Bradley G. Klein

Basal Ganglia Accumulation and Motor Assessment Following Manganese Chloride Exposure in the C57BL/6 Mouse

Equivocal clinical evidence for involvement of manganese in development of Parkinson’s disease necessitates experimental studies on this issue. The aged, 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine–treated C57BL/6 mouse is one of the most common models for Parkinson’s disease. However, there is little information on brain bioaccumulation of manganese, and little or no information on clinical/behavioral manifestations of manganese neurotoxicity, in this strain. Male C57BL/6 retired breeder mice were given a single subcutaneous injection of either 0, 50, or 100 mg/kg of MnCl2 (single-dose regimen) or three injections of either of these doses over 7 days (multiple-dose regimen). Behavioral assessment was performed 24 h after final injection, followed by sacrifice, and body weight was recorded each day. There was a 105% increase in striatal manganese concentration 1 day after a single 100 mg/kg injection, and 421% and 647% increases, respectively, 1 day after multiple doses of 50 or 100 mg/kg of MnCl2. One day after a single injection, there were respective 30.9% and 38.9% decreases in horizontal movement (grid crossing) for the 50 and 100 mg/kg doses and a 43.2% decrease for the multiple dose of 100 mg/kg. There was no significant main effect of dose level on rearing, swimming, grip strength, or grip fatigue. Unlike previous work with the C57BL/6 strain using smaller intraperitoneal doses, this study established dosing regimens that produced significant increases in basal ganglia manganese concentration reminiscent of brain increases in the CD-1 mouse following subcutaneous doses close to our lowest. A decrease in locomotor behavior, significant but not severe in this study, has been reported following manganese exposure in other mouse strains. These data, particularly the significant increase in basal ganglia manganese concentration, provide guidance for designing studies of the potential role of manganese in Parkinson’s disease using the most common animal model for the disorder.

Immunohistochemical Changes in the Mouse Striatum Induced by the Pyrethroid Insecticide Permethrin

Epidemiological studies have linked insecticide exposure and Parkinsons disease. In addition, some insecticides produce damage or physiological disruption within the dopaminergic nigrostriatal pathway of non-humans. This study employed immunohistochemical analysis in striatum of the C57BL/6 mouse to clarify tissue changes suggested by previous pharmacological studies of the pyrethroid insecticide permethrin. Dopamine transporter, tyrosine hydroxylase, and glial fibrillary acidic protein immunoreactivities were examined in caudate-putamen to distinguish changes in amount of dopamine transporter immunoreactive protein from degeneration or other damage to dopaminergic neuropil. Weight-matched pairs of pesticide-treated and vehicle-control mice were dosed and sacrificed on the same days. Permethrin at 0.8, 1.5 and 3.0 mg/kg were the low doses and at 200 mg/kg the high dose. Brains from matched pairs of mice were processed on the same slides using the avidin-biotin technique. Four fields were morphometrically located in each of the serial sections of caudateputamen, digitally photographed, and immunopositive image pixels were counted and compared between members of matched pairs of permethrin-treated and vehicle-control mice. For low doses, only 3.0 mg/kg produced a significant decrease in dopamine transporter immunostaining. The high dose of permethrin did not produce a significant change in dopamine transporter or tyrosine hydroxylase immunostaining, but resulted in a significant increase in glial fibrillary acidic protein immunostaining. These data suggest that a low dose of permethrin can reduce the amount of dopamine transporter immunoreactive protein in the caudate-putamen. They also suggest that previously reported reductions in dopamine uptake of striatal synaptosomes of high-dose mice may be due to nondegenerative tissue damage within this region as opposed to reductions of dopamine transporter protein or death of nigrostriatal terminals. These data provide further evidence that insecticides can affect the primary neurodegenerative substrate of Parkinsons disease.

Examination of Concurrent Exposure to Repeated Stress and Chlorpyrifos on Cholinergic, Glutamatergic, and Monoamine Neurotransmitter Systems in Rat Forebrain Regions

Repeated stress has been reported to cause reversible impairment in the central nervous system (CNS). It was proposed that alterations in glutamatergic, cholinergic, and monoamine neurotransmitter systems after exposure to stress are initial CNS events contributing to this impairment and that exacerbation could occur with concurrent exposure to cholinesterase inhibitors. Effects of concurrent exposure to repeated stress and chlorpyrifos on activities of acetylcholinesterase (AChE), carboxylesterase, and choline acetyltransferase (ChAT); concentrations of excitatory amino acids, monoamines, and their metabolites; and maximum binding densities (B max) and equilibrium dissociation rate constants (K d) of glutamatergic N-methyl-d-aspartate (NMDA) and total muscarinic cholinergic receptors were studied in the blood, hippocampus, cerebral cortex, or hypothalamus of adult Long-Evans rats. Stress treatments extended over 28 days included (1) control rats handled 5 days/week; (2) rats restrained 1 h/day for 5 days/week; (3) rats swum 30 min for 1 day/week; or (4) rats restrained 4 days/week and swum for 1 day/week. On day 24, each stress treatment group was randomly divided and injected either with corn oil or chlorpyrifos, 160 mg/kg subcutaneously (sc) (60% of the maximum tolerated dose), 4 h after restraint. Blood and brain tisssues were collected on day 28. Rats restrained and swum had a statistical trend toward increasing concentrations of glutamate in the hippocampus when compared to rats only swum (p = .064). Chlorpyrifos administration decreased restraint-induced elevated aspartate in the hippocampus, and decreased B max of total muscarinic receptors in the cerebral cortex. In addition, chlorpyrifos decreased B max and K d of total muscarinic receptors in the cerebral cortex of swum rats. Results demonstrated that chlorpyrifos inhibited AChE activity in blood, cerebral cortex, and hippocampus, but stress did not affect AChE activity. Carboxylesterase activity was inhibited by chlorpyrifos and by repeated restraint with swim. Swim stress decreased concentrations of norepinephrine in the hippocampus and hypothalamus, and increased concentrations of dopamine and its metabolite, DOPAC, in the hypothalamus. Both stress and chlorpyrifos altered serotonin concentrations, and the interactions of repeated stress and chlorpyrifos on serotonin approached significance in the hippocampus (p = .06) and hypothalamus (p = .08). Therefore, stress models were demonstrated to alter glutamatergic and monoamine responses, whereas chlorpyrifos alone had effects on cholinergic and monoamine systems in the rat CNS. However, the interactions between stress and chlorpyrifos significant at p < 0.05 were restricted to attenuation of elevated aspartate in the hippocampus of restrained with swim rats and decreased K d of acetylcholine receptors in the cerebral cortex of swum rats and restrained rats.

Topography of the facial musculature within the facial (VII) motor nucleus of the neonatal rat.

SummaryWGA-HRP, HRP and fluorescent tracers were used to determine the representation of the facial muscles in the facial motor nuclear complex (FMNC) of the newborn rat. Tracer injections of the superficial cervical and anterior mandibular portions of platysma, the orbicularis oculi muscle, the nasolabial musculature and the posterior auricular musculature revealed an adultlike topographic organization across FMNC subnuclei. Tracer delivery to individual vibrissa follicle loci of the whiskerpad also demonstrated an adult-like musculotopic organization within the lateral subnucleus.

Chronic Functional Consequences of Adult Infraorbital Nerve Transection for Rat Trigeminal Subnucleus Interpolaris

In adult rats, transection of the infraorbital nerve and subsequent regeneration have been shown to result in altered somatotopic organization and changes in response properties of primary afferents within the trigeminal ganglion. The present study examined how these changes affect the postsynaptic targets of these neurons within subnucleus interpolaris of the trigeminal brainstem. Extracellular recordings were made from 330 cells in normal rats and 424 cells in rats surviving 57-290 days after transection of the infraorbital nerve in adulthood. Adult infraorbital nerve transection resulted in significant functional reorganization within subnucleus interpolaris. Relative to normal rats, the major changes can be summarized as follows: (1) a decrease in the dorsoventral extent of infraorbital representation; (2) a disruption of inter- and intradivisional somatotopic organization; (3) an increase in the proportion of cells with no discernible receptive field; (4) an increase in receptive field size for cells with infraorbital receptive field components; (5) the appearance of a significant proportion of cells with discontinuous receptive fields; (6) an increase in the proportion of cells exhibiting interdivisional convergence; (7) significant changes in the types of receptor surfaces activating local-circuit neurons with infraorbital receptive field components; (8) the appearance of a significant proportion of cells exhibiting convergence of different receptor surfaces; (9) significant changes in the dynamic response characteristics of cells with infraorbital receptive field components; and (10) an increase in the proportion of spontaneously active infraorbital-responsive cells. The changes observed were quite similar to those reported in adult subnucleus interpolaris following neonatal infraorbital nerve transection. The majority of changes observed in both studies can be most parsimoniously explained by alterations of primary afferents. However, central mechanisms may be more likely substrates for others. Regardless of the mechanism, the mature rodent trigeminal system appears capable of considerable functional reorganization following peripheral nerve damage.

Trigeminal projections to contralateral dorsal horn originate in midline hairy skin.

The present study tested the hypothesis that the trigeminal (V) primary afferent projection to the contralateral dorsal horn originates in midline hairy skin. A prior study (Jacquin et al., 1990) showed that this crossed projection is heaviest to ophthalmic regions of medullary and cervical dorsal horns, and that it does not arise from V ganglion cells that innervate cornea, nasal mucosa, or cerebral dura mater. Here, retrograde double-labeling methods were used to show that many ophthalmic ganglion cells that innervate midline hairy skin via the supraorbital nerve project to the contralateral medullary and upper cervical dorsal horns. Diamidino yellow injections into the right dorsal horn labeled an average of 104 cells in the left V ganglion. Of these contralaterally projecting ganglion cells, an average of 45% were also labeled by horseradish peroxidase (HRP) injections into the left supraorbital nerve, and 25% were also labeled by HRP injections into the midline opthalmic hairy skin. However, only 2% were labeled by HRP injections restricted to left supraorbital vibrissae follicle nerves. Almost all of the double-labeled cells were located in the dorsal one-half of the V ganglion, and they did not differ in size from single-labeled cells. On the basis of these and prior data, we conclude that a high percentage of contralaterally projecting V ganglion cells originate in midline hairy skin. It is also likely that the contralaterally projecting V ganglion cells serve a low-threshold mechanoreceptive function, given the relatively large ganglion cells and axons giving rise to this pathway and their central terminations in dorsal horn laminae III-V.

Corticosterone in drinking water: altered kinetics of a single oral dose of corticosterone and concentrations of plasma sodium, albumin, globulin, and total protein.

Effects of chronic exposure to corticosterone in drinking water on corticosterone kinetics, blood chemistry, and concentrations of catecholamines in parts of brain were studied in Long-Evans rats. Rats were randomly grouped into 3-2 treatments (n-4), with three treatments of drinking water (tap water, or 2.5% ethanol, or 400 mg/mL of corticosterone in 2.5% ethanol) for 28 days and two treatments of gavage with a single dose of either corn oil or corticosterone 20 mg/kg on day 28. Blood samples were collected at 0, 15, 30, 60, 120, 240, 480, and 720 min after dosing to determine plasma corticosterone concentrations. Blood samples were collected for clinical pathology on day 42. Hippocampus, cerebral cortex, caudate-putamen, and pons were examined to determine concentrations of catecholamines and activities of esterases. Concentrations of plasma corticosterone before gavage of the corticosterone-drinking rats (47.619 ± 1.13 ng/mL) were lower than the water (418.479 ± 1.13 ng/mL) or the ethanol rats (383.719 ± 1.13 ng/mL, P <0.0001). Plasma corticosterone rose to peak concentrations by 15 min after gavage in all three groups of drinking rats. Corticosterone- drinking rats had concentrations of plasma corticosterone that returned to basal levels slower than water- and ethanol-drinking rats. Plasma sodium and chloride concentrations were lower in the corticosterone-drinking rats than the water-drinking rats P <0.01). Plasma albumin, globulin, and total protein were highest in the corticosterone-drinking rats when compared to the other groups of drinking rats P <0.001, P <0.05, and P <0.001, respectively). Corticosterone in drinking water did not affect activities of brain neurotoxic esterase, carboxylesterase, acetylcholinesterase, or concentrations of monoamines and their metabolites. A single oral dose of corticosterone reduced neurotoxic esterase activity in the cerebral cortex P <0.05) and increased norepinephrine concentrations in the hippocampus P <0.05).

Orofacial pain sensitivity in adult rats following neonatal infraorbital nerve transection

A modification of the formalin test was used to assess orofacial pain sensitivity in adult rats that received infraorbital nerve transection at birth. Normal and neonatally lesioned adult animals received an injection of either 5% formalin or saline vehicle into the whiskerpad and the duration of whiskerpad rubbing was observed for 45 min. Normal rats given formalin exhibited the previously reported biphasic pattern of rubbing. Neonatally lesioned rats given formalin did not exhibit this pattern, and were indistinguishable from either of the saline control groups.

Pyrethroid and organophosphate insecticide exposure in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease: an immunohistochemical analysis of tyrosine hydroxylase and glial fibrillary acidic protein in dorsolateral striatum.

The pyrethroid insecticide permethrin and the organophosphate insecticide chlorpyrifos can experimentally produce Parkinson’s disease (PD)-associated changes in the dopaminergic nigrostriatal pathway, short of frank degeneration, although at doses considerably higher than from a likely environmental exposure. The ability of permethrin (200 mg/kg), chlorpyrifos (50 mg/kg), or combined permethrin + chlorpyrifos to facilitate nigrostriatal damage in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg/kg) C57BL/6 mouse model of PD was investigated in three separate experiments. Tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) immunohistochemistry assessed nigrostriatal degeneration or nigrostriatal damage more subtle than frank degeneration. Four fields in the dorsolateral caudate-putamen were examined at two rostrocaudal locations. The dopaminergic neurotoxin MPTP decreased striatal TH immunopositive neuropil and increased GFAP immunopositive neuropil. Neither permethrin nor chlorpyrifos, alone or in combination, altered the effects of MPTP upon TH or GFAP immunostaining. Permethrin alone increased striatal GFAP immunopositive neuropil but not when combined with chlorpyrifos treatment. Therefore, combined administration of the two insecticides appeared to protect against an increase in a neuropathological indicator of striatal damage seen with permethrin treatment alone. Differences compared with analysis of entire striatum emphasize the value of varying the topographic focus used to assess nigrostriatal degeneration in studies of insecticides in PD.

Biochemical and anatomical consequences of adult infraorbital nerve transection for serotonergic afferents within rat trigeminal subnuclei interpolaris and caudalis

Immunocytochemistry and high-performance liquid chromatography with electrochemical detection (HPLC-ED) were used, more than 76 days after infraorbital nerve (ION) transection, to examine the distribution and density of serotonin-immunoreactive (5-HTIR) axons, as well as serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content, within the infraorbital (IO) regions of subnuclei caudalis (SpVc) and interpolaris (SpVi). In SpVi, increases in 5-HT concentration and in density of 5-HTIR axonal varicosities were observed on the lesioned side. No changes were seen in SpVc.