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Following the postnatal decline of cell proliferation in the mammalian central nervous system, the adult brain retains progenitor cells with stem cell-like properties in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampus. Brain injury can stimulate proliferation and redirect the migration pattern of SVZ precursor cells to the injury site. Sublethal exposure to the neurotoxicant trimethyltin (TMT) causes dose-dependent necrosis and apoptosis in the hippocampus dentate gyrus and increases SGZ stem cell proliferation to generate new granule cells. To determine whether SVZ cells also contribute to the repopulation of the TMT-damaged dentate gyrus, 6-8 week old male C3H mice were injected with the carbocyanine dye spDiI and bromodeoxyuridine (80mg/kg; ip.) to label ventricular cells prior to TMT exposure. The presence of labeled cells in hippocampus was determined 7 and 28days after TMT exposure. No significant change in the number of BrdU(+) and spDiI(+) cells was observed in the dentate gyrus 7days after TMT treatment. However, 28days after TMT treatment there was a 3-4 fold increase in the number of spDiI-labeled cells in the hippocampal hilus and dentate gyrus. Few spDiI(+) cells stained positive for the mature phenotypic markers NeuN or GFAP, suggesting they may represent undifferentiated cells. A small percentage of migrating cells were BrdU(+)/spDiI(+), indicating some newly produced, SVZ- derived precursors migrated to the hippocampus. Taken together, these data suggest that TMT-induced injury of the hippocampus can stimulate the migration of ventricular zone-derived cells to injured dentate gyrus., (Copyright © 2016. Published by Elsevier B.V.)

Developmental exposure to methylmercury (MeHg) induces a spectrum of neurological impairment characterized by cognitive disturbance, sensory/motor deficit, and diffuse structural abnormalities of the brain. These alterations may arise from neural path-finding errors during brain development, resulting from disturbances in the function of morphoregulatory guidance molecules. The Eph family of tyrosine kinase receptors and their ligands, the ephrins, guide neuronal migration and neurite pathfinding mainly via repulsive intercellular interactions. The present study examined the effects of MeHg on mRNA and protein expression profiles of Ephs and ephrins in the P19 embryonal carcinoma (EC) cell line and its neuronal derivatives. Undifferentiated control P19 cells displayed low- to undetectable levels of mRNA for ephrins or Ephs, with the sole exception of EphA2 which was highly expressed. Upon differentiation into neurons, the ephrin expression increased progressively through day 10. Similarly, expression of the Ephs, including EphsA3, -A4, -A8, -B2, -B3, -B4, and -B6, increased significantly. In contrast, EphA2 expression decreased in day 2, 6 and 10 control neurons. Treatment with MeHg did not affect the expression of mRNA for ephrins or Ephs in undifferentiated P19 cells. However, treatment of differentiating neurons with MeHg for 24 h caused consistent increases in ligand mRNA expression, particularly ephrin-A5, -A6, -B1, and -B2. Similarly, MeHg induced variable increases in mRNA expression of receptors EphA2, -A3, -B3, and -B6. A trend toward a concentration-response relationship was observed for the alterations in Eph receptor mRNA expression although increases at the low and mid concentrations did not reach statistical significance. Immunoblots for ligand and receptor proteins mirrored the increases in the mRNA levels at the 0.5 and 1.5 microM MeHg concentrations but showed decreased protein levels compared to controls at the 3.0 microM concentration. Alterations in the Eph/ephrin family of repulsion molecules may represent an important mechanism in developmental MeHg neurotoxicity.

We examined the constitutive and inducible levels of microsomal cytochromes P450 1A1 and 1A2 (CYP1A) in rat cerebral cortex and cerebellum at the level of proteins by western blot analysis, and by catalytic activities via ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD). In the cerebral cortex, cytochrome P450 1A1 (CYP1A1) protein was more abundant than cytochrome P450 1A2 (CYP1A2) protein. Treatment with beta-naphthoflavone (beta-NF) caused a slight decrease in the level of the former but induced the latter 5.8-fold. In the cerebellum, in contrast to the cerebral cortex, CYP1A1 protein was less abundant than CYP1A2 protein in untreated rats, and while beta-NF treatment caused a 3.3-fold induction of CYP1A1 protein, it resulted in a 10-fold decrease in CYP1A2 protein. The CYP1A-preferential activity EROD was 2.3-fold higher in the cerebellum than in the cerebral cortex, and was induced 1.5-fold and 1.9-fold in the cerebellum and cerebral cortex, respectively, by beta-NF treatment. The CYP1A2-preferential activity MROD was 3-fold higher in the cerebellum than in the cerebral cortex, and was repressed 2.2-fold in the cerebellum but induced 3.7-fold in the cerebral cortex following beta-NF treatment. The results show that CYP1A1 and CYP1A2 proteins and catalytic activities are constitutively expressed in brain but are differentially inducible in the rat cerebral cortex and cerebellum.

Cytochromes P450 1A1 and 1A2 are involved in the oxidation of a wide spectrum of endogenous compounds and xenobiotics. Although their presence has been repeatedly confirmed in brain tissue, reports regarding their distribution in the brain are often contradictory. In the present study the possibility was examined that CYP1A1 and CYP1A2 are localized and inducible in the brain-CSF barrier and regions with a leaky blood brain barrier, where they may serve as a protective metabolic barrier. CYP1A1 and CYP1A2 levels were determined in subcellular fractions of multiple brain regions, as well as tissue homogenates of circumventricular organs, and the meninges by Western blotting and catalytic activity in control male rats and rats treated with the inducer beta-naphthoflavone (BNF). In control animals CYP1A1 immunoreactive protein was undetectable in regional brain microsomes or whole tissue homogenates of the arachnoid, dura mater, choroid plexus, pineal gland, median eminence, and pituitary. However, low levels of ethoxyresorufin O-deethylase (EROD) activity were observed in homogenates of the arachnoid, dura mater, choroid plexus, pineal gland, and pituitary. Western blotting revealed only low levels of CYP1A2 immunoreactive protein in brain microsomes from the cortex, cerebellum, brainstem, thalamus, hippocampus, and striatum from control animals. Following BNF treatment, EROD activity was induced 12-42-fold in the arachnoid, choroid plexus, dura mater, pineal gland, pituitary, and median eminence. Western blot analysis revealed CYP1A1 to be induced in the arachnoid, dura mater, choroid plexus, pineal gland, and pituitary, while CYP1A2 was undetectable. No induction of CYP1A1 or CYP1A2 protein was observed in brain microsomes from the olfactory bulb, cortex, striatum, hippocampus, cerebellum, or brainstem following BNF treatment, providing that the arachnoid membranes and choroid plexus had been carefully removed prior to brain dissection. Neither CYP1A1, 1A2 protein, nor EROD activity were detected in purified brain mitochondria, regardless of treatment or region. In conclusion, catalytically active CYP1A1 is located in the meninges as well as certain circumventricular organs, is inducible by BNF, and appears to be absent or expressed constitutively at very low levels in the majority of the brain parenchyma. The localization of CYP1A1 in the blood-CSF barrier and circumventricular tissues likely plays a role in protecting the brain from xenobiotics., (Copyright 1998 Academic Press.)

The role of striatal astrocytes in the metabolic processing (by deamination) of methamphetamine-released dopamine is not known. To investigate the relationship between methamphetamine and dopamine metabolism, we measured 6-hydroxydopamine, dopamine and, 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations following methamphetamine treatment of cultured striatal astrocytes prepared from 1-2 day-old rats. Addition of low concentrations of dopamine (5 x 10(-5) to 5 x 10(-4) M) to cultured astrocytes increased DOPAC levels in a dose-dependent fashion while higher concentrations (5 x 10(-3) to 10(-2) M) inhibited its metabolism and induced formation of 6-hydroxydopamine. Under the same experimental conditions, 10(-4) M dopamine in combination with methamphetamine (10(-5) to 10(-3) M) inhibited DOPAC formation and increased dopamine levels in a dose-dependent fashion, but the formation of intracellular 6-hydroxydopamine was not evident. Deprenyl (10(-5) or 10(-4) M), an inhibitor of monoamine oxidase B, and pargyline (10(-5) or 10(-4) M), a non-selective monoamine oxidase inhibitor, completely inhibited DOPAC formation and increased dopamine levels, while clorgyline (10(-5) or 10(-4) M), an inhibitor of monoamine oxidase-A, only partially inhibited DOPAC formation (42 or 45% of control, respectively). These results support the hypothesis that methamphetamine inhibits monoamine oxidase and causes increases in dopamine levels in cultured striatal astrocytes.

The distribution of glutathione (GSH) and glutathione S-transferases (GSTs) in the adult rat brain is cell-type specific, but their cellular distribution in the developing central nervous system is unknown. In the present study, GSH distribution in the mouse nervous system was visualized by mercury orange histochemistry and class-specific GSTs were localized by immunohistochemistry at ages E13 to PN30. Both neuronal and glial progenitor cells stain uniformly positive for GSH at E13. Spinal anterior horn neurons become GSH-negative by E17, at which time neurons and glia in other CNS regions are still GSH-positive. By PN5, most neurons have lost GSH staining and are surrounded by GSH-rich neuropil, ependyma, and vasculature. Olfactory mitral and granule cells, cerebellar granule cells, and dorsal root ganglion (DRG) neurons retain consistently high levels of GSH throughout development and into adulthood. Immunoreactivity to alpha-class GST antisera is not observed in the CNS until PN10, when very weak staining becomes apparent in the pia, ependyma, choroid plexus and neurons throughout the brain and spinal cord. Immunoreactivity to mu-GST is observed in neurons and astrocytes (but not oligodendrocytes), pia, ependyma, and choroid plexus throughout the brain by PN10. pi-GST immunoreactivity is observed in all cells of the embryonic nervous system. Postnatally, it is found in neurons and oligodendrocytes (but not astrocytes) in all regions of the brain and spinal cord as well as in pia, ependyma, and choroid plexus. The neurons and satellite cells of the DRG are immunoreactive to alpha-, mu-, and pi-GST antisera at all time points examined. The developmental changes in the cellular distribution of GSH and GSTs suggest that enzymatic conjugation and antioxidant activities may also be cell specific during brain development.

Glutathione and its related enzymes play a major role in the detoxification of toxic chemicals. In rat brain the pattern of distribution of reduced glutathione exhibits cellular heterogeneity, suggesting also the possibility of cellular differences in glutathione conjugating capacity. To understand the potential role of GSH in detoxification of neurotoxicants, the distributions of the glutathione conjugating and metabolizing enzymes, glutathione S-transferase (GST; alpha-, mu- and pi-classes) and gamma-glutamyl transpeptidase (gamma-GT) were determined immunohistochemically in brain, lumbar spinal cord and dorsal root ganglia (DRG) of adult Sprague-Dawley rats using polyclonal antibodies. The influence of tissue fixation on apparent distribution was also examined. Glial cells and neurons throughout the nervous system were only weakly positive with alpha-GST in frozen sections. No immunoreactivity for the alpha-class GSTs was observed in any of the paraformaldehyde-fixed neural specimens examined. In microwave-fixed frozen sections, immunoreactivity to mu-GST was found in astrocytes and neurons throughout the brain and spinal cord, and in the neurons and satellite cells of the DRG. Immunoreactivity for pi-GST was seen in oligodendrocytes but not in astrocytes in any region of the CNS examined. Similarly, satellite cells of the DRG were positive for pi-GST. Neuronal perikarya of the entire neopallium, hippocampus, cerebellum, brainstem, spinal cord and DRG were also positively stained for pi-GST. The differential staining of astrocytes and oligodendrocytes with pi- and mu-GST was unaltered in paraformaldehyde fixed tissues, but the neuronal immunostaining was lost. The ependyma, pia and choroid plexus stained positively with all three GST antibodies regardless of fixation. Gamma-Glutamyl transpeptidase-like immunoreactivity was confined to non-neuronal elements of both central and peripheral nervous systems. Ependymal cells throughout the central nervous systems stained intensely with antibodies directed against gamma-GT. Satellite and Schwann cells of the DRG and glial cells of the spinal cord and brain exhibited moderate to intense immunoreactivity for gamma-GT. The heterogeneous cellular distribution of glutathione and its metabolizing enzymes may reflect cellular differences in capacity for metabolic processing of both endogenous compound and xenobiotics.

The formation of 6-hydroxydopamine (6-OHDA) from dopamine (DA) was investigated in the striatum of male Sprague-Dawley rats following a single administration of methamphetamine hydrochloride (100 mg/kg, sc). Rats were sacrificed 30, 60, and 90 min, and 1 wk after injection, and striatal 6-OHDA, DA, and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured by HPLC with electrochemical detection. Methamphetamine decreased striatal DA and DOPAC levels (to 65 and 50% at 90 min, respectively) in the time-course study and also resulted in a long-lasting dopamine depletion (34%) 1 wk after its administration. However, endogenous 6-OHDA formation proved difficult to detect after administration of the methamphetamine alone. Pretreatment with the monoamine oxidase (MAO) inhibitor pargyline (100 mg/kg, ip) and the catechol-O-methyltransferase (COMT) inhibitor pyrogallol (25 mg/kg, ip) resulted in the HPLC detection of a 6-OHDA-like substance 30 min after methamphetamine administration when the oxidizing potential was set at 0.5 V, but not when it was set at 0.2 V. Moreover, pargyline (25 mg/kg, ip) alone or in combination with pyrogallol exacerbated the long-lasting dopamine depletion induced by methamphetamine (50 mg/kg, sc). These results indicate that simultaneous inhibition of MAO and COMT provides a cellular environment that encourages the autoxidation of dopamine to a 6-OHDA-like substance.

Cellular heterogeneity and structural complexity of the nervous system, coupled with regional and cellular differences in the metabolic capabilities of neurons, glia and other non-neuronal elements, may underlie the selective cellular involvement following exposure to neurotoxicants. Determination of the role of biotransformation of xenobiotics in neural pathoclisis requires an understanding of the cellular distribution of both phase I and phase II enzyme systems in the brain. While ependyma, choroid plexus and endothelial linings of blood vessels throughout the nervous system appear to contain appreciable amounts of several isoforms of cytochrome P450 (CYP450), glia and neurons tend to be highly specific in which forms of CYP450 they express. Regional and cellular heterogeneity similarly characterize the distribution of glutathione (GSH) and the conjugating enzyme glutathione S-transferase (GST) in the brain. While all cells of the embryonic nervous system express high levels of GSH and pi-GST (with lesser amounts of alpha- and mu-class), by adulthood neurons and non-neuronal cells differ in the distribution of GSH and isoforms of GST. Neurons (except the dorsal root ganglia and the cerebellar granule cells) become GSH-negative but contain alpha-, mu-, and pi-GST. Glia, ependyma, choroid plexus and neurovascular cells are rich in GSH and variously express GSTs. The differences found in the cellular distribution of GSH and GSTs may contribute to changes in the vulnerability of the nervous system to neurotoxicants at different ages. A comprehensive understanding of the neurotoxicological and pharmacological consequences of the cellular heterogeneity in the localization the CYP450s and glutathione S-transferases pre- and postnatally will require systematic study of the distribution, substrate specificity, and inducibility of the various isoforms of these enzyme systems.

Microtubule (MT) assembly and stability are thought to be dependent on intracellular glutathione for the maintenance of critical sulfhydryl groups. Since methyl mercury (MeHg) is a sulfhydryl-binding toxicant, it is possible that alteration of intracellular glutathione status might enhance the toxic effects of MeHg on microtubules. The influence of MeHg on the relationship between intracellular glutathione and the structural integrity of interphase microtubules was assessed in embryonal carcinoma cells by immunofluorescence microscopy, using antibodies to tyrosinated and acetylated alpha-tubulins. Intracellular glutathione concentrations were reduced by treatment with 10 microM buthionine sulfoximine (BSO; an inhibitor of gamma-glutamyl cysteine synthetase) for 18-24 hr. BSO-treated cells displayed little change in the pattern of microtubule staining, despite reduction of glutathione levels to less than 10% of control levels. Similarly, a combination of BSO and the nonspecific glutathione-depleting agent diethylmaleimide (DEM) had little effect on microtubule networks, except at the highest concentrations of DEM where nonspecific cytotoxicity was observed. The susceptibility of microtubules to MeHg-induced disassembly was determined in normal and glutathione-depleted cells incubated with 1.0 to 7.5 microM MeHg for 2 hr. MeHg treatment alone resulted in concentration-dependent disassembly of microtubules; depletion of glutathione with BSO prior to MeHg treatment did not enhance MT damage. Further, BSO-pretreated cells exposed to MeHg still showed substantial recovery of microtubule networks following removal of MeHg from culture media, even when glutathione levels remained less than 5% of control levels. These data indicate that the integrity of interphase microtubules is largely unaffected by reductions in glutathione concentration and that susceptibility of microtubules to MeHg-induced disassembly is not directly dependent on intracellular glutathione content.

3-Acetylpyridine (3-AP), an analogue of nicotinamide, produces highly selective CNS lesions, the severity of which may be influenced by prior alterations in the metabolic activity of the affected neurons. The present study was undertaken to determine whether prior axotomy modified the response of dorsal root ganglia (DRG) and anterior horn (AH) neurons to 3-AP. A single administration (50 or 80 mg/kg i.p.) of 3-AP to adult rats resulted in degeneration of primarily small-dark DRG neurons by 24 h. The AH neurons were not affected by either dose of 3-AP. Light and electron microscopy of the DRG revealed a spectrum of damage ranging from loss of Nissl substance and cytoplasmic degradation to frank necrosis with neuronophagia. Frequently, injured neurons exhibited perinuclear aggregation of cytoplasmic organelles with dissolution of Nissl substance, clearing of the peripheral cytoplasm, and formation of large peripheral vacuoles. Occasionally, a second pattern of 3-AP injury was observed in which the nuclear chromatin of the neurons was condensed and there was formation of small vacuoles throughout the cytoplasm without peripheral clearing or perinuclear aggregation of cytoplasmic organelles. Axotomy induced typical axon reactions in both large-pale and small-dark DRG neurons. The combination of axotomy followed by 3-AP 4 days later produced morphological features characteristic of both axotomy and 3-AP exposure, but did not appear to alter the incidence of neuronal cell death. The almost exclusive vulnerability of the small dorsal root ganglion neurons to 3-AP neurotoxicity make this model potentially useful for the study of small fibre neuropathies.

Conjugation with glutathione (GSH) is a mechanism of detoxification of acrylamide (ACR); hence, prior depletion of GSH might be expected to exacerbate ACR's neurotoxicity. GSH levels in female rats were reduced by ip administration of styrene oxide (SO; 250 mg/kg), diethylmaleate (DEM; 0.5 ml/kg), or 2-vinylpyridine (VP; 100 mg/kg) 1.5 or 2 hr prior to a single dose of ACR (100 mg/kg). The time course of GSH depletion following treatment with SO/ACR, DEM/ACR, or VP/ACR showed that all three regimens were equally effective in reducing GSH in liver, cerebellum, cerebral cortex, and hippocampus. GSH levels in the liver were reduced to 4-22% of control levels between 2 and 4 hr after treatment and to 38-57% of control levels in all brain regions between 4 and 8 hr. ACR alone (100 mg/kg) reduced both brain and liver GSH to about 60% of normal. The administration of a second dose of ACR (also 100 mg/kg) 12 hr later further depleted brain and liver GSH to 33% of control. Brains were examined 2, 4, 7, 14, and 30 days after treatment by light and electron microscopy. The administration of SO plus ACR (in either order) produced lesions consisting of pyknotic granule cells confined to the anterior portions of the cerebellum and some of the small neurons of lamina II and III of the cerebral cortex. Electron microscopy revealed condensation of the granule cell chromatin and dissolution of the cytoplasm with the formation of large pericellular spaces. The granule cell lesion was not produced when the time between SO and ACR injections was either 4 or 24 hr. No pathology was observed following treatment with DEM/ACR, VP/ACR, ACR/ACR, vehicle (peanut oil), SO, or ACR alone. It appears that the neurotoxicity in animals treated with SO plus ACR is not directly the result of reduced cellular GSH levels per se, but may involve other detoxification pathways of ACR and SO.

Electrophysiological function and morphology of dorsal root ganglion (DRG) neurons were investigated in female Wistar rats following administration of methylmercury (5 mg/kg/day for 10 days). Neurons were classified according to the conduction velocities of their peripheral axons and perikaryal action potential characteristics as types A alpha, A beta A delta and C. In methylmercury-treated rats, the majority of DRG neuron action potential characteristics were not significantly altered. However, axonal conduction velocities were slowed in all type A ganglion cells, leading to significant delays in action potential onset in types A beta and A delta neurons. An initial complex-soma inflection was observed on the rising phase of the action potential of some cells. These inflection potentials had longer conduction times and lower amplitudes in treated animals. Repetitive discharge, characterized by 3-5 action potentials upon single peripheral nerve stimulation, was observed in 8 of 38 cells recorded in treated animals, predominantly in type A beta (4/8) and A beta (3/12) neurons; direct current injection into repetitively discharging cells via the recording electrode elicited only single action potentials. Extracellular recordings from dorsal root filaments confirmed that the repetitive action potentials arising in the DRG were conducted to the spinal cord. Morphological examination of DRG revealed damage in up to 10% of neurons, consisting of loss of Nissl staining, cytoplasmic vacuolization, and proliferation of satellite cells. Large, pale neurons appeared preferentially involved. Rarely, neuronophagia was observed. Further microscopic examination of proximal and distal sections of sciatic and tibial nerves revealed only occasional degeneration of myelinated axons. While morphological alterations did not appear to offer a structural basis for the multiple firing, it is speculated that repetitive action potential discharge may contribute to the paresthesias associated with MeHg intoxication.

beta,beta'-Iminodipropionitrile (IDPN) impairs axonal transport of neurofilaments; their accumulation leads to the formation of proximal swellings in motor axons. Similar proximal swellings are a feature of some cases of motor neuron disease such as amyotrophic lateral sclerosis (ALS). Motor units in IDPN-treated animals were assessed to determine their relative susceptibilities to impaired function and whether the functional changes resulting from proximal axonal swellings share certain electromyographic features with ALS. Intrinsic properties of medial gastrocnemius motoneurones (MN) and contractile responses of their motor units were examined during the evolution of proximal axonal swellings in cats administered IDPN (50 mg/kg once weekly) for 7, 14 or 35 days. While conduction velocities were significantly decreased in all motor unit types by 35 days, the conduction slowing was greater in fast fatigable (types FF and FI) motor units than in fatigue resistant (types FR and S) motor units. Normal correlations between axonal conduction velocity and MN input resistance (Rin) and the inverse relationship between Rin and rheobase were lost with progression of the neuropathy. Twitch and maximum tetanic tension developed by fast-fatigable motor units declined early in the neuropathy, whereas fatigue-resistant units did not show similar changes until later stages of the intoxication. In some motor units, irregular and abnormal tetanic tensions were elicited by repetitive MN discharge. At 14 and 35 days, a novel, intermediate motor unit response classified as slow and fatigable (SF) was observed. Conduction block, characterized by repetitive MN firing without a corresponding contractile response, was observed in some type FF and S units by 35 days. Morphometric analysis of muscle fiber types showed significant atrophy, particularly in the type I fibers at 14-35 days; the atrophy reversed following cessation of IDPN administration. The influence of proximal axonal swellings on motor unit function in IDPN neuropathy is discussed in terms of reported electrophysiological alterations in motoneurone disease.

We examined the histochemical distribution of glucose-6-phosphate dehydrogenase (G6PD) activity in neural tissue using different diffusion barriers. Although polyvinyl alcohol and agar overlays permitted regional localization of G6PD, a semipermeable membrane revealed cellular differences in G6PD activity within populations of neurons. Distribution of G6PD activity in selected regions of the nervous system was examined using the membrane technique. White matter usually exhibited strong G6PD activity. The neuronal somata of the dorsal root ganglia (L4-L6) and anterior horns of the spinal lumbar enlargement demonstrated a variation in activity which was independent of somal size. Satellite cells showed intense activity when the membrane technique was used. Hippocampal pyramidal and granular cells of the dentate gyrus exhibited moderate, uniform G6PD activity, but only weak activity was seen in hippocampal and dentate molecular layers. High levels of activity were observed in the vascular endothelial cells of the brain, spinal cord, and choroid plexus, and in the ependymal cells of the spinal central canal and ventricles of the brain. The superior vestibular nucleus appeared to have little G6PD activity in either the neuron cell bodies or the surrounding parenchyma. The use of a semipermeable membrane for localization of G6PD activity in neural tissues permits enhanced resolution of neuron elements and may provide a more accurate assessment of G6PD activity in histological preparations.

Differences in the cellular distribution of antioxidant defense mechanisms in heterogeneous tissue such as the nervous system are likely critical determinants of differential sensitivity to toxicants. Regional and cellular localization of reduced glutathione (GSH) in central and peripheral nervous tissue was determined from the pattern of fluorescence observed in tissue sections stained with mercury orange; localization was confirmed using a novel histofluorochromatic staining method, o-phthaldialdehyde (OPT). Excellent concordance between the distribution of fluorescence obtained with mercury orange and OPT staining was observed. Depletion of GSH by treatment with diethyl maleate resulted in a diminution in both mercury orange and OPT histofluorescence. Generally, strong staining of the CNS neuropil was seen with little or no observable fluorescence in neuronal somata. The cerebellar granular cells were an exception, exhibiting fluorescence with both mercury orange and OPT. Cerebellar Purkinje cells exhibited nonuniform fluorescence with mercury orange but generally uniform staining with OPT. In contrast to the patterns observed in the CNS, the sciatic nerve and the sensory cell bodies of the lumbar dorsal root ganglia exhibited prominent fluorescence with both mercury orange and OPT. Reduced glutathione in the central nervous system appears primarily localized in the neuropil and white matter tracts; with a few exceptions, the neuronal somata do not appear to contain appreciable amounts of GSH. The heterogeneous distribution of GSH and enzymes involved in the detoxification and/or excretion of xenobiotics in the nervous system may form a basis for selective cellular and/or regional expression of neurotoxicity.

Repetitive exposure to low doses of acrylamide results in extensive pathological changes at the neuromuscular junction (NMJ), but it remains undetermined if a single exposure to a larger dose will produce a similar neuropathological outcome. In the present study, morphometric and ultrastructural analyses of rat soleus NMJ were performed to determine early pathological effects of an intraperitoneal injection of 100 mg/kg acrylamide. Widespread nerve terminal degeneration, terminal sprouting, and endplate lengthening were evident as early as 4 days after injection. Degenerating terminal branches were swollen and exhibited enhanced argyrophilia. Ultrastructurally, the majority of terminals exhibited axolemmal abnormalities, neurofilament accumulations, and a paucity of synaptic vesicles; occasional swollen terminals lacked neurofilaments but contained increased numbers of tubulovesicular profiles. This early morphological pattern of nerve terminal changes suggests that acrylamide may disrupt both synaptic vesicle recycling and neurofilament degradation. These findings indicate that a single high dose of acrylamide triggers pathological lesions and remodeling in motor nerve terminals virtually identical to those resulting from multiple low doses.

Prolonged acrylamide administration produces motor nerve-terminal branch degeneration and impairs axonal outgrowth following nerve crush. It is unclear how early terminal branch degeneration is initiated and whether there is a compensatory regenerative response at the neuromuscular junction (NMJ). A modified Pestronk and Drachman silver-acetylcholinesterase strain was used to carry out a detailed morphometric analysis of the NMJ in soleus and lumbrical muscles. Rats were given 3, 5, or 10 doses of acrylamide, 35 mg/kg/day, by intraperitoneal injection, 5 days/week, and killed 4, 7, or 14 days after the first dose, respectively. Degenerating terminal branches were evident in soleus NMJ after only three doses of acrylamide. Diminished synaptic vesicle content, neurofilament accumulations and tubulo-vesicular profiles were evident after three doses. At later time points, degenerating terminals contained few synaptic vesicles and were engorged with neurofilaments. Endplate lengthening, indicative of denervation supersensitivity, accompanied degeneration. Terminal sprouting proliferated after 3 and 5 doses but was less prominent after 10 doses. Although similar changes occurred in the lumbrical muscle, they were not initiated until after 5 doses. These experiments reveal that pathological changes in terminal branches commence earlier and after a lower cumulative dose of acrylamide than previously reported and suggest that acrylamide exerts a primary effect at motor nerve-terminal branches. Early, vigorous terminal sprouting indicates that acrylamide does not prevent the initiation of regeneration, but with prolonged treatment does cause degeneration of maturing sprouts.

Cats were given vincristine sulfate (50 micrograms/kg i.v. every 4 days) and studied after 7-29 injections when neurological deficits became evident. The conduction velocity spectrum of individual afferent nerves from soleus muscles was shifted toward slower velocities. Relatively few functional muscle spindles or other proprioceptors which responded to muscle stretch were encountered. Those primary endings of soleus muscle spindles which did respond were significantly reduced in dynamic but not length sensitivity. Length sensitivity of secondary endings was unchanged. Thresholds of secondary but not primary endings were elevated. The average conduction velocity of soleus motor axons was reduced 30% but no deficit was detected in motor nerve terminal function. Indirectly-elicited contractile tension of the soleus muscles of the neuropathic cats was not significantly lower than that in untreated animals. Amplitudes of spinal monosynaptic reflexes were unaffected. These data indicate, that in the cat, neurological impairment results partly from dysfunction in muscle spindles and peripheral nerves.

Depressed or lost tendon reflexes commonly observed in patients with peripheral neuropathies may result partly from attenuation of the dynamic discharge from the primary endings of muscle spindles. This possibility was investigated in cats with an experimental neuropathy induced with acrylamide (30 mg/kg/day intramuscularly). Achilles tendon reflexes and the dynamic discharge from primary muscle spindles were evaluated after five or ten injections of acrylamide. After five injections the animals were moderately impaired neurologically, the Achilles tendon reflex was difficult to elicit in 2 of 5 animals, and the dynamic responses of primary endings of soleus muscle spindles to stretch were depressed. Following ten injections of acrylamide the cats were severely impaired neurologically; tendon responses were either absent or difficult to elicit, and the dynamic responses of their muscle spindles to stretch were reduced by 50% (p less than 0.01). Additionally, the spindels responded to stretch with only 20 to 30% the normal number of afferent impulses. These data suggest that lost tendon responses in acrylamide neuropathy result in part from inadequate activation of motoneurons by spindle afferent discharge.

A delayed localized neuropathy of peripheral nerves in a single hind leg of the cat develops after a single intraarterial 2 mg/kg injection of diisopropylfluorophosphate (DFP). This neuropathy is manifested by a maximum loss of the capacity of soleus alpha-motor nerve terminals to generate stimulus-bound repetition 21 days after DFP exposure. Phenylmethanesulfonyl fluoride (PMSF) is a protective inhibitor of the neurotoxic esterase which is associated with the development of the delayed organophosphorus neuropathy. Pretreatment of cats with PMSF (30 mg/kg i.p.) 24 h before the DFP injection protected the cats from the delayed neuropathy. No clinical neurotoxic signs were observed at 21 days after DFP. The stimulus-bound repetitive capacity of soleus alpha-motor nerve terminals was not lost at this time and its incidence was much greater than that which occurred in cats not pretreated with PMSF.

The effect of monomeric acrylamide, a potent neurotoxic agent, on total and neuron specific enolase activity was studied in vitro and in vivo. Acrylamide (10 mM) completely inhibited total enolase activity of rat brain soluble fractions. The I50 concentration was 3.7 mM. In rats chronically treated with acrylamide (550 mg/kg total) and exhibiting marked symptoms of neurotoxicity, neuron specific enolase activity was not detectable in sciatic nerves and was only 60% of control activity in brain. Total enolase activity in both central and peripheral nervous tissues was unchanged from control. The results suggest that inhibition of neuron specific enolase may be an important factor in the development of acrylamide neuropathy by interfering with glycolysis in neuronal tissue.

beta, beta'-Iminodipropionitrile (IDPN) produces neurofilamentous giant axonal swellings in proximal internodes of large myelinated axons. Secondary demyelinative changes result from the production of these axonal enlargements. Electrophysiological studies have demonstrated profound alterations in the electrical properties of motor neurons (MN) within the spinal cord. On the basis of intracellular recordings, it has been suggested that electrical contacts may exist between swollen axons and neighboring MN. In addition, the possibility remained that synaptic contacts develop on demyelinated axonal swellings. In the present study, we report the lack of either synapses on demyelinated axonal swellings or direct electrical contacts between neighboring MN. Axonal swellings are surrounded by attenuated processes of glial cells (probably fibrillary astrocytes), a finding discussed in terms of its possible role in the production of ephaptic transmission. There was considerable variation in the degree of axonal enlargements and in the extent of secondary (passive and active) demyelination. It is suggested that these morphological changes may represent structural correlates of some electrophysiological alterations observed in IDPN neuropathy.

In previous investigations acrylamide was found to inhibit several enzymes of glycolysis both in vitro and in vivo. The present study examines the characteristics of the in vitro inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and compares the in vivo effects of acrylamide on GAPDH activity to other analogs. Inhibition of GAPDH produced by acrylamide was characteristic of an irreversible or slowly reversible mechanism. In vivo, GAPDH activity was determined in sciatic nerve, brain, skeletal muscle and liver after cumulative doses of 250, 350 or 500 mg/kg of acrylamide. Specific activities were significantly lower in extensor muscle and liver after the 250 mg/kg dose. Activities in brain and sciatic nerve tended to be decreased but the differences were not statistically significant. Specific activity of GAPDH was decreased in medulla pons, cerebellum and the rest of the brain after a 350 mg/kg cumulative dose of acrylamide, although protein concentrations were not different from those in controls. The maximum decrease was about 20%. Treatment with acrylamide, methylene-bis-acrylamide (non-neurotoxic), or N-isopropylacrylamide (neurotoxic) significantly decreased the weight of the cortex and associated brain areas as well as general body weights. No signs of developing neuropathy were observed during treatment with methylene-bis-acrylamide to a cumulative dose (8.1 mmoles/kg) equivalent to that of acrylamide causing frank paralysis. Although the compound exhibited some ability to inhibit GAPDH in vitro, no decrease in GAPDH activity was found in rat brain. Treatment with N-isopropylacrylamide resulted in progressive neurologic impairment. After treatment to a cumulative dose of the compound causing a severe hind-limb paralysis (9.2 mmoles/kg), a small but significant decrease in GAPDH was found in the three areas of brain examined.

beta-beta'-iminodipropionitrile (IDPN) neurotoxicity is morphologically characterized by the presence of giant axon swellings in the first proximal internodes of motor axons. The electrophysiological consequences of these proximal giant axon swellings on monosynaptic reflexes and recurrent inhibition were investigated along dorsal root-ventral root, medial gastrocnemius-ventral root and soleus-ventral root pathways of IDPN-intoxicated cats 35, 50 and 100 days following initial administration of the toxin (50 mg/kg/wk for 5 weeks). Monosynaptic reflex action potentials, normally relatively synchronous spike potentials, frequently appeared as doublet potentials which did not represent temporal fractionation of the spike potential. Latencies to the monosynaptic reflex action potentials were prolonged. Amplitudes of unconditioned monosynaptic reflex action potentials were significantly decreased at all time points, although there was a tendency for the amplitudes to recover with time. Post-tetanic potentiation was variously altered. Recurrent inhibition was reduced by 42-49% along monosynaptic reflex pathways. These results demonstrate that electrophysiological function in the spinal cord of IDPN-intoxicated cats is profoundly altered and the dysfunction partly results from the presence of the giant axon swellings.

An experimental neuropathy was induced in cats by injections of acrylamide (7.5, 15 or 30 mg/kg/day) for 2 to 10 days. The responses of primary and secondary endings of soleus muscle spindles to stretch were evaluated and correlated with the appearance of ataxia and incoordinated motor movements. Animals that received 15 or 30 mg/kg/day became ataxic and demonstrated poor motor coordination on the 7th or 4th day, respectively. At these times, both primary and secondary endings of muscle spindles had elevated thresholds and diminished discharge frequencies. Continued acrylamide administration resulted in exacerbation of the clinical symptoms and further attenuation of spindle responses. The discontinuation of acrylamide was followed by slow recovery. Only those cats which received a total dose of 75 mg/kg or less remained asymptomatic and had normal spindle function. The coincidence of onset of motor coordination deficits and spindle dysfunction, coupled with a lack of demonstrable motor defect at the same time, suggests that the initial clinical features of acrylamide neuropathy may be partly the consequence of impaired spindle function.

Group la primary afferent terminal (PAT) function was assessed in cats with acrylamide neuropathy. Spinal monosynaptic input-output relationships, critical input, the ability to maintain repetitive discharge at low frequencies, and apparent transmitter turnover parameters were determined in animals with subthreshold, moderate and severe degrees of acrylamide neuropathy (75, 150, and 300 mg/kg, respectively. The monosynaptic pathways of the soleus and medial gastrocnemius motoneuron pools were elicited by stimulation of their respective muscle nerves. The threshold for activation of the monosynaptic reflex (MSR) evoked by both SOL and MG nerves was increased 50% (critical input), regardless of dose regimen. Repetitive stimulation of the MSR at low frequencies (2-10 Hz) produces a slow decline in amplitude until it reaches a plateau. The height of the plateau attained in neuropathic cats was considerably lower than the controls. Determination of apparent transmitter turnover parameters in neuropathic cats showed that the constant fraction of neurotransmitter released from group la PAT remained the same while the rate of replenishment of transmitter available for release was attenuated in both SOL-VR and MG-VR pathways. The increase in the critical input and decrease in the rate of replenishment points up a functional defect in a group la PAT in cats with acrylamide neuropathy.

Administration of beta,beta'-iminodipropionitrile (IDPN) to rodents has previously been shown to produce neurofilament-filled axonal swellings in the proximal regions of motor and sensory nerve fibers. Because of the distinctive distribution of these swellings, IDPN has been classed as a proximal axonopathy and thereby distinguished from other disorders in which similar axonal swellings occur in the distal parts of the axon (distal axonopathies). This report describes the pathology in the peripheral nerves of cats which received intermittent injections of IDPN and calls attention to two previously undescribed pathological changes. First, in addition to the typical proximal swellings associated with IDPN, these animals developed numerous axonal swellings within the distal branches of the sciatic nerve. Distal swellings were present as early as 23 days after initiation of intoxication, indicating that they formed locally (rather than developing in the proximal axon and undergoing transport into the distal regions). The second finding was Wallerian-like degeneration within the affected nerve branches. These changes in the distal sciatic nerve and its branches closely resembled the pathology of the distal axonopathies produced by agents such as the neurotoxic hexacarbons and carbon disulfide. The pathological similarities suggest that IDPN may share with these agents pathogenetic mechanisms to an extent not previously suspected.

This study explored the prejunctional actions of vecuronium using the in vivo cat soleus nerve-muscle preparation. Vecuronium doses of 1 to 5 micrograms/kg iv suppressed the repetitive firing of the motor nerve endings, and the obligatory potentiation of the twitch response following high-frequency conditioning at 400 Hz for 10 s without attenuating neuromuscular transmission at 0.4 Hz. It was also found that extremely low doses of vecuronium had excitatory effects at the cat soleus motor nerve endings: doses of 0.5 and 1 microgram/kg iv evoked a modest postdrug repetitive firing of the nerve endings and a concomitant potentiation of the muscle responses. These dose-related agonist and antagonist activities suggest that at the motor nerve endings, vecuronium is a weak partial agonist. The major action of vecuronium at the motor nerve endings, however, was suppressive, and this antagonist action contributed to the neuromuscular blocking action of this muscle relaxant.

Acrylamide was found to be tissue selective in its toxicity. Doses which were clearly neurotoxic to rats were without effect on hepatic UDP-glucuronyltransferase, total hepatic protein or microsomal protein.

The effects of lidocaine (1--20 microgram/ml) on afferent discharge patterns of primary endings of muscle spindles in cat tenuissimus muscle were investigated. Discharge from the endings, recorded in Ia afferent axons, was evoked by ramp stretch of the muscle, stimulation of single static or dynamic fusimotor axons or by a combination of stretch and fusimotor stimulation. Spontaneous discharge of the endings at the initial length of the muscles was reduced by 2--5 microgram/ml and abolished by 10--15 microgram/ml lidocaine. The static but not the dynamic discharge elicited by muscle stretch was blocked by concentrations of 10--15 microgram/ml. The same concentrations abolished static and dynamic fusimotor influences on primary ending discharge. However, in one experiment where the spindle was microscopically observed, fusimotor stimulation still resulted in contraction of the intrafusal muscle even when fusimotor stimulation failed to elicit changes in discharge response patterns of the primary endings. These findings indicate that lidocaine interferes with the encoding mechanism prior to block of impulse conduction in either the fusimotor or afferent axons.

The in vitro effects of the neurotoxic compounds, acrylamide and 2,5-hexanedione, on several glycolytic enzymes including enolase, phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactic dehydrogenase (LDH) were studied in rat brain. A differential sensitivity of the enzymes to the inhibitory effects of the neurotoxins was observed. The order of increasing sensitivity to 2,5-hexanedione was enolase -- GAPDH -- PFK and to acrylamide the order was PFK -- enolase -- GAPDH. Neither neurotoxin inhibited LDH. The inhibition of enolase by acrylamide exhibited a mixed type pattern in double reciprocal plots. The inhibition could be completely reversed by dialysis indicating that it did not involve covalent bond formation. In the presence of dithiothreitol (DTT) or glutathione the inhibition of enolase by either acrylamide or 2,5-hexanedione was potentiated. Activity of enolase inhibited by both acrylamide and DTT could not be restored to pre-inhibition rates following dialysis indicating that an irreversible interaction between acrylamide and enolase had taken place. The results suggest that neurotoxic compounds which produce distal axonopathies have a common pattern of attack on glycolytic enzymes and that interruption of glycolysis is the underlying biochemical basis for both the physiological and morphological damage caused by these compounds.

beta,beta'-Iminodipropionitrile (IDPN) was given to cats (50 mg/kg/week for 5 weeks) to induce giant axonal swellings in the proximal internodes of motor axons. Conventional intracellular recording techniques were used to investigate the influence of the axon swellings on axonal impulse conduction and generation of action potentials in unidentified lumbosacral motoneurons (MN). Action potentials recorded from axon swellings, verified by lack of orthodromically or antidromically elicited EPSPs or IPSPs, afterhyperpolarization potentials or initial segment-somaldendritic (IS-SD) inflections, were variable in shape. Some were indistinguishable from recordings in normal axons. Component or extra potentials occurred in 45% of recordings from axon swellings; their position on the action potential depended on the direction of impulse invasion into the swelling. Many action potentials were broad, with amplitudes less than 30 mV. Impulse conduction was estimated to be blocked in 19% of motor axons tested. Action potentials recorded in MN of IDPN treated cats resembled in many aspects those recorded in chromatolytic MN, with increased latencies upon antidromic stimulation and decreased IS conduction times and SD thresholds; other parameters did not differ significantly. The minimal interval between two stimuli which each evoked action potentials increased from 3.3 +/- 0.1 to 5.8 +/- 0.5 ms.(ABSTRACT TRUNCATED AT 250 WORDS)

Motoneurons (MNs) exhibit characteristic electrophysiological alterations following axotomy which are concomitants of perikaryal remodeling induced by the axonal injury. beta,beta'-Iminodipropionitrile (IDPN) neurotoxicity, which produces proximal axonal swellings in the first internodes of motor fibers, was studied as a model of perikaryal electrophysiological properties in axonal pathologies without axonal degeneration. Similarities between parameters of MN excitability (delayed depolarizations, repetitive discharge, and the afterhyperpolarization potential, AHP) known to occur in axotomized MN and those in IDPN neuropathy were examined in type-identified spinal MN of cats during the evolution (7 to 35 days) of proximal axonal swellings. Delayed depolarization potentials were observed frequently in fast MN types throughout the neuropathy but only at 35 days in slow MN types. Similarly, repetitive firing occurred most prominently in fast MNs early in the neuropathy. Concomitantly, AHP duration decreased as early as 7 days in all MN types and was significantly shortened in types FF, FR, and S motoneurons. AHP peak amplitude and current declined continuously from 7 to 35 days of the neuropathy and were significantly (p less than 0.05) decreased at 35 days in types FF and S MNs. These results suggest that not only are fast MN types vulnerable early in IDPN neuropathy, but also that all MN types exhibit electrophysiological changes strikingly similar to those following mechanical axotomy. The possibility is raised that IDPN may initiate electrophysiological changes, analogous to perikaryal remodeling, by mechanism(s) unrelated to axonal degeneration.

Previous studies of electrophysiological function of motoneurons (MNs) in animals with beta, beta'-iminodipropionitrile (IDPN) neuropathy revealed alterations in action potential discharge indicative of enhanced MN excitability. These changes, particularly manifested in increased numbers of delayed depolarizations and repetitive firing upon single stimulation, are strikingly similar to those observed in axotomized MNs. The present study extends the parallels between axotomized and neuropathic conditions by assessing rhythmic firing responses of MNs to direct current injection in cats treated with IDPN (50 mg/kg/week for 5 weeks). Changes in excitability and frequency-current relationships were studied on Day 35 of IDPN intoxication and compared to those reported for axotomized (chromatolytic) MNs. In treated animals, lumbar MNs had increased thresholds for sustained discharge, tended to achieve higher maximum discharge frequencies, and adapted less to continuous current injection. In untreated cats 74% of MNs exhibited firing in both primary and secondary ranges, whereas only 39% of those in treated animals made the transition to secondary range firing. Most fired monotonically in the primary range with unchanged slope of the frequency-current relationships. When achieved, secondary range slopes were one-third of normal. MN afterhyperpolarization peak amplitude, duration, and conductance were reduced while input resistance was increased. The findings are discussed in terms of altered MN and dendritic excitability induced by the neuropathy, and similarities to electrophysiological changes in axotomized MNs.

Dantrolene sodium (DS) was investigated for its effects on cat soleus muscle contractile properties and motor nerve terminal activity in particular. DS, 0.1-1.5 mg/kg i.v., caused a dose-dependent depression of indirectly elicited contractile strength which was more pronounced at lower frequencies of stimulation. Maximum tetanic strength at frequencies of 10-400 Hz was depressed to a lesser degree than contractile responses evoked by lower frequencies of stimulation; the twitch/tetanus contraction ratios were reduced with increasing dose, primarily because of diminished twitch. DS was without effect on motor nerve terminals as evidenced by normal post-tetanic repetition in the nerves following DS administration. Post-tetanic potentiation became relatively larger in amplitude as contractile strength was diminished. These data suggest that DS depresses neuromuscular function at a site other than the neural apparatus at the neuromuscular junction.

A localized peripheral neuropathy was induced in cats with di-isopropyl fluorophosphate (DFP). Soleus nerve-muscle preparations, and the motor nerve terminals in particular, were evaluated for responsiveness to edrophonium (200 mug/kg i.v.). Potentiation of contractile strength was absent 24 hr after DFP, and showed a trend toward recovery 7-14 days post-DFP; it then fell to about 25% of normal 3 weeks following DFP administration. During the ensuing 5 weeks this aspect of edrophonium responsiveness was largely regained. The underlying post-drug repetition which gives rise to the potentiated responses was not demonstrable in either the nerve or muscle 3 weeks after DFP, but was again observed 8 weeks after poisoning. These findings suggest a delayed peripheral neuropathy indicative of a trophic deprivation and further illustrate a motor nerve terminal deficit as the initial function alteration in DFP neuropathy.

Post-tetanic potentiation and the underlying post-tetanic repetition in cat soleus muscle require normal motor nerve terminals. These indices of nerve terminal viability are depressed 10 days and absent 15 days after tenotomy of the soleus muscle.

Beta,beta'-iminodipropionitrile (IDPN) is a synthetic compound known to induce massive focal accumulations of neurofilaments almost exclusively in the proximal segments of motor and sensory axons. The present paper deals with a cat intoxicated with IDPN for five weeks. Swellings filled with skeins of maloriented neurofilaments were observed in the initial (non-myelinated) segment of intraparenchymal spinal axons in addition to the swellings consistently located in the first internodes. Filamentous masses in the initial segment were in some cases intermingled with focal accumulations of mitochondria and smooth vesicles to give the proximal axon the classical appearance of 'spheroid'. In some motoneurons, clumps of neurofilaments occupied the axon hillock and extended well into the perikaryon and some dendritic stems, making the borderlines between cell body and processes undistinguishable. Cytoplasmic organelles were displaced and rearranged in the presence of somal neurofibrillary changes. In a few neurons, regardless of the presence of swellings in the axon initial segment, signs of central chromatolysis were noticed, in the form of nuclear caps of Nissl substance and eccentricity of the nucleus. These findings are discussed with reference to other neurotoxic models of neurofibrillary pathology and the immunochemical differences recently detected between neurofilaments in perikarya, dendrites and axons.

Spinal cords of cats treated with the neurotoxic compound beta,beta'-iminodipropionitrile (IDPN) were observed to contain rounded homogeneous bodies, 1-12 microns in diameter, termed "dense microspheres" (DMS). These bodies, absent in control animals, were consistently found only in the ventral horns. No relationship with blood vessels was evident. When stained with PAS and a modified von Kossa's silver nitrate technique, DMS remained negative, showing only very slight metachromasia in some toluidine blue-stained sections. They were consistently acidophilic as evidenced by destaining and differentiation investigations. DMS were observed more frequently in the proximity of nerve cell bodies or closely adjacent to dendrites and their location was mainly extracytoplasmic; with the electron microscope, however, some DMS were also found in glial processes. Rounded osmiophilic bodies, 0.1-0.8 microns in diameter, were noticed in mitochondria of both neurons and glial cells; however, whether they were special forms of DMS or different inclusions was not assessed. Both intra- and extracytoplasmic DMS were similar in ultrastructure, appearing as single membrane-bound spherical or pear-shaped bodies containing a cottony or finely granular matrix. Additionally, both perikaryon and processes of large motoneurons were found to contain pigment granules identified as lipofuscin, which seemed to increase in number and to spread centrifugally in the processes in correlation with duration of the intoxication and size of axonal swellings induced by IDPN.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of cats with acrylamide, either 7.5 or 15 mg/kg IM, once a day for 10 days, resulted in increases of 31 and 47% in the phospholipid content of sciatic nerve, respectively, from a control level of 41.1 +/- 2.7 mg/kg wet weight. Determination of the distribution of individual phospholipids indicated no significant differences between control cats and those receiving a cumulative dose of 150 mg/kg acrylamide. In a separate experiment, cats were treated with the 150 mg/kg dose of acrylamide and the sciatic nerve was divided into proximal and distal portions at the level of the triceps surae nerve. Significant increases in phospholipid content were observed in both the proximal and distal portions of peripheral nerve of the acrylamide-intoxicated cats. This effect was present even when the phospholipid content was expressed in terms of total protein, dry weight or total lipid. Total weight of nerve segments, however, was significantly decreased in the neuropathic animals. The data are consistent with a focal degeneration of axons with relative sparing of phospholipids.

Vincristine is a vinca alkaloid widely utilized in cancer chemotherapy. Its major clinical limitation is due to a drug induced sensory-motor neuropathy, the pathogenesis of which is poorly understood. This communication describes the morphological basis of a vincristine neuropathy in cats. Major pathological lesions were focal axonal swellings (giant axon formations) due to malaligned accumulations of neurofilaments and secondary demyelination of the paranodal type. These were primarily confined to the proximal portions of the peripheral nerves. The distal portions of the peripheral nerves contained only a few giant axon formations. Wallerian degeneration was noted to involve a small number of nerve fibers in the distal regions.

beta,beta'-Iminodipropionitrile (IDPN) causes formation of axonal swellings in the proximal internodes of spinal motor axons. The swellings enlarge and demyelinate with the progression of the neuropathy. The correlation between axonal swellings and electrophysiologic function of motoneurons was examined in cats 2 to 35 days after initial administration of IDPN. Morphologic changes in intraspinal motor axons, occasionally observed 2 days after the first injection, became progressively more evident at later times, with enlargement at the first internode in some axons and appearance of fusiform or balloon-like axonal swellings. At 7 days axonal swellings were infrequently observed and the main structural feature was a reduction in myelin thickness in affected nerve fibers. Despite scant histopathologic changes, motoneuron action potential discharge at this time was significantly altered in latency to onset of spike and rate of rise. Abnormal motoneuron firing patterns were observed at this time. As the neuropathy progressed, both the frequency of occurrence and the size of axonal swellings increased markedly but at no time was there morphologic evidence of chromatolysis. At 14 and 35 days, (after two or five IDPN injections) action potential discharge became further altered in latency to spike onset, rate of rise, initial segment conduction time and somal-dendritic threshold. The incidence of repetitive firing increased and axonal conduction block was observed in several motoneuron recordings. The electrophysiologic changes closely resemble those reported in chromatolytic motoneurons after axotomy. The axonal swellings induced by IDPN may produce an axotomy-like condition which becomes more prominent as the neuropathy progresses but without morphologic evidence of chromatolysis.

To assess motor nerve and motor nerve terminal function in acrylamide neuropathy, cats were given i.m. injections of acrylamide (15 mg/kg) daily for 10 days to induce a peripheral neuropathy. Tests of function were performed on the day of the 10th injection (day 0) and 7, 21 and 35 days thereafter. In untreated animals tetanic conditioning evoked stimulus-bound repetition (SBR) in 85% of soleus alpha-motoneurones. Following administration of acrylamide, the percent of axons elaborating SBR were: day 0 -- 79%, day 7 -- 71%, day 21 -- 31%, day 35 -- 22%. The response of soleus muscle to SBR is normally a post-tetanic potentiation (PTP) of contractile tension which is proportional to the tetanic conditioning frequency; during the development of the neuropathy, PTP in response to all tetanic frequencies progressively declined, concomitant with and as a result of the declining incidence of SBR. These data indicate that initial functional alterations in motor nerves during acrylamide neuropathy occurs at the level of the nerve terminal, preceding alterations in conduction velocities in the axons. However, the motor nerve deficit is not adequate, in either time to onset or severity, to account for the clinical manifestations of the neuropathy. The possible contribution to clinical signs of the neuropathy made by lesions to other peripheral nerves is discussed.

Acrylamide was administered to rats for 10 days (50 mg/kg/day) after ligation of either proximal (sciatic) or distal (posterior tibial) nerves. Morphologic evaluation of the nerves revealed severe degeneration 0.5 cm proximal to the ligature, with progressively less pronounced change at successively higher levels above the ligature, when compared with ligated non-treated controls. Although severe degeneration of the nerves was observed in both proximal- and distal-ligated groups of animals, the distal-ligated group showed slightly more change (at 0.5 cm above the ligature), suggesting a minor effect of distance of the site of nerve injury from the nerve cell body.