Capturing Photoelectron Motion with Guiding Centers.

Electron motion in combined strong laser and Coulomb fields is central to laser-matter interactions. By mapping this problem onto the motion of a guiding center, we derive a reduced model which naturally embeds important Coulomb effects such as focusing and asymmetry, and clearly distinguishes direct versus rescattered electron ionization processes. We demonstrate the power of this tool by unraveling the bifurcation in photoelectron momentum distributions seen in experiments.

Differential responses of human neuroblastoma and glioblastoma to apoptosis.

Staurosporine, a protein kinase and etoposide, a topoisomerase II inhibitor, are known to enhance apoptosis. The differential effects of these agents on T98G glioblastoma and SK-N-SH neuroblastoma, cell lines both derived from human tumors, have not been determined. We assessed cellular viability, DNA fragmentation and laddering, chromatin condensation, and Poly(ADP-ribose) polymerase (PARP) cleavage induced by these agents at a series of concentrations and times. In addition, to gain an understanding of the mechanism by which these agents work, we measured Protein Kinase C (PKC) activity. Staurosporine induced significant alterations in all apoptotic parameters tested in both cell lines. Etoposide induced apoptotic alterations similar to those caused by staurosporine in neuroblastoma but produced no detectable apoptotic changes in glioblastoma cells. Etoposide induced membrane but not cytosolic PKC activity in neuroblastoma but had no effect on PKC activity in glioblastoma. Our results show that the induction of apoptosis is cell type dependent. PKC activity appears to be crucial in the initiation of apoptosis.

Neurology patient education materials: do our educational aids fit our patients' needs?

The purpose of this study was to assess the suitability of various neurology patient education brochures, as well as neurology information on the World Wide Web, to determine whether they were appropriate for patients with low literacy. A convenience sample of 520 patients in university-based public and private neurology clinics participated. In this cross-sectional study, the patients' reading levels were assessed by using the Rapid Estimate of Adult Literacy in Medicine. Forty-three patient education materials were assessed for reading level by using the Fog Index and for suitability characteristics (including content, type and relevance of graphics, layout, type size and fonts, and motivating qualities) by using the Suitability Assessment of Materials. The mean education level of patients was 12th grade; however, the mean reading level was 7th-8th grade. Of the assessed materials, 91% were written at a 9th-grade level or above, even though only 58% of patients read at a 9th-grade level. Only 14% of the materials were considered to have superior suitability, whereas 58% were adequate, and 28% were determined not suitable. Thus, the available neurology patient education aids are not matched to the patient population or to the average U.S. reading levels. Ideally, information needs to be written at a 9th-grade level or below. Instructional graphics, simpler words, patient interaction, and cultural sensitivity improve material suitability.

Poly (ADP-ribose) polymerase induction is an early signal of apoptosis in human neuroblastoma.

Poly (ADP-ribose) polymerase (PARP) is an abundant chromatin associated protein important in DNA repair, maintenance of chromosomal stability and programmed cell death. Here we report that an increase in caspase 3-activity and cleavage of PARP serves as an early execution phase signal in human neuroblastoma. Human neuroblastoma SK-N-SH cells were exposed to a protein kinase inhibitor, staurosporine, or a topoisomerase II inhibitor, etoposide, at various concentrations and time points. Cells exposed to staurosporine (0.1 microM) for 30 min showed an increase in caspase 3-activity and by 1 h an increase in PARP 116-kDa band and an 85-kDa cleavage product, which further increased in density with time after treatment. Quantitative analysis for condensed chromatin material using bisbenzimide, and DNA fragmentation enzyme immunoassays showed a significant increase in apoptosis 5 h after staurosporine treatment. This was further confirmed with a Klenow fragment of DNA polymerase I assay which primarily detects single-stranded DNA breaks. A significant decrease in mitochondrial metabolism occurred within 8-12 h after treatment. Studies using Trypan Blue exclusion, and lactic dehydrogenase (LDH) release revealed a significant increase in membrane permeability 8 h after staurosporine (0.1 microM) or etoposide (10 microM) treatments. Cleavage of lamin B1, a protein important in maintaining the nuclear envelope integrity was observed 12 h after staurosporine treatment. Our results show that activation of caspase 3 followed by PARP cleavage occur at much earlier time point than any other morphological or biochemical parameters of apoptosis or cytotoxicity.

Overexpression in neurons of human presenilin-1 or a presenilin-1 familial Alzheimer disease mutant does not enhance apoptosis.

Programmed cell death, or apoptosis, has been implicated in Alzheimer's disease (AD). DNA damage was assessed in primary cortical neurons infected with herpes simplex virus (HSV) vectors expressing the familial Alzheimer's disease (FAD) gene presenilin-1 (PS-1) or an FAD mutant of this gene, A246E. After infection, immunoreactivity for PS-1 was shown to be enhanced in infected cells. The infected cells exhibited no cytotoxicity, as evaluated by trypan blue exclusion and mitochondrial function assays. Quantitative analysis of cells that were immunohistochemically labeled using a Klenow DNA fragmentation assay or the TUNEL method revealed no enhancement of apoptosis in PS-1-infected cells. This result was confirmed using assays for chromatin condensation and for DNA fragmentation. Expression of PS-1 protected against induction of apoptosis in the cortical neurons by etoposide or staurosporine. The specificity of this phenotype was demonstrated by the fact that cortical cultures infected with recombinant HSV vectors expressing the amyloid precursor protein (APP-695) showed, in contrast, a significant increase in the number of apoptotic cells and an increase in DNA fragmentation for all parameters tested. Our results indicate that overexpression of wild-type or A246E mutant PS-1 does not enhance apoptosis in postmitotic cortical cells and suggest that the previously reported enhancement of apoptosis by presenilins may be dependent on cell type.

Nuclear clustering in myotubes: a proposed role in acetylcholine receptor mRNA expression.

We investigated the functional relationship between nuclear topology, as expressed by degree and type of nuclear aggregation, and appearance of acetylcholine receptor (AChR) subunit mRNAs. Embryonic chick muscle cell cultures treated with the muscle activity blocking agents decamethonium (DCM), d-tubocurare (TBC), and tetrodotoxin (TTX) or co-cultured with cholinergic neurons were examined for the influence of muscle activity on nuclear aggregation and its effects on AChR alpha-, gamma-, and delta-subunit message expression. mRNA was measured by in situ hybridization and nuclei were visualized by bis-benzimide DNA staining. DCM and TBC treatments, as well as neuronal co-culture, resulted in increased nuclear clustering within myotubes and a per nucleus upregulation in mRNA expression relative to control for each subunit. The pattern of nuclear aggregation was treatment dependent, with more and larger aggregates found when myotubes were co-cultured with neurons. Moreover, as nuclear aggregates became larger: (1) nearly all nuclei within active aggregates expressed mRNA and (2) local accumulation (mRNA per unit area) was elevated relative to single nuclei, while per nucleus mRNA production decreased. To determine whether mRNA expression was transient and did not result in steady-state upregulation of AChR receptor protein, we performed a double labeling of surface AChRs with 125I-alpha-bungarotoxin (125I-alpha-BTX) concomitant to the in situ hybridization for mRNA quantification on TTX treated muscle cells. Surface receptor expression tracked mRNA expression forall types of nuclear topology observed, indicating that message levels are in fact reliable indicators of receptor population on the plasma membrane surface in myotubes. We propose that nuclear clustering is an organelle-level, accessory mechanism whereby cells concentrate relatively large amounts of AChR mRNA/protein in specific myotube regions.

Persistent vegetative state in Alzheimer disease. Does it exist?

OBJECTIVE: To determine if the published criteria for diagnosis of the persistent vegetative state could be applied to patients suffering from Alzheimer disease. DESIGN AND METHODS: Eighty-eight institutionalized patients with a diagnosis of possible or probable Alzheimer disease were evaluated for the presence of persistent vegetative state. Initial screening excluded patients who were able to do any of the following: feed themselves, respond to command, walk, or maintain continence of bowel and bladder. A sample of 12 of 28 patients unable to perform any of these functions was examined independently by 3 of us. RESULTS: During the first examination, 2 patients were diagnosed as being in a vegetative state by 2 of us and 3 additional patients by 1 of us. One of us did not diagnose any patient as being in a vegetative state. A second evaluation of the same patients was performed 2 months later, after holding a consensus meeting to standardize the evaluation procedure. During the second evaluation, the vegetative state was diagnosed in 6 patients but only by 1 of us. CONCLUSION: The diagnostic disagreement between the neurologists indicate that Alzheimer disease may only rarely progress to the persistent vegetative state.

Involvement of three glutamate receptor epsilon subunits in the formation of N-methyl-D-aspartate receptors mediating excitotoxicity in primary cultures of mouse cerebellar granule cells.

The N-methyl-D-aspartate receptors have been implicated in neuronal plasticity and their overactivation leads to neurotoxicity. Molecular cloning and co-expression of various glutamate receptor zeta and epsilon complementary DNAs support a heteromeric structural organization for N-methyl-D-aspartate receptors. In this study, we show that cerebellar granular neurons in primary culture of mouse express glutamate receptor zeta1 and at least three glutamate receptor epsilon (epsilon1, epsilon2, and epsilon3) protein subunits. In vitro, the temporal patterns of glutamate receptor epsilon1, epsilon2, and epsilon3 subunit expression depend on culture stages. By day 9, a somatic and neuritic immunolocalization for all N-methyl-D-aspartate subunits was clearly identified in most neuronal, but not glial cells. The role of particular subunits in N-methyl-D-aspartate-mediated excitotoxicity was probed by exposing the cerebellar granule cells to antisense oligodeoxynucleotides generated against specific N-methyl-D-aspartate receptor subunits. Antisense oligodeoxynucleotide treatments significantly down-regulated the amounts of the corresponding N-methyl-D-aspartate subunits. The decrease in N-methyl-D-aspartate subunit protein correlated with a reduction in N-methyl-D-aspartate-induced calcium influx and N-methyl-D-aspartate-mediated excitotoxicity in cerebellar cultures. In contrast, antisense oligodeoxynucleotide treatment failed to protect neurons from 1-methyl-4-phenylpyridinium-induced metabolic cell toxicity. Antisense oligodeoxynucleotide treatment targeted at N-methyl-D-aspartate glutamate receptor epsilon subunits demonstrate that glutamate receptor epsilon1, epsilon2, and epsilon3 proteins form N-methyl-D-aspartate receptors responsible for neurotoxic effects on cerebellar neurons. This study provides direct evidence for the existence of distinct N-methyl-D-aspartate receptor subunit proteins in cerebellar granule cells developing in vitro that may trigger N-methyl-D-aspartate-dependent excitotoxicity.

A novel in situ double-labeling method for simultaneous detection of mRNA and expressed protein or two different mRNAs.

We have developed a novel double-labeling method to investigate multiple gene expression in single cells. The method relies on the use of a radioactive probe followed by a colorimetric probe. Unique to this method, the radioactive signal is first captured on an emulsion pre-coated slide, which totally separates it from the process of color development and prevents any interference with the radiolabeled probe. We cite two applications of the new procedure: (1) to study the correlation between acetylcholine receptor (AChR) alpha-subunit mRNA and protein expression in cultured chick myoblasts and (2) to investigate the co-expression of (AChR) alpha and gamma mRNAs in nascent myotubes. In the former case, the radioactive signal is generated by incubation of live cells with 125I-alpha-bungarotoxin, in the latter, by an in situ hybridization (ISH) with 35S-labeled DNA probe. Colorimetric labeling is accomplished in a second step by ISH using digoxigenin-labeled oligos. Analysis of 203 myoblasts showed that AChR alpha-subunit protein and mRNA are co-expressed. Examination of 4-day-old myotubes suggested that most, but not all, nuclear clusters co-express alpha and gamma mRNAs. These results demonstrate that the described protocol has high sensitivity and specificity for detection of protein and message, or two different mRNAs on a single cell level.

Injury zone denervation in traumatic quadriplegia in humans.

In order to obtain an electrophysiological characterization of the injury zone in traumatic quadriplegia, we performed electromyography and nerve conduction studies on the upper limbs of 15 patients with cervical cord trauma. Evidence of significant axonal loss was found in multiple myotomes of all patients. In most cases, the level of the most severe denervation, as determined by the absence or diminution of the compound motor action potential and the density of fibrillation potentials, was 2-5 spinal segments below the clinically and radiologically defined injury levels. In patients with injuries, the rostral extent of which is at C5 or higher, the most obvious clinical and electromyographic denervation was seen in the intrinsic hand muscles (C8/T1), with complete loss of C8/T1 motor axons in a subset of these patients. Our results document that spinal cord trauma can cause loss of motor axons in regions several segments caudal to the rostral level of injury. This finding may have implications for the pathophysiology of secondary injury, for recovery potential, and for the design of rehabilitation strategies.

DNA strand breaks induced by sustained glutamate excitotoxicity in primary neuronal cultures.

We developed a new approach to study single- and double-stranded DNA breaks during chronic, moderate excitotoxicity resulting from the inhibition of the glutamate transporter in cerebellar granule cell primary cultures. A 24 hr treatment of 2-week-old cultures with L-alpha-amino adipate (LAA), an inhibitor of the cerebellar glutamate uptake transporter, caused a gradual extracellular accumulation of endogenous glutamate that induced reversible morphological change of granule neurons but no neuronal cell death despite sustained, but moderate, elevations of the free intracellular calcium concentrations. Nick translation experiments on isolated nuclei or cells from cerebellar cultures chronically exposed to LAA revealed increased radioactive nucleotide incorporation indicative of DNA nicking. This LAA effect was dose-dependent and suppressed by NMDA receptor antagonists. Cultures treated for 24 hr with LAA and subjected to in situ nick translation showed an intense nuclear labeling of neurons but not glia, which could be abolished by MK801. A similar labeling was also observed in altered nuclei of granule neurons acutely exposed to high glutamate concentrations or undergoing an apoptotic cell death. Although the TUNEL labeling method detected no DNA double-strand breaks in LAA-treated cerebellar cultures, it displayed clear evidence of DNA damage during acute glutamate excitotoxicity or during apoptosis. However, Southern blot analysis of nuclear DNA revealed a DNA laddering only in apoptotic cell death. Our results demonstrate that DNA damage, characterized by DNA single-strand breaks, is an early event in chronic, moderate excitotoxicity. This type of DNA degradation, which appears before any nuclear morphological changes, is distinct from the massive DNA single- and/or double-strand damages observed during acute glutamate excitotoxicity or apoptosis.

Differential expression and co-assembly of NMDA zeta 1 and epsilon subunits in the mouse cerebellum during postnatal development.

The differential distribution of NMDA receptor subunit mRNAs in the developing mammalian cerebellum has been previously described. In this study, we investigated the temporal expression of NMDA receptor proteins in the postnatal murine cerebellum using antibodies specific for the NMDA zeta 1 and NMDA epsilon subunits. Our results showed a gradual increase during the first three weeks of life in the relative amount of NMDA zeta 1 and NMDA epsilon 1 proteins. In contrast, NMDA epsilon 2 increased transiently during this period, reaching a maximum around postnatal day 9 and decreasing thereafter to nearly undetectable levels by the end of the third week of life. The level of NMDA epsilon 3 increased dramatically between postnatal days 9 and 15 and thereafter remained constant. Immunoprecipitation of native proteins revealed that a large fraction of NMDA epsilon 2 was associated with NMDA zeta 1 and epsilon 1. At later developmental stages, NMDA epsilon 3 was predominantly assembled with NMDA zeta 1 but not with NMDA epsilon 1 or NMDA epsilon 2. These results demonstrate that NMDA receptor subtypes, formed by the assembly of different NMDA epsilon subunits with NMDA zeta 1, are sequentially expressed in the developing mouse cerebellum. The time course of their expression suggest, that these NMDA receptor subtypes may contribute to specific aspects of granule cell differentiation in the cerebellum.

Expression of the alpha 4 neuronal nicotinic acetylcholine receptor subunit in the developing mouse hippocampus.

Neurotransmitters such as acetylcholine can control neuritogenesis of hippocampal cells. The timing of its receptors expression consequently may influence synaptogenesis and neuronal activity in the developing hippocampus. We investigated the mRNA expression of the nicotinic acetylcholine-gated ion channel receptor (nAChR) alpha 4 subunit in the embryonic and postnatal hippocampal formation. Although its expression level is low in the adult hippocampus, this protein constitutes the major nAChR subunit in the central nervous system. We carried out in-situ hybridization experiments to determine whether or not the alpha 4 AChR subunit mRNA distributions show evidence of regional and developmental regulation during hippocampal maturation. Our studies reveal that alpha 4 AChR mRNA expression was low at the embryonic stage, but increased transiently during postnatal development reaching a maximum during the second week of life and decreasing thereafter, to a minimum at adulthood. In hippocampal regions, the peak values of alpha 4 AChR expression were between 400 and 800% of adult alpha 4 messenger levels. In the postnatal hippocampus, most of the cells from the pyramidal layer of the CA3 and CA2 areas displayed a strong hybridization signal for the alpha 4 AChR subunit. In the hilus and the CA1 regions, the localization of the alpha 4 transcripts seemed to be restricted to some interneurons and pyramidal cells, respectively. Moderate and uniform in-situ hybridization signals were observed in granular cells from the dentate gyrus. The transient profile of alpha 4 expression suggests that nAChRs may participate in the early postnatal maturation of hippocampal circuity.

Characterization of nicotinic acetylcholine receptors expressed in primary cultures of cerebellar granule cells.

Nicotinic acetylcholine receptors (nAChRs), like other calcium permeable channel receptors, may play a crucial role during neuronal development. We have characterized nAChRs in developing mouse cerebellar granule cells in primary culture. L-[3H]Nicotine, [3H]cytisine and [125I]alpha-bungarotoxin binding experiments revealed the presence of a single class of saturable and specific high affinity binding sites for each ligand. The expression of these nicotinic binding sites followed a developmental pattern reaching a maximum during the establishment of excitatory amino acid synaptic contacts. Immunolabeling with monoclonal antibodies to nAChR subunits revealed the presence of alpha 4 and beta 2 subunits in most neurons. Moreover, some neuronal cells displayed a somatic as well as a neuritic localization for the alpha 7 subunit as shown by [125I]alpha-bungarotoxin autoradiography. The reverse transcription-polymerase chain reaction (RT-PCR) detected the presence of mRNAs for alpha 3, alpha 4, alpha 5, alpha 7, beta 2 and beta 4 nAChR subunits. Non-neuronal cells did not express nAChRs, as shown by [3H]nicotine and [125I]alpha-bungarotoxin binding, immunocytochemistry and PCR. Maximum Ca2+ influx elicited by nicotine, and partly sensitive to alpha-bungarotoxin, was observed around 10-14 days after plating. This correlated with the time period at which the highest number of nicotine binding sites was detected. Sensitivity to several NMDA receptor antagonists as well as to removal of endogenous glutamate by pyruvate transaminase treatment revealed a glutamatergic component in the nicotine stimulated calcium influx. The time-dependent specific nAChR expression and the potential association between nAChRs and NMDA receptor activation suggest that nAChRs may regulate glutamatergic activity during synaptogenesis in cerebellar granule cells.

Myoblast and myotube nuclei display similar patterns of heterogeneous acetylcholine receptor subunit mRNA expression.

Muscle progenitor cells differentiate to myoblasts, and subsequently myotubes, upon expression of muscle specific genes. We and others have previously shown that myotube nuclei, even in the absence of nerve, express AChR alpha subunit RNA at varying levels, with a small subset (about ten percent) of the nuclei expressing at high levels. These findings raised two important questions: 1) is the observed heterogeneity a unique property of the alpha subunits, and 2) when does the heterogeneity begin? In particular, is it induced only at or after the time of fusion, or does it exist at the myoblast stage? We have, therefore, extended our observations to the gamma and delta subunits and we also have examined the distributions of AChR alpha, gamma, and delta subunit RNAs in both myoblasts and myotubes. We used intron and intron-exon probes to detect prespliced transcripts or mature mRNAs in the cells. Because intron-containing transcripts are not transported out of the nuclei, the distributions of these transcripts can indicate their expression patterns among nuclei in the same myotubes. Our results show that both myotubes and myoblasts have distributions of the AChR alpha, gamma, and delta subunit RNAs which differ sharply from that of the U1 RNA or Myo D. Thus, the heterogeneous expression of AChR genes is not only an intrinsic property of muscle cell nuclei (in the sense that it does not require the presence of nerves), but it also exists prior to fusion. Our results suggest that muscle nuclei attain individualized capacities for AChR subunit mRNA production early in their development. Conceptual models consistent with such individuality imply an additional level of regulation beyond the known diffusible transcriptional factors.

Gene expression and cellular content of cathepsin D in Alzheimer's disease brain: evidence for early up-regulation of the endosomal-lysosomal system.

In Alzheimer's disease brains, more than 90% of pyramidal neurons in lamina V and 70% in lamina III displayed 2- to 5-fold elevated levels of cathepsin D (Cat D) mRNA by in situ hybridization compared with neurologically normal controls. Most of these cells appeared histologically normal. The less vulnerable nonpyramidal neuron population in lamina IV had relatively normal message levels. Neuronal populations expressing more Cat D mRNA also displayed quantitatively increased Cat D immunoreactive protein. Cat D mRNA expression was only moderately increased in astrocytes. Degenerating neurons exhibited intense immunoreactivity but lowered Cat D mRNA levels. The upregulation of Cat D synthesis and accumulation of hydrolase-laden lysosomes indicate an early activation of the endosomal-lysosomal system in vulnerable neuronal populations, possibly reflecting early regenerative or repair processes. These abnormalities also represent a basis for altered regulation of amyloid precursor protein processing.

Optimized chemiluminescent detection of DNA amplified in the exponential phase of PCR.

We describe a procedure for quantitative detection of nucleic acids by coupling PCR with improved chemiluminescent detection techniques. After performing PCR in the exponential phase in the presence of a trace amount of digoxigenin-11-dUTP, the amplified products are transferred to a positively charged nylon membrane. The membrane is cleaned with a modified method involving sodium dodecyl sulfate and ethanol washing steps to ensure low backgrounds in chemiluminescent detection. The membrane is exposed to two stacked x-ray films to increase the dynamic response in a film exposure. This sensitive and quantitative procedure is useful for molecular biology studies.

The effect of nicotine on recurrent inhibition in the spinal cord.

Recurrent inhibition via Renshaw cells provides a mechanism by which spinal and supraspinal centers exert control over movement. The conditioned H-reflex technique of Pierrot-Deseilligny and Bussel permits noninvasive assessment of recurrent inhibitory pathways. We employed this technique to investigate changes in Renshaw cell activity due to nicotine (a potent CNS cholinergic agonist that excites Renshaw cells in animals) contained in inhaled tobacco smoke. In 10 normal subjects, cigarette smoking caused a large, rapid drop in the conditioned H-response amplitude, implying increased activation of Renshaw cells. The time course of the change in conditioned H-response amplitude closely approximated the known pharmacokinetics of inhaled nicotine. Nicotine administered via chewing gum had a much slower and less dramatic effect, probably due to the slower rise in blood levels with this mode of administration. Increased activity in Renshaw cells may contribute to spasticity in spinal cord-injured patients, raising the possibility that cigarette smoking could cause further increases in tone in such patients.

Differential distribution of vesicular carriers during differentiation and synapse formation.

Coated and noncoated vesicles participate in cellular protein transport. Both acetylcholine receptors (AChR) and acetylcholinesterase (AChE) are transported via coated vesicles, some of which accumulate beneath the neuromuscular synapse where AChRs cluster. To investigate the mechanisms by which these proteins are transported during postsynaptic remodeling, we purified coated vesicles from the bovine brain via column chromatography (Sephacryl S-1000) and raised monoclonal antibodies to epitopes of the vesicular membranes enriched in AChE. We assayed for AChE (coated vesicle enriched), hexosaminidase (lysosomal contaminants), NADH cytochrome C reductase (mitochondrial containing), and protein and demonstrated electron microscopically using negative staining that the vesicular fraction contained 95% pure coated vesicles. We then injected coated vesicle fractions and the fractions from which the coat was removed intraperitoneally into mice and obtained three monoclonal antibodies: C-33, C-172, and F-22. On immunoblots of purified vesicles and cultured skeletal muscle, mAb C-33 stained a 180 Kd band and mAb C-172 stained a 100 kd band. MAb F-22 stained 50 kd and 55 kd bands and was not characterized further. Immunofluorescent microscopy with C-33 and C-172 revealed punctate fluorescence whose distribution depends upon the stage of myotube development. Four days after plating, myotubes showed punctate fluorescence throughout the myotube, whereas those stained 8 days after plating showed a punctate perinuclear distribution. Myotubes innervated by ciliary neurons show punctate fluorescence limited to the nuclear periphery and most concentrated around nuclei which line up beneath neuronal processes. This differential vesicular distribution, observed during myotube differentiation and innervation, suggests that these vesicles participate in vesicular membrane traffic.

Axonal branching and growth cone structure depend on target cells.

Growth cones provide crucial guidance to neurons in response to appropriate targets and other environmental cues. We have cocultured ciliary neurons with myotubes and utilized antibodies to GAP-43 (a neuron-specific, growth-associated phosphoprotein) and MAP-2 (a cytoskeletal marker for dendrites) together with immunofluorescence microscopy to characterize the changes in patterns and polarity that ciliary nerve growth cones undergo when they contact a "proper" target. Our results show that ciliary neurons plated alone or cocultured with fibroblasts have one or two axons, but, when cocultured with myotubes, most cells have 4 or 5 axons showing GAP-43 immunoreactivity. The mean number of axons per cell soma was 1.9 +/- 0.9 micron2 (SEM) when ciliary neurons were plated alone and 3.4 +/- 0.1 when ciliary neurons were cocultured with myotubes. Differences in growth cone size were readily apparent in these two types of cultures. In coculture with myotubes the neuronal growth cone lamelopodia occupied 20-30 microns2 with an average area of 25.0 +/- 2.3 microns2 (SEM) whereas those neurons plated alone or in the presence of fibroblasts occupied an average area of 56.3 microns2 +/- 4.1 microns2 (SEM). The number of growth cone filopodia depended on culture condition. In nerve-muscle cocultures, the average number of filopodia per growth cone was 3.6 +/- 0.2 (SEM), whereas in ciliary cultures plated in the absence of myotubes the number of filopodia was 6.8 +/- 0.4 (SEM). These results indicate that muscle cells or the factors they release can regulate the growth and topography of axons and their growth cones.