Kyoji Taguchi

Effect of paclitaxel on transient receptor potential vanilloid 1 in rat dorsal root ganglion

&NA; The paclitaxel treatment increases TRPV1 expression in DRG neurons and may contribute to functional peripheral neuropathic pain. &NA; Peripheral neuropathy is a common adverse effect of paclitaxel treatment. To analyze the contribution of transient receptor potential vanilloid 1 (TRPV1) in the development of paclitaxel‐induced thermal hyperalgesia, TRPV1 expression in the rat dorsal root ganglion (DRG) was analyzed after paclitaxel treatment. Behavioral assessment using the tail‐flick test showed that intraperitoneal administration of 2 and 4 mg/kg paclitaxel induced thermal hyperalgesia after days 7, 14, and 21. Paclitaxel‐induced thermal hyperalgesia after day 14 was significantly inhibited by the TRP antagonist ruthenium red (3 mg/kg, s.c.) and the TRPV1 antagonist capsazepine (30 mg/kg, s.c.). Paclitaxel (2 and 4 mg/kg) treatment increased the expression of TRPV1 mRNA and protein in DRG neurons. Immunohistochemistry showed that paclitaxel (4 mg/kg) treatment increased TRPV1 protein expression in small and medium DRG neurons 14 days after treatment. Antibody double labeling revealed that isolectin B4‐positive small DRG neurons co‐expressed TRPV1. TRPV1 immunostaining was up‐regulated in paw skin day 14 after paclitaxel treatment. Moreover, in situ hybridization histochemistry revealed that most of the TRPV1 mRNA‐labeled neurons in the DRG were small or medium in size. These results suggest that paclitaxel treatment increases TRPV1 expression in DRG neurons and may contribute to functional peripheral neuropathic pain.

Neurophysiological and immunohistochemical studies on Guillain-Barré syndrome with IgG anti-GalNAc-GD1a antibodies—effects on neuromuscular transmission

We investigated the epitopes and functional role of IgG anti-GalNAc-GD1a antibodies appearing in serum from a patient with Guillain-Barre syndrome (GBS) and IgG anti-GalNAc-GD1a antibody that was produced by immunization of a rabbit with GalNAc-GD1a. Both sera blocked neuromuscular transmission in muscle-spinal cord co-culture cells. The acetylcholine-induced potential did not reduce by adding sera, suggesting that the blockade is presynaptic. The effect was complement-independent and purified IgG from serum of the patient or the rabbit had the same effects. The epitope with both anti-GalNAc-GD1a antibodies was observed in the soma of large neurons in the anterior horns of the adult rat spinal cord and their motor axons of rat ventral roots. Both anti-GalNAc GD1a antibodies reacted strongly with the motor nerve terminals in rats. The anti-GalNAc-GD1a antibodies may block neuromuscular transmission by attacking on presynaptic motor axon, probably affecting the ion channels in the presynaptic motor axon.

Localization of verotoxin receptors in nervous system.

We use immunohistochemistry to show the existence of verotoxin receptor in small sensory neurons in DRG of human, rabbit, rat and mouse. In capillary in nervous system, the verotoxin receptor exists in human and rabbit, but the receptor could not be demonstrated in rat and mouse, by this method. The receptors in sensory neuron of rat and in capillary in rabbit brain are determined as galactosylglobotriaosylceramide (GalGb3) and globotriaosylceramide (Gb3,), respectively. Although verotoxin was reported to bind to glycolipid receptors that possess the terminal disaccharide Galalpha1-4Galbeta (galactobiose), the binding to toxin to galabiosylceramide was half of that of GalGb3 which has galactobiose internally.

Cholinergic Mechanisms Responsible for Blood Pressure Regulation on Sympathoexcitatory Neurons in the Rostral Ventrolateral Medulla of the Rat

We examined whether reticulospinal sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM) have muscarinic receptors and ACh inputs, and whether these cholinergic mechanisms on RVLM neurons are involved in the pressor response induced by peripheral administration of physostigmine. Microiontophoretic application of ACh and carbachol enhanced the firing rate of RVLM sympathoexcitatory neurons and the enhancement of RVLM neurons by these cholinoceptor agonists was abolished by the nonselective muscarinic receptor antagonist scopolamine and/or by the M2 muscarinic receptor antagonist methoctramine. Physostigmine and the ACh releaser 3,4-diaminopyridine also enhanced the firing rate of RVLM neurons. Intravenous administration of physostigmine enhanced RVLM sympathoexcitatory neuronal activity and the physostigmine-induced response was reversed by iontophoretic application of scopolamine onto the neurons. These results are consistent with the hypothesis that M2 muscarinic receptors responsible for blood pressure regulation are present on RVLM sympathoexcitatory neurons and these receptors receive ACh inputs. Physostigmine injected systemically may exert a portion of its hypertensive effect through a direct enhancement of cholinergic mechanisms on RVLM sympathoexcitatory neurons.

Biochemical and pathological study of endogenous 1-benzyl-1,2,3,4-tetrahydroisoquinoline-induced parkinsonism in the mouse.

We administered 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BnTIQ; 80 mg/kg, i.p.), an endogenous neurotoxin known to cause bradykinesia, the Parkinsons disease-like symptom, in order to obtain biochemical and pathological evidence of behavioral abnormalities. Immunohistochemical analysis demonstrated that 1-BnTIQ did not decrease the number of tyrosine hydroxylase-positive cells in the substantia nigra. Biochemical analysis demonstrated significantly increased striatal dopamine (DA) content, while DA metabolites in the striatum remained at control levels. We concluded that the 1-BnTIQ-induced bradykinesia has a different mechanism of action than that underlying the MPTP-induced depletion of striatal DA neurons.

Stereoselective effect of (R)- and (S)-1-methyl-1,2,3,4-tetrahydroisoquinolines on a mouse model of Parkinson’s disease

We carried out behavioral, pathological, and biochemical studies in order to determine whether the stereo-structure of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ) affects the onset of Parkinsons disease-like symptoms, which are induced by 1,2,3,4-tetrahydroisoquinoline (TIQ) in mice. Pretreatment with (R)-1-MeTIQ or its racemate (RS)-1-MeTIQ prevented the TIQ-induced bradykinesia. Pretreatment with a combination of L-DOPA and carbidopa significantly prevented subsequent TIQ-induced bradykinesia. Furthermore, the pathological study demonstrated that either (R)-1-MeTIQ or its racemate protected against TIQ-induced loss of tyrosine hydroxylase-positive cells of the substantia nigra pars compacta. (R)-1-MeTIQ and its racemate also prevented the TIQ-induced reduction in the levels of dopamine and its metabolites in the striatum. Serotonin and its metabolite were not affected by repeated administration of (RS)-1-MeTIQ or its derivatives. On the other hand, (S)-1-MeTIQ induced moderate but significant bradykinesia, whereas (R)-1-MeTIQ did not induce this behavioral abnormality at all. In addition, (S)-enantiomer prevented the onset of TIQ-induced bradykinesia, though to a lesser extent than did either (R)-enantiomer or its racemate. However, (S)-enantiomer did not prevent the loss of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. We concluded that (R)-1-MeTIQ, and not (S)-enantiomer, plays a crucial role in protection against TIQ-induced parkinsonism, a fact which suggests that enantiomeric biochemical events such as 1-MeTIQ biosynthesis may participate in the pathogenesis of Parkinsons disease.

Evaluation of neurotoxicity of TIQ and MPTP and of parkinsonism‐preventing effect of 1‐MeTIQ by in vivo measurement of pre‐synaptic dopamine transporters and post‐synaptic dopamine D2 receptors in the mouse striatum

Parkinsonism‐inducing neurotoxicity of 1,2,3,4‐tetrahydroisoquinoline (TIQ), as contrasted to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), and parkinsonism‐preventing effect of 1‐methyl‐1,2,3,4‐tetrahydroisoquinoline (1‐MeTIQ) have been investigated in mice by measuring their effects on the in vivo binding of radioligand to pre‐synaptic dopamine transporters (DATs) or to dopamine D2 receptors (D2R) in the striatum. A significant reduction of the ligand‐DATs binding was found in the mice treated with MPTP, but not with TIQ, under the dosage inducing behavioral abnormality and loss of tyrosine hydroxylase‐positive cells in the substantia nigra. A slight decrease in the ligand‐DATs binding was observed in the mice given a larger dose of TIQ. Compensatory up‐regulation in the post‐synaptic D2Rs was found in the MPTP‐treated mice. Pre‐treatment with (S)‐enantiomer, but not (R)‐enantiomer, of 1‐MeTIQ prevented the degeneration of DATs to some extent. We concluded that the TIQ‐induced parkinsonism model is different from the MPTP‐induced model as evaluated by the radioligand‐DATs binding and that (S)‐1‐MeTIQ has a preventing effect for the degeneration of the DATs to a certain extent.

Effects of morphine on release of acetylcholine in the rat striatum: an in vivo microdialysis study

SummaryWe examined the effect of morphine on the release of acetylcholine (ACh) in the striatum of freely moving rats using the in vivo microdialysis method. The basal level of ACh was 3.01 ± 0.51 pmol/30 μl/15 min in the presence of neostigmine (10 μM). Tetrodotoxin (1 μM), a selective blocker of voltage-dependent Na+ channels, rapidly decreased the release of ACh in the striatal perfusates. Morphine at a dose of 10 mg/kg (i.p.) caused a reduction of ACh release in the striatum at 90–150 min. However, a lower dose of morphine (5 mg/kg, i.p.) did not affect ACh release in the striatum. The reduction following intraperitoneal administration of morphine was abolished by naloxone (1.0 mg/kg).After microinjection of the neurotoxin 6-hydroxydopamine (6 μg/3 μl, 7 days before) in the substantia nigra, the morphine (10 mg/kg)-induced decrease of ACh was attenuated, and a similar result occurred following reserpine (2 mg/kg, i.p.) 24 h before combined with α-methyl-p-tyrosine (300 mg/kg, i. p.) 2.5 h before.These findings indicate that morphine exerts an inhibitory influence on striatal ACh release in freely moving rats and that this inhibitory effect is mediated by the nigro-striatal dopaminergic system.

Molecular mimicry : Sensitization of lewis rats with Campylobacter jejuni lipopolysaccharides induces formation of antibody toward GD3 ganglioside

Recently we have reported cases of demyelinating inflammatory neuropathy showing elevated titers of anti‐GD3 antibodies, which occurs rarely in Guillain‐Barré syndrome. To examine the correlation between the anti‐GD3 antibody titer and Campylobacter jejuni infection, we sensitized female Lewis rats with lipopolysaccharides (LPSs) from serotype HS19 of C. jejuni and examined changes in nerve conduction velocity and nerve conduction block (P/D ratio). After 16 weeks of sensitization, animals revealed decreases of nerve conduction velocity and conduction block (P/D ratio) and high titer of anti‐GD3 antibodies. These anti‐GD3 antibodies also blocked transmission in neuromuscular junctions of spinal cord‐muscle cells cocultures. The GD3 epitope was verified to be located on the Schwann cell surface and nodes of Ranvier in rat sciatic nerve. To determine the target epitope for GD3 antibodies in causing nerve dysfunction, the LPS fraction containing the GD3 epitope was purified from the total LPS by using an anti‐GD3 monoclonal antibody‐immobilized affinity column. Subsequently, chemical analysis of the oligosaccharide portion was performed and confirmed the presence of a GD3‐like epitope as having the following tetrasaccharide structure: NeuAcα2‐8NeuAc2‐3Galβ1‐4Hep. Our data thus support the possibility of a contribution of GD3 mimicry as a potential pathogenic mechanism of peripheral nerve dysfunction.

Effect of rabbit anti-asialo-GM1 (GA1) polyclonal antibodies on neuromuscular transmission and acetylcholine-induced action potentials: Neurophysiological and immunohistochemical studies

We produced anti–asialo-GM1 (GA1) polyclonal antibodies by sensitizing New Zealand rabbits with GA1 and investigated the epitopes and pathogenic role of anti-GA1 antibodies that appeared in serum. The serum blocked neuromuscular transmission, but not acetylcholine (ACh)-induced potentials, in muscle–spinal cord cocultured cells. The effect was complement independent. The antibodies inhibited voltage-gated Ca2+ channel (VGCC). The epitopes recognized by the antibodies were located in the outer membrane of Schwann cells and motor axons of Wistar rat ventral roots and on motor axons extended from spinal cord to muscle cells in muscle–spinal cocultured cells. The ACh-induced potential was not reduced by the addition of sera, suggesting the blockade is presynaptic. Thus, anti-GA1 antibodies may block neuromuscular transmission by suppressing VGCC on axonal terminals of motor nerves.