Whole cell calcium currents in acutely isolated olfactory bulb output neurons of the rat.

1. Voltage-gated whole cell Ca2+ currents have been investigated in olfactory bulb (OB) output neurons acutely isolated from neonatal rats. 2. Identification of OB output neurons, mitral or tufted cells, was based on morphology and size and validated by their retrograde labeling with rhodamine or Fast Blue. Of labeled neurons, 45% exhibited either phasic or nonphasic spontaneous firing that was blocked by 10(-7) M tetrodotoxin, 0.5 mM Cd2+, or 1 mM Co2+ in the bathing solution. 3. Whole cell Ca2+ currents displayed holding potential sensitivity indicative of low voltage-activated (LVA) and high voltage-activated (HVA) currents, which exhibited similar dependence on extracellular Ca2+ concentration and could be completely abolished by bathing in 500 microM Cd2+ or in Ca(2+)-free solution. 4. A T-type LVA Ca2+ current, detected in 65% of OB output neurons tested, was activated by depolarizing to -57 mV from holding potential -86 mV and fully inactivated at holding potentials more positive than -60 mV. It was permeated equally by 2.6 mM Ca2+, Sr2+ and Ba2+. The half-activation potential was -35 mV with a slope factor of 7 mV. Depolarizing to -26 mV from different holding potentials in a 2.6-mM Ca2+ solution gave a steady-state half-inactivation potential of -82 mV with a slope factor of 10.7 mV. This LVA current was not sensitive to 5 microM omega-conotoxin (omega-CgTx) or 5 microM Bay K 8644 and was resistant to block by 30 microM Cd2+, by 50 microM verapamil or by 5 microM nifedipine. 5. HVA Ca2+ currents, detected in 97% of OB output cells, activated at around -30 to -20 mV, with maximum peak current at approximately 4 mV in 2.6 mM Ca2+ external solution. They showed similar permeability to 2.6 mM Ca2+ and Sr2+, but the maximum peak current was increased 40% in 2.6 mM Ba2+. Depolarizing to 4 mV from different holding potentials yielded a half-inactivation potential of -67 mV with a slope factor of 13.2 mV. Two components, as suggested by their sensitivities to 5 microM Bay K 8644, nifedipine. omega-CgTx and to voltage, may resemble the L-type and N-type currents described in other neural preparations. However, 5 microM omega-CgTx seemed to block both components, being more effective at more positive potentials. There was a residual component of Cd(2+)-sensitive current not affected by cumulative addition of nifedipine and omega-CgTx. 6. omega-Agatoxin IVA (omega-Aga), a selective P-type Ca2+ channel blocker, had no detectable effect at 50 or 200 nM and 1 microM doses on whole cell Ca2+ currents elicited by 200-ms voltage steps to 4 mV from holding potential -86 mV. 7. We conclude that both LVA and HVA Ca2+ currents exist in neonatal rat OB output neurons, showing distinct kinetic and pharmacological characteristics. The HVA Ca2+ currents contain at least two components, probably resembling L- and N-type currents. Another fast-inactivating HVA component, insensitive to nifedipine, omega-CgTx and omega-Aga, could represent the newly established R-type Ca2+ current.

Whole-cell K+ currents in identified olfactory bulb output neurones of rats.

1. Voltage-gated whole-cell K+ currents have been investigated in olfactory bulb (OB) output (mitral/tufted) neurones from neonatal rats, which were retrogradely labelled by rhodamine or Fast Blue and identified after enzymatic dissociation. Forty-five per cent of labelled neurones exhibited either phasic or non-phasic spontaneous firing in cell-attached configuration. 2. Four outward K+ currents have been identified in all such identified OB output neurones. They are the transient (IA), the delayed rectifier (IDK), and two Ca(2+)-dependent (IK(Ca)) currents. No inward rectifier was detected. 3. The IA was activated at around -45 mV and reached its peak within 3-10 ms. The decay phase could be described by single exponential distribution with the time constant of 45.2 +/- 3.8 ms at depolarizations 10-60 mV from a holding potential of -70 mV. Its activation and steady-state inactivation processes could be fitted with Boltzmann equations yielding half-maximal activation potentials of 7.6 +/- 0.4 and -47.4 +/- 0.2 mV, respectively. It was sensitive to block by 4-AP (1 mM) and by Zn2+ (1 mM). 4. The IDK was activated at potentials more positive than -30 mV, with half-maximal activation at 21 mV. It was sustained during 1 s test pulses without apparent decay. It was blocked by TEA at a concentration of 20 mM. About 8% of the sustained current, in 11/24 cells tested, was found to resist block by a combination of all pharmacological agents tested. 5. Apamin at 100 nM blocked a TEA-insensitive component which accounted for about 23% of the maximal sustained currents. Iberiotoxin (IbTX), which has been found to block maxi K+ currents more selectively than does charybdotoxin, reversibly blocked Ca(2+)-activated K+ current, with a half-maximal dose of about 100 nM in 8/13 OB output neurones tested. This accounted for 20% of the maximal sustained K+ current. The effect of IbTX was not observed in the presence of 20 mM external TEA. 6. Direct evidence is provided in this study regarding kinetic and pharmacological properties of four types of outward K+ channels in OB output neurones.

Ba(2+)-sensitive K+ channels in basal membrane of confluent Madin-Darby canine kidney cells.

A method is described for gaining access to the basolateral membranes of confluent Madin-Darby canine kidney (MDCK) cells by surgical reflection of the cell layer overlying fluid-filled domes. Single-channel recordings from cell-attached inside-out and outside-out configurations revealed two K+ channels located in the basal membranes of the highly differentiated monolayers. With 140 mmol/l KCl in pipette, the intermediate-conductance K+ channel displayed outward rectification in cell-attached configuration with channel conductances of 65 pS for outward part and 17 pS for inward part. In excised-patch recording, this channel had a conductance of 92 pS with 140 mmol/l KCl on the extracellular side of the patch and 5 mmol/l KCl on the cytosolic side. The maximum conductance obtained in symmetrical KCl (140 mmol/l) solution was 140 pS. Ba2+ (1 mmol/l) and tetraethylammonium (5 mmol/l) blocked this channel reversibly. Channel open probability (Po) was reduced from 0.41 at cytosolic pH 7.4 to 0.14 at pH 6.8 and increased to 0.64 at pH 8.0. The channel activity was significantly inhibited by elevation of intracellular Ca2+. A small-conductance K+ channel was also observed mainly in excised patches with single-channel conductance of 48 pS in symmetrical KCl solutions. However, the activity of this channel was partially obscured by the intermediate-conductance K+ channel and further analysis was not possible. A physiological role of these channels in mediating K+ recycling through the monolayer is suggested.

P-glycoprotein expression in classical multi-drug resistant leukaemia cells does not correlate with enhanced chloride channel activity.

1. P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump responsible for classical multi-drug resistance (MDR). 2. Pgp is part of a supergene family of membrane transport proteins that includes the cystic fibrosis gene product. 3. Transfection of cells with the MDR1 gene has been previously shown to generate volume-regulated chloride channel activity in association with Pgp expression. 4. We have used whole-cell patch clamping to examine the drug-sensitive T lymphoblastic cell line CEM-CCRF and its classical MDR derivative CEM/VLB100. The results suggest that expression of Pgp is not associated with increased chloride channel activity in this multi-drug resistant cell line. 5. We were unable to confirm previously reported results in MDR1 transfected cell lines that suggested that Pgp was associated with the presence of volume-regulated chloride channels.

Olfactory bulb output neurons excited from a basal forebrain magnocellular nucleus.

We present intracellular data which demonstrates a unique facilitatory centrifugal influence on the output cells of the olfactory bulb; the source being the lateral component of the nucleus of the horizontal limb of the diagonal band (HDB), part of the basal forebrain magnocellular complex. Damage to this facilitatory HDB influence may explain the loss of olfactory sensitivity seen early in Alzheimer's disease in which pathological changes occur in the basal forebrain.

Effect of stimulating the nucleus of the horizontal limb of the diagonal band on single unit activity in the olfactory bulb.

The effects of centrifugal afferents on single unit discharge in the main olfactory bulb were studied in anaesthetized rats. Recording with extracellular micropipettes revealed spontaneous firing in all bulb layers. Units were located to different laminae using evoked field-potential profiles and histological verification. Output neurons were identified by antidromic response to stimulation of the lateral olfactory tract. Single- or brief multiple-pulse stimulation in the nucleus of the horizontal limb of the diagonal band, but not in adjacent regions, facilitated 17 out of 27 mitral cells with no effect on 10, but inhibited 21 out of 33 granule cell layer units with no effect on 12. Of 13 presumed tufted cells, six were facilitated and the rest unaffected. In contrast, stimulation of olfactory cortex inhibited mitral cells and facilitated most granule layer cells. The results are consistent with an inhibition of tonic granule cell discharge by the horizontal diagonal band nucleus, with resultant disinhibition of mitral cells via the dendrodendritic synapses of granule cells on mitral cell secondary dendrites.

Some properties of spinal gamma-motoneurones in the cat, determined by micro-electrode recording.

1. Micro-electorde recordings were made from motoneurones in the lumbo-sacral region of the cat spinal cord whose axonal conduction velocities were 10--55 m/sec. Most of these may be presumed to be fusimotor in function. 2. Intracellular records from twelve gamma-motoneurones revealed six with short (2--4 msec) and six with long (30--100 msec) duration after-hyperpolarizations following an antidromically conducted action potential. 3. Using extracellular recording, the excitability of eighty-nine other gamma-motoneurones following an antidromic impulse was tested with a second antidromic action potential. In eighty-four of these neurones, the minimum antidromic response interval was short, 1.5--3.5 msec, implying that in most gamma-motoneurones, after-hyperpolarization was of limited effectiveness and of short duration. In the remaining five neurones, the minimum response interval was longer, 20--80 msec. 4. There was a lack of monosynaptic excitation from group 1 afferent axons in the dorsal roots in eleven of the twelve motoneurones from which intracellular records were obtained. Polysynaptic excitation was commonly observed. 5. In these anaesthetized preparations, there was a lack of recurrent i.p.s.p.s even though such evidence of Renshaw inhibition could be found in the neighbouring alpha-motoneurones. 6. The mean input resistance of gamma-motoneurones was shown to be 1.55 Momega and the principal time constant 8.5 msec by passing hyperpolarizing current through the recording micro-electrode in a bridge circuit. These values are open to error because of the small numbers of neurones investigated, and of the use of the single micro-electrode method. 7. Depolarizing current passed through the recording micro-electrode caused a maintained discharge of action potentials at a high rate. After-hyperpolarization had little effect on discharge rate. The threshold for injected current to cause discharge was very low, and the discharge rate increased rapidly with the magnitude of the current. 8. These properties of gamma-motoneurones arediscussed in relation to their function.