In format provided by Birmingham et al. (JUNE 2014) NATURE REVIEWS | DRUG DISCOVERY www.nature.com/reviews/drugdisc Box S1 | Bioelectronic medicines: the detailed research roadmap Creation of a visceral nerve atlas Structural mappingOverall objective: Create organ-centric wiring diagrams in models representative of human anatomy. Research imperatives:• Generate tools for high-resolution tracing of the fibre anatomy and taxonomy to and from individual organs o Build a library of tracers to visualize the full length of pre-ganglionic axons, ganglionic cell bodies, post-ganglionic axons, and intrinsic neurons 1 , with particular focus on tracing that starts at the target organ 2-5 o Advance approaches for high-resolution labelling of peripheral neurotransmitters, their receptors and co-receptors, and for imaging of myelination, peri-and epineurium o Develop and adapt micrometer-resolution imaging and 3D reconstruction techniques for visceral organs and peripheral nerves (for example, automated tissue slicing, clearing, in situ hybridization, multi-photon imaging) 6-12 • Explore inter-and intra-species variation in neuroanatomy and establish the optimal animal models for detailed mapping of each organ o Update and extend the macro-level innervation map of the major visceral organs in key animal model species and establish the extent to which this map is conserved in humans o Characterise the variability in different parts of these maps between individuals • Build organ-centric high-resolution maps for each visceral organ in their most representative animal model o Conduct high-resolution nerve tracing, labelling, and imaging in the animal model of choice, taking the organ as the starting point o Standardise and coordinate mapping, data management and 3D visualisation across organs • Establish methods to image and find nerves in the clinical setting o Develop tracers and associated imaging techniques that can be used in human preoperative and intra-operative settings to identify and localize peripheral nerves o Identify anatomical landmarks associated with putative intervention points for surgery Functional mappingOverall objective: Map the neural signalling patterns that control individual organ functions. Research imperatives:• Generate simultaneous recordings of neural signal pattern and organ function o Record both afferent (sensory) and efferent (motor) neural signals and associated end-organ biomarkers at a range of physiological stimuli

The provided electrophysiological and immunohistochemical data provide strong support to the viability of the developed probe technology. Furthermore, the obtained data provide insights into further optimization of the probe design, including tip geometry, use of neurotrophic and anti-inflammatory drugs in the Matrigel coating, and placement of the recording sites.

Activated Iridium microelectrodes were implanted for 450 to 1282 days in the sensorimotor cortex of 7 adult domestics cats and then pulsed for 240 hours (8 hours per day for 30 days) at 50 Hz. Continuous stimulation at 2 nC/phase and with a geometric charge density of 100 μC/cm2 produced no detectable change in neuronal density in the tissue surrounding the microelectrode tips. However, pulsing with a continuous (100% duty cycle) at 4 nC/ph and with a geometric charge density of 200 μC/cm2 induced loss of cortical neurons over a radius of at least 150 μm from the electrode tips. The same stimulus regimen, but with a duty cycle of 50% (1 sec of stimulation, then 1 second without stimulation repeated for 8 hours) produced neuronal loss within a smaller radius, approximately 60 μm from the center of the electrode tips. However, there also was significant loss of neurons surrounding the unpulsed electrodes, presumably as a result of mechanical injury due to their insertion into and long-term residence in the tissue, and this was responsible for most of the neuronal loss within 150 μm of the electrodes pulsed with the 50% duty cycle.

Recovery of urinary tract function after spinal cord injury (SCI) is important in its own right and may also serve as a model for studying mechanisms of functional recovery after injury in the CNS. Normal micturition requires coordinated activation of smooth muscle of the bladder (detrusor) and striated muscle of the external urethral sphincter (EUS) that is controlled by spinal and supraspinal circuitry. We used a clinically relevant rat model of thoracic spinal cord contusion injury to examine the effect of varying the degree of residual supraspinal connections on chronic detrusor-EUS coordination. Urodynamic evaluation at 8 weeks after SCI showed that detrusor contractions of the bladder recovered similarly in groups of rats injured with a 10 gm weight dropped 12.5, 25, or 50 mm onto the spinal cord. In contrast, the degree of coordinated activation of the EUS varied with the severity of initial injury and the degree of preservation of white matter at the injury site. The 12.5 mm SCI resulted in the sparing of 20% of the white matter at the injury site and complete recovery of detrusor-EUS coordination. In more severely injured rats, the chronic recovery of detrusor-EUS coordination was very incomplete and correlated to decreased innervation of lower motoneurons by descending control pathways and their increased levels of mRNA for glutamate receptor subunits NR2A and GluR2. These results show that the extent of recovery of detrusor-EUS coordination depends on injury severity and the degree of residual connections with brainstem control centers.

Conus medullaris and/or cauda equina forms of spinal cord injury commonly result in a permanent loss of bladder function. Here, we developed a cauda equina injury and repair rodent model to investigate whether surgical implantation of avulsed lumbosacral ventral roots into the spinal cord can promote functional recovery of the lower urinary tract. Adult female rats underwent sham surgery (n ϭ 6), bilateral L5-S2 ventral root avulsion (VRA) injury (n ϭ 5), or bilateral L5-S2 VRA followed by an acute implantation of the avulsed L6 and S1 ventral roots into the conus medullaris (n ϭ 6). At 12 weeks after operation, the avulsed group demonstrated urinary retention, absence of bladder contractions and external urethral sphincter (EUS) electromyographic (EMG) activation during urodynamic recordings, increased bladder size, and retrograde death of autonomic and motoneurons in the spinal cord. In contrast, the implanted group showed reduced urinary retention, return of reflexive bladder voiding contractions coincident with EUS EMG activation, anatomical reinnervation of the EUS demonstrated by retrograde neuronal labeling, normalization of bladder size, and a significant neuroprotection of both autonomic and motoneurons. In addition, a positive correlation between motoneuronal survival and voiding efficiency was observed in the implanted group. Our results show that implantation of avulsed lumbosacral ventral roots into the spinal cord promotes reinnervation of the urinary tract and return of functional micturition reflexes, suggesting that this surgical repair strategy may also be of clinical interest after conus medullaris and cauda equina injuries.

Our findings indicate an evolving interaction between changes in the tissue surrounding the microelectrodes and the microelectrode's electrical properties. Ongoing loss of neurons around recording microelectrodes, and the interactions between their delayed electrical deterioration and early tissue scarring around the tips appear to pose the greatest threats to the microelectrodes' long-term functionality.

Aims/hypothesis A new class of treatments termed bioelectronic medicines are now emerging that aim to target individual nerve fibres or specific brain circuits in pathological conditions to repair lost function and reinstate a healthy balance. Carotid sinus nerve (CSN) denervation has been shown to improve glucose homeostasis in insulin-resistant and glucose-intolerant rats; however, these positive effects from surgery appear to diminish over time and are heavily caveated by the severe adverse effects associated with permanent loss of chemosensory function. Herein we characterise the ability of a novel bioelectronic application, classified as kilohertz frequency alternating current (KHFAC) modulation, to suppress neural signals within the CSN of rodents. Methods Rats were fed either a chow or high-fat/high-sucrose (HFHSu) diet (60% lipid-rich diet plus 35% sucrose drinking water) over 14 weeks. Neural interfaces were bilaterally implanted in the CSNs and attached to an external pulse generator. The rats were then randomised to KHFAC or sham modulation groups. KHFAC modulation variables were defined acutely by respiratory and cardiac responses to hypoxia (10% O 2 + 90% N 2 ). Insulin sensitivity was evaluated periodically through an ITT and glucose tolerance by an OGTT. Results KHFAC modulation of the CSN, applied over 9 weeks, restored insulin sensitivity (constant of the insulin tolerance test [K ITT ] HFHSu sham, 2.56 ± 0.41% glucose/min; K ITT HFHSu KHFAC, 5.01 ± 0.52% glucose/min) and glucose tolerance (AUC HFHSu sham, 1278 ± 20.36 mmol/l × min; AUC HFHSu KHFAC, 1054.15 ± 62.64 mmol/l × min) in rat models of type 2 diabetes. Upon cessation of KHFAC, insulin resistance and glucose intolerance returned to normal values within 5 weeks. Conclusions/interpretation KHFAC modulation of the CSN improves metabolic control in rat models of type 2 diabetes. These positive outcomes have significant translational potential as a novel therapeutic modality for the purpose of treating metabolic diseases in humans.

As millimeter waves (MMWs) are being increasingly used in communications and military applications, their potential effects on biological tissue has become an important issue for scientific inquiry. Specifically, several MMW effects on the whole-nerve activity were reported, but the underlying neuronal changes remain unexplored. This study used slices of cortical tissue to evaluate the MMW effects on individual pyramidal neurons under conditions mimicking their in vivo environment. The applied levels of MMW power are three orders of magnitude below the existing safe limit for human exposure of 1 mW cm(-2). Surprisingly, even at these low power levels, MMWs were able to produce considerable changes in neuronal firing rate and plasma membrane properties. At the power density approaching 1 microW cm(-2), 1 min of MMW exposure reduced the firing rate to one third of the pre-exposure level in four out of eight examined neurons. The width of the action potentials was narrowed by MMW exposure to 17% of the baseline value and the membrane input resistance decreased to 54% of the baseline value across all neurons. These effects were short lasting (2 min or less) and were accompanied by MMW-induced heating of the bath solution at 3 degrees C. Comparison of these results with previously published data on the effects of general bath heating of 10 degrees C indicated that MMW-induced effects cannot be fully attributed to heating and may involve specific MMW absorption by the tissue. Blocking of the intracellular Ca(2+)-mediated signaling did not significantly alter the MMW-induced neuronal responses suggesting that MMWs interacted directly with the neuronal plasma membrane. The presented results constitute the first demonstration of direct real-time monitoring of the impact of MMWs on nervous tissue at a microscopic scale. Implication of these findings for the therapeutic modulation of neuronal excitability is discussed.