publications

2024

1. Role of Local Angiotensin II Signaling in Bladder Function

   Hannah A Anderson , Gabriella L Robilotto , and Aaron D Mickle

   Am. J. Physiol. Renal Physiol., 2024

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   Local renin-angiotensin signaling is present within various organ systems of the body. Angiotensin II signaling acts as a crucial mechanism of the renin-angiotensin signaling system. In this manuscript, we review current literature regarding local angiotensin II signaling in the urinary bladder. Our interest in this pathway is due to its potential role in interstitial cystitis/bladder pain syndrome and other bladder diseases, which are chronic in nature and negatively affect patients’ quality of life. Current treatments for bladder diseases are generally ineffective due to our lack of understanding of their pathophysiology. We evaluate literature investigating angiotensin II signaling in the bladder across several perspectives, including local expression and production, functional properties, tissue morphology, and clinical implications. Further, we identify gaps in knowledge and suggest areas where we can improve our understanding of angiotensin II signaling in the bladder.

2. Hypertension and urologic chronic pelvic pain syndrome: An analysis of MAPP-I data

   Rosalynn R Z Conic , Terrie Vasilopoulos , Karthik Devulapally , Rene Przkora , Andrew Dubin , Kimberly T Sibille , and Aaron D Mickle

   BMC Urol., Jan 2024

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   BACKGROUND: Urologic chronic pelvic pain syndrome (UCPPS), which includes interstitial cystitis/bladder pain syndrome (IC/BPS) and chronic prostatitis (CP/CPPS), is associated with increased voiding frequency, nocturia, and chronic pelvic pain. The cause of these diseases is unknown and likely involves many different mechanisms. Dysregulated renin-angiotensin-aldosterone-system (RAAS) signaling is a potential pathologic mechanism for IC/BPS and CP/CPPS. Many angiotensin receptor downstream signaling factors, including oxidative stress, fibrosis, mast cell recruitment, and increased inflammatory mediators, are present in the bladders of IC/BPS patients and prostates of CP/CPPS patients. Therefore, we aimed to test the hypothesis that UCPPS patients have dysregulated angiotensin signaling, resulting in increased hypertension compared to controls. Secondly, we evaluated symptom severity in patients with and without hypertension and antihypertensive medication use. METHODS: Data from UCPPS patients (n = 424), fibromyalgia or irritable bowel syndrome (positive controls, n = 200), and healthy controls (n = 415) were obtained from the NIDDK Multidisciplinary Approach to the Study of Chronic Pelvic Pain I (MAPP-I). Diagnosis of hypertension, current antihypertensive medications, pain severity, and urinary symptom severity were analyzed using chi-square test and t-test. RESULTS: The combination of diagnosis and antihypertensive medications use was highest in the UCPPS group (n = 74, 18%), followed by positive (n = 34, 17%) and healthy controls (n = 48, 12%, p = 0.04). There were no differences in symptom severity based on hypertension in UCPPS and CP/CPPS; however, IC/BPS had worse ICSI (p = 0.031), AUA-SI (p = 0.04), and BPI pain severity (0.02). Patients (n = 7) with a hypertension diagnosis not on antihypertensive medications reported the greatest severity of pain and urinary symptoms. CONCLUSION: This pattern of findings suggests that there may be a relationship between hypertension and UCPPS. Treating hypertension among these patients may result in reduced pain and symptom severity. Further investigation on the relationship between hypertension, antihypertensive medication use, and UCPPS and the role of angiotensin signaling in UCPPS conditions is needed.

3. Acute ampakines increase voiding function and coordination in a rat model of SCI

   Sabhya Rana , Firoj Alom , Robert C Martinez , David D Fuller , and Aaron D Mickle

   Elife, Jan 2024

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   Neurogenic bladder dysfunction causes urological complications and reduces the quality of life in persons with spinal cord injury (SCI). Glutamatergic signaling via AMPA receptors is fundamentally important to the neural circuits controlling bladder voiding. Ampakines are positive allosteric modulators of AMPA receptors that can enhance the function of glutamatergic neural circuits after SCI. We hypothesized that ampakines can acutely stimulate bladder voiding that has been impaired due to thoracic contusion SCI. Adult female Sprague–Dawley rats received a unilateral contusion of the T9 spinal cord (n = 10). Bladder function (cystometry) and coordination with the external urethral sphincter (EUS) were assessed 5 d post-SCI under urethane anesthesia. Data were compared to responses in spinal-intact rats (n = 8). The ‘low-impact’ ampakine CX1739 (5, 10, or 15 mg/kg) or vehicle (2-hydroxypropyl-beta-cyclodextrin [HPCD]) was administered intravenously. The HPCD vehicle had no discernible impact on voiding. In contrast, following CX1739, the pressure threshold for inducing bladder contraction, voided volume, and the interval between bladder contractions were significantly reduced. These responses occurred in a dose-dependent manner. We conclude that modulating AMPA receptor function using ampakines can rapidly improve bladder-voiding capability at subacute time points following contusion SCI. These results may provide a new and translatable method for therapeutic targeting of bladder dysfunction acutely after SCI.

2023

1. Optogenetic urothelial cell stimulation induces bladder contractions and pelvic nerve afferent firing

   Gabriella L Robilotto , Olivia J Yang , Firoj Alom , Richard D Johnson , and Aaron D Mickle

   Am. J. Physiol. Renal Physiol., Aug 2023

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   Urothelial cells, which play an essential role in barrier function, are also thought to play a sensory role in bladder physiology by releasing signaling molecules in response to sensory stimuli that act upon adjacent sensory neurons. However, it is challenging to study this communication due to the overlap in receptor expression and proximity of urothelial cells to sensory neurons. To overcome this challenge, we developed a mouse model where we can directly stimulate urothelial cells using optogenetics. We crossed a uroplakin II (UPK2) cre mouse with a mouse that expresses the light-activated cation channel channelrhodopsin-2 (ChR2) in the presence of cre expression. Optogenetic stimulation of urothelial cells cultured from UPK2-ChR2 mice initiates cellular depolarization and release of ATP. Cystometry recordings demonstrated that optical stimulation of urothelial cells increases bladder pressure and pelvic nerve activity. Increases in bladder pressure persisted, albeit to a lesser extent, when the bladder was excised in an in vitro preparation. The P2X receptor antagonist PPADS significantly reduced optically evoked bladder contractions in vivo and ex vivo. Furthermore, corresponding nerve activity was also inhibited with PPADS. Our data suggest that urothelial cells can initiate robust bladder contractions via sensory nerve signaling or contractions through local signaling mechanisms. These data support a foundation of literature demonstrating communication between sensory neurons and urothelial cells. Importantly, with further use of these optogenetic tools, we hope to scrutinize this signaling mechanism, its importance for normal micturition and nociception, and how it may be altered in pathophysiological conditions.NEW & NOTEWORTHY Urothelial cells play a sensory role in bladder function. However, it has been particularly challenging to study this communication as both sensory neurons and urothelial cells express similar sensory receptors. Here we demonstrate using an optogenetic technique, that specific urothelial stimulation alone resulted in bladder contractions. This approach will have a long-lasting impact on how we study urothelial-to-sensory neuron communication and the changes that occur under disease conditions.

2. Evaluating the transduction efficiency of systemically delivered AAV vectors in the rat nervous system

   Olivia J Yang , Gabriella L Robilotto , Firoj Alom , Karla Alemán , Karthik Devulapally , Abigail Morris , and Aaron D Mickle

   Front. Neurosci., Jan 2023

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   Gene delivery or manipulation with viral vectors is a frequently used tool in basic neuroscience studies. Adeno-associated viruses (AAV) are the most widely used vectors due to their relative safety and long-term efficacy without causing overt immunological complications. Many AAV serotypes have been discovered and engineered that preferentially transduce different populations of neurons. However, efficient targeting of peripheral neurons remains challenging for many researchers, and evaluation of peripheral neuron transduction with AAVs in rats is limited. Here, we aimed to test the efficiency of systemic AAVs to transduce peripheral neurons in rats. We administered AAV9-tdTomato, AAV-PHP.S-tdTomato, or AAV-retro-GFP systemically to neonatal rats via intraperitoneal injection. After 5 weeks, we evaluated expression patterns in peripheral sensory, motor, and autonomic neurons. No significant difference between the serotypes in the transduction of sensory neurons was noted, and all serotypes were more efficient in transducing NF200 + neurons compared to smaller CGRP + neurons. AAV-retro was more efficient at transducing motor neurons compared to other serotypes. Moreover, PHP.S was more efficient at transducing sympathetic neurons, and AAV-retro was more efficient at transducing parasympathetic neurons. These results indicate that specific AAV serotypes target peripheral neuron populations more efficiently than others in the neonatal rat.

2022

1. Role of Src kinase in regulating protein kinase C mediated phosphorylation of TRPV1

   Gabriella L Robilotto , Durga P Mohapatra , Andrew J Shepherd , and Aaron D Mickle

   Eur. J. Pain, Oct 2022

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   BACKGROUND: Transient receptor potential vanilloid-1 (TRPV1), activated by heat, acidic pH, endogenous vanilloids and capsaicin, is essential for thermal hyperalgesia. Under inflammatory conditions, phosphorylation of TRPV1 by protein kinase C (PKC) can sensitize the channel and decrease the activation threshold. Src kinase also phosphorylates TRPV1, promoting channel trafficking to the plasma membrane. These post-translational modifications are important for several chronic pain conditions. This study presents a previously undescribed relationship between Src and PKC phosphorylation of TRPV1, influencing the thermal hypersensitivity associated with TRPV1 activation. METHODS: We assessed TRPV1 channel activity using intracellular calcium imaging and patch-clamp electrophysiology in mouse dorsal root ganglion cultures. Additionally, we used behavioural experiments to evaluate plantar thermal sensitivity following intraplantar injections of activators of known modulators of TRPV1 with and without an Src antagonist. RESULTS: Using calcium imaging and patch-clamp techniques, we demonstrated that pharmacological inhibition of Src kinase or mutation of the Src phosphorylation site on TRPV1 prevented PKC but not PKA-mediated sensitization of TRPV1 in vitro. We found that intraplantar injection of the PKC activator phorbol 12-myristate 13-acetate (PMA) or bradykinin produces thermal hypersensitivity that can be attenuated by pharmacological inhibition of Src. Additionally, complete Freund’s Adjuvant (CFA)-induced inflammatory hypersensitivity could also be attenuated by local Src kinase inhibition. CONCLUSIONS: Our data demonstrate that Src phosphorylation is critical for PKC-mediated sensitization of TRPV1. Further, in a model of inflammatory pain, CFA, Src kinase inhibition could reduce thermal hypersensitivity. Targeting of Src kinase may have analgesic benefits in inflammatory pain conditions. SIGNIFICANCE: Src kinase-mediated phosphorylation of TRPV1 is a critical regulator of the PKC-induced sensitization induced by multiple inflammatory mediators. This suggest a new regulatory mechanism governing TRPV1 function and a potential therapeutic target for inflammatory type pain, including cancer pain where Src antagonists are currently utilized.

2. Open source timed pressure control hardware and software for delivery of air mediated distensions in animal models

   Trishna Patel , Jamie Hendren , Nathan Lee , and Aaron D Mickle

   HardwareX, Apr 2022

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   Studying the visceral sensory component of peripheral nervous systems can be challenging due to limited options for consistent and controlled stimulation. One method for mechanical stimulation of hollow organs, including the colon and bladder, is controlled distensions mediated by compressed air. For example, distension of the bladder can be used as an assay for bladder nociception. Bladder distension causes a corresponding increase in abdominal electromyography, which increases with distension pressure and is attenuated with analgesics. However, the hardware used to control these distensions are primarily all one-off custom builds, without clear directions on how to build your own. This has made it difficult for these methods to be fully utilized and replicated as not everyone has the access, knowledge, and resources required to build this controller. Here we show an open-source Arduino-based system for controlling a solenoid valve to deliver timed pressure distensions in the experimental model. This device can be controlled by one of two methods through direct TTL pulses from the experimenter’s data acquisition software (ex. CED Spike2) or by a graphical user interface, where the user can set the time before, during, and after distension as well as the number of cycles. This system’s low cost and relative ease to build will allow more groups to utilize timed pressure distensions in their experiments.

2021

1. Characterization of a method to study urodynamics and bladder nociception in male and female mice

   Paulome Srivastava , Henry H Lai , and Aaron D Mickle

   Low. Urin. Tract Symptoms, Apr 2021

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   OBJECTIVES: Abdominal electromyogram or visceromotor response (VMR) elicited by bladder distension is a validated as a measure of bladder nociception in mice, however it is not without its limitations. The aim of this study is to address some of these limitations and validate voiding evoked VMR as a measure of bladder nociception mice. METHODS: Using both male and female C57BL/6 mice we assessed the VMR response to cytometry- induced voiding before and after instillation of 0.5% acetic acid into the bladder. We then delivered intravesical lidocaine to confirm the VMR response as nociceptive. VMR and correlative cystometric bladder pressures were analyzed. RESULTS: We found that the VMR can be evoked by continuous fluid infusion into the bladder of both male and female mice. This response is potentiated after bladder injury and can be attenuated by administration of a local anesthetic, providing strong evidence that this method can be used to evaluate bladder nociception. Further, evaluation of cystometric pressure traces obtained during VMR recording revealed that intercontraction intervals were not altered after bladder injury in either male or female mice. However, we did observe a decrease in peak threshold pressures after bladder injury in female mice, which could be rescued by lidocaine administration. CONCLUSIONS: In conclusion, this technique can measure the VMR and bladder nociception associated with voiding in both female and male mice. Although confounds still exist with the use of anesthesia, further exploration of non-anesthetized voiding-evoked VMR is warranted.

2019

1. Battery-free, fully implantable optofluidic cuff system for wireless optogenetic and pharmacological neuromodulation of peripheral nerves

   Yi Zhang , Aaron D Mickle , Philipp Gutruf , Lisa A McIlvried , Hexia Guo , Yixin Wu , Judith P Golden , Yeguang Xue , Jose G Grajales-Reyes , Xueju Wang , Siddharth Krishnan , Yiwen Xie , Dongsheng Peng , Chun-Ju Su , Fengyi Zhang , Jonathan T Reeder , Sherri K Vogt , Yonggang Huang , John A Rogers , and Robert W Gereau

   Sci. Adv., Jul 2019

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   Studies of the peripheral nervous system rely on controlled manipulation of neuronal function with pharmacologic and/or optogenetic techniques. Traditional hardware for these purposes can cause notable damage to fragile nerve tissues, create irritation at the biotic/abiotic interface, and alter the natural behaviors of animals. Here, we present a wireless, battery-free device that integrates a microscale inorganic light-emitting diode and an ultralow-power microfluidic system with an electrochemical pumping mechanism in a soft platform that can be mounted onto target peripheral nerves for programmed delivery of light and/or pharmacological agents in freely moving animals. Biocompliant designs lead to minimal effects on overall nerve health and function, even with chronic use in vivo. The small size and light weight construction allow for deployment as fully implantable devices in mice. These features create opportunities for studies of the peripheral nervous system outside of the scope of those possible with existing technologies.

2. A wireless closed-loop system for optogenetic peripheral neuromodulation

   Aaron D Mickle , Sang Min Won , Kyung Nim Noh , Jangyeol Yoon , Kathleen W Meacham , Yeguang Xue , Lisa A McIlvried , Bryan A Copits , Vijay K Samineni , Kaitlyn E Crawford , Do Hoon Kim , Paulome Srivastava , Bong Hoon Kim , Seunghwan Min , Young Shiuan , Yeojeong Yun , Maria A Payne , Jianpeng Zhang , Hokyung Jang , Yuhang Li , H Henry Lai , Yonggang Huang , Sung-Il Park , Robert W Gereau , and John A Rogers

   Nature, Jan 2019

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   The fast-growing field of bioelectronic medicine aims to develop engineered systems that can relieve clinical conditions by stimulating the peripheral nervous system1-5. This type of technology relies largely on electrical stimulation to provide neuromodulation of organ function or pain. One example is sacral nerve stimulation to treat overactive bladder, urinary incontinence and interstitial cystitis (also known as bladder pain syndrome)4,6,7. Conventional, continuous stimulation protocols, however, can cause discomfort and pain, particularly when treating symptoms that can be intermittent (for example, sudden urinary urgency)8. Direct physical coupling of electrodes to the nerve can lead to injury and inflammation9-11. Furthermore, typical therapeutic stimulators target large nerve bundles that innervate multiple structures, resulting in a lack of organ specificity. Here we introduce a miniaturized bio-optoelectronic implant that avoids these limitations by using (1) an optical stimulation interface that exploits microscale inorganic light-emitting diodes to activate opsins; (2) a soft, high-precision biophysical sensor system that allows continuous measurements of organ function; and (3) a control module and data analytics approach that enables coordinated, closed-loop operation of the system to eliminate pathological behaviours as they occur in real-time. In the example reported here, a soft strain gauge yields real-time information on bladder function in a rat model. Data algorithms identify pathological behaviour, and automated, closed-loop optogenetic neuromodulation of bladder sensory afferents normalizes bladder function. This all-optical scheme for neuromodulation offers chronic stability and the potential to stimulate specific cell types.

2018

1. Parathyroid hormone-related peptide activates and modulates TRPV1 channel in human DRG neurons

   A J Shepherd , A D Mickle , L A McIlvried , R W Gereau , and D P Mohapatra

   Eur. J. Pain, Oct 2018

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   Parathyroid hormone-related peptide (PTHrP) is associated with advanced tumor growth and metastasis, especially in breast, prostate and myeloma cancers that metastasize to bones, resulting in debilitating chronic pain conditions. Our recent studies revealed that the receptor for PTHrP, PTH1R, is expressed in mouse DRG sensory neurons, and its activation leads to flow-activation and modulation of TRPV1 channel function, resulting in peripheral heat and mechanical hypersensitivity. In order to verify the translatability of our findings in rodents to humans, we explored whether this signalling axis operates in primary human DRG sensory neurons. Analysis of gene expression data from recently reported RNA deep sequencing experiments performed on mouse and human DRGs reveals that PTH1R is expressed in DRG and tibial nerve. Furthermore, exposure of cultured human DRG neurons to PTHrP leads to slow-sustained activation of TRPV1 and modulation of capsaicin-induced channel activation. Both activation and modulation of TRPV1 by PTHrP were dependent on PKC activity. Our findings suggest that functional PTHrP/PTH1R-TRPV1 signalling exists in human DRG neurons, which could contribute to local nociceptor excitation in the vicinity of metastatic bone tumor microenvironment.

2. A bright future? Optogenetics in the periphery for pain research and therapy

   Aaron D Mickle , and Robert W Gereau

   Pain, Sep 2018
3. Macrophage angiotensin II type 2 receptor triggers neuropathic pain

   Andrew J Shepherd , Aaron D Mickle , Judith P Golden , Madison R Mack , Carmen M Halabi , Annette D Kloet , Vijay K Samineni , Brian S Kim , Eric G Krause , Robert W Gereau , and Durga P Mohapatra

   Proc. Natl. Acad. Sci. U. S. A., Aug 2018

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   Peripheral nerve damage initiates a complex series of structural and cellular processes that culminate in chronic neuropathic pain. The recent success of a type 2 angiotensin II (Ang II) receptor (AT2R) antagonist in a phase II clinical trial for the treatment of postherpetic neuralgia suggests angiotensin signaling is involved in neuropathic pain. However, transcriptome analysis indicates a lack of AT2R gene (Agtr2) expression in human and rodent sensory ganglia, raising questions regarding the tissue/cell target underlying the analgesic effect of AT2R antagonism. We show that selective antagonism of AT2R attenuates neuropathic but not inflammatory mechanical and cold pain hypersensitivity behaviors in mice. Agtr2-expressing macrophages (M\Phis) constitute the predominant immune cells that infiltrate the site of nerve injury. Interestingly, neuropathic mechanical and cold pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral M\Phis and AT2R-null hematopoietic cell transplantation. Our study identifies AT2R on peripheral M\Phis as a critical trigger for pain sensitization at the site of nerve injury, and therefore proposes a translatable peripheral mechanism underlying chronic neuropathic pain.

4. Angiotensin II triggers peripheral macrophage-to-sensory neuron redox crosstalk to elicit pain

   Andrew J Shepherd , Bryan A Copits , Aaron D Mickle , Páll Karlsson , Suraj Kadunganattil , Simon Haroutounian , Satya M Tadinada , Annette D Kloet , Manouela V Valtcheva , Lisa A McIlvried , Tayler D Sheahan , Sanjay Jain , Pradipta R Ray , Yuriy M Usachev , Gregory Dussor , Eric G Krause , Theodore J Price , Robert W Gereau , and Durga P Mohapatra

   J. Neurosci., Aug 2018

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   Injury, inflammation, and nerve damage initiate a wide variety of cellular and molecular processes that culminate in hyperexcitation of sensory nerves, which underlies chronic inflammatory and neuropathic pain. Using behavioral readouts of pain hypersensitivity induced by angiotensin II (Ang II) injection into mouse hindpaws, our study shows that activation of the type 2 Ang II receptor (AT2R) and the cell-damage-sensing ion channel TRPA1 are required for peripheral mechanical pain sensitization induced by Ang II in male and female mice. However, we show that AT2R is not expressed in mouse and human dorsal root ganglia (DRG) sensory neurons. Instead, expression/activation of AT2R on peripheral/skin macrophages (M\Phis) constitutes a critical trigger of mouse and human DRG sensory neuron excitation. Ang II-induced peripheral mechanical pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral M\Phis. Furthermore, AT2R activation in M\Phis triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on mouse and human DRG sensory neurons via cysteine modification of the channel. Our study thus identifies a translatable immune cell-to-sensory neuron signaling crosstalk underlying peripheral nociceptor sensitization. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic pain and thus identifies multiple druggable analgesic targets.SIGNIFICANCE STATEMENT Pain is a widespread health problem that is undermanaged by currently available analgesics. Findings from a recent clinical trial on a type II angiotensin II receptor (AT2R) antagonist showed effective analgesia for neuropathic pain. AT2R antagonists have been shown to reduce neuropathy-, inflammation- and bone cancer-associated pain in rodents. We report that activation of AT2R in macrophages (M\Phis) that infiltrate the site of injury, but not in sensory neurons, triggers an intercellular redox communication with sensory neurons via activation of the cell damage/pain-sensing ion channel TRPA1. This MΦ-to-sensory neuron crosstalk results in peripheral pain sensitization. Our findings provide an evidence-based mechanism underlying the analgesic action of AT2R antagonists, which could accelerate the development of efficacious non-opioid analgesic drugs for multiple pain conditions.

5. Parathyroid hormone-related peptide elicits peripheral TRPV1-dependent mechanical hypersensitivity

   Andrew J Shepherd , Aaron D Mickle , Suraj Kadunganattil , Hongzhen Hu , and Durga P Mohapatra

   Front. Cell. Neurosci., Feb 2018

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   Bone metastasis in breast, prostate and lung cancers often leads to chronic pain, which is poorly managed by existing analgesics. The neurobiological mechanisms that underlie chronic pain associated with bone-metastasized cancers are not well understood, but sensitization of peripheral nociceptors by tumor microenvironment factors has been demonstrated to be important. Parathyroid hormone-related peptide (PTHrP) is highly expressed in bone-metastasized breast and prostate cancers, and is critical to growth and proliferation of these tumors in the bone tumor microenvironment. Previous studies have suggested that PTHrP could sensitize nociceptive sensory neurons, resulting in peripheral pain hypersensitivity. In this study, we found that PTHrP induces both heat and mechanical hypersensitivity, that are dependent on the pain-transducing transient receptor potential channel family vanilloid, member-1 (TRPV1), but not the mechano-transducing TRPV4 and TRPA1 ion channels. Functional ratiometric Ca2+ imaging and voltage-clamp electrophysiological analysis of cultured mouse DRG neurons show significant potentiation of TRPV1, but not TRPA1 or TRPV4 channel activation by PTHrP. Interestingly, PTHrP exposure led to the slow and sustained activation of TRPV1, in the absence of any exogenous channel agonist, and is dependent on the expression of the type-1 parathyroid hormone receptor (PTH1), as well as on downstream phosphorylation of the channel by protein kinase C (PKC). Accordingly, local administration of specific small-molecule antagonists of TRPV1 to mouse hindpaws after the development of PTHrP-induced mechanical hypersensitivity led to its significant attenuation. Collectively, our findings suggest that PTHrP/PTH1-mediated flow activation of TRPV1 channel contributes at least in part to the development and maintenance of peripheral mechanical pain hypersensitivity, and could therefore constitute a mechanism for nociceptor sensitization in the context of metastatic bone cancer pain.

6. Miniaturized, battery-free optofluidic systems with potential for wireless pharmacology and optogenetics

   Kyung Nim Noh , Sung Il Park , Raza Qazi , Zhanan Zou , Aaron D Mickle , Jose G Grajales-Reyes , Kyung-In Jang , Robert W Gereau , Jianliang Xiao , John A Rogers , and Jae-Woong Jeong

   Small, Jan 2018

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   Combination of optogenetics and pharmacology represents a unique approach to dissect neural circuitry with high specificity and versatility. However, conventional tools available to perform these experiments, such as optical fibers and metal cannula, are limited due to their tethered operation and lack of biomechanical compatibility. To address these issues, a miniaturized, battery-free, soft optofluidic system that can provide wireless drug delivery and optical stimulation for spatiotemporal control of the targeted neural circuit in freely behaving animals is reported. The device integrates microscale inorganic light-emitting diodes and microfluidic drug delivery systems with a tiny stretchable multichannel radiofrequency antenna, which not only eliminates the need for bulky batteries but also offers fully wireless, independent control of light and fluid delivery. This design enables a miniature (125 mm3 ), lightweight (220 mg), soft, and flexible platform, thus facilitating seamless implantation and operation in the body without causing disturbance of naturalistic behavior. The proof-of-principle experiments and analytical studies validate the feasibility and reliability of the fully implantable optofluidic systems for use in freely moving animals, demonstrating its potential for wireless in vivo pharmacology and optogenetics.

7. Natural wax for transient electronics

   Sang Min Won , Jahyun Koo , Kaitlyn E Crawford , Aaron D Mickle , Yeguang Xue , Seunghwan Min , Lisa A McIlvried , Ying Yan , Sung Bong Kim , Seung Min Lee , and  others

   Advanced Functional Materials, Jan 2018

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   Emerging classes of bioresorbable electronic materials serve as the basis for active biomedical implants that are capable of providing sensing, monitoring, stimulating, and other forms of function over an operating period matched to biological processes such as wound healing. These platforms are of interest because subsequent dissolution, enzymatic degradation, and/or bioresorption can eliminate the need for surgical extraction. This report introduces natural wax materials as long-lived, hydrophobic encapsulation layers for such systems, where biodegradation eventually occurs by chain scission. Studies of wax stability as an encapsulation material demonstrate the ability to retain operation of underlying biodegradable electronic systems for durations between a few days to a few weeks during complete immersion in aqueous solutions in ex-vivo physiological conditions. Electrically conductive composites result from the addition of tungsten micro/nanoparticles, as a conductive, printable paste with similar lifetimes. Demonstrations of these materials in partially biodegradable wireless light-emitting diodes and near-field communication circuits illustrate their use in functional bioresorbable electronic systems. Investigations in animal models reveal no signs of toxicity or other adverse biological responses.

2017

1. Optogenetic silencing of nociceptive primary afferents reduces evoked and ongoing bladder pain

   Vijay K Samineni , Aaron D Mickle , Jangyeol Yoon , Jose G Grajales-Reyes , Melanie Y Pullen , Kaitlyn E Crawford , Kyung Nim Noh , Graydon B Gereau , Sherri K Vogt , H Henry Lai , John A Rogers , and Robert W Gereau

   Sci. Rep., Nov 2017

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   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 nociceptive sensory afferents could be used to modulate bladder pain. The light-activated inhibitory proton pump Archaerhodopsin (Arch) was expressed under control of the sensory neuron-specific sodium channel (sns) gene to selectively silence these neurons. Optically silencing nociceptive sensory 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 nociceptive sensory afferents 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 nociceptive sensory afferents reduced both ongoing pain and evoked cutaneous hypersensitivity in the context of cystitis, but had no effect in uninjured, naı̈ve mice. These results suggest that selective optogenetic silencing of nociceptive bladder afferents may represent a potential future therapeutic strategy for the treatment of bladder pain.

2. Fully implantable, battery-free wireless optoelectronic devices for spinal optogenetics

   Vijay K Samineni , Jangyeol Yoon , Kaitlyn E Crawford , Yu Ra Jeong , Kajanna C McKenzie , Gunchul Shin , Zhaoqian Xie , Saranya S Sundaram , Yuhang Li , Min Young Yang , Jeonghyun Kim , Di Wu , Yeguang Xue , Xue Feng , Yonggang Huang , Aaron D Mickle , Anthony Banks , Jeong Sook Ha , Judith P Golden , John A Rogers , and Robert W Gereau

   Pain, Nov 2017

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   The advent of optogenetic tools has allowed unprecedented insights into the organization of neuronal networks. Although recently developed technologies have enabled implementation of optogenetics for studies of brain function in freely moving, untethered animals, wireless powering and device durability pose challenges in studies of spinal cord circuits where dynamic, multidimensional motions against hard and soft surrounding tissues can lead to device degradation. We demonstrate here a fully implantable optoelectronic device powered by near-field wireless communication technology, with a thin and flexible open architecture that provides excellent mechanical durability, robust sealing against biofluid penetration and fidelity in wireless activation, thereby allowing for long-term optical stimulation of the spinal cord without constraint on the natural behaviors of the animals. The system consists of a double-layer, rectangular-shaped magnetic coil antenna connected to a microscale inorganic light-emitting diode (μ-ILED) on a thin, flexible probe that can be implanted just above the dura of the mouse spinal cord for effective stimulation of light-sensitive proteins expressed in neurons in the dorsal horn. Wireless optogenetic activation of TRPV1-ChR2 afferents with spinal μ-ILEDs causes nocifensive behaviors and robust real-time place aversion with sustained operation in animals over periods of several weeks to months. The relatively low-cost electronics required for control of the systems, together with the biocompatibility and robust operation of these devices will allow broad application of optogenetics in future studies of spinal circuits, as well as various peripheral targets, in awake, freely moving and untethered animals, where existing approaches have limited utility.

3. Flexible near-field wireless optoelectronics as subdermal implants for broad applications in optogenetics

   Gunchul Shin , Adrian M Gomez , Ream Al-Hasani , Yu Ra Jeong , Jeonghyun Kim , Zhaoqian Xie , Anthony Banks , Seung Min Lee , Sang Youn Han , Chul Jong Yoo , Jong-Lam Lee , Seung Hee Lee , Jonas Kurniawan , Jacob Tureb , Zhongzhu Guo , Jangyeol Yoon , Sung-Il Park , Sang Yun Bang , Yoonho Nam , Marie C Walicki , Vijay K Samineni , Aaron D Mickle , Kunhyuk Lee , Seung Yun Heo , Jordan G McCall , Taisong Pan , Liang Wang , Xue Feng , Tae-Il Kim , Jong Kyu Kim , Yuhang Li , Yonggang Huang , Robert W Gereau , Jeong Sook Ha , Michael R Bruchas , and John A Rogers

   Neuron, Feb 2017

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   In vivo optogenetics provides unique, powerful capabilities in the dissection of neural circuits implicated in neuropsychiatric disorders. Conventional hardware for such studies, however, physically tethers the experimental animal to an external light source, limiting the range of possible experiments. Emerging wireless options offer important capabilities that avoid some of these limitations, but the current size, bulk, weight, and wireless area of coverage is often disadvantageous. Here, we present a simple but powerful setup based on wireless, near-field power transfer and miniaturized, thin, flexible optoelectronic implants, for complete optical control in a variety of behavioral paradigms. The devices combine subdermal magnetic coil antennas connected to microscale, injectable light-emitting diodes (LEDs), with the ability to operate at wavelengths ranging from UV to blue, green-yellow, and red. An external loop antenna allows robust, straightforward application in a multitude of behavioral apparatuses. The result is a readily mass-producible, user-friendly technology with broad potential for optogenetics applications.

2016

1. Stretchable multichannel antennas in soft wireless optoelectronic implants for optogenetics

   Sung Il Park , Gunchul Shin , Jordan G McCall , Ream Al-Hasani , Aaron Norris , Li Xia , Daniel S Brenner , Kyung Nim Noh , Sang Yun Bang , Dionnet L Bhatti , Kyung-In Jang , Seung-Kyun Kang , Aaron D Mickle , Gregory Dussor , Theodore J Price , Robert W Gereau , Michael R Bruchas , and John A Rogers

   Proc. Natl. Acad. Sci. U. S. A., Dec 2016

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   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.

2. Nociceptive TRP channels: Sensory detectors and transducers in multiple pain pathologies

   Aaron D Mickle , Andrew J Shepherd , and Durga P Mohapatra

   Pharmaceuticals (Basel), Nov 2016

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   Specialized receptors belonging to the transient receptor potential (TRP) family of ligand-gated ion channels constitute the critical detectors and transducers of pain-causing stimuli. Nociceptive TRP channels are predominantly expressed by distinct subsets of sensory neurons of the peripheral nervous system. Several of these TRP channels are also expressed in neurons of the central nervous system, and in non-neuronal cells that communicate with sensory nerves. Nociceptive TRPs are activated by specific physico-chemical stimuli to provide the excitatory trigger in neurons. In addition, decades of research has identified a large number of immune and neuromodulators as mediators of nociceptive TRP channel activation during injury, inflammatory and other pathological conditions. These findings have led to aggressive targeting of TRP channels for the development of new-generation analgesics. This review summarizes the complex activation and/or modulation of nociceptive TRP channels under pathophysiological conditions, and how these changes underlie acute and chronic pain conditions. Furthermore, development of small-molecule antagonists for several TRP channels as analgesics, and the positive and negative outcomes of these drugs in clinical trials are discussed. Understanding the diverse functional and modulatory properties of nociceptive TRP channels is critical to function-based drug targeting for the development of evidence-based and efficacious new generation analgesics.

2015

1. Induction of thermal and mechanical hypersensitivity by parathyroid hormone-related peptide through upregulation of TRPV1 function and trafficking

   Aaron D Mickle , Andrew J Shepherd , Lipin Loo , and Durga P Mohapatra

   Pain, Sep 2015

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   The neurobiological mechanisms underlying chronic pain associated with cancers are not well understood. It has been hypothesized that factors specifically elevated in the tumor microenvironment sensitize adjacent nociceptive afferents. We show that parathyroid hormone-related peptide (PTHrP), which is found at elevated levels in the tumor microenvironment of advanced breast and prostate cancers, is a critical modulator of sensory neurons. Intraplantar injection of PTHrP led to the development of thermal and mechanical hypersensitivity in both male and female mice, which were absent in mice lacking functional transient receptor potential vanilloid-1 (TRPV1). The PTHrP treatment of cultured mouse sensory neurons enhanced action potential firing, and increased TRPV1 activation, which was dependent on protein kinase C (PKC) activity. Parathyroid hormone-related peptide induced robust potentiation of TRPV1 activation and enhancement of neuronal firing at mild acidic pH that is relevant to acidic tumor microenvironment. We also observed an increase in plasma membrane TRPV1 protein levels after exposure to PTHrP, leading to upregulation in the proportion of TRPV1-responsive neurons, which was dependent on the activity of PKC and Src kinases. Furthermore, co-injection of PKC or Src inhibitors attenuated PTHrP-induced thermal but not mechanical hypersensitivity. Altogether, our results suggest that PTHrP and mild acidic conditions could induce constitutive pathological activation of sensory neurons through upregulation of TRPV1 function and trafficking, which could serve as a mechanism for peripheral sensitization of nociceptive afferents in the tumor microenvironment.

2. Sensory TRP channels: the key transducers of nociception and pain

   Aaron D Mickle , Andrew J Shepherd , and Durga P Mohapatra

   Prog. Mol. Biol. Transl. Sci., Feb 2015

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   Peripheral detection of nociceptive and painful stimuli by sensory neurons involves a complex repertoire of molecular detectors and/or transducers on distinct subsets of nerve fibers. The majority of such molecular detectors/transducers belong to the transient receptor potential (TRP) family of cation channels, which comprise both specific receptors for distinct nociceptive stimuli, as well as for multiple stimuli. This chapter discusses the classification, distribution, and functional properties of individual TRP channel types that have been implicated in various nociceptive and/or painful conditions.

2014

1. NMDA receptor mediates chronic visceral pain induced by neonatal noxious somatic stimulation

   Adrian Miranda , Aaron Mickle, Mitchell Bruckert , Pradeep Kannampalli , Banani Banerjee , and Jyoti N Sengupta

   Eur. J. Pharmacol., Dec 2014

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   NMDA receptors (NMDAR) are important in the development and maintenance of central sensitization. Our objective was to investigate the role of spinal neurons and NMDAR in the maintenance of chronic visceral pain. Neonatal rats were injected with acidic saline adjusted to pH 4.0 in the gastrocnemius muscle every other day for 12 days. In adult rats, NR1 and NR2B subunits were examined in the lumbo-sacral (LS) spinal cord. A baseline, visceromotor response (VMR) to graded colorectal distension (CRD) was recorded before and after administration of the NMDA antagonist, CGS-19755. Extracellular recordings were performed from CRD-sensitive LS spinal neurons and pelvic nerve afferents (PNA) before and after CGS-19755. Rats that received pH 4.0 saline injections demonstrated a significant increase in the expression NR2B subunits and VMR response to CRD>20 mmHg. CGS-19755 (i.v. or i.t.) had no effect in naı̈ve rats, but significantly decreased the response to CRD in pH 4.0 saline injected rats. CGS-19755 had no effect on the spontaneous firing of SL-A, but decreased that of SL-S. Similarly, CGS-19755 attenuates the responses of SL-S neurons to CRD, but had no effect on SL-A neurons or on the response characteristics of PNA fibers. Neonatal noxious somatic stimulation results in chronic visceral hyperalgesia and sensitizes a specific subpopulation of CRD-sensitive spinal neurons. The sensitization of these SL-S spinal neurons is attenuated by the NMDAR antagonist. The results of this study suggest that spinal NMDARs play an important role in the development of hyperalgesia early in life.

2. Interference with peroxisome proliferator-activated receptor-γin vascular smooth muscle causes baroreflex impairment and autonomic dysfunction

   Giulianna R Borges , Donald A Morgan , Pimonrat Ketsawatsomkron , Aaron D Mickle , Anthony P Thompson , Martin D Cassell , Durga P Mohapatra , Kamal Rahmouni , and Curt D Sigmund

   Hypertension, Sep 2014

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   S-P467L mice expressing dominant negative peroxisome proliferator-activated receptor-γselectively in vascular smooth muscle exhibit impaired vasodilation, augmented vasoconstriction, hypertension, and tachycardia. We hypothesized that tachycardia in S-P467L mice is a result of baroreflex dysfunction. S-P467L mice displayed increased sympathetic traffic to the heart and decreased baroreflex gain and effectiveness. Carotid arteries exhibited inward remodeling but no changes in distensibility or stress/strain. Aortic depressor nerve activity in response to increased arterial pressure was blunted in S-P467L mice. However, the arterial pressure and heart rate responses to aortic depressor nerve stimulation were unaltered in S-P467L mice, suggesting that the central and efferent limbs of the baroreflex arc remain intact. There was no transgene expression in nodose ganglion and no change in expression of the acid-sensing ion channel-2 or -3 in nodose ganglion. There was a trend toward decreased expression of transient receptor potential vanilloid-1 receptor mRNA in nodose ganglion, but no difference in the immunochemical staining of transient receptor potential vanilloid-1 receptor in the termination area of the left aortic depressor nerve in S-P467L mice. Although there was no difference in the maximal calcium response to capsaicin in cultured nodose neurons from S-P467L mice, there was decreased desensitization of transient receptor potential vanilloid-1 receptor channels. In conclusion, S-P467L mice exhibit baroreflex dysfunction because of a defect in the afferent limb of the baroreflex arc caused by impaired vascular function, altered vascular structure, or compromised neurovascular coupling. These findings implicate vascular smooth muscle peroxisome proliferator activated receptor-γas a critical determinant of neurovascular signaling.

3. Visceral analgesic effect of 5-HT(4) receptor agonist in rats involves the rostroventral medulla (RVM)

   Jyoti N Sengupta , Aaron Mickle, Pradeep Kannampalli , Russell Spruell , John McRorie , Reza Shaker , and Adrian Miranda

   Neuropharmacology, Apr 2014

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   The 5-HT(4) receptor agonist tegaserod (TEG) has been reported to modulate visceral pain. However, the underlying mechanism remains unknown. The objective of the present study was to examine the analgesic mechanism and site of action of TEG. In male rats, visceral pain was assessed by measuring visceromotor response (VMR) to colorectal distension (CRD). Inflammation was induced by intracolonic injection of tri-nitrobenzene sulfonic acid (TNBS). The effect of TEG on the VMR was tested by injecting intraperitoneal (i.p.), intrathecal (i.t.), intracerebroventricular (i.c.v) or in the rostroventral medulla (RVM). The effect of the drug was also tested on responses of CRD-sensitive pelvic nerve afferents (PNA) and lumbo-sacral (LS) spinal neurons. Systemic injection of TEG attenuated VMR in naive and TNBS-treated rats. Similarly, supraspinal, but not spinal, injection of TEG attenuated the VMR. While GR113808, (selective 5-HT(4) antagonist) blocked the effect, naloxone (NLX) an opioid receptor antagonist reversed the effect of TEG. Although i.t. NLX did not block the inhibitory effect of TEG in VMR study, i.t. injection of \alpha2-adrenergic receptor antagonist yohimbine blocked the effect of TEG when given systemically. While TEG had no effect on the responses of CRD-sensitive PNA, it inhibited the responses of CRD-sensitive LS neurons in spinal intact condition. This inhibition was blocked by GR113808, NLX and β-funaltrexamine (β-FNA) when injected into the RVM. Results indicate that TEG produces analgesia via activation of supraspinal 5-HT(4) receptors which triggers the release of opioids at supraspinal site, which activates descending noradrenergic pathways to the spinal cord to produce analgesia.

2012

1. Distinct modifications in Kv2.1 channel via chemokine receptor CXCR4 regulate neuronal survival-death dynamics

   Andrew J Shepherd , Lipin Loo , Raeesa P Gupte , Aaron D Mickle , and Durga P Mohapatra

   J. Neurosci., Dec 2012

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   The chemokine stromal cell-derived factor-1α(SDF-1α) has multiple effects on neuronal activity, survival, and death under conditions that generate a proinflammatory microenvironment within the brain, via signaling through C-X-C-type chemokine receptor 4 (CXCR4), although the underlying cellular/molecular mechanisms are unclear. Using rat hippocampal neurons, we investigated distinct modifications in the voltage-gated K⁺ (Kv) channel Kv2.1 in response to short- and long-term SDF-1α/CXCR4-mediated signaling as an underlying mechanism for CXCR4-dependent regulation of neuronal survival and death. Acute exposure of neurons to SDF-1αled to dynamic dephosphorylation and altered localization of Kv2.1 channel, resulting in enhanced voltage-dependent activation of Kv2.1-based delayed-rectifier Kv currents (I(DR)). These changes were dependent on CXCR4- and/or NMDA receptor-mediated activation of calcineurin and provide neuroprotection. However, prolonged SDF-1αtreatment leads to CXCR4-mediated activation of p38 mitogen-activated protein kinase, resulting in phosphorylation of Kv2.1 at S800 and enhanced surface trafficking of the channel protein, resulting in increased I(DR)/Kv2.1 current density. This, in combination with sustained dephosphorylation-induced enhancement of the voltage-dependent activation of I(DR)/Kv2.1, predisposed neurons to excessive K⁺ efflux, a vital step for the neuronal apoptotic program. Such apoptotic death was dependent on CXCR4 and Kv2.1 function and was absent in cells expressing the Kv2.1-S800A mutant channel. Furthermore, similar modifications in Kv2.1 and CXCR4/Kv2.1-dependent apoptosis were observed following treatment of neurons with the human immunodeficiency virus-1 (HIV-1) glycoprotein gp120. Therefore, distinct modifications in Kv2.1 in response to short- and long-term CXCR4-mediated signaling could provide a basis for neuroprotection or apoptosis in neuropathologies, such as neuroinflammation, stroke, brain tumors, and HIV-associated neurodegeneration.

2. Pronociceptive effect of 5-HT(1A) receptor agonist on visceral pain involves spinal N-methyl-D-aspartate (NMDA) receptor

   A Mickle , P Kannampalli , M Bruckert , A Miranda , B Banerjee , and J N Sengupta

   Neuroscience, Sep 2012

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   The functional role of serotonergic 5-HT(1A) receptors in the modulation of visceral pain is controversial. The objective of this study was to systematically examine the mechanism and site of action of a selective 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (DPAT) on visceral pain. In the behavioral model of visceral pain, systemic injection (5-250 \mug/kg) of DPAT produced a significant increase in the viscero-motor response (VMR) to colorectal distension (CRD) and this effect was blocked by the selective 5-HT(1A) receptor antagonist WAY-100135 (5 mg/kg, s.c.). Similarly, intrathecal (i.t.) injection (5 \mumol) of DPAT into the lumbo-sacral (L6-S1) spinal cord produced a significant increase in VMR. The administration of N-methyl D-aspartate (NMDA) receptor antagonist AP5 (50 \mug/kg) prior to DPAT injection completely blocked the pronociceptive effect of DPAT. Similarly, DPAT failed to increase VMR in rats chronically treated with NR1 subunit-targeted antisense oligonucleotide (ON), whereas the drug increased VMR in rats treated with mismatched-ON. Chronic i.t. injection of allylglycine (AG), a γ-amino decarboxylase (GAD) enzyme inhibitor, produced significant increase in VMRs, suggesting that the inhibition of GABA synthesis produces pronociception. In AG-treated rats, i.t. injection of DPAT failed to further increase in VMR, suggesting that the DPAT action is linked to GABA release. Similarly, WAY-100135 failed to attenuate VMR in AG-treated rats, suggesting that unlike DPAT, AG action is not via the activation of 5-HT(1A) receptors. In electrophysiology experiments, DPAT (50 \mug/kg) significantly increased the responses of spinal neurons to CRD, but did not influence the mechanotransduction property of CRD-sensitive pelvic nerve afferent fibers. The effect of DPAT on spinal neurons remained unaffected when tested in spinal-transected (C1-C2) rats. These results indicate that the 5-HT(1A) receptor agonist DPAT produces pronociceptive effects, primarily via the activation of presynaptic 5-HT(1A) receptors in GABAergic neuron to restrict GABA release and thereby disinhibits the excitatory glutamatergic neurons in the spinal cord.

3. The C-type natriuretic peptide induces thermal hyperalgesia through a noncanonical Gβγ-dependent modulation of TRPV1 channel

   Lipin Loo , Andrew J Shepherd , Aaron D Mickle , Ramón A Lorca , Leonid P Shutov , Yuriy M Usachev , and Durga P Mohapatra

   J. Neurosci., Aug 2012

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   Natriuretic peptides (NPs) control natriuresis and normalize changes in blood pressure. Recent studies suggest that NPs are also involved in the regulation of pain sensitivity, although the underlying mechanisms remain essentially unknown. Many biological effects of NPs are mediated by guanylate cyclase (GC)-coupled NP receptors, NPR-A and NPR-B, whereas the third NP receptor, NPR-C, lacks the GC kinase domain and acts as the NP clearance receptor. In addition, NPR-C can couple to specific Gα(i)-Gβγ-mediated intracellular signaling cascades in numerous cell types. We found that NPR-C is coexpressed in transient receptor potential vanilloid-1 (TRPV1)-expressing mouse dorsal root ganglia (DRG) neurons. NPR-C can be coimmunoprecipitated with Gα(i), and C-type natriuretic peptide (CNP) treatment induced translocation of protein kinase Cε(PKCε) to the plasma membrane of these neurons, which was inhibited by pertussis toxin pretreatment. Application of CNP potentiated capsaicin- and proton-activated TRPV1 currents in cultured mouse DRG neurons and increased their firing frequency, an effect that was absent in DRG neurons from TRPV1(-/-) mice. CNP-induced sensitization of TRPV1 activity was attenuated by pretreatment of DRG neurons with the specific inhibitors of Gβγ, phospholipase C-β(PLCβ), or PKC, but not of protein kinase A, and was abolished by mutations at two PKC phosphorylation sites in TRPV1. Furthermore, CNP injection into mouse hindpaw led to the development of thermal hyperalgesia that was attenuated by administration of specific inhibitors of Gβγor TRPV1 and was also absent in TRPV1(-/-) mice. Thus, our work identifies the Gβγ-PLCβ-PKC-dependent potentiation of TRPV1 as a novel signaling cascade recruited by CNP in mouse DRG neurons that can lead to enhanced nociceptor excitability and thermal hypersensitivity.

2011

1. Neonatal cystitis-induced colonic hypersensitivity in adult rats: a model of viscero-visceral convergence

   A Miranda , A Mickle , J Schmidt , Z Zhang , R Shaker , B Banerjee , and J N Sengupta

   Neurogastroenterol. Motil., Jul 2011

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   BACKGROUND: The objective of this study was to determine if neonatal cystitis alters colonic sensitivity later in life and to investigate the role of peripheral mechanisms. METHODS: Neonatal rats received intravesical zymosan, normal saline, or anesthesia only for three consecutive days [(postnatal (PN) days 14-16)]. The estrous cycle phase was determined prior to recording the visceromotor response (VMR) to colorectal distension (CRD) in adult rats. Eosinophils and mast cells were examined from colon and bladder tissues. CRD- or urinary bladder distension (UBD)-sensitive pelvic nerve afferents (PNAs) were identified and their responses to distension were examined. The relative expression of N-methyl-d-aspartic acid (NMDA)-NR1 subunit in the lumbo-sacral (L6-S1) spinal cord was examined using Western blot. KEY RESULTS: The VMR to CRD (\geq10mmHg) in the neonatal zymosan group was significantly higher than control in both the diestrus, estrus phase and in all phases combined. There was no difference in the total number of eosinophils, mast cells or number of degranulated mast cells between groups. The spontaneous firing of UBD, but not CRD-sensitive PNAs from the zymosan-treated rats was significantly higher than the saline-treated control. However, the mechanosensitive properties of PNAs to CRD or UBD were no different between groups (P>0.05). The expression of spinal NR1 subunit was significantly higher in zymosan-treated rats compared with saline-treated rats (P<0.05). CONCLUSIONS & INFERENCES: Neonatal cystitis results in colonic hypersensitivity in adult rats without changing tissue histology or the mechanosensitive properties of CRD-sensitive PNAs. Neonatal cystitis does result in overexpression of spinal NR1 subunit in adult rats.

2010

1. Antinociceptive effects of melatonin in a rat model of post-inflammatory visceral hyperalgesia: a centrally mediated process

   Aaron Mickle, Manu Sood , Zhihong Zhang , Golbon Shahmohammadi , Jyoti N Sengupta , and Adrian Miranda

   Pain, Jun 2010

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   Previous reports suggest that melatonin may play an important role in visceral nociception and neurogenic inflammation. We aimed to examine the role of melatonin on visceral hypersensitivity and to explore the site of action using a rat model of post-inflammatory visceral hyperalgesia. In all rats, a baseline viscero-motor response (VMR) to graded colorectal distension (CRD; 10-60mmHg) was recorded prior and 1 week following tri-nitrobenzenesulfonic acid (TNBS) induced colonic inflammation. Melatonin (30, 45 or 60mg/kg, ip) was given 20min before testing the VMR in naı̈ve and TNBS-treated rats. Extracellular single-unit recordings were made from CRD-sensitive pelvic nerve afferent (PNA) fibers and lumbosacral (LS) spinal neurons in TNBS-treated animals. The effect of melatonin (60mg/kg) was examined on responses of PNAs and spinal neurons to graded CRD. In separate experiments, luzindole (non-specific MT(1)/MT(2) receptor antagonist) or naltrexone (non-specific opiod receptor antagonist) was injected prior to melatonin. Following TNBS, there was a significant increase in the VMR to CRD compared to baseline. This increase was attenuated by melatonin (60mg/kg) at pressures >20mmHg. The same dose of melatonin had no effect on the VMR in naı̈ve animals. In TNBS-treated rats, melatonin significantly attenuated the responses of CRD-sensitive spinal neurons to CRD, but had no effect in spinal transected rats or PNA fibers. Both luzindole and naltrexone blocked melatonin’s effect on the VMR and LS spinal neurons. Results indicate melatonin’s antinociceptive effects are not via a peripheral site of action but rather a supra-spinal process linked to the central opioidergic system.

2009

1. Altered mechanosensitive properties of vagal afferent fibers innervating the stomach following gastric surgery in rats

   A Miranda , A Mickle , B Medda , Z Zhang , R J Phillips , N Tipnis , T L Powley , R Shaker , and J N Sengupta

   Neuroscience, Sep 2009

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   BACKGROUND AND AIMS: Several types of gastric surgeries have been associated with early satiety, dyspepsia and food intolerances. We aimed to examine alterations in gastric vagal afferents following gastric surgery-fundus ligation. METHODS: Six week old, male Sprague-Dawley rats underwent chronic ligation (CL) of the fundus. Sham rats underwent abdominal surgery, but without ligation. Another group of rats underwent acute ligation (AL) of the fundus immediately prior to experiments. CL and sham rats were allowed to grow to age 3-4 months. Food intake and weights were recorded post-operatively. Gastric compliance and gastric wall thickness was measured at baseline and during gastric distension (GD). Extracellular recordings were made to examine response characteristics of vagal afferent fibers to GD and to map the stomach receptive field (RF). The morphological structures of afferent terminals in the stomach were examined with retrograde tracings from the nodose ganglion. RESULTS: The CL group consumed significantly less food and weighed less than sham control. The mean compliance of the CL group was significantly less than control, but higher than the AL group. The spontaneous firing and responses to GD of afferent fibers from the CL rats were significantly higher than AL rats. There was a marked expansion of the gastric RF in the CL rats with significant reorganization and regeneration of intramuscular array (IMA) terminals. There was no difference in total wall or muscle thickness among the groups. CONCLUSION: CL results in aberrant remodeling of IMAs with expansion of the gastric RF and alters the mechanotransduction properties of vagal afferent fibers. These changes could contribute to altered sensitivity following gastric surgery.