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J P Walsh

Publications and source records attributed to J P Walsh.

At least 19 recordsLinked to original sources
PubMed1996

'Intact' dopaminergic midbrain neurons of the rat display unclamped dendritic Ca2+ currents.

Calcium channels play an important role in generating the complex electrophysiology of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNc). To directly study these calcium currents in dendritically intact neurons, two issues needed to be addressed: the identity of the neuron (DAergic versus non-DAergic) and control of putative dendritic calcium conductances. A measure of the anomalous rectifier (Ih) was used to identify DAergic neurons. Both groups of neurons produced large (approximately 1 nA), sustained inward currents that persisted as 'plateau currents' for hundreds of milliseconds after the termination of the depolarizing voltage clamp step. We conclude that intact DAergic neurons can be identified under conditions optimal for recording calcium currents (i.e. Cs+ internal solution), but powerful dendritic currents limit rigorous biophysical analysis.

Animals↗
PubMed1996

Effects of central administration of highly selective opioid mu-, delta- and kappa-receptor agonists on plasma luteinizing hormone (LH), prolactin, and the estrogen-induced LH surge in ovariectomized ewes.

A reduction in endogenous opioid inhibition (disinhibition) of GnRH secretion is thought to be permissive for the preovulatory GnRH/LH surge. There are no published studies of the effects of highly specific opioid receptor agonists on the LH surge in any species, and the relative importance of the opioid receptor subtypes mu, delta and kappa in the mechanism of disinhibition is unknown. In sheep, attempts to block the LH surge with opiates have been largely unsuccessful, and there is little evidence for reduced opioid inhibition during the GnRH/LH surge. The opioid receptor subtypes regulating PRL secretion in sheep are also unknown. Conscious, ovariectomized ewes with permanent third ventricular cannulae were injected with estradiol benzoate (EB) 50 micrograms or oil im (t = 0 h). In this model, EB elicits a time-delayed surge in LH secretion after 13-18 h. Jugular venous blood was sampled at half hourly intervals between-2 and 0 h and 10 and 26 h. From 12-20 h, infusions were made into the third ventricle of either the highly specific mu-agonist DAGO (10, 20 or 40 nmol/h), the delta-agonist DPDPE (40 nmol/h), the kappa-agonist U50488 (40 nmol/h) or saline (vehicle). In oil-treated animals (n = 4-6), DAGO infusion at 20 and 40 nmol/h reduced plasma LH whereas DPDPE or U50488 had no effect. In EB-treated animals (n = 6), DAGO (40 nmol/h) delayed the LH surge (mean +/- SEM time to surge onset 21.4 +/- 0.3 h vs. 14.0 +/- 0.4 h in controls, P < 0.0001). DAGO at 10 nmol/h did not alter surge onset and at 20 nmol/h had variable effects. DPDPE or U50488 did not affect LH surge timing or amplitude. All doses of DAGO increased plasma PRL, whereas DPDPE and U50488 had no effect. We conclude that, in ovariectomized ewes, activation of opioid mu-receptors, but not delta- or kappa-receptors, inhibits GnRH secretion, can block the estrogen-induced GnRH/LH surge and increases PRL secretion. The results are consistent with the disinhibition hypothesis.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1995

Arachidonoyl-diacylglycerol kinase. Specific in vitro inhibition by polyphosphoinositides suggests a mechanism for regulation of phosphatidylinositol biosynthesis.

We previously described the purification of a membrane-bound diacylglycerol kinase highly selective for sn-1-acyl-2-arachidonoyl diacylglycerols (Walsh, J. P., Suen, R., Lemaitre, R. N., and Glomset, J. A. (1994) J. Biol. Chem. 269, 21155-21164). This enzyme appears to be responsible for the rapid clearance of the arachidonate-rich pool of diacylglycerols generated during stimulus-induced phosphoinositide turnover. We have now shown phosphatidylinositol 4,5-bisphosphate to be a potent and specific inhibitor of arachidonoyl-diacylglycerol kinase. Kinetic analyses indicated a Ki for phosphatidylinositol 4,5-bisphosphate of 0.04 mol %. Phosphatidic acid also was an inhibitor with a Ki of 0.7 mol %. Other phospholipids had only small effects at these concentrations. A series of multiply phosphorylated lipid analogs also inhibited the enzyme, indicating that the head group phosphomonoesters are the primary determinants of the polyphosphoinositide effect. However, these compounds were not as potent as phosphatidylinositol 4,5-bisphosphate, indicating some specificity for the polyphosphoinositide additional to its total charge. Five other diacylglycerol kinases were activated to varying degrees by phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, suggesting that inhibition by acidic lipids may be specific for the arachidonoyl-DAG kinase isoform. Given the presumed role of arachidonoyl-diacylglycerol kinase in the phosphoinositide cycle, this inhibition may represent a mechanism for polyphosphoinositides to regulate their own synthesis.

3T3 Cells↗
PubMed1994

Arachidonoyl-diacylglycerol kinase from bovine testis. Purification and properties.

Previous work in our laboratory demonstrated the existence of a membrane-bound diacylglycerol kinase highly selective for diacylglycerols containing arachidonate as the sn-2 fatty acyl moiety (MacDonald, M. L., Mack K. F., Richardson, C. N., and Glomset, J. A. (1988) J. Biol. Chem. 263, 1575-1583). We now report the purification of arachidonoyl-diacylglycerol kinase 34,400-fold to apparent homogeneity from bovine testis. High concentrations of both salt and detergent were required to extract the enzyme from membranes and stabilize its activity, suggesting that in vivo the enzyme is part of a complex with other membrane or cytoskeletal proteins. Arachidonoyl-diacylglycerol kinase had an apparent M(r) of 58,000 both on SDS-polyacrylamide gels and by size exclusion chromatography. The enzyme appeared to be an integral membrane protein. In a mixed micellar assay, arachidonoyl-diacylglycerol kinase followed surface dilution kinetics with respect to diacylglycerol. The purified enzyme retained the arachidonate selectivity observed previously in membranes. Kinetic analyses indicated a Km for sn-1-stearoyl-2-arachidonoylglycerol of 2.4 mol %, as compared to 43 mol % for sn-1-palmitoyl-2-oleoylglycerol. Calcium, an activator of some other diacylglycerol kinases, had no apparent effect on the arachidonate-specific enzyme. Guanosine triphosphate could effectively substitute for ATP as the phosphoryl donor and Mg2+ could be replaced by Mn2+ or Ca2+. Phosphatidylserine and, to a lesser extent, phosphatidylinositol inhibited the purified enzyme. Phosphatidylcholine and phosphatidylethanolamine had only small effects.

3T3 Cells↗
PubMed1994

Loss of paired-pulse facilitation at the corticostriatal synapse of the aged rat.

Changes in calcium (Ca2+) homeostasis have been proposed to contribute to the aging process. Paired-pulse facilitation, a form of synaptic enhancement that relies upon an accumulation of Ca2+ in the presynaptic terminal, was used to examine the effect of aging at the corticostriatal synapse. Intracellular recordings in striatal neurons from young rats demonstrated a consistent enhancement in the second of two synaptic responses evoked by stimulation of the corpus callosum. In contrast, neurons from aged rats showed a consistent depression of the second synaptic response at identical pairing intervals. These differences were not explained by an age-dependent increase in synaptic depression and demonstrate an alteration in the Ca(2+)-mediated process of presynaptic facilitation.

Aging↗
PubMed1994

Neurophysiological, pharmacological and morphological properties of human caudate neurons recorded in vitro.

Tissue samples from the caudate nucleus were obtained from eight children (eight to 172 months of age) who underwent hemispherectomies for the relief of intractable seizures. Neurophysiological, pharmacological and morphological properties of caudate neurons were characterized by intracellular recordings in an in vitro slice preparation. These properties were compared with those of tissue obtained from animal studies. Electrophysiological properties of human caudate neurons that were similar to those of cat caudate and rat neostriatal cells included resting membrane potential, input resistance, action potential rise time, fall time, duration and action potential afterhyperpolarization amplitude, as well as the general characteristics of locally evoked synaptic responses. Properties that were different included action potential amplitudes and time-constants. Human caudate neurons also displayed responses similar to those of cat caudate or rat neostriatal cells to manipulation of excitatory amino acid receptor systems and to dopamine application. Kynurenic acid, a broad-spectrum excitatory amino acid receptor antagonist, decreased the amplitude of evoked synaptic responses, indicating that they were partially mediated by excitatory amino acids. In Mg2+ free Ringer's solution, the amplitudes and durations of postsynaptic responses were increased and bursts of action potentials were induced. These effects were mediated by activation of N-methyl-D-aspartate receptors since they were blocked by 2-amino-5-phosphonovalerate, a specific N-methyl-D-aspartate-receptor antagonist. Iontophoretic application of N-methyl-D-aspartate also induced membrane oscillations and bursts in almost all caudate neurons. Dopamine decreased the amplitude of postsynaptic responses, an effect antagonized by domperidone, a selective D2 dopamine receptor antagonist. Developmentally, the greatest change was an increase in action potential amplitude, although input resistance decreased and action potential afterhyperpolarization amplitude increased. Postsynaptic responses were similar across age. All but one of the caudate neurons identified by intracellular injection of biocytin or Lucifer Yellow were medium-sized spiny cells. These experiments show that human caudate neurons display a number of electrophysiological properties similar to rat neostriatal or cat caudate neurons recorded in brain slices. Furthermore, few electrophysiological parameters changed significantly over the age period examined suggesting that the human caudate at eight months displays many of the neuronal functions of the more mature caudate nucleus.

Adolescent↗
PubMed1993

Depression of excitatory synaptic input in rat striatal neurons.

Intracellular recording of rat striatal neurons was performed in vitro to investigate posttetanic changes in the excitatory post-synaptic potential (EPSP) elicited by stimulation of the corpus callosum. Tetanic stimulation induced posttetanic potentiation (PTP) in 11 of 12 cells. The PTP decayed in 1-5 min and was followed by either a short or long duration depression of the EPSP in 10 of 12 neurons. The remaining two neurons examined showed a slight enhancement of the EPSP that lasted for 30 min after the tetanus. The group of cells demonstrating short-duration depression (n = 3) were characterized by a decay of the depression to the control level by 15-20 min post-tetanus. The EPSP in the remaining cells showing depression (n = 7) showed a maintained depression for the entire recording session. Blockade of GABAA receptors with bicuculline (30 microM) or NMDA receptors with APV (50 microM) did not effect the induction of long-lasting depression of the striatal EPSP. The data indicates that the corticostriatal pathway can undergo enduring forms of use-dependent synaptic plasticity. This type of synaptic modification may participate in the refinement of movement and contribute to striatal related learning and memory.

Animals↗
PubMed1993

Evidence for enhanced synaptic excitation in transplanted neostriatal neurons.

Fetal neostriatal tissue was transplanted into either the neostriatum or substantia nigra of adult rats. One to 6 months after transplantation, coronal brain slices were taken through the rostrocaudal extent of the transplants and neurons were characterized electrophysiologically using an in vitro slice preparation. When compared to control neurons taken from intact adult neostriata, transplanted neostriatal neurons (TSNs) had higher input resistances and longer time constants. All other passive and active membrane properties assessed were comparable between transplanted and control neostriatal neurons. Regardless of the transplantation site, local extracellular stimulation outside the graft elicited high-amplitude, long-duration depolarizing synaptic potentials that typically triggered bursts of action potentials. These synaptic potentials contrast with lower amplitude, shorter duration synaptic potentials consistently elicited in control neostriatal neurons. The depolarizing synaptic potentials evoked in the TSNs appeared to be mediated by a combined activation of N-methyl-D-aspartate (NMDA) and non-NMDA excitatory amino acid receptors. Both the broad-spectrum excitatory amino acid antagonist kynurenic acid and the specific non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione significantly reduced postsynaptic potentials elicited in TSNs. The specific NMDA antagonist 2-amino-5-phosphonovalerate had less effect on the amplitude but markedly reduced the duration of the synaptic potentials. The duration and amplitude of the bursts were augmented by the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline methiodide, indicating that inhibition occurred in TSNs. TSNs were also more sensitive than control neurons to direct application of glutamate or NMDA. These findings demonstrate that TSNs express altered electrophysiological properties. The pharmacological analysis indicates that depolarizing postsynaptic potentials were mediated by activation of excitatory amino acid receptors, suggesting either innervation of the graft by host fibers which contain excitatory amino acids or development of novel local excitatory interactions intrinsic to the graft. Furthermore, the occurrence of high-amplitude, long-duration depolarizing synaptic potentials in TSNs, regardless of the site of transplantation, suggests that grafted neostriatal neurons become hyperexcitable to synaptic input.

2-Amino-5-phosphonovalerate↗
PubMed1993

Synaptic activation of N-methyl-D-aspartate receptors induces short-term potentiation at excitatory synapses in the striatum of the rat.

We examined the effect of augmenting the activation of N-methyl-D-aspartate receptors during tetanic stimulation of the corpus callosum. Excitatory postsynaptic potentials were recorded from striatal neurons in an in vitro brain slice bathed in either normal or Mg(2+)-free artificial cerebrospinal fluid. Post-tetanic potentiation was followed by a long-lasting depression of the excitatory postsynaptic potential in normal artificial cerebral spinal fluid. Mg(2+)-free artificial cerebrospinal fluid increased the duration of the pretetanus excitatory postsynaptic potential and the amplitude and duration of the direct response to the tetanus. Mg(2+)-free artificial cerebrospinal fluid also enabled the expression of a short-term potentiation of the excitatory postsynaptic potential amplitude and duration. N-methyl-D-aspartate receptor antagonists blocked the induction of all tetanus-induced excitatory postsynaptic potential changes specific to the Mg(2+)-free artificial cerebral spinal fluid. These results indicate that maintained activation of N-methyl-D-aspartate receptors by synaptically released glutamate can produce a sustained enhancement of the excitatory postsynaptic potential. This use-dependent increase in synaptic efficacy may contribute to basal ganglia-related motor performance.

Animals↗
PubMed1992

Awareness of heart attack signals and cardiac risk markers amongst the general public in Dublin.

Following myocardial infarction, early access to medical care is essential. In order to assess the ability to recognise and manage heart attack and its risk factors, 302 members of the public were surveyed. A surprisingly comprehensive knowledge base was revealed, together with apparently appropriate ideas about the management of myocardial infarction and its risk factors. The significance of this finding is discussed in the light of the well documented delays by members of the public in summoning help when they suspect heart attack.

Adult↗
PubMed1991

Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate.

We have synthesized phosphatidylinositol 3-phosphate from phosphatidylinositol 4-phosphate by using diisopropylcarbodiimide to promote migration of the 4-phosphate via a cyclic phosphodiester intermediate. The product was isolated by a thin-layer chromatographic method that depends on the ability of phosphatidylinositol 4-phosphate, but not phosphatidylinositol 3-phosphate, to form complexes with boric acid. The final yield of the procedure was 8% phosphatidylinositol 3-phosphate, which was approximately 80% pure. The product was shown to be phosphatidylinositol 3-phosphate by the following criteria: (i) cochromatography with an authentic standard on borate thin-layer chromatography, (ii) cochromatography of the deacylated product with glycerophosphoinositol 3-phosphate on high-performance liquid chromatography, (iii) conversion of the product to phosphatidylinositol by homogeneous phosphatidylinositol 3-phosphate 3-phosphatase, and (iv) deacylation and deglyceration of the product to a compound that comigrates with inositol 1,3-bisphosphate on high-performance liquid chromatography. The availability of mass amounts of phosphatidylinositol 3-phosphate will allow further elaboration of reactions in this recently discovered pathway of phosphatidylinositol metabolism.

Autoradiography↗
PubMed1991

Neurophysiological maturation of cat caudate neurons: evidence from in vitro studies.

The membrane properties and synaptic physiology of developing cat caudate (Cd) nucleus neurons were studied in in vitro slice preparations. Recordings were obtained from 98 cells in kittens from fetal day (F) 56 to postnatal day (P) 90. With increasing age, the following maturational changes occurred; resting membrane potentials became more negative, action potential rise times decreased, action potential amplitudes increased, and action potential durations and input resistances decreased. The frequency of occurrence of afterhyperpolarizations and of anomalous rectification increased with age. The primary response to local extracellular stimulation was a depolarization usually accompanied by an action potential. Evoked hyperpolarizing responses were seen after P28 but only occurred if the membrane was depolarized by intracellular current injection. Cells identified by intracellular injection of Lucifer yellow were primarily medium-sized spiny neurons although it was not always possible to determine the cell type in slices from animals less than P5. Somatic diameter, dendritic length, and spine density increased with age. Dye-coupling occurred in slices less than P20. Its frequency decreased with age. These results show that Cd neurons undergo significant maturation during late prenatal and early postnatal periods. In contrast, substantia nigra neurons mature more rapidly and should be capable of influencing the less mature Cd neurons during development.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1991

Neurophysiological maturation of cat substantia nigra neurons: evidence from in vitro studies.

The membrane properties and synaptic physiology of developing cat substantia nigra (SN) neurons were studied in in vitro slice preparations. Stable intracellular recordings were obtained from 46 neurons in 20 kittens ranging in age from fetal day (F) 51 to postnatal day (P) 120. Only two of these properties changed with development. The percentage of cells displaying inward rectification and the percentage of cells that generated low-threshold Ca++ spikes increased with age. Properties that did not change included resting membrane potentials, action potential amplitudes and durations, and input resistances. At all ages locally evoked synaptic responses consisted of sequences of excitatory postsynaptic potentials followed by inhibitory postsynaptic potentials. Most of the cells recorded had the electrophysiological properties which have been attributed to SN dopamine-containing neurons. To identify neurons morphologically, and verify the recording site, cells were filled with Lucifer yellow at the end of each experiment. Somatic shapes varied widely from oval to fusiform to triangular. Somatic diameters and dendritic length increased with development. Filopodial processes and growth cones were present up to the first postnatal month. Dye-coupling occurred only in the fetal group. These results indicate that cat SN neurons have many mature physiological properties during late fetal and early postnatal development. This contrasts with the significant maturation that occurs in cat caudate neurons during the same developmental period.

Action Potentials↗
PubMed1990

sn-1,2-diacylglycerol kinase of Escherichia coli. Diacylglycerol analogues define specificity and mechanism.

A detailed structure/function analysis of the substrate specificity of Escherichia coli sn-1,2-diacylglycerol kinase was performed with three goals in mind: (a) to define the substrate specificity; (b) to discover inhibitors; and (c) to elucidate the specificity of diacylglycerol-dependent inactivation. Forty-seven structural analogues of sn-1,2-diacylglycerol were prepared and examined as substrates, inhibitors, and irreversible inactivators of the enzyme using mixed micellar assay methods. Modification of the acyl chains or the sn-2 ester affected the apparent Km but had only small effects on Vm; modifications of the sn-1 ester, sn-3 methylene, or sn-3 hydroxyl had large effects on the apparent Vm and smaller effects on Km. Consistent with these observations, diacylglycerol analogues modified only in the acyl chains or sn-2 ester were not diacylglycerol kinase inhibitors, whereas analogues with substitutions of the sn-1 ester or sn-3 hydroxyl frequently caused inhibition. A hydrogen bond-donating group was required for an analogue to be a diacylglycerol kinase inhibitor. Studies of diacylglycerol kinase inactivation by the various analogues were consistent with the previous conclusion that this process involves an interaction of diacylglycerols with an enzyme conformation different from that active in catalysis (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 15062-15069). Studies with a water-soluble diacylglycerol, sn-1,2-dibutyrylglycerol, allowed direct comparison of diacylglycerol kinase activity in mixed micelles with that in native membranes. The results are discussed in relation to the structural requirements of other diacylglycerol-dependent enzymes.

Diacylglycerol Kinase↗