Malcolm S. Brodwick

Effects of nystatin on membrane conductance and internal ion activities inAplysia neurons

SummaryTwo methods were used to study effects of the antibiotic, nystatin, on giant neurons ofAplysia. In the first method the effects of various concentrations of nystatin on the current-voltage relationship were evaluated at a fixed time after exposure to the antibiotic using a two-microelectrode voltage clamp. Nystatin increased membrane conductance in a dose-dependent manner. The dose-response relation was very steep, with little or no effect below 15 mg/liter and an effect too large to measure at concentrations greater than 30 mg/liter. Upon return to antibiotic-free solution, membrane conductance returned to pre-treatment levels within 30 minutes. The second type of experiment involved use of ion-specific microelectrodes to measure changes of intracellular univalent ion activities which attended the nystatin-induced permeability. meability was also increased. Nystatin may therefore be used to selectively rearrange the internal ionic milieu to study the effect of such a change on membrane tranpsort or electrical properties.

The Na+/H+ exchanger is a major pH regulator in GABAergic presynaptic nerve terminals synapsing onto rat CA3 pyramidal neurons

The effects of pHi on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were studied in mechanically dissociated CA3 pyramidal neurons, by use of ammonium prepulse and whole‐cell patch‐clamp techniques, under the voltage‐clamp condition. NH4Cl itself, which is expected to alkalinize pHi, increased GABAergic mIPSC frequency in a concentration‐dependent manner. In contrast, NH4Cl decreased mIPSC frequency, either in the presence of 200 µm Cd2+ or in Ca2+‐free external solution, suggesting that intraterminal alkalosis decreased GABAergic mIPSC frequency while [NH4+] itself may activate Ca2+ channels by depolarizing the terminal. On the other hand, GABAergic mIPSC frequency was greatly increased immediately after NH4Cl removal, a condition expected to acidify pHi, and recovered to the control level within 2 min after NH4Cl removal. This explosive increase in mIPSC frequency observed after NH4Cl removal was completely eliminated after depletion of Ca2+ stores with 1 µm thapsigargin in the Ca2+‐free external solution, suggesting that acidification increases in intraterminal Ca2+ concentration via both extracellular Ca2+ influx and Ca2+ release from the stores. However, the acidification‐induced increase in mIPSC frequency had not recovered by 10 min after NH4Cl removal either in the Na+‐free external solution or in the presence of 10 µm 5‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIPA), a specific Na+/H+ exchanger (NHE) blocker. The present results suggest that NHEs are major intraterminal pH regulators on GABAergic presynaptic nerve terminals, and that the NHE‐mediated regulation of pHi under normal physiological or pathological conditions might play an important role in the neuronal excitability by increasing inhibitory tones.

Sustained depolarizing shift of the GABA reversal potential by glutamate receptor activation in hippocampal neurons

The inhibitory action of GABA is a consequence of a relatively hyperpolarized Cl(-) reversal potential (E(Cl)), which results from the activity of K(+)-Cl(-) cotransporter (KCC2). In this study we investigated the effects of glutamate and glutamatergic synaptic activity on E(Cl). In dissociated culture of mature hippocampal neurons, the application of glutamate caused positive E(Cl) shifts with two distinct temporal components. Following a large transient depolarizing state, the sustained depolarizing state (E(Cl)-sustained) lasted more than 30 min. The E(Cl)-sustained disappeared in the absence of external Ca(2+) during glutamate application and was blocked by both AP5 and MK801, but not by nifedipine. The E(Cl)-sustained was also induced by NMDA. The E(Cl)-sustained was blocked by furosemide, a blocker of both KCC2 and NKCC1, but not bumetanide, a blocker of NKCC1. On the other hand, in immature neurons having less expression of KCC2, NMDA failed to induce the sustained depolarizing E(Cl) shift. In organotypic slice cultured neurons, repetitive activation of glutamatergic afferents also generated a sustained depolarizing E(Cl) shift. These results suggest that Ca(2+) influx through NMDA receptors causes the down-regulation of KCC2 and gives rise to long lasting positive E(Cl) shifts, which might contribute to hyperexcitability, LTP, and epileptiform discharges.

Effects of osmotic stress on mast cell vesicles of the beige mouse

SummaryThe large size of the vesicles of beige mouse peritoneal mast cells (4 μm in diameter) facilitated the direct observation of the individual osmotic behavior of vesicles. The vesicle diameter increased as much as 73% when intact cells were perfused with a 10 mM pH buffer solution; the swelling of the vesicle membranes exceeded that of the insoluble vesicle gel matrix, which resulted in the formation of a clear space between the optically dense gel matrix and the vesicle membrane. Hypertonic solutions shrank intact vesicles of lysed cells in a nonideal manner, suggesting a limit to the compressibility of the gel matrix. The nonideality at high osmotic strengths can be adequately explained as the consequence of an excluded volume and/or a three-dimensional gel-matrix spring. The observed osmotic activity of the vesicles implies that the great majority of the histamine known to be present is reversibly bound to the gel matrix. This binding allows vesicles to store a large quantity of transmitter without doing osmotic work. The large size of the vesicles also facilitated the measurement of the kinetics of release as a collection of individual fusion events. Capacitance measurements in beige mast cells revealed little difference in the kinetics of release in hypotonie, isotonic, and hypertonic solutions, thus eliminating certain classes of models based on the osmotic theory of exocytosis for mast cells.

Tea blocks potassium current in squid axon

Abstract 1. 1. Internal perfusion of squid axon with an infusion of tea (orange pekoe and pekoe) caused an irreversible loss of voltage-activated potassium conductance ( g K ) in squid axon. 2. 2. Internal application of tannic acid similarly reduced g K , while theophylline had little effect on potassium currents. 3. 3. Both tea and tannic acid reduced sodium inactivation.

On the time course of surgically induced compensatory muscle hypertrophication of the rat plantaris muscle

1. The time course of the hypertrophy and hyperplasia of the rat plantaris muscle was determined from measurements of total muscle mass and cross-section analysis of fixed muscle. 2. Muscle enlargement was induced by the surgical removal of the plantaris synergist muscles, the gastrocnemius and the soleus. 3. From the date of surgery through the third post-operative week, muscle enlargement is due to fiber hypertrophication (approximately 100% increase in diameter). After post-operative week three, muscle enlargement is due to a combination of hyperplasia and hypertrophy. At week four the cross-sectional areas return to control values. 4. The neuromuscular junction area was determined by measuring Karnovsky stained post-synaptic membrane. Only a modest 10-30% increase was noted at weeks 2 and 3 with a return to control levels at week 4. The differences were not statistically different.