Ruth E. Brooke

Neuronal P2X7 Receptors Are Targeted to Presynaptic Terminals in the Central and Peripheral Nervous Systems

The ionotropic ATP receptor subunits P2X1–6 receptors play important roles in synaptic transmission, yet the P2X7receptor has been reported as absent from neurons in the normal adult brain. Here we use RT-PCR to demonstrate that transcripts for the P2X7 receptor are present in extracts from the medulla oblongata, spinal cord, and nodose ganglion. Using in situ hybridization mRNA encoding, the P2X7 receptor was detected in numerous neurons throughout the medulla oblongata and spinal cord. Localizing the P2X7 receptor protein with immunohistochemistry and electron microscopy revealed that it is targeted to presynaptic terminals in the CNS. Anterograde labeling of vagal afferent terminals before immunohistochemistry confirmed the presence of the receptor in excitatory terminals. Pharmacological activation of the receptor in spinal cord slices by addition of 2′- and 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP; 30 μm) resulted in glutamate mediated excitation of recorded neurons, blocked by P2X7 receptor antagonists oxidized ATP (100 μm) and Brilliant Blue G (2 μm). At the neuromuscular junction (NMJ) immunohistochemistry revealed that the P2X7 receptor was present in motor nerve terminals. Furthermore, motor nerve terminals loaded with the vital dye FM1–43 in isolated NMJ preparations destained after application of BzATP (30 μm). This BzATP evoked destaining is blocked by oxidized ATP (100 μm) and Brilliant Blue G (1 μm). This indicates that activation of the P2X7 receptor promotes release of vesicular contents from presynaptic terminals. Such a widespread distribution and functional role suggests that the receptor may be involved in the fundamental regulation of synaptic transmission at the presynaptic site.

Properties of interneurones in the intermediolateral cell column of the rat spinal cord : Role of the potassium channel subunit KV3.1

Sympathetic preganglionic neurones located in the intermediolateral cell column (IML) are subject to inputs descending from higher brain regions, as well as strong influences from local interneurones. Since interneurones in the IML have been rarely studied directly we examined their electrophysiological and anatomical properties. Whole cell patch clamp recordings were made from neurones in the IML of 250 microM slices of the thoracic spinal cord of the rat at room temperature. Action potential durations of interneurones (4.2+/-0.1 ms) were strikingly shorter than those of sympathetic preganglionic neurones (9.4+/-0.2 ms) due to a more rapid repolarisation phase. Low concentrations of tetraethylammonium chloride (TEA) (0.5 mM) or 4-aminopyridine (4-AP) (30 microM) affected interneurones but not sympathetic preganglionic neurones by prolonging the action potential repolarisation as well as decreasing both the afterhypolarisation amplitude and firing frequency. Following recordings, neurones sensitive to TEA and 4-AP were confirmed histologically as interneurones with axons that ramified extensively in the spinal cord, including the IML and other autonomic regions. In contrast, all cells that were insensitive to TEA and 4-AP were confirmed as sympathetic preganglionic neurones. Both electrophysiological and morphological data are therefore consistent with the presence of the voltage-gated potassium channel subunit Kv3.1 in interneurones, but not sympathetic preganglionic neurones. Testing this proposal immunohistochemically revealed that Kv3.1b was localised in low numbers of neurones within the IML but in higher numbers of neurones on the periphery of the IML. Kv3.1b-expressing neurones were not sympathetic preganglionic neurones since they were not retrogradely labelled following intraperitoneal injections of Fluorogold. Since Kv3.2 plays a similar role to Kv3.1 we also tested for the presence of Kv3.2 using immunohistochemistry, but failed to detect it in neuronal somata in the spinal cord. These studies provide electrophysiological and morphological data on interneurones in the IML and indicate that the channels containing the Kv3.1 subunit are important in setting the firing pattern of these neurones.

Input-Specific Modulation of Neurotransmitter Release in the Lateral Horn of the Spinal Cord via Adenosine Receptors

Activation of adenosine A2A receptors (A2ARs) in the CNS produces a variety of neuromodulatory actions dependent on the region and preparation examined. In autonomic regions of the spinal cord, A1R activation decreases excitatory synaptic transmission, but the effects of A2AR stimulation are unknown. We sought to determine the location and function of the A2ARs in the thoracic spinal cord, focusing on the intermediolateral cell column (IML). A2AR immunoreactivity was observed throughout the gray matter, with particularly dense immunostaining in regions containing sympathetic preganglionic neurons (SPNs), namely, the IML and intercalated nucleus. Electron microscopy revealed A2AR immunoreactivity within presynaptic terminals and in postsynaptic structures in the IML. To study the functional relevance of these A2ARs, visualized whole-cell patch-clamp recordings were made from electrophysiologically identified SPNs and interneurons within the IML. The A2AR agonist c2-[p-(carboxyethyl)phenethylamino]-5′-N-ethylcarboxyamidoadenosine (CGS 21680) had no significant effect on EPSPs but increased the amplitude of IPSPs elicited by stimulation of the lateral funiculus. These effects were attributable to activation of presynaptic A2ARs because CGS 21680 application altered the paired pulse ratio. Furthermore, neurons in the IML that have IPSPs increased via A2AR activation also receive excitatory inputs that are inhibited by A1R activation. These data show that activating A2ARs increase inhibitory but not excitatory transmission onto neurons in the IML. Simultaneous activation of A1Rs and A2ARs therefore could facilitate inhibition of the postsynaptic neuron, leading to an overall reduction of sympathetic nervous activity.

Association of potassium channel Kv3.4 subunits with pre- and post-synaptic structures in brainstem and spinal cord

Voltage-gated K+ channels (Kv) are divided into eight subfamilies (Kv1-8) and play a major role in determining the excitability of neurones. Members of the Kv3 subfamily are highly abundant in the CNS, with each Kv3 gene (Kv3.1-Kv3.4) exhibiting a unique pattern of expression, although single neurones can express more than one subtype. Of the Kv3 subunits relatively little is known of the Kv3.4 subunit distribution in the nervous system, particularly in the brainstem and spinal cord of the rat. We performed immunohistochemistry to determine both the cellular and sub-cellular distribution of the Kv3.4 subunit in these areas. Kv3.4 subunit immunoreactivity (Kv3.4-IR) was widespread, with dense, punctate staining in many regions including the intermediolateral cell column (IML) and the dorsal vagal nucleus (DVN), nucleus ambiguus (NA) and nucleus tractus solitarius (NTS). In the ventral horn a presynaptic location was confirmed by co-localization of Kv3.4-IR with the synaptic vesicle protein, SV2 and also with the glutamate vesicle markers vesicular glutamate transporter (VGluT) 1, VGluT2 or the glycine transporter GlyT2, suggesting a role for the channel in both excitatory and inhibitory neurotransmission. Electron microscopy confirmed a presynaptic terminal location of Kv3.4-IR in the VH, IML, DVN, NA and NTS. Interestingly however, patches of Kv3.4-IR were also revealed postsynaptically in dendritic and somatic structures throughout these areas. This staining was striking due to its localization at synaptic junctions at terminals with morphological features consistent with excitatory functions, suggesting an association with the postsynaptic density. Therefore the pre and postsynaptic localization of Kv3.4-IR suggests a role both in the control of transmitter release and in regulating neuronal excitability.

Kv3 voltage-gated potassium channels regulate neurotransmitter release from mouse motor nerve terminals.

Voltage‐gated potassium (Kv) channels are critical to regulation of neurotransmitter release throughout the nervous system but the roles and identity of the subtypes involved remain unclear. Here we show that Kv3 channels regulate transmitter release at the mouse neuromuscular junction (NMJ). Light‐ and electron‐microscopic immunohistochemistry revealed Kv3.3 and Kv3.4 subunits within all motor nerve terminals of muscles examined [transversus abdominus, lumbrical and flexor digitorum brevis (FDB)]. To determine the roles of these Kv3 subunits, intracellular recordings were made of end‐plate potentials (EPPs) in FDB muscle fibres evoked by electrical stimulation of tibial nerve. Tetraethylammonium (TEA) applied at low concentrations (0.05–0.5 mm), which blocks only a few known potassium channels including Kv3 channels, did not affect muscle fibre resting potential but significantly increased the amplitude of all EPPs tested. Significantly, this effect of TEA was still observed in the presence of the large‐conductance calcium‐activated potassium channel blockers iberiotoxin (25–150 nm) and Penitrem A (100 nm), suggesting a selective action on Kv3 subunits. Consistent with this, 15‐µm 4‐aminopyridine, which blocks Kv3 but not large‐conductance calcium‐activated potassium channels, enhanced evoked EPP amplitude. Unexpectedly, blood‐depressing substance‐I, a toxin selective for Kv3.4 subunits, had no effect at 0.05–1 µm. The combined presynaptic localization of Kv3 subunits and pharmacological enhancement of EPP amplitude indicate that Kv3 channels regulate neurotransmitter release from presynaptic terminals at the NMJ.

Immunohistochemical localisation of the voltage gated potassium ion channel subunit Kv3.3 in the rat medulla oblongata and thoracic spinal cord

Voltage gated K+ channels (Kv) are a diverse group of channels important in determining neuronal excitability. The Kv superfamily is divided into 12 subfamilies (Kv1-12) and members of the Kv3 subfamily are highly abundant in the CNS, with each Kv3 gene (Kv3.1-Kv3.4) exhibiting a unique expression pattern. Since the localisation of Kv subunits is important in defining the roles they play in neuronal function, we have used immunohistochemistry to determine the distribution of the Kv3.3 subunit in the medulla oblongata and spinal cord of rats. Kv3.3 subunit immunoreactivity (Kv3.3-IR) was widespread but present only in specific cell populations where it could be detected in somata, dendrites and synaptic terminals. Labelled neurones were observed in the spinal cord in laminae IV and V, in the region of the central canal and in the ventral horn. In the medulla oblongata, labelled cell bodies were numerous in the spinal trigeminal, cuneate and gracilis nuclei whilst rarer in the lateral reticular nucleus, hypoglossal nucleus and raphe nucleus. Regions containing autonomic efferent neurones were predominantly devoid of labelling with only occasional labelled neurones being observed. Dual immunohistochemistry revealed that some Kv3.3-IR neurones in the ventral medullary reticular nucleus, spinal trigeminal nucleus, dorsal horn, ventral horn and central canal region were also immunoreactive for the Kv3.1b subunit. The presence of Kv3.3 subunits in terminals was confirmed by co-localisation of Kv3.3-IR with the synaptic vesicle protein SV2, the vesicular glutamate transporter VGluT2 and the glycine transporter GlyT2. Co-localisation of Kv3.3-IR was not observed with VGluT1, tyrosine hydroxylase, serotonin or choline acetyl transferase. Electron microscopy confirmed the presence of Kv3.3-IR in terminals and somatic membranes in ventral horn neurones, but not motoneurones. This study provides evidence supporting a role for Kv3.3 subunits in regulating neuronal excitability and in the modulation of excitatory and inhibitory synaptic transmission in the medulla oblongata and spinal cord.

Spinal cord interneurones labelled transneuronally from the adrenal gland by a GFP-herpes virus construct contain the potassium channel subunit Kv3.1b

Interneurones in the spinal cord are likely to play an important role in the generation of activity in sympathetic preganglionic neurones (SPNs) and, therefore, sympathetic outflow. Although the properties of these interneurones have rarely been studied directly, here we show that neurones antecedent to SPNs contain the voltage-gated potassium channel subunit Kv3.1b, while SPNs do not. SPNs and interneurones were labelled by injection of a green fluorescent protein expressing herpes simplex virus (HSV-GFP) into the adrenal gland. SPNs identified by concomitant tracing with Fluorogold did not contain Kv3.1b immunoreactivity. Significantly, neurones that did not contain Fluorogold and which were unlikely to be SPNs were double labelled for Kv3.1b and GFP. This indicates that spinal cord intemeurones antecedent to SPNs contain Kv3.1b. To test the role of Kv3.1b whole cell patch clamp recordings were made from SPNs and interneurones in spinal cord slices. Selective blockade of Kv3.1b containing channels with 30 microM 4-amino-pyridine (4-AP) or 500 microM tetraethylammonium chloride (TEA) revealed that this Kv subunit contributes to fast repolarisation and fast firing frequencies of interneurones in the vicinity of the IML, allowing them to fire action potentials at much higher frequencies than SPNs. This is the first time that transneuronal labelling with this viral construct has been combined with immunohistochemical detection of ion channels. In conjunction with our electrophysiological data, this highlights a role for the Kv3.1b subunit in shaping the activity of intemeurones involved in sympathetic control.

Kv3.3 immunoreactivity in the vestibular nuclear complex of the rat with focus on the medial vestibular nucleus: Targeting of Kv3.3 neurones by terminals positive for vesicular glutamate transporter 1

Kv3 voltage-gated K(+) channels are important in shaping neuronal excitability and are abundant in the CNS, with each Kv3 gene exhibiting a unique expression pattern. Mice lacking the gene encoding for the Kv3.3 subunit exhibit motor deficits. Furthermore, mutations in this gene have been linked to the human disease spinocerebellar ataxia 13, associated with cerebellar and extra-cerebellar symptoms such as imbalance and nystagmus. Kv subunit localisation is important in defining their functional roles and thus, we investigated the distribution of Kv3.3-immunoreactivity in the vestibular nuclear complex of rats with particular focus on the medial vestibular nucleus (MVN). Kv3.3-immunoreactivity was widespread in the vestibular nuclei and was detected in somata, dendrites and synaptic terminals. Kv3.3-immunoreactivity was observed in distinct neuronal populations and dual labelling with the neuronal marker NeuN revealed 28.5+/-1.9% of NeuN labelled MVN neurones were Kv3.3-positive. Kv3.3-immunoreactivity co-localised presynaptically with the synaptic vesicle marker SV2, parvalbumin, the vesicular glutamate transporter VGluT2 and the glycine transporter GlyT2. VGluT1 terminals were scarce within the MVN (2.5+/-1.1 per 50 microm(2)) and co-localisation was not observed. However, 85.4+/-9.4% of VGluT1 terminals targeted and enclosed Kv3.3-immunoreactive somata. Presynaptic Kv3.3 co-localisation with the GABAergic marker GAD67 was also not observed. Cytoplasmic GlyT2 labelling was observed in a subset of Kv3.3-positive neurones. Electron microscopy confirmed a pre- and post-synaptic distribution of the Kv3.3 protein. This study provides evidence supporting a role for Kv3.3 subunits in vestibular processing by regulating neuronal excitability pre- and post-synaptically.

Balance appointment information leaflets: employing performance-based user-testing to improve understanding.

Abstract Objective: To use performance-based user-testing to evaluate the effectiveness of balance appointment patient information leaflets (PILs) in conveying important information. Design: The study used a sequential groups design. Twenty participants were asked to find and demonstrate understanding of 11 key points of information contained within two NHS leaflets, A and B (10 participants each), through individual structured-interviews. Participants’ views of the leaflets were explored through a short semi-structured interview. Following analysis, a revised leaflet was developed and tested on a further 20 participants. Study sample: 40 participants (25F/15M, aged 46–72) with no experience of balance problems or balance assessment appointments. Results: Participants exhibited difficulties with finding and/or understanding 5/11 and 6/11 points of information within leaflets A and B, respectively. Five out of eleven points of the revised leaflet also posed problems. Ten out of eleven points were understood by > 90% of participants testing the revised leaflet compared with 6/11 points for leaflets A and B. Conclusions: Some balance appointment PILs contain information which is difficult to find and/or understand for some readers. PILs should be evaluated prior to use using performance-based methods, since poor information provision may lead to increased patient anxiety and appointment non-attendance, cancellation, or postponement.

Effect of Varying Phase Between Frequency and Amplitude Modulation on Bone Conduction Auditory Steady State Responses

Objectives: Auditory steady state response (ASSR) testing provides a means to objectively estimate hearing levels in newborns and adults for whom behavioral tests prove difficult. When testing these patient groups, it is preferable that clear responses to both air and bone conduction stimuli are obtained in a short amount of time. Much of the literature addressing ASSRs, such as investigations of stimulus and recording parameters, have focused on air conduction ASSRs. The aim of this investigation was to study the amplitudes, latencies, and test times of bone conduction ASSRs elicited using amplitude- (AM), frequency- (FM), and mixed-modulated (MM) stimuli and provide suggestions for optimum recording parameters. Design: Bone and air conduction multiple ASSRs were recorded from two groups of 20 normal-hearing adults using the Multiple Auditory Steady State Response research system. AM, FM, and MM sinusoidal tones were used (0.5-, 1-, 2-, and 4-kHz carrier frequencies), which were modulated between 78 and 92 Hz. AM depth was 100% and FM depth was 20%. ASSR amplitudes and latencies (calculated using the “preceding cycles” technique) were analyzed for MM phase settings across the cycle from 0° at 45° intervals and compared with AM responses. Optimum phase settings for bone and air conduction ASSRs were calculated using a sinusoidal model based on the amplitude data. Results: Similar effects of stimulus type and carrier frequency were observed for bone and air conduction ASSRs. AM responses were larger in amplitude compared with FM responses. MM (at all phase settings tested) and AM response latencies increased with decreasing carrier frequency. MM phase setting had a significant (p < 0.01) sinusoidal effect on ASSR amplitudes, compared with AM responses, at 1, 2, and 4 kHz but not 0.5 kHz for air conduction and 1 and 2 kHz but not 0.5 and 4 kHz for bone conduction. Using a sinusoidal function to model this effect, MM phase settings (±95% confidence intervals) of 318° (295 to 350°) and 295° (290 to 310°) are predicted to evoke the largest responses for bone conduction ASSRs at 1 and 2 kHz, respectively. Phase settings of 293° (285 to 310°), 300° (280 to 310°), and 280° (255 to 330°) are predicted for air conduction ASSRs at 1, 2, and 4 kHz, respectively. MM phase setting had little effect on estimated latency. Test times were significantly (p < 0.01) affected by phase setting with both increases and decreases being observed. Test times for ASSRs at 1, 2, and 4 kHz could be significantly reduced if the estimated optimum phase settings are used. Conclusions: Different stimuli can significantly affect the amplitudes of bone conduction ASSRs. These effects are similar to those observed for air conduction ASSRs. MM stimuli with specific phase settings evoke larger bone conduction ASSRs compared with AM and FM stimuli alone, and calculations show that the time taken to obtain these responses is reduced. Implementation of the suggested optimum settings will promote efficient collection of bone conduction, and indeed air conduction, ASSR data.