1. In this, and the accompanying paper (Duchen & Biscoe, 1992), we test the hypothesis that the oxygen sensitivity of mitochondrial electron transport forms a basis for transduction in the carotid body, the primary peripheral arterial oxygen sensor. We here describe for isolated type I cells the changes in autofluorescence of mitochondrial NAD(P)H that accompany changes in PO2. 2. NAD(P)H autofluorescence (excitation, 340‐360 nm; emission peak, 450 nm) increased with anoxia, reflecting a rise in the NAD(P)H/NAD(P) ratio. Graded increases in autofluorescence were seen in response to graded decreases in PO2, suggesting that mitochondrial function is progressively altered below a PO2 of about 60 mmHg. 3. A mitochondrial origin for the NAD(P)H autofluorescence was suggested by the mutual exclusion of the responses to anoxia and cyanide. 4. Oxidized flavoproteins fluoresce when excited at 450 nm with an emission peak at 550 nm. The small signals obtained under these conditions increased with uncoupler and showed a graded decrease with falling PO2 reflecting a rise in the FADH/FAD ratio. 5. Hypoxia raises [Ca2+]i. The hypoxia‐induced changes in mitochondrial function were not secondary to this rise. A brief K(+)‐induced depolarization leads to a transient increase in [Ca2+]i. At the same time there is a rapid decrease in NAD(P)H autofluorescence followed by an increase that far outlasts the rise in [Ca2+]i. This delayed increase in autofluorescence was smaller than was the increase with anoxia, even though K(+)‐induced depolarization raised [Ca2+]i more than does anoxia. In Ca(2+)‐free solutions the depolarization‐induced changes were abolished, while those associated with hypoxia were maintained. 6. The changes of autofluorescence with K(+)‐induced depolarization appear to reflect (i) oxidation of NAD(P)H by stimulation of respiration following mitochondrial Ca2+ uptake and (ii) reduction of NAD(P) by the Ca(2+)‐dependent activation of mitochondrial dehydrogenases. This activation could last several minutes following only 100 ms depolarization, while the changes accompanying hypoxia closely followed the time course of the change in PO2. 7. In similarly isolated rat or mouse chromaffin cells and mouse dorsal root ganglion neurons under identical conditions, no measurable change in autofluorescence or in [Ca2+]i was seen until the PO2 fell below about 5 mmHg. 8. Carbonyl cyanide p‐trifluoromethoxy‐phenylhydrazone (FCCP) increases O2 consumption, oxidizing mitochondrial NADH and hence decreasing autofluorescence, (delta FFCCP). Blockade of electron transport by anoxia or CN‐ decreases O2 consumption, increasing mitochondrial NADH/NAD and autofluorescence (delta FCN). The fractional change in autofluorescence with FCCP, delta FFCCP/delta FFCCP+FCN), is thus a measure of resting O2 consumption.(ABSTRACT TRUNCATED AT 400 WORDS)

1. In the accompanying paper (Duchen & Biscoe, 1992) we have described graded changes in autofluorescence derived from mitochondrial NAD(P)H in type I cells of the carotid body in response to changes of PO2 over a physiologically significant range. These observations suggest that mitochondrial function in these cells is unusually sensitive to oxygen and could play a role in oxygen sensing. We have now explored further the relationships between hypoxia, mitochondrial membrane potential (delta psi m) and [Ca2+]i. 2. The fluorescence of Rhodamine 123 (Rh 123) accumulated within mitochondria is quenched by delta psi m. Mitochondrial depolarization thus increases the fluorescence signal. Blockade of electron transport (CN‐, anoxia, rotenone) and uncoupling agents (e.g. carbonyl cyanide p‐trifluoromethoxy‐phenylhydrazone; FCCP) increased fluorescence by up to 80‐120%, while fluorescence was reduced by blockade of the F0 proton channel of the mitochondrial ATP synthase complex (oligomycin). 3. delta psi m depolarized rapidly with anoxia, and was usually completely dissipated within 1‐2 min. The depolarization of delta psi m with anoxia (or CN‐) and repolarization on reoxygenation both followed a time course well characterized as the sum of two exponential processes. Oligomycin (0.2‐2 micrograms/ml) hyperpolarized delta psi m and abolished the slower components of both the depolarization with anoxia and of the subsequent repolarization. These data (i) illustrate the role of the F1‐F0 ATP synthetase in slowing the rate of dissipation of delta psi m on cessation of electron transport, (ii) confirm blockade of the ATP synthetase by oligomycin at these concentrations, and (iii) indicate significant accumulation of intramitochondrial ADP during 1‐2 min of anoxia. 4. Depolarization of delta psi m was graded with graded changes in PO2 below about 60 mmHg. The stimulus‐response curves thus constructed strongly resemble those for [Ca2+]i and NAD(P)H with PO2. The change in delta psi m closely followed changes in PO2 with time. 5. The rate of rise of [Ca2+]i in response to anoxia is strongly temperature sensitive. The rate of depolarization of delta psi m with anoxia similarly increased at least two‐ to fivefold on warming from 22 to 36 degrees C. The change with FCCP was not significantly altered by temperature. 6. These data show that the mitochondrial membrane potential changes over a physiological range of PO2 values in type I cells. This contrasts with the behaviour in dissociated chromaffin cells and sensory neurons, in which no change was measurable until the PO2 fell close to zero.(ABSTRACT TRUNCATED AT 400 WORDS)

The carotid body is a major oxygen sensor in the mammal. Measurements of cell electrophysiology, (Ca2+)i, mitochondrial membrane potential, and intracellular NADH suggest that specialized mitochondrial electron transport underlies sensory transduction.

1. The carotid body chemoreceptors are stimulated in situ by hypoxia. We have studied type I cells freshly dissociated from the carotid body of the rabbit. We have used microfluorimetric and patch clamp techniques to examine the responses to hypoxia, to anoxia, and to metabolic inhibition. 2. NADH autofluorescence measured at both 400 and 500 nm increased rapidly and reversibly in response to anoxia or to cyanide (CN‐), reflecting a change in mitochondrial metabolism. 3. Indo‐1 was used to measure changes in intracellular calcium, [Ca2+]i. Anoxia reversibly increased [Ca2+]i from approximately 50‐100 to approximately 200‐450 nM in all cells tested. The response showed a striking temperature sensitivity. Responses to hypoxic stimuli were barely detectable at 17‐20 degrees C, and were dramatically increased on warming to 36 degrees C. In contrast, responses to K(+)‐induced depolarization were only slightly increased in rate of onset and recovery by warming. 4. The rise in [Ca2+]i originated largely from an intracellular store which was slowly depleted by exposure to nominally Ca2(+)‐free solutions. Responses were unaffected by blockade of Ca2+ channels with organic (D600, verapamil) or inorganic (Co2+) blockers, by blockade of Na+ channels with tetrodotoxin (TTX), or by increasing action potential duration with tetraethylammonium (TEA). Responses to anoxia were increased by the increased [Ca2+]i loading that follows prior exposure to Ca2(+)‐free solutions. 5. Responses to anoxia, to blockade of electron transport by CN‐, and to the mitochondrial uncoupler, carbonyl cyanide p‐trifluoromethoxy‐phenylhydrazone (FCCP), were equivalent in amplitude. The response to anoxia was occluded by concurrent application of FCCP, suggesting that the Ca2+ originates from the same pool in each case. 6. At 35‐36 degrees C, responses to graded levels of PO2 were also graded. Thresholds varied between cells, but were typically 30‐50 mmHg. Stimulus‐responses curves were essentially hyperbolic, increasing dramatically as the PO2 approached 0 mmHg. 7. The sensitivity of cells to hypoxic solutions was increased by acidification of the superfusate over the pH range from 7.3 to 6.85. 8. Cell‐attached patch clamp recordings showed depression of spontaneous action potentials associated with a rise in [Ca2+]i during exposure to anoxic solutions. Whole‐cell recordings showed that anoxia increased a voltage‐gated gK as described previously for CN‐, while producing no change in resting conductance. 9. These data suggest that the rise in [Ca2+]i originates largely from Ca2+ efflux from a mitochondrial pool.(ABSTRACT TRUNCATED AT 400 WORDS)

Understanding transduction mechanisms is central to much of sensory physiology. The carotid chemoreceptors monitor the PO2 of arterial blood en route to the brain and are powerfully excited when the arterial PO2 falls to less than 60 mmHg. The type I cell is generally believed to be the transducer. These cells release catecholamines in response to agents that excite the receptor (hypoxia, cyanide, K(+)-induced depolarization, etc.). Adherent to the cells are the saucer-shaped nerve endings of the axons of the sinus nerve. We and others have used patch-clamp techniques to study the electrophysiological properties of the type I cells. We have also investigated type I cell chemistry with microfluorometric techniques, to measure intracellular Ca2+ concentration ([Ca2+]i), mitochondrial NADH, and mitochondrial membrane potential (delta psi m). During hypoxia there are graded increases in NADH, [Ca2+]i (which presumably will promote transmitter release), and graded depolarization of delta psi m. These results suggest that the Ca2+ is largely derived from an intracellular store, probably from mitochondria, and that release is entrained to delta psi m. Comparative studies with other cells indicate an increased sensitivity of the mitochondria of type I cells to changes in PO2. The data suggest that the electrophysiological responses of type I cells to hypoxia are not central to the response, although the excitability of the cells may provide a mechanism for the modulation of the response by varying voltage-gated Ca2+ influx.

1. The carotid body is the major peripheral sensor of arterial PO2 in the mammal and is excited by cyanide (CN‐). Type I cells, the presumed sites for transduction, were freshly dissociated from the carotid body of the adult rabbit and studied with the whole‐cell patch clamp technique. 2. Type I cells were hyperpolarized by CN‐, the action potential was shortened, and there was an increased after‐hyperpolarization. 3. Under voltage clamp control, CN‐ increased a voltage‐dependent outward current, which showed pronounced outward rectification. Tail currents increased by CN‐ reversed close to the predicted EK, the reversal potential of the CN‐‐induced current depended on extracellular [K+], and the current was blocked by intracellular TEA+ and Cs+. 4. The i‐V relation of the CN‐‐induced conductance strongly mirrored that of voltage‐gated Ca2+ entry, and the response was abolished by removal of extracellular Ca2+. We conclude that the increased gK is Ca2+ ‐dependent (gK(Ca]. 5. The Ca2+ current was attenuated by CN‐, and showed an increased rate of inactivation. Thus, the increased gK(Ca) must result from an alteration in Ca2+ homeostasis independent of the Ca2+ current, and not an increased Ca2+ entry through voltage‐activated channels. 6. Carbachol also hyperpolarized cells and increased a K+ conductance. 7. At depolarized holding potentials a steady‐state outward current was increased by CN‐. The current reversed close to EK, and was associated with increased current fluctuations. Noise analysis showed that a channel conductance of 3 pS carries the current. 8. The response to CN‐ was not impaired by the inclusion of 5 mM‐MgATP in the patch pipette. 9. If signals to the CNS are initiated by the calcium‐dependent release of transmitters from type I cells, transduction would appear to be the direct consequence of the energy dependence of Ca2+ homeostasis.

Quarterly Journal of Experimental PhysiologyVolume 74, Issue 7 p. 1-2 ArticleFree Access THE SCHOOL OF BERNARD KATZ PREFACE T. J. BISCOE, T. J. BISCOE Department of Physiology, University College London, Gower Street, London WC1E 6BTSearch for more papers by this author T. J. BISCOE, T. J. BISCOE Department of Physiology, University College London, Gower Street, London WC1E 6BTSearch for more papers by this author First published: 07 December 1989 https://doi.org/10.1113/expphysiol.1989.sp003375AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume74, Issue7December 7, 1989Pages 1-2 RelatedInformation

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.

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.

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)

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.

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)

Binding of gamma-aminobutyrate and benzodiazepine receptor ligands has been studied in the cerebellum of adult normal (C3H) and Lurcher mutant mice. The adult mutant has lost all Purkinje cells and more than 90% of the granule cells in the cerebellar cortex. When compared with their normal littermates Lurcher mice displayed large decreases in the number of high-affinity binding sites for [3H]muscimol, a synaptic gamma-aminobutyrate receptor ligand, in washed cerebellar homogenates. This observation was consistent with the extensive loss of gamma-aminobutyrate receptive Purkinje and granule cells from the Lurcher cerebellum. However, specific binding of the benzodiazepine-receptor ligand [3H]flunitrazepam to Lurcher cerebellum remained unchanged. Indeed quantitative autoradiography, employing [3H]flunitrazepam as a photoaffinity label, showed no significant differences in the density of labelling between Lurcher and normal littermate mice in any region of the cerebellum. These benzodiazepine binding sites in washed homogenates or tissue sections displayed a gamma-aminobutyrate-induced enhancement of [3H]flunitrazepam binding which occurred to the same extent in both Lurcher and normal cerebellum, a facilitatory effect which could be blocked by the addition of bicuculline methobromide. Our results suggest that a large proportion of the high-affinity, specific benzodiazepine binding sites in mouse cerebellum are not coupled to the synaptic gamma-aminobutyrate receptors thought to be labelled by high affinity [3H]muscimol binding. Further, that benzodiazepine binding sites do not appear to be enriched on either the soma or dendrites of Purkinje cells, as has been suggested from previous studies. Investigations at the electron microscope level are now required to elucidate the cellular location of benzodiazepine binding sites in the cerebellar cortex and to examine whether or not they are likely to be exposed to gamma-aminobutyrate in vivo.

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.