Showing papers in "The Journal of Physiology in 1996"

Interaction between intracortical inhibition and facilitation in human motor cortex.

Abstract: 1. In seven normal subjects, subthreshold transcranial magnetic conditioning stimuli (using a figure-of-eight coil) were applied over the motor cortex in order to evoke activity in intracortical neuronal circuits. The net effect on cortical excitability was evaluated by measuring the effect on the size of EMG responses elicited in the abductor digiti minimi (ADM) muscle by a subsequent suprathreshold test stimulus. 2. A single conditioning stimulus suppressed the size of the test response at interstimulus intervals (ISIs) of 1-4 ms whereas the response was facilitated at ISIs of 6-20 ms. The facilitation could be augmented if pairs of conditioning stimuli were given. 3. Inhibition and facilitation appeared to have separate mechanisms. The threshold for inhibition (0.7 active motor threshold) was slightly lower than that for facilitation (0.8 active threshold). Similarly, the inhibitory effect was independent of the direction of current flow induced in the cortex by the conditioning shock, whereas facilitation was maximal with posterior-anterior currents and minimal with lateromedial current. 4. Direct corticospinal effects were probably not responsible for the results since facilitation of cortical test responses could be produced by conditioning stimuli which had no effect on the amplitude of H reflexes elicited in active ADM muscle. 5. Inhibition and facilitation appeared to interact in a roughly linear manner, consistent with separate inputs to a common neurone. 6. We suggest that subthreshold transcranial magnetic stimulation is capable of activating separate populations of excitatory and inhibitory interneurones in the motor cortex.

Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors.

Abstract: 1. To investigate the origin and functional significance of a recently described tonic GABA(A) receptor-mediated conductance in cerebellar granule cells we have made recordings from cells in cerebellar slices from rats of different ages (postnatal days P4 to P28).2. During development there was a dramatic change in the properties of GABA-mediated synaptic transmission. The contribution to GABA(A) receptor-mediated charge transfer from the tonic conductance (G(GABA)), relative to that resulting from discrete spontaneous postsynaptic currents (sPSCs), was increased from 5% at P7 to 99% at P21. G(GABA) was reduced by bicuculline, tetrodotoxin and by lowering extracellular Ca2+, and was initially present only in those cells which exhibited sPSCs.3. At P7 sPSCs were depolarizing, occasionally triggering a single action potential. By P18 the GABA reversal potential was shifted close to the resting potential and G(GABA) produced a shunting inhibition. Removal of G(GABA) by bicuculline increased granule cell excitability in response to current injection.4. This novel tonic inhibition is present despite the low number of Golgi cell synapses on individual granule cells and appears to result from 'overspill' of synaptically released GABA leading to activation of synaptic and extrasynaptic GABA(A) receptors.

Supraspinal factors in human muscle fatigue: evidence for suboptimal output from the motor cortex.

Abstract: 1. Voluntary activation of elbow flexor muscles can be optimal during brief maximal voluntary contractions (MVCs), although central fatigue, a progressive decline in the ability to drive the muscle maximally, develops during sustained or repeated efforts. We stimulated the motor cortex and motor point in human subjects to investigate motor output during fatigue. 2. The increment in force (relative to the voluntary force) produced by stimulation of the motor point of biceps brachii increased during sustained isometric MVCs of the elbow flexors. Motoneuronal output became suboptimal during the contraction, i.e. central fatigue developed and accounted for a small but significant loss of maximal voluntary force. During 3 min MVCs, voluntary activation of biceps fell to an average of 90.7% from an average of > 99%. 3. The increment in force (relative to the voluntary force) produced by magnetic cortical stimulation was initially small (1.0%) but also increased during sustained MVCs to 9.8% (with a 2 min MVC). Thus, cortical output was not optimal at the time of stimulation nor were sites distal to the motor cortex already acting maximally. 4. A sphygmomanometer cuff around the upper arm blocked blood supply to brachioradialis near the end of a sustained MVC and throughout subsequent brief MVCs. Neither maximal voluntary force nor voluntary activation recovered during ischaemia after the sustained MVC. However, fatigue-induced changes in EMG responses to magnetic cortical stimulation recovered rapidly despite maintained ischaemia. 5. In conclusion, during sustained MVCs, voluntary activation becomes less than optimal so that force can be increased by stimulation of the motor cortex or the motor nerve. Complex changes in excitability of the motor cortex also occur with fatigue, but can be dissociated from the impairment of voluntary activation. We argue that inadequate neural drive effectively 'upstream' of the motor cortex must be one site involved in the genesis of central fatigue.

Analysis of gamma rhythms in the rat hippocampus in vitro and in vivo.

Abstract: 1 We have shown previously, with experimental and computer models, how a ‘40 Hz’ (gamma) oscillation can arise in networks of hippocampal interneurones, involving mutual GABAA-mediated synaptic inhibition and a source of tonic excitatory input Here, we explore implications of this model for some hippocampal network phenomena in the rat in vitro and in vivo 2 A model network was constructed of 1024 CA3 pyramidal cells and 256 interneurones AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), NMDA (N-methyl-D-aspartate), GABAA and GABAB receptors were simulated on pyramidal cells and on interneurones 3 In both model and experiment, the frequency of network oscillations, in the gamma range, depended upon three parameters: GABAA conductance and decay time constant in interneurone-->interneurone connections, and the driving current to the interneurones 4 The model of gamma rhythm predicts an average zero phase lag between firing of pyramidal cells and interneurones, as observed in the rat hippocampus in vivo The model also reproduces a gamma rhythm whose frequency changes with time, at theta frequency (about 5 Hz) This occurs when there is 5 Hz modulation of a tonic signal to chandelier and basket cells 5 Synchronized bursts can be produced in the model by several means, including partial blockade of GABAA receptors or of AMPA receptors on interneurones, or by augmenting AMPA-mediated EPSCs In all of these cases, the burst can be followed by a ‘tail’ of transiently occurring gamma waves, a phenomenon observed in the hippocampus in vivo following sharp waves This tail occurs in the model because of delayed excitation of the interneurones by the synchronized burst A tail of gamma activity was found after synchronized epileptiform bursts both in the hippocampal slice (CA3 region) and in vivo 6 Our data suggest that gamma-frequency EEG activity arises in the hippocampus when pools of interneurones receive a tonic or slowly varying excitation The frequency of the oscillation depends upon the strength of this excitation and on the parameters regulating the inhibitory coupling between the interneurones The interneurone network output is then imposed upon pyramidal neurones in the form of rhythmic synchronized IPSPs

PGE2 modulates the tetrodotoxin-resistant sodium current in neonatal rat dorsal root ganglion neurones via the cyclic AMP-protein kinase A cascade.

Abstract: 1. In current-clamp recordings, 1 microM prostaglandin E2 (PGE2) increased the excitability of neonatal rat dorsal root ganglion neurones. The current threshold for firing was reduced, and the response to a constant suprathreshold stimulation was modified such that a single evoked action potential was converted to a train of action potentials. The excitatory action of PGE2 was still apparent when action potentials were evoked in the presence of 500 nM tetrodotoxin. 2. In voltage-clamp experiments 1 microM PGE2 frequently increased the magnitude of the peak currents recorded, and caused a hyperpolarizing shift (of approximately 6 mV) in the activation curve for the tetrodotoxin-resistant sodium current (TTX-R INa). In some cells, the hyperpolarizing shift in the activation curve was accompanied by a decrease in peak conductance. PGE2 also caused a hyperpolarizing shift in the steady-state inactivation curve for the sodium current. 3. Extracellular application of the cAMP analogue dibutyryl cAMP (dbcAMP) at a concentration of 1 mM produced effects on both the current-voltage relationship and the steady-state inactivation curve for the TTX-R INa which were indistinguishable from those observed with PGE2. Prior exposure of the neurones to dbcAMP occluded the effect of a subsequent treatment with PGE2. 4. Forskolin (10 microM), a direct activator of adenylate cyclase, mimicked the effects of PGE2 and dbcAMP on TTX-R INa. The inactive congener of forskolin, 1, 9-dideoxyforskolin (10 microM), reduced the amplitude of TTX-R INa, but did not evoke a hyperpolarizing shift in the activation curve. 5. Intracellular perfusion of the neurones with an inhibitor of protein kinase A inhibited the effect of PGE2 on TTX-R INa. 6. PGE2 also reduced the amplitude of voltage-gated potassium currents (IK), which will contribute to the excitatory action. The mechanisms underlying the changes in IK have yet to be elucidated. 7. We propose that the PGE2-mediated increase in excitability in sensory neurones may be due, at least in part, to the cAMP-protein kinase A-dependent modulation of the tetrodotoxin-resistant sodium channel.

In vivo human gastrocnemius architecture with changing joint angle at rest and during graded isometric contraction

Abstract: 1. Human gastrocnemius medialis architecture was analysed in vivo, by ultrasonography, as a function of joint angle at rest and during voluntary isometric contractions up to the maximum force (MCV). maximum force (MVC). 2. At rest, as ankle joint angle increased from 90 to 150 deg, pennation increased from 15.8 to 27.7 deg, fibre length decreased from 57.0 to 34.0 mm and the physiological cross-sectional area (PCSA) increased from 42.1 to 63.5 cm2. 3. From rest to MVC, at a fixed ankle joint angle of 110 deg, pennation angle increased from 15.5 to 33.6 deg and fibre length decreased from 50.8 to 32.9 mm, with no significant change in the distance between the aponeuroses. As a result of these changes the PCSA increased by 34.8%. 4. Measurements of pennation angle, fibre length and distance between the aponeuroses of the gastrocnemius medialis were also performed by ultrasound on a cadaver leg and found to be in good agreement with direct anatomical measurements. 5. It is concluded that human gastrocnemius medialis architecture is significantly affected both by changes of joint angle at rest and by isometric contraction intensity. The remarkable shortening observed during isometric contraction suggests that, at rest, the gastrocnemius muscle and tendon are considerably slack. The extrapolation of muscle architectural data obtained from cadavers to in vivo conditions should be made only for matching muscle lengths.

Paired-pulse facilitation and depression at unitary synapses in rat hippocampus: quantal fluctuation affects subsequent release.

Abstract: 1. Excitatory synaptic transmission between pairs of monosynaptically coupled pyramidal cells was examined in rat hippocampal slice cultures. Action potentials were elicited in single CA3 pyramidal cells impaled with microelectrodes and unitary excitatory postsynaptic currents (EPSCs) were recorded in whole-cell voltage-clamped CA1 or CA3 cells. 2. The amplitude of successive unitary EPSCs in response to single action potentials varied. The amplitude of EPSCs was altered by adenosine or changes in the [Mg2+]/[CA2+] ratio. We conclude that single action potentials triggered the release of multiple quanta of glutamate. 3. When two action potentials were elicited in the presynaptic cell, the amplitude of the second EPSC was inversely related to the amplitude of the first. Paired-pulse facilitation (PPF) was observed when the first EPSC was small, i.e. the second EPSC was larger than the first, whereas paired-pulse depression (PPD) was observed when the first EPSC was large. 4. The number of trials displaying PPD was greater when release probability was increased, and smaller when release probability was decreased. 5. PPD was not postsynaptically mediated because it was unaffected by decreasing ionic flux with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or receptor desensitization with aniracetam. 6. PPF was maximal at an interstimulus interval of 70 ms and recovered within 500 ms. Recovery from PPD occurred within 5 s. 7. We propose that multiple release sites are formed by the axon of a CA3 pyramidal cell and a single postsynaptic CA1 or CA3 cell. PPF is observed if the first action potential fails to release transmitter at most release sites. PPD is observed if the first action potential successfully triggers release at most release sites. 8. Our observations of PPF are consistent with the residual calcium hypothesis. We conclude that PPD results from a decrease in quantal content, perhaps due to short-term depletion of readily releasable vesicles.

Force-velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.

Abstract: 1. A large population (n = 151) of human skinned skeletal muscle fibres has been studied. Force-velocity curves of sixty-seven fibres were obtained by load-clamp manoeuvres at 12 degrees C. In each fibre maximum shortening velocity (Vmax), maximum power output (Wmax), optimal velocity (velocity at which Wmax is developed, Vopt), optimal force (force at which Wmax is developed, Popt), specific tension (Po/CSA, isometric tension/cross-sectional area) were assessed. Unloaded shortening velocity (Vo) was also determined at 12 degrees C in a different group (n = 57) of fibres by slack-test procedure. 2. All fibres used for mechanical experiments were characterized on the basis of the myosin heavy chain (MHC) isoform composition by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and divided into five types: type I (or slow), types IIA and IIB (or fast), and types I-IIA and IIA-IIB (or mixed types). 3. Vmax, Wmax, Vopt, Popt, Vopt/Vmax ratio, Po/CSA and Vo were found to depend on MHC isoform composition. All parameters were significantly lower in type I than in the fast (type IIA and IIB) fibres. Among fast fibres, Vmax, Wmax, Vopt and Vo were significantly lower in type IIA and than in IIB fibres, whereas Popt, Po/CSA and Vopt/Vmax were similar. 4. The temperature dependence of Vo and Po/CSA was assessed in a group of twenty-one fibres in the range 12-22 degrees C. In a set of six fibres temperature dependence of Vmax was also studied. The Q10 (5.88) and activation energy E (125 kJ mol-1) values for maximum shortening velocity calculated from Arrhenius plots pointed to a very high temperature sensitivity. Po/CSA was very temperature dependent in the 12-17 degrees C range, but less dependent between 17 and 22 degrees C.

The hyperpolarization-activated current (Ih) and its contribution to pacemaker activity in rat CA1 hippocampal stratum oriens-alveus interneurones.

Abstract: 1. The hyperpolarization-activated current (Ih) and its role in pacemaking activity in rat hippocampal stratum oriens-alveus interneurones was studied using whole-cell and perforated patch-clamp configurations. 2. Voltage-clamp recordings revealed Ih as a slowly activating, inward current, activated by hyperpolarizing steps (holding potential, Vh = -40 mV), with a reversal potential close to -30 mV. Its activation curve ranged from approximately -50 to -120 mV with a mid-activation point of -84.1 mV. 3. Ih was blocked by external application of Cs+ (2-5 mM) and ZD7288 (100 microM), but not by Ba2+ (1 mM). 4. Ih was potentiated by both noradrenaline and isoprenaline by a mechanism consistent with a shift in the Ih activation curve. 5. Under current-clamp conditions (Vh = -60 mV), ZD7288 induced a membrane hyperpolarization concomitant with an increase in the membrane input resistance and abolished the voltage sag generated by hyperpolarizing current injection. 6. Analysis of the current-discharge relationship revealed that block of Ih differentially increased the firing frequency of spikes occurring early in the train compared with those occurring late in the discharge. 7. When applied to spontaneously firing cells, ZD7288 reduced the firing frequency by selectively altering the time course of the interspike interval, while minimally affecting other action potential characteristics. Similarly, isoprenaline increased the spontaneous firing frequency by an effect exclusively on the after-hyperpolarization and interspike interval. 8. These results provide evidence for the involvement of Ih in the excitability and generation of spontaneous firing in hippocampal stratum oriens-alveus interneurones.

Kinematic determinants of human locomotion

Abstract: 1. The aim of this study was to find kinematic patterns that are invariant across the normal range of locomotion speeds. Subjects walked at different, freely chosen speeds ranging from 0.9 to 2.1 m s-1, while motion and ground reaction forces on the right side of the body were recorded in three-dimensional space. 2. The time course of the anatomical angles of flexion-extension at the hip and ankle was variable not only across subjects, but even from trial to trial in the same subject. By contrast, the time course of the changes in the angles of elevation of each limb segment (pelvis, thigh, shank and foot) relative to the vertical was stereotyped across subjects. 3. To compare the waveforms across speeds, data were scaled in time relative to gait cycle duration. The pattern of ground reaction forces was highly speed dependent. Several distinct families of curves could be recognized in the flexion-extension angles at the hip and ankle. Instead, the waveforms of global length and elevation of the limb, elevation angles of all limb segments and flexion-extension at the knee were invariant with speed. 4. When gait trajectories at all speeds are plotted in the position space defined by the elevation angles of the limb segments, they describe regular loops on a plane. The statistical characteristics of these angular covariations were quantified by means of principal component analysis. The first two principal components accounted together for > 99% of the total experimental variance, and were quantitatively comparable in all subjects. 5. This constraint of planar covariation of the elevation angles is closely reminiscent of that previously described for the control of posture. The existence of laws of intersegmental co-ordination, common to the control of posture and locomotion, presumably assures the maintenance of dynamic equilibrium during forward progression, and the anticipatory adaptation to potentially destabilizing factors by means of co-ordinated kinematic synergies of the whole body.

A mechanism for altered flexibility in human skeletal muscle.

Abstract: 1. We investigated the effect of a long-term stretching regimen on the tissue properties and stretch tolerance of human skeletal muscle. 2. Resistance to stretch was measured as torque (in N m) offered by the hamstring muscle group during passive knee extension while electromyographic (EMG) activity, knee joint angle and velocity were continuously monitored during a standardized stretch manoeuvre. Seven healthy subjects were tested before and after a 3 week training period using two separate protocols. Protocol 1 consisted of a slow stretch at 0.087 rad s-1 to a predetermined angle followed by a 90 s holding phase. Subjects were brought to the same angle before and after the training period. Protocol 2 was a similar stretch, but continued to the point of pain. 3. During protocol 1 the torque rose during the stretch and then declined during the holding phase. EMG activity was small and did not change significantly during the protocol. No significant differences in stiffness, energy and peak torque about the knee joint were seen as a result of the training. During protocol 2 the angle to which the knee could be extended was significantly increased as a result of the training. This was accompanied by a comparable increase in peak torque and energy. EMG activity was small and not affected by training. 4. It is concluded that reflex EMG activity does not limit the range of movement during slow stretches and that the increased range of motion achieved from training is a consequence of increased stretch tolerance on the part of the subject rather than a change in the mechanical or viscoelastic properties of the muscle.

Impaired sympathetic vascular regulation in humans after acute dynamic exercise.

Abstract: 1. The reduction in vascular resistance which accompanies acute dynamic exercise does not subside immediately during recovery, resulting in a post-exercise hypotension. This sustained vasodilatation suggests that sympathetic vascular regulation is altered after exercise. 2. Therefore, we assessed the baroreflex control of sympathetic outflow in response to arterial pressure changes, and transduction of sympathetic activity into vascular resistance during a sympatho-excitatory stimulus (isometric handgrip exercise) after either exercise (60 min cycling at 60% peak aerobic power (VO2,peak)) or sham treatment (60 min seated rest) in nine healthy subjects. 3. Both muscle sympathetic nerve activity and calf vascular resistance were reduced after exercise (-29.7 +/- 8.8 and -25.3 +/- 9.1%, both P < 0.05). The baroreflex relation between diastolic pressure and sympathetic outflow was shifted downward after exercise (post-exercise intercept, 218 +/- 38 total integrated activity (heartbeat)-1; post-sham intercept, 318 +/- 51 total integrated activity (heartbeat)-1, P < 0.05), indicating less sympathetic outflow across all diastolic pressures. Further, the relation between sympathetic activity and vascular resistance was attenuated after exercise (post-exercise slope, 0.0031 +/- 0.0007 units (total integrated activity)-1 min; post-sham slope, 0.0100 +/- 0.0033 units (total integrated activity)-1 min, P < 0.05), indicating less vasoconstriction with any increase in sympathetic activity. 4. Thus, both baroreflex control of sympathetic outflow and the transduction of sympathetic activity into vascular resistance are altered after dynamic exercise. We conclude that the vasodilation which underlies post-exercise hypotension results from both neural and vascular phenomena.

Changes in motor cortical excitability during human muscle fatigue

Abstract: 1. The excitability of the motor cortex was investigated during fatiguing con of the elbow flexors in human subjects. During sustained contractions at 30 and 1 voluntary force (MVC), the short-latency electromyographic responses (EMG) evoke brachii and brachioradialis by transcranial magnetic stimulation increased in si EMG in the elbow flexors following the evoked muscle potential (silent period), duration during a sustained MVC but not during 30% MVCs nor during a sustained M muscle (adductor pollicis). 2. When the blood supply to brachioradialis was blocked with sphygmomanometer cuff sustained MVC, the changes in EMG responses to transcranial stimulation rapidly control values, This suggests that changes in these responses during fatigue wer small-diameter muscle afferents. 3. Tendon vibration during sustained MVCs indicated that the changes in the resp cortial stimulation were not mediated by reduced muscle spindle inputs. 4. Muscle action potentials evoked in brachioradialis by electrical stimulation cervicomedullary junction did not increase in size during sustained MVCs. Thus, cortically evoked responses during sustained MVCs reflects a change in cortical Although the silent period following cervicomedullary stimulation lengthened, it substantially shorter than the cortically evoked silent period. 5. The altered EMG responses to transcranial stimulation during fatigue suggest exitation and increased inhibition in the motor cortex. As these changes were un manipulation of afferent input they presumably result from intrinsic cortical pr altered voluntary drive to the motor cortex.

Relationship of firing intervals of human motor units to the trajectory of post-spike after-hyperpolarization and synaptic noise.

Abstract: 1. Interspike interval distributions from human motor units of a variety of muscles were analysed to assess the role of synaptic noise in excitation. The time course of the underlying post-spike after-hyperpolarization (AHP) was deduced by applying a specially developed transform to the interval data. Different firing rates were studied both by varying the firing voluntarily, and by selecting subpopulations of spikes for a given firing rate from long recordings with slight variations in frequency. 2. At low firing rates the interval histograms had an exponential tail. Thus at long intervals, the motoneurone was randomly excited by noise and its post-spike AHP was complete. This contrasts with the firing produced by intracellular current injection in the cat, when the membrane potential increases linearly until threshold is reached. The interval histogram was therefore analysed with the aid of a model of synaptic excitation to deduce the mean ‘trajectory’ of membrane voltage in the last part of the interspike interval. 3. The computer model, described in the Appendix, was used to determine the effect of the mean level of membrane potential on the probability of a spike being excited, per unit time, during an on-going interspike interval. All variables were treated as voltages, with synaptic noise simulated by time-smoothed Gaussian noise. This enabled an interval distribution to be transformed into a segment of the underlying trajectory of the membrane potential; the potential was expressed in terms of the noise amplitude and the spike threshold. 4. At low firing rates, the equilibrium value of the membrane voltage trajectory lay well below threshold; the deviation typically corresponded to the standard deviation of the noise or more. The noise standard deviation was estimated to be about 2 mV. 5. With increasing mean firing rate, the near-threshold portion of the trajectory obtainable from the histogram occurred earlier, was steeper and rose to a higher level. Trajectories for different firing rates fell on the same curve after shifting them vertically by varying amounts. The curve was taken to represent the AHP of the motoneurone and was closely exponential. The shift of the trajectory gave its mean synaptic drive. The duration of the AHP varied between units and was longer than average for units from soleus muscle. 6. Further modelling showed that summation of noise with the AHP can explain the well-known changes in discharge variability that occur as firing rate increases. 7. It is concluded that synaptic noise plays a major role in the excitation of tonically firing human motoneurones and that the noiseless motoneurone with a linear trajectory provides an inadequate model for the conscious human. This is of interest in relation to various standard measures of human motor unit activity such as short-term synchronization.

Activation of metabotropic glutamate receptor type 2/3 suppresses transmission at rat hippocampal mossy fibre synapses.

Abstract: 1. The effects of metabotropic glutamate receptor (mGluR) agonists on excitatory transmission at mossy fibre-CA3 synapses were studied in rat hippocampal slice preparations using both extracellular and whole-cell clamp recording techniques. 2. Application of a novel and potent mGluR2/mGluR3-specific agonist (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV, 0.1 microM) reversibly suppressed field excitatory postsynaptic potentials evoked by mossy fibre stimulation. DCG-IV at the same concentration did not affect other glutamatergic excitatory transmissions at the commissural/associational input to CA3 or at the Schaffer collateral/commissural input to CA1 regions. 3. This suppressing effect of DCG-IV on mossy fibre transmission was dose dependent and partly antagonized by a competitive mGluR antagonist (+)-methyl-4-carboxylphenylglycine (1 mM). 4. The field potential changes induced by pressure application of glutamate (0.1 mM) to the stratum lucidum of the CA3 region was unaffected by 0.1 microM DCG-IV. 5. In whole-cell clamp experiments, 0.1 microM DCG-IV suppressed excitatory postsynaptic currents evoked by mossy fibre stimulation without inducing detectable inward current in CA3 neurons, and paired-pulse facilitation was enhanced by DCG-IV application. 6. These results suggest that mGluR2/mGluR3 are specifically expressed at mossy fibre synapses in the hippocampal CA3 region, and activation of the receptor suppresses synaptic transmission by an action on a presynaptic site.

Mechanisms of long-lasting hyperpolarizations underlying slow sleep oscillations in cat corticothalamic networks.

Abstract: 1. To explore the nature of the long-lasting hyperpolarizations that characterize slow oscillations in corticothalamic circuits in vivo, intracellular recordings were obtained under ketamine-xylazine anaesthesia from cortical (Cx) cells of the cat precruciate motor cortex, thalamic reticular (RE) cells from the rostrolateral sector, and thalamocortical (TC) cells from the ventrolateral (VL) nucleus. 2. Measurements in the three cell types showed input resistance (Rin) to be highest during the long-lasting hyperpolarizations that correspond to depth-positive waves of the cortical EEG. Rin was lowest during the early phase of high-amplitude depth-negative EEG waves and increased thereafter until the next cycle of the slow oscillation. 3. Spontaneous long-lasting hyperpolarizations were compared with those evoked by dorsal thalamic stimulation. Voltage versus current (V-I) plots showed similar membrane potential (Vm) ranges and slopes for spontaneous and evoked hyperpolarizations in both Cx and RE cells. V-I plots from TC cells had similar slopes, but Vm during evoked hyperpolarizations was displaced towards more negative values. 4. Intracellular injection of constant hyperpolarizing current in Cx cells increased the amplitude of the initial part of the depolarizing plateau of the slow oscillation, but decreased the amplitude of the last part. 5. These results suggest disfacilitation to be the dominant mechanism in the membrane of cortical and thalamic cells during the spontaneous long-lasting hyperpolarizations, which shape and synchronize slow oscillations in corticothalamic networks. In Cx and RE cells, the same mechanism underlies thalamically evoked long-lasting hyperpolarizations. By contrast, evoked responses in TC cells show a strong additional hyperpolarizing factor. We propose that GABAB processes are stronger in TC than in Cx neurones, thus rendering the thalamus an easier target for absence-type epileptic phenomena through potentiation of thalamic rebound capabilities.

Changes in muscle strength, relaxation rate and fatiguability during the human menstrual cycle.

Abstract: 1. The effect of the different phases of the menstrual cycle on skeletal muscle strength, contractile properties and fatiguability was investigated in ten young, healthy females. Results were compared with a similar group on the combined (non-phasic) oral contraceptive pill (OC). Cycle phases were divided into the early and mid-follicular, mid-cycle (ovulatory) and mid- and late luteal. Cycle phases were estimated from the first day of the menstrual bleed. 2. Subjects were studied weekly through two complete cycles. Measurements included quadriceps and handgrip maximum voluntary isometric force and the relaxation times, force-frequency relationship and fatigue index of the quadriceps during percutaneous stimulation at a range of frequencies from 1 to 100 Hz. 3. In the women not taking the OC there was a significant increase of about 11% in quadriceps and handgrip strength at mid-cycle compared with both the follicular and luteal phases. Accompanying the increases in strength there was a significant slowing of relaxation and increase in fatiguability at mid-cycle. No changes in any parameter were found in the women taking the OC. 4. The changes in muscle function at mid-cycle may be due to the increase in oestrogen that occurs prior to ovulation.

Slow inactivation of Na+ current and slow cumulative spike adaptation in mouse and guinea-pig neocortical neurones in slices.

Abstract: 1. Spike adaptation of neocortical pyramidal neurones was studied with sharp electrode recordings in slices of guinea-pig parietal cortex and whole-cell patch recordings of mouse somatosensory cortex. Repetitive intracellular stimulation with 1 s depolarizing pulses delivered at intervals of < 5 s caused slow, cumulative adaptation of spike firing, which was not associated with a change in resting conductance, and which persisted when Co2+ replaced Ca2+ in the bathing medium. 2. Development of slow cumulative adaptation was associated with a gradual decrease in maximal rates of rise of action potentials, a slowing in the post-spike depolarization towards threshold, and a positive shift in the threshold voltage for the next spike in the train; maximal spike repolarization rates and after-hyperpolarizations were unchanged. 3. The data suggested that slow adaptation reflects use-dependent removal of Na+ channels from the available pool by an inactivation process which is much slower than fast, Hodgkin-Huxley-type inactivation. 4. We therefore studied the properties of Na+ channels in layer II-III mouse neocortical cells using the cell-attached configuration of the patch-in-slice technique. These had a slope conductance of 18 +/- 1 pS and an extrapolated reversal potential of 127 +/- 6 mV above resting potential (Vr) (mean +/- S.E.M.; n = 5). Vr was estimated at -72 +/- 3 mV (n = 8), based on the voltage dependence of the steady-state inactivation (h infinity) curve. 5. Slow inactivation (SI) of Na+ channels had a mono-exponential onset with tau on between 0.86 and 2.33 s (n = 3). Steady-state SI was half-maximal at -43.8 mV and had a slope of 14.4 mV (e-fold)-1. Recovery from a 2 s conditioning pulse was bi-exponential and voltage dependent; the slow time constant ranged between 0.45 and 2.5 s at voltages between-128 and -68 mV. 6. The experimentally determined parameters of SI were adequate to simulate slow cumulative adaptation of spike firing in a single-compartment computer model. 7. Persistent Na+ current, which was recorded in whole-cell configuration during slow voltage ramps (35 mV s-1), also underwent pronounced SI, which was apparent when the ramp was preceded by a prolonged depolarizing pulse.

Extracellular K(+)-induced hyperpolarizations and dilatations of rat coronary and cerebral arteries involve inward rectifier K(+) channels.

Abstract: 1. The hypothesis that inward rectifier K(+) channels are involved in the vasodilatation of small coronary and cerebral arteries (100-200 microm diameter) in response to elevated [K+]o was tested. The diameters and membrane potentials of pressurized arteries from rat were measured using a video-imaging system and conventional microelectrodes, respectively. 2. Elevation of [K+]o from 6 to 16 mM caused the membrane potential of pressurized (60 mmHg) arteries to hyperpolarize by 12-14 mV. Extracellular Ba(2+) (Ba2+(o)) blocked K(+)-induced membrane potential hyperpolarizations at concentrations (IC(50), 6 microM) that block inward rectifier K(+) currents in smooth muscle cells isolated from these arteries. 3. Elevation of [K+]o from 6 to 16 mM caused sustained dilatations of pressurized coronary and cerebral arteries with diameters increasing from 125 to 192 microm and 110 to 180 microm in coronary and cerebral arteries, respectively. Ba2+(o) blocked K(+)-induced dilatations of pressurized coronary and cerebral arteries (IC50, 3-8 microM). 4. Elevated [K+]o-induced vasodilatation was not prevented by blockers of other types of K(+) channels (1 mM 4-aminopyridine, 1 mM TEA+, and 10 mu M glibenclamide), and blockers of Na(+)-K(+)-ATPase. Elevated [K+]o-induced vasodilatation was unaffected by removal of the endothelium. 5. These findings suggest that K+(o) dilates small rat coronary and cerebral arteries through activation of inward rectifier K(+) channels. Furthermore, these results support the hypothesis that inward rectifier K(+) channels may be involved in metabolic regulation of coronary and cerebral blood flow in response to changes in [K+]o.

Molecular determinants for activation and inactivation of HERG, a human inward rectifier potassium channel.

Abstract: 1. The human eag-related potassium channel, HERG, gives rise to inwardly rectifying K+ currents when expressed in Xenopus oocytes. 2. The apparent inward rectification is caused by rapid inactivation. In extracellular Cs+ solutions, large outward currents can be recorded having an inactivation time constant at 0 mV of about 50 ms with an e-fold change every 37 mV. 3. HERG channel inactivation is not caused by an amino-terminal ball structure, as a deletion of the cytoplasmic amino terminus (HERG delta 2-373) did not eliminate inactivation. However, channel deactivation was accelerated about 12-fold at -80 mV. 4. Mutation of S631 to A, the homologous residue of eag channels, in the outer mouth of the HERG pore completely abolished channel inactivation. 5. Activity of HERG channels depended on extracellular cations, which are effective for channel activation, in the order Cs+ > K+ > > Li+ > Na+. The point mutation S631A strongly reduced this channel regulation. 6. By analogy to functional aspects of cloned voltage-gated potassium channels, rectification of HERG, as well as its kinetic properties during the course of an action potential, are presumably governed by a mechanism reminiscent of C-type inactivation.

Single axon IPSPs elicited in pyramidal cells by three classes of interneurones in slices of rat neocortex.

Abstract: 1. Using dual intracellular recordings in slices of adult rat neocortex, twenty-four IPSPs activated by single presynaptic interneurones were studied in simultaneously recorded pyramidal cells. Fast spiking interneurones inhibited one in four or five of their close pyramidal neighbours. No reciprocal connections were observed. After recordings neurones were filled with biocytin. 2. Interneurones that elicited IPSPs were classified as classical fast spiking (n = 10), as non-classical fast spiking (n = 3, including one burst-firing interneurone), as unclassified, or slow interneurones (n = 8), or as regular spiking interneurones (n = 3), i.e. interneurones whose electrophysiological characteristics were indistinguishable from those of pyramidal cells. 3. All of the seven classical fast spiking cells anatomically fully recovered had aspiny, beaded dendrites. Their partially myelinated axons ramified extensively, varying widely in shape and extent, but randomly selected labelled axon terminals typically innervated somata and large calibre dendrites on electron microscopic examination. One 'autapse' was demonstrated. One presumptive regular spiking interneurone axon made four somatic and five dendritic connections with unlabelled targets. 4. Full anatomical reconstructions of labelled classical fast spiking interneurones and their postsynaptic pyramids (n = 5) demonstrated one to five boutons per connection. The two recorded IPSPs that were fully reconstructed morphologically (3 and 5 terminals) were, however, amongst the smallest recorded ( 20 spikes (> 150 Hz), however, resulted in an additional, more slowly decaying component (latency > 50 ms, duration > 200 ms). The possible involvement of GABAB receptors in this component is discussed. 9. It is suggested that three broad classes of interneurones may activate GABAA receptors on relatively proximal portions of neocortical pyramidal neurones. The different time courses of the IPSPs elicited by the three classes may reflect different types of postsynaptic receptor rather than dendritic location. An additional class, burst firing, spiny interneurones appear to activate GABAA receptors on more distal sites.

Excitatory actions of gaba in developing rat hypothalamic neurones

Abstract: 1. Gramicidin-perforated patch clamp recording was employed to study GABA-mediated responses in rat hypothalamic neurones (n = 102) with an intracellular Cl- concentration unaltered by the pipette solution. 2. In young cultures after 1-7 days in vitro (DIV), GABA induced depolarizing membrane potentials (+16.5 +/- 1.3 mV) that often surpassed the threshold for the firing of action potentials (-42 +/- 1 mV) and resulted in an increase in neuronal activity. The depolarizing responses to GABA in young cultures were dose dependent. The concentration of GABA necessary to evoke the half-maximal depolarization (EC50) was 2.8 microM. In contrast, GABA induced hyperpolarizing membrane potentials (-12.0 +/- 1.4 mV) and a decrease in neuronal activity in older neurones (20-33 DIV). Both the depolarization and the hyperpolarization induced by GABA were blocked by bicuculline, indicating a mediation by GABAA receptors. 3. The reversal potentials of the GABA-evoked currents were between -40 to -50 mV during the first week of culture, and shifted to below -70 mV after 3 weeks of culture. In parallel, neurones that were dissociated from older animals (postnatal day 5) had a more negative reversal potential for the GABA-evoked currents than cells from younger animals (embryonic day 15), suggesting that the negative shift of the reversal potential occurs both in vitro and in vivo. Our data suggest that the mechanism for GABA-induced depolarization is the depolarized Cl- reversal potential found in young but not older neurones. 4. Consistent with the depolarizing response to exogenous application of GABA, some spontaneous depolarizing postsynaptic potentials in young cultures were insensitive to AP5-CNQX, but were eliminated by bicuculline, indicating that synaptically released GABA mediated excitatory synaptic transmission in early development. 5. By combining a rapid computer-controlled delivery of GABA with subthreshold positive current injections into recorded neurones, we found in young cultures that the GABA-evoked depolarization could directly trigger action potentials, facilitate some depolarizing input to fire action potentials, and shunt other depolarizing input. Whether the GABA-induced depolarization is excitatory or inhibitory would be determined by the reversal potential of the GABA-evoked current, and the temporal relationship between GABA-evoked depolarizations and other excitatory events. 6. We conclude that the reversal potential of the GABA-evoked current shifts negatively from depolarizing to hyperpolarizing in developing hypothalamus. Consequently, GABA neurotransmission may serve both excitatory and inhibitory roles during early development.

Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells.

Abstract: 1. The M2 protein of influenza A virus is implicated in transmembrane pH regulation during infection. Whole-cell patch clamp of mouse erythroleukaemia cells expressing the M2 protein in the surface membrane showed a conductance due to M2 which was specifically blocked by the anti-influenza drug rimantadine. 2. The ion selectivity of the rimantadine-sensitive current through M2 was determined. Reversal potentials were close to equilibrium potentials for transmembrane pH gradients and not to those for Na+, K+ or Cl- concentration gradients. M2 permeability to Na+ relative to H+ was estimated to be less than 6 x 10(-7). 3. The M2 conductance increased as external pH decreased below 8.5 and approached saturation at an external pH of 4, effects attributable to increased permeability due to increased driving potential and to activation by low external pH. Both activation and permeation could be described by interaction of protons with sites on M2, with apparent dissociation constants of approximately 0.1 microM and 1 microM, respectively, under physiological conditions. 4. The M2 protein can transfer protons selectively across membranes with the H+ electrochemical gradient, properties consistent with its role in modifying virion and trans-Golgi pH during virus infection.

Subtype-specific regulation of recombinant NMDA receptor-channels by protein tyrosine kinases of the src family.

Abstract: 1. Tyrosine kinases regulate NMDA receptor-channel activity in cultured neurons, and NMDA receptor subunits are tyrosine phosphorylated in the brain. 2. Heteromeric NMDA receptor-channels were transiently expressed in human embryonic kidney (HEK) 293 cells and glutamate (100 microM)-activated whole-cell currents (500 ms) were studied when tyrosine kinases of the src gene family were included in the pipette solution. 3. Glutamate-activated currents (evoked every 20 s for up to 20 min) were increased by src and fyn kinases without affecting the desensitization and deactivation kinetics in NR1-NR2A but the kinases had no effects in NR1-NR2B, NR1-NR2C and NR1-NR2D receptor-channels, suggesting that a phosphorylation site in NR2A is targeted. 4. In a mutant channel consisting of NR1 and a C-terminal deletion mutant of NR2A (NR2A delta C), src and fyn kinases lost their potentiating effects indicating that the phosphorylation of tyrosine(s) in the C-terminal domain of NR2A affects the current flux through native NMDA receptor-channels.

Spindle oscillation in cats : the role of corticothalamic feedback in a thalamically generated rhythm

Abstract: 1. Spindles represent an oscillatory activity (7-14 Hz) of the electroencephalogram (EEG) originating in the thalamus and appearing during early stages of sleep. We investigated: (i) the phase relations between thalamic and cortical neurons during this rhythm; (ii) the patterns of spindles under different anaesthetics and their modifications at various levels of the membrane potential (Vm); and (iii) the potentiating role of the corticothalamic feedback in the genesis of spindles. Intra- and extracellular recordings were performed in cats from reticular and dorsal thalamic nuclei, as well as from various cortical areas. 2. In thalamic reticular neurons, spindles were sequences of waves at 7-14 Hz, riding on a prolonged depolarizing plateau and occurring in phase with depth-negative cortical EEG waves. In thalamocortical cells, spindles consisted of inhibitory postsynaptic potentials (IPSPs) in phase with depth-positive cortical EEG waves and occasionally leading to rebound spike bursts. In cortical cells, spindle waves were rhythmic (7-14 Hz) excitatory postsynaptic potentials (EPSPs) that sometimes gave rise to action potentials. Spindles occurred in phase among thalamic reticular, thalamocortical and neocortical neurons. 3. In thalamic reticular neurons, spindle waves and their depolarizing plateaux increased in amplitude with slight cellular hyperpolarization, but at a Vm more negative than -80 or -85 mV they decreased in amplitude. No frequency alterations were observed with these Vm changes. 4. The waxing-and-waning pattern of spontaneous spindles under barbiturate anaesthesia was distinct from the waning pattern under ketamine-xylazine anaesthesia. Under all anaesthetics, spindles had a waning pattern when elicited by cortical stimuli. The amplitude of cortical-evoked spindle waves diminished with the decrease in stimulation intensity. 5. Under urethane or ketamine-xylazine anaesthesia, spindle sequences were grouped by a cortically generated slow oscillation (< 1 Hz) and were preceded by a depth-positive EEG wave that corresponded to a prolonged hyperpolarization in all three investigated (cortical, thalamic reticular, and thalamocortical) cellular types. 6. We propose that the waxing pattern of spindle oscillation is due to a progressive entrainment of units into the oscillation until a maximum number is reached, depending on the background activity in the network. The phase relations between cortical, thalamic reticular and thalamocortical neurons are ascribed to distributed excitatory signals from thalamocortical neurons to both cortical and reticular neurons at each cycle of the oscillation. In turn, cortical neurons provide a powerful drive to potentiate the genesis of thalamic spindles.

Hypoxia increases persistent sodium current in rat ventricular myocytes.

Abstract: 1. A persistent inward current activated by depolarization was recorded using the whole-cell, tight seal technique in rat isolated cardiac myocytes. The amplitude of the inward current increased when cells were exposed to a solution with low oxygen tension. 2. The persistent inward current had the characteristics of the persistent Na+ current described previously in rat ventricular myocytes: it was activated at negative potentials (-70 mV), reversed close to the equilibrium potential for Na+ (ENa), was blocked by TTX and was resistant to inactivation. 3. Persistent single Na+ channel currents activated by long (200-400 ms) depolarizations were recorded in cell-attached patches on isolated ventricular myocytes. Hypoxia increased the frequency of opening of the persistent Na+ channels. 4. Persistent Na+ channels recorded during hypoxia had characteristics similar to those of persistent Na+ channels recorded at normal oxygen tensions. They had a null potential at ENa, their amplitude varied with [Na+], they were resistant to inactivation and their mean open time increased with increasing depolarization. 5. The persistent Na+ channels in cell-attached patches were blocked by TTX (50 microM) in the patch pipette and by lidocaine (100 microM). 6. It was concluded that hypoxia increases the open probability of TTX-sensitive, inactivation-resistant Na+ channels. The voltage dependence of these channels, and their greatly increased activity during hypoxia, suggest that they may play an important role in the generation of arrhythmias during hypoxia.

Non-NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites.

Abstract: 1. Excitatory postsynaptic currents (EPSCs) were recorded under whole-cell voltage clamp from granule cells in slices of rat cerebellum. EPSCs from individual mossy fibre inputs were identified by their all-or-none appearance in response to a graded stimulus. Excitatory synaptic transmission was investigated at room temperature (approximately 24 degrees C) and at near-physiological temperature (approximately 34 degrees C) by analysing current fluctuations in the peak and decay of the non-N-methyl-D-aspartate (non-NMDA) component of EPSCs. 2. In a subset of synapses the mean EPSC amplitude remained unchanged as the probability of transmitter release was substantially lowered by raising the extracellular [Mg2+] and lowering [Ca2+]. These synapses were considered to have only one functional release site. Single-site synapses had small EPSCs (139 +/- 16 pS, n = 5, at 24 degrees C) with a large coefficient of variation (c.v. = 0.23 +/- 0.02, n = 5) and an amplitude distribution that was well fitted by a Gaussian distribution in four out of five cases. The EPSC latency had a unimodal distribution and its standard deviation had a temperature dependence with a temperature coefficient (Q10; range, 24-35 degrees C) of 2.4 +/- 0.4 (n = 4). 3. Peak-scaled non-stationary fluctuation analysis of single-site EPSCs indicated that the mean conductance of the underlying non-NMDA channels was 12 +/- 2 pS (n = 4) at 35 degrees C. Upper and lower limits for mean channel open probability (Po), calculated from fluctuations in the EPSC peak amplitude, were 0.51 and 0.38, respectively. These estimates, together with the open probability of the channel when bound by transmitter, suggest that only about 50% of the non-NMDA channels were occupied following the release of a quantum of transmitter. 4. At some multi-site synapses EPSCs had a low c.v. (0.4 +/- 0.01, n = 5) at 34 degrees C and non-stationary fluctuation analysis gave a parabolic variance-mean current relationship. This suggests that practically all of the non-NMDA receptors were occupied by glutamate at the peak of EPSC. The channel open probability (Po = 0.84 +/- 0.03, n = 5) at these 'saturated' multi-site synapses will therefore equal the open probability of the channel when bound by transmitter (Po,max). 5. Non-stationary fluctuation analysis of EPSCs from 'saturating' multi-site synapses indicated that 170 +/- 40 postsynaptic non-NMDA channels were exposed to transmitter at the peak of the EPSC. The mean conductance of the synaptic channels was 10 +/- 2 pS (n = 5) at 34 degrees C. 6. At synapses with multiple release sites the EPSC decay time became faster when release probability was lowered (by reducing the external [Ca2+]/[Mg2+] ratio), indicating that the transmitter concentration profile depended on release probability. No such speeding of the EPSC decay was observed at single-site synapses. 7. Our results suggest that release of a packet of transmitter from a single release site does not saturate postsynaptic non-NMDA receptors at cerebellar mossy fibre-granule cell synapses. However, at multi-site synapses transmitter released from neighbouring sites can overlap, changing the transmitter concentration profile in the synaptic cleft. We conclude that the level of postsynaptic receptor occupancy can depend on the probability of transmitter release at individual multi-site synapses.

Influence of actinomycin d, a rna synthesis inhibitor, on long-term potentiation in rat hippocampal neurons in vivo and in vitro

Abstract: 1. Hippocampal long-term potentiation (LTP) may serve as an elementary process underlying certain forms of learning and memory in vertebrates. As is the case with behavioural memory, hippocampal LTP in the rat CA1 region and in the dentate gyrus occurs in stages, which can be separated by an inhibitor of RNA synthesis. 2. Experiments have been performed in two brain regions, in the hippocampal CA1 region in vitro and in the dentate gyrus in vivo. 3. Maintenance of hippocampal LTP in the CA1 region in vitro was influenced by the RNA synthesis inhibitor actinomycin D from 4 h onwards. 4. The effect of actinomycin D on the time course of the population spike potentiation was more pronounced than the effect on the time course of the EPSP component, suggesting different mechanisms for the two forms of potentiation. 5. Intrahippocampal and intracerebroventricular injection of actinomycin D into rats prevented a late stage of LTP in the dentate gyrus in vivo measured as the population spike amplitude. 6. Since actinomycin D was only effective in influencing the maintenance of LTP when applied before tetanization, the requirement for transcription during LTP may have a critical time window. 7. Actinomycin D influenced the maintenance of LTP specifically, since the drug did not alter any potentials in control experiments after its removal or when it was administered shortly after tetanization. A second, structurally different RNA synthesis inhibitor, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole, mimicked the effect of actinomycin D in vitro.

Myofibrillar ATPase activity in skinned human skeletal muscle fibres: fibre type and temperature dependence.

Abstract: 1. Myofibrillar ATP consumption and isometric tension (P0) were determined in chemically skinned skeletal muscle fibres from human rectus abdominis and vastus lateralis muscle. Fibres were classified in four groups (I, IIA, IIB, IIA/B or mixed) based on myosin heavy chain composition. 2. ATP consumption (+/- S.E.M.) at 20 degrees C varied from 0.41 +/- 0.06 mmol l-1 s-1 in type IIB fibres (n = 5) to 0.10 +/- 0.01 mmol l-1 s-1 in type I fibres (n = 13). 3. The ratio between ATPase activity and P0 (tension cost) differed significantly between fast type II and slow type I fibres. At 12 degrees C tension cost was lower than the values found previously in corresponding fibre types in the rat. 4. The relative increase in ATPase activity for a 10 degrees C temperature change (Q10), determined in the range from 12 to 30 degrees C, was temperature independent and amounted to 2.60 +/- 0.06. The increase in P0 with temperature was smaller and declined when the temperature increased. 5. From these measurements, estimates were obtained for the maximum rate of isometric ATP consumption and force development at muscle temperature in vivo (35 degrees C).

Skin blood flow responses to the iontophoresis of acetylcholine and sodium nitroprusside in man: possible mechanisms.

Abstract: 1. The mechanisms involved in the human skin blood flow responses to iontophoretic application of acetylcholine (ACH; delivered using an anodal charge) or sodium nitroprusside (SNP; administered with a cathodal charge) are unclear. The aims of this study were to investigate possible contributions of prostaglandin production to the increase in skin blood flow induced following the iontophoresis of ACh and to investigate possible contributions from local sensory nerves to the perfusion responses induced by ACh, SNP and their vehicles. 2. The contribution of prostaglandins to the ACh response was determined in a randomized double-blind study of eight healthy subjects, who were studied on two occasions. Basal responses to ACh were measured before the oral administration of 600 mg soluble aspirin in diluted orange juice (1 occasion or orange juice (1 occasion) and again 30 min after the drink. The contribution of local sensory nerve activation to the responses to ACh and ACh vehicle (8 subjects) and to SNP and SNP vehicle (7 subjects) was assessed. EMLA (5%) (a eutectic mixture of lignocaine and prilocaine) and placebo cream were applied to two separate areas on the forearm in a double-blind randomized manner 2 h before drug responses were measured. In all studies the skin microcirculation responses to iontophoretically applied drug vehicle (1 site) and drug (2 sites) were recorded by laser Doppler perfusion imaging. 3. The increase in forearm skin perfusion (P < 0.001) in response to the iontophoresis of ACh minus the response to ACh vehicle was not significantly different following placebo or aspirin administration. The increase in forearm skin red blood cell flux (P < 0.001) in response to the iontophoresis of ACh minus the response to ACh vehicle was not significantly different at the placebo-compared with the EMLA-treated site. THe small increase in perfusion (P < 0.001) in response to the iontophoresis of ACh vehicle was significantly inhibited at the EMLA-compared with the placebo-treated site (P < 0.05). The marked increase in perfusion (P < 0.001) in response to the iontophoresis of SNP vehicle was significantly inhibited at the EMLA-compared with the placebo-treated site (P < 0.01). 4. These data suggest that in healthy volunteers: (1) mechanisms other than prostaglandin production and local sensory nerve activation may be involved in the increase in skin perfusion observed following the iontophoretic application of ACh; and (2) stimulation of local sensory nerves may be responsible for the increase in tissue perfusion observed following the iontophoretic application of either ACh vehicle or SNP vehicle.