Validation of the i.t50 method for assessing neuronal responsiveness to microiontophoretic applications: A single-cell recording study

doi:10.1016/0160-5402(88)90041-1

Journal of Pharmacological Methods

Volume 19, Issue 1, March 1988, Pages 23-30

https://doi.org/10.1016/0160-5402(88)90041-1 Get rights and content

Abstract

The I.T₅₀ method consists of determining the charge required to obtain a 50% depression of firing activity of neurons recorded extracellularly with microiontophoretic applications of inhibitory agents. This method has been used successfully to detect modification of neuronal responsiveness, but the limits of its validity had never been determined. In the present study, it was found that the use of microiontophoretic currents greater than 3 nA yielded consistent I.T₅₀ values when serotonin (5-HT) was applied to rat hippocampal pyramidal neurons. The departure from linearity of I.T₅₀ values measured from applications carried out with a very low current (0.5 nA) of 5-HT is probably due to the relatively important contribution of the leak when a minimal ejecting current is used. The responsiveness to 5-HT was not altered by the activation of the recorded neuron produced by acetylcholine.

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Neonatal destruction of the nigrostriatal dopamine projection by intraventricular 6-hydroxydopamine leads to a serotonin (5-hydroxytryptamine, 5-HT) hyperinnervation of the adult neostriatum accompanied by increased radioligand binding to 5-HT_1B, 5-HT_1nonAB and 5-HT₂ receptors. The consequences of such 5-HT receptor changes on neuronal responsiveness to 5-HT and corresponding receptor agonists were assessed with a quantitative iontophoretic approach. For comparative purposes, similar data were also obtained from rats 6-hydroxydopamine lesioned as adults, showing severe neostriatal dopamine denervation but no 5-HT hyperinnervation. In controls, 5-HT and its receptor agonists, m-chlorophenylpiperazine (mCPP; 5-HT_1B/2C agonist) and dimethoxy-iodophenyl-aminopropane (DOI; 5-HT_2A/2C agonist), depressed the firing rate of a majority of the units tested. Three months after neonatal 6-hydroxydopamine lesion (5-HT-hyperinnervated tissue), inhibitory responses to all three agents were significantly increased and comparable results were obtained for 5-HT and DOI in the rostral versus caudal neostriatum. After 6-hydroxydopamine lesion in adults, neither responsiveness to 5-HT, mCPP or DOI nor the density of 5-HT_1B or 5-HT_2A binding were significantly different from control. Thus, the up-regulation of 5-HT_1B, 5-HT_2A and possibly 5-HT_2C receptors accompanying the 5-HT hyperinnervation after neonatal but not after adult dopamine denervation was associated with increased responsiveness (IT₅₀) of neostriatal neurons to iontophoresed 5-HT and its receptor agonists. Under these conditions, neostriatal 5-HT transmission might be enhanced in spite of a basal release seemingly comparable to normal (Jackson and Abercrombie, 1992, J. Neurochem. 58, 890). The elevetad number of 5-HT_2A receptors might also explain why systemic treatment with 5-HT_2A receptor antagonists reverses the spontaneous hyperactivity observed in these rats (Luthman et al., 1991, J. Psychopharmacol. 5, 418).
Changes of D<inf>1</inf> and D<inf>2</inf> receptors in adult rat neostriatum after neonatal dopamine denervation: Quantitative data from ligand binding, in situ hybridization and iontophoresis
1993, Neuroscience
The specific binding of [³H]SCH23390 to D₁ and of [³H]raclopride to D₂ dopamine receptors was measured by autoradiography in the rostral and caudal halves of neostriatum and in the substantia nigra of adult rats subjected to near total destruction of nigrostriatal dopamine neurons by intraventricular 6-hydroxydopamine soon after birth. Three months after this lesion, [³H]SCH23390 binding (D₁ receptors) was slightly but significantly decreased in the rostral neostriatum (22%), but unchanged in its caudal half and in the substantia nigra. In contrast, [³H]raclopride binding (D₂ receptors) was considerably increased throughout the neostriatum (10–40%), while markedly decreased in the substantia nigra (80%). In the rostral neostriatum, there were no parallel changes in D₂ receptor messenger RNA levels, as measured byin situ hybridization on adjacent sections. Caudally, however, slight but significant increases in D₂ messenger RNA could be observed (10–20%). As assessed by quantitative iontophoresis, there was a marked enhancement (63%) of the inhibitory responsiveness of spontaneously firing units in the rostral neostriatum to dopamine and the D₁ agonist, SKF38393, in neonatally lesioned compared to control rats. On the other hand, responsiveness to PPHT, a potent D₂ agonist, appeared to be unchanged.
Such opposite changes in the number of D₁ and D₂ binding sites, dissociated from the expression of D₂ receptor messenger RNA and from the sensitivity to dopamine and D₁ and D₂ agonists, suggested independent adaptations of these various parameters following the neonatal dopamine denervation of neostriatum. They also provided further evidence for mechanisms other than the dopamine innervation in the control of the expression of neostriatal D₂ receptor messenger RNA during ontogenesis, and emphasized that the effects of dopamine and its D₁ and D₂ agonists in neostriatum do not depend strictly on the number of D₁ and D₂ primary ligand recognition sites.
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Effects of iontophoresed γ-aminobutyric acid (GABA) and two GABA agonists, 4,5,6,7-tetrahydrotsooxazoIo-[5,4-c]pyridine-3-ol (THIP) and baclofen were quantitatively compared in the anterior cingulate, frontal, and parietal cortex of urethaneanesthetized intact rats after catecholamine (CA) depletion with α-methyl-p-tyrosine (α-MPT) or selective dopamine (DA) denervation with 6-hydroxydopamine (6-OHDA). As assessed with to the IT₅₀ index, the postsynaptic sensitivity to GABA was significantly higher in anterior cingulate than in frontal and parietal cortex. The responsiveness to GABA was also greater in frontal than in parietal cortex. Sensitivity to GABA was significantly reduced in both anterior cingulate and frontal cortex after CA depletion, and similarly, after DA denervation with 6-OHDA. The difference in the sensitivity to GABA between the three cortical regions in intact rats as well as after CA depletion did not seem to be correlated with either GABA_A or GABA_B receptors since the responsiveness to both GABA agonists in every region examined was comparable in intact rats, and remained unchanged after α-MPT treatment. This finding raises the possibility that some GABA receptors in the cerebral cortex may be pharmacologically distinct from the two main subtypes of GABA receptors, GABA_A and GABA_B. When GABA was administered by iontophoresis in the anterior cingulate cortex during continuous applications of subthreshold currents of DA, the inhibition induced by GABA was either increased or decreased. As DA innervation density is nearly two-fold greater in anterior cingulate than in frontal cortex, and 30-fold greater in anterior cingulate than in parietal cortex, these results suggest that responsiveness to GABA may be correlated with the regional density of DA innervation and that elevated levels of DA may enhance the sensitivity to GABA.
Chronic caffeine exposure enhances adenosinergic inhibition of cerebral cortical neurons
1990, Brain Research
Chronic administration of caffeine (s.c. for a period of 14 days in escalating doses of 10–70 mg/kg) increased the sensitivity of rat cerebral cortical neurons to the inhibitory action of microiontophoretically applied adenosine. The sensitivity of spontaneously firing rat cerebral cortical neurons in caffeine-treated animals was compared with that of saline-treated controls using the same multiple-barrel micropepettes tested on the same day. Adenosine sensitivity was determined by the I·T₅₀ method. The I·T₅₀ value for 134 neurons in the caffeine-treated rats of 130.77 ± 4.33 (S.E.M.) was significantly (P < 0.001) different to that of 136 neurons in the saline-treated control rats (222.16 ± 6.68), indicating a supersensitivity to adenosine in neurons which had been chronically exposed to caffeine.
Cortical neuron sensitivity to neurotransmitters following neonatal noradrenaline depletion
1990, International Journal of Developmental Neuroscience
In order to test the functional significance of rapid eye movement (REM)-sleep and noradrenergic activity for cerebral cortex maturation, rat pups were daily injected with clonidine from 8 to 21 days of life. Previous studies have shown that this treatment reduces the amount of time spent in REM-sleep and the level of noradrenaline turnover in the brain. For long-term consequences of such treatment in adulthood, cortical neuron responses to micro-iontophoretically applied neurotransmitters were studied. No significant differences were found in the single cell responses to glutamate, GABA or noradrenaline in the cerebral cortex of clonidine treated rats as compared with age matched controls. However, the magnitude of GABAergic depression of glutamate induced neuronal responses was greater in the clonidine than in the control group.
Electrophysiological characterization of adrenoceptors in the rat dorsal hippocampus. II. Receptors mediating the effect of synaptically released norepinephrine
1988, Brain Research
The present studies were undertaken to determine the nature of the receptors mediating the effects of endogenous norepinephrine (NE) released by stimulation of the locus coeruleus (LC) on the firing activity of dorsal hippocampus pyramidal neurons in the rat. Unitary activity of CA3 pyramidal neurons were recorded extracellularly. In most neurons, the LC stimulation pyoduced a period of suppression, followed by a period of activation. The suppression was selectively blocked by prazosin, anα₁-adrenoceptor antagonist, whereas the activation was selectively blocked by propranolol, a β-adrenoceptor antagonist. Idazoxan, anα₂-adrenoceptor antagonist, increased the period of suppression without affecting the period of activation. The effectiveness of microiontophoretic applications of NE on the same neurons was reduced by idazoxan, but was modified neither by propranolol nor prazosin. Lesion of the central noradrenergic system by intracerebroventricular 6-hydroxydopamine markedly decreased the NE content in the hippocampus in all rats but the effectiveness of the LC stimulation was reduced only in rats with a depletion greater than 90%. These results demonstrate that the suppressant effect of endogenous NE released by LC stimulation on hippocampus pyramidal neurons is mediated by anα₁-adrenoceptor and suggest that its late excitatory effect might involve β-adrenoceptors. Since the effect of microiontophoretically applied NE on the same neurons is mediated byα₂-adrenoceptors, these data provide evidence that, in the rat hippocampus, postsynapticα₁-adrenoceptors are intrasynaptic, whereas postsynapticα₂-adrenoceptors are extrasynaptic.

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Validation of the i.t50 method for assessing neuronal responsiveness to microiontophoretic applications: A single-cell recording study

Abstract

Neuropharmacology

Neuropharmacology

Brain Res Bull

Neuropharmacology

Eur J Pharmacol

Eur J Pharmacol

Brain Res Bull

J Ther Biol

Brain Res

Brain Res

Effects of the two antidepressant drugs mianserin and indalpine on the serotonergic system: Single cell studies in the rat

Psychopharmacology

Microiontophoretic release of drugs from micropipettes: Use of 24Na as a model

Br J Pharmacol

Central synaptic transmission: Microelectrophoretic studies

Annu Rev Pharmacol

Electroconvulsive shock treatments enhance responsiveness of forebrain neurons to serotonin

J Pharmacol Exp Ther

Validation of the i.t₅₀ method for assessing neuronal responsiveness to microiontophoretic applications: A single-cell recording study

Microiontophoretic release of drugs from micropipettes: Use of ²⁴Na as a model