Tertiapin-Q blocks recombinant and native large conductance K+ channels in a use-dependent manner

Tertiapin, a short peptide from honey bee venom, has been reported to specifically block the inwardly rectifying K+ (Kir) channels, including G protein-coupled inwardly rectifying potassium channel (GIRK) 1 + GIRK4 heteromultimers and ROMK1 homomultimers. In the present study, the effects of a stable and functionally similar derivative of tertiapin, tertiapin-Q, were examined on recombinant human voltage-dependent Ca2+-activated large conductance K+ channel (BK or MaxiK; alpha-subunit or hSlo1 homomultimers) and mouse inwardly rectifying GIRK1 + GIRK2 (i.e., Kir3.1 and Kir3.2) heteromultimeric K+ channels expressed in Xenopus oocytes and in cultured newborn mouse dorsal root ganglion (DRG) neurons. In two-electrode voltage-clamped oocytes, tertiapin-Q (1-100 nM) inhibited BK-type K+ channels in a use- and concentration-dependent manner. We also confirmed the inhibition of recombinant GIRK1 + GIRK2 heteromultimers by tertiapin-Q, which had no effect on endogenous depolarization- and hyperpolarization-activated currents sensitive to extracellular divalent cations (Ca2+, Mg2+, Zn2+, and Ba2+) in defolliculated oocytes. In voltage-clamped DRG neurons, tertiapin-Q voltage- and use-dependently inhibited outwardly rectifying K+ currents, but Cs+-blocked hyperpolarization-activated inward currents including I-H were insensitive to tertiapin-Q, baclofen, barium, and zinc, suggesting absence of functional GIRK channels in the newborn. Under current-clamp conditions, tertiapin-Q blocked the action potential after hyperpolarization (AHP) and increased action potential duration in DRG neurons. Taken together, these results demonstrate that the blocking actions of tertiapin-Q are not specific to Kir channels and that the blockade of recombinant BK channels and native neuronal AHP currents is use-dependent. Inhibition of specific types of Kir and voltage-dependent Ca2+-activated K+ channels by tertiapin-Q at nanomolar range via different mechanisms may have implications in pain physiology and therapy.

Block of voltage-gated potassium channels by Pacific ciguatoxin-1 contributes to increased neuronal excitability in rat sensory neurons

The present study investigated the actions of the polyether marine toxin Pacific ciguatoxin-1 (P-CTX-1) on neuronal excitability in rat dorsal root ganglion (DRG) neurons using patch-clamp recording techniques. Under current-clamp conditions, bath application of 2-20 nM P-CTX-1 caused a rapid, concentration-dependent depolarization of the resting membrane potential in neurons expressing tetrodotoxin (TTX)-sensitive voltage-gated sodium (Na-v,.) channels. This action was completely suppressed by the addition of 200 nM TTX to the external solution, indicating that this effect was mediated through TTX-sensitive Na-v channels. In addition, P-CTX-1 also prolonged action potential and afterhyperpolarization (AHP) duration. In a subpopulation of neurons, P-CTX-1 also produced tonic action potential firing, an effect that was not accompanied by significant oscillation of the resting membrane potential. Conversely, in neurons expressing TTX-resistant Na-v currents, P-CTX-1 failed to alter any parameter of neuronal excitability examined in this study. Under voltage-clamp conditions in rat DRG neurons, P-CTX-1 inhibited both delayed-rectifier and 'A-type' potassium currents in a dose-dependent manner, actions that Occurred in the absence of alterations to the voltage dependence of activation. These actions appear to underlie the prolongation of the action potential and AHP. and contribute to repetitive firing. These data indicate that a block of potassium channels contributes to the increase in neuronal excitability, associated with a modulation of Na-v. channel gating, observed clinically in response to ciguatera poisoning. (c) 2004 Elsevier Inc. All rights reserved.

The stimulus-response curve and motor unit variability in normal subjects and subjects with amyotrophic lateral sclerosis

The behavior and stability of motor units (MUs) in response to electrical stimulation of different intensities can be assessed with the stimulus-response curve, which is a graphical representation of the size of the compound muscle action potential (CMAP) in relation to stimulus intensity. To examine MU characteristics across the whole stimulus range, the variability of CMAP responses to electrical stimulation, and the differences that occur between normal and disease states, the curve was studied in 11 normal subjects and 16 subjects with amyotrophic lateral sclerosis (ALS). In normal subjects, the curve showed a gradual increase in CMAP size with increasing stimulus intensity, although one or two discrete steps were sometimes observed in the upper half of the curve, indicating the activation of large MUs at higher intensities. In ALS subjects, large discrete steps, due to loss of MUs and collateral sprouting, were frequently present. Variability of the CMAP responses was greater than baseline variability, indicating variability of MU responses, and at certain levels this variability was up to 100 mu Vms. The stimulus-response curve shows differences between normal and ALS subjects and provides information on MU activation and variability throughout the curve.

Attenuation of cardiovascular remodeling in DOCA-salt rats by the vasopeptidase inhibitor, omapatrilat

Omapatrilat, a vasopeptidase inhibitor, inhibits both neutral endopeptidase and angiotensin-converting enzyme with similar potency. The aim of this study was to investigate whether omapatrilat prevents or reverses cardiovascular remodeling and hypertension in deoxycorticosterone acetate (DOCA)-salt rats. Male Wistar rats (313 2 g, n=114) were uninephrectomized (UNX) with or without further treatment with DOCA and 1% NaCl in the drinking water. Compared with UNX control rats, DOCA-salt rats developed hypertension, cardiovascular hypertrophy, perivascular and interstitial cardiac fibrosis and inflammation, endothelial dysfunction, and the prolongation of ventricular action potential duration within four weeks. The administration of omapatrilat (40 mg/kg/day po) for two weeks commencing two weeks after surgery attenuated the development of cardiovascular hypertrophy, inflammation, fibrosis, and ventricular action potential prolongation. In contrast, omapatrilat treatment did not lower systolic blood pressure nor improve endothelial dysfunction. This study concludes that the renin-angiotensin-aldosterone, natriuretic peptide, and bradykinin systems are directly involved in the pathogenesis of cardiovascular remodeling in the DOCA-salt model of hypertension in rats, which may be independent of their effects on blood pressure.

Improved cardiovascular function with aminoguanidine in DOCA-salt hypertensive rats

1 The ability of aminoguanidine (AG), an inhibitor of collagen crosslinking, to prevent changes in cardiac and vascular structure and function has been determined in the deoxycorticosterone acetate (DOCA)-salt hypertensive rat as a model of the cardiovascular remodelling observed in chronic human hypertension. 2 Uninephrectomized rats (UNX) administered DOCA (25 mg every fourth day s.c.) and 1% NaCl in drinking water for 28 days developed cardiovascular remodelling shown as systolic hypertension, left ventricular hypertrophy, increased thoracic aortic and left ventricular wall thickness, increased left ventricular inflammatory cell infiltration together with increased interstitial collagen and increased passive diastolic stiffness, impaired contractility, prolongation of the action potential duration and vascular dysfunction. 3 Treatment with AG (0.05-0.1% in drinking water; average 182 +/- 17 mg kg(-1) day(-1) in DOCA-salt rats) decreased blood pressure (DOCA-salt 176 +/- 4; + AG 144 +/- 5 mmHg; *P < 0.05 vs DOCA-salt), decreased left ventricular wet weights (DOCA-salt 3.17 +/- 0.07; + AG 2.66 +/- 0.08 mg g(-1) body wt*), reduced diastolic stiffness constant (DOCA-salt 30.1 +/- 1.2; + AG 24.3 +/- 1.2* (dimensionless)), improved cardiac contractility (DOCA-salt 1610 +/- 130; + AG 2370 +/- 100 mmHg s(-1)*) and vascular reactivity (3.4-fold increase in maximal contractile response to noradrenaline, 3.2-fold increase in maximal relaxation response to acetylcholine, twofold increase in maximal relaxation response to sodium nitroprusside) and prolonged the action potential duration at 50% repolarization without altering collagen content or inflammatory cell infiltration. 4 Thus, cardiovascular function in DOCA-salt hypertensive rats can be improved by AG independent of changes in collagen content. This suggests that collagen crosslinking is an important cause of cardiovascular dysfunction during cardiovascular remodelling in hypertension.

Rosuvastatin attenuates hypertension-induced cardiovascular remodeling without affecting blood pressure in DOCA-salt hypertensive rats

The pleiotropic effects of statins represent potential mechanisms for the treatment of end-organ damage in hypertension. This study has investigated the effects of rosuvastatin in a model of cardiovascular remodeling, the DOCA-salt hypertensive rat. Male Wistar rats weighing 300 to 330 g were uninephrectomized (UNX) or UNX and treated with DOCA (25 mg subcutaneously every fourth day) and 1% NaCl in the drinking water. Compared with UNX controls, DOCA-salt rats developed hypertension, cardiovascular hypertrophy, inflammation with perivascular and interstitial cardiac fibrosis, endothelial dysfunction, and prolongation of ventricular action potential duration at 28 days. Rosuvastatin-treated rats received 20mg/kg/d of the drug in 10% Tween 20 by oral gavage for 32 days commencing 4 days before uninephrectomy. UNX and DOCA-salt controls received vehicle only. Rosuvastatin therapy attenuated the development of cardiovascular hypertrophy, inflammation, fibrosis, and ventricular action potential prolongation, but did not modify hypertension or vascular dysfunction. We conclude that the pleiotropic effects of rosuvastatin include attenuation of aspects of cardiovascular remodeling in the DOCA-salt model of hypertension in rats without altering systolic blood pressure.

Simulation of Brugada syndrome using cellular and three-dimensional whole-heart modeling approaches

Brugada syndrome (BS) is a genetic disease identified by an abnormal electrocardiogram ( ECG) ( mainly abnormal ECGs associated with right bundle branch block and ST-elevation in right precordial leads). BS can lead to increased risk of sudden cardiac death. Experimental studies on human ventricular myocardium with BS have been limited due to difficulties in obtaining data. Thus, the use of computer simulation is an important alternative. Most previous BS simulations were based on animal heart cell models. However, due to species differences, the use of human heart cell models, especially a model with three-dimensional whole-heart anatomical structure, is needed. In this study, we developed a model of the human ventricular action potential (AP) based on refining the ten Tusscher et al (2004 Am. J. Physiol. Heart Circ. Physiol. 286 H1573 - 89) model to incorporate newly available experimental data of some major ionic currents of human ventricular myocytes. These modified channels include the L-type calcium current (ICaL), fast sodium current (I-Na), transient outward potassium current (I-to), rapidly and slowly delayed rectifier potassium currents (I-Kr and I-Ks) and inward rectifier potassium current (I-Ki). Transmural heterogeneity of APs for epicardial, endocardial and mid-myocardial (M) cells was simulated by varying the maximum conductance of IKs and Ito. The modified AP models were then used to simulate the effects of BS on cellular AP and body surface potentials using a three-dimensional dynamic heart - torso model. Our main findings are as follows. (1) BS has little effect on the AP of endocardial or mid-myocardial cells, but has a large impact on the AP of epicardial cells. (2) A likely region of BS with abnormal cell AP is near the right ventricular outflow track, and the resulting ST-segment elevation is located in the median precordium area. These simulation results are consistent with experimental findings reported in the literature. The model can reproduce a variety of electrophysiological behaviors and provides a good basis for understanding the genesis of abnormal ECG under the condition of BS disease.

Motor unit number estimation - A Bayesian approach

All muscle contractions are dependent on the functioning of motor units. In diseases such as amyotrophic lateral sclerosis (ALS), progressive loss of motor units leads to gradual paralysis. A major difficulty in the search for a treatment for these diseases has been the lack of a reliable measure of disease progression. One possible measure would be an estimate of the number of surviving motor units. Despite over 30 years of motor unit number estimation (MUNE), all proposed methods have been met with practical and theoretical objections. Our aim is to develop a method of MUNE that overcomes these objections. We record the compound muscle action potential (CMAP) from a selected muscle in response to a graded electrical stimulation applied to the nerve. As the stimulus increases, the threshold of each motor unit is exceeded, and the size of the CMAP increases until a maximum response is obtained. However, the threshold potential required to excite an axon is not a precise value but fluctuates over a small range leading to probabilistic activation of motor units in response to a given stimulus. When the threshold ranges of motor units overlap, there may be alternation where the number of motor units that fire in response to the stimulus is variable. This means that increments in the value of the CMAP correspond to the firing of different combinations of motor units. At a fixed stimulus, variability in the CMAP, measured as variance, can be used to conduct MUNE using the "statistical" or the "Poisson" method. However, this method relies on the assumptions that the numbers of motor units that are firing probabilistically have the Poisson distribution and that all single motor unit action potentials (MUAP) have a fixed and identical size. These assumptions are not necessarily correct. We propose to develop a Bayesian statistical methodology to analyze electrophysiological data to provide an estimate of motor unit numbers. Our method of MUNE incorporates the variability of the threshold, the variability between and within single MUAPs, and baseline variability. Our model not only gives the most probable number of motor units but also provides information about both the population of units and individual units. We use Markov chain Monte Carlo to obtain information about the characteristics of individual motor units and about the population of motor units and the Bayesian information criterion for MUNE. We test our method of MUNE on three subjects. Our method provides a reproducible estimate for a patient with stable but severe ALS. In a serial study, we demonstrate a decline in the number of motor unit numbers with a patient with rapidly advancing disease. Finally, with our last patient, we show that our method has the capacity to estimate a larger number of motor units.

Calcium channels controlling acetylcholine release from preganglionic nerve terminals in rat autonomic ganglia

Little is known about the nature of the calcium channels controlling neurotransmitter release from preganglionic parasympathetic nerve fibres. In the present study, the effects of selective calcium channel antagonists and amiloride were investigated on ganglionic neurotransmission. Conventional intracellular recording and focal extracellular recording techniques were used in rat submandibular and pelvic ganglia, respectively. Excitatory postsynaptic potentials and excitatory postsynaptic currents preceded by nerve terminal impulses were recorded as a measure of acetylcholine release from parasympathetic and sympathetic preganglionic fibres following nerve stimulation. The calcium channel antagonists omega-conotoxin GVIA (N type), nifedipine and nimodipine (L type), omega-conotoxin MVIIC and omega-agatoxin IVA (P/Q type), and Ni2+ (R type) had no functional inhibitory effects on synaptic transmission in both submandibular and pelvic ganglia. The potassium-sparing diuretic, amiloride, and its analogue, dimethyl amiloride, produced a reversible and concentration-dependent inhibition of excitatory postsynaptic potential amplitude in the rat submandibular ganglion. The amplitude and frequency of spontaneous excitatory postsynaptic potentials and the sensitivity of the postsynaptic membrane to acetylcholine were unaffected by amiloride. In the rat pelvic ganglion, amiloride produced a concentration-dependent inhibition of excitatory postsynaptic currents without causing any detectable effects on the amplitude or configuration of the nerve terminal impulse. These results indicate that neurotransmitter release from preganglionic parasympathetic and sympathetic nerve terminals is resistant to inhibition by specific calcium channel antagonists of N-, L-, P/Q- and R-type calcium channels. Amiloride acts presynaptically to inhibit evoked transmitter release, but does not prevent action potential propagation in the nerve terminals, suggesting that amiloride may block the pharmacologically distinct calcium channel type(s) on rat preganglionic nerve terminals. (C) 1999 IBRO. Published by Elsevier Science Ltd.

Functional interconnectivity between the globus pallidus and the subthalamic nucleus in the mouse brain slice

In accordance with its central role in basal ganglia circuitry, changes in the rate of action potential firing and pattern of activity in the globus pallidus (GP)-subthalamic nucleus (STN) network are apparent in movement disorders. In this study we have developed a mouse brain slice preparation that maintains the functional connectivity between the GP and STN in order to assess its role in shaping and modulating bursting activity promoted by pharmacological manipulations. Fibre-tract tracing studies indicated that a parasagittal slice cut 20 deg to the midline best preserved connectivity between the GP and the STN. IPSCs and EPSCs elicited by electrical stimulation confirmed connectivity from GP to STN in 44/59 slices and from STN to GP in 22/33 slices, respectively. In control slices, 74/76 (97%) of STN cells fired tonically at a rate of 10.3 ± 1.3 Hz. This rate and pattern of single spiking activity was unaffected by bath application of the GABAA antagonist picrotoxin (50 μM, n = 9) or the glutamate receptor antagonist (6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) 10 μM, n = 8). Bursting activity in STN neurones could be induced pharmacologically by application of NMDA alone (20 μM, 3/18 cells, 17%) but was more robust if NMDA was applied in conjunction with apamin (20-100 nM, 34/77 cells, 44%). Once again, neither picrotoxin (50 μM, n = 5) nor CNQX (10 μM, n = 5) had any effect on the frequency or pattern of the STN neurone activity while paired STN and GP recordings of tonic and bursting activity show no evidence of coherent activity. Thus, in a mouse brain slice preparation where functional GP-STN connectivity is preserved, no regenerative synaptically mediated activity indicative of a dynamic network is evident, either in the resting state or when neuronal bursting in both the GP and STN is generated by application of NMDA/apamin. This difference from the brain in Parkinson's disease may be attributed either to insufficient preservation of cortico-striato-pallidal or cortico-subthalamic circuitry, and/or an essential requirement for adaptive changes resulting from dopamine depletion for the expression of network activity within this tissue complex. © The Physiological Society 2005.

Independent neuronal oscillators of the rat globus pallidus

In vivo, neurons of the globus pallidus (GP) and subthalamic nucleus (STN) resonate independently around 70 Hz. However, on the loss of dopamine as in Parkinson's disease, there is a switch to a lower frequency of firing with increased bursting and synchronization of activity. In vitro, type A neurons of the GP, identified by the presence of Ih and rebound depolarizations, fire at frequencies (≤80 Hz) in response to glutamate pressure ejection, designed to mimic STN input. The profile of this frequency response was unaltered by bath application of the GABAA antagonist bicuculline (10 μM), indicating the lack of involvement of a local GABA neuronal network, while cross-correlations of neuronal pairs revealed uncorrelated activity or phase-locked activity with a variable phase delay, consistent with each GP neuron acting as an independent oscillator. This autonomy of firing appears to arise due to the presence of intrinsic voltage- and sodium-dependent subthreshold membrane oscillations. GABAA inhibitory postsynaptic potentials are able to disrupt this tonic activity while promoting a rebound depolarization and action potential firing. This rebound is able to reset the phase of the intrinsic oscillation and provides a mechanism for promoting coherent firing activity in ensembles of GP neurons that may ultimately lead to abnormal and pathological disorders of movement.

Seeing and believing: three episodes in the history of neurophysiology

In this contribution I look at three episodes in the history of neurophysiology that bring out the complex relationship between seeing and believing. I start with Vesalius in the mid-sixteenth century who writes that he can in no way see any cavity in nerves, even in the optic nerves. He thus questions the age-old theory (dating back to the Alexandrians in the third century BC) but, because of the overarching psychophysiology of his time, does not press his case. This conflict between observation and theory persisted for a quarter of a millennium until finally resolved at the beginning of the nineteenth century by the discoveries of Galvani and Volta. The second case is provided by the early history of retinal synaptology. Schultze in 1866 had represented rod spherules and bipolar dendrites in the outer plexiform layer as being separated by a (synaptic) gap, yet in his written account, because of his theoretical commitments, held them to be continuous. Cajal later, 1892, criticized Schultze for this pusillanimity, but his own figure in La Cellule is by no means clear. It was only with the advent of the electron microscopy in the mid-twentieth century that the true complexity of the junction was revealed and it was shown that both investigators were partially right. My final example comes from the Hodgkin-Huxley biophysics of the 1950s. Their theory of the action potential depended on the existence of unseen ion pores with quite complex biophysical characteristics. These were not seen until the Nobel-Prize-winning X-ray diffraction analyses of the early twenty-first century. Seeing, even at several removes, then confirmed Hodgkin and Huxley’s belief. The relation between seeing and believing is by no means straightforward.

The neurotoxicity of acrylamide

The experiments described in this thesis compared conventional methods of screening for neurotoxins with potential electrophysiological and pharmacological tests in an attempt to improve the sensitivity of detection of progressive distal neuropathy. Adult male albino mice were dosed orally with the neurotoxicant acylamide and subjected to a test of limb strength and co-ordination and a functional observational battery. These methods established a no observable effect level of 10 mg/kg. A dose of 200 mg/kg resulted in abnormalities of gait and reduced limb strength and/or co-ordination. Analysis of the in vitro 'jitter' of the latency of trains of action potentials evoked at a frequency of 30 Hz in the mouse phrenic nerve/hemidiaphragm preparation showed this technique to be unsuitable for detection of the early phases of acrylamide induced peripheral neuropathy (l00 mg/kg). The evoked and spontaneous twitch responses of the hemidiaphragm preparation following in vitro exposure to the organophosphorous anticholinesterase compound ecothiopate were altered by in vivo pre treatment with acrylamide. Acrylamide caused an increase in the time course of the potentiation of stimulated twitches and a decrease in the maximum potentiation. Spontaneous twitches were reduced in amplitude and frequency. These effects occurred at an acrylamide dose level insufficient to cause clinical signs of neuropathy. Investigations into the mechanisms underlying these observations yielded the following observations. Analysis of miniature endplate potentials at this dose level indicated prolongation of the life of acetylcholine in the synaptic cleft but the implied decrease in cholinesterase activity could not be demonstrated biochemically or histologically. The electrical excitability of the nerve terminal region of phrenic motor nerves was reduced following acrylamide although a possible compromise of antidromic action potential conduction could not be confirmed. There was no histopathological evidence of neuropathy at this dose level. Further exploration of this phenomenon is desirable in order to ascertain whether the effect is specific to acrylamide and/or ecothiopate and to elucidate the mechanisms behind these novel observations.

Functional CB2 type cannabinoid receptors at CNS synapses

To date, it has been thought that cannabinoid receptors in CNS are primarily of the CB1R subtype, with CB2R expressed only in glia and peripheral tissues. However, evidence for the expression of CB2 type cannabinoid receptors at neuronal sites in the CNS is building through anatomical localization of receptors and mRNA in neurons and behavioural studies of central effects of CB2R agonists. In the medial entorhinal area of the rat, we found that blockade of CB1R did not occlude suppression of GABAergic inhibition by the non-specific endogenous cannabinoid 2-AG, suggesting that CB1R could not account fully for the effects of 2-AG. Suppression could be mimicked using the CB2R agonist JWH-133 and reversed by the CB2R inverse agonist AM-630, indicating the presence of functional CB2R. When we reversed the order of drug application AM-630 blocked the effects of the CB2R agonist JWH-133, but not the CB1R inverse agonist LY320135. JTE-907, a CB2R inverse agonist structurally unrelated to AM-630 elicited increased GABAergic neurotransmission at picomolar concentrations. Analysis of mIPSCs revealed that CB2R effects were restricted to action potential dependent, but not action potential independent GABA release. These data provide pharmacological evidence for functional CB2R at CNS synapses.

Differential control of two forms of glutamate release by group III metabotropic glutamate receptors at rat entorhinal synapses

Neurotransmitter release at CNS synapses occurs via both action potential-dependent and independent mechanisms, and it has generally been accepted that these two forms of release are regulated in parallel. We examined the effects of activation of group III metabotropic glutamate receptors (mGluRs) on stimulus-evoked and spontaneous glutamate release onto entorhinal cortical neurones in rats, and found a differential regulation of action potential-dependent and independent forms of release. Activation of presynaptic mGluRs depressed the amplitude of stimulus-evoked excitatory postsynaptic currents, but concurrently enhanced the frequency of spontaneous excitatory currents. Moreover, these differential effects on glutamate release were mediated by pharmacologically separable mechanisms. Application of the specific activator of adenylyl cyclase, forskolin, mimicked the effect of mGluR activation on spontaneous, but not evoked release, and inhibition of adenylyl cyclase with 9-tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) blocked mGluR-mediated enhancement of spontaneous release, but not depression of evoked release. Occlusion studies with calcium channel blockers suggested that the group III mGluRs might depress evoked release through inhibition of both N and P/Q, but not R-type calcium channels. We suggest that the concurrent depression of action potential-evoked, and enhancement of action potential-independent glutamate release operate through discrete second messenger/effector systems at excitatory entorhinal terminals in rat brain. © 2007 IBRO.