Neuronal Growth Cone Dynamics are Regulated by a Nitric Oxide-Initiated Second Messenger Pathway.

During development, neurons must find their way to and make connections with their appropriate targets. Growth cones are dynamic, motile structures that are integral to the establishment of appropriate connectivity during this wiring process. As growth cones migrate through their environment, they encounter guidance cues that direct their migration to their appropriate synaptic targets. The gaseous messenger nitric oxide (NO), which diffuses across the plasma membrane to act on intracellular targets, is a signaling molecule that affects growth cone motility. However, most studies have examined the effects of NO on growth cone morphology when applied in large concentrations and to entire cells. In addition, the intracellular second messenger cascade activated by NO to bring about these changes in growth cone morphology is not well understood. Therefore, this dissertation addresses the effects that a spatially- and temporally-restricted application of physiological amounts of NO can have on individual growth cone morphology, on the second messenger pathway that is activated by this application of NO, and on the calcium cascades that result and ultimately affect growth cone morphology. Helisoma trivolvis, a pond snail, is an excellent model system for this type of research because it has a well-defined nervous system and cultured neurons form large growth cones. In the present study, local application of NO to Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number, and an increase in the intracellular calcium concentration ([Ca2+]i). In B5 neurons, the effects of NO on growth cone behavior and [Ca2+]i are mediated via sGC, protein kinase G, cyclic adenosine diphosphate ribose, and ryanodine receptor-mediated intracellular calcium release. This study demonstrates that neuronal growth cone pathfinding in vitro is affected by a single spatially- and temporally-restricted exposure to NO. Furthermore, NO acts via a second messenger cascade, resulting in a calcium increase that leads to cytoskeletal changes. These results suggest that NO may be a signal that promotes appropriate pathfinding and/or target recognition within the developing nervous system. Taken together, these data indicate that NO may be an important messenger during the development of the nervous system in vivo

An Investigation of Lipid Modulation of Low Voltage Activated Currents in Spiral Ganglion Neurons

Type I spiral ganglion neurons (SGNs) synapse onto cochlear inner hair cells and constitute the majority of afferent fibres in the auditory nerve (AN). Better characterisation of their biophysical properties may identify therapeutic targets for optimising AN sensitivity. This study aimed to characterise the membrane physiology underlying the firing properties of post-hearing onset SGNs and investigated whether their properties could be modified by the presence of native and synthetic lipids. In dissociated ganglionic cultures, SGNs displayed an intrinsic variation in their firing properties; this could be correlated with the magnitudes of specific membrane currents. SGNs were categorised by their response to depolarising current injection; SGNs either adapted to the stimulus rapidly, slowly or not at all. Rapid adaptation, a mechanism that preserves temporal precision throughout the auditory system, was found to be regulated by a dendrotoxin-K (DTX-K) and tityustoxin-Kα (TsTx)-sensitive low-threshold voltage-activated (LVA) K+ current, suggesting contribution by Kv1.1 and Kv1.2 subunits. As Kv1.2 channels were known to be positively modulated by membrane phosphoinositides, we investigated the influence of phosphatidylinositol-4,5- bisphosphate (PIP2) availability on SGN K+ currents. Inhibiting PIP2 production using wortmannin, or sequestration using a palmitoylated peptide (PIP2-PP), slowed or abolished adaptation in SGNs. PIP2-PP specifically reduced SGN LVA currents in a manner that was partly rescued by intracellular dialysis with diC8PIP2, a nonhydrolysable analogue of PIP2. PIP2-PP application induced similar levels of current inhibition in Kv1.1/Kv1.2 channels heterologously expressed in HEK293 cells. Accordingly, the lipid sensitivity of the Kv1.2 channel was further explored with a range of native and synthetic free fatty acids. Polyunsaturated fatty acids were found to be strong inhibitors of Kv1.2 currents, offering further potential candidates for SGN modulation. Collectively, this data identifies Kv1.1 and Kv1.2 containing K+ channels as key regulators of excitability in the AN, and potential targets for pharmacological modulation

Forced-Exercise Alleviates Neuropathic Pain in Experimental Diabetes: Effects on Voltage-Gated Calcium Channels

Exercise is now established as an integral adjunct to the management of diabetes. Diabetic polyneuropathy, a painful complication of diabetes, remains untreatable, emphasizing a critical need for improved therapeutic strategies. Recent evidence suggests that exercise may facilitate recovery of peripheral nerve function in diabetes. However, the mechanism by which exercise protects against diabetes-induced nerve dysfunction is unknown. In this dissertation we hypothesized that forced-exercise protects against experimental DPN by preventing glucose-associated alterations of voltage-gated calcium currents (VGCC) in small diameter dorsal root ganglion (DRG) neurons. Using behavioral, nerve-electrophysiology and patch-clamp methodology we examined the functional consequences of forced-exercise (treadmill, 5.4 km/week) on VGCC in dissociated small diameter DRG neurons from rats conferred diabetic by streptozotocin (STZ) treatment. Exercised-STZ rats in comparison to sedentary-STZ rats, demonstrated a 4 week delay in the onset of tactile hyperalgesia that was independent of changes in blood glucose levels. Interestingly, forced-exercise induced protection against diabetes-induced tactile hyperalgesia was reversed in a dose dependent manner by the opioid antagonist, naloxone. Forced-Exercise also prevented peripheral nerve conduction deficits in STZ-treated rats. Small diameter DRG neurons harvested from sedentary-STZ rats with demonstrated hyperalgesia exhibited 2-fold increase in peak high-voltage activated (HVA) Ca2+ current density and low-voltage activated (LVA) Ca2+ current component. The steady-state inactivation (SSI) (measure of channel availability) of LVA currents demonstrated a rightward shift in sedentary-STZ rats (+7.5 mV shift; V50 = -50.9 ± 0.6 mV; vehicle treated rats V50 = -58.4 ± 0.9 mV). Forced-exercise prevented the increase in both, peak HVA Ca2+ current density and LVA SSI shift (V50 = -58.2 ± 1.4 mV), but did not alter LVA current component. We conclude that forced-exercise delayed the onset of diabetic tactile hyperalgesia by preventing the alteration of VGCCs in small diameter DRG neurons, possibly by decreasing total calcium influx and dampening neuronal over-excitability

Modelling changes in excitability of the peripheral nervous system using compartmentalised microfluidic culture

This thesis describes the use of compartmentalised microfluidic devices to investigate changes in neuronal excitability. All studies carried out in this work were completed in line with principles of the NC3Rs (reduction, replacement and refinement). Particular interest was given to the study of the excitability of dorsal root ganglion neurons (DRGs) in the context of pain-based signalling. This also included the in vitro culture and characterisation of non-neuronal cells involved in inflammation and nociception. Current methods for In vitro modelling of pain pathways often fails to replicate the unique morphology of the DRG neurons. These pseudo-unipolar neurons detect nociceptive stimuli at the peripheral terminals, and transduce long range action-potentials to higher processing centres in the central nervous system. Unlike in vivo modelling of pain behaviours, in vitro models of nociception provide the capacity to monitor changes in neuronal function at a cellular and molecular level. However, until the development of technology such as microfluidics, the standard methods of culture failed to isolate the axons from the soma. The primary aim of this project was to develop a model capable of replicating the complex microenvironment that terminals of the DRG neurons encounter during the development and onset of pain. This involved the optimisation of cell culture methods for inflammatory cells used to induce changes in neuronal excitability, both from the context of the peripheral terminals, or from the CNS if desired. At a molecular level, the microfluidic model was also used to investigate the role of small non-coding RNA (microRNAs) on regulating DRG excitability in the context of nociception. This Thesis hypothesises that voltage-gated potassium channels form an interesting target for a microRNA of interest. However, it is widely acknowledged that microRNAs regulate the expression of multiple mRNAs. The use of functional studies using the microfluidic model have shown here that there are differences in the way in which a neuron responds to a stimulus, dependent on whether it is applied locally to the axon or the soma. Live cell imaging was used to measure evoked changes in Ca2+ transients as a proxy for cell excitability. As well as significant differences in the response to depolarising agents such as potassium chloride (KCL), the use of biologically relevant stimuli to the study of nociception was also developed. The culture of inflammatory cells such as bone marrow derived macrophages led to the development of cytokine-rich media which was used to evoke changes in neuronal excitability. By exploiting the microfluidic nature of the device, subsequent investigations to the role of microRNA 138-5p in regulating neuronal excitability were undertaken. The use of cell permeable microRNA inhibition showed a reduction in cell excitability if applied locally to the axons. Bioinformatics led to the development of Kv1.2 as a potential target for miR-138-5p in vivo, which could explain the effects of miR-138-5p in modulating excitability of the DRGs. The findings in this work have demonstrated the potential for development of more biologically relevant in vitro models using microfluidic compartmentalised cell culture. For example, fluidic isolation has characterised the role of miR-138-5p in regulating DRG excitability at the axons

The Use of Microfluidic Chambers to Study Action Potential Propagation and Stimulus Transduction in Sensory Neurons in Vitro

Primary afferent sensory neurons can be incredibly long single cellular structures, often traversing distances of over one metre in the human. Cutaneous sensory stimuli are transduced in the periphery by specialised end-organs or free nerve endings which enable the coding of the stimulus into electrical action potentials that propagate towards the central nervous system. Despite significant advances in our knowledge of sensory neuron physiology and ion channel expression, many commonly used techniques fail to accurately model the primary afferent neuron in its entirety. In vitro experiments often focus on the cell somata and neglect the fundamental processes of peripheral stimulus transduction and action potential propagation. Despite this, these experiments are frequently used as a model for cellular investigations of the receptive terminals. Crucially, somal responses may not represent the functional expression of ion channels in the axon and end terminals. The aim of this thesis was to develop a system using compartmentalised culture chambers and ratiometric calcium imaging to directly and accurately compare the sensitivity and functional protein expression of isolated neuronal regions in vitro. Using this preparation I demonstrate that the nerve terminals of cultured DRG neurons can be depolarised to induce action potential propagation, which has both a TTX-resistant and TTX-sensitive component. Furthermore, I show that there is a differential regulation of proton sensitivity between the sensory terminals and somata in cultured sensory neurons. I also go on to show that capsaicin sensitivity is highly dependent on embryonic dissection age. This novel approach enables a comprehensive method to study the excitability characteristics and regional sensitivity differences of cultured sensory neurons on a single cell level. Examination of the sensory terminals is crucial to further understand the properties and diversity of DRG sensory neurons

Modelling changes in excitability of the peripheral nervous system using compartmentalised microfluidic culture

This thesis describes the use of compartmentalised microfluidic devices to investigate changes in neuronal excitability. All studies carried out in this work were completed in line with principles of the NC3Rs (reduction, replacement and refinement). Particular interest was given to the study of the excitability of dorsal root ganglion neurons (DRGs) in the context of pain-based signalling. This also included the in vitro culture and characterisation of non-neuronal cells involved in inflammation and nociception. Current methods for In vitro modelling of pain pathways often fails to replicate the unique morphology of the DRG neurons. These pseudo-unipolar neurons detect nociceptive stimuli at the peripheral terminals, and transduce long range action-potentials to higher processing centres in the central nervous system. Unlike in vivo modelling of pain behaviours, in vitro models of nociception provide the capacity to monitor changes in neuronal function at a cellular and molecular level. However, until the development of technology such as microfluidics, the standard methods of culture failed to isolate the axons from the soma. The primary aim of this project was to develop a model capable of replicating the complex microenvironment that terminals of the DRG neurons encounter during the development and onset of pain. This involved the optimisation of cell culture methods for inflammatory cells used to induce changes in neuronal excitability, both from the context of the peripheral terminals, or from the CNS if desired. At a molecular level, the microfluidic model was also used to investigate the role of small non-coding RNA (microRNAs) on regulating DRG excitability in the context of nociception. This Thesis hypothesises that voltage-gated potassium channels form an interesting target for a microRNA of interest. However, it is widely acknowledged that microRNAs regulate the expression of multiple mRNAs. The use of functional studies using the microfluidic model have shown here that there are differences in the way in which a neuron responds to a stimulus, dependent on whether it is applied locally to the axon or the soma. Live cell imaging was used to measure evoked changes in Ca2+ transients as a proxy for cell excitability. As well as significant differences in the response to depolarising agents such as potassium chloride (KCL), the use of biologically relevant stimuli to the study of nociception was also developed. The culture of inflammatory cells such as bone marrow derived macrophages led to the development of cytokine-rich media which was used to evoke changes in neuronal excitability. By exploiting the microfluidic nature of the device, subsequent investigations to the role of microRNA 138-5p in regulating neuronal excitability were undertaken. The use of cell permeable microRNA inhibition showed a reduction in cell excitability if applied locally to the axons. Bioinformatics led to the development of Kv1.2 as a potential target for miR-138-5p in vivo, which could explain the effects of miR-138-5p in modulating excitability of the DRGs. The findings in this work have demonstrated the potential for development of more biologically relevant in vitro models using microfluidic compartmentalised cell culture. For example, fluidic isolation has characterised the role of miR-138-5p in regulating DRG excitability at the axons

Lycium barbarum (wolfberry) polysaccharide facilitates ejaculatory behaviour in male rats

Poster Session AOBJECTIVE: Lycium barbarum (wolfberry) is a traditional Chinese medicine, which has been considered to have therapeutic effect on male infertility. However, there is a lack of studies support the claims. We thus investigated the effect of Lycium barbarum polysaccharide (LBP), a major component of wolfberry, on male rat copulatory behavior. METHOD: Sprague-Dawley rats were divided into two groups (n=8 for each group). The first group received oral feeding of LBP at dosage of 1mg/kg daily. The control group received vehicle (0.01M phosphate-buffered saline, served as control) feeding daily for 21 days. Copulatory tests were conducted at 7, 14 and 21 days after initiation of treatment. RESULTS: Compared to control animals, animals fed with 1mg/kg LBP showed improved copulatory behavior in terms of: 1. Higher copulatory efficiency (i.e. higher frequency to show intromission rather than mounting during the test), 2. higher ejaculation frequency and 3. Shorter ejaculation latency. The differences were found at all time points (Analyzed with two-tailed student’s t-test, p<0.05). There is no significant difference found between the two groups in terms of mount/intromission latency, which indicates no difference in time required for initiation of sexual activity. Additionally, no difference in mount frequency and intromission frequency was found. CONCLUSION: The present study provides scientific evidence for the traditional use of Lycium barbarum on male sexual behavior. The result provides basis for further study of wolfberry on sexual functioning and its use as an alternative treatment in reproductive medicine.postprintThe 30th Annual Meeting of the Australian Neuroscience Society, in conjunction with the 50th Anniversary Meeting of the Australian Physiological Society (ANS/AuPS 2010), Sydney, Australia, 31 January-3 February 2010. In Abstract Book of ANS/AuPS, 2010, p. 177, abstract no. POS-TUE-19

Studies on Cellular Mechanisms and Single-Cell Transcriptional Profiling

학위논문 (박사) -- 서울대학교 대학원 : 자연과학대학 뇌인지과학과, 2020. 8. 오석배이인아.Pain sensations experienced in the body, as well as in the orofacial region, convey to the central nervous system (CNS) by peripheral sensory neurons such as the dorsal root ganglion (DRG) and the trigeminal ganglion (TG). Nociceptive pain, called nociception, begins at peripheral nerve endings of specialized peripheral sensory neurons known as nociceptors, which exclusively respond to noxious stimuli. Up to date, it has been well studied how the distinct nociceptors respond to various noxious stimuli such as heat, cold, chemical, or mechanical. However, it remains relatively little understood on mechanisms for how innocuous or non-painful stimuli, which would never be considered noxious, can cause pain. In this study, I have explored these polymodalities of nociception that may be elicited by innocuous stimuli or endogenous mediators of inflammation in several pathophysiological conditions as below. In the first chapter, I examined the contribution of peripheral γ–aminobutyric acid type A receptor (GABAAR) that may be activated by endogenous GABA produced in the inflamed tissue to inflammatory pain. Using complete Freunds adjuvant (CFA)-induced persistent inflammatory pain mouse model, I demonstrated that CFA-induced spontaneous nociceptive behavior and mechanical hypersensitivity were inhibited by intraplantar (i.pl.) injection of GABAAR antagonists. Moreover, local blockage of endogenous GABA with injection (i.pl.) of anti-GABA antibody attenuated CFA-induced mechanical hypersensitivity, whereas, i.pl. injection of a positive allosteric modulator of GABAAR facilitated mechanical allodynia in naïve mice. These findings suggest that peripheral GABAAR signaling contributes to CFA-induced hypersensitivity, and its modulation can potentially be a therapeutic target for inflammatory pain alleviation. In the second chapter, I investigated cellular mechanisms by which sweet substances excite dental primary afferent (DPA) neurons, thereby leading dentin hypersensitivity. Sweet substances contain high sugar concentrations so that they cause hyperosmolar conditions at the teeth. I thus demonstrated that the transient receptor potential cation channel, subfamily M, member 8 (TRPM8), a well-known cold- and menthol-receptor, also served as a hyperosmosensor in DPA neurons. By applying a hyperosmolar sucrose solution to the mouse exposed tooth dentin, I investigated whether TRPM8 in DPA neurons mediates upregulation of c-fos expression as a marker of hyperosmolarity-induced nociception in the CNS level. I showed that hyperosmolarity-induced dental nociception was significantly attenuated by a selective TRPM8 antagonist, implying innocuous stimuli such as sweet substances can be sufficient to result in dental nociception via TRPM8 channels. In recent years, an increasing number of studies have successfully applied single-cell transcriptomics to characterize a population of cells and to identify rare subtypes or novel therapeutic targets in heterogeneous sensory system. In the third chapter, I thus employed transcriptional profiling using single-cell RNA sequencing (scRNA-seq) and specific gene-expression validation with in situ hybridization in order to identify unique molecular signatures in the adult mouse DPA neurons. The single-cell transcriptome analysis detected six distinct clusters of DPA neurons. Interestingly, a particular cluster of DPA neurons was characterized by high expression of a low-threshold mechanosensitive Piezo2 ion channel and a pain-related neuropeptide Calca encoding CGRP (calcitonin gene-related peptide). These findings provide an insight into one of the previously proposed mechanisms underlying dentin hypersensitivity (i.e., hydrodynamic theory; Brännström and Astroem 1964), which is a common occurrence by innocuous mechanical irritations such as brush or air puffs. I further discussed mechanosensitive ion channels that may play critical roles in generating pain within the tooth pulp and their clinical implications.신체와 구강악안면 영역에서 경험되는 통증 감각은 후근신경절(dorsal root ganglion)과 삼차신경절(trigeminal ganglion)과 같은 말초 감각 신경을 통해 중추 신경계로 전달된다. 일반적인 통증 감각은 유해감수기(nociceptor)로 불리는 특수한 말초 감각 신경의 말단에서 시작하고 이러한 유해감수기는 유해 자극에 주로 반응한다. 지금까지 열, 저온, 화학 물질 또는 기계적 자극과 같은 다양한 유해 자극에 반응하는 유해감수기에 대해서는 잘 연구되었지만 특수한 병태생리학적 상황에서는 무해한 자극도 통증을 일으킬 수 있는데, 이에 대한 기전은 아직 완전하게 규명되지 않았다. 따라서 본 학위 논문은 여러 유형의 병태생리 상태에서 무해 자극 또는 염증으로 인한 내생적 매개체(endogenous mediator)에 의해서 활성화될 수 있는 통증 감각의 기전을 세포, 분자 생물학 및 동물행동학적 수준에서 다음과 같이 탐구하고자 했다. 첫 번째 장에서는 염증 상태에서 내생적 감마아미노 낙산(GABA)에 의해 활성화 될 수 있는 감마아미노 낙산 유형 A 수용체(GABAAR)의 역할을 조사했다. CFA로 유도된 염증성 통증 마우스 모델을 사용하여, 후근신경절의 말단에 발현하고 있는 감마아미노 낙산 유형 A 수용체의 활성을 줄이기 위한 선택적 억제제의 사용 또는 항-감마아미노 낙산 항체를 염증 부위에 주사하여 내생적 감마아미노 낙산을 직접적으로 차단함으로써 조절했을 때, CFA로 유도된 자발적 통증 행동(spontaneous nociceptive behavior)과 기계적 자극에 대한 통각과민(mechanical hypersensitivity)이 감소했다. 이와는 반대로 감마아미노 낙산 유형 A 수용체의 활성을 양성 입체다른자리 조절자로 증가시켰을 때는 정상 마우스에서도 기계적 자극에 대한 이질통(mechanical allodynia)이 증가했다. 이를 통해 말초 감각 신경의 감마아미노 낙산 유형 A 수용체를 매개로 하는 신호 전달이 염증성 통증 조절을 위한 새로운 치료 표적이 될 수 있음을 입증했다. 두 번째 장에서는 단 물질이 어떻게 치수유래 일차 구심성 신경(dental primary afferent neuron)을 자극하여 상아질 과민증(dentin hypersensitivity)을 유발하는지에 대한 세포 기전을 탐색했다. 설탕 농도가 높은 단 물질은 삼투압이 높기 때문에 우리가 단 음식을 섭취할 때 치아는 고삼투압 상태가 되며, 단 음식은 치통을 빈번하게 유발한다. 따라서, TRPM8 이온 채널-저온과 멘톨 수용체로 잘 알려져 있지만 특별히 각막 신경에서는 각막의 건조와 같은 고삼투압 상태에서 눈의 깜빡임을 조절하는 것으로 보고됨-이 치수유래 일차 구심성 신경에서 고삼투압 자극에 활성화되어 통증 감각을 전달할 수 있음을 확인하고자 했다. 마우스의 치수유래 일차 구심성 신경 세포에는 TRPM8이 기능적으로 발현하고 있었으며, 살아있는 마우스의 치아 상아질을 노출시키고 고삼투압성의 설탕 용액을 적용함으로써 유발된 통증 감각이 TRPM8의 선택적 억제제에 의해서 현저하게 감소되는 것을 중추신경계 수준에서 확인했다. 따라서, 단 물질과 같은 무해한 자극이 TRPM8 채널을 통해 치통을 유발할 수 있음을 증명했다. 최근에 점점 더 많은 연구가 단일 세포 수준의 전사유전체학(single-cell transcriptomics)을 사용하여 혼합된 세포 집단 또는 복잡한 감각 신경 시스템에서 세포의 이종성, 희귀한 세포 유형, 새로운 치료 표적을 성공적으로 발견하고 있다. 세 번째 장에서는 마우스의 치수유래 일차 구심성 신경 세포에서 보이는 독특한 유전자 발현을 탐색하기 위해 단일 세포 수준의 RNA 시퀀싱(single-cell RNA sequencing) 기술과 현장혼성화(in situ hybridization) 조직 염색 기법을 도입했다. 단일 세포 수준의 전사체 프로파일링을 통해 치아 신경에서 여섯 개의 구별된 세포 집단(cluster)들이 존재하는 것을 발견했다. 흥미롭게도 가장 큰 집단은 약한 기계적 자극에 활성화 되는 Piezo2 이온 채널과 CGRP(칼시토닌 연관 단백질)를 부호화(encoding)하는 통증 관련 신경전달물질인 Calca의 높은 발현을 특징으로 했다. 이러한 발견은 치아의 신경세포가 피부 영역에 분포 되어 있는 다른 감각 신경과는 달리, 칫솔질 또는 공기 분사와 같은 약한 기계적 자극이 빈번하게 상아질 과민증을 발생시키는 기전을 설명하며, 이는 기존에 중요하게 제안된 유체역학 이론(hydrodynamic theory)을 입증했다. 추가적으로 나는 치통에 중요하게 관여할 수 있는 기계적 감각 이온 채널들과 본 연구의 임상적 적용에 대해서 논의했다.CHAPTER 1: Functional Roles of Peripheral GABAA Receptors in Persistent Inflammatory Hypersensitivity 29 Abstract 30 Introduction 32 Materials and methods 34 Results 39 Discussion 56 CHAPTER 2: Cellular Mechanisms of TRPM8 Channels Contributing to Dentin Hypersensitivity 62 Abstract 63 Introduction 64 Materials and methods 66 Results 74 Discussion 87 CHAPTER 3: Gene-Expression Signatures of the Adult Mouse Dental Sensory System 93 Abstract 94 Introduction 95 Materials and methods 98 Results 104 Discussion 117 Conclusion 123 Bibliography 125 국문초록 142Docto

An electrophysiological study of the interaction between fenamate NSAIDs and the GABA(_A) receptor

The effects of certain NSAIDs were determined on agonist-evoked responses recorded from rat neurones maintained in vitro using electrophysiological techniques. Initially, the rat isolated vagus and optic nerves were employed. Alphaxalone, pentobarbitone, propofol and the NSAID, mefenamic acid (MFA), potentiated GABA-evoked responses of the vagus nerve. Propofol (1-100µM) selectively potentiated GABA and glycine-evoked responses of the rat vagus and optic nerves, but had little effect on nicotinic acetylcholine-, a,β-methylene-ATP or 5-hydroxytryptamine-mediated responses. The interaction between MFA and ligand-gated receptors was investigated further using voltage-clamped rat hippocampal neurones maintained in culture. MFA (3-100µM) selectively, concentration-dependently and reversibly potentiated GABA-evoked responses, consistent with the observations made using the vagus nerve. MFA (3-100|aM) however had little or no effect on glycine, AMPA, kainate or NMDA-receptor mediated responses. A final series of experiments investigated the site and molecular mechanism of the interaction between MFA and the GABA-gated chloride ion channel. The potentiating effects of MFA (and other fenamates) were not the result of prostaglandin synthesis inhibition, since other NSAIDs did not modulate the GABA(_A) receptor (GR). The actions of MFA were not mediated via the benzodiazepine site of the GR, nor where they due to inhibition of GABA- uptake or membrane perturbation. The modulatory effects of MFA were not use-dependent, but the potentiating effects of MFA were voltage-dependent, where the potentiation was 3-fold greater at -100mV than at +40mV, with no change in the equilibrium potential for GABA. MFA activated a current, in the absence of GABA. Hippocampal neurones varied in sensitivity to modulation by MFA and the anticonvulsant, loreclezole, which may indicate a degree of sub- unit selectivity. These data are discussed in relation to the possible site and mechanism of action of fenamates at the GR, their similarities with other positive modulators of the GR and the neurophysiological implications of these findings