Aaron P. Fox

Calcium current variation between acutely isolated adult rat dorsal root ganglion neurons of different size.

1. The distribution of pharmacologically and/or biophysically unique Ca2+ current subtypes was studied in different diameter rat dorsal root ganglion (DRG) neuron cell bodies. DRG cells which fell into three diameter ranges, small (20‐27 microns), medium (33‐38 microns) and large (45‐51 microns), were studied. T‐type Ca2+ current was defined as low‐threshold, rapidly inactivating current evoked by a weak test depolarization (‐50 mV) from negative holding potentials (‐80 to ‐100 mV), and which was sensitive to changes in holding potential. L‐type Ca2+ current was defined as peak high‐threshold Ca2+ current evoked from a holding potential of ‐60 mV and sensitive to blockade by 2 microM‐nimodipine. N‐type Ca2+ current was defined as peak high‐threshold Ca2+ current evoked from a holding potential of ‐60 mV and sensitive to blockade by 0.9 microM‐omega‐conotoxin GVIA. 2. T‐type Ca2+ currents were observed in small and medium diameter, but not in large diameter, DRG cell bodies. Large diameter DRG cell bodies had a small amount of low‐threshold Ca2+ current but this current did not inactivate and was insensitive to a change in holding potential from ‐80 to ‐90 mV, and thus did not appear to be conducted through T‐type Ca2+ channels. The T‐type Ca2+ currents observed in medium diameter DRG cell bodies were considerably larger in amplitude (1‐6 nA) than those observed in small diameter DRG cell bodies (100 pA‐1 nA). This difference could not be accounted for by the difference in membrane surface area of small versus medium diameter DRG cell bodies. 3. The T‐type Ca2+ currents observed in medium diameter DRG cells were sensitive to blockade by amiloride. Amiloride (500 microM) blocked 79.4 +/‐ 0.9% (mean +/‐ S.E.M.) of T‐type Ca2+ current amplitude in six medium diameter DRG cell bodies which were held at ‐80 mV and depolarized to ‐50 or ‐40 mV. Amiloride (500 microM) failed to block high‐threshold current in five medium diameter DRG cell bodies, indicating that it was specific for T‐type Ca2+ current in these cells. 4. The percentage of peak whole‐cell L‐type Ca2+ current was significantly larger in small diameter DRG cell bodies (52.9 +/‐ 4.7% of total whole‐cell Ca2+ current) than in medium diameter DRG cell bodies (6.6 +/‐ 3.9% of total whole‐cell Ca2+ current) or large diameter DRG cell bodies (19.4 +/‐ 5.7% of total whole‐cell Ca2+ current).(ABSTRACT TRUNCATED AT 400 WORDS)

Differential activation of adenosine receptors decreases N-type but potentiates P-type Ca2+ current in hippocampal CA3 neurons.

Adenosine is released in the brain in significant quantities in response to increased cellular activity. Adenosine has been shown either to decrease synaptic transmission or to produce an excitatory response in hippocampal synapses, resulting in increased glutamate release. Previous reports have shown that adenosine or its analogs reduced Ca2+ current in dorsal root ganglion and hippocampal neurons. Here we show that the selective activation of adenosine receptor subtypes has different effects on Ca2+ channels from acutely isolated pyramidal neurons from the CA3 region of guinea pig hippocampus. Activation of A1 receptors inhibited primarily N-type Ca2+ current. In contrast, activation of A2b receptors resulted in significant potentiation of P-type but not N-type Ca2+ current. This potentiation could be inhibited by blocking the cAMP-dependent protein kinase. Because of the ubiquity of adenosine, the differential effects on Ca2+ channels of adenosine receptor subtype activation may have significant implications for neuronal excitability.

Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion

To facilitate the study of trophic interactions between mesencephalic dopaminergic neurons and their target cells, clonal hybrid cell lines have been developed from rostral mesencephalic tegmentum (RMT) of the 14-day-old embryonic mouse employing somatic cell fusion techniques. Among the hybrid cell lines obtained, one contains a high level of dopamine (DA), another predominantly 3,4-dihydroxyphenylalanine (DOPA), and a third no detectable catecholamines. The hybrid nature of the cell lines is supported by karyotype analysis and by the expression of adhesion molecules as assessed by aggregation in rotation-mediated cell culture. The DA cell line shows neuronal properties including catecholamine-specific histofluorescence, neurite formation with immunoreactivity to neurofilament proteins, and large voltage-sensitive sodium currents with the generation of action potentials. In contrast to the pheochromocytoma cell line (PC12), the dopamine content of the DA hybrid cell line is depleted by low concentrations of N-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Reflections on Ca2+-channel diversity, 1988–1994

partners throughout the CNS. Added to this, the coupling of the NO-cGMP pathway to ectlvatlon of glutamate [especially N-methyl-n-aspartate (NMDA)] receptors, first demonstrated In the cerebellum*, appears to be widely applicable. Flnally, the early avallablllty of pharmacologlcal tools, notably lnhlbltors of NO synthase, NO scav. engers and drugs that donate NO, provlded the means to test for the lnvolvement of NO In CNS function. It turns out to be lmpllcated In a bewIlder. lng array of phenomena, ranging from synaptic plastlclty In several of Its forms (for example, long4erm potentlatlon and depresslon of syneptlc transmls= slon, synaptogenesls and development, drug tolerance and dependence and hyperalgesla) to the moduIatlon of sensory nnd motor pathways, the local regulation of cerebral blood flow, neuroendocrlne reguiatlon, leernlng, feedlng and sexual behavlour’“, Under more pathologlcal condltlons, excessive productlon of NO by neurones or gllrl cells might be an lmportnnt medlator of cell denth nnoI thur, contrlbute to ncurodcgeneratlve states, for cnar~ple, followlng cerebral lschaemla~4~~~, The field Is relatively young, and It Is still expand. Ing rapldly: the past few years have seen an annual twoto threefold Increase In the number of papers publlshed on the subject, Dssplte the Increase In knowledge, our level of understandlng of NO slgnalllng In the braln remains crude, Many key Issues surroundlng Its proposed functions remeln unresolved and steeped In controversy, for example, Its role (or otherwlse) In Ion&term potentlatlon and Its supposed whole-animal correlates of leemlng and memory. The dust should settle, In tlme. One of the most Important goals for the future Is to elucidate the cellular end molecular mechenlsms performed by NO end Its second messenger, cGMP, In neural cells, Wlth respect to the latter, and wlth a few notable exceptlons, we are not much wiser than we were when It was first found In the braln, 25 years ago,

Whole-cell recordings of inwardly rectifying K+ currents activated by 5-HT1A receptors on dorsal raphe neurones of the adult rat.

1. An inwardly rectifying K+ current activated by serotonin (5‐HT) was recorded from acutely isolated adult dorsal raphe (DR) neurones using the whole‐cell recording mode of the patch clamp technique. 2. The 5‐HT‐induced K+ current (I5‐HT) was only visible at an [K+]0 > 5 mM and it was observed in 69% of the cells. 3. The reversal potential for I5‐HT was close to the potassium equilibrium potential and was shifted by 51 mV per 10‐fold change in [K+]0 indicating that I5‐HT was carried predominantly by K+. The chord conductance of I5‐HT at ‐90 mV was proportional to the external [K+] raised to a fractional power. 4. A dose‐response relationship revealed that I5‐HT was activated with an ED50 of 30 nM. Ba2+ (0.1 mM) blocked I5‐HT completely. Spiperone reversibly antagonized the response to 5‐HT and 8‐OHDPAT (8‐hydroxy‐2‐(di‐n‐propylamino)tetralin) mimicked the response indicating that the receptor activated was of the 5‐HT1A subtype. 5. The response to 5‐HT was largely prevented by in vitro pretreatment of the cells with pertussis toxin (PTX) indicating the involvement of a PTX‐sensitive G‐protein in the transduction mechanism. 6. cAMP and lipoxygenase metabolites, both implicated in the modulation of similar currents in other preparations, were found not to alter the effectiveness of 5‐HT. 7. Glibenclamide and tolbutamide, blockers of the ATP‐regulated K+ channel, did not reduce the effect of 5‐HT in DR neurones. 8. These results show that in acutely isolated adult DR neurones 5‐HT activates an inwardly rectifying K+ current and this involves a PTX‐sensitive G‐protein in the transduction pathway which may interact with the K+ channel directly.

Evidence for multiple types of Ca2+ channels in acutely isolated hippocampal CA3 neurones of the guinea-pig

1. Current through Ca2+ channels was studied in acutely isolated guinea‐pig pyramidal neurones from the CA3 region of the hippocampus. Both the whole‐cell and single‐channel patch‐clamp configuration were used. 2. Both whole‐cell and single‐channel currents displayed holding potential sensitivity indicative of two high‐threshold currents similar to L‐ and N‐type Ca2+ currents. 3. A low‐threshold whole‐cell current, similar to T‐type current seen in dorsal root ganglion (DRG) neurones, activated at ‐60 to ‐50 mV and was blocked by nickel (100 microM) and amiloride (500 microM). Exposure to 50 microM‐cadmium left a fraction of the T‐type current intact but blocked N‐ and L‐type current. This T‐like component needed extremely negative holding potentials to be completely reprimed. 4. Whole‐cell N‐type Ca2+ channel current was blocked by omega‐conotoxin (1 microM). From a holding potential of ‐90 mV, omega‐conotoxin decreased the peak whole‐cell current by 33%. 5. A slowly inactivating high‐threshold Ca2+ current (L‐type) that was present at depolarized holding potentials, displayed dihydropyridine sensitivity. From a holding potential of ‐50 mV, addition of the dihydropyridine Ca2+ channel antagonist nimodipine (2 microM) to the bath decreased whole‐cell peak current by 45%. Interestingly, at negative holding potentials nimodipine worked as an agonist. From a holding potential of ‐90 mV, nimodipine (2 microM) increased peak current at test potentials from ‐50 to ‐20 mV and shifted the peak of the current‐voltage relationship in the hyperpolarizing direction similar to the effect of Ca2+ channel agonist Bay K 8644. Exposure to Bay K 8644 (2 microM) increased peak current and single channel open probability independent of holding potential while shifting the peak of the whole‐cell current‐voltage relationship 11 mV in the hyperpolarizing direction. Our experiments suggest that there are approximately the same number of L‐type as omega‐conotoxin sensitive N‐type Ca2+ channels in CA3 neurones. 6. A high‐voltage‐activated whole‐cell current was still present in cells exposed to both nimodipine and omega‐conotoxin (2 and 1 microM, respectively) suggesting the existence of a fourth type of Ca2+ channel in these neurones or that a population of either L‐type or N‐type Ca2+ channels did not respond to dihydropyridine antagonists or omega‐conotoxin, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuronal alpha-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors

We have characterized the α-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of α7-containing receptors that bind α-Bgt with high affinity (Kd = 94 pm). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and α-Bgt block ∼90% of the whole-cell current. The binding of nicotinic agonists to cell-surface BgtRs was highly cooperative with four different agonists showing Hill coefficients in the range of 2.3–2.4. A similar agonist binding cooperativity was observed for BgtR homomers formed from chimeric α7/5HT3 subunits expressed in tsA 201 cells. Two classes of agonist binding sites, in the ratio of 4:1 for PC12 cell BgtRs and 3:1 for α7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and α7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and α7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.

Two types of Ca2+ currents are found in bovine chromaffin cells: facilitation is due to the recruitment of one type.

1. Whole‐cell Ca2+ currents in cultured bovine chromaffin cells were studied using patch‐clamp electrophysiology. With Ba2+ or Ca2+ as the current carriers, two separate components of whole‐cell current could be distinguished by biophysical and pharmacological criteria. These components of Ca2+ current were different from T‐ or N‐type Ca2+ channels previously described, as they were not inactivated at a holding potential of ‐60 mV. 2. Depolarization of the cells past ‐20 mV in 10 mM‐Ba2+ activated a single component of Ca2+ current, called the standard’ current. This current showed no detectable voltage‐dependent inactivation, but did show marked current‐dependent inactivation as steady‐state inactivation (H‐infinity) plots obtained in the presence of Ba2+ were quite different from those obtained from Ca2+. 3. In most chromaffin cells large pre‐depolarizations or repetitive depolarizations in the physiological range activated a second component of Ca2+ current called ‘facilitation’. Facilitation was observed with either Ca2+ or Ba2+ as the charge carrier. Recruiting facilitation increased whole‐cell currents by an average of 60%. 4. Pre‐pulses to +120 mV lasting 200 ms completely activated facilitation. Pre‐pulses longer than 800 ms started to inactivate facilitation, while pre‐pulses longer than 2500 ms completely inactivated this component of Ca2+ current. Because only outward currents were recorded at +120 mV, it is likely that facilitation inactivated in a voltage‐dependent manner. 5. When the extracellular Ba2+ concentration was increased in the range from 2 to 90 mM activation of both facilitation and standard Ca2+ currents shifted in the depolarizing direction. In 2 mM‐Ba2+ facilitation activated at potentials 10 mV more negative than the standard component, while in 90 mM‐Ba2+, facilitation activated at a potential about 10 mV more depolarized than the standard component. Thus, the voltage sensor for the facilitation Ca2+ current appeared to sense more surface charge than did the standard Ca2+ current. 6. Tail currents measured at ‐20 and ‐30 mV in the absence of facilitation (without pre‐pulses) showed one time constant for current deactivation. Tail currents measured with both facilitation and standard currents activated showed a significantly slower deactivation rate than that seen with the standard current alone. 7. The dihydropyridine antagonist nisoldipine (1 microM) completely suppressed the facilitation Ca2+ current even when cells were held at negative holding potentials (‐80 mV). In contrast, the standard current was unaffected by 1 microM‐nisoldipine, even at depolarized holding potentials (‐20 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

Epigenetic regulation of hypoxic sensing disrupts cardiorespiratory homeostasis

Recurrent apnea with intermittent hypoxia is a major clinical problem in preterm infants. Recent studies, although limited, showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension, suggesting that apnea of prematurity predisposes to autonomic dysfunction in adulthood. Here, we demonstrate that adult rats that were exposed to intermittent hypoxia as neonates exhibit exaggerated responses to hypoxia by the carotid body and adrenal chromaffin cells, which regulate cardio-respiratory function, resulting in irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity was associated with elevated oxidative stress, decreased expression of genes encoding antioxidant enzymes, and increased expression of pro-oxidant enzymes. Decreased expression of the Sod2 gene, which encodes the antioxidant enzyme superoxide dismutase 2, was associated with DNA hypermethylation of a single CpG dinucleotide close to the transcription start site. Treating neonatal rats with decitabine, an inhibitor of DNA methylation, during intermittent hypoxia exposure prevented oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction. These findings implicate a hitherto uncharacterized role for DNA methylation in mediating neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood.

Three types of bovine chromaffin cell Ca2+ channels: facilitation increases the opening probability of a 27 pS channel.

1. Cell‐attached patch recordings from bovine chromaffin cells were performed with 90 mM‐Ba2+ in the patch pipette and with isotonic potassium aspartate in the bathing solution to zero the membrane potential. Three different types of unitary Ca2+ channel activity could be distinguished in these recordings. 2. A 27 pS Ca2+ channel was distinguished by constructing amplitude histograms and measuring slope conductance. This channel activated over a broad range of potentials (depolarizations greater than ‐10 mV). 3. A second Ca2+ channel with a slope conductance of 14 pS could also be detected with amplitude histograms. This channel activated with depolarizations greater than ‐20 mV. 4. An 18 pS Ca2+ channel was observed infrequently indicating that this channel may carry only a small amount of the whole‐cell current. This 18 pS channel was sensitive to changes in holding potential. Depolarizing the patch to +10 mV from a holding potential of ‐80 mV elicited robust unitary activity. Changing the patch holding potential to ‐40 mV while maintaining test depolarizations to +10 mV completely inactivated the 18 pS channel. Neither the 25 pS nor the 14 pS Ca2+ channels were affected by changes in holding potential in the range from ‐80 mV to ‐40 mV, indicating the 18 pS channel was a different type of channel. As the 18 pS channel was observed so infrequently, no detailed studies of it were possible. 5. Chromaffin cell Ca2+ currents exhibited facilitation. Large pre‐depolarizations greatly augmented whole‐cell currents observed in these cells. Whole‐cell currents could double or triple after recruiting facilitation. The application of large pre‐depolarizations altered the gating behaviour of the 27 pS Ca2+ channel manifested as dramatically increased channel opening probabilities measured during subsequent test pulses. Large pre‐depolarizations induced unitary activity in the 27 pS Ca2+ channel similar to the long‐lived openings exhibited by L‐type Ca2+ channels in the presence of Bay K 8644. Large pre‐depolarizations did not change the gating behaviour of the 14 pS Ca2+ channel. 6. Repetitive depolarizations in the physiological range could also induce facilitation. At the single‐channel level facilitation was manifested as a striking increase in opening probability of the 27 pS Ca2+ channel. No effect of repetitive activity was observed on 14 pS channel gating. At the whole‐cell level, repetitive depolarizations dramatically increased the current observed. 7. Facilitation of 27 pS Ca2+ channel activity could be induced by changing the holding potential to a depolarized level (greater than or equal to ‐10 mV).(ABSTRACT TRUNCATED AT 400 WORDS)