Slices Of Guinea-pig Olfactory Cortex Research Articles

<b>THE CYTOCHROME REDOX STATE IN GUINEA-PIG CORTICAL BRAIN SLICES ELICITED BY TETANIC </b><b>STIMULATION </b>

The relationship between the production and consumption systems of energy-rich phosphates (ATP, adenosine triphosphate) was investigated in slices of guinea-pig olfactory cortex by an electrical method and spectrophotometry. Potentials from brain slices were evoked by the orthodromic and repetitive stimulation of the lateral olfactory tract. The changes in the evoked potential amplitudes induced by various modes of electrical stimulus frequency and duration, were regarded as the consumptive index of ATP. On the other hand, the nervous tissues became hypoxic when ATP was aerobically produced during the acceleration of oxygen consumption in mitochondrial oxidative phosphorylation process. This hypoxic state caused the redox state of the cytochromes to shift to the reduction side, so the shift was thought to reflect the kinetics of the production of bioenergic metabolites

Muscarinic inhibition of excitatory neurotransmission in guinea-pig olfactory cortex slices: Weak antagonism by M 3-muscarinic receptor antagonists

Dose-dependent depression of the electrically evoked surface-negative field potential (N-wave) produced by bath-superfusion of carbachol was measured in guinea-pig olfactory cortex slices maintained in vitro. The possible involvement of M 3 (smooth muscle/glandular) type muscarinic receptors in partly mediating this response was investigated by testing the effectiveness of the muscarinic M 3 receptor antagonists hexahydro-sila-difenidol (HHSiD) and p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD). Low doses of HHSiD (10–100 nM) or p-F-HHSiD (up to 1 μM), pre-applied for 30 min, produced no obvious antagonism of carbachol responses. However, a clear competitive-type inhibition of carbachol effects was observed in 250 nM-1 μM HHSiD or 10–50 μM p-F-HHSiD respectively. Schild plot analysis (regression slope constrained to unity) of pooled data yielded pA 2 values of 6.6 for HHSiD (n = 6 slices) and 5.5 for p-F-HHSiD (n = 6 slices) respectively, suggesting a weak competitive antagonism by both compounds. In addition, combination experiments using either HHSiD or p-F-HHSiD with atropine, produced dose-ratio shifts close to those predicted for two antagonists competing for a common receptor site. By comparison, another suggested M 3-receptor antagonist, 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) was a potent competitive blocker of carbachol responses. Schild analysis for 4-DAMP versus carbachol gave a pA 2 of 7.9 (n = 6 slices). It is concluded that the muscarinic receptors involved in the suppression of the olfactory cortical N-wave possess a low affinity for HHSiD and particularly for p-F-HHSiD, but not 4-DAMP. We therefore suggest that M 3 type receptors may not be significantly involved in mediating this muscarinic response.

Carbachol-induced suppression of the surface N-wave in guinea-pig olfactory cortex slices: Weak antagonism by M3-selective muscarinic antagonists, HHSiD and p-F-HHSiD

Carbachol-induced suppression of the surface N-wave in guinea-pig olfactory cortex slices: Weak antagonism by M3-selective muscarinic antagonists, HHSiD and p-F-HHSiD

Effects of coenzymes Q 2 and Q 10 on the field potential of guinea pig olfactory cortex slices maintained in hypoxia

The effects of exogenous CoQ 2 and CoQ 10 on field potentials were studied while brain slice preparations of guinea pig olfactory cortex were incubated in hypoxia. The potential amplitude, which diminished in hypoxia, recovered when the slices were superfused with solutions containing CoQ 2 and CoQ 10. Adenosine triphosphate (ATP) levels in the slices duplicated the experimental results of the potential amplitude. The effects of these ubiquinones on potentials measured in hypoxia were suppressed when the slices were incubated with a barbiturate solution.

Muscarinic suppression of the evoked N-wave by oxotremorine-M-recorded in the guinea-pig olfactory cortex slice

The effect of the muscarinic agonist oxotremorine-M has been studied on the surface-negative field potential (N-wave) evoked by orthodromic stimulation of the lateral olfactory tract in slices of guinea-pig olfactory cortex. Bath-application of oxotremorine-M (5–80 μM) or carbachol (10–300 μM) produced a reversible depression of the N-wave amplitude without affecting the lateral olfactory tract compound action potential, Oxotremorine-M was ∼ 5 times more potent than carbachol in this respect, and the effects of both agonists were competitively blocked by telenzepine (5–100 nM), a selective M 1-receptor antagonist. In contrast, methoctramine or AF-DX 116, two ‘cardioselective’ M 2-receptor antagonists, had little or no blocking effect on the agonist responses, it is suggested that oxotremorine-M (like carbachol) inhibits the evoked field potential by activating presynaptic M 1-type muscarinic receptors in the olfactory cortex slice.

Properties of K-currents in unmyelinated presynaptic axons of brain revealed by extracellular polarisation

Thin pial surface slices of guinea-pig olfactory cortex contain unmyelinated axons derived from the lateral olfactory tract (LOT). The severed ends of the groups of these axons were drawn into a suction electrode to record the currents resulting from propagated action potentials. On stimulating these unmyelinated axons, a tetrodotoxin-sensitive positive current of22.5 ± 3.0nA was recorded by the suction electrode. The positive current was often followed by a small negative current. A 100 mV positive polarisation applied to the suction electrode revealed a large negative after-current (25.8 ± 3.5nAs. In contrast, the positive current was slightly reduced by the polarisation. The early phases of the negative after-current induced by the electrode polarisation were blocked by 3,4-diaminopyridine, 4-aminopyridine or 2,3-diaminopyridine (in order of potency). The entire negative after-current was blocked by prolonged (3 h) equilibration in a medium containing 3.5 mmol/l Cs and 1.5 mmol/l K. Tetraethylammonium (TEA) or Ba 2+ by themselves had little effect. In aminopyridine, the residual negative after-current was blocked by TEA (10 mmol/l) or 1 mmol/l Ba 2+. Muscarinic agonists had no effect on these currents. These experiments show that some axonal currents can be revealed by extracellular polarisation and that these axons rely on an ‘A’ type of current for the rapid repolarisation of the membrane although slower K-channels are present.

A phorbol diester‐induced enhancement of synaptic transmission in olfactory cortex

1. Extracellular field synaptic potentials were recorded from pial surface slices of guinea-pig olfactory cortex maintained in vitro. 2. Phorbol 12,13-dibutyrate (0.1-10 microM) enhanced the amplitude of the evoked potential (by 51.2 +/- 10.4% with 1 microM) in normal solution. When the evoked potential was partially depressed by Cd, Co, Mn or a reduced Ca concentration, phorbol 12,13-dibutyrate (1 microM) induced a much larger enhancement of the evoked potential (196.5 +/- 24.4% increase). Phorbol 12,13-diacetate and mezerein had similar effects but were less potent. 4 beta-Phorbol (10 microM) had no effect. 3. The diacylglycerol analogues, dioctanoylglycerol (100-1000 microM), 1-oleoyl-2-acetylglycerol (100-500 microM) or diolein (100 microM) had no effect on the evoked potentials, either alone or in the presence of Cd. 4. The isoquinolinylsulphonamide inhibitor (H-7) of protein kinase C slightly enhanced the e.p.s.p. and had no effect on the potentiation produced by phorbol ester. Another protein kinase C inhibitor, acridine orange (100-1000 microM), had no effect on the action of phorbol ester. 5. These results show that transmitter release, as at other synapses, is enhanced by phorbol esters but Ca did not potentiate this action. The pharmacological profile of the effect on transmitter release differed from that of protein kinase C in cell-free preparations and therefore it is unclear whether protein kinase C was involved in the present study.

A quantitative study of the effects of some muscarinic antagonists on the guinea-pig olfactory cortex slice.

1. Muscarinic depression of the electrically-evoked surface-negative field potential (N-wave) was measured in guinea-pig olfactory cortex slices maintained in vitro. 2. The effects of three muscarinic receptor antagonists, pirenzepine, atropine and gallamine on this muscarinic response were analysed in detail. 3. Pirenzepine was a potent competitive antagonist of carbachol (CCh)-evoked responses. Schild plot analysis yielded a pA2 value of 7.9 (Schild slope constrained to unity). A similar analysis for atropine versus CCh responses gave a pA2 of 8.9. 4. Combination experiments using pirenzepine and atropine produced dose-ratio shifts close to those expected for two antagonists competing for a similar receptor site. 5. Gallamine was only a weak antagonist of responses to CCh. 6. Oxotremorine behaved as a competitive antagonist at this muscarinic receptor (pA2 = 6.1). 7. It is concluded that the presynaptic muscarinic receptor mediating depression of the N-wave in the olfactory cortex slice is of the M1-subtype.

Quantitative effects of some muscarinic agonists on evoked surface‐negative field potentials recorded from the guinea‐pig olfactory cortex slice

1. The effects of muscarinic receptor agonists on the electrically-evoked surface-negative field potential (N-wave) were measured in the guinea-pig olfactory cortex slice maintained in vitro. 2. Bath-superfusion of (+/-)-muscarine, acetylcholine (ACh), carbachol (CCh), or methacholine (MCh) (10-200 microM) produced reversible, dose-dependent depressions of the N-wave (ACh and MCh effects were observed in the presence of 10 microM neostigmine). The order of potencies (based on agonist dose causing 50% field depression: IC50) was: ACh greater than or equal to muscarine greater than CCh greater than MCh. All four agonists depressed the field potential by 100% at doses greater than 500 microM. 3. Pilocarpine and bethanechol were weak agonists and only produced measurable effects at high doses (1-2 mM). Neither agonist evoked a maximum response at doses up to 10 mM. 4. The muscarinic ganglion stimulant, McN-A-343 yielded inconsistent results, depressing the field potential in some slices, but having no effect in others. Pre-application of a conditioning dose (100 microM) of McN-A-343 reduced subsequent responses to CCh, suggesting possible partial agonist properties. 5. Oxotremorine (up to 100 microM) did not depress the field potential, but it reversibly antagonized the effects of CCh. 6. It is concluded that reproducible, quantifiable responses to muscarinic agonists can be evoked in the olfactory cortex slice. We suggest this preparation may be useful for conducting pharmacological studies of 'intact' central muscarinic receptors.

Presynaptic Na/Ca action potentials in unmyelinated axons of olfactory cortex.

(1) Pial surface slices of guinea-pig olfactory cortex were cut to have a thickness of 150 micron. Action potentials were recorded from the sectioned ends of the unmyelinated afferent axons originating from the lateral olfactory tract (LOT). These potentials were prolonged by the K-channel blocker 3,4-diaminopyridine (0.1 mmol/l) and further lengthened by tetraethylammonium (10 mmol/l). The action potential was also greatly prolonged by partly replacing the K+ in the bathing solution by Cs+. (2) These prolonged action potentials were shortened by Cd2+; Gd3+ (gadolinium); Ni2+; Mn2+; Co2+, in order of potency. The residual early component of the action potential was tetrodotoxin (TTX) sensitive. In contrast, the LOT action potential was little affected by Ca-channel blockade. (3) Organic Ca-channel blockers either had no effect (0.05 mmol/l nifedipine), or depressed the early and later phases of the prolonged action potential equally (0.05-0.5 mmol/l verapamil or 0.05-0.2 mmol/l diltiazem). (4) A propagated action potential was also obtained in solution containing TTX and low Na+. This potential was supported by Ca2+, Sr2+ or Ba2+ and completely suppressed by Cd2+. (5) The later parts of the action potential, after K-channel blockade, had a pharmacological sensitivity towards Ca-channel blockers matching that of synaptic transmission. This suggests the falling phase of the action potential is caused by charge carrier (mainly Ca2+) passing through Ca-channels that have similar properties to, or are the same as those which open prior to transmitter release.

Cellular mechanisms of potassium homeostasis in the mammalian nervous system.

Double-barrelled ion-sensitive microelectrodes were used to measure changes in the intracellular activities of K+, Na+, and Cl- (aKi, aNai, aCli) in neurones of rat sympathetic ganglia and in glial cells of slices from guinea-pig olfactory cortex. In sympathetic neurones, carbachol and gamma-aminobutyric acid (GABA) produced a reversible decrease of aKi. The decrease of aKi during carbachol was accompanied by a rise of aNai, whereas in the presence of GABA decreases of aKi and aCli were seen. The reuptake of K+ released during the action of carbachol was completely blocked by ouabain, whereas furosemide inhibited the aKi recovery after the action of GABA. In glial cells, in contrast to the observations in the sympathetic neurones, aKi and aCli increased, whereas aNai decreased when neuronal activity was enhanced by repetitive stimulation of the lateral olfactory tract. It was found that barium ions and ouabain strongly reduced the activity-related rise of intraglial aKi in slices of guinea-pig olfactory cortex. These data show that mammalian neurones as well as glial cells possess several K+ uptake mechanisms that contribute to potassium homeostasis. Ouabain, furosemide, and Ba2+ are useful pharmacological tools to separate these mechanisms.

Ion activities and potassium uptake mechanisms of glial cells in guinea‐pig olfactory cortex slices.

1. Double-barrelled ion-sensitive micro-electrodes were used to measure changes in the intracellular activities of K+, Na+ and Cl- (aiK, aiNa, aiCl) in glial cells of slices from guinea-pig olfactory cortex during repetitive stimulation of the lateral olfactory tract. 2. Base-line levels of aiK, aiNa and aiCl were about 66, 25 and 6 mM, respectively, for cells with resting potentials higher than -80 mV. During stimulation, intraglial aiK and aiCl increased, whereas aiNa decreased. Within about 2 min after stimulation the ion activities returned to their base-line levels. 3. The Cl- equilibrium potential was found to be close to the membrane potential (Em). There was also a strong correlation between changes of Em and aiCl. These observations indicate a high Cl- conductance of the glial cell membrane. 4. In the presence of Ba2+, the usual depolarizing response of the glial cells to a rise of the extracellular K+ activity (aeK) reversed into a membrane hyperpolarization. Furthermore, Ba2+ strongly reduced the stimulus-related rise of intraglial aiK. An additional application of ouabain blocked both the membrane hyperpolarization as well as the remaining rise of aiK. 5. In conclusion, our data show that glial cells in guinea-pig olfactory cortex slices possess at least two mechanisms of K+ accumulation. One mechanism is sensitive to the K+ channel blocker Ba2+ and might be a passive KCl influx. The other appears to be the electrogenic Na+/K+ pump, which can be activated by excess extracellular K+.

Ca-channel blockers and the electrophysiology of synaptic transmission of the guinea-pig olfactory cortex

Slices of guinea-pig olfactory cortex have been used to compare the potency of various Ca-blockers on the electrophysiology of synaptic transmission. Listed in the order of potency, the divalent cations Cd 2+, Ni 2+, Mn 2+, Co 2+, La 3+ and Mg 2+ depressed synaptic transmission. The organic Ca-blockers, nifedipine or nimodipine or verapamil and diltiazem were ineffective up to 0.01 nmol/l. Verapamil, D600 or diltiazem (0.1–0.3 mmol/l) depressed both synaptic transmission and the sodium-mediated presynaptic action potential. These results reaffirm the idea that “organic Ca-antagonist’ do not block all Ca-channels in brain and the high Cd 2+ sensitivity suggests the Ca-channels in post- and presynaptic membranes have dissimilar pharmacological profiles.

An analysis of the action of pentobarbitone on the excitatory postsynaptic potentials and membrane properties of neurones in the guinea-pig olfactory cortex.

Intracellular recordings were made from neurones in slices of guinea-pig olfactory cortex maintained in vitro at 37 degrees C. The average membrane potential was 63 +/- 12 mV and the input resistance of these cells was 42 +/- 20 M omega (mean +/- s.d.). Stimulation of the lateral olfactory tract (l.o.t.) generated a transient depolarization in these cells which had the characteristics of an excitatory postsynaptic potential (e.p.s.p.). If the e.p.s.p. was of sufficient amplitude it culminated in an action potential. The e.p.s.p. was potentiated by repetitive stimulation at 10-50 Hz and showed post-tetanic potentiation after a prolonged period of high frequency stimulation (50-100 Hz for 30-60 s). Pentobarbitone (0.1-0.5 mM) depressed the e.p.s.p. reversibly but was without effect on the resting membrane potential, input resistance or time constant of the neurones. Pentobarbitone did not inhibit potentiation of the e.p.s.p. by a preceding conditioning shock. It is concluded that pentobarbitone does not affect the passive membrane properties of neurones in the olfactory cortex. The depressant action of pentobarbitone on synaptic transmission results from a decrease in the amount of transmitter released in response to a nerve impulse, or a decrease in the sensitivity of the postsynaptic membrane to the transmitter or a combination of both effects.

γ-Aminobutyric acid partly mediates the pentobarbitone depression of synaptic excitation in the guinea-pig olfactory cortex in vitro

Pentobarbitone depresses synaptic excitation in the guinea-pig olfactory cortex slice in vitro. A study has been made to elucidate the possible role of γ-aminobutyric acid (GABA) in this depression by testing pentobarbitone in the presence of high concentrations of the GABA blockers, i.e. picrotoxin or bicuculline. These blockers reduced the action of pentobarbitone; the dose-depression curve for pentobarbitone was shifted to the right by a factor of 2.3. It is concluded that pentobarbitone has a bimodal action, one action via GABA and another unrelated to GABA or Cl − conductances.

Muscarinic depression of evoked surface-negative field potentials recorded from guinea-pig olfactory cortex in vitro

The action of some cholinergic drugs has been studied on the field potentials evoked by orthodromic stimulation of the lateral olfactory tract (LOT) in guinea-pig olfactory cortex slices maintained in vitro. A reversible depression of the electrically evoked surface-negative field potential (N-wave) was seen following superfusion of muscarine (10–200 μM) or mixed-agonist choline esters but not nicotinic agonists. This depression was blocked by atropine and pirenzepine, but not d-tubocurarine or by antagonists active at γ-aminobutyric acid or adenosine receptors. Little effect of muscarinic agonists was observed on the compound action potential recorded from the LOT, or on pial surface DC potential. A possible presynaptic site of action of muscarinic agonists in the olfactory cortex is discussed.

Inhibition of GABA uptake potentiates the conductance increase produced by GABA-mimetic compounds on single neurones in isolated olfactory cortex slices of the guinea-pig.

Membrane potential and input conductance were recorded in single neurones in slices of guinea-pig olfactory cortex in vitro. gamma-Aminobutyric acid (GABA) and GABA-mimetic compounds were applied by bath-perfusion. Potency was measured as the concentration required to double the input conductance. The potency of GABA was increased (i.e. the equi-effective concentrations were reduced) by 15.5 +/- 2.3 times (mean +/- s.e. mean) on reducing external [Na+] from 144 to 20 mmol l-1, by replacement with Mg2+. Corresponding potency changes for other agonists were + 10.8 +/- 2.5 for 3-aminopropanesulphonic acid (3-APS); 3.25 +/- 1.06 for isoguvacine and 2.43 +/- 0.69 for muscimol. Nipecotic acid (0.5 mM) produced the following increases in potency: GABA 2.68 +/- 0.02; 3-aminopropanesulphonic acid, 3.11 +/- 0.07; isoguvacine, 1.92 +/- 0.34; muscimol, 2.24 +/- 0.17. The concentration of GABA in the bathing fluid necessary to double input conductance increased with increasing depth of the recording site from the cut surface. The apparent potency fell 10 times for each 60 micron depth increment up to 150 micron. The recording depth also affected the apparent potency of muscimol and 3-APS but to a lesser extent. Reduction of external [Na+] reduced the depth-dependence of both GABA and 3-APS potency. No clear change in the duration of the recurrent inhibitory postsynaptic conductance could be detected in the presence of 0.5 mmol l-1 nipecotic acid. 6 It is suggested that agonist uptake by a Na+-dependent, nipecotic acid-sensitive mechanism severely attenuates the responses of olfactory neurones to exogenous GABA and to its analogues 3-APS, muscimol and isoguvacine, but has little immediate influence on the duration of the GABA-mediated inhibitory postsynaptic conductance.

Detection of neurotoxicities of clioquinol by in vitro brain slices and cultured nerve cells

In order to know acute toxicities of environmental pollutants and drugs on human brain, mammalian brain slices have been proposed as a suitable assay system(1). Clioquinol is known to cause a subacute myelo-optico neuropathy (SMON). Clioquinol and related compounds; 5-chloro,7-iodo, 8-hydroxyquinoline(CI·I-HQ), 5,7-dichloro,8-hydroxyquinoline(Cl·Cl·HQ), 5-chloro,8-hydroxyquinoline (Cl·HQ), 8-hydroxyquinoline(HQ), 8-hydroxyquinoline, 5-sulfonic acid(HQ-S) were applied to the guinea pig olfactory cortex slices in vitro,and amplitudes of the postsynaptic potentials evoked by the electrical stimulation of lateral clfactory tracts were observed. These compounds inhibited the synaptic transmission in the slices at the concentration of 100 μM. Percent inhibitions at 20 min after the additions were: Cl-I-HQ,40%; Cl·Cl·HQ,38 % ; Cl·HQ,35%; HQ,25%; HQ-S,14%. To test chronic toxicities, they were added to cell cultures of neuroblastoma originated from rat brain. Cl-I-HQ and C1-C1-HQ (each 100 pM) did not change the viability and morphology until 1 hr after the addition. The result suggests that the acute toxicity of clioquinol and related compounds on the synaptic transmission in brain slices does not come from their cyto-toxicity.

Convulsant actions of 4-aminopyridine on the guinea-pig olfactory cortex slice

The effects of bath-applied 4-aminopyridine on neurones and extracellular potassium and calcium concentrations were recorded in slices of guinea-pig olfactory cortex. Neurones were orthodromically activated by stimulating the lateral olfactory tract. 4-Aminopyridine (3–10 μM) had the following effects: (1) an increase in the frequency and amplitude of spontaneous postsynaptic potentials: (2) a prolongation and oscillatory behaviour or orthodromically evoked postsynaptic potentials; (3) induction of spontaneous or stimulus-evoked seizure-type discharges which were accompanied by large rises in extracellular potassium and falls in calcium concentration; (4) a prolongation of the lateral olfactory tract population fibre spike. Prior to paroxysmal depolarization, membrane potential, input resistance and soma spike duration were unaffected. In the seconds before seizure discharges, a late hyperpolarizing potential (evoked by orthodromic stimulation) was reduced in amplitude or abolished. Diphenylhydantoin (50 μM) or magnesium ions (5 mM) prevented paroxysmal activity. Our results whow that 4-aminopyridine can produce seizure-type discharges in a brain slice preparation. The role of increased spontaneous potentials and possible loss of synaptic inhibition as causal factors for such discharges is discussed.

Effects of heating on electrical activities of guinea pig olfactory cortical slices.

We examined the effect of heating on electrical activity of neurons in the guinea pig olfactory cortex slice. At the control temperature (37 degree C) the potential evoked by stimulation of the lateral olfactory tract consisted of an initial spike (IS) potential and a negative (N) potential. The IS potential is considered to be presynaptic and the other transsynaptic. The IS potential decreased in amplitude on heating and completely disappeared at 49 degree C. However, it recovered when the temperature was lowered to 37 degree C after five minutes of incubation at 49 degree C. In contrast, the N potential increased in amplitude at 39 degree C, was completely suppressed at 47 degree C and did not recover when the temperature was dropped to the control temperature. The maximum temperature from which the N potential recovered was 43 degree C. Unit activity was extracellularly recorded from neurons in the slice. On heating the brain slice some neurons showed an increase in activity others a decrease, and the rest were unaffected. We conclude that neurons in the olfactory cortex have different thermal sensitivities.