Axon-like Processes Research Articles

Axonal regeneration and development of de novo axons from distal dendrites of adult feline commissural interneurons after a proximal axotomy

Following proximal axotomy, several types of neurons sprout de novo axons from distal dendrites. These processes may represent a means of forming new circuits following spinal cord injury. However, it is not know whether mammalian spinal interneurons, axotomized as a result of a spinal cord injury, develop de novo axons. Our goal was to determine whether spinal commissural interneurons (CINs), axotomized by 3-4-mm midsagittal transection at C3, form de novo axons from distal dendrites. All experiments were performed on adult cats. CINs in C3 were stained with extracellular injections of Neurobiotin at 4-5 weeks post injury. The somata of axotomized CINs were identified by the presence of immunoreactivity for the axonal growth-associated protein-43 (GAP-43). Nearly half of the CINs had de novo axons that emerged from distal dendrites. These axons lacked immunoreactivity for the dendritic protein, microtubule-associated protein2a/b (MAP2a/b); some had GAP-43-immunoreactive terminals; and nearly all had morphological features typical of axons. Dendrites of other CINs did not give rise to de novo axons. These CINs did, however, each have a long axon-like process (L-ALP) that projected directly from the soma or a very proximal dendrite. L-ALPs were devoid of MAP2a/b immunoreactivity. Some of these L-ALPs projected through the lesion and formed bouton-like swellings. These results suggest that proximally axotomized spinal interneurons have the potential to form new connections via de novo axons that emerge from distal dendrites. Others may be capable of regeneration of their original axon.

Interaction of receptor-activity-modifying protein1 with tubulin

Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified β-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and β-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and β-tubulin. Interestingly, α-tubulin was co-extracted with β-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated α- and β-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors.

Inhibition of protein phosphatases induces transport deficits and axonopathy

The activity of protein phosphatase (PP)-2A and PP-1 decreased in the brains of Alzheimer's disease and inhibition of the phosphatases led to spatial memory deficit in rats. However, the molecular basis underlying memory impairment of the phosphatase inhibition is elusive. In the present study, we observed a selective inhibition of PP-2A and PP-1 with Calyculin A (CA) not only caused hyperphosphorylation of cytoskeletal proteins, but also impaired the transport of pEGFP-labeled neurofilament-M subunit in the axon-like processes of neuroblastoma N2a cells and resulted in accumulation of neurofilament in the cell bodies. To analyze the morphological alteration of the cells during inhibition of the phosphatases, we established a cell model showing steady outgrowth of axon-like cell processes and employed a stereological system to analyze the retraction of the processes. We found CA treatment inhibited outgrowth of the cell processes and prolonged treatment with CA caused retraction of the processes and meanwhile, the early neurodegenerative varicosities were also obvious in the CA-treated cells. We conclude suppression of PP-2A and PP-1 by CA not only damages intracellular transport but also leads to cell degeneration, which may serve as the functional and structural elements for the memory deficits induced by suppression of the phosphatases.

The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines

Diazinon and cypermethrin are pesticides extensively used in sheep dipping. Diazinon is a known anti-cholinesterase, but there is limited information regarding its molecular mechanism of action. This paper describes the effects of diazinon and cypermethrin at a morphological and molecular level on differentiating mouse N2a neuroblastoma and rat C6 glioma cell lines. Concentrations up to 10 microM of both compounds and their mixture had no effect on the viability of either cell line, as determined by methyl blue tetrazolium reduction and total protein assays. Microscopic analysis revealed that 1 microM and 10 microM diazinon but not cypermethrin inhibited the outgrowth of axon-like processes in N2a cells after a 24-h exposure but neither compound affected process outgrowth by differentiating C6 cells at these concentrations. Under these conditions, 10 microM diazinon inhibited AChE slightly compared to the control after a 4-h exposure but not after 24 h. Western blotting analysis showed that morphological changes were associated with reduced cross-reactivity with antibodies that recognize the neurofilament heavy chain (NFH), microtubule associated protein MAP 1B and HSP-70 compared to control cell extracts, whereas reactivity with anti-alpha-tubulin antibodies was unchanged. Aggregation of NFH was observed in cell bodies of diazinon-treated N2a cells, as determined by indirect immunofluorescence staining. These data demonstrate that diazinon specifically targets neurite outgrowth in neuronal cells and that this effect is associated with disruption of axonal cytoskeleton proteins, whereas cypermethrin has no effect on the same parameters.

Agonist-Dependent Postsynaptic Effects of Opioids on Miniature Excitatory Postsynaptic Currents in Cultured Hippocampal Neurons

Although chronic treatment with morphine is known to alter the function and morphology of excitatory synapses, the effects of other opioids on these synapses are not clear. Here we report distinct effects of several opioids (morphine, [d-ala(2),me-phe(4),gly(5)-ol]enkephalin (DAMGO), and etorphine) on miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons: 1) chronic treatment with morphine for >3 days decreased the amplitude, frequency, rise time and decay time of mEPSCs. In contrast, "internalizing" opioids such as etorphine and DAMGO increased the frequency of mEPSCs and had no significant effect on the amplitude and kinetics of mEPSCs. These results demonstrate that different opioids can have distinct effects on the function of excitatory synapses. 2) mu opioid receptor fused with green fluorescence protein (MOR-GFP) is clustered in dendritic spines in most hippocampal neurons but is concentrated in axon-like processes in striatal and corticostriatal nonspiny neurons. It suggests that MORs might mediate pre- or postsynaptic effects depending on cell types. 3) Neurons were cultured from MOR knock-out mice and were exogenously transfected with MOR-GFP. Chronic treatment with morphine suppressed mEPSCs only in neurons that contained postsynaptic MOR-GFP, indicating that opioids can modulate excitatory synaptic transmission postsynaptically. 4) Morphine acutely decreased mEPSC amplitude in neurons expressing exogenous MOR-GFP but had no effect on neurons expressing GFP. It indicates that the low level of endogenous MORs could only allow slow opioid-induced plasticity of excitatory synapses under normal conditions. 5) A theoretical model suggests that morphine might affect the function of spines by decreasing the electrotonic distance from synaptic inputs to the soma.

Neuronal polarity is regulated by glycogen synthase kinase-3 (GSK-3β) independently of Akt/PKB serine phosphorylation

An essential step during the development of hippocampal neurons is the polarised outgrowth of a single axon. Recently, it has been suggested that inhibition of glycogen synthase kinase-3beta (GSK-3beta) via Akt/PKB-dependent phosphorylation of Ser9, specifically at the tip of the presumptive axon, is required for selective axonal outgrowth. We now report that, by using neurons from double knock-in mice in which Ser9 and Ser21 of the two GSK-3beta isoforms have been replaced by Ala, polarity develops independently of phosphorylation at these sites. Nevertheless, global inhibition of GSK-3beta disturbs polarity development by leading to the formation of multiple axon-like processes in both control and knock-in neurons. This unpolarised outgrowth is accompanied by the symmetric delivery of membrane components to all neurites. Finally, the adenomatous polyposis coli (APC) protein accumulates at the tip of one neurite before and during axon elongation, but global inhibition of GSK-3beta leads to APC protein accumulation in all neurites. We conclude that GSK-3beta inhibition promotes the development of neuronal polarity, but that this is not mediated by Akt/PKB-dependent phosphorylation.

Phenotypes ofDrosophilaBrain Neurons in Primary Culture Reveal a Role for Fascin in Neurite Shape and Trajectory

Subtle cellular phenotypes in the CNS may evade detection by routine histopathology. Here, we demonstrate the value of primary culture for revealing genetically determined neuronal phenotypes at high resolution. Gamma neurons of Drosophila melanogaster mushroom bodies (MBs) are remodeled during metamorphosis under the control of the steroid hormone 20-hydroxyecdysone (20E). In vitro, wild-type gamma neurons retain characteristic morphogenetic features, notably a single axon-like dominant primary process and an arbor of short dendrite-like processes, as determined with microtubule-polarity markers. We found three distinct genetically determined phenotypes of cultured neurons from grossly normal brains, suggesting that subtle in vivo attributes are unmasked and amplified in vitro. First, the neurite outgrowth response to 20E is sexually dimorphic, being much greater in female than in male gamma neurons. Second, the gamma neuron-specific "naked runt" phenotype results from transgenic insertion of an MB-specific promoter. Third, the recessive, pan-neuronal "filagree" phenotype maps to singed, which encodes the actin-bundling protein fascin. Fascin deficiency does not impair the 20E response, but neurites fail to maintain their normal, nearly straight trajectory, instead forming curls and hooks. This is accompanied by abnormally distributed filamentous actin. This is the first demonstration of fascin function in neuronal morphogenesis. Our findings, along with the regulation of human Fascin1 (OMIM 602689) by CREB (cAMP response element-binding protein) binding protein, suggest FSCN1 as a candidate gene for developmental brain disorders. We developed an automated method of computing neurite curvature and classifying neurons based on curvature phenotype. This will facilitate detection of genetic and pharmacological modifiers of neuronal defects resulting from fascin deficiency.

GABAB receptors in the centromedian/parafascicular thalamic nuclear complex: An ultrastructural analysis of GABABR1 and GABABR2 in the monkey thalamus

Strong gamma-aminobutyric acid type B (GABA(B)) receptor binding has been shown throughout the thalamus, but the distribution of the two GABA(B) receptor subunits, GABA(B) receptor subunit 1 (GABA(B)R1) and GABA(B) receptor subunit 2 (GABA(B)R2), remains poorly characterized. In primates, the caudal intralaminar nuclei, centromedian and parafascicular (CM/PF), are an integral part of basal ganglia circuits and a main source of inputs to the striatum. In this study, we analyzed the subcellular and subsynaptic distribution of GABA(B) receptor subunits by using light and electron microscopic immunocytochemical techniques. Quantitative immunoperoxidase and immunogold analysis showed that both subunits display a similar pattern of distribution in CM/PF, being expressed largely at extrasynaptic and perisynaptic sites in neuronal cell bodies, dendrites, and axon-like processes and less abundantly in axon terminals. Postsynaptic GABA(B)R1 labeling was found mostly on the plasma membrane (70-80%), whereas GABA(B)R2 was more evenly distributed between the plasma membrane and intracellular compartments of CM/PF neurons. A few axon terminals forming symmetric and asymmetric synapses were also labeled for GABA(B)R1 and GABA(B)R2, but the bulk of presynaptic labeling was expressed in small axon-like processes. About 20% of presynaptic vesicle-containing dendrites of local circuit neurons displayed GABA(B)R1/R2 immunoreactivity. Vesicular glutamate transporters (vGluT1)-containing terminals forming asymmetric synapses expressed GABA(B)R1 and/or displayed postsynaptic GABA(B)R1 at the edges of their asymmetric specialization. Overall, these findings provide evidence for multiple sites where GABA(B) receptors could modulate GABAergic and glutamatergic transmission in the primate CM/PF complex.

β1,4-N-Acetylglucosaminyltransferase III potentiates β1 integrin-mediated neuritogenesis induced by serum deprivation in Neuro2a cells

Aspects of the biological significance of the bisecting N-acetylglucosamine (GlcNAc) structure on N-glycans introduced by beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in Neuro2a cell differentiation are demonstrated. The overexpression of GnT-III in the cells led to the induction of axon-like processes with numerous neurites and swellings, in which beta1 integrin was localized, under conditions of serum starvation. This enhancement in neuritogenesis was suppressed by either the addition of a bisecting GlcNAc-containing N-glycan or erythroagglutinating phytohemagglutinin (E(4)-PHA), which preferentially recognizes the bisecting GlcNAc. GnT-III-promoted neuritogenesis was also significantly perturbed by treatment with a functional blocking anti-beta1 integrin antibody. In fact, beta1 integrin was found to be one of the target proteins of GnT-III, as confirmed by a pull-down assay with E(4)-PHA. These data suggest that N-glycans with a bisecting GlcNAc on target molecules, such as beta1 integrin, play important roles in the regulation of neuritogenesis.

Inhibition of neurite outgrowth in differentiating mouse N2a neuroblastoma cells by phenyl saligenin phosphate: Effects on MAP kinase (ERK 1/2) activation, neurofilament heavy chain phosphorylation and neuropathy target esterase activity

Sub-lethal concentrations of the organophosphate phenyl saligenin phosphate (PSP) inhibited the outgrowth of axon-like processes in differentiating mouse N2a neuroblastoma cells (IC 50 2.5 μM). A transient rise in the phosphorylation state of neurofilament heavy chain (NFH) was detected on Western blots of cell extracts treated with 2.5 μM PSP for 4 h compared to untreated controls, as determined by a relative increase in reactivity with monoclonal antibody Ta51 (anti-phosphorylated NFH) compared to N52 (anti-total NFH). However, cross-reactivity of PSP-treated cell extracts was lower than that of untreated controls after 24 h exposure, as indicated by decreased reactivity with both antibodies. Indirect immunofluorescence analysis with these antibodies revealed the appearance of neurofilament aggregates in the cell bodies of treated cells and reduced axonal staining compared to controls. By contrast, there was no significant change in reactivity with anti-α-tubulin antibody B512 at either time point. The activation state of the MAP kinase ERK 1/2 increased significantly after PSP treatment compared to controls, particularly at 4 h, as indicated by increased reactivity with monoclonal antibody E-4 (anti-phosphorylated MAP kinase) but not with polyclonal antibody K-23 (anti-total MAP kinase). The observed early changes were concomitant with almost complete inhibition of the activity of neuropathy target esterase (NTE), one of the proposed early molecular targets in organophosphate-induced delayed neuropathy (OPIDN).

Axon‐like processes in type III cells of taste organs

Type III cells of the taste organs are widely considered to be chemoreceptors. The present study was performed on the frog taste disk and describes an axon-like process in type III cells, which often contains a bundle of densely-packed parallel microfilaments. These processes pass through the basal membrane of the gustatory epithelium, running into the lamina propria (transbasal membrane processes, tBMPs). In their intraepithelial tract, tBMPs contain dense-cored vesicles revealing their origin from type III cells. Type III cells showing both classic nonrigid processes (with vesicles and nerve contacts) and tBMPs are present. The connective tract of a tBMP usually contains dense-cored vesicles only in its proximal portion. In some cases, the connective tract of tBMPs is almost perpendicular to the basal lamina. In other cases, it runs parallel to and below the basal lamina. Some tBMPs contact nerve fibers running in the subepithelial connective tissue; the contact area is rather wide but evident synapse-like junctions were never detected. Contacts between tBMPs and nerve fibers innervating basal cells are also found. In conclusion, the data demonstrate the existence of epithelial cells resembling primitive neurons that display an apical dendrite and axon-like basal processes. Until now, it was not considered possible that epithelial receptor cells extend processes out of the epithelium.

Role of extracellular-regulated kinase and c-jun NH2-terminal kinase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurofilament phosphorylation

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes selective degeneration of dopaminergic neurons in which the c-Jun NH2-terminal kinase (JNK) signalling cascade has been implicated. We have employed a differentiated mouse neuroblastoma N2a cell model to investigate the involvement of JNK and extracellular-regulated kinase (ERK) in MPTP-mediated toxicity and their role in neurofilament heavy chain (NF-H) phosphorylation. Acute treatment with a cytotoxic MPTP concentration (5 mM) caused rapid and sustained JNK phosphorylation and ERK dephosphorylation, accompanied by cell death. In contrast, subcytotoxic concentrations of 10 microM MPTP resulted in lower, transient JNK activation in the presence of sustained ERK activity. This resulted in an aberrant increase in a phosphorylation-dependent NF-H epitope, perikaryal accumulation of NF-H, and loss of axon-like processes, prior to cell death. Inhibition of MEK kinase, using PD98059, showed that MEK 1/2 or the downstream kinase, ERK, is required for N2a cell differentiation, NF-H phosphorylation and survival. Indeed, MPTP-induced cell death was exacerbated by the presence of PD98059. However, in the presence of MPTP, reducing JNK activity by using an upstream specific mixed-lineage kinase inhibitor (CEP-11004) significantly attenuated aberrant NF-H phosphorylation and perikaryal NF-H accumulation and maintained axon-like processes, in addition to attenuating cell death. This study reports a switch in the predominant kinase involved in NF phosphorylation in a neuronal cell model and may have implications for the formation of inclusions. Our studies provide further evidence that modulation of the JNK pathway could have a role in alleviating neuronal cell death.

The fate of neural progenitor cells expressing astrocytic and radial glial markers in the postnatal rat dentate gyrus

In the dentate gyrus neurons continue to be generated from late embryonic to adult stage. Recent extensive studies have unveiled several key aspects of the adult neurogenesis, but only few attempts have so far been made on the analysis of the early postnatal neurogenenesis, a transition state between the embryonic and adult neurogenesis. Here, we focus on the early postnatal neurogenesis and examine the nature and development of neural progenitor cells in Wistar rats. Immunohistochemistry for Ki67, a cell cycle marker, and 5-bromo-2-deoxyuridine (BrdU) labelling show that cell proliferation occurs mainly in the hilus and partly in the subgranular zone. A majority of the proliferating cells express S100beta and astrocyte-specific glutamate transporter (GLAST) and the subpopulation are also positive for glial fibrillary acidic protein (GFAP) and nestin. Tracing with BrdU and our modified retrovirus vector carrying enhanced green fluorescent protein (GFP) indicate that a substantial population of the proliferating cells differentiate into proliferative neuroblasts and immature neurons in the hilus, which then migrate to the granule cell layer (66.8%), leaving a long axon-like process behind in the hilus, and the others mainly become star-shaped astrocytes (12.0%) and radial glia-like cells (4.7%) in the subgranular zone. These results suggest that the progenitors of the granule cells expressing astrocytic and radial glial markers, proliferate and differentiate into neurons mainly in the hilus during the early postnatal period.

Distribution of P2Y6 and P2Y12 receptor: their colocalization with calbindin, calretinin and nitric oxide synthase in the guinea pig enteric nervous system

The distribution of P2Y(6) and P2Y(12) receptor-immunoreactive (ir) neurons and fibers and their coexistence with calbindin, calretinin and nitric oxide synthase (NOS) has been investigated with single and double labeling immunostaining methods. The results showed that 30-36% of the ganglion cells in the myenteric plexus are strongly P2Y(6) receptor-ir neurons; they are distributed widely in the myenteric plexus of stomach, jejunum, ileum and colon, but not in the submucosal plexus, with a typical morphology of multipolar neurons with a long axon-like process. About 42-46% of ganglion cells in both the myenteric and submucosal plexuses show P2Y(12) receptor-ir. About 28-35% of P2Y(6) receptor-ir neurons were found to coexist with NOS and 41-47% of them coexist with calretinin, but there was no coexistence of P2Y(6) receptor-ir with calbindin. In contrast, all P2Y(12) receptor-ir neurons were immunopositive for calbindin, although occasionally P2Y(12) receptor-ir neurons without calbindin immunoreactivity were found, while none of the P2Y(12) receptor-ir neurons were found to coexist with calretinin or NOS in the gastrointestinal system of guinea pig. The P2Y(12) receptor-ir neurons coexpressing calbindin-ir in the small intestine are Dogiel type II/AH, intrinsic primary afferent neurons.

Comparison of immunoreactivity to serotonin, FMRFamide and SCPb in the gut and visceral nervous system of larvae, pupae and adults of the yellow fever mosquito Aedes aegypti

In all life stages, the gut of the mosquito is innervated by a small number (typically 4) of central neurons immunoreactive to serotonin (SI). The serotonergic system appears to pass through metamorphosis largely intact, despite extensive remodeling of the gut. Axons immunoreactive to antibodies raised against molluscan FMRFamide (RF-I) constitute peptidergic innervation that anatomically parallels the serotonergic system. In the larva, two clusters of 3 neurons project to the anterior regions of the gut, whereas in the pupa and adult, typically two large RF-I neurons located next to the esophagus send several processes posteriorly. In adults, these neurons branch throughout the diverticula and anterior stomach. In pupae, but not in larvae or adults, the gut RF-l system coexpresses reactivity to antibodies raised against a member of another peptide family, molluscan small cardioactive peptide b (SCP-I). SCP-I immunoreactivity is localized independently of RF-l immunoreactivity in the ganglia of all stages and in neurons that project along the gut of the adult. We did not find any colocalization of S-I and the peptide markers. Distinct populations of enteroendocrine cells populate different regions of the gut at different life stages. Changes in staining pattern suggest that these cells are replaced at metamorphosis along with the other gut cells during the extensive remodeling of the tract. Distributed in the gut epithelium are subpopulations that express either RF-I or SCP-I; a small fraction of these cells bind antibodies to both peptides. The stomachs of adult females are larger than those of males, and the numbers of SCP-I and RF-I enteroendocrine cells are proportionately greater in females. In all the life stages, the junctions between different regions of the gut are the focus of regulatory input. The larval cardiac valve possesses a ring of cells, the necklace cells, which appear to receive extensive synaptic inputs from both the serotonergic system and the peptidergic system. Another focus of control is the pyloric valve, which is encircled by axon-like processes. The immunoreactive pattern of this region differs across life stages, expressing SCP-I in larvae, S-I in pupae, and both SCP-I and RF-I in adults.S-I serotonin-like immunoreactivity RF-I FMRFamide-like immunoreactivity SCP-I small cardioactive peptide b-like immunoreactivity

Phosphatidyl‐inositide signalling proteins in a novel class of sensory cells in the mammalian olfactory epithelium

Ciliated sensory neurons, supporting cells and basal stem cells represent major cellular components of the main olfactory epithelium in mammals. Here we describe a novel class of sensory cells in the olfactory neuroepithelium. The cells express phospholipase C beta-2 (PLC beta2), transient receptor potential channels 6 (TRPC6) and inositol 3, 4, 5-trisphosphate receptors type III (InsP3R-III). Unlike ciliated olfactory neurons, they express neither olfactory marker protein nor centrin, adenylyl cyclase or cyclic nucleotide-gated cation channels. Typical components of the cytoskeleton of microvilli, ezrin and actin are found co-localized with PLC beta2 and TRPC6 in apical protrusions of the cells. In Ca2+-imaging experiments, the cells responded to odours. They express neuronal marker proteins and possess an axon-like process, but following bulbectomy the cells do not degenerate. Our results suggest a novel class of microvillous secondary chemosensory cells in the mammalian olfactory system. These cells, which utilize phosphatidyl-inositides in signal transduction, represent about 5% of all olfactory cells. Their abundance indicates that they play an important role in stimulus-dependent functions and/or the regeneration of the olfactory system.

Co-Transplantation of Schwann Cells Promotes the Survival and Differentiation of Neural Stem Cells Transplanted into the Injured Spinal Cord

The present study investigates whether Schwann cells (SCs) could promote the survival and differentiation of neural stem cells in the injured spinal cord. Neural stem cells were dissociated and cloned from the hippocampal tissue of newborn rats. SCs were also dissociated and purified simultaneously from the sciatic nerves of 4-day-old rats. The results showed that the number of surviving neural stem cells and differentiated neuron-like cells was significantly increased in the co-grafted (SCs and neural stem cells) group compared with the control group (neural stem cells only). Neuron-like cells that developed axon-like processes were observed more commonly in the co-grafted group. These results demonstrate that SCs can promote the survival and differentiation of transplanted neural stem cells in the injured spinal cord.

Effects of Chlorpyrifos and Chlorpyrifos-Methyl on the Outgrowth of Axon-Like Processes, Tubulin, and GAP-43 in N2a Cells

The aim of this work was to study the neurodegenerative effects of the organophosphate (OP) pesticides chlorpyrifos (CPF) and chlorpyrifos-methyl (CHM) on cultured mouse N2a neuroblastoma cells. CPF or CHM, at a subcytotoxic concentration of 3 μM, were added to the cells either at the time of the induction of cell differentiation (codifferentiation) or 16 h after the induction of differentiation (postdifferentiation). CPF and CHM were similar in inhibiting significantly the outgrowth of axon-like processes from N2a cells after only 4 h exposure under both co- and postdifferentiation exposure conditions. Densitometric scanning of Western blots of extracts of cells treated with CPF or CHM for 4 h revealed significantly decreased cross-reactivity with a monoclonal antibody recognizing the protein GAP-43 under post- but not under codifferentiation exposure conditions. Exposure to CPF or CHM for 4 h under postdifferentiation conditions also resulted in reduced fluorescence of N2a cell body staining with anti-GAP-43. Cross-reactivity of Western blots with a monoclonal antibody recognizing α-tubulin was not significantly affected by OP treatment. These data indicate that a disturbance in GAP-43 may be important in the retraction of axons in predifferentiated N2a cells and support the notion that the mechanisms involved in CPF-and CHM-induced inhibition of axonal outgrowth may be different under co- and postdifferentiation exposure conditions.

Characterization of genetically labeled catecholamine neurons in the mouse retina.

Mouse neurons were labeled transgenically with red fluorescent protein (RFP) driven by the tyrosine hydroxylase (TH) promoter and observed in living retinas and brain slices. Two types of retinal amacrine cells expressed TH::RFP. One type had large cell bodies, processes that ramified in S1 of the inner plaxiform layer (IPL) and were TH immunoreactive, identifying them as dopaminergic neurons. A second type had smaller somas, ramified in S3 and lacked TH. Dopaminergic cells had large dendritic fields and exceptionally long axon-like processes, whereas type 2 cells were more compact. Neither cell type exhibited tracer coupling. Thus, murine retinal dopaminergic neurons exhibit functional anatomy similar to their primate counterparts and TH::RFP mice are useful for in situ characterization of catecholaminergic neurons.

Lower rhombic lip-derived cells undergo transmedian tangential migration followed by radial migration in the chick embryo brainstem.

Migration behaviour and fate of cells originated from the lower rhombic lip (LRL) was examined in the chick embryo hindbrain. LRL-derived cells tangentially migrate along the pial surface of the brainstem and form a transient subpial migratory stream. In the initial stages of migration, LRL-derived cells appose each other or axon-like processes, which is indicative of mode of homophilic chain migration and/or axophilic migration. Some LRL-derived cells relocate rostroventrally towards the pontine region, although the majority of them migrate circumferentially to the ventral medulla oblongata. Depending on the stage of generation, LRL-derived cells undergo transmedian migration; late-generated LRL-derived cells preferentially colonize the contralateral brainstem compared with early generated cells. Thus, latecomer neuron precursors may migrate past their predecessors in the migratory stream. When LRL-derived cells leave the subpial migratory stream, they change their migratory direction to a radial one and relocate inwardly, with a profile that resembles a tangential-to-radial change seen in cerebellar granule cell precursors. After they enter the parenchymal region of the brainstem, they exhibited morphological differentiation, and some differentiate into excitatory neurons. The present results suggest that LRL-derived cells migrate across boundaries such as midline or rhombomere, which may facilitate to build up cellular and functional architectures of the hindbrain.