Electrophysiological properties of inferior olive neurons: A compartmental model.
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As a step in exploring the functions of the inferior olive, we constructed a biophysical model of the olivary neurons to examine their unique electrophysiological properties. The model consists of two compartments to represent the known distribution of ionic currents across the cell membrane, as well as the dendritic location of the gap junctions and synaptic inputs. The somatic compartment includes a low-threshold calcium current (I(Ca_l)), an anomalous inward rectifier current (I(h)), a sodium current (I(Na)), and a delayed rectifier potassium current (I(K_dr)). The dendritic compartment contains a high-threshold calcium current (I(Ca_h)), a calcium-dependent potassium current (I(K_Ca)), and a current flowing into other cells through electrical coupling (I(c)). First, kinetic parameters for these currents were set according to previously reported experimental data. Next, the remaining free parameters were determined to account for both static and spiking properties of single olivary neurons in vitro. We then performed a series of simulated pharmacological experiments using bifurcation analysis and extensive two-parameter searches. Consistent with previous studies, we quantitatively demonstrated the major role of I(Ca_l) in spiking excitability. In addition, I(h) had an important modulatory role in the spike generation and period of oscillations, as previously suggested by Bal and McCormick. Finally, we investigated the role of electrical coupling in two coupled spiking cells. Depending on the coupling strength, the hyperpolarization level, and the I(Ca_l) and I(h) modulation, the coupled cells had four different synchronization modes: the cells could be in-phase, phase-shifted, or anti-phase or could exhibit a complex desynchronized spiking mode. Hence these simulation results support the counterintuitive hypothesis that electrical coupling can desynchronize coupled inferior olive cells. (+info)
Kinetic and pharmacological properties of human brain Na(+)/H(+) exchanger isoform 5 stably expressed in Chinese hamster ovary cells.
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The recently cloned Na(+)/H(+) exchanger isoform 5 (NHE5) is expressed predominantly in brain, yet little is known about its functional properties. To facilitate its characterization, a full-length cDNA encoding human NHE5 was stably transfected into NHE-deficient Chinese hamster ovary AP-1 cells. Pharmacological analyses revealed that H(+)(i)-activated (22)Na(+) influx mediated by NHE5 was inhibited by several classes of drugs (amiloride compounds, 3-methylsulfonyl-4-piperidinobenzoyl guanidine methanesulfonate, cimetidine, and harmaline) at half-maximal concentrations that were intermediate to those determined for the high affinity NHE1 and the low affinity NHE3 isoforms, but closer to the latter. Kinetic analyses showed that the extracellular Na(+) dependence of NHE5 activity followed a simple hyperbolic relationship with an apparent affinity constant (K(Na)) of 18.6 +/- 1.6 mM. By contrast to other NHE isoforms, NHE5 also exhibited a first-order dependence on the intracellular H(+) concentration, achieving half-maximal activation at pH 6.43 +/- 0.08. Extracellular monovalent cations, such as H(+) and Li(+), but not K(+), acted as effective competitive inhibitors of (22)Na(+) influx by NHE5. In addition, the transport activity of NHE5 was highly dependent on cellular ATP levels. Overall, these functional features distinguish NHE5 from other family members and closely resemble those of an amiloride-resistant NHE isoform identified in hippocampal neurons. (+info)
Comparative study on the vasorelaxant effects of three harmala alkaloids in vitro.
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Three psychological active principles from the seeds of Peganum harmala L., harmine, harmaline and harmalol, showed vasorelaxant activities in isolated rat thoracic aorta preparations precontracted by phenylephrine or KCl with rank order of relaxation potency of harmine > harmaline > harmalol. The vasorelaxant effects of harmine and harmaline (but not harmalol) were attenuated by endothelium removal or pretreatment with a nitric oxide (NO) synthase Nomega-nitro-L-arginine methyl ester. In cultured rat aortic endothelial cells, harmine and harmaline (but not harmalol) increased NO release, which was dependent on the presence of external Ca2+. In endothelium-denuded preparations, pretreatment of harmine, harmaline or harmalol (3-30 microM) inhibited phenylephrine-induced contractions in a non-competitive manner. Receptor binding assays indicated that all 3 compounds interacted with cardiac alpha1-adrenoceptors with comparable affinities (Ki value around 31 - 36 microM), but only harmine weakly interacted with the cardiac 1,4-dihydropyridine binding site of L-type Ca2+ channels (Ki value of 408 microM). Therefore, the present results suggested that the vasorelaxant effects of harmine and harmaline are attributed to their actions on the endothelial cells to release NO and on the vascular smooth muscles to inhibit the contractions induced by the activation of receptor-linked and voltage-dependent Ca2+ channels. The vasorelaxant effect of harmalol was not endothelium-dependent. (+info)
Rhythmicity without synchrony in the electrically uncoupled inferior olive.
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Neurons of the inferior olivary nucleus (IO) form the climbing fibers that excite Purkinje cells of the cerebellar cortex. IO neurons are electrically coupled through gap junctions, and they generate synchronous, subthreshold oscillations of membrane potential at approximately 5-10 Hz. Experimental and theoretical studies have suggested that both the rhythmicity and synchrony of IO neurons require electrical coupling. We recorded from pairs of IO neurons in slices of mouse brainstem in vitro. Most pairs of neurons from wild-type (WT) mice were electrically coupled, but coupling was rare and weak between neurons of knock-out (KO) mice for connexin36, a neuronal gap junction protein. IO cells in both WT and KO mice generated rhythmic membrane fluctuations of similar frequency and amplitude. Oscillations in neighboring pairs of WT neurons were strongly synchronized, whereas the oscillations of KO pairs were uncorrelated. Spontaneous oscillations in KO neurons were not blocked by tetrodotoxin. Spontaneously oscillating neurons of both WT and KO mice generated occasional action potentials in phase with their membrane rhythms, but only the action potentials of WT neuron pairs were synchronous. Harmaline, a beta-carboline derivative thought to induce tremor by facilitating rhythmogenesis in the IO, was injected systemically into WT and KO mice. Harmaline-induced tremors were robust and indistinguishable in the two genotypes, suggesting that gap junction-mediated synchrony does not play a role in harmaline-induced tremor. We conclude that electrical coupling is not necessary for the generation of spontaneous subthreshold oscillations in single IO neurons, but that coupling can serve to synchronize rhythmic activity among IO neurons. (+info)
Attenuation of activity-induced increases in cerebellar blood flow in mice lacking neuronal nitric oxide synthase.
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We used mice deficient in neuronal nitric oxide (NO) synthase (nNOS) to specifically investigate the role of neuronal NO in the increase of cerebellar blood flow (BFcrb) produced by neural activation. Crus II, a region of the cerebellar cortex that receives trigeminal sensory afferents, was activated by low-intensity stimulation of the upper lip (5-25 V, 4-16 Hz) in anesthetized mice. BFcrb was recorded in Crus II by using a laser-Doppler flow probe. In wild-type mice, upper lip stimulation increased BFcrb in the Crus II by 28 +/- 3% (25 V, 10 Hz, n = 6). The rise in BFcrb was attenuated by 73 +/- 3% in nNOS-/- mice (P < 0.05, n = 6). The increases in BFcrb produced by superfusion of Crus II with glutamate or by systemic administration of harmaline were also attenuated in nNOS-/- mice (P < 0.05). In contrast, the increases in BFcrb produced by topical superfusion of Crus II with acetylcholine or adenosine and the increase in BFcrb produced by hypercapnia were not affected (P > 0.05). The field potentials evoked in the Crus II by upper lip stimulation did not differ between wild-type and nNOS-null mice. These data provide the first nonpharmacological evidence that nNOS-derived NO is a critical link between glutamatergic synaptic activity and blood flow in the activated cerebellum. (+info)
Contribution of individual cytochrome P450 isozymes to the O-demethylation of the psychotropic beta-carboline alkaloids harmaline and harmine.
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The psychotropic beta-carboline alkaloids, showing high affinity for 5-hydroxytryptamine, dopamine, benzodiazepine, and imidazoline receptors and the stimulation of locus coeruleus neurons, are formed endogenously from tryptophan-derived indolealkylamines through the Pictet-Spengler condensation with aldehydes in both plants and mammals. Cytochromes P450 1A1 (18.5), 1A2 (20), and 2D6 (100) catalyzed the O-demethylation of harmaline, and CYP1A1 (98.5), CYP1A2 (35), CYP2C9 (16), CYP2C19 (30), and CYP2D6 (115) catalyzed that of harmine (relative activities). The dehydrogenation/aromatization of harmaline to harmine was not carried out by aromatase (CYP19), CYP1A2, CYP2C9, CYP2D6, CYP3A4, pooled recombinant cytochromes P450, or human liver microsomes (HLMs). Kinetic parameters were calculated for the O-demethylations mediated by each isozyme and by pooled HLMs. K(cat) (min(-1)) and K(m) Awake M) values for harmaline were: CYP1A1, 10.8 and 11.8; CYP1A2, 12.3 and 13.3; CYP2C9, 5.3 and 175; CYP2C19, 10.3 and 160; and CYP2D6, 39.9 and 1.4. Values for harmine were: CYP1A1, 45.2 and 52.2; CYP1A2, 9.2 and 14.7; CYP2C9, 11.9 and 117; CYP2C19, 21.4 and 121; and CYP2D6, 29.7 and 7.4. Inhibition studies using monoclonal antibodies confirmed that CYP1A2 and CYP2D6 were the major isozymes contributing to both harmaline (20% and 50%, respectively) and harmine (20% and 30%) O-demethylations in pooled HLMs. The turnover numbers for CYP2D6 are among the highest ever reported for a CYP2D6 substrate. Finally, CYP2D6-transgenic mice were found to have increased harmaline and harmine O-demethylase activities as compared with wild-type mice. These findings suggest a role for polymorphic CYP2D6 in the pharmacology and toxicology of harmine and harmaline. (+info)
Attenuation of activity-induced increases in cerebellar blood flow by lesion of the inferior olive.
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We sought to define the contribution of the climbing fibers (CF), one of the major inputs to Purkinje neurons, to the increase in cerebellar blood flow (BFcrb) produced by activation of the cerebellar cortex. The neurotoxin 3-acetylpyridine was used to lesion the inferior olive, the site from which the CF originate. Crus II, an area of the cerebellar cortex that receives sensory afferents from the perioral region, was activated by low-intensity stimulation of the upper lip (5-25 V and 4-16 Hz) in sham-lesioned and lesioned mice. BFcrb was recorded in crus II using a laser-Doppler flow probe. The increase in BFcrb produced by harmaline, an alkaloid that activates the CF, was abolished in lesioned mice (P > 0.05 vs. BFcrb before harmaline, n = 6), attesting to the effectiveness of the lesion. In sham-lesioned animals, upper lip stimulation increased BFcrb in crus II by 25 +/- 2% (25 V and 10 Hz, n = 6). The rise in BFcrb was attenuated by 63 +/- 7% (25 V and 10 Hz) in lesioned mice (P < 0.05, n = 6). In contrast, the increase in BFcrb produced by hypercapnia was not affected (P > 0.05). These data suggest that CF are responsible for a substantial portion of the increase in BFcrb produced by crus II activation. Thus the hemodynamic response evoked by functional activation of the cerebellar cortex reflects, in large part, CF activity. (+info)
Microbial metabolites of harman alkaloids.
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Several microorganisms showed the ability to transform the harman alkaloids, harmaline (1), harmalol (2) and harman (5). Harmaline (1) and harmalol (2) were converted by Rhodotorula rubra ATCC 20129 into the tryptamines, 2-acetyl-3-(2-acetamidoethyl)-7-methoxyindole (3) and 2-acetyl-3-(2-acetamidoethyl)-7-hydroxyindole (4), respectively. Harman (5) was biotransformed by Cunninghamella echinulata NRRL 3655 into 6-hydroxyharman (6) and harman-2-oxide (7). (+info)