M. Pullen

Soft, stretchable, fully implantable miniaturized optoelectronic systems for wireless optogenetics

Optogenetics allows rapid, temporally specific control of neuronal activity by targeted expression and activation of light-sensitive proteins. Implementation typically requires remote light sources and fiber-optic delivery schemes that impose considerable physical constraints on natural behaviors. In this report we bypass these limitations using technologies that combine thin, mechanically soft neural interfaces with fully implantable, stretchable wireless radio power and control systems. The resulting devices achieve optogenetic modulation of the spinal cord and peripheral nervous system. This is demonstrated with two form factors; stretchable film appliqués that interface directly with peripheral nerves, and flexible filaments that insert into the narrow confines of the spinal epidural space. These soft, thin devices are minimally invasive, and histological tests suggest they can be used in chronic studies. We demonstrate the power of this technology by modulating peripheral and spinal pain circuitry, providing evidence for the potential widespread use of these devices in research and future clinical applications of optogenetics outside the brain.

Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures

Primary cultures of rodent sensory neurons are widely used to investigate the cellular and molecular mechanisms involved in pain, itch, nerve injury and regeneration. However, translation of these preclinical findings may be greatly improved by direct validation in human tissues. We have developed an approach to extract and culture human sensory neurons in collaboration with a local organ procurement organization (OPO). Here we describe the surgical procedure for extraction of human dorsal root ganglia (hDRG) and the necessary modifications to existing culture techniques to prepare viable adult human sensory neurons for functional studies. Dissociated sensory neurons can be maintained in culture for >10 d, and they are amenable to electrophysiological recording, calcium imaging and viral gene transfer. The entire process of extraction and culturing can be completed in <7 h, and it can be performed by trained graduate students. This approach can be applied at any institution with access to organ donors consenting to tissue donation for research, and is an invaluable resource for improving translational research.

Metabotropic Glutamate Receptor 2/3 (mGluR2/3) Activation Suppresses TRPV1 Sensitization in Mouse, But Not Human, Sensory Neurons

Abstract The use of human tissue to validate putative analgesic targets identified in rodents is a promising strategy for improving the historically poor translational record of preclinical pain research. We recently demonstrated that in mouse and human sensory neurons, agonists for metabotropic glutamate receptors 2 and 3 (mGluR2/3) reduce membrane hyperexcitability produced by the inflammatory mediator prostaglandin E2 (PGE2). Previous rodent studies indicate that mGluR2/3 can also reduce peripheral sensitization by suppressing inflammation-induced sensitization of TRPV1. Whether this observation similarly translates to human sensory neurons has not yet been tested. We found that activation of mGluR2/3 with the agonist APDC suppressed PGE2-induced sensitization of TRPV1 in mouse, but not human, sensory neurons. We also evaluated sensory neuron expression of the gene transcripts for mGluR2 (Grm2), mGluR3 (Grm3), and TRPV1 (Trpv1). The majority of Trpv1 + mouse and human sensory neurons expressed Grm2 and/or Grm3, and in both mice and humans, Grm2 was expressed in a greater percentage of sensory neurons than Grm3. Although we demonstrated a functional difference in the modulation of TRPV1 sensitization by mGluR2/3 activation between mouse and human, there were no species differences in the gene transcript colocalization of mGluR2 or mGluR3 with TRPV1 that might explain this functional difference. Taken together with our previous work, these results suggest that mGluR2/3 activation suppresses only some aspects of human sensory neuron sensitization caused by PGE2. These differences have implications for potential healthy human voluntary studies or clinical trials evaluating the analgesic efficacy of mGluR2/3 agonists or positive allosteric modulators.

Optogenetic silencing of primary afferents reduces evoked and ongoing bladder pain

Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) suffer from chronic pain that severely affects quality of life. Although the underlying pathophysiology is not well understood, inhibition of bladder sensory afferents temporarily relieves pain. Here, we explored the possibility that optogenetic inhibition of bladder sensory afferents could be used to modulate bladder pain. Specifically, we chose to study the role of Nav1.8+ sensory afferents before and after induction of a mouse model of bladder pain. The light-activated inhibitory proton pump Archaerhodopsin (Arch) was expressed under control of the Nav1.8+ promoter to selectively silence these neurons. Optically silencing Nav1.8+ afferents significantly blunted the evoked visceromotor response to bladder distension and led to small but significant changes in bladder function. To study of the role of these fibers in freely behaving mice, we developed a fully implantable, flexible, wirelessly powered optoelectronic system for the long-term manipulation of bladder afferent expressed opsins. We found that optogenetic inhibition of Nav1.8+ fibers reduced both ongoing pain and evoked cutaneous hypersensitivity in the context of cystitis, but had no effect in uninjured, naïve mice. These results suggest that selective optogenetic silencing of bladder afferents may represent a potential future therapeutic strategy for the treatment of bladder pain.

PD70-04 OPTOGENETIC MODULATION OF BLADDER FUNCTION

dysregulation of S-nitrosylation, a process that mediates proteins activity, contributes to the pathogenesis of this disorder. We aim to investigate detrusor function and cAMP activation as a possible treatment for detrusor overactivity in an experimental model lacking a key denitrosylation enzyme, S-nitrosoglutathione reductase (GSNOR). METHODS: GSNOR-deficient (GSNOR-/-) (n1⁄430) and wildtype (WT) mice (n1⁄426) were treated for 7 days with the cAMP activator, Colforsin (1mg/kg), or vehicle intraperitoneally. Cystometric studies or molecular analyses of bladder specimens were performed. Bladder function indices and expression levels of proteins that regulate detrusor relaxation (nitric oxide synthase pathway) or contraction (RhoA/Rhokinase pathway) and oxidative stress were assessed. Student t-test and one-way ANOVA were used. RESULTS: GSNOR-/mice showed a significant increase (p<0.05) in voiding and non-voiding contraction frequencies compared to WT mice (figure 1, arrows indicate voiding contractions). Colforsin normalized these abnormalities. Western blot analyses showed an upregulation of the RhoA/Rho-kinase pathway reflected by a significant increase (p<0.05) of phosphorylated-MYPT1 expression in GSNOR-/mouse bladders, which was reversed by Colforsin treatment (figure 2). An increased level (p<0.05) of gp91phox expression in bladders of GSNOR-/mice was observed without significant change after Colforsin treatment. Neuronal and endothelial nitric oxide synthase phosphorylation on Ser-1412 and Ser-1177, respectively, did not differ between GSNOR-/and WT mouse bladders irrespective of Colforsin treatment. CONCLUSIONS: Impaired denitrosylation contributes to detrusor overactivity in association with upregulated RhoA/Rho-kinase signaling. Colforsin reverses physiologic and molecular abnormalities. This study describes a novel model of detrusor overactivity and suggests a possible basis for its treatment.