Methylmercury alters Eph and ephrin expression during neuronal differentiation of P19 embryonal carcinoma cells.

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.

Enhanced resolution of histochemical distribution of glucose-6-phosphate dehydrogenase activity in rat neural tissue by use of a semipermeable membrane.

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.

Occurrence of lipofuscin pigment granules and "dense microspheres" in the spinal cord of young cats treated with beta,beta'-iminodipropionitrile (IDPN).

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)

Muscle spindle function in organophosphorus neuropathy.

The contribution of muscle spindle dysfunction to the neurological signs o-delayed organophosphorus neuropathy was investigated in the hindlimbs of cats intra-arterially injected with 2 mg/kg of diisopropylfluorophosphate (DFP). In this model of a delayed peripheral neuropathy, the animals exhibit a peculiar high-step gait and a sluggish response to noxious stimuli. These signs initially appear 14 days and are maximum 21--28 days after DFP administration. The position sensitivities of secondary but not primary soleus muscle spindles were depressed at 14 days after DFP. At 21 days after DFP, both primary and secondary endings had attenuated position sensitivities and significantly elevated thresholds. Spindle function was normal at 56 days after DFP exposure. The onset, peak and recovery of soleus muscle spindle dysfunction corresponded to those in alpha-motor axons, indicating the mixed sensory motor nature of organophosphorus neuropathy. Thus, impairment of both sensory and motor functions contributes to the neurological signs of this neuropathy.

Electrophysiological investigation of beta,beta'-iminodipropionitrile neuropathy: intracellular recordings in spinal cord.

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)

The etiology of acrylamide neuropathy: possible involvement of neuron specific enolase.

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.

The etiology of toxic peripheral neuropathies: in vitro effects of acrylamide and 2,5-hexanedione on brain enolase and other glycolytic enzymes.

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.

Structural correlates of physiological abnormalities in beta, beta'-iminodipropionitrile.

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.

Dantrolene effects on neuromuscular function in cat soleus muscle.

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.

Studies on drug-induced neuropathies. III. Motor nerve deficit in cats with experimental acrylamide neuropathy.

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.

Motor nerve terminal response to edrophonium in delayed DFP neuropathy.

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.

Modification by lidocaine of the discharges of primary endings of muscle spindles in cat tenuissimus muscle.

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.

Motor unit function during evolution of proximal axonal swellings.

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.

Group Ia primary afferent terminal defect in cats with acrylamide neuropathy.

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.

Electrophysiological investigation of beta, beta'-iminodipropionitrile neurotoxicity: monosynaptic reflexes and recurrent inhibition.

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.

Inhibition of glyceraldehyde-3-phosphate dehydrogenase in tissues of the rat by acrylamide and related compounds.

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.

Electrophysiological investigation of IDPN neuropathy--initial studies.

beta,beta'-iminodipropionitrile (IDPN) causes giant axon swellings in proximal internodes of spinal motor axons and in stem processes of dorsal root ganglia neurons. The electrophysiological consequences of the swellings were investigated in cats given IDPN (50 mg/kg i.p.) weekly for 0 (control), 1 (7 days), 2 (14 days), or 5 (35 days) weeks; some additional animals were studied 15 (50 days) or 65 (100 days) days following the fifth injection. Extracellular recordings of the spinal monosynaptic reflex revealed amplitudes of efferent responses to be equally reduced at 35, 50 and 100 days. Single stimulation of dorsal roots, or soleus or medial gastrocnemius afferents often evoked doublet efferent responses which arose intraspinally. Similar repetitive responses were observed in dorsal root (afferent) input upon single stimulation of peripheral nerves. Latencies to monosynaptic responses became progressively prolonged over 100 days; spinal cord contribution to the increased latencies was maximal by 35 days. Conduction velocities in single soleus and medial gastrocnemius axons declined to 70 and 64% of normal by 50 days and to 56 and 50% of normal by 100 days respectively. Maximal recurrent inhibition was reduced 42-49% by 35 days and quantitatively similar at 50 and 100 days. Intracellular recordings from spinal motoneurons revealed that, at 35 days, action potentials could be elicited by orthodromic but not by antidromic stimulation in 14% of cells tested. This was taken as evidence of block of impulse conduction by the intervening axonal swelling upon antidromic stimulation. The possibility of abnormal electrical interactions between swollen and demyelinated intraparenchymal axons was tested by determining the number of ventral root fibers via which antidromic stimulation would elicit an action potential in the same motoneuron perikaryon. Electrical crosstalk, never seen in normal animals, varied in incidence during the evolution of the neuropathy (7-35 days), reaching a maximum of 30% at 14 days. Many motoneuron action potentials were remarkably similar to those observed in chromatolytic motoneurons, exhibiting, among other features, decreased SD thresholds and IS conduction times, reduced amplitudes and durations of afterhyperpolarization, delayed depolarizations and an enhanced propensity to fire repetitively upon single stimulation. There was no morphological evidence of chromatolysis in the motoneurons. The concept of "functional axotomy" is introduced to accommodate these findings and discussed in terms of altered dendritic excitability. Not all motoneuron types are equally compromised in the neuropathy.(ABSTRACT TRUNCATED AT 400 WORDS)

Ectopic impulse generation in dorsal root ganglion neurons during methylmercury intoxication: an electrophysiological and morphological study.

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.

Substrates for neural metabolism of xenobiotics in adult and developing brain.

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.

Developmental changes in the cellular distribution of glutathione and glutathione S-transferases in the murine nervous system.

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.