in peripheral axons innervating skin are required for capsaicin-induced heat hypersensitivity in male and female mice...

Bioluminescent optogenetics (BL-OG) allows activation of photosensory proteins, such as opsins, by either fiberoptics or by administering a luciferin. BL-OG thus confers both optogenetic and chemogenetic access within the same genetically targeted neuron. This bimodality offers a powerful approach for non-invasive chemogenetic manipulation of neural activity during brain development and adult behaviors with standard optogenetic spatiotemporal precision. We detail protocols for bioluminescent stimulation of neurons in postnatally developing brain and its validation through bioluminescence imaging and electrophysiological recording in mice...

Ion channels underlie all forms for electrical signaling including the transmission of information about harmful events. Voltage-gated calcium ion channels have dual function, they support electrical signaling as well as intracellular calcium signaling through excitation-dependent calcium entry across the plasma membrane. Mechanisms that regulate ion channel forms and actions are essential for myriad cell functions and these are targeted by drugs and therapeutics. When disrupted, the cellular mechanisms that control ion channel activity can contribute to disease pathophysiology...

In vivo fluorescence miniature microscopy has recently proven a major advance, enabling cellular imaging in freely behaving animals. However, fluorescence imaging suffers from autofluorescence, phototoxicity, photobleaching and non- homogeneous illumination artifacts. These factors limit the quality and time course of data collection. Bioluminescence provides an alternative kind of activity-dependent light indicator. Bioluminescent calcium indicators do not require light input, instead generating photons through chemiluminescence...

Cell-specific alternative splicing modulates myriad cell functions and is disrupted in disease. The mechanisms governing alternative splicing are known for relatively few genes and typically focus on RNA splicing factors. In sensory neurons, cell-specific alternative splicing of the presynaptic CaV channel Cacna1b gene modulates opioid sensitivity. How this splicing is regulated is unknown. We find that cell and exon-specific DNA hypomethylation permits CTCF binding, the master regulator of mammalian chromatin structure, which, in turn, controls splicing in a DRG-derived cell line...

There is a pressing need to increase the rigor of research in the life and biomedical sciences. To address this issue, we propose that communities of 'rigor champions' be established to campaign for reforms of the research culture that has led to shortcomings in rigor. These communities of rigor champions would also assist in the development and adoption of a comprehensive educational platform that would teach the principles of rigorous science to researchers at all career stages.