Marina Sciancalepore

Intracellular calcium stores modulate miniature GABA‐mediated synaptic currents in neonatal rat hippocampal neurons

The whole‐cell configuration of the patch clamp technique was used to record miniature γ‐aminobutyric acidA (GABAA) receptor‐mediated currents (in tetrodotoxin, 1 μm and kynurenic acid 1 mm) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6–9) old rats. Switching from a Ca2+‐containing to a nominally Ca2+‐free medium (in which Ca2+ was substituted with Mg2+, in the presence or in the absence of 100 μm EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration‐dependent increase in frequency but not in amplitude of tetrodotoxin‐resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 μm over control) was 1.8 ± 0.5, (n = 9). In nominally Ca2+‐free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mm), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mm) reversibly reduced the amplitude of the current induced by GABA (100 μm) indicating a clear postsynaptic effect on GABAA receptor. Superfusion of ryanodine (30 μm), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine–ryanodine‐sensitive stores facilitates GABA release.

Developmental Changes in Spontaneous GABAA-mediated Synaptic Events in Rat Hippocampal CA3 Neurons

Ongoing spontaneous postsynaptic potentials (SPSPs) were intracellularly recorded at 34‐36°C from hippocampal CA3 neurons in slices obtained from postnatal days (P) 0 – 6 and 7 – 31. SPSPs occurred randomly, and their frequency distribution was fitted by a single exponential function. They were little affected by kynurenic acid, but were reversibly blocked by bicuculline, implying that they were mediated by GABAA receptors. The mean amplitude was 4.53 ± 0.89 mV in control conditions and 4.07 ± 0.79 mV in kynurenic acid. In kynurenic acid (with CsCl‐filled microelectrodes), SPSPs reversed polarity at 2.4 ± 2 mV. When tetrodotoxin (1 μM) was added to kynurenic acid solution, GABAA‐mediated miniature postsynaptic potentials (MPSPs) were recorded. Under these conditions large events disappeared. The mean amplitude of MPSPs was 2.51 ± 0.43 mV. The mean frequency decreased from 2.96 ± 1.04 Hz in kynurenic acid to 0.4 ± 0.15 Hz in kynurenic acid plus tetrodotoxin. In contrast with P0 – P6, at P7 – P31 SPSPs were significantly affected by kynurenic acid. The mean amplitude of SPSPs shifted from 4.71 ± 0.82 mV in control conditions to 3.79 ± 0.76 mV in kynurenic acid. At this developmental stage, the reversal potential of GABAA‐mediated SPSPs shifted towards more negative values (‐23.7 ± 1.3 mV). Addition of tetrodotoxin to kynurenic acid solution abolished larger events and revealed GABAergic MPSPs. The mean amplitude of MPSPs was 2.72 ± 0.5 mV, a value very close to that observed at P0 – P6. Synaptic currents were recorded at 22 – 24°C from voltage‐clamped CA3 pyramidal neurons (at P6) using the tight‐seal whole‐cell recording technique. Cells were dialysed with CsCl solution and held at ‐70 mV. Spontaneous GABAA‐mediated miniature postsynaptic currents (MPSCs) were recorded in the presence of kynurenic acid and tetrodotoxin. The decay time of MPSCs was fitted with a single exponential and was 29 ± 3 ms. No correlation was found between the peak amplitude of individual events and their rise or decay time constant. The mean amplitude distribution of MPSCs was 12 ± 4.3 pA. In outside‐out patches from acutely dissociated CA3 hippocampal neurons, GABA (50 μM) activated single‐channel events of 24 and 35 pS conductance. Therefore one quantal current represents the simultaneous opening of five to seven GABAA receptor channels on the postsynaptic cell. These data show also that in the immediate postnatal period spontaneous glutamatergic potentials are poorly developed and start appearing towards the end of the first postnatal week concomitant with the shift of GABA from the depolarizing to the hyperpolarizing direction.

γ-Aminobutyric acidA ρ receptor subunits in the developing rat hippocampus

The RT‐PCR approach was used to estimate the expression of γ‐aminobutyric acid (GABA)A ρ receptor subunits in the hippocampus of neonatal and adult rats. All three ρ subunits were detected at postnatal day (P) 2, the ρ3 subunit being expressed at an extremely low level. The ρ1 and ρ2 products appeared to be developmentally regulated; they were found to be more pronounced in adulthood. In another set of experiments, to correlate gene expression with receptor function, GABAA ρ subunit mRNAs were detected with single‐cell RT‐PCR in CA3 pyramidal cells (from P3–P4 hippocampal slices), previously characterized with electrophysiological experiments for their bicuculline‐sensitive or ‐insensitive responses to GABA. In 6 of 19 cells (31%), pressure application of GABA evoked at –70 mV inward currents that persisted in the presence of 100 μM bicuculline (314 ± 129 pA). RT‐PCR performed in two of these neurons revealed the presence of ρ1 and ρ2 subunits, the latter being present with the α2 subunit. A ρ2 subunit was also found in 1 neuron (among 9) exhibiting a response to GABA, which was completely abolished by bicuculline. This might be due to the lack of putative accessory GABAA subunits that can coassemble with ρ2 to make functional receptors. Similar experiments from 10 P15 CA3 pyramidal cells failed to reveal any ρ1–3 transcripts. However, these neurons abundantly express α3 subunits. It is likely that in CA3 pyramidal cells of neonatal and adult hippocampus GABAA ρ subunits are present but at very low levels of expression.

Ionic permeability of the mitochondrial outer membrane

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM)), derived from mouse liver, showed the presence of currents corresponding to low conductances (< 50 pS), as well as of four distinct conductances of 99 pS,152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).

Changes of N-methyl-D-aspartate activated channels of cerebellar granule cells with days in culture

N-Methyl-D-Aspartate (NMDA) activated channels were studied in enzymatically dissociated cerebellar granule cells primary cultures. Measurements of single channel currents were made on different days in culture. Changes in the electrophysiological behavior of NMDA-activated channels, which were dependent on the time in culture, were found. The variations of single channel maximum conductance during the developement of the cells in culture were detected. Three different characteristic periods could be distinguished: the first period (1-3 days) in which the conductance assumed a value of 15.5 pS; the second one (5-8 days) characterized by a value of 35.7 pS and the last one (9-11 days) in which the conductance reached values of 46.8 pS. Moreover mean open time of NMDA-activated channels was less than 1 msec during the first two days in culture and stabilized at 3 to 6 msec around the fifth day.

Protein kinase A-dependent increase in frequency of miniature GABAergic currents in rat CA3 hippocampal neurons☆

The whole-cell configuration of the patch clamp technique was used to study the effect of an intracellular increase in cAMP on the frequency of GABA-mediated miniature post synaptic currents (MPSCs) in neonatal rats from (P6 to P12) CA3 hippocampal neurons in slices. In the presence of tetrodotoxin (1 microM), and kynurenic acid (1 mM) to block ionotropic glutamatergic currents, forskolin, an activator of adenylate cyclase, markedly increased the frequency of MPSCs without affecting their amplitude or kinetics. The inactive forskolin analog, 1,9-dideoxyforskolin (30 microM), had no effect on the frequency of MPSCs. The effect of forskolin was prevented by the specific protein kinase A (PKA) antagonist Rp-cAMP (30 microM). It is concluded that stimulation of PKA potentiates spontaneous GABA release in immature hippocampal neurons.

Voltage dependent calcium channels in cerebellar granule cell primary cultures

Voltage activated calcium channels were studied in rat cerebellar granule cells in primary culture. Macroscopic currents, carried by 20 mM Ba2+, were measured in the whole-cell configuration. Slowly inactivating macroscopic currents, with a maximum value at a membrane potential around 5 mV were recorded between the 1st and the 4th day in culture. These currents were completely blocked by 5 mM Co2+ , partially blocked by 10 μM nifedipine, and increased by 2 to 5 μM BAY K-8644. Two types of channels, in the presence of 80 mM Ba2+, were identified by single channel recording in cell-attached patches. The first type, which was dihydropyridine agonist sensitive, had a conductance of 18 pS, a half activation potential of more than 10 mV and did not inactivate. This type of channel was the only type found during the first four days in culture, although it was also present up to the 11th day. The second type of channel was dihydropyridine insensitive, had a conductance of 10 pS, a half activation potential less than −15 mV and displayed voltage dependent inactivation. This second type of channel was found in cells for more than four days in culture.

Kainate activated single channel cnrrents as revealed by domoic acid

We have studied the properties of kainic acid receptor-activated channels using domoic acid as an agonist. Similarities of the electrophysiological, pharmacological and noise properties of domoic acid and kainic acid-evoked currents confirm that domoate is a potent and specific agonist of the kainate receptor. Single-channel properties of domoic acid-evoked currents were directly determined from outside-out membrane patches for the first time, and results were compared with those obtained by fluctuation analysis of macroscopic currents. Small conductance cationic-selective channels of ≈4 pS and a mean open time of 2 to 3 ms were detected using both methods.

Patch-clamp study of neurones in rat olfactory cortex slices: properties of a slow post-stimulus afterdepolarizing current (IADP)

WHOLE-CELL recordings were made from neurones in slices of rat olfactory cortex (10–19 days old), using potassium methylsulphate-filled pipettes. Positive commands applied from —60 mV in the presence of oxotremorine-M (10–20 μM, 20 of 30 cells) or trans-ACPD (10–50μM, 4 of 9 cells) evoked a slow inward tail current similar to the K+-mediated tail current (IK,ADP) recorded with sharp electrodes. IADP was reduced by hyperpolarization, showed a 27% decrease in input conductance at its peak and was depressed by Cd2+, 4AP or high K+ but unaffected by Cs+, Ba2+ or TEA (5mM). IADP was significantly larger in cells between postnatal days 13 and 15 than in younger neurones (10–12 days). These data show that stable whole-cell recordings of IADP can be made from olfactory neurones in cortical slices, and support our hypothesis that IADP is a slowly reactivating, Ca2+-sensitive K+ conductance.

Single-channel currents of NMDA type activated by l- and d-homocysteic acid in cerebellar granule cells in culture

The whole-cell and the outside-out configurations of the patch-clamp technique were used to study macroscopic and single-channel currents evoked by the enantiomers of homocysteic acid, the sulphur containing analogue of glutamate, in cerebellar granule cells in culture. L-Homocysteic acid (L-HC, 15 microM) and D-homocysteic acid (D-HC, 50 microM) induced whole-cell currents of comparable amplitude, that were abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (AP-5) (20 microM). AMPA (100 microM) induced whole-cell currents which were not modified by AP-5 (20 microM) but were blocked by CNQX. In the outside-out configuration, both L-HC and D-HC (15 microM) elicited single-channel currents of the same conductance, mean open time and reversal potential as the NMDA-induced single-channel events. In the presence of Mg2+ (2 mM), D- and L-HC-induced single-channel currents were voltage-dependent. These data suggest that in cerebellar granule cells in culture, both L-HC and D-HC activate the same NMDA receptor channel complex.