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J F MacDonald

Publications and source records attributed to J F MacDonald.

At least 91 records · Page 5Linked to original sources
PubMed1989

The regulation of NMDA receptors in the central nervous system.

1. Excitatory amino acids are major transmitters within the CNS. 2. Postsynaptic receptors for these transmitters, particularly those of the NMDA subtype are important in a variety of physiological and pathological processes. Therefore psychoactive drugs and potentially therapeutic drugs may target these receptors. 3. NMDA receptors can be regulated by competitive antagonists or by dissociative anaesthetics which act as use dependent blockers of the NMDA channel. 4. Alternatively drugs could modify intracellular second messenger systems that control expression of NMDA receptors.

Allosteric Regulation↗
PubMed1989

Excitatory amino acid blockers differentially affect bursting of in vitro hippocampal neurons in two pharmacological models of epilepsy.

The role of excitatory amino acid neurotransmitters in generating two distinct types of epileptiform discharge in the CA3 region of hippocampal slices was examined in the guinea-pig using a variety of different excitatory amino acid blockers. In magnesium-free medium the N-methyl-D-aspartate receptor antagonist, DL-aminophosphonovaleric acid, and the putative N-methyl-D-aspartate channel blockers phencyclidine and ketamine, reduced the amplitude and duration of spontaneous bursts and blocked their afterdischarges. In marked contrast none of these compounds significantly depressed spontaneous bursts recorded in the presence of 2 mM Mg2+ and penicillin. This was confirmed for individual slices where Mg2+-free bursts had previously been suppressed by the same concentration of blocker. Therefore, the N-methyl-D-aspartate receptor mechanism does not contribute significantly to the generation of this type of epileptiform discharge. The channel blockers lost their effect on Mg2+-free bursts in the presence of penicillin; however, the receptor blockers did not. This is probably due to the unique mechanisms of action of the channel blockers. This implies that different types of N-methyl-D-aspartate blockers might be effective anticonvulsants only in specific conditions. While the non-specific excitatory amino acid blocker, kynurenic acid, was effective against both burst types it appeared to suppress them by different mechanisms. Kynurenic acid depressed the amplitude and duration of Mg2+-free bursts but its only effect on penicillin bursts was to reduce their frequency. This suggests that neither N-methyl-D-aspartate nor non-N-methyl-D-aspartate receptors play a major role in the production of the paroxysmal depolarizing shift resulting from the block of GABA-mediated inhibition by penicillin. However, these receptors may be involved in generating the spontaneous activity which triggers the bursts.

2-Amino-5-phosphonovalerate↗
PubMed1989

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

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)

Adenosine Triphosphate↗
PubMed1988

Requirement of NMDA receptor/channels for intracellular high-energy phosphates and the extent of intraneuronal calcium buffering in cultured mouse hippocampal neurons.

Whole-cell patch-clamp recordings were undertaken in cultured mouse hippocampal neurons in order to investigate time-dependent changes in: (i) currents evoked by L-aspartic acid (Asp) and kainic acid (KAI), two excitatory amino acids active at N-methyl-D-aspartic acid (NMDA) and KAI receptor sites respectively, and (ii) tetrodotoxin (TTX) resistant voltage-dependent inward currents carried by Ca2+. Consistent with previous observations, Ca2+ currents gradually run down unless a support system containing Mg-ATP, phosphocreatine and creatinine phosphokinase is added to the intracellular medium. Here we report that, in addition to suppressing the rundown of currents through voltage-gated Ca2+ channels, such a support system is also necessary to prevent rundown of ionic currents through excitatory amino acid-gated channels of the NMDA type. When this support system was omitted from the recording pipette, currents induced by Asp, but not KAI, progressively declined over a period of 20 min and stabilized at values of about 50% of the initial. This progressive decline occurred regardless of the extent of intraneuronal Ca2+ buffering, indicating that it was not due to accumulation of cytosolic Ca2+. After the rundown, reversal potentials of ASP-induced currents were the same whether recorded with or without the intracellular support system and the Asp induced currents could be blocked by the specific NMDA channel blocker ketamine. We conclude that ionic currents through NMDA gated channels have two components: one requires high-energy phosphates and will run down if these are not supplied; the other requires no such supply and remains steady.

Adenosine Triphosphate↗
PubMed1988

A potassium conductance contributes to the action of somatostatin-14 to suppress ACTH secretion.

Somatostatin activates an inwardly rectifying potassium conductance in AtT-20 clonal corticotrophs, a cell line derived from the mouse pituitary gland. The action of somatostatin is blocked by pertussis toxin indicating that a GTP-binding protein couples the somatostatin receptor to the potassium channel. The potassium conductance is depressed by cesium. Cesium also attenuates the suppression of adrenocorticotropin hormone secretion by somatostatin suggesting that the increase in potassium conductance plays a role in this action of somatostatin.

Adrenocorticotropic Hormone↗
PubMed1987

Effects of reduced magnesium on hippocampal synchrony.

Spontaneous, synchronous burst discharges originating in the CA2-CA3 region of guinea pig hippocampal slices occur when the concentration of magnesium in the perfusate is reduced below 250 microM. The burst frequency is greater than that seen with other convulsants and afterdischarge is common. Cyclic periods of bursting resembling ictal discharges occur spontaneously in some slices and with repetitive stimulation (0.5-2 Hz) in most slices. The spontaneous bursts are blocked by 2-APV suggesting the involvement of NMDA receptors in their generation.

2-Amino-5-phosphonovalerate↗
PubMed1987

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2.

Dispersed brain cells from 12-14 day old mouse embryos were loaded with the Ca2+-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca2+ concentration ([Ca2+]i) of 158 nM (SE +/- 5) in the presence of 1 mM [Ca2+]o. When external [Ca2+] was raised from 0 to 1 mM there was an increase of [Ca2+]i of 70 nM; with further additions of Ca to greater than or equal to 10 mM [Ca2+]o the level of [Ca2+]i increased by less than or equal to 25 nM. Releasable intracellular Ca2+ stores, estimated from the increase in [Ca2+] produced by 4-Br A23187 in the absence of extracellular Ca2+, were 24 fmol/10(6) cells. A small increase in [Ca2+]i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K+ was raised by 10-20 mM, intracellular Ca2+ levels increased from 152 (SE +/- 7) to 204 nM (SE +/- 10). These K+-induced increases in [Ca2+]i were blocked by verapamil, did not occur in the absence of extracellular Ca2+, and presumably reflect the activation of voltage-dependent Ca2+ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca2+]i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2-aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca2+-dependent and -independent mechanisms of action of these excitatory amino acids.

Amino Acids↗
PubMed1987

Signal transduction mechanisms in cultured CNS neurons and clonal pituitary cells.

The experimental accessibility of monolayer culture has been used to study signal transduction mechanisms in primary CNS neurons and clonal pituitary cells. Here we review results on two signals representative of the emerging diversity of mechanisms discovered in all species studied thus far. One is mediated by micromolar concentrations of the amino acid GABA at postsynaptic membranes throughout the mammalian CNS and involves transient activation of Cl- ion channels whose distribution of conducting periods accounts for the millisecond time course of the signal. This signal serves to depress the probability that the target cell will trigger an action potential. The signal intensifies as the postsynaptic membrane is depolarized and can be modulated by clinically important drugs, primarily through changes in channel kinetics. The other signal involves nanomolar concentrations of the peptide TRH, which stimulates secretion of prolactin from clonal "GH3" pituitary cells. Intracellular recordings of GH3B6 cells show that TRH triggers a complex electrical response lasting several minutes. The response consists of Ca2+-activated K+ conductance followed by Ca2+-action potential activity. Whole-cell patch recordings, which rapidly dialyze the cell, can eliminate the TRH-induced changes in membrane excitability. Inclusion of aqueous lysates of the GH3B6 clone or the soluble second messenger factors inositol trisphosphate (IP3) or protein kinase (PKC) can restore various aspects of the change in membrane excitability. Thus, TRH alters ion conductance mechanisms through a second messenger cascade likely to involve IP3-mediated mobilization of Ca2+ from the endoplasmic reticulum and transient translocation of PKC from cytoplasm to plasma membrane. These synaptic and extrasynaptic signals reflect some of the diversity of transduction mechanisms involved in intercellular communication.

Amino Acids↗
PubMed1987

Intracellular calcium recordings from isolated cells of the mammalian central nervous system.

Measurements made with two different techniques of intracellular calcium levels from small isolated cells of the mammalian central nervous system are described and compared. Recordings in cultured mouse embryo spinal cord and dorsal root ganglion neurons, made with double-barrelled borosilicate Ca2+-selective microelectrodes yielded a mean Ca2+ level of 2.3 (SE +/- 0.54) microM for the lowest values recorded in 24 out of 46 cells. Intracellular Ca2+ dependence on membrane potential was apparent with levels of calcium greater than or equal to 4 microM (r = 0.371, n = 29). Both cyclic fluctuations induced by tetraethylammonium and an apparent increase in Ca2+ evoked by the depolarizing excitatory amino acid, L-aspartate, were observed. In contrast, estimates of intracellular Ca2+ obtained by spectrofluorimetry of suspensions of mouse embryo brain cells, loaded with the intracellular Ca-binding fluorescent probe, quin2 provided a approximately equal to 10-fold lower value, 152 (SE +/- 7) nM. This more closely resembles levels reported for large neurons where large-tip microelectrodes with greater sensitivity were used, and in spite of the heterogeneity of the cells this value is presumed to be a more accurate estimate of intraneuronal Ca2+ concentration. In these fluorescence studies KCl readily evoked increases in intracellular Ca2+ which could be blocked by verapamil and Cd2+ and were not induced in the absence of Ca2+. Increases were also produced by N-methyl-D-aspartate, but not by the kainate-like Lathyrus neurotoxin, L-3-oxalylamino-2-aminopropionic acid. These results provide preliminary evidence for both voltage-sensitive and receptor-activated Ca channels in embryonic brain cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Measurements of intracellular free Ca2+ in mammalian central neurons.

Neutral carrier-containing Ca2+-selective microelectrodes were used to record the cytoplasmic free Ca2+ concentration [( Ca2+]i) in spinal cells in cats and in hippocampal cells of rats (in situ). The mean [Ca2+]i in motoneurons was close to 1 microM. Antidromic or direct stimulation for 30 s at 10 Hz increased [Ca2+]i by a mean of 90 nM. Such a small increase in [Ca2+]i and its slow decay (with a mean half-time of 23 (SD +/- 14.5) s) indicate very effective intracellular sequestration of Ca2+. Orthodromic stimulation consistently evoked smaller increases in [Ca2+]i. A much larger rise of interneuronal [Ca2+]i was evoked by stimulation of dorsal roots: by contrast intra-axonal recording (in motor or sensory fibres) failed to reveal any increase in [Ca2+]i in response to stimulation at 100 Hz. In the hippocampus, presumably because of poorer recording conditions, resting values of [Ca2+]i were higher (mean 8.5 microM). Repetitive stimulation of the fimbria--commissure at 5-20 Hz for 30 Hz, had variable effects on [Ca2+]i. Very large increases (to greater than 200 microM) were elicited repeatedly in some cells, either near the end of the tetanic stimulation or after a 20-30 s delay. Such major increases, which were associated with population cell discharges in bursts, may be related to long-term changes in hippocampal neuronal properties that are evoked by tetanic stimulation. Both in the spinal cord and the hippocampus, probable intraglial recordings showed relatively high mean levels of [Ca2+]i (about 30 microM).

Animals↗
PubMed1987

Opposing actions of the enantiomers of BAY-K-8644 on calcium currents and ACTH secretion in clonal pituitary corticotrophs.

BAY-K-8644 in low concentrations is known to stimulate, and in higher concentrations, to depress calcium-dependent ACTH secretion from mouse clonal (tumor) pituitary corticotrophs, AtT-20/D16-16 (AtT-20). In the present study, voltage-dependent inward calcium currents in these cells were potentiated by low concentrations of this compound and depressed by higher concentrations consistent with its actions on ACTH secretion. A similar relationship was demonstrated for a different but related compound, CGP 28,392. Each of BAY-K-8644's enantiomers, BAY-R(-)5417 and BAY-R(+)4407, had opposing effects upon these inward calcium currents and ACTH secretion. The (+)isomer antagonized both inward calcium currents and ACTH secretion. In contrast, the (-)enantiomer was responsible for the stimulatory effects of BAY-K-8644. Nevertheless, some antagonistic properties were noted with high concentrations of this latter enantiomer. The stimulation of ACTH secretion in AtT-20 cells by low concentrations of BAY-K-8644 can be attributed to a potentiation of voltage-activated calcium currents by one of its enantiomers, BAY-R-(-)5417. In contrast, the depression of secretion that occurs at higher concentrations is likely to be the result of the reduction of these currents by the other enantiomer (BAY-R(+)4407).

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Use-dependent block of excitatory amino acid currents in cultured neurons by ketamine.

1. Mouse hippocampal neurons grown in dissociated cell culture were patch clamped using a whole cell voltage clamp (discontinuous switching clamp) technique. The currents generated by pressure applications of excitatory amino acids were studied over a wide range of holding potentials, and current-voltage curves were plotted. Excitatory amino acids that activated the N-methyl-D-aspartic acid (NMDA) receptor demonstrated some degree of desensitization with repeated applications, whereas the currents observed in response to kainic acid (KAI) did not. Desensitization could be minimized by keeping the frequency of application sufficiently low (i.e., less than 0.1 Hz). 2. The short-acting dissociative anaesthetic, ketamine (2-50 microM), selectively blocked L-aspartic acid (L-Asp), NMDA, and L-glutamic acid (L-Glu) currents while sparing those in response to KAI. Therefore, ketamine is a relatively selective blocker of the NMDA response versus that (those) activated by KAI. 3. The block by ketamine of excitatory amino acid currents is highly voltage dependent. Concentrations of ketamine that had little effect on outward current responses at depolarized potentials were quite effective at blocking inward current responses at hyperpolarized potentials. In contrast, DL-2-amino-5-phosphonovaleric acid (APV) was equally effective at blocking both inward and outward currents (voltage independent). The voltage dependence of ketamine (a positively charged molecule) could be accounted for if ketamine blocked the NMDA response by binding to a site that experienced 55% of the membrane field. 4. In the presence of ketamine, peak inward currents evoked by repeated applications of NMDA, L-Asp, or L-Glu progressively declined to a steady-state level of block (use-dependent block). This decrement occurred at frequencies much lower than those that were employed to demonstrate desensitization (in the absence of ketamine). Moving the membrane potential to depolarized values did not, in itself, relieve the ketamine block. However, if the appropriate excitatory amino acid (L-Asp, NMDA, L-Glu) was applied during the period of depolarization, a relief of the block could be demonstrated. No recovery from the blockade occurred with periods of rest (no amino acid application) as long as 5 min. Furthermore, no recovery was observed even when ketamine was washed out of the bathing solution until the appropriate agonist was applied. Thus recovery from blockade, like development of blockade, was use dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1986

Voltage-dependent block of excitatory amino acid currents by pentobarbital.

The blocking of excitatory amino acid currents by pentobarbital was studied in cultured hippocampal neurons. Peak amino acid currents were plotted against holding potential prior to and following bath applications of pentobarbital. The divalent cation Mg2+ was not added either to the recording or extracellular solution. As a consequence, current-voltage curves were approximately linear regardless of the excitatory amino acid employed. Kainic acid (KAI) currents were more sensitive to blockade by pentobarbital than were those activated by L-aspartic acid (L-Asp). However, blockade of all currents (including those for KAI) was more pronounced at hyperpolarized values than at comparable depolarized potentials (i.e. in the presence of the same driving force). Furthermore, the voltage-dependence of the pentobarbital blockade was greater for L-Asp than for KAI. Since pentobarbital is neutrally charged (or weakly negative) under the present recording conditions, the voltage-sensitivity of its block must arise from the influence of membrane field upon the site of the block rather than upon the access of the blocking molecule to this site.

Animals↗
PubMed1986

Frequency-dependent decay of calcium spikes in cultured spinal cord neurons.

Calcium-dependent action potentials were recorded from cultured (dissociated) spinal cord neurons in the presence of extracellular tetrodotoxin and tetraethylammonium in the mouse. The plateau portion of these action potentials underwent pronounced frequency-dependent decay with repetitive depolarizing current injections. Interstimulus intervals as long as 5 min were often required to ensure reproducible spike durations. This decay was not due to recurrent inhibition or intracellular calcium accumulation. It may be due to accumulation of either voltage-dependent inactivation of the calcium spike or of voltage-dependent outward currents. Any manipulation that prolonged the duration of the spike resulted in an apparent enhancement of the decay; however, this may simply be a consequence of the hyperbolic relationship between spike duration and the slope of the spike decay. It does not necessarily imply an effect on the mechanisms underlying frequency-dependent decay. Caution should, therefore, be exercised in interpreting the effects of drugs upon these calcium-dependent spikes.

Action Potentials↗
PubMed1985

Ketamine and phencyclidine cause a voltage-dependent block of responses to L-aspartic acid.

Excitatory amino acids depolarize central mammalian neurons by increasing membrane conductance. This increase in conductance can be voltage-dependent (i.e. N-methyl-D-aspartate or L-aspartic acid (L-ASP)) or voltage-independent (i.e. kainic acid (KA)) depending on whether or not the channel is blocked by Mg2+ [8,9]. Intracellular recordings were made from dissociated mouse spinal cord cells and conductance was calculated using constant current techniques. The dissociative anesthetics, ketamine and phencyclidine caused a selective depression in the change in conductance evoked by L-ASP but not that by KA. Under whole cell voltage-clamp (in the absence of extracellular Mg2+) this depression of responses to L-ASP was found to be highly voltage-dependent suggesting a blockade of the channel.

Animals↗
PubMed1985

Changes in intracellular free Ca ion concentration evoked by electrical activity in cat spinal neurons in situ.

In cats under allobarbitone anaesthesia, Ca2+-sensitive microelectrodes were inserted into the lumbosacral spinal neurons to measure intracellular free Ca2+ concentration [Ca]i. In 72 resting motoneurons, the global mean [Ca]i was 7.9 microM (SD +/- 25.9). In the 36 "best" cells (with resting and action potentials better than 60 mV), mean [Ca]i was 1.6 microM (SD +/- 1.64). Activation of motoneurons by antidromic or direct stimulation evoked mean increases in [Ca]i of about 90 nM when stimulating for 30 s at 10 Hz, and 170 nM at 20 Hz. The mean time to half-recovery was 23 s (SD +/- 14.5). Orthodromic stimulation consistently produced smaller increases in [Ca]i. Measurements in motor axons showed a comparable resting level of [Ca]i, but only minimal changes during stimulation, even at 100 Hz. Sensory axons (also recorded within the spinal cord) similarly failed to show any increase in [Ca]i during high frequency stimulation. In some interneurons, however, particularly large and rapid increases in [Ca]i could be evoked by dorsal root stimulation at 1-5 Hz. Unresponsive cells (presumably neuroglia), with a typically high and stable resting potential, had a variable [Ca]i giving a mean of 32 microM (SD +/- 63.0). A tentative theoretical analysis of the magnitude and time course of delta [Ca]i evoked in motoneurons by tetanic stimulation is consistent with remarkably slow apparent diffusion of intracellular Ca2+ (1/250 of rate of diffusion in water), such as might be expected in the presence of very efficient mechanisms of Ca2+ sequestration.

Animals↗