Cultured Rat Neocortical Neurons Research Articles

Effect of acute stretch injury on action potential and network activity of rat neocortical neurons in culture

The basis for acute seizures following traumatic brain injury (TBI) remains unclear. Animal models of TBI have revealed acute hyperexcitablility in cortical neurons that could underlie seizure activity, but studying initiating events causing hyperexcitability is difficult in these models. In vitro models of stretch injury with cultured cortical neurons, a surrogate for TBI, allow facile investigation of cellular changes after injury but they have only demonstrated post-injury hypoexcitability. The goal of this study was to determine if neuronal hyperexcitability could be triggered by in vitro stretch injury. Controlled uniaxial stretch injury was delivered to a spatially delimited region of a spontaneously active network of cultured rat cortical neurons, yielding a region of stretch-injured neurons and adjacent regions of non-stretched neurons that did not directly experience stretch injury. Spontaneous electrical activity was measured in non-stretched and stretch-injured neurons, and in control neuronal networks not subjected to stretch injury. Non-stretched neurons in stretch-injured cultures displayed a three-fold increase in action potential firing rate and bursting activity 30–60min post-injury. Stretch-injured neurons, however, displayed dramatically lower rates of action potential firing and bursting. These results demonstrate that acute hyperexcitability can be observed in non-stretched neurons located in regions adjacent to the site of stretch injury, consistent with reports that seizure activity can arise from regions surrounding the site of localized brain injury. Thus, this in vitro procedure for localized neuronal stretch injury may provide a model to study the earliest cellular changes in neuronal function associated with acute post-traumatic seizures.

AMPA glutamate receptor subunits 1 and 2 regulate dendrite complexity and spine motility in neurons of the developing neocortex

Within neurons of several regions of the CNS, mature dendrite architecture is attained via extensive reorganization of arbor during the developmental period. Since dendrite morphology determines the firing patterns of the neuron, morphological refinement of dendritic arbor may have important implications for mature network activity. In the neocortex, a region of brain that is sensitive to activity-dependent structural rearrangement of dendritic arbor, the proportion of AMPA receptors increases over the developmental period. However, it is unclear whether changes in AMPA receptor expression contribute to maturation of dendritic architecture. To determine the effects of increasing AMPA receptor expression on dendrite morphology and connectivity within the neocortex, and to determine whether these effects are dependent on specific AMPA receptor subunits, we overexpressed the AMPA glutamate receptor subunit 1 (GluR1) and glutamate receptor subunit 2 (GluR2) in cultured rat neocortical neurons at the time that AMPA receptors would normally be incorporated into synapses. Following expression of GluR1 or GluR2 we observed increases in the length and complexity of dendritic arbor of cortical neurons, and a concurrent reduction in motility of spines. In addition, expression of either subunit was associated with an increased density of excitatory postsynaptic puncta. These results suggest that AMPA receptor expression is an important determinant of dendrite morphology and connectivity in neocortical neurons, and further, that contrary to other regions of the CNS, the effects of AMPA receptors on dendrite morphology are not subunit-specific.

NKCC1 activity modulates formation of functional inhibitory synapses in cultured neocortical neurons

Intracellular Cl(-) concentration ([Cl(-)](i)) in immature neurons is higher than that expected for a passive distribution, therefore the equilibrium potential for chloride is more positive than the resting membrane potential, and the resulting GABA renders immature neurons depolarization. The higher [Cl(-)](i) in immature neurons is thought to be attributed to the uptake of Cl(-) mediated by NKCC1 (Na(+), K(+)-2Cl(-) cotransporter). Thus, a dysfunction of this transporter could affect synaptic development through a GABA(A) receptor-mediated pathway. To test this possibility, we examined the effects of a Cl(-)-uptake inhibitor on the development of synaptic activities of rat neocortical neurons in culture. Chronic treatment with bumetanide at 10 microM during the culture diminished the amplitude of synaptically-driven rhythmic depolarizing potentials (RDPs) in neurons and also decreased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but not of spontaneous excitatory postsynaptic currents (sEPSCs). Chronic treatment with bumetanide decreased vesicular GABA transporter (VGAT)-immunopositive particles without affecting paired-pulse ratio of evoked IPSCs (eIPSCs), indicating decrease in the number of functional GABAergic synapses. Acute treatment with bumetanide (10 microM) decreased neuronal [Cl(-)](i), the amplitude of RDPs, and neuronal excitability, while bumetanide had no effect on RDPs and neuronal excitability in the presence of bicuculline. These results suggest that the uptake of Cl(-) by NKCC1 affects the development of inhibitory synapses by promoting a depolarizing GABA-mediated response.

Identification of Neurite Outgrowth-promoting Domains of Neuroglycan C, a Brain-specific Chondroitin Sulfate Proteoglycan, and Involvement of Phosphatidylinositol 3-Kinase and Protein Kinase C Signaling Pathways in Neuritogenesis

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. We report that the recombinant ectodomain of NGC core protein enhances neurite outgrowth from rat neocortical neurons in culture. Both protein kinase C (PKC) inhibitors and phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated the NGC-mediated neurite outgrowth in a dose-dependent manner, suggesting that NGC promotes neurite outgrowth via PI3K and PKC pathways. The active sites of NGC for neurite outgrowth existed in the epidermal growth factor (EGF)-like domain and acidic amino acid (AA)-domain of the NGC ectodomain. The EGF-domain caused cells to extend preferentially one neurite from a soma, whereas the AA-domain caused several neurites to develop. The EGF-domain also enhanced neurite outgrowth from GABA-positive neurons, but the AA-domain did not. These results suggest that the EGF-domain and AA-domain have distinct functions in terms of neuritogenesis. From these findings, NGC can be considered to be involved in neuritogenesis in the developing central nervous system.

Identification of Endophilins 1 and 3 as Selective Binding Partners for VGLUT1 and Their Co-Localization in Neocortical Glutamatergic Synapses: Implications for Vesicular Glutamate Transporter Trafficking and Excitatory Vesicle Formation

1. Selective protein-protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation. 2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1-endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3. 3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.

Quantal Characteristics of Synaptic GABA Release under Conditions of Stimulation of Single Synaptic Terminals of Cultured Cortical Cells of the Rat

We studied evoked inhibitory postsynaptic currents (eIPSC) using local electrical stimulation of single presynaptic terminals of cultured rat neocortical neurons. According to pharmacological and kinetic properties, these currents were qualified as GABAA-activated. Using autocorrelation analysis of distributions of the eIPSC amplitudes, which were in all cases polymodal, we examined quantal characteristics of the above eIPSC. These results were compared with the values of quantal parameters (N, p, Q, and m) of the current families obtained using approximation by binomial distribution. Amplitude histograms of spontaneous miniature IPSC recorded under conditions of the minimum quantal release of the neurotransmitter were normal (close to Gaussian) with the mode within a 10 pA range, which is very close to analogous parameters calculated using autocorrelation and binomial techniques.

Neocortical potassium currents are enhanced by the antiepileptic drug lamotrigine.

We used field-potential recordings in slices of rat cerebral cortex along with whole-cell patch recordings from rat neocortical cells in culture to test the hypothesis that the antiepileptic drug (AED) lamotrigine (LTG) modulates K+-mediated, hyperpolarizing currents. Extracellular field-potential recordings were performed in neocortical slices obtained from Wistar rats aged 25-50 days. Rat neocortical neurons in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated K+ currents. In the in vitro slice preparation, LTG (100-400 microM) reduced and/or abolished epileptiform discharges induced by 4-aminopyridine (4AP, 100 microM; n = 10), at doses that were significantly higher than those required to affect epileptiform activity recorded in Mg2+-free medium (n = 8). We also discovered that in cultured cortical cells, LTG (100-500 microM; n = 13) increased a transient, 4AP-sensitive, outward current elicited by depolarizing commands in medium containing voltage-gated Ca2+ and Na+ channel antagonists. Moreover, we did not observe any change in a late, tetraethylammonium-sensitive outward current. Our data indicate that LTG, in addition to the well-known reduction of voltage-gated Na+ currents, potentiates 4AP-sensitive, K+-mediated hyperpolarizing conductances in cortical neurons. This mechanism of action contributes to the anticonvulsant effects exerted by LTG in experimental models of epileptiform discharge, and presumably in clinical practice.

Glutamate-induced increases in intracellular Ca2+ in cultured rat neocortical neurons.

In neurons, changes in intracellular Ca2+ concentration ([Ca2+]i) trigger neurotransmitter release, regulate membrane excitability, affect gene expression, and govern short- and long-term forms of synaptic plasticity. Rises in cytoplasmic Ca2+ are thought to underlie the various effects of glutamatergic neurotransmitters within the central nervous system. In the present study, we applied a calcium imaging technique using a confocal laser scanning microscope to investigate the effects of excitatory amino acids on glutamate induced calcium influx in primary cultured neocortical neurons. Glutamate (5 microM) induced increases in [Ca2+]i in both the soma and dendritic processes of the cells. The increase was partially blocked by 10 microM DL-2-amino-5-phosphovaleric acid (APV), a NMDA antagonist. The reduction was higher in the dendritic process than in the cell body: the reduction was 58% in the cell body and 67% in the dendritic processes. In contrast, 5 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA antagonist, had less effect on the response. We observed an 11% reduction in the dendritic processes, but no change in the cell bodies. The results demonstrated the heterogeneous distribution of APV- and CNQX-sensitive channels in primary cultured neocortical neurons. In both the cell body and dendritic processes, [Ca2+]i increase induced by low concentrations of glutamate was mainly due to the activation of NMDA receptors.

Intracellular [Cl −] modulates synchronous electrical activity in rat neocortical neurons in culture by way of GABAergic inputs

The influence of GABAergic neurons on spontaneous electrical activities of neocortical neurons in culture, which was estimated to be about 9.5% of the total neurons by immunohistochemistry, was examined using dual whole-cell recording. Synchronized depolarization or hyperpolarization was observed in recorded neurons with pipettes containing low [Cl −] solution, while synchronized bursting of action potentials (APs) was observed with pipettes containing high [Cl −] solution. Spontaneous currents (SCs) were synchronous in all pairs tested with either pipettes containing low or high [Cl −] solution and spontaneous outward currents (SOCs) observed at around −30 mV were sensitive to the GABA-A receptor antagonist, bicuculline. Their reversal potential ( V rev) was linearly related to the logarithm of Cl − activity in the pipette (−56.9 mV/decade). The intracellular chloride concentration was estimated from the V rev of SCs with gramicidin perforated-patch recordings and was between 5.9 and 28.1 mM (mean: 13.0 mM). These results suggest that GABA depolarized some neurons and hyperpolarized others, depending on the E Cl. Bicuculline decreased the frequency of periodic depolarized potentials and increased their amplitudes. However, perfusion with low [Cl −] bath solution did not decrease the frequency. Our data indicate that recurrent subthreshold electrical activities by GABAergic inputs along with glutamatergic inputs take part in deterring synchronized bursting and that intracellular [Cl −] can modulate this bursting.

Distribution of nifedipine- and ω-conotoxin GVIA-sensitive Ca 2+ channels in cultured rat neocortical neurons

L- and N-type voltage-dependent calcium channels are widely distributed in neurons of the CNS. To investigate their subcellular distributions on CNS neurons, intracellular calcium concentration ([Ca 2+] i) increase in response to high potassium ([K +]) solution was detected in primary cultured rat neocortical neurons using the calcium indicator dye Oregon Green with a confocal laser scanning microscope. Extracellular application of 90 mM [K +] solution induced fluorescence increase in a manner dependent on extracellular [Ca 2+]. The increase was partially blocked by 10 μM nifedipine, and the reduction was higher in cell bodies compared to dendritic processes. In contrast, ω-conotoxin GVIA reduced the 90 mM [K +] induced fluorescence increase more in the dendritic processes. The results demonstrated the heterogeneous distribution of nifedipine- and ω-conotoxin GVIA-sensitive calcium channels, which may suggest a functional difference in nifedipine- and ω-conotoxin GVIA-sensitive channels in cultured neocortical neurons.

Mutual regulation between the intercellular messengers nitric oxide and brain-derived neurotrophic factor in rodent neocortical neurons.

The diffusible factors, nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) are both suggested to be intercellular messengers that have similar synaptic activities and developmental influences in the brain. In the present study, we have analysed their mutual regulation with respect to their production in rodent neocortical neurons. Some of the cultured rat neocortical neurons exhibited immunoreactivity for both neuronal NO synthase (NOS) and the BDNF receptor trkB. Neuronal NOS appeared to be activated autonomously and produced NO in culture as monitored by nitrite accumulation. Inhibition of the endogenous NO production in culture by a NOS inhibitor, NG-monomethyl-L-arginine (NMMA), enhanced basal expression of BDNF mRNA and protein. Similarly, cerebroventricular administration of another NOS inhibitor, N-omega-nitro-L-arginine methylester (L-NAME), but not D-NAME or saline, increased BDNF content in the neocortex. In the opposite direction, however, BDNF appeared to function as a positive regulator for NO synthesis. Addition of BDNF upregulated the neuronal NOS expression as well as NO production in neocortical culture. In agreement, BDNF knock-out mice exhibited significant impairment of neuronal NOS expression in the neocortex. Taken together, these observations suggest that the trans-synaptic signalling molecules, NO and BDNF, influence the production of each other and mutually regulate the strength of their intercellular communications.

Brain-derived neurotrophic factor regulates the expression of AMPA receptor proteins in neocortical neurons

The role of the neurotrophins; nerve growth factor, [22]brain-derived neurotrophic factor, [2]neurotrophin-3 [16, 24]and neurotrophin-4/5, [3]in synaptic development and plasticity has been extensively investigated. [23, 33]The neurotrophins regulate synaptic transmission [1, 13, 19, 34]as well as neural development [4, 5, 12, 18, 25]in the brain. However, the mechanisms underlying these processes are unknown. In this study we show that brain-derived neurotrophic factor triggers an increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (GluR) proteins without significant changes in their messenger RNA levels. Brain-derived neurotrophic factor treatment specifically increased the protein levels of GluR1 (193±22%) and GluR2/3 (182±11%) in cultured rat neocortical neurons. In contrast, nerve growth factor and neurotrophin-3 failed to alter the protein levels of these neurons, and brain-derived neurotrophic factor effects on N-methyl- D-aspartate-type glutamate receptors were either modest or negligible. Immunocytochemical studies indicated that the increase in AMPA receptor proteins reflects the induction of their neuronal expression, but not selective neuronal survival. In agreement with these results, cortical neurons from brain-derived neurotrophic factor-knockout mice exhibited a reduction in AMPA receptor proteins in the cytoskeletal fraction containing postsynaptic proteins. Thus, the neurotrophin plays a crucial role in modulating the expression of AMPA receptors presumably at translational or post-translation levels and is implicated in synaptic development and plasticity.

Influence of bcl-2 and p53 on Neuronal Survival During Hypoxia-Induced Neuronal Apoptosis • 181

During hypoxic-ischemic episodes in the brain, the resulting neuronal death is due, in part, to apoptosis, a genetically controlled form of cell death.p53, a tumor suppressor gene, is believed to promote apoptosis.bcl-2, a proto-oncogene, is believed to inhibit apoptosis. However, the role of these two genes in neuronal survival or death during hypoxia is not clear. We hypothesized that: a) neuronal expression of p53 and bcl-2 is altered during hypoxia, b) altered expression of p53 is associated with decreased neuronal survival during hypoxia, and c) altered expression of bcl-2 is associated with prolonged survival during hypoxia. Methods. 7-10 day old cultured rat neocortical neurons were exposed to 0.1,1, or 3% oxygen. Culture dishes were removed at 24-hour intervals for quantitative immunofluorescence staining and immunoblot analyses. Apoptosis was assessed by changes in cell morphology and in situ and genomic DNA fragmentation. To assess the impact of p53 and bcl-2 expression upon neuronal survival during hypoxia, expression of these genes was attenuated by treating selected neuronal culture plates with 3 μM antisense, sense, and nonsense oligonucleotides 24 hours prior to exposure to 0.1% oxygen. Neuronal survival was assessed by Trypan blue exclusion at 24-hour intervals during hypoxic exposure. Results. At each level of hypoxia studied, morphologic changes (cellular shrinkage, nuclear condensation) and in situ and genomic DNA fragmentation consistent with apoptosis was detected. Immunoblot and quantitative immunofluorescence analyses revealed an increase in both neuronal p53 and bcl-2 during hypoxic exposure. After 48 hours at 0.1% oxygen, the proportion of viable neurons was significantly greater in plates treated with p53 antisense oligonucleotide (≈60%) than in untreated plates(35-40%)(n=5;p 0.05). Conclusions.p53 and bcl-2 are increased in neurons during hypoxic exposure. Expression of p53 is, in part, necessary for hypoxia-induced neuronal apoptosis.bcl-2 expression, in part, prolongs neuronal survival during hypoxia.

State of actin filaments is changed by anoxia in cultured rat neocortical neurons

Cultured necortical neurons respond rapidly to oxygen deprivation. Within minutes they demonstrate an increase in intracellular calcium and pronounced changes in their morphology. These changes include swelling, bleb formations, process retraction and a change in shape of the soma from pyramidal or ellipsoidal to round. Since the cytoskeleton is responsible for the maintenance of cell shape, we investigated the changes in state of a major component of the neuronal cytoskeleton, the actin filaments. Actin exists in a dynamic equilibrium between the monomeric and filamentous states. This equilibrium is dependent, in part, upon intracellular ATP, which is reduced during anoxia. We differentially labelled monomeric actin with Texas Red-tagged DNase-1 and the polymeric form with Bodipy–phallicidin. Using confocal microscopy and image reconstruction, we have found that the ratio of filamentous to monomeric actin increases on average three-fold following 10 min of anoxia. In addition, filamentous actin redistributes within the soma and appears to have a more homogeneous distribution than in normoxic neurons. Our results show that, in cultured neocortical neurons, actin filaments are modulated by anoxia. We believe that, although the filamentous/monomeric ratio is modulated, at least in part, by the levels of ATP and ADP, the relative distribution and amount of the filamentous to monomeric form within neurons are likely regulated by other factors such as calcium-sensitive actin-binding and severing proteins.

Synchronization of neuronal activity promotes survival of individual rat neocortical neurons in early development.

Neural activity is thought to play a significant role during the development of the cerebral cortex. In this study, we examined the effects of global activity block or enhancement and the effects of patterned firing on the ability of cultured rat neocortical neurons to survive during the second week in vitro, beyond the beginning of synaptogenesis. Blockade of neuronal activity by adding tetrodotoxin (TTX) and increasing magnesium concentration in the medium strongly reduced the survival of cortical cells. Increasing neuronal activity by raising the external potassium concentration significantly improved the survival of cortical neurons. We postulated that in a developing neuronal network the survival of nerve cells is regulated by synaptically mediated events that involve changes in the intracellular calcium concentration. To examine this question further, we monitored the activity of the developing network by optically recording the intracellular calcium signals of many neurons simultaneously. These recordings show that in low magnesium neocortical neurons express synchronized oscillation of their intracellular calcium concentration. The ability of a network to synchronize the changes in intracellular calcium of multiple cells appeared gradually during the second week in culture, paralleled by both an increase in the synaptic density and a decline in the number of surviving neurons. By examining the fate of identified cells several days after a recording session, we found that those nerve cells that were co-activated with other neurons had a significantly higher chance to survive than cells that did not participate in synchronized events. These experiments demonstrate that during early cortical network development cortical neurons show synchronized firing activity and that the survival of neurons is at least partially dependent on this pattern of neuronal activity.

Secreted form of -amyloid precursor protein activates protein kinase C in cultured rat neocortical neurons

Secreted form of -amyloid precursor protein activates protein kinase C in cultured rat neocortical neurons

816 Secreted form of β-amyloid precursor protein activates protein kinase C in cultured rat neocortical neurons

Hybrid biomedical structures have been used widely in various tissue-regenerating materials because they effectively induce exceptional physical and cellular responses. In this study, a new hybrid process was used to design a three-dimensional (3D) biomedical hybrid scaffold with a controlled pore-structure and high mechanical strength. A melt-dispensing method was used to obtain mechanical properties and electrohydrodynamic direct-jet (EHD-DJ) printing was used to provide microsized fibrous structures for the scaffold. Furthermore, the poly(ε-caprolactone) (PCL) hybrid scaffolds were coated biomimetically with type-I collagen to increase bioactive interactions between cells and scaffolds. The fabricated scaffolds showed similar mechanical properties to the two control scaffolds; however, the results of culturing osteoblast-like (MG63) cells showed significant increases in in vitro cellular activities (cell viability > twofold and calcium deposition > sevenfold). Based on these results, we propose a newly designed hybrid scaffold that can support significant in vitro cellular activities at the interface between cells and the 3D micro-pore structure for soft and hard tissue regeneration.

Molecular Cloning and Characterization of Annexin V‐Binding Proteins with Highly Hydrophilic Peptide Structure

We previously reported that annexin V promoted the survival of cultured rat neocortical neurons. In an effort to elucidate the mechanism underlying this neurotrophic activity of annexin V, we have attempted to identify the target or binding proteins of annexin V in neuronal cells. Herein, we screened an embryonic day 17 rat brain cDNA library by western blot using glutathione S-transferase-annexin V fusion protein as a probe and then isolated four clones showing binding to annexin V in a Ca2+ - and phospholipid-dependent manner. Although these cDNAs encoded different polypeptides, all four polypeptides shared the unique feature of containing highly hydrophilic stretches with high Lys, Glu, and Ser contents. Deduced amino acid sequences of two clones showed high homology with human X-linked Helicase2 (XH2) and DNA (cytosine-5) methyltransferase (DMTase) sequences, whereas the other two were not related to any known peptide sequence. These results suggest that XH2 and DMTase are candidates for annexin V-binding proteins and thus may mediate the biological activity of annexin V.

Development of low magnesium-induced spontaneous synchronized bursting and GABAergic modulation in cultured rat neocortical neurons

Development of spontaneous synchronized bursting in the early stages of rat neocortical neuronal cultures was studied by whole-cell and extracellular recordings. Neocortical neurons from rat embryos were cultured on planar electrode arrays, and low Mg2+-induced spontaneous activity was recorded from 5 to 16 days in vitro (DIV). At 5–6 DIV the current synchronized to the bursting had only a slow component lasting 3–5 s, whereas in older cultures a fast transient component was dominant. A γ-aminobutyric acid (GABA)A receptor antagonist, bicuculline methiodide, had little effect on the spontaneous activity at 5–6 DIV, whereas in older cultures it had a marked effect on the slow current component. These results suggest a role of GABAergic transmission in the development of synchronized activities.

Neurotrophic and neuroprotective effects of neuron-specific enolase on cultured neurons from embryonic rat brain

We previously reported that the γγ-isozyme of enolase (neuron-specific enolase, NSE), one of the glycolytic enzymes, promoted the survival of embryonic rat neocortical neurons in culture, but αα-isozyme (non-neuronal enolase) had no effect. In the present study, the neurotrophic effects of NSE on cultured mesencephalic and spinal neurons from rat embryo were examined. NSE promoted the survival of neurons not only in neocortical cultures but also in mesencephalic and spinal cord cultures. Furthermore, NSE showed neuroprotective action on cultured neocortical neurons in a low-oxygen atmosphere. By contrast, non-neuronal enolase did not show any neurotrophic or neuroprotective activities. To clarify the mechanism of the neurotrophic effect of NSE, the binding of NSE to cultured neurons was determined by radio-receptor assay using 125I-labelled NSE. The specific binding, which was dose-dependent, saturable, and calcium-dependent, could be detected. These results suggest that NSE has neurotrophic and neuroprotective effects on rather a broad spectrum of neurons in the central nervous system. The existence of specific binding of NSE to cultured neurons suggests the possibility that receptor-like or carrier-like molecules on the neuronal surface are involved in the neurotrophic activity of NSE.