Daniel S. Brenner

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.

A Novel Behavioral Assay for Measuring Cold Sensation in Mice

Behavioral models of cold responses are important tools for exploring the molecular mechanisms of cold sensation. To complement the currently cold behavioral assays and allow further studies of these mechanisms, we have developed a new technique to measure the cold response threshold, the cold plantar assay. In this assay, animals are acclimated on a glass plate and a cold stimulus is applied to the hindpaw through the glass using a pellet of compressed dry ice. The latency to withdrawal from the cooled glass is used as a measure of the cold response threshold of the rodents, and the dry ice pellet provides a ramping cold stimulus on the glass that allows the correlation of withdrawal latency values to rough estimates of the cold response threshold temperature. The assay is highly sensitive to manipulations including morphine-induced analgesia, Complete Freunds Adjuvant-induced inflammatory allodynia, and Spinal Nerve Ligation-induced neuropathic allodynia.

TRPV4 is necessary for trigeminal irritant pain and functions as a cellular formalin receptor.

Summary Formalin‐evoked trigeminal pain behavior depends on TRPV4 calcium‐permeable channels, also on MEK‐ERK signaling in trigeminal ganglion neurons. ABSTRACT Detection of external irritants by head nociceptor neurons has deep evolutionary roots. Irritant‐induced aversive behavior is a popular pain model in laboratory animals. It is used widely in the formalin model, where formaldehyde is injected into the rodent paw, eliciting quantifiable nocifensive behavior that has a direct, tissue‐injury‐evoked phase, and a subsequent tonic phase caused by neural maladaptation. The formalin model has elucidated many antipain compounds and pain‐modulating signaling pathways. We have adopted this model to trigeminally innervated territories in mice. In addition, we examined the involvement of TRPV4 channels in formalin‐evoked trigeminal pain behavior because TRPV4 is abundantly expressed in trigeminal ganglion (TG) sensory neurons, and because we have recently defined TRPV4’s role in response to airborne irritants and in a model for temporomandibular joint pain. We found TRPV4 to be important for trigeminal nocifensive behavior evoked by formalin whisker pad injections. This conclusion is supported by studies with Trpv4−/− mice and TRPV4‐specific antagonists. Our results imply TRPV4 in MEK‐ERK activation in TG sensory neurons. Furthermore, cellular studies in primary TG neurons and in heterologous TRPV4‐expressing cells suggest that TRPV4 can be activated directly by formalin to gate Ca2+. Using TRPA1‐blocker and Trpa1−/− mice, we found that both TRP channels co‐contribute to the formalin trigeminal pain response. These results imply TRPV4 as an important signaling molecule in irritation‐evoked trigeminal pain. TRPV4‐antagonistic therapies can therefore be envisioned as novel analgesics, possibly for specific targeting of trigeminal pain disorders, such as migraine, headaches, temporomandibular joint, facial, and dental pain, and irritation of trigeminally innervated surface epithelia.

Stretchable multichannel antennas in soft wireless optoelectronic implants for optogenetics

Significance Soft, multichannel antennas enable wireless, battery-free operation of fully implantable optoelectronic systems designed for use in studies of brain function. These systems support independent, remote control of multiple light-emitting diodes that inject into targeted regions of the deep brain, where they separately stimulate activity in genetically and spatially discrete neural circuits, via the use of the techniques of optogenetics. These capabilities represent significant advancements over alternative technology approaches for this important branch of neuroscience research. In vivo studies using optimized systems demonstrate wireless control of two different brain regions and distinct activation of subpopulations of neurons using separately activated light sources associated with these subdermal devices. Optogenetic methods to modulate cells and signaling pathways via targeted expression and activation of light-sensitive proteins have greatly accelerated the process of mapping complex neural circuits and defining their roles in physiological and pathological contexts. Recently demonstrated technologies based on injectable, microscale inorganic light-emitting diodes (μ-ILEDs) with wireless control and power delivery strategies offer important functionality in such experiments, by eliminating the external tethers associated with traditional fiber optic approaches. Existing wireless μ-ILED embodiments allow, however, illumination only at a single targeted region of the brain with a single optical wavelength and over spatial ranges of operation that are constrained by the radio frequency power transmission hardware. Here we report stretchable, multiresonance antennas and battery-free schemes for multichannel wireless operation of independently addressable, multicolor μ-ILEDs with fully implantable, miniaturized platforms. This advance, as demonstrated through in vitro and in vivo studies using thin, mechanically soft systems that separately control as many as three different μ-ILEDs, relies on specially designed stretchable antennas in which parallel capacitive coupling circuits yield several independent, well-separated operating frequencies, as verified through experimental and modeling results. When used in combination with active motion-tracking antenna arrays, these devices enable multichannel optogenetic research on complex behavioral responses in groups of animals over large areas at low levels of radio frequency power (<1 W). Studies of the regions of the brain that are involved in sleep arousal (locus coeruleus) and preference/aversion (nucleus accumbens) demonstrate the unique capabilities of these technologies.

A dynamic set point for thermal adaptation requires phospholipase C-mediated regulation of TRPM8 in vivo

&NA; We measure the rapid adaptation of mice to changing environmental conditions. This process that preserves temperature responsiveness is TRPM8 dependent, and mediated by phospholipase C. &NA; The ability to sense and respond to thermal stimuli at varied environmental temperatures is essential for survival in seasonal areas. In this study, we show that mice respond similarly to ramping changes in temperature from a wide range of baseline temperatures. Further investigation suggests that this ability to adapt to different ambient environments is based on rapid adjustments made to a dynamic temperature set point. The adjustment of this set point requires transient receptor potential cation channel, subfamily member 8 (TRPM8), but not transient receptor potential cation channel, subfamily A, member 1 (TRPA1), and is regulated by phospholipase C (PLC) activity. Overall, our findings suggest that temperature response thresholds in mice are dynamic, and that this ability to adapt to environmental temperature seems to mirror the in vitro findings that PLC‐mediated hydrolysis of phosphoinositol 4,5‐bisphosphate modulates TRPM8 activity and thereby regulates the response thresholds to cold stimuli.

A technique to measure cold adaptation in freely behaving mice

BACKGROUND Adaptation to environmental temperature is essential for survival in seasonal areas. The mechanisms of adaptation have been studied in vitro, but it has not been quantified in vivo. NEW METHOD The extended Cold Plantar Assay (eCPA) cools the entire testing environment. Once the desired environmental temperature has been reached, a separate focal cold stimulus is applied to the hindpaw and the latency to withdrawal is recorded as a proxy for cold sensitivity. RESULTS Using this technique, we can test the cold responsiveness of freely behaving mice at ambient temperatures ranging from 5°C to 30°C. The responses are consistent and unambiguous, and the environmental temperatures generated are reproducible. We are also able to measure cold responsiveness as animals are in the process of adapting to cold environments. COMPARISON WITH EXISTING METHOD(S) Existing methods, such as the dynamic cold plate and the 2-plate preference assay test how mice respond to cold environments, but cannot assess how the thresholds for response are changed by acclimation in cold environments. Additionally, the eCPA requires very little specialized equipment, can test many mice at the same time on one apparatus, and has an objective readout. CONCLUSIONS The extended Cold Plantar assay is a significant methodological improvement, allowing the assessment of cold responsiveness in freely behaving mice at a wide range of environmental temperature conditions and during cold adaptation.

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity. Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 °C to 5 °C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above. This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Postinflammatory hyperpigmentation after human cold pain testing

Abstract Changes in cold temperature sensitivity are often associated with chronic pain conditions. Progress in understanding the neurobiological mechanism underlying these changes and resulting development of effective therapies has been slowed by the accessibility and affordability of devices used to measure thermal sensitivity in humans. To address this gap, we developed an inexpensive method to measure cold pain thresholds in healthy adult volunteers using dry ice and a thermode. However, early in preliminary testing, a subject presented with epidermal postinflammatory hyperpigmentation that lasted for >200 days. Although this response was unique among the small number of subjects in development of the assay, it raised questions as to the safety of the assay design.

The Molecular and Cellular Basis for Cold Sensation

OF THE DISSERTATION xiii Chapter 1: Introduction to pain and temperature sensation 1 Why study pain? 2 What is pain? 3 How do pain and nociception happen? 4 Why study mice to learn about human pain? 8 Behavioral assays for thermal and cold sensation in rodents 10 Molecular mechanisms of temperature responsiveness 13 Adaptation to changes in environmental temperature 20 The functions of individual dorsal root ganglia neurons have been studied with ablation studies 21 Novel methods for studying the roles of individual neuronal populations 23 Conclusions 26