Biomedical subjects

M R Duchen

Publications and source records attributed to M R Duchen.

47 records · Page 3Linked to original sources

PubMed1989

Measurements of intracellular Ca2+ in dissociated type I cells of the rabbit carotid body.

1. The carotid body chemoreceptors are stimulated in situ by cyanide (CN-), which mimics the effect of hypoxia. We have shown that CN- increases a calcium-dependent potassium conductance (gK(Ca)) in single type I cells dissociated from the carotid body of the rabbit. We have now used the Ca2(+)-sensitive fluorophore, Fura-2, to measure intracellular Ca2+ directly in single type I cells. 2. CN- reversibly increased [Ca2+]i from approximately 90 nM to a mean of approximately 200 nM. Some of this Ca2+ originated from an intracellular store, which was depleted by exposure to Ca2(+)-free solutions. Prolonged application of CN- caused a sustained increase in [Ca2+]i, suggesting that CN- impairs the removal or sequestration of Ca2+. 3. pHi measured with the dye BCECF (2,7-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein) did not change consistently in response to CN-, although pHi changed predictably in response to both ammonium chloride and to acidification of the superfusate with CO2. 4. Potassium-induced depolarization (35 mM-K+) caused a large, cadmium-sensitive rise in [Ca2+]i. The K(+)-induced Ca2+ load was used to study the regulation of [Ca2+]i. 5. The clearance of a Ca2+ load was slowed either by removal of [Na+]o or by application of CN-. This shows that both a Na+-Ca2+ exchange and an energy-dependent process or processes contribute to the regulation of [Ca2+]i. 6. Carbachol (CCh, 10-100 microM), which also hyperpolarizes type I cells, caused a small transient rise in [Ca2+]i, indicating release from an exhaustible intracellular pool. The response to CN- was unaffected by prior or continued exposure to CCh, suggesting that the two stimuli operate by distinct mechanisms. 7. The increased gK(Ca) seen in type I cells in response to CN- thus reflects a change in cellular Ca2+ homeostasis. The rise in [Ca2+]i presumably underlies the documented increase in transmitter release from the carotid body in response to CN-. If chemotransduction is a consequence of the release of transmitters from the type I cell, the response of the carotid body to CN-, and possibly also to hypoxia, is thus a direct consequence of the energy dependence of Ca2+ homeostasis in the type I cell.

Animals↗

PubMed1988

Biophysical studies of the cellular elements of the rabbit carotid body.

The carotid body is a major sensor of oxygen partial pressure in the arterial blood, and plays a role in the control of respiration. Despite extensive investigation of the structure, the cellular basis of the transduction mechanism remains poorly understood. We have developed a preparation of freshly dissociated cells from the rabbit carotid body, in which two cell types may be identified using morphological criteria. The preparation allows application of the patch clamp technique to characterize the properties of the cells which have otherwise proved difficult to study in situ. Carotid bodies of rabbits were dissociated using a combination of enzymatic and mechanical procedures. The dissociated preparation obtained consisted of clusters of spherical or ovoid cells of 12-15 microns in diameter and a distinct population of spherical cells of 8-10 microns diameter. Electron microscopic techniques were used to identify the cells present in the preparation. Again two populations of cells could be distinguished. A population of cells 10-12 microns in diameter, often found in clusters, possessed the dense-cored vesicles characteristic of Type I cells, while a population of smaller cells (diameter 5-7 microns) had peripherally condensed nuclear chromatin and fine cytoplasmic surface extensions characteristic of Type II cells. Patch clamp study of the cells showed that they represent two electrophysiologically distinct populations. The larger cells, corresponding to Type I cells, were found to be excitable, generating fast, sodium-dependent action potentials that were recorded both in the cell attached and whole cell recording configurations. The smaller Type II cells did not generate action potentials. Voltage clamp study of Type I cells allowed definition of a range of voltage-gated currents. These included an inactivating, tetrodotoxin-sensitive inward sodium current, a high threshold sustained inward calcium current, and outward potassium currents. A component of the outward current showed a dependence on voltage-gated calcium entry, and was blocked by cobalt or cadmium. Of the calcium-dependent current, a component was sensitive to apamin, and the remaining current was blocked by tetraethylammonium. Type II cells showed only a high threshold outward potassium current. These studies have thus revealed an electrophysiological differentiation that parallels the morphological differentiation of the cells of the carotid body. The Type I cell is essentially neuron-like in its properties, while the Type II cell appears to have properties resembling those of glial elements elsewhere in the nervous system.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1987

Primary afferent terminal excitability in the normal and spastic mutant mouse spinal cord.

A microcomputer-based system has been used to apply the technique of excitability testing to the study of the actions of a range of pharmacological agents on the excitability of single primary afferent terminals in the mouse spinal cord in vitro. GABAA analogues all evoked increases in excitability that were bicuculline sensitive. GABA itself also evoked biphasic changes in excitability, or occasionally only suppressed terminal excitability. This latter effect was often enhanced in the presence of bicuculline, and resembled the action of the GABAB agonist, baclofen. The GABAA action could be enhanced by concurrent application of either benzodiazepine, midazolam or flurazepam. Bicuculline alone frequently decreased excitability. This action could be abolished by blocking synaptic activity with a low Ca2+ high Mg2+ superfusate, and was therefore considered to be due to reduction of the tonic action of GABA released at synaptic connections. Comparison of the action of these agents on terminals in the spastic mutant mouse showed an increased sensitivity of the GABA response to the benzodiazepines in mutant animals.

Animals↗

PubMed1986

Excitation of mouse motoneurones by GABA-mediated primary afferent depolarization.

Observations on the reflex properties of a mouse spinal cord preparation in vitro are reported. The findings show that the synaptically evoked, GABA-mediated, discharge of action potentials in primary afferent fibres, monitored as the dorsal root reflex, may lead to the excitation of motoneurones. Subthreshold, bicuculline-sensitive increases in motoneuronal excitability, followed by prolonged inhibition, may be seen in preparations in which the delayed reflex is not seen. Thus, primary afferent depolarization may both increase motoneuronal excitability and also cause presynaptic inhibition of afferent input.

Action Potentials↗

PubMed1986

Synaptic physiology of spinal motoneurones of normal and spastic mice: an in vitro study.

Spinal cord reflexes have been examined in a preparation of the mouse spinal cord maintained in vitro. Responses of the motoneurone population of normal and spastic mutant mice to stimulation of a segmental dorsal root were compared. In the normal spinal cord, a monosynaptic response with very little polysynaptic excitation was typical. In the mutant, the monosynaptic response was typically followed by a depolarizing wave on which asynchronous compound action potentials were superimposed. In some spastic cords, an oscillating depolarizing wave was seen, lasting up to 500 ms. The stimulus range from threshold to maximal response was the same for the normal and mutant. The dorsal root reflex (d.r.r.) and dorsal root potential (d.r.p.) were prominent in both normal and mutant, and no consistent difference could be identified. Intracellular recordings were made from motoneurones using electrodes filled with potassium acetate. Mean resting potentials and input resistances were not significantly different in mutant and normal mice. The voltage-dependent conductances, seen as the after-depolarization and after-hyperpolarizations following antidromic action potentials and the responses of motoneurones to depolarizing current injection were similar in both populations. The synaptic responses of motoneurones following stimulation of the segmental dorsal root were clearly abnormal in the mutant. In the normal mice, a monosynaptic excitatory post-synaptic potential (e.p.s.p.), seen at low stimulus intensities, was followed at higher stimulus intensities by polysynaptic activity lasting up to 100 ms, which rarely reached threshold for action potential discharge. In the mutant mice, the monosynaptic response was typically followed by depolarizing synaptic responses which often evoked action potentials before the monosynaptic response reached threshold. At higher stimulus intensities, the monosynaptic response was followed by at least one and often multiple action potentials generated on prolonged depolarizing synaptic activity. When cells were impaled with potassium-acetate-filled electrodes, very little spontaneous synaptic activity was seen in either normal or mutant mice. Spontaneous depolarizing post-synaptic potentials (p.s.p.s) were prominent in normal motoneurones when potassium chloride was used to fill electrodes and were increased in amplitude by ionophoresis of chloride into the cells. Under these conditions stimulation of a ventral root evoked a depolarizing p.s.p. and the Renshaw i.p.s.p. reversed. The spontaneous p.s.p.s were blocked by ionophoresis or bath application of the glycine antagonist strychnine.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetates↗

PubMed1985

An intracellular study of the interactions of N-methyl-DL-aspartate with ketamine in the mouse hippocampal slice.

Intracellular recordings were made from dentate and CA1 pyramidal cells of the mouse hippocampal slice preparation. N-methyl-DL-aspartate (NMDLA), quisqualate and kainate and the anaesthetic agent, ketamine, were applied by microelectrophoresis. Excitation by NMDLA but not by the other amino acids, was associated with increased outward rectification. Ketamine had no effect on the resting potential or current/voltage relation of the cells, but selectively antagonised the responses to NMDLA. Action potentials evoked by NMDLA were characteristically broader than those evoked by the other amino acids or by the passage of depolarising current through the electrode.

Action Potentials↗

PubMed1985

An intracellular study of dentate, CA1 and CA3 neurones in the mouse hippocampal slice.

Intracellular recordings were made from CA1 and CA3 pyramidal cells and from dentate granule cells of the mouse hippocampal slice preparation. The passive electrical properties of the cells and their responses to electrical stimulation of the major antidromic and orthodromic pathways were explored. The majority of cells were impaled between 60 and 100 micron from the surface of the slice. Mean resting potentials were about -66 mV for dentate and CA1 cells and -61 mV for CA3 cells. Mean input resistances were 87, 78 and 73 M omega respectively, with a range of 30-160 M omega for all three populations. Action potential amplitudes ranged from 70 to 110 mV and were typically about 90 mV. Current-voltage (I-V) plots for all three populations were ohmic within a range 10-20 mV negative to the resting potentials. The chord resistance of the I-V relation was lower at more negative potentials and higher at more positive potentials than at the resting potential. Antidromic stimulation at intensities subthreshold for action potential invasion of the impaled cell gave rise to inhibitory post-synaptic potentials (i.p.s.p.s) in CA1 and CA3 cells. The reversal potential of the i.p.s.p.s lay between -65 and -75 mV. They were chloride dependent and could be attenuated by application of bicuculline methiodide. No recurrent i.p.s.p. was seen in dentate cells when using potassium-acetate-filled electrodes. If potassium-chloride-filled intracellular electrodes were used, thus raising the intracellular chloride ion concentration, an antidromically evoked, bicuculline-sensitive depolarizing post-synaptic potential (p.s.p.) could be evoked. Thus, a gamma-aminobutyric acid (GABA)-mediated recurrent inhibitory pathway was present in the slice in all three cell populations but appeared to be difficult to evoke reliably in the dentate gyrus. Orthodromic excitation of CA1 and CA3 cells evoked an excitatory post-synaptic potential (e.p.s.p.) followed by a biphasic hyperpolarization. The early hyperpolarization, lasting about 50 ms, reversed at about -65 mV and was chloride dependent. The later hyperpolarization lasted up to 400 ms, reversed at about -85 mV, and was chloride independent. The e.p.s.p. evoked in dentate cells by stimulation of the perforant path was biphasic and was followed by a hyperpolarization lasting 300-600 ms. The hyperpolarization resembled the late hyperpolarization described above. The two components of the e.p.s.p. may have been produced by the combined activation of the medial and lateral components of the perforant path. Small-amplitude regenerative potentials have been seen in all three cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1985

The anion selectivity of GABA-mediated post-synaptic potentials in mouse hippocampal cells.

Intracellular recordings were made from CA1 and dentate cells of the mouse hippocampal slice. When potassium acetate was used to fill the electrodes, spontaneous synaptic activity was rarely seen in CA1 or dentate cells. In contrast, when electrodes filled with potassium chloride were used, spontaneous depolarizing post-synaptic potentials (p.s.p.s) were prominent. That these p.s.p.s were mediated by gamma-aminobutyric acid (GABA) was supported by the following: they were blocked by electrophoretic application of bicuculline methiodide; their appearance was associated with the reversal of the evoked GABA-mediated recurrent inhibitory post-synaptic potential (i.p.s.p.) and their appearance was associated with the positive displacement of the reversal potential for the response to GABA applied by electrophoresis close to the cell soma. The interval distribution for the p.s.p.s could not be fitted by a Poisson distribution. The anionic selectivity of the conductance evoked by GABA was investigated by using the potassium salts of a range of anions to fill the electrodes. Depolarizing spontaneous p.s.p.s were seen in all cells impaled with electrodes containing potassium chloride, iodide, bromide, nitrate, or chlorate. In these cells, the reversal potential for the response to electrophoretically applied GABA was displaced in the depolarizing direction and the evoked recurrent i.p.s.p. was reversed. When anions with hydrated shells larger than chlorate (bicarbonate, sulphate, chromate, acetate or citrate) were used, spontaneous depolarizing p.s.p.s were not seen, and the reversal potential for the response to somatic GABA application and for the evoked recurrent i.p.s.p. lay between -70 and -75 mV.

Action Potentials↗

PubMed1985

Actions and interactions of GABA and benzodiazepines in the mouse hippocampal slice.

Intracellular recordings have been made from CA1, CA3 and dentate cells of the mouse hippocampal slice. Gamma-aminobutyric acid (GABA) and the water-soluble benzodiazepines, midazolam and flurazepam were applied close to the impaled cell somata by microelectrophoresis. GABA always caused a fall in input resistance, although the associated changes in membrane potential were variable. These were consistent with reports which have defined a hyperpolarizing response to somatic and a depolarizing response to dendritic application of GABA to CA1 and CA3 cells in the rat and guinea-pig. In this study, the phenomenon was seen in CA1, CA3 and dentate cells. The reversal potential for the hyperpolarizing, somatic response to GABA lay between -70 and -75 mV, similar to the reversal potential of the evoked recurrent inhibitory post-synaptic potential (i.p.s.p.). The change in cell input conductance caused by GABA was larger at membrane potentials positive to the resting potential and smaller at hyperpolarized membrane potentials. Extracellular recordings of action potential frequency were made from ten cells in which the application of either midazolam or flurazepam increased the inhibitory potency of GABA. In three of these cells, the benzodiazepine reduced action potential frequency slightly when applied alone. Neither midazolam nor flurazepam had a consistent effect on membrane potential, resting input resistance or the current-voltage (I-V) relations of the cells when ejected alone. In twenty-eight of forty-one cells examined in detail, ejection of either midazolam or flurazepam was found to increase the response to GABA. In six cells, the response to GABA was significantly reduced by the benzodiazepine tested whilst in the remainder, no interaction of the drugs could be demonstrated. Examination of the dose-response relation for GABA alone and in the presence of midazolam or flurazepam showed that the maximal response to GABA was increased by the benzodiazepine in some cells while it was unchanged in others. When intracellular electrodes were filled with potassium chloride, spontaneous depolarizing GABA-mediated post-synaptic potentials (p.s.p.s) were seen. Measurement of the interval and amplitude distributions of these events showed that flurazepam increased their amplitude but not their frequency.

Action Potentials↗

PubMed1980

Hyperprolactinaemia and long-term lactational amenorrhoea.

Clinical details of lactational and menstrual history post-partum have been correlated with measurements of serum prolactin, luteinizing hormone, follicle stimulating hormone, oestradiol and progesterone in Transkei women in order to gain further insight into the role of prolactin in the maintenance of post-partum lactational amenorrhoea. Of seventy-four breast-feeding mothers studied, sixty-six had elevated serum levels of prolactin. Forty-four women were amenorrhoeic with minimal or absent endocrine evidence of ovarian activity. Of the twenty-seven lactating women who reported menstruation, twelve had high levels of prolactin and no apparent ovarian activity, eleven had lower prolactin levels and increased oestradiol levels suggesting ovarian follicular development, while only four women had increased progesterone levels indicating definite luteal activity. Three lactating women were pregnant. The results support previous reports that lactational amenorrhoea is associated with hyperprolactinaemia and a possible sequence of endocrine events resulting in the return of post-partum fertility is proposed.

Amenorrhea↗

PubMedDate not supplied

Mitochondria and Ca(2+)in cell physiology and pathophysiology.

There is now a consensus that mitochondria take up and accumulate Ca(2+)during physiological [Ca(2+)](c)signalling. This contribution will consider some of the functional consequences of mitochondrial Ca(2+)uptake for cell physiology and pathophysiology. The ability to remove Ca(2+)from local cytosol enables mitochondria to regulate the [Ca(2+)] in microdomains close to IP3-sensitive Ca(2+)-release channels. The [Ca(2+)] sensitivity of these channels means that, by regulating local [Ca(2+)](c), mitochondrial Ca(2+)uptake modulates the rate and extent of propagation of [Ca(2+)](c)waves in a variety of cell types. The coincidence of mitochondrial Ca(2+)uptake with oxidative stress may open the mitochondrial permeability transition pore (mPTP). This is a catastrophic event for the cell that will initiate pathways to cell death either by necrotic or apoptotic pathways. A model is presented in which illumination of an intramitochondrial fluorophore is used to generate oxygen radical species within mitochondria. This causes mitochondrial Ca(2+)loading from SR and triggers mPTP opening. In cardiomyocytes, mPTP opening leads to ATP consumption by the mitochondrial ATPase and so results in ATP depletion, rigor and necrotic cell death. In central mammalian neurons exposed to glutamate, a cellular Ca(2+)overload coincident with NO production also causes loss of mitochondrial potential and cell death, but mPTP involvement has proven more difficult to demonstrate unequivocally.

Biological Transport↗