BCL-2 and BAX protect adult mice from lethal Sindbis virus infection but do not protect spinal cord motor neurons or prevent paralysis

Cellular proteins that regulate apoptotic cell death can modulate the outcome of Sindbis virus (SV) encephalitis in mice. Both endogenous and overexpressed BCL-2 and BAX proteins protect newborn mice from fatal SV infection by blocking apoptosis in infected neurons. To determine the effects of these cellular factors on the course of infection in older animals, a more neurovirulent SV vector (dsNSV) was constructed from a viral strain that causes both prominent spinal cord infection with hind-limb paralysis and death in weanling mice. This vector has allowed assessment of the effects of BCL-2 and BAX on both mortality and paralysis in these hosts. Similar to newborn hosts, weanling mice infected with dsNSV encoding BCL-2 or BAX survived better than animals infected with control viruses. This finding indicates that BCL-2 and BAX both protect neurons that mediate host survival. Neither cellular factor, however, could suppress the development of hind-limb paralysis or prevent the degeneration of motor neurons in the lumbar spinal cord. Infection of BAX knockout mice with dsNSV demonstrated that endogenous BAX also enhances the survival of animals but has no effect on paralysis. These findings for the spinal cord are consistent with earlier data showing that dying lumbar motor neurons do not exhibit an apoptotic morphology. Thus, divergent cell death pathways are activated in different target populations of neurons during neurovirulent SV infection of weanling mice.     

The prostate apoptosis response-4 protein participates in motor neuron degeneration in amyotrophic lateral sclerosis.

Prostate apoptosis response-4 (Par-4), a protein containing a leucine zipper domain within a death domain, is up-regulated in prostate cancer cells and hippocampal neurons induced to undergo apoptosis. Here, we report higher Par-4 levels in lumbar spinal cord samples from patients with amyotrophic lateral sclerosis (ALS) than in lumbar spinal cord samples from neurologically normal patients. We also compared the levels of Par-4 in lumbar spinal cord samples from wild-type and transgenic mice expressing the human Cu/Zn-superoxide dismutase gene with a familial ALS mutation. Relative to control samples, higher Par-4 levels were observed in lumbar spinal cord samples prepared from the transgenic mice at a time when they had hind-limb paralysis. Immunohistochemical analyses of human and mouse lumbar spinal cord sections revealed that Par-4 is localized to motor neurons in the ventral horn region. In culture studies, exposure of primary mouse spinal cord motor neurons or NSC-19 motor neuron cells to oxidative insults resulted in a rapid and large increase in Par-4 levels that preceded apoptosis. Pretreatment of the motor neuron cells with a Par-4 antisense oligonucleotide prevented oxidative stress-induced apoptosis and reversed oxidative stress-induced mitochondrial dysfunction that preceded apoptosis. Collectively, these data suggest a role for Par-4 in models of motor neuron injury relevant to ALS.     

Motor neuronal protection by l-arginine prolongs survival of mutant SOD1 (G93A) ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Despite the fact that many different therapeutic strategies have been applied to prevent disease progression, no cure or effective therapy is currently available for ALS. We found that l-arginine protects cultured motor neurons from excitotoxic injury. We also found that l-arginine supplementation both prior to and after the onset of motor neuron degeneration in mtSOD1 (G93A) transgenic ALS mice significantly slowed the progression of neuropathology in lumbar spinal cord, delayed onset of motor dysfunction, and prolonged life span. Moreover, l-arginine treatment was associated with preservation of arginase I activity and neuroprotective polyamines in spinal cord motor neurons. Our findings show that l-arginine has potent in vitro and in vivo neuroprotective properties and may be a candidate for therapeutic trials in ALS.     

Erythropoietin attenuates the sequels of ischaemic spinal cord injury with enhanced recruitment of CD34+ cells in mice

Erythropoietin has been shown to promote tissue regeneration after ischaemic injury in various organs. Here, we investigated whether Erythropoietin could ameliorate ischaemic spinal cord injury in the mouse and sought an underlying mechanism. Spinal cord ischaemia was developed by cross-clamping the descending thoracic aorta for 7 or 9 min. in mice. Erythropoietin (5000 IU/kg) or saline was administrated 30 min. before aortic cross-clamping. Neurological function was assessed using the paralysis score for 7 days after the operation. Spinal cords were histologically evaluated 2 and 7 days after the operation. Immunohistochemistry was used to detect CD34(+) cells and the expression of brain-derived neurotrophic factor and vascular endothelial growth factor. Each mouse exhibited either mildly impaired function or complete paralysis at day 2. Erythropoietin-treated mice with complete paralysis demonstrated significant improvement of neurological function between day 2 and 7, compared to saline-treated mice with complete paralysis. Motor neurons in erythropoietin-treated mice were more preserved at day 7 than those in saline-treated mice with complete paralysis. CD34(+) cells in the lumbar spinal cord of erythropoietin-treated mice were more abundant at day 2 than those of saline-treated mice. Brain-derived neurotrophic factor and vascular endothelial growth factor were markedly expressed in lumbar spinal cords in erythropoietin-treated mice at day 7. Erythropoietin demonstrated neuroprotective effects in the ischaemic spinal cord, improving neurological function and attenuating motor neuron loss. These effects may have been mediated by recruited CD34(+) cells, and enhanced expression of brain-derived neurotrophic factor and vascular endothelial growth factor.     

Activation of divergent neuronal cell death pathways in different target cell populations during neuroadapted sindbis virus infection of mice

Infection of adult mice with neuroadapted Sindbis virus (NSV) results in a severe encephalomyelitis accompanied by prominent hindlimb paralysis. We find that the onset of paralysis parallels morphologic changes in motor neuron cell bodies in the lumbar spinal cord and in motor neuron axons in ventral nerve roots, many of which are eventually lost over time. However, unlike NSV-induced neuronal cell death found in the brain of infected animals, the loss of motor neurons does not appear to be apoptotic, as judged by morphologic and biochemical criteria. This may be explained in part by the lack of detectable caspase-3 expression in these cells.     

Ablation of the Ferroptosis Inhibitor Glutathione Peroxidase 4 in Neurons Results in Rapid Motor Neuron Degeneration and Paralysis

Glutathione peroxidase 4 (GPX4), an antioxidant defense enzyme active in repairing oxidative damage to lipids, is a key inhibitor of ferroptosis, a non-apoptotic form of cell death involving lipid reactive oxygen species. Here we show that GPX4 is essential for motor neuron health and survival in vivo. Conditional ablation of Gpx4 in neurons of adult mice resulted in rapid onset and progression of paralysis and death. Pathological inspection revealed that the paralyzed mice had a dramatic degeneration of motor neurons in the spinal cord but had no overt neuron degeneration in the cerebral cortex. Consistent with the role of GPX4 as a ferroptosis inhibitor, spinal motor neuron degeneration induced by Gpx4 ablation exhibited features of ferroptosis, including no caspase-3 activation, no TUNEL staining, activation of ERKs, and elevated spinal inflammation. Supplementation with vitamin E, another inhibitor of ferroptosis, delayed the onset of paralysis and death induced by Gpx4 ablation. Also, lipid peroxidation and mitochondrial dysfunction appeared to be involved in ferroptosis of motor neurons induced by Gpx4 ablation. Taken together, the dramatic motor neuron degeneration and paralysis induced by Gpx4 ablation suggest that ferroptosis inhibition by GPX4 is essential for motor neuron health and survival in vivo.     

Maintenance of motor neuron progenitors in Xenopus requires a novel localized cyclin

The ventral spinal cord contains a pool of motor neuron progenitors (pMNs), which sequentially generate motor neurons and oligodendrocytes in the embryo. The mechanisms responsible for the maintenance of pMNs are not clearly understood. We have identified a novel cyclin, cyclin Dx (ccndx), which is specifically expressed in pMNs in Xenopus. Here, we show that inhibition of ccndx causes paralysis in embryos. Furthermore, we show that maintenance of pMNs requires ccndx function. In addition, inhibition of ccndx results in the specific loss of differentiated motor neurons. However, the expression of interneuron or sensory neuron markers is unaffected in these embryos, suggesting that the role of ccndx is specifically to maintain pMNs. Thus, we have identified, for the first time, a tissue-specific cell-cycle regulator that is essential for the maintenance of a pool of neural progenitors in the vertebrate spinal cord.     

SV2 mediates entry of tetanus neurotoxin into central neurons

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.     

Poliovirus induces indoleamine-2,3-dioxygenase and quinolinic acid synthesis in macaque brain.

Accumulation of the neurotoxin quinolinic acid within the brain occurs in a broad spectrum of patients with inflammatory neurologic disease and may be of neuropathologic significance. The production of quinolinic acid was postulated to reflect local induction of indoleamine 2,3-dioxygenase by cytokines in reactive cells and inflammatory cell infiltrates within the central nervous system. To test this hypothesis, macaques received an intraspinal injection of poliovirus as a model of localized inflammatory neurologic disease. Seventeen days later, spinal cord indoleamine 2,3-dioxygenase activity and quinolinic acid concentrations in spinal cord and cerebrospinal fluid were both increased in proportion to the degree of inflammatory responses and neurologic damage in the spinal cord, as well as the severity of motor paralysis. The absolute concentrations of quinolinic acid achieved in spinal cord and cerebrospinal fluid exceeded levels reported to kill spinal cord neurons in vitro. Smaller increases in indoleamine 2,3-dioxygenase activity and quinolinic acid concentrations also occurred in parietal cortex, a poliovirus target area. In frontal cortex, which is not a target for poliovirus, indoleamine 2,3-dioxygenase was not affected. A monoclonal antibody to human indoleamine 2,3-dioxygenase was used to visualize indoleamine 2,3-dioxygenase predominantly in grey matter of poliovirus-infected spinal cord, in conjunction with local inflammatory lesions. Macrophage/monocytes in vitro synthesized [13C6]quinolinic acid from [13C6]L-tryptophan, particularly when stimulated by interferon-gamma. Spinal cord slices from poliovirus-inoculated macaques in vitro also converted [13C6]L-tryptophan to [13C6]quinolinic acid. We conclude that local synthesis of quinolinic acid from L-tryptophan within the central nervous system follows the induction of indoleamine-2,3-dioxygenase, particularly within macrophage/microglia. In view of this link between immune stimulation and the synthesis of neurotoxic amounts of quinolinic acid, we propose that attenuation of local inflammation, strategies to reduce the synthesis of neuroactive kynurenine pathway metabolites, or drugs that interfere with the neurotoxicity of quinolinic acid offer new approaches to therapy in inflammatory neurologic disease.     

The transneuronal spread phenotype of herpes simplex virus type 1 infection of the mouse hind footpad.

The mouse hind footpad inoculation model has served as a standard laboratory system for the study of the neuropathogenesis of herpes simplex virus type 1 (HSV-1) infection. The temporal and spatial distribution of viral antigen, known as the transneuronal spread phenotype, has not previously been described; nor is it understood why mice develop paralysis in an infection that involves sensory nerves. The HSV-as-transneuronal-tracer experimental paradigm was used to define the transneuronal spread of HSV-1 in this model. A new decalcification technique and standard immunocytochemical staining of HSV-1 antigens enabled a detailed analysis of the time-space distribution of HSV-1 in the intact spinal column. Mice were examined on days 3, 4, 5, and 6 postinoculation (p.i.) of a lethal dose of wild-type HSV-1 strain 17 syn+. Viral antigen was traced retrograde into first-order neurons in dorsal root ganglia on day 3 p.i., to the dorsal spinal roots on days 4 and 5 p.i., and to second- and third-order neurons within sensory regions of the spinal cord on days 5 and 6 p.i. HSV-1 antigen distribution was localized to the somatotopic representation of the footpad dermatome within the dorsal root ganglia and spinal cord. Antigen was found in the spinal cord gray and white matter sensory neuronal circuits of nociception (the spinothalamic tract) and proprioception (the dorsal spinocerebellar tract and gracile fasciculus). Within the brain stems and brains of three paralyzed animals examined late in infection (days 5 and 6 p.i.), HSV antigen was restricted to the nucleus subcoeruleus region bilaterally. Since motor neurons were not directly involved, we postulate that hindlimb paralysis may have resulted from intense involvement of the posterior column (gracile fasciculus) in the thoracolumbar spinal cord, a region known to contain the corticospinal tract in rodents.     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis.

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by approximately 30%. The death of motor neurons was confirmed using the terminal transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl-modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord.     

Age-dependent poliomyelitis of mice: expression of endogenous retrovirus correlates with cytocidal replication of lactate dehydrogenase-elevating virus in motor neurons.

The widespread presence of endogenous retroviruses in the genomes of animals and humans has suggested that these viruses may be involved in both normal and abnormal developmental processes. Previous studies have indicated the involvement of endogenous ecotropic murine leukemia virus (MuLV) in the development of age-dependent poliomyelitis caused by infection of old C58 or AKR mice by lactate dehydrogenase-elevating virus (LDV). The only genetic components which segregate with susceptibility to LDV-induced paralytic disease are multiple proviral copies of ecotropic MuLV and the permissive allele, at the Fv-1 locus, for N-tropic, ecotropic virus replication (Fv-1n/n). Using in situ hybridization and Northern (RNA) blot hybridization, we have correlated the expression of the endogenous MuLV, both temporally and spatially, with LDV infection of anterior horn motor neurons and the development of paralysis. Our data indicate that treatment of 6- to 7-month-old C58/M mice with cyclophosphamide, which renders these mice susceptible to LDV-induced paralytic disease, results in transient increases in ecotropic MuLV RNA levels in motor neurons throughout the spinal cord. Peripheral inoculation of C58/M mice with LDV, at the time of elevated MuLV RNA levels, results in a rapid spread of LDV to some spinal cord motor neurons. LDV infections then spread slowly but progressively throughout the spinal cord, involving an increasing number of motor neurons. LDV replication is cytocidal and results in neuron destruction and paralysis of the infected animals 2 to 3 weeks postinfection. The slow replication of LDV in the spinal cord contrasts sharply with the rapid replication of LDV in macrophages, the normal host cells for LDV, during the acute phase of infection. The data indicate that the interaction between the endogenous MuLV with the generally nonpathogenic murine togavirus LDV occurs at the level of the motor neuron. We discuss potential mechanisms for the novel dual-virus etiology of age-dependent poliomyelitis of mice.     

Inhibition of murine retrovirus-induced neurodegeneration in the spinal cord by explant culture.

The neurovirulent chimeric mouse ecotropic retrovirus FrCasE causes a rapid neurodegenerative disease of the central nervous system (CNS) characterized by the appearance of spongiform lesions in motor areas 10 days after neonatal inoculation. To study the details of the pathogenic process, we examined the ability of an ex vivo spinal cord model to recapitulate disease. Organotypic spinal cord slice cultures were established from IRW mice 7 days after neonatal inoculation. This corresponds to a time when virus expression in the CNS is first detectable but spongiform changes have yet to evolve. Infectivity associated with these cultures peaked at 7 days in vitro and persisted at this level for 6 weeks. FrCasE infection of the spinal cord slices was primarily found associated with microglial cells. Infection of neurons, astrocytes, oligodendroglia, and endothelial cells was not observed; however, significant astrogliosis was found. Despite the presence of extensive microglial infection in close association with spinal motor neurons in organotypic cultures, no virus-specific spongiform degenerative changes were observed. These results suggest that removal of motor neurons from the developing CNS, despite maintaining the local cytoarchitectural relationships, prevents the virus from eliciting its pathological effects. Possible reasons for the interruption of lesion development are discussed.     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by ∼30%. The death of motor neurons was confirmed using the terminal transferase‐mediated deoxyuridine triphosphate‐biotin nick‐end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl‐modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 185–201, 1999     

Electrophysiological Properties of Motor Neurons in a Mouse Model of Severe Spinal Muscular Atrophy: In Vitro versus In Vivo Development

We examined the electrophysiological activity of motor neurons from the mouse model of severe spinal muscular atrophy (SMA) using two different methods: whole cell patch clamp of neurons cultured from day 13 embryos; and multi-electrode recording of ventral horns in spinal cord slices from pups on post-natal days 5 and 6. We used the MED64 multi-electrode array to record electrophysiological activity from motor neurons in slices from the lumbar spinal cord of SMA pups and their unaffected littermates. Recording simultaneously from up to 32 sites across the ventral horn, we observed a significant decrease in the number of active neurons in 5–6 day-old SMA pups compared to littermates. Ventral horn activity in control pups is significantly activated by serotonin and depressed by GABA, while these agents had much less effect on SMA slices. In contrast to the large differences observed in spinal cord, neurons cultured from SMA embryos for up to 21 days showed no significant differences in electrophysiological activity compared to littermates. No differences were observed in membrane potential, frequency of spiking and synaptic activity in cells from SMA embryos compared to controls. In addition, we observed no difference in cell survival between cells from SMA embryos and their unaffected littermates. Our results represent the first report on the electrophysiology of SMN-deficient motor neurons, and suggest that motor neuron development in vitro follows a different path than in vivo development, a path in which loss of SMN expression has little effect on motor neuron function and survival.     

Upregulation of metallothionein-I mRNA expression in a rodent model for amyotrophic lateral sclerosis

Metallothionein (MT) mRNA expression was investigated in a rodent model (G93A SOD1 transgenic mouse) for a lethal motor neuron disease, amyotrophic lateral sclerosis (ALS). In 8-wk-old mice that did not yet exhibit motor paralysis, MT-I mRNA expression was already significantly upregulated in the region of the spinal cord responsible for motor paralysis. The expression of another isoform, MT-III, was not changed. In the cerebellum, which is not responsible for motor paralysis in ALS, neither the expression profiles of MT-I nor MT-III were altered. In 16-wk-old mice exhibiting motor paralysis, the expression of MT-I mRNA remained upregulated and the MT-III level tended to be elevated. Although no significant differences were found in the levels of both isoforms in the liver or kidney of 8-wk-old mice, the MT-I mRNA expression level was significantly upregulated in the kidney and liver of 16-wk-old mice. These results indicated that the MT-I isoform, but not the MT-III isoform, is associated with motor neuron death in ALS and suggested that the disease might be a systemic disorder to which the spinal cord is particularly susceptible.     

The role of CD4(+) T cells in biphasic hind limb paralysis induced by the D variant of encephalomyocarditis virus (EMC-D) in DBA/2 mice.

DBA/2 CrSlc mice infected with the D variant of encephalomyocarditis virus (EMC-D) (10 PFU/head) developed biphasic hind limb paralysis due to spinal cord lesion. The early phase lesion was characterized by demyelination with infiltration of macrophages in the funiculus lateraris and the late phase lesion by degeneration of motor neurons with infiltration of CD4(+) T cells in the cornu ventrale. In the present study, treatment with anti-Mac1 monoclonal antibody (MAb) or anti-CD4 MAb prior to virus infection (-3 to -1 days) reduced the early phase lesion and the incidence of the first paralysis. Signals of viral RNAs were observed only in a few oligodendrocytes in the funiculus lateraris. Treatment with anti-CD4 MAb from 31 to 33 days post infection when mice showed recovery from the first paralysis reduced the late phase lesion and prevented the second paralysis. Signals of viral RNAs were still detected in a few degenerated neurons in the cornu ventrale. These results indicate that while macrophages and CD4(+) T cells participate in the early phase lesion and paralysis and only CD4(+) T cells in the late phase lesion and paralysis.     

Regulation of cholinergic expression in cultured spinal cord neurons

Factors regulating development of cholinergic spinal neurons were examined in cultures of dissociated embryonic rat spinal cord. Levels of choline acetyltransferase (CAT) activity in freshly dissociated cells decreased rapidly, remained low for the first week in culture, and then increased. The decrease in enzyme activity was partially prevented by increased cell density or by treatment with spinal cord membranes. CAT activity was also stimulated by treatment with MANS, a molecule solubilized from spinal cord membranes. The effects of MANS were greatest in low-density cultures and in freshly plated cells, suggesting that the molecule may substitute for the effects of elevated density and cell-cell contact. CAT activity in ventral (motor neuron-enriched) spinal cord cultures was similarly regulated by elevated density or treatment with MANS, whereas enzyme activity was largely unchanged in mediodorsal (autonomic neuron-enriched) cultures under these conditions. These observations suggest that development of cholinergic motor neurons and autonomic neurons are not regulated by the same factors. Treatment of ventral spinal cord cultures with MANS did not increase the number of cholinergic neurons detected by immunocytochemistry with a monoclonal CAT antibody, suggesting that MANS did not increase motor neuron survival but rather stimulated levels of CAT activity per neuron. These observations indicate that development of motor neurons can be regulated by cell-cell contact and that the MANS factor may mediate the stimulatory effects of cell-cell contact on cholinergic expression.