Showing papers in "The Journal of Physiology in 1989"

Giant synaptic potentials in immature rat CA3 hippocampal neurones.

Abstract: 1. Intracellular recordings were made from rat CA3 hippocampal neurones in vitro during the first eighteen days of postnatal life. The cells had resting membrane potentials more negative than -51 mV, action potentials greater than 55 mV and membrane input resistances of 117 +/- 12 M omega. An unusual characteristic of these cells was the presence of spontaneous giant depolarizing potentials (GDPs) which were observed during the first eight postnatal (P) days in over 85% of neurones. They were less frequent between P9 and P12 (48%) and disappeared after P12. 2. The GDPs were synchronously generated by a population of neurones; they reversed polarity at -27 mV when recorded with KCl-containing electrodes and at -51 mV with potassium acetate- or potassium methylsulphate-filled electrodes. 3. The GDPs were blocked by bath application of bicuculline (10 microM) or picrotoxin (100-200 microM). Exogenously applied gamma-aminobutyric acid (GABA; 0.2-1 mM) induced at resting membrane potential a bicuculline-sensitive membrane depolarization which reversed polarity at -25 and -51 mV when recorded with KCl- or potassium methylsulphate-filled electrodes respectively. 4. The GDPs were reduced in frequency or blocked by the N-methyl-D-aspartate (NMDA) receptor antagonists DL-2-amino-7-phosphonoheptanoate (AP-7; 50 microM), D(-)2-amino-5-phosphonovalerate (AP-5, 10-50 microM) and (+-)3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 10-50 microM) or NMDA channel blockers phencyclidine (2 microM) and ketamine (20 microM). 5. Stimulation of the hilus during the first week of life evoked a GDP followed by a hyperpolarization. The GDPs were generated by a population of synchronized neurones and reversed polarity at -27 mV with KCl-filled electrodes and at -52 mV with potassium acetate- or potassium methylsulphate-containing electrodes. 6. Bath application of bicuculline (1-10 microM) or picrotoxin (100-200 microM) reversibly blocked the evoked GDPs in the majority of cells. The NMDA receptor antagonists AP-5 (50 microM), AP-7 (50 microM) and CPP (30 microM) usually reduced the amplitude and the duration of the evoked GDPs. In neurones in which evoked GDPs were blocked by bicuculline, a NMDA-mediated component was revealed by increasing the strength or the frequency of stimulation. 7. During the second week of postnatal life, when spontaneous GDPs were extremely rare or absent, superfusion with bicuculline (10 microM) induced, as in adult slices, interictal discharges. These reversed polarity near 0 mV with KCl- or potassium acetate-containing electrodes and were reduced in amplitude and duration by AP-5 (50 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Electric and magnetic stimulation of human motor cortex: surface EMG and single motor unit responses.

Abstract: 1. The effects of different forms of brain stimulation on the discharge pattern of single motor units were examined using the post-stimulus time histogram (PSTH) technique and by recording the compound surface electromyographic (EMG) responses in the first dorsal interosseous (FDI) muscle. Electrical and magnetic methods were used to stimulate the brain through the intact scalp of seven normal subjects. Electrical stimuli were applied either with the anode over the lateral central scalp and the cathode at the vertex (anodal stimulation) or with the anode at the vertex and the cathode lateral (cathodal stimulation). Magnetic stimulation used a 9 cm diameter coil centred at the vertex; current in the coil flowed either clockwise or anticlockwise when viewed from above. 2. Supramotor threshold stimuli produced one or more narrow (less than 2 ms) peaks of increased firing in the PSTH of all thirty-two units studied. Anodal stimulation always produced an early peak. The latencies of the peaks produced by other forms of stimulation, or by high intensities of anodal stimulation, were grouped into four time bands relative to this early peak, at intervals of -0.5 to 0.5, 1-2, 2.5-3.5 and 4-5.5 ms later. Peaks occurring within these intervals are referred to as P0 (the earliest anodal), P1, P2 and P3 respectively. 3. At threshold, anodal stimulation evoked only the P0 peak; at higher intensities, the P2 or more commonly the P3 peak also was recruited. The size of the P0 peak appeared to saturate at high intensities. 4. In five of six subjects, cathodal stimulation behaved like anodal stimulation, except that there was a lower threshold for recruitment of the P2 or P3 peak relative to that of the P0 peak. In the other subject, the P3 peak was recruited before the P0 peak. 5. Anticlockwise magnetic [corrected] stimulation, at threshold, often produced several peaks. These always included a P1 peak, and usually a P3 peak. A P0 peak in the PSTH was never produced by an anticlockwise stimulation [corrected] at intensities which we could explore with the technique. 6. Clockwise magnetic [corrected] stimulation never recruited a P1 peak; in most subjects a P3 peak was recruited first and at higher intensities was accompanied by P0 or P2 peaks. 7. On most occasions when more than one peak was observed in a PSTH, the unit fired in only one of the preferred intervals after each shock. However, double firing was seen in five units when high intensities of stimulation were used.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective recruitment of high-threshold human motor units during voluntary isotonic lengthening of active muscles.

Abstract: 1. We have investigated the possibility that voluntary muscle lengthening contractions can be performed by selective recruitment of fast-twitch motor units, accompanied by derecruitment of slow-twitch motor units. 2. The behaviour of motor units in soleus, gastrocnemius lateralis and gastrocnemius medialis muscles was studied during (a) controlled isotonic plantar flexion against a constant load (shortening contraction, S), maintained plantar flexion, or dorsal flexion resisting the load and gradually yielding to it (lengthening contraction, L), (b) isometric increasing or decreasing plantar torque accomplished by graded contraction or relaxation of the triceps surae muscles, (c) isometric or isotonic ballistic contractions, and (d) periodic, quasi-sinusoidal isotonic contractions at different velocities. The above tasks were performed under visual control of foot position, without activation of antagonist muscles. The motor units discharging during foot rotation were grouped on the basis of the phase(s) during which they were active as S, S + L and L. The units were also characterized according to both the level of isometric ramp plantar torque at which they were first recruited and the amplitude of their action potential. 3. S units were never active during dorsal flexion; some of them were active during the sustained contraction between plantar and dorsal flexion. Most S + L units were active also during the maintenance phase and were slowly derecruited during lengthening; their behaviour during foot rotations was similar to that during isometric contractions or relaxations. L units were never active during either plantar or maintained flexion, but discharged during lengthening contraction in a given range of rotation velocities; the velocity of lengthening consistently influenced the firing frequency of these units. Such dependence on velocity was not observed in S + L units. 4. A correlation was found between the amplitude of the action potential and the threshold torque of recruitment among all the units. In addition, the amplitudes of both the action potential and the threshold torque were higher in the case of L units than in the case of S and S + L units. Most L units could be voluntarily recruited only in the case of ballistic isometric or isotonic contraction. 5. Occasionally, L units were directly activated by electrical stimulation of motor fibres and their conduction velocity was in the higher range for alpha-axons. In contrast, nerve stimulation could induce a reflex activation of S and S + L units.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonin-induced bistability of turtle motoneurones caused by a nifedipine-sensitive calcium plateau potential.

Abstract: 1. The effect of serotonin on the firing properties of motoneurones was studied in transverse sections of the adult turtle spinal cord in vitro with intracellular recording techniques. 2. In normal medium, turtle motoneurones adapt from an initial high frequency to a low steady firing during a depolarizing current pulse. In the presence of serotonin (4-100 microM) motoneurones responded with accelerated firing and a frequency jump during a depolarizing current pulse followed by an after-depolarization outlasting the stimulus. From a depolarized holding potential motoneuronal activity was shifted between two stable states by brief depolarizing and hyperpolarizing current pulses. As an expression of this bistable firing behaviour, the frequency-current relation in response to a triangular current injection was counter-clockwise in serotonin while clockwise in normal medium. 3. The delay to onset of the frequency jump was shortened as the amplitude of the activation pulse was increased. From a positive holding potential the after-depolarization exceeded spike threshold and its duration increased with an increase in steady bias current. The effect of serotonin on turtle motoneurones could be blocked by methysergide (10 microM). 4. When action potentials were depressed by tetrodotoxin, a voltage-dependent, non-inactivating plateau potential, intrinsic to the motoneurone, was revealed. Activation of this voltage plateau provides the motoneurones with two stable states of firing. The apparent input resistance was 2-4-fold lower during the plateau than at rest. 5. The serotonin-induced plateau potential was Ca2+-dependent and was blocked when Ca2+ was replaced by either Co2+ (3 mM) or Mn2+ (3 mM). 6. The Ca2+ plateau was blocked by nifedipine (1-15 microM). 7. Serotonin reduced the slow after-hyperpolarization following action potentials. The change in balance between inward and outward currents seems to be sufficient to reveal the plateau response. 8. Although a small plateau response was induced by Bay K 8644 (1-15 microM), this L-channel agonist could not reproduce the pronounced effect of serotonin. 9. It is concluded that serotonin induces a Ca2+-dependent and nifedipine-sensitive plateau potential in turtle motoneurones primarily by reducing a K+-current responsible for the slow after-hyperpolarization.

Calcium currents in rat thalamocortical relay neurones: kinetic properties of the transient, low‐threshold current.

Abstract: 1. Calcium currents were recorded with whole-cell voltage-clamp procedures in relay neurones of the rat thalamus which had been acutely isolated by an enzymatic dissociation procedure. 2. Low-threshold and high-threshold Ca2+ currents were elicited by depolarizing voltage steps from holding potentials more negative than -60 mV. A transient current, analogous to the T-current in sensory neurones, was activated at low threshold near -65 mV and was completely inactivating at command steps up to -35 mV. Voltage steps to more depolarized levels activated a high-threshold current that inactivated slowly and incompletely during a 200 ms step depolarization. 3. The high-threshold current contained both non-inactivating and slowly inactivating components which were insensitive and sensitive to holding potential, respectively. 4. A 'T-type' current was prominent in relay neurones, in both absolute terms (350 pA peak current average) and in relation to high-threshold currents. The average ratio of maximum transient to maximum sustained current was greater than 2. 5. T-current could be modelled in a manner analogous to that employed for the fast Na+ current underlying action potential generation, using the m3h format. The rate of activation of T-current was voltage dependent, with a time constant (tau m) varying between 8 and 2 ms at command potentials of -60 to -10 mV at 23 degrees C. The rate of inactivation was also voltage dependent, and the time constant tau h varied between 50 and 20 ms over the same voltage range. With command potentials more positive than -35 mV, the inactivation of Ca2+ current could no longer be fitted by a single exponential. 6. Steady-state inactivation of T-current could be well fitted by a Boltzman equation with slope factor of 6.3 and half-inactivated voltage of -83.5 mV. 7. Recovery from inactivation of T-current was not exponential. The major component of recovery (70-80% of total) was not very voltage sensitive at potentials more negative than -90 mV, with tau r of 251 ms at -92 mV and 23 degrees C, compared to 225 ms at -112 mV. A smaller, voltage-sensitive component accounted for the remainder of recovery. 8. All kinetic properties, including rates of activation, inactivation, and recovery from inactivation, as well as the amplitude of T-current, were temperature sensitive with Q10 (temperature coefficient) values of greater than 2.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Frequency‐dependent depression of inhibition in guinea‐pig neocortex in vitro by GABAB receptor feed‐back on GABA release.

Abstract: 1. The mechanisms involved in the lability of inhibition at higher frequencies of stimulation were investigated in the guinea-pig in vitro neocortical slice preparation by intracellular recording techniques. We attempted to test the possibility of a feedback depression of GABA on subsequent release. 2. At resting membrane potential (Em, -75.8 +/- 5.2 mV) stimulation of either the pial surface or subcortical white matter evoked a sequence of depolarizing and hyperpolarizing synaptic components in most neurones. An early hyperpolarizing component (IPSPA) was usually only obvious as a pronounced termination of the EPSP, followed by a later hyperpolarizing event (IPSPB). Current-voltage relationships revealed two different conductances of about 200 and 20 nS and reversal potentials of -73.0 +/- 4.4 and -88.6 +/- 6.1 mV for the early and late component, respectively. 3. The conductances of IPSPA and IPSPB were fairly stable at a stimulus frequency of 0.1 Hz. At frequencies between 0.5 and 2 Hz both IPSPs were attenuated with the second stimulus and after about five stimuli a steady state was reached. Concomitantly IPSPs were shortened. The average decrease in synaptic conductance between 0.1 and 1 Hz was 80% for the IPSPA and 60% for the IPSPB. At these frequencies the reversal potentials decreased by 5 and 2 mV, respectively; Em and input resistance (Rin) were not consistently affected. 4. The amplitudes of field potentials, action potentials and EPSPs of pyramidal cells were attenuated less than 10% at stimulus frequencies up to 1 Hz, suggesting that alterations in local circuits between the stimulation site and excitatory input onto inhibitory interneurones may play only a minor role in the frequency-dependent decay of IPSPs. 5. Localized application of GABA produced multiphasic responses. With low concentrations and application near the soma an early hyperpolarization prevailed followed by a depolarizing late component. Brief application of GABA at low frequencies induced constant responses; at higher frequencies, the responses sometimes declined. The current-voltage relationships of the two GABA responses were similar to each other and to the early IPSP. An apparently fivefold higher conductance was estimated at lower Ems, suggesting that the GABA response had a voltage sensitivity. The slope conductance of IPSPs was decreased by up to 50% for tens of seconds after postsynaptically detectable effects of GABA had dissipated. 6. Application of the GABA uptake inhibitor nipecotic acid (50-500 microM) reduced the conductance of both components of orthodromically evoked inhibition and shortened the IPSP at low frequencies, but had no additional effects at higher stimulation rates.(ABSTRACT TRUNCATED AT 400 WORDS)

Dihydropyridine-sensitive low-threshold calcium channels in isolated rat hypothalamic neurones.

Abstract: 1. Low-voltage-activated Ca2+ channels which produce a transient inward current were studied in neurones freshly isolated from the ventromedial hypothalamic region of the rat. Membrane currents were recorded using a suction-pipette technique which allows for internal perfusion under a single-electrode voltage clamp. A concentration-jump technique was also used for rapid drug application. 2. In most cells superfused with 10 mM-Ca2+, a transient inward Ca2+ current was evoked by a step depolarization to potentials more positive than -65 mV from a holding potential of -100 mV. Such a low-threshold Ca2+ current could easily be separated from a high-threshold, steady type of Ca2+ current by selecting the holding and test potential levels, as well as by resistance to the wash-out during cell dialysis. 3. Activation and inactivation processes of the low-threshold Ca2+ current were highly potential dependent at 20-22 degrees C. For a test potential change from -60 to +20 mV, the time to peak of the current decreased from 45 to 9 ms, and the time constant of the current decay decreased from 90 to 40 ms. The steady-state inactivation occurred at very negative potentials, reaching a 50% level at -93 mV. Recovery from inactivation showed a time constant between 2.63 and 0.94 s for a potential change from -80 to -120 mV. 4. The amplitude of the low-threshold Ca2+ current depended on the external Ca2+ concentration [( Ca2+]o), approaching saturation at 100 mM [Ca2+]o. Ba2+ substituted for Ca2+ reduced the current amplitude by 30-50% while Sr2+ produced no definite changes in the current amplitude. 5. The low-threshold Ca2+ current was blocked by various di- or trivalent cations in the sequence of La3+ greater than Zn2+ greater than Cd2+ greater than Ni2+ greater than Co2+. The corresponding apparent dissociation constants (KD) were 7 x 10(-7), 1 x 10(-4), 3 x 10(-4), 6 x 10(-4) and 3 x 10(-3) M. 6. Various organic Ca2+ antagonists were effective in blocking the low-threshold Ca2+ current in the following sequence: flunarizine greater than nicardipine greater than nifedipine greater than nimodipine greater than D600 (methoxyverapamil) greater than diltiazem. The corresponding KDs were 7 x 10(-7), 3.5 x 10(-6), 5 x 10(-6), 7 x 10(-6), 5 x 10(-5) and 7 x 10(-5) M. These Ca2+ antagonists induced a use-dependent decrease in the current amplitude.(ABSTRACT TRUNCATED AT 400 WORDS)

The organization of heterogenic reflexes among muscles crossing the ankle joint in the decerebrate cat.

Abstract: 1. Mechanical actions of heterogenic (intermuscular) reflexes arising from proprioceptors in flexor and extensor ankle muscles were measured in intercollicular and premammillary decerebrate cats. Length inputs were applied to the freed tendons of one of a pair of muscles crossing the ankle joint and resulting changes in force in both muscles were measured. Interactions between autogenic and heterogenic reflexes were studied by applying length changes to both muscles. 2. A consistent asymmetry was observed in the heterogenic inhibition between the single-joint antagonists soleus and tibialis anterior (TA). Inhibition from soleus to TA was weak or absent during the reflex activation of TA. In contrast, a strong heterogenic inhibition was consistently observed from TA to soleus during the activation of soleus by a crossed-extension reflex. The effect of this inhibition in the intact joint is to increase the apparent mechanical stiffness of soleus. 3. Mutual synergism among soleus, medial gastrocnemius (MG) and lateral gastrocnemius (LG) was demonstrated only at low to moderate forces by the observation of excitatory reflexes among them. During a naturally or electrically evoked crossed-extension reflex, however, a unidirectional inhibitory reflex from MG and LG to soleus was observed. This inhibition increased with force in MG or LG. These results suggest that the knee and ankle joints become more tightly linked mechanically at high forces since the stiffness of the biarticular gastrocnemius muscle predominates over that of the uniarticular soleus. 4. Under quiescent conditions (no resting muscle activation), mutual synergism was obeyed among the ankle extensors soleus, LG and MG and also between the pretibial flexors TA and extensor digitorum longus (EDL). Moreover, inhibition was generally observed between a pretibial flexor and an ankle extensor. Departures from this expected pattern of heterogenic reflexes occurred when the muscle groups were activated by crossed-extension and flexion reflexes. Reflexes onto soleus, TA and EDL reversed in sign or increased in magnitude. 5. The observed patterns of reflex connectivity among the ankle flexors and extensors were similar in both intercollicular and premammillary preparations, although changes in reflex strength were sometimes noted in cases where a second, lower transection was performed during the experiment. 6. It is argued from the large magnitudes of certain heterogenic reflexes that the mechanical response properties of muscles crossing the ankle joint in the intact animal are not dominated by autogenic reflexes and intrinsic mechanical properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes of intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle.

Abstract: 1. Maximal calcium-activated force (Fmax) and calcium sensitivity were markedly decreased in detergent-skinned fibres from skeletal and cardiac muscle by solutions that mimicked the total milieu changes associated with fatigue and hypoxia. Further experiments determined the relative contribution of each of the individual changes in milieu. 2. Both Ca2+ sensitivity and Fmax of skeletal and cardiac fibres were decreased with increased [H+] or inorganic phosphate (Pi). These effects were greater in cardiac muscle. 3. Decreasing MgATP over the range observed with fatigue and hypoxia (6.8-4.7 mM) had no effect on Fmax or Ca2+ sensitivity of either muscle type. 4. Decreasing phosphocreatine (PCr: 15-1 mM) increased Fmax but had little effect on Ca2+ sensitivity in both muscle types. In cardiac fibres, the effect on Fmax could be mimicked by inhibition of endogenous creatine kinase. 5. ADP (0.7 mM) increased Fmax and Ca2+ sensitivity, while AMP (0.06 mM) slightly increased Fmax but had no effect on Ca2+ sensitivity of either skeletal or cardiac fibres. 6. Creatine (25 mM) had no significant effect on either Ca2+ sensitivity or Fmax of skeletal and cardiac muscle fibres. At higher levels (50 mM), however, creatine depressed Fmax and slightly altered Ca2+ sensitivity. 7. Thiophosphorylation of myosin P light chains (phosphorylatable light chains of myosin) in rabbit psoas fibres had no effect on Ca2+ sensitivity, yet slightly but significantly increased Fmax under fatigue conditions. 8. Reducing the affinity for ATP hydrolysis (by adding ADP, AMP and creatine) over the range calculated for fatigue/hypoxia (60-45 kJ/mol) produced the enhancement in Fmax expected from added ADP and AMP in cardiac but not skeletal muscle, indicating that changes in affinity influence Fmax of skeletal muscle. Reducing affinity produced little change in Ca2+ sensitivity of skeletal muscle. In contrast, the change produced in cardiac muscle was greater than that expected from addition of ADP and AMP; i.e. decreasing affinity increases calcium sensitivity of the heart. 9. Simple summation of all significant changes expected from each constituent altered by fatigue/hypoxia adequately predicted the observed changes in Fmax and Ca2+ sensitivity in both cardiac and skeletal muscle fibres with but one exception (the change in Ca2+ sensitivity of skeletal muscle at pH 7 was slightly overestimated).

An after-hyperpolarization of medium duration in rat hippocampal pyramidal cells.

Abstract: 1. In hippocampal pyramidal cells, action potentials are followed by three after-hyperpolarizations (AHPs): a fast AHP (fAHP) lasting 2-5 ms, a medium AHP (mAHP) lasting 50-100 ms, and a slow AHP (sAHP) lasting more than 1 s. The mechanism underlying the mAHP was studied in CA1 cells (n = 46) in rat hippocampal slices, using injection of depolarizing current to elicit discharge. 2. The current underlying the mAHP was studied by single-electrode voltage clamp in two ways. Either the voltage clamp was activated following a burst of spikes, thus recording the early tail current underlying the mAHP (hybrid clamp), or, after blocking the spikes with tetrodotoxin, the early tail current following a depolarizing voltage clamp command (to -20 to -45 mV for 100-400 ms) was measured. In both cases, the early tail current (measured at -60 mV) showed the following characteristics: (a) it decayed exponentially with a time constant of about 50 ms; (b) it was substantially reduced by the muscarinic agonist carbachol (40-50 microM); (c) it was moderately reduced (by 20% or less) by Ca2+-free medium and Ca2+ channel blockers (Cd2+, Mn2+), which abolished the fAHP and the sAHP; (d) it was partly blocked by tetraethylammonium (TEA, 1-10 mM) both before and during Ca2+ channel blockade; (e) it was resistant to noradrenaline (5-10 microM), which blocked the sAHP, and to apamin (100 nM). 3. The mAHP itself, recorded under current clamp, showed properties corresponding to those of the early tail current. 4. Unlike the current underlying the sAHP, which was reduced and reversed by hyperpolarization, the early tail current appeared to be reduced only at potentials down to -80 mV, and to increase at more negative potentials. The early tail current and mAHP-like undershoot at hyperpolarized potentials was blocked by external Cs+, but not by carbachol, in contrast to the early tail current and mAHP at -60 mV. 5. It was concluded that two currents contribute to the mAHP: IM (a voltage-gated muscarine-sensitive K+ current) and IC (a Ca2+-dependent TEA-sensitive K+ current). TEA reduced both the IM (5 mM) and the IC (1 mM) component of the mAHP. When the cell is hyperpolarized, a third current, IQ (a Ca+-sensitive mixed Na+-K+ inward current activated by hyperpolarization), masks the reversal of the mAHP by causing a depolarizing sag which resembles the decay of the mAHP.

Nucleotide modulation of the activity of rat heart ATP-sensitive K+ channels in isolated membrane patches.

Abstract: 1. We have measured the ATP dependence of KATP channel activity, and the effect of various metabolites on this relationship, in inside-out membrane patches isolated from rat ventricular myocytes. 2. The inhibition of KATP channel activity by ATP could be described as a sigmoid function of [ATP] with a Hill coefficient (HATP) of 2 and a half-maximal inhibition at an ATP concentration (Ki, ATP) of 25 microM, in the presence of 0 mM, or 0.5 mM, total [Mg2+]. The non-hydrolysable ATP analogue, AMP-PNP, also inhibited the channel with Ki, AMP-PNP = 60 microM and HAMP-PNP = 2. 3. Acidosis caused a small, but significant, increase in Ki, ATP from 25 microM at pH 7.25 to 50 microM at pH 6.25, but phosphate and lactate were without effect (at 20 mM) on channel activity. 4. In the absence of ATP or Mg2+, ADP3- inhibited channel activity with Ki, ADP = 275 microM, and HADP = 1.2. Other purine and pyrimidine triphosphates, diphosphates and monophosphates also inhibited the channel with apparent order of inhibitory effectiveness ATP greater than AMP-PNP greater than ADP greater than CTP greater than GDP = AMP = ITP. 5. In the absence of Mg2+, but in the presence of 40 microM-ATP, channel inhibition by GTP, ITP, CTP, GDP, ADP or AMP was additive with inhibition by ATP. 6. In the presence of 0.5 mM-Mg2+ and 40 microM-ATP, inhibition by GTP, GMP and AMP was still additive with inhibition by ATP. The diphosphates ADP and GDP, however, paradoxically increased channel activity in the presence of ATP. This increase in channel activity appeared to result from a competitive increase in Ki, ATP, MgADP did not appear to cause any inhibition of channel activity. 7. We conclude that, in cardiac tissue, KATP channels are regulated by [ATP], and that this regulation is sensitive to other intracellular nucleotides, Mg2+, and pH, but not to phosphate or lactate. A simple, interactive two binding-site model is consistent with the nucleotide-dependent regulation that we observe.

Sensations evoked by intraneural microstimulation of C nociceptor fibres in human skin nerves.

Abstract: 1. Seventy-one C polymodal nociceptors supplying glabrous and hairy skin in limbs of awake human volunteers were identified on the basis of cutaneous stimulus-response characteristics recorded intraneurally by microneurography (MNG). The large majority of such units were primarily detected during intraneural microstimulation (INMS) on the basis of subjective quality and cutaneous localization of evoked painful sensation. Electrophysiological studies were supplemented with rigorous psychophysical testing during microstimulation delivered at intraneural C recording sites. 2. The conduction velocity of single C nociceptor units could be shown to become transiently slowed following repetitive INMS at threshold intensity for conscious sensation. Such ‘marking’ witnessed that particular C units, identified by recording, had been effectively activated during INMS and psychophysical testing. 3. Cognitive attributes of sensations evoked from C recording sites by INMS at threshold intensity for perception were estimated psychophysically for subjective quality, temporal attributes and localized projection. There was remarkable matching of physiological unit type (C polymodal nociceptor) with subjective quality of evoked sensation (dull or burning pain). Further, there was remarkable spatial matching of receptive field of given C nociceptors with projected field of the pain sensation evoked from the C recording site by INMS delivered at threshold intensity for conscious sensation. 4. Dissociated A nerve fibre blocks caused by compression-ischaemia did not abolish the sensation of burning pain projected to hairy skin, evoked by INMS delivered at C recording sites. 5. While the double matching of (a) subjective quality and spatial localization with (b) objective physiological unitary type and receptor location, coupled with the results of A blocks, provide evidence that C nociceptor fibres can be fairly selectively activated during INMS, the results also attest that C polymodal nociceptors from human skin evoke delayed dull or burning pain, accurately projected to a defined locus in skin, even after spatial summation is reduced to a minimum.

Sensitivity of macaque retinal ganglion cells to chromatic and luminance flicker.

Abstract: 1 We have studied the sensitivity of macaque retinal ganglion cells to sinusoidal flicker Contrast thresholds were compared for stimuli which alternated only in luminance ('luminance flicker') or chromaticity ('chromatic flicker'), or which modulated only the middle- or long-wavelength-sensitive cones ('silent substitution') 2 For luminance flicker, the lowest thresholds were those of phasic, non-opponent ganglion cells Sensitivity was maximal near 10 Hz 3 Tonic, cone-opponent ganglion cells were relatively insensitive to luminance flicker, especially at low temporal frequencies, but were sensitive to chromatic flicker, thresholds changing little from 1 to 20 Hz Those with antagonistic input from middle- and long-wavelength-sensitive (M- and L-) cones had a low threshold to chromatic flicker between red and green lights Those with input from short-wavelength-sensitive (S-) cones had a low threshold to chromatic flicker between blue and green Expressed in terms of cone contrast, the S-cone inputs to blue on-centre cells had higher thresholds than M- and L-cone inputs to other cell types 4 Phasic, non-opponent cells responded to high-contrast red-green chromatic flicker at twice the flicker frequency This frequency-doubled response is due to a non-linearity of summation of M- and L-cone mechanisms It was only apparent at cone contrasts which were above threshold for most tonic cells 5 M- or L-cones were stimulated selectively using silent substitution Thresholds of M- and L-cone inputs to both red and green on-centre cells were similar This implies that these cells' sensitivity to chromatic flicker is derived in equal measure from centre and surround Thresholds of the isolated cone inputs could be used to predict sensitivity to chromatic flicker The high threshold of these cells to achromatic contrast is thus, at least in part, due to mutual cancellation by opponent inputs rather than intrinsically low sensitivity 6 Thresholds of M- and L-cone inputs to phasic cells were similar at 10 Hz, and were comparable to those of tonic cells, suggesting that at 1400 td cone inputs to both cell groups are of similar strength 7 The modulation transfer function of phasic cells to luminance flicker was similar to the detection sensitivity curve of human observers who viewed the same stimulus For chromatic flicker, at low temporal frequencies thresholds of tonic cells (red or green on-centre cells in the case of red-green flicker or blue on-centre cells in the case of blue-green flicker) approached that of human observers We propose the different cell types are the substrate of different channels which have been postulated on the basis of psychophysical experiments 8 At frequencies of chromatic flicker above 2 Hz, human sensitivity falls off steeply whereas tonic cell sensitivity remained the same or increased This implies that high-frequency signals in the chromatic, tonic cell pathway are not available to the central pathway respons

Physiological mechanisms adopted by chondrocytes in regulating longitudinal bone growth in rats.

Abstract: 1. Chondrocyte activities within growth plate cartilage are the principal determinants of longitudinal bone growth, and it was the aim of this investigation to assess how these cell activities are modulated under various growth rate conditions. Using proximal tibial growth plates from rats of different ages, growth rate was determined by fluorochrome labelling and incident light fluorescence microscopy. Various cellular parameters contributing to longitudinal bone growth were quantified by light microscopic stereology. The size of the proliferating cell population ('growth fraction') was estimated by autoradiography (using [3H]thymidine labelling). 2. A comparison between data for suckling (21-day-old) and fast-growing (35-day-old) rats revealed that growth acceleration is achieved almost exclusively by cell-shape modelling, namely by an increase in final cell height and a decrease in lateral diameter, whereas final cell volume and surface area are slightly reduced. Cell proliferation rate in the longitudinal direction and net matrix production per cell remain unchanged. The physiological increase in linear growth rate thus appears to be based principally upon a controlled structural modulation of the chondrocyte phenotype. On the other hand, a physiological reduction in growth rate (i.e. growth deceleration) effected during the transition from pre-puberty (35-day-old rats) to maturity (80-day-old rats) is achieved by simultaneous decreases in several chondrocyte parameters, including cell height (i.e. phenotype modulation), cell volume and proliferation rate (in the longitudinal direction). However, chondrocytes continue to produce matrix at a level comparable to that attained during the period characterized by high growth rates (i.e. at 21 and 35 days). Cartilage matrix thus appears to play a subordinate role in regulating longitudinal bone growth rate. The duration of the hypertrophic cell activity (i.e. phenotype modulation) phase remains constant (at approximately 2 days) under the various growth rate conditions. 3. The findings presented in this study indicate that measurement of bulk parameters such as [35S]sulphate incorporation into matrix components, [3H]thymidine uptake by cells and growth plate height are of limited value as estimators of longitudinal bone growth, since changes in the parameters that these measurements quantify bear little relationship to changes in linear growth rate, and may be useful only as indicators of total growth plate activity.

Activation and adaptation of transducer currents in turtle hair cells.

Abstract: 1. Transducer currents were recorded in turtle cochlear hair cells during mechanical stimulation of the hair bundle. The currents were measured under whole-cell voltage clamp in isolated cells that were firmly stuck to the floor of the recording chamber. 2. Stimuli were calibrated by projecting the image of the hair bundle onto a rapidly scanned 128 photodiode array. This technique showed that, while the cell body was immobilized, the tip of the bundle would follow faithfully the motion of an attached glass probe up to frequencies of more than 1 kHz. 3. The relationship between inward transducer current and bundle displacement was sigmoidal. Maximum currents of 200-400 pA were observed for deflections of the tip of the bundle of 0.5 microns, equivalent to rotating the bundle by about 5 deg. 4. In response to a step deflection of the bundle, the current developed with a time constant (about 0.4 ms for small stimuli) that decreased with the size of displacement. This suggests that the onset of the current was limited by the gating kinetics of the transduction channel. The onset time course was slowed about fourfold for a 20 degrees C drop in temperature. 5. For small maintained displacements, the current relaxed to about a quarter of the peak level with a time constant of 3-5 ms. This adaptation was associated with a shift of the current-displacement relationship in the direction of the stimulus. The rate and extent of adaptation were decreased by lowering external Ca2+. 6. Adaptation was strongly voltage sensitive, and was abolished at holding potentials positive to the reversal potential of the transducer current of about 0 mV. It was also diminished by loading cells with 10 mM of the Ca2+ chelator BAPTA. These observations suggest that adaptation may be partly controlled by influx of Ca2+ through the transducer channels. 7. Removal of adaptation produced asymmetric responses, with fast onsets but slow decays following return of the bundle to its resting position; the offset time course depended on both the magnitude and duration of the prior displacement. 8. In some experiments, hair bundles were deflected with a flexible glass fibre whose motion was monitored using a dual photodiode arrangement. Positive holding potentials abolished adaptation of the transducer currents, but had no influence on the time course of motion of the fibre. We have no evidence therefore that adaptation is caused by a mechanical reorganization within the bundle.

Modulation of excitatory amino acid receptors by group IIB metal cations in cultured mouse hippocampal neurones.

Abstract: 1. Responses to the excitatory amino acids kainate, quisqualate, N-methyl-D-aspartate (NMDA), L-glutamate and L-aspartate were recorded in mouse hippocampal neurones in cell culture, using the whole-cell configuration of the patch clamp technique. Agonists were applied rapidly from an array of flow pipes each of 250 microns diameter, positioned within 100 microns of the nerve cell body. 2. Responses to NMDA, L-aspartate and to low concentrations of L-glutamate, recorded with glycine in the extracellular fluid, were strongly antagonized by 50 microM-zinc. Responses to kainate, quisqualate, and in glycine-free solution, responses to L-glutamate, were potentiated by 50 microM-zinc, but partially antagonized by 1 mM-zinc. On average, with 50 microM-zinc, responses to NMDA were reduced to 0.19 times control, while responses to kainate and quisqualate were increased to 1.09 and 1.14 times control. With 1 mM-zinc responses to kainate and quisqualate were reduced to 0.54 and 0.42 times control. 3. Cadmium had a similar, though less potent action, and at 50 microM antagonized responses to NMDA but potentiated responses to kainate and quisqualate. On average, with 50 microM-cadmium, responses to NMDA were reduced to 0.39 times control, while responses to kainate and quisqualate were increased to 1.08 and 1.15 times control. With 1 mM-cadmium responses to NMDA were reduced to 0.04 times control while responses to kainate and quisqualate were reduced to 0.79 and 0.60 times control. Mercury was neurotoxic and increased the leakage current; however, no reduction of the response to NMDA was produced by 5 microM-mercury. 4. The equilibrium dissociation constant (Kd) for zinc antagonism of responses to NMDA, estimated from fit of a single binding site adsorption isotherm, was 13 microM; cadmium was about 4 times less potent than zinc. These effects of zinc and cadmium were nearly voltage independent. In contrast the antagonism of responses to NMDA by 150 microM-magnesium was highly voltage dependent, such that the Kd for magnesium increased e-fold per 17.6 mV depolarization. 5. The potency of zinc as an NMDA antagonist did not vary with the concentration of NMDA, and was not greatly influenced by a 1000-fold variation in the concentration of the NMDA-modulator glycine. This suggests that zinc acts as a non-competitive antagonist, and does not directly interfere with the binding of NMDA to the agonist recognition site nor with the binding of glycine to an allosteric site on the NMDA receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of an electrical synapse between solitary pairs of catfish horizontal cells by dopamine and second messengers.

Abstract: 1 Retinas from channel catfish were dissociated and the cells maintained in culture Horizontal cells that normally receive input from cone photoreceptors were identified The conductance of the electrical junction formed between a pair of 'cone' horizontal cells was measured by controlling the membrane voltage of each cell with a voltage clamp maintained through either a micropipette or a patch pipette The two techniques yielded similar results 2 Transjunctional current was measured while transjunctional voltage was stepped to values between +/- 60 mV The current (measured 5 ms after a step) was proportional to voltage over the range tested For steps to voltages greater than +/- 45 mV, the current exhibited a slight time-dependent decline 3 Dopamine decreased junctional conductance in a dose-dependent fashion A 50% reduction was obtained with 10 nM-dopamine The D1 agonist fenoldopam (100 nM) also decreased junctional conductance The uncoupling produced by either agent was rapid and reversible 4 The introduction of 100 microM-cyclic AMP into one cell of a pair decreased junctional conductance by, on average, 40% Forskolin (1-10 microM), an activator of adenylate cyclase, decreased junctional conductance 50-90% 5 The introduction of 80 microM-cyclic GMP into one cell of a pair decreased junctional conductance by, on average, 40% Nitroprusside (1-10 microM), an activator of guanylate cyclase, reduced junctional conductance 40-65% 6 The introduction of a peptide inhibitor specific for the cyclic AMP-dependent protein kinase reversed a decrease in junctional conductance produced by superfusion with either dopamine (1 microM), fenoldopam (100 nM) or forskolin (5-10 microM) 7 Intracellular Ca2+ concentration was measured with the fluorescent indicator Fura-2 The intracellular Ca2+ concentration was increased by activation of a Ca2+ current Junctional conductance remained constant as the internal Ca2+ concentration changed from 100 to 700 nM 8 Intracellular pH was measured with the fluorescent indicator bis-carboxyethylcarboxyfluorescein The application of acetate (25 mM) reduced intracellular pH by 02-03 units and decreased junctional conductance by approximately 50% A subsequent application of fenoldopam did not alter intracellular pH, but decreased junctional conductance by more than 50% 9 The sensitivity of the junctional conductance between isolated horizontal cells to dopamine is consistent with dopamine having a direct effect on coupling in intact retina Dopamine regulates the activity of a cyclic AMP-dependent protein kinase which in turn modulates junctional conductance Changes in intracellular pH and Ca2+ concentration are not involved in mediating the effect of dopamine on coupling Cyclic GMP and intracellular pH may participate in regulatory pathways independent of that used by cyclic AMP

A T-type Ca2+ current underlies low-threshold Ca2+ potentials in cells of the cat and rat lateral geniculate nucleus.

Abstract: 1. The characteristics of a transient inward Ca2+ current (IT) underlying low-threshold Ca2+ potentials were studied in projection cells of the cat and rat dorsal lateral geniculate nucleus (LGN) in vitro using the single-electrode voltage-clamp technique. 2. In cat LGN slices perfused at 25 degrees C with a solution which included 1 mM-Ca2+ and 3 mM-Mg2+, IT could be evoked by depolarizing voltage steps to -55 mV from a holding potential (Vh) of -95 mV and was abolished by reducing [Ca2+]o from 1 to 0.1 mM. IT was also blocked by 8 mM-Mg2+ and 500 microM-Ni2+, but 500 microM-Cd2+ was a significantly less effective antagonist. 3. The inactivation of IT, which occurred at Vh positive to -65 mV, was removed as Vh approached -100 mV. The process of inactivation removal was also time dependent, with 800-1000 ms needed for total removal. Activation curves for IT showed a threshold of -70 mV and illustrated that IT was extremely voltage sensitive over the voltage range from -65 to -55 mV. 4. The decay phase of IT followed a single-exponential time course with a time constant of decay which was voltage sensitive and ranged from 20 to 100 ms. The mean peak conductance increase associated with IT was 8.4 nS (+/-0.9, S.E.M.). 5. In more 'physiological' conditions (35 degrees C and 1.5 mM-Ca2+, 1 mM-Mg2+) the voltage dependence of activation and inactivation were unaffected. However, the development and decay of IT proceeded more rapidly and only 500-600 ms were needed for total removal of inactivation. Under these conditions, the use of voltage ramps showed that depolarization rates of greater than 30 mV/s were necessary for IT activation. 6. The use of multiple voltage-step protocols illustrated that the process of inactivation removal was rapidly reversed by brief returns to a Vh of -50 mV. Furthermore, any delay in IT activation, once the LGN cell membrane potential was in the IT activation range, resulted in a current of reduced amplitude. 7. Although IT in rat LGN cells was briefer and had a shorter latency to peak, it was otherwise similar to that seen in cat LGN cells. 8. The characteristics of IT are very similar to those of the T-type Ca2+ currents of other excitable membranes. The properties of IT are discussed with respect to its role in generating the low-threshold Ca2+ potentials which are central to the oscillatory behaviour of thalamic projection cells.

Spinal mechanisms in man contributing to reciprocal inhibition during voluntary dorsiflexion of the foot.

Abstract: 1 The inhibition of the soleus Hoffmann reflex (H reflex) during voluntary dorsiflexion of the foot--henceforth referred to as ‘natural’ reciprocal inhibition--was found to be initiated 50 ms before the onset of the EMG activity in the tibialis anterior muscle and to increase gradually during a ramp-and-hold dorsiflexion There was a positive correlation between strength of tonic dorsiflexion and amount of ‘natural’ reciprocal inhibition 2 The change of activity in the disynaptic and a long-latency group Ia inhibitory pathway and the change in presynaptic inhibition of the Ia fibres mediating the soleus H reflex were tested separately during ramp-and-hold dorsiflexion as well as during tonic dorsiflexion of the foot, and the results were compared with the development of the ‘natural’ reciprocal inhibition of the unconditioned soleus H reflex 3 The disynaptic group I inhibition of soleus motoneurones was increased, as compared to rest, during the dynamic phase of a ramp-and-hold dorsiflexion movement, but the inhibition generally did not increase during tonic dorsiflexion of the foot 4 The long-latency group I inhibition was seen only during dorsiflexion of the foot It appeared around 50 ms before tibial anterior EMG activity and there was a positive correlation between strength of tonic dorsiflexion and amount of this long-latency inhibition 5 Presynaptic inhibition of Ia afferents terminating on soleus motoneurones was estimated by an indirect method The increase of presynaptic inhibition started soon after the onset of the ramp-and-hold dorsiflexion, and gradually became more pronounced during the ramp phase The amount of presynaptic inhibition was positively correlated with strength of tonic dorsiflexion 6 It is concluded that all investigated mechanisms may contribute to the ‘natural’ reciprocal inhibition and it seems that the different pathways are used differentially during different types of movement

Intracellular calcium and tension during fatigue in isolated single muscle fibres from Xenopus laevis.

Abstract: 1. Single muscle fibres were dissected from Xenopus lumbrical muscles and microinjected with the photoprotein aequorin in order to measure the myoplasmic free calcium concentration ([Ca2+]i). Fatigue was produced by repeated intermittent tetanic stimulation continued until tension had declined to approximately 50% of the initial level. Fibres were then allowed to recover by giving tetani at less frequent intervals. Aequorin light (a measure of [Ca2+]i) and tension were measured during fatiguing stimulation and recovery. 2. During fatiguing stimulation, tetanic tension declined steadily, but peak aequorin light first increased before declining substantially. The largest light signal was about 155% of initial control while at the end of fatiguing stimulation the tetanic light fell to about 14% of control. 3. Fibres showed a characteristic slowing of relaxation in the fatigued state. This was associated with a slowing of the rate of decline of the aequorin light signal. 4. Intracellular acidosis produced by equilibrating the Ringer solution with either 5 or 15% CO2 caused an increase in the light signal associated with a tetanus. Carbon dioxide also caused a reduction of tension and a slowing of relaxation. 5. In vivo pCa-tension curves were constructed by exposing the fibres to a series of K+ concentrations which produced contractures of different sizes. Light and tension were measured during periods when both were relatively stable and the light signal was subsequently converted to pCa. 6. Exposure of fibres to 5 or 15% CO2 caused the pCa-tension curve to be shifted to the right of the control curve. This indicates a reduced Ca2+ sensitivity of the contractile proteins, which is in agreement with results from skinned fibre studies. 7. The pCa-tension points obtained from tetani during the early part of fatiguing stimulation also deviated to the right of the control pCa-tension curve, suggesting a reduced Ca2+ sensitivity of the contractile proteins. At the end of fatiguing stimulation, however, pCa-tension points did not differ greatly from the control pCa-tension curve, suggesting that Ca2+ sensitivity was approximately normal. Thus the reduced [Ca2+]i during tetani at the end of fatiguing stimulation (when tension was reduced to approximately 50%) could explain all of the reduction in tension. 8. After fatiguing stimulation, tension and light recovered monotonically in some fibres; however, in the majority of fibres, tension and light showed a secondary decline followed by a slower recovery (post-contractile depression). 9. During post-contractile depression, caffeine contractures or tetani in the presence of caffeine gave increased aequorin light signals and the tension developed was close to that produced in an unfatigued tetanus.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanical influences on long-lasting human muscle fatigue and delayed-onset pain.

Abstract: 1. The influence of three mechanical factors, force, muscle length and passive lengthening, on long-lasting changes in voluntary force generation, the force:frequency relationship and the development of tenderness has been studied in healthy human skeletal muscle. The elbow flexors were used in all studies. The effect of muscle length was also investigated in the quadriceps and adductor pollicis muscles. Eighty maximal voluntary contractions (MVCs) were performed: one contraction, lasting approximately 2 s, every 15 s. The MVC and force:frequency relationships were measured before and immediately after the exercise and, together with an assessment of tenderness, at 24 h intervals thereafter. 2. In a series of experiments designed to investigate the effects of force, eccentric (lengthening) contractions were found to cause greater fatique and delayed-onset muscle pain than either isometric or concentric (shortening) contractions. There were, however, no substantial differences between the effects of isometric and concentric contractions. Changes in MVC took 24-48 h to return to normal while the low-frequency fatigue required 3-4 days to recover. 3. Passive lengthening with a comparable number of movements over the full range had no effect on the force generation of the muscle, nor did it cause any muscle pain. 4. In the series of experiments designed to investigate the effects of length, isometric MVCs were performed at either short or long length and the muscles subsequently tested at an intermediate length. The contractions at long length resulted in greater low-frequency fatigue and pain, despite the fact that they generated less force than those at the short length. 5. The results demonstrate that there is no simple relationship between the force generated during exercise and the development of long-lasting muscle fatigue and pain. Furthermore, there is a length-dependent component in the generation of low-frequency fatigue and muscle pain.

Kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture.

Abstract: 1. The kinetic properties of the main conductance state of gamma-aminobutyric acidA (GABA) receptor channels from somata of mouse spinal cord neurones in cell culture were investigated using patch clamp techniques. 2. Whole-cell GABA receptor currents increased in a concentration-dependent manner from 0.5 to 5 microM. 3. Single-channel currents were recorded with a main conductance state of 27.2 pS and a less frequent conductance state of 15.9 pS. The main conductance state opened singly and in bursts of several openings. 4. Mean open times of GABA receptor main conductance currents were increased and open-time frequency histograms were shifted to longer times as GABA concentration was increased from 0.5 to 5 microM. Three exponential functions were required to fit the histograms at all GABA concentrations, suggesting that the channel opened into at least three open states (O1, O2 and O3). The three functions had the same time constants (1.0 +/- 0.2, 3.7 +/- 0.4 and 11.3 +/- 0.5 ms; mean +/- S.D.) at each concentration. The increase in long open times with concentration was due to a shift in relative frequency of occurrence of openings from the shortest (O1) to the two longest (O2 and O3) open states. 5. Closed-time distributions of closures between main conductance state openings were fitted with multiple exponential functions, suggesting that the channel had several closed states. The two shortest time constants (0.24 +/- 0.03 and 2.0 +/- 0.3 ms) were concentration independent (0.5 to 5 microM). Three longer time constants decreased as concentration increased. 6. Bursts were defined as groups of openings surrounded by closures greater than a critical closed time (tc = 5 ms). Mean burst durations were increased and burst duration frequency histograms were shifted to longer times as GABA concentration was increased from 0.5 to 5 microM. Burst-duration frequency histograms were best fitted with three exponential functions. The time constants were concentration independent and were 1.0 +/- 0.2, 5.5 +/- 0.2 and 29.8 +/- 1.6 ms. The increase in burst duration with concentration was due to a relative shift from short duration bursts to longer duration bursts. 7. The shortest burst time constant was similar to the shortest open time constant suggesting that there was a population of single openings of short duration. The two longest burst time constants were longer than the two longest open time constants, suggesting that the bursts from the two longest burst components were composed of two or more openings.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of N‐methyl‐D‐aspartate receptors revealed by intracellular dialysis of murine neurones in culture.

Abstract: 1. The whole-cell patch clamp recording technique was employed to investigate the intracellular regulation of N-methyl-D-aspartate (NMDA) receptors in cultured murine hippocampal neurones. Excitatory amino acids were repeatedly applied at regular intervals during intracellular dialysis with solutions of various composition. 2. Currents evoked by L-aspartate, an agonist of NMDA receptors, gradually 'washed out' to approximately 50% of their initial amplitude during dialysis with an intracellular solution containing CsCl and EGTA as a calcium buffer. In contrast, responses to kainate did not wash out. The wash-out of L-aspartate currents followed an exponential time course with a time constant of about 150 s. Wash-out did not appear to be related to desensitization of NMDA receptors. 3. Following wash-out, L-aspartate responses were blocked by Mg2+, ketamine or D-2-amino-5-phosphonovalerate indicating that these responses were still mediated by NMDA receptors. Furthermore, responses to NMDA itself showed wash-out to the same extent and with a time course similar to that for L-aspartate responses. 4. Neither the time course nor the extent of the wash-out of responses to L-aspartate was affected when the Ca2+ concentration of the dialysate was varied from zero to 1.5 x 10(-5) M. In addition, wash-out was unaffected by substitution of BAPTA for EGTA, indicating that wash-out was not a consequence of changes in intracellular pH related to the binding of Ca2+ to the buffer or to the kinetics of this binding. Therefore, the wash-out of NMDA currents could not be attributed to a gradual elevation of the concentration of intracellular Ca2+. 5. The extent of the wash-out of L-aspartate currents was similar for cells held at +40 versus -60 mV although the rate of wash-out was slower at the depolarized potential. In addition, the reversal potential of these currents was not altered, demonstrating that a change in driving force did not account for a component of the wash-out. 6. Inclusion of an ATP regeneration solution (Forscher & Oxford, 1985) in the dialysate prevented the wash-out of L-aspartate currents. ATP alone was less effective in preventing wash-out whereas phosphocreatine and creatine phosphokinase were ineffective by themselves. Wash-out also occurred when ATP was replaced with the non-hydrolysable analogue, beta, gamma-methyleneATP, or with GTP. In cells where wash-out of L-aspartate currents had been established, subsequent dialysis with the ATP regenerating solution partially reversed this wash-out.(ABSTRACT TRUNCATED AT 400 WORDS)

Barbiturate regulation of kinetic properties of the GABAA receptor channel of mouse spinal neurones in culture.

Abstract: 1 Barbiturate regulation of the kinetic properties of gamma-aminobutyric acidA (GABA) receptor channel chloride currents from somata of mouse spinal cord neurones were investigated using whole-cell and excised outside-out patch-clamp recording techniques 2 GABA (2 microM), GABA (2 microM) plus phenobarbitone (PhB) (500 microM) and GABA (2 microM) plus pentobarbitone (PB) (50 microM), applied by pressure ejection from blunt perfusion micropipettes, evoked inward chloride currents when neurones or patches were voltage clamped at -75 mV and the chloride equilibrium potential was 0 mV GABA receptor channel currents were increased by PhB and PB 3 Single GABA receptor channel currents were recorded with a main conductance state of 27 pS and a less frequent subconductance state of 165 pS The conductances of the two states were unchanged by the barbiturates 4 The main conductance state kinetics were analysed GABA alone or with the barbiturates gated the channel open singly and in groups of openings 5 The barbiturates increased GABA receptor channel mean open time and shifted frequency histograms of channel open times to longer times 6 Three exponential functions were required to fit the frequency histograms of GABA receptor channel open times, suggesting that the channel has at least three open states (O1, O2, O3) The time constants for the exponential functions (09, 27 and 78 ms, respectively) were unchanged by the barbiturates The increases in mean open times and the shifts of the open-time frequency histograms by the barbiturates were due to a reduction in relative frequency of occurrence of the two short open states (O1 and O2) and to an increase in the relative frequency of occurrence of the longest open state (O3) 7 Frequency histograms of GABA receptor channel closed times were fitted with five exponential functions, suggesting that the channel has multiple closed states None of the time constants nor areas of the exponential functions were significantly changed by the barbiturates 8 For analysis, a burst was defined as openings surrounded by closures greater than a critical closed time, tc, of 5 ms For GABA (2 microM), frequency histograms of GABA receptor channel bursts were fitted with three exponential functions, suggesting that the channel has three burst states (B1, B2, B3) The B1 burst state was probably a single opening to the O1 open state while the B2 and B3 burst states were probably composed of multiple openings to the O2 and O3 open states(ABSTRACT TRUNCATED AT 400 WORDS)

Some electrical properties of the endothelium-dependent hyperpolarization recorded from rat arterial smooth muscle cells.

Abstract: 1. Electrical responses produced by acetylcholine (ACh) and histamine were recorded from smooth muscle cells of the intralobular small pulmonary artery (SPA), main pulmonary artery (MPA) and thoracic aorta of rats. 2. In MPA and SPA, ACh and histamine produced a transient hyperpolarization of the membrane, and the potential decayed exponentially with a time constant of 2-3 min. In aorta, ACh produced a sustained and histamine produced a transient hyperpolarization. 3. The ACh- and histamine-induced hyperpolarizations were blocked by atropine and mepyramine, respectively, or by removing the endothelial cells. 4. The amplitude of the hyperpolarization was increased in low [K+]o solutions and decreased in high [K+]o solutions. The ionic conductance of the membrane was increased during the hyperpolarization, suggesting an involvement of the increased potassium conductance. 5. A reproducible amplitude of hyperpolarization was generated when ACh or histamine was applied at intervals of over 10 or 30 min, respectively. 6. In aorta, after the transient hyperpolarization had ceased during continued application of histamine, ACh again produced a hyperpolarization, i.e. the transient nature of the hyperpolarization was not due to desensitization of the receptor upon which the hyperpolarizing substance acted, assuming histamine and ACh release the same hyperpolarizing substance. 7. ACh and histamine relaxed the tissues from SPA, MPA and aorta during the noradrenaline (NA)- or high [K+]o solution-induced contraction, in a concentration-dependent manner, only when the endothelial cells were intact. Both ACh and histamine were potent relaxants in MPA and aorta, but showed weak relaxing actions in SPA. 8. In aorta, ACh and histamine produced a sustained relaxation for up to 10 min, and Methylene Blue diminished and altered it to a transient relaxation (for histamine) or an initial large, followed by a small sustained (for ACh), relaxation. 9. In the presence of NA and NA plus Methylene Blue, ACh and histamine also produced a hyperpolarization similar to that seen in the control. 10. It is concluded that in arteries of the rat, ACh and histamine release a hyperpolarizing substance from the endothelial cells. This substance may be different from the endothelium-derived relaxing factor (EDRF), and is released mainly transiently. The hyperpolarization is generated by an increase in potassium conductance of the membrane, and this has some contribution to the endothelium-dependent relaxation.

Changes in force and intracellular metabolites during fatigue of human skeletal muscle.

Abstract: 1. The relationship between intracellular metabolites and the generation of force during fatigue has been examined in the first dorsal interosseous muscle of the hand. With the arm made ischaemic, the muscle was fatigued by three bouts of maximal voluntary contraction, leaving approximately three minutes ischaemic rest between contractions. During one series of experiments intracellular phosphorus metabolites were measured by nuclear magnetic resonance during the intervals between the fatiguing contractions: in the second series contractile properties were tested with brief electrical stimulation during the rest intervals. 2. The relationships between loss of force and change in metabolite concentrations obtained with four normal subjects were compared with those from one subject with myophosphorylase deficiency (MPD) who could not utilize muscle glycogen and therefore produced no hydrogen ion from glycolysis during exercise. 3. For both the MPD and normal subjects the relationship between relative force loss and inorganic phosphate (Pi) concentration was curvilinear, force changing little in the early stages of the contraction when the intracellular Pi was accumulating rapidly but falling faster when the Pi was above 25 mM and increasing relatively slowly. 4. In the normal subjects intracellular pH fell from a mean of 7.03 +/- 0.01 (mean +/- S.E. of mean, n = 19) in the fresh muscle to 6.51 +/- 0.02 at the end of the fatiguing exercise; force, as a percentage of the initial value, fell in proportion to the increase in H+ concentration. In the MPD subject pH did not change and force loss was therefore independent of H+ accumulation. In the normal subjects the force of the fatiguing muscle showed an approximately linear relationship with the concentration of the monobasic form of inorganic phosphate. However, the MPD subject showed a quite different relationship, with force loss being much greater for a given concentration of monobasic phosphate. This result indicates that monobasic phosphate is not a unique determinant of force loss in fatigued muscle. 5. During the first 60 s of recovery in the normal subjects, pH remained low while force recovered, indicating a mechanism of force loss that was independent of H+ accumulation. However, the recovery of force was not complete, so that for comparable phosphocreatine contents the recovering, more acid, muscle generated less force than the muscle that was being fatigued. It was estimated that H(+)-dependent and independent mechanisms contributed roughly equally to the observed force loss. The relationship between force and the concentration of monobasic phosphate differed in fatiguing and recovering muscle.

The effects of metabolic inhibition on intracellular calcium and pH in isolated rat ventricular cells.

Abstract: 1. Intracellular calcium concentration [( Ca2+]i) and pH (pHi) were measured in single, isolated rat ventricular myocytes using, respectively, the fluorescent indicators Fura-2 and BCECF (2',7'-bis(carboxyethyl-5(6)-carboxyfluorescein). Contraction was measured simultaneously. The intracellular calibration of BCECF is demonstrated. In a HEPES-buffered bathing solution of pH 7.4, pHi had a mean value of 7.16 +/- 0.05 (mean +/- S.E.M.). 2. Addition of NH4Cl (5-20 mM) produced an intracellular alkalosis that was associated with an increase of contraction amplitude. Removal of NH4Cl produced an acidosis and decrease of contraction. 3. The addition of 2 mM-cyanide (CN-) to inhibit oxidative phosphorylation had variable effects on contraction amplitude. Changes of contraction amplitude could largely be accounted for by changes in the systolic Ca2+ transient. 4. CN- addition increased lactic acid production. However, in the majority of experiments, this was not accompanied by an intracellular acidosis. 5. Anaerobic glycolysis was inhibited by either removal of glucose, addition of deoxyglucose, or addition of iodoacetate. Under these conditions the application of CN- decreased systolic [Ca2+]i and contraction amplitude. This was sometimes preceded by a transient increase of systolic [Ca2+]i and contraction amplitude. 6. When glycolysis was inhibited, the subsequent addition of CN- always increased diastolic [Ca2+]i and produced a contracture. The increase of [Ca2+]i occurred before the contracture. However, once the contracture had developed, decreasing [Ca2+]i (by removal of external Ca2+) did not cause relaxation. 7. With glycolysis inhibited, addition of CN- resulted in a large (0.51 +/- 0.05 pH unit) acidosis that was sometimes preceded by an alkalosis. This acidosis was unaffected by removal of external Ca2+ or external alkalinization. Calculations show that some of this acidosis may result from protons released by ATP hydrolysis. 8. If the acidosis produced by metabolic blockade was partly reversed by adding NH4Cl then a contracture immediately developed. This suggests that the acidosis delays the onset of the contracture. 9. We conclude that metabolic inhibition increases diastolic [Ca2+]i. The accompanying acidosis prevents contraction. Once the contracture has developed it is maintained by factors other than increased [Ca2+]i, possibly by a fall of [ATP].

Gating of the afferent volley of the monosynaptic stretch reflex during movement in man.

Abstract: 1. The time course of the changes in presynaptic inhibition of Ia fibres to soleus motoneurones has been investigated during a voluntary ramp-and-hold plantar flexion. 2. Monosynaptic Ia facilitation of the soleus H reflex was evoked by stimulation of the inferior soleus (homonymous) and femoral (heteronymous) nerves. Changes in presynaptic inhibition of the Ia fibres mediating the conditioning volleys were inferred from changes in the amount of reflex facilitation evoked by such constant conditioning stimulations. 3. At the beginning of the voluntary contraction, both homonymous and heteronymous Ia facilitations were markedly increased with respect to their rest values. Then, they dropped abruptly in the middle of the ramp, whatever the ramp duration. 4. The stronger the contraction at the end of the ramp, the larger the initial increase in reflex facilitation. 5. These results indicate that presynaptic inhibition of Ia terminals on motoneurones of the contracting muscle is decreased during the first half of the ramp and then returns to its rest level. It is argued that these changes in presynaptic inhibition are centrally programmed, and it is suggested that, under certain conditions, presynaptic inhibition can be modulated according to both the strength and timing of the contraction. 6. The functional role of the resulting changes in the gain of the stretch reflex is discussed.

Modulation by intracellular Ca2+ of the hyperpolarization-activated inward current in rabbit single sino-atrial node cells.

Abstract: 1. The sensitivity to internal Ca2+ of the hyperpolarization-activated inward current (Ih or If) in rabbit single sino-atrial node cells was investigated by the whole-cell voltage-clamp method. 2. When the patch pipette contained an internal solution of pCa 10, the amplitude of If decreased by 74.8 +/- 3.3% in 10 min (n = 7) after rupture of the patch membrane. When the pipette contained an internal solution of pCa 7, If increased by 43.7 +/- 8.7% within 10 min (n = 5). 3. Increase of If by the higher Ca2+ internal solution was confirmed in the same cell using the cell dialysis method. Both If and its tail current were increased at every membrane potential. The amplitude of If increased most markedly between pCa 8 and 7. 4. The reversal potential and kinetics of If were unaffected by the internal Ca2+ concentration. Increase of If by the high internal Ca2+ concentration was sensitively blocked by Cs+. These findings confirm that the increased current is indeed If and not a newly activated If-like current due to elevation of internal Ca2+. 5. The activation curve of If shifted approximately 13 mV in a positive direction by elevating Ca2+ from pCa 10 to 7 (n = 21), indicating that the voltage dependence of If was modulated by internal Ca2+. 6. beta-Agonists also modulated If, but the underlying mechanisms of their effects on If differed from those of the internal Ca2+. The former affected the If kinetics rather than its amplitude, whereas the latter acted on the If conductance rather than on its kinetics. 7. The increase in If by the internal Ca2+ was unaffected by protein kinase inhibitor or calmodulin inhibitor, suggesting that the internal Ca2+ directly modulates If. 8. When the patch pipette contained pCa 7 internal solution, the maximum diastolic potential shifted towards a positive potential but the heart rate remained almost constant.

Calcium‐sensitive and insensitive transient outward current in rabbit ventricular myocytes.

Abstract: 1. A suction pipette whole-cell voltage-clamp technique was used to record membrane currents and potentials of isolated ventricular myocytes from rabbit hearts. 2. Transient outward current (Ito) was activated by voltage steps positive to -20 mV, increasing in amplitude with further depolarization to reach a maximum around +70 mV. The current attained its peak within 10 ms and then it inactivated for 100-200 ms. 3. A large portion of Ito still remained after the calcium current (ICa) was blocked when depolarizing pulses were applied at a frequency of 0.1 Hz or less. Therefore, this current component is referred to as calcium-insensitive Ito or It. 4. It showed voltage- and time-dependent inactivation similar to that observed in Purkinje fibres and other cardiac preparations. 5. The reversal potential of It depended on external K+ concentration, [K+]o, with a slope of 32 mV per 10-fold change in the presence of a normal [Na+]o (143 mM), while the slope was 48 mV per 10-fold change in low [Na+]o (1.0 mM). 6. It was completely inhibited by 2-4 mM-4-aminopyridine. Ito in the presence of ICa was also partially blocked by 4-aminopyridine and the remainder was abolished by 5 mM-caffeine. 7. The calcium-insensitive and caffeine-sensitive Ito differed in their decay rates as well as in their recovery time courses. The former was predominantly available at a slow pulsing rate, while the latter increased its amplitude with high-frequency depolarization. 8. The caffeine-sensitive Ito was inhibited by a blockade of ICa, by replacing Ca2+ with Sr2+, by external application of ryanodine and by internal application of EGTA. This indicates that the current is calcium-sensitive and is dependent on increased myoplasmic Ca2+ through Ca2+ influx via the sarcolemma and Ca2+ release from the sarcoplasmic reticulum. The current is therefore designated as IK, Ca. 9. The physiological functions of IK, Ca and It are indicated by their contribution to ventricular repolarization at fast and slow heart rates, respectively.