BACKGROUND
the use of live animals or primary neurons for neurotoxicity research is complicated by technical, ethical and economic considerations.
an alternative strategy is the use of a cultured cell line that can be induced to exhibit neuronal characteristics.
to date, the most common of these “neurogenic” cell lines are human and mouse neuroblastomas, which often exhibit poor sensitivity to neurotoxins, are comprised of heterogeneous phenotypes and may not form functioning synapses, making it questionable how accurately they model neuronal mechanisms of pathogenesis  <cit> .
an effective cell-based model system for neurotoxin research would combine the biological relevance of primary neurons with the flexibility of continuous cell lines.
such a model would exhibit normal neurogenic progression, faithfully recapitulate the full range of interactions between primary neurons and neurotoxins, be compatible with modern cell and molecular techniques and facilitate moderate-throughput screening applications  <cit> .
early efforts to differentiate escs into neurons produced cells with morphological and functional characteristics similar to primary neurons  <cit> .
these cultures also contained a high percentage of glial cells and multiple neuron subtypes, which significantly detracted from their suitability as a basic research tool or therapeutic screening platform.
recently, a variation of the 4/ <dig> method  from esc aggregates for  <dig> days followed by supplementation with retinoic acid  for  <dig> d) was described with significantly improved neuronal yield and purity  <cit> .
widespread application of this protocol was limited by several factors: custom media formulations were required; multiple manipulations were needed to isolate escs from feeder cell populations; and neurons were not produced at yields amenable to moderate-throughput screening.
here we demonstrate that the robust production of enriched glutamatergic neurons from suspension-cultured mouse escs using a variant of the 4/ <dig> method offers a facile neuron model that exhibits high sensitivity to a variety of biological neurotoxins and replicates stages of neurogenesis observed in dissociated primary neuron cultures.
we developed a facile, cost-effective procedure that enables the rapid production of large numbers of highly enriched glutamatergic esns from continuous suspension cultures of murine escs.
immunological, genomic, morphological and functional evaluations suggest that esn maturation and behavior recapitulate those described in primary neurons.
we further demonstrate that esns appropriately respond to different neurotropic toxins  at doses similar to primary neurons.
these data provide compelling evidence that suspension culture-derived esns comprise a novel cell model for neurotoxicity research that combines the verisimilitude of primary neurons with the flexibility of continuous cell lines.
the identification of a robust, scalable and sensitive neuron-based research platform amenable to genetic modification and compatible with moderate-throughput techniques should enable detailed biochemical and molecular approaches that are not feasible in neurogenic cells or primary neurons.
we anticipate this platform to expedite the identification and validation of novel therapeutic approaches to a variety of neurotoxins as well as the elucidation of molecular aspects of pathogenesis following intoxication.
RESULTS
suspension-adapted escs remain pluripotent and mitotically active
escs are traditionally maintained on feeder cells in the presence of lif and fetal calf serum to preserve germline competency  <cit> .
to adapt esc lines to feeder cell-free suspension culture, r <dig>  d <dig> and c57bl/ <dig> escs that had been co-cultured with mitotically inactivated mouse embryonic fibroblasts were dissociated and cultivated in bacterial dishes until suspended aggregates developed.
mitotic rates of the suspension-adapted escs stabilized by five passages  and mean doubling times were  <dig>  ±  <dig>  h,  <dig>  ±  <dig>  h and  <dig>  ±  <dig>  h over  <dig> passages .
expression of the pluripotency marker oct3/ <dig> did not vary through  <dig> passages, whereas withdrawal of lif resulted in a loss of oct3/ <dig> immunoreactivity within  <dig> d .
theoretical estimates of cumulative yield over  <dig> passages from a single dish were  <dig> escs .
suspension-adapted escs were differentiated into neurons using a modified 4/ <dig> protocol, with increased ra concentrations and incubation under rotary conditions in low-attachment dishes   <cit> .
we initially observed that differentiation under static conditions resulted in large, agglomerated complexes by div  <dig> .
hypothesizing that agglomeration might limit recovery of npcs at div  <dig>  npc yields were compared between static differentiation conditions versus differentiation on a rotary shaker at  <dig> rpm.
rotary conditions eliminated agglomeration and increased average yield by 290% to 97 ×  <dig> npcs per 10-cm dish .
no differences were observed in npc yield between 5– <dig> passages in any esc line.
in one instance, an r <dig> culture maintained for  <dig> passages produced  <dig> ×  <dig> npcs, which underwent normal neuronal development, indicating that extended periods in suspension culture may not interfere with neurogenic competence.
differentiated cells express transcriptional, morphological and immunological markers of neurogenesis
neuronal maturation was characterized between div 1– <dig> using the dendritic marker map <dig>  the axonal marker map-tau and the pre-synaptic marker synapsin- <dig> .
although map <dig> was uniformly present at div  <dig>  map-tau expression was not widely observed in the majority of neurons until div  <dig>  axonal arborization increased extensively between div 3– <dig>  while dendritic extension occurred predominantly after div  <dig> .
weak synapsin puncta were widely dispersed along axons at div  <dig>  primarily in the absence of proximal dendrites, but as of div  <dig>  synapsin- <dig> staining accumulated at axodendritic interfaces.
by div  <dig>  an extensive “lawn” of neurites was apparent, with robust axonal arborization and elongated dendrites appearing in close proximity to single or fasciculated axons .
greater than 99% of surviving cells expressed neuron-specific markers at div  <dig>  and less than one gfap+ glial cell was observed per mm <dig> at div  <dig> .
glial cells that did survive were most often observed at regions of high neuron density , suggesting that a supportive microenvironment  may be permissive for glial persistence in the absence of serum.
a presumptive neuronal phenotype was further evaluated by expression profiling at div  <dig>  an average of  <dig>  ±  <dig> nuclear mrna transcripts were detected at a single copy or higher , of which about  <dig>  were present in high abundance   <cit> .
overall, esns expressed a broad range of neurotypic genes , and 28% of the most abundant transcripts coded for neuron-specific proteins .
gene expression was highly enriched for a neuronal phenotype and strongly associated with neuron-specific canonical pathways and functions .
esns expressed high levels of glutamatergic markers , with low-to-moderate levels of gabaergic markers  and virtually no markers  of cholinergic, serotonergic, dopaminergic or motor neuron differentiation.
a variety of neurotransmitter receptors were expressed, including those for glycine, gaba, acetylcholine  and glutamate .
esns also expressed transcripts essential for synaptic activity and electrochemical signal propagation, including subunits of the neuronal n- and p/q-type voltage-dependent ca2+ channels , snare proteins, synaptic vesicle-associated proteins, na+/k+ pumps, and a large number of voltage-gated na+, k+ and cl- channels .
depolarizing stimuli evoke reversible ca2+ uptake
the expression of gated ion channels and pumps important in maintaining and altering membrane polarity suggested that application of depolarizing stimuli might elicit ca2+ uptake.
we altered the plasma membrane potential using indirect  and direct  methods and found that both methods of depolarization elicited reversible ca2+ influxes .
the functional verification of ca2+ uptake in response to classical depolarizing stimuli confirms intracellular recordings and neurotransmitter release assays  <cit> , and suggests that esns may be sensitive to neurotoxic stimuli whose mechanism of action involves dysregulation of electrochemical signal propagation.
esns are sensitive to glutamatergic excitotoxicity
glutamatergic excitotoxicity has been attributed to the pathologic internalization of ca2+ through post-synaptic nmda receptors, compounded by activation of vdccs in response to excitatory post-synaptic currents  from ampa and ka receptors  <cit> .
in primary neuron cultures, neurotoxicity has been reported over a wide range of glutamate  doses and exposure durations  <cit> .
esns express transcripts for nmda, ka and ampa receptor subunits and the nmda-associated protein grina at high levels , suggesting that esns may be functionally sensitive to glu treatment.
treatment of esns with  <dig> μm glu resulted in acute levels of ca2+ uptake similar to those caused by k+, whereas the inhibitory neurotransmitter gaba or vehicle controls had no effect .
glu treatment resulted in significant time-  and dose-dependent  toxicity, further confirmed by morphological evidence of neurite degeneration  <dig> h after a  <dig> μm treatment .
co-administration of the glur antagonists apv and cnqx afforded complete protection against toxicity after a  <dig> h exposure to  <dig>  and  <dig>  μm glu, and 50% protection against  <dig> μm glu.
these results indicate that esns undergo a time- and dose-dependent glu toxicity that is mediated by ionotropic glurs.
neuron viability and ca2+ influx following exposure to alpha-latrotoxin
unlike glu exposure, which induces epscs in post-synaptic compartments, ltx forms ca2+-permissive pores in the presynaptic membrane that result in fulminant neurotransmitter release and activation of non-synaptic ca2+-sensitive intracellular pathways  <cit> .
esns express transcripts of known ltx receptors , and ltx treatment of esns evokes unregulated ca2+ influx, followed by morphological and biochemical indicators of neurotoxicity  <cit> .
to better characterize ltx toxicity, we evaluated dose-dependent relationships between ltx, onset and magnitude of ca2+ uptake and neuron cell death.
the ec <dig> for ca2+ uptake at  <dig> min was determined to be  <dig>  pm , with positive correlations between dose, onset and magnitude of ca2+ uptake .
we also demonstrated a strong correlation between ltx dose and inhibition of metabolic activity after a  <dig> min exposure .
these findings illustrate the compatibility of esns with moderate-throughput screening approaches, confirm that esn sensitivity and response to ltx are similar to those of primary neurons, and demonstrate that esns offer a novel platform with which to screen for therapeutics that prevent ltx toxicity using multi-well formats  <cit> .
esns are a biologically relevant model of bont intoxication
esns strongly express transcripts and protein for proteolytic targets  of the seven bont serotypes and known protein receptors for presynaptic uptake   <cit> ).
we have shown that esns are sensitive to femtomolar concentrations of bont/a and /e  <cit> .
to determine whether esns are a suitable model for the remaining serotypes, we evaluated cleavage of the target snare proteins after a  <dig> h exposure to bonts /b, /c, /d, /f and /g .
esns exhibited similar or improved sensitivities to all bont serotypes tested compared to primary mouse spinal cord or cerebellar granule cell neurons, and several orders of magnitude improved sensitivity over neuroblastoma cells   <cit> .
bont/c is the only holotoxin that targets multiple snare proteins; interestingly, we found that intoxication of esns with bont/c resulted in cleavage of snap- <dig> as well as syntaxin- <dig> with roughly equivalent ec <dig> values.
notes: primary neurons’ values are compiled from two sources.
annotation by  represents 50% inhibition of neurotransmitter release in rat cerebellar neuron cultures exposed to bonts for  <dig> h   <cit> , whereas annotation by  represents cleavage of 50% of snap- <dig> protein in fetal mouse spinal cord cultures exposed to bont for  <dig> h  <cit> .
sh-sy5y neuroblastomas’ values indicate inhibition of 50% of noradrenalin release in sh-sy5y human neuroblastomas cells after  <dig> h incubation  <cit> .
esn values represent 50% cleavage of snare proteins after a  <dig> h tonic intoxication.
esn ec <dig> values for bont/b, /c, /d, /f and /g are from this manuscript, while bont/a and /e are from mcnutt et al.
.
nr, not reported.
bont holotoxin binds to pre-synaptic receptors and is internalized via synaptic endocytosis.
following cell entry, the proteolytically active light chain is released to the synaptic compartment through pores formed by the heavy chain in the endosome membrane  <cit> .
bont uptake and/or activation has been reported to be enhanced following intoxication under depolarizing conditions   <cit> .
we found that a  <dig> min intoxication of esns by bont/a in depolarizing media increased snap- <dig> cleavage by five-fold after  <dig> h as compared to two different basal media .
discussion
esc lines remain mitotically active, pluripotent and neurogenic following adaptation to feeder-independent suspension culture
with the aim of developing a more facile, robust and efficient method for the production of homogenous neuronal cultures from mouse esc lines, we evaluated the neurogenic capacity of escs adapted to feeder-independent suspension culture and differentiated under rotary conditions.
mitotic rates of suspension-adapted r <dig>  d <dig> and c57bl/ <dig> esc lines were stabilized by five passages, and all esc lines remained mitotically active and expressed the pluripotency marker oct3/ <dig> in the absence of feeder cells.
germline competence was not evaluated, so it is unclear whether these culture conditions restrict non-neurogenic developmental fates.
adaptation to suspension culture streamlined and economized esc culture and neuronal differentiation by obviating the need for feeder cells and allowing the initiation of neuronal differentiation directly from suspension cultures every  <dig> h. suspension-cultured escs remained neurogenic, and differentiated neurons exhibited a multitude of neurotypic characteristics when maintained in relatively inexpensive, commercially available media.
we found that the simple addition of a rotary step during differentiation significantly increased neuron yield to levels sufficient for moderate-throughput screening techniques.
these improvements resulted in the generation of sufficient npcs from one 10-cm dish to plate  <dig> cm <dig> of tissue culture surface  at  <dig>  cells per cm <dig>
morphological and immunological characterization of neurogenesis
the neurotypic character of esns was confirmed using morphology, transcriptional profiling, immunocytochemistry and functional characterization.
temporal changes in map <dig>  map-tau and synapsin- <dig> expression and localization demonstrated a characteristic progression through the first  <dig> stages of neurogenesis, as described by dotti et al.
in embryonic hippocampal neurons, culminating in a dense axodendritic arbor with punctate localization of synapsin- <dig> at axodendritic interfaces.
as soon as div  <dig>  a majority of esns produced short map2+ processes that were indistinguishable from one another, characteristic of development stage   <dig> neurons  <cit> .
between div 1– <dig> most esns underwent rapid extension of minor neurites and increased expression of axonal markers, characteristic of ds  <dig> .
axons then underwent rapid extension and arborization , accompanied by the onset of dendritic outgrowth and synaptogenesis at ds  <dig> .
the earliest appearance of synapsin accumulation at axodendritic interfaces appeared between div 7– <dig>  consistent with previous demonstrations of characteristic pre- and post-synaptic architectures at div  <dig>  <cit> .
it should be noted that the plating density is important in producing a neuronal population with few contaminating, non-neuronal cells.
not only does serum withdrawal at div  <dig> initiate the final stages of neuronal differentiation, but it also eliminates the β3-tubulin non-expressing cells, which are likely to be glial cells  <cit> .
glia that survive beyond div  <dig> tend to be localized to regions of high neuron density.
thus, if npcs are plated at high density, then increased numbers of glia survive serum-starvation.
if unchecked, they can proliferate and eventually overwhelm the esn culture.
we have found that glial contamination due to overplating can be suppressed without detectibly altering neuron viability or function by addition of 5-fluoro-deoxyuridine/uridine  from div 8– <dig>
during synaptogenesis, presynaptic proteins are bundled and transported along axons in mobile precursors of presynaptic sites known as cytoplasmic transport packets   <cit> .
because synapsin- <dig> has been identified as a component of ctps, the abundant distribution of weak synapsin- <dig> puncta along axons in the absence of proximal map2+ processes at div  <dig> may represent the presence of ctps  <cit> .
interestingly, mobile puncta have been demonstrated to undergo synaptic vesicle recycling in the absence of post-synaptic partners  <cit> , possibly accounting for the observation that esns are highly sensitive to bont/a at div  <dig>  prior to the formation of established synapses  <cit> .
transcriptional characterization of neurogenesis
expression profiling and gene enrichment analyses of rna isolated from div  <dig> esns were consistent with immunocytological evidence of neuronal phenotype.
esns express a large set of neurotypic transcripts, including the post-mitotic nuclear marker neun, and abundant transcripts indicative of a vglut2+/vglut1- glutamatergic neuron subtype, with a smaller fraction expressing gabaergic genes.
these findings corroborate previous reports indicating that ≫ 95% of esns are vglut2+ and release glutamate in ca2+-dependent fashion under depolarizing stimuli  <cit> .
consistent with evidence of synaptogenesis, transcripts of genes essential to synapse formation and function are also abundant.
for example, snap- <dig>  vamp <dig> and syntaxin 1a/b are all expressed in the top 2% of all transcripts, emphasizing the importance of neurotransmitter release to neuronal function.
esns also express a large number of genes essential for neurotransmitter reception and intracellular electrochemical signal propagation, providing a mechanistic basis for previous demonstrations of ca2+-dependent, k+-evoked glutamate release and tetrodotoxin-sensitive measurements of action potentials at div  <dig>  <cit> .
functional characterization of esn responses to neuron-specific stimuli
a relevant neuron-based research platform must exhibit functional responses and sensitivities to neurotropic stimuli that are consistent with primary neurons.
we first demonstrated that two classical methods of membrane depolarization  rapidly induced reversible ca2+ uptake, confirming that esns functionally express a wide range of voltage-gated ion channels.
esns exposed to glutamate or ltx exhibited acute ca2+ influx followed by cell death at doses and timescales similar to those reported in primary neuron populations  <cit> .
these data suggest that esns offer a scalable and genetically tractable neuron-based model system that is amenable to characterization of excitotoxic mechanisms and novel identification of therapeutic targets.
furthermore, the ability to evaluate multiple metrics of toxicity in multi-well formats will facilitate moderate-throughput screening for antagonists of nmda receptor activity, antagonists of ltx pore formation/function or small molecules that mitigate acute or chronic aspects of excitotoxic progression.
unlike the excitogenic neurotoxins, the bonts inhibit neurotransmitter release by cleaving snare proteins with exquisite sensitivity.
since intoxication by bont holotoxins is highly specific and critically dependent on the function of a large set of neuron-specific proteins within the presynaptic compartment, the sensitivity of a cell population is therefore a reflection of presynaptic function  <cit> .
in combination with previous work, we have now shown that esns are sensitive to all seven classical bont serotypes, with ec <dig> values that are similar to those of primary neuron cultures and several orders of magnitude improved over neurogenic cell lines .
to our knowledge, this is the first time that a cell line has been shown to be responsive to all serotypes, including the dual-specificity of serotype /c.
these data indicate that esns functionally express the necessary receptors, co-factors and substrates for the internalization and activation of all of the bont serotypes, and suggest that bont is trafficked and processed in a fashion similar to primary neurons.
although the general mode of action is understood for each of these neurotoxins, efforts to precisely characterize cellular mechanisms of action have been complicated by the lack of a neuromimetic model system that is amenable to modern molecular and biochemical techniques.
for the first time, we demonstrate a single model system that is highly sensitive to these neurotoxins at physiologically meaningful concentrations, providing compelling evidence that esns are a biologically relevant, functional model for neurotoxicity research.
given that no other neurogenic model has demonstrated comparable sensitivity and neuronal homogeneity to the botulinum neurotoxins  <cit> , esns appear to be a novel and relevant model in which to conduct basic research, evaluate therapeutic candidates, determine potencies and diagnose the presence of multiple bont serotypes.
CONCLUSIONS
we demonstrate a variant of the 4/ <dig> neuronal differentiation process that results in high yields of enriched neurons, which respond to a variety of neurotropic stimuli in a sensitive and reproducible manner.
the robust production of a highly enriched population that exhibits morphological and functional evidence of a neuronal phenotype resolves problems that have hindered moderate-throughput drug screening approaches based on primary neurons  and neurogenic cells .
esns do not suffer from allelic variability and are genetically tractable, allowing the interrogation of the role of specific genes in neurogenesis.
repeated production of large quantities of esns is relatively simple and offers a highly reductionist model amenable to basic investigations of developmental and functional questions.
unlike dissociated primary cultures derived from neonatal or postnatal cns tissues, esn production does not involve disruption of previously established neuronal interactions.
neurogenesis is synchronized and neuron populations are highly enriched, reducing intra- and inter-experimental variability.
we anticipate that the ability to recapitulate pathophysiological responses in a relatively homogeneous, scalable and genetically tractable neuron model system will facilitate new approaches to neurotoxicity and neurogenesis research, including detailed investigation into molecular mechanisms of action, “omics”-based discovery and diagnostic screening.
although more work is needed to characterize neurogenesis and neuron maturation, the apparent similarities between esns and dissociated primary neuron cultures suggest that esns may also serve as a model system to explore mechanistic aspects of neuron development, synaptogenesis, neurotransmission and plasticity.
the same features that make esns a productive model for neurotoxicity make them attractive platforms for mechanistic exploration of neurogenic mechanisms.

 the use of live animals or primary neurons for neurotoxicity research is complicated by technical, ethical and economic considerations. an alternative strategy is the use of a cultured cell line that can be induced to exhibit neuronal characteristics. to date, the most common of these “neurogenic” cell lines are human and mouse neuroblastomas, which often exhibit poor sensitivity to neurotoxins, are comprised of heterogeneous phenotypes and may not form functioning synapses, making it questionable how accurately they model neuronal mechanisms of pathogenesis. an effective cell-based model system for neurotoxin research would combine the biological relevance of primary neurons with the flexibility of continuous cell lines. such a model would exhibit normal neurogenic progression, faithfully recapitulate the full range of interactions between primary neurons and neurotoxins, be compatible with modern cell and molecular techniques and facilitate moderate-throughput screening applications. early efforts to differentiate escs into neurons produced cells with morphological and functional characteristics similar to primary neurons. these cultures also contained a high percentage of glial cells and multiple neuron subtypes, which significantly detracted from their suitability as a basic research tool or therapeutic screening platform. recently, a variation of the 4/ method from esc aggregates for days followed by supplementation with retinoic acid for d) was described with significantly improved neuronal yield and purity. widespread application of this protocol was limited by several factors: custom media formulations were required; multiple manipulations were needed to isolate escs from feeder cell populations; and neurons were not produced at yields amenable to moderate-throughput screening. here we demonstrate that the robust production of enriched glutamatergic neurons from suspension-cultured mouse escs using a variant of the 4/ method offers a facile neuron model that exhibits high sensitivity to a variety of biological neurotoxins and replicates stages of neurogenesis observed in dissociated primary neuron cultures. we developed a facile, cost-effective procedure that enables the rapid production of large numbers of highly enriched glutamatergic esns from continuous suspension cultures of murine escs. immunological, genomic, morphological and functional evaluations suggest that esn maturation and behavior recapitulate those described in primary neurons. we further demonstrate that esns appropriately respond to different neurotropic toxins at doses similar to primary neurons. these data provide compelling evidence that suspension culture-derived esns comprise a novel cell model for neurotoxicity research that combines the verisimilitude of primary neurons with the flexibility of continuous cell lines. the identification of a robust, scalable and sensitive neuron-based research platform amenable to genetic modification and compatible with moderate-throughput techniques should enable detailed biochemical and molecular approaches that are not feasible in neurogenic cells or primary neurons. we anticipate this platform to expedite the identification and validation of novel therapeutic approaches to a variety of neurotoxins as well as the elucidation of molecular aspects of pathogenesis following intoxication.  suspension-adapted escs remain pluripotent and mitotically active escs are traditionally maintained on feeder cells in the presence of lif and fetal calf serum to preserve germline competency. to adapt esc lines to feeder cell-free suspension culture, r d and c57bl/ escs that had been co-cultured with mitotically inactivated mouse embryonic fibroblasts were dissociated and cultivated in bacterial dishes until suspended aggregates developed. mitotic rates of the suspension-adapted escs stabilized by five passages and mean doubling times were  ±  h,  ±  h and  ±  h over passages. expression of the pluripotency marker oct3/ did not vary through passages, whereas withdrawal of lif resulted in a loss of oct3/ immunoreactivity within d. theoretical estimates of cumulative yield over passages from a single dish were escs. suspension-adapted escs were differentiated into neurons using a modified 4/ protocol, with increased ra concentrations and incubation under rotary conditions in low-attachment dishes. we initially observed that differentiation under static conditions resulted in large, agglomerated complexes by div. hypothesizing that agglomeration might limit recovery of npcs at div npc yields were compared between static differentiation conditions versus differentiation on a rotary shaker at rpm. rotary conditions eliminated agglomeration and increased average yield by 290% to 97 ×  npcs per 10-cm dish. no differences were observed in npc yield between 5– passages in any esc line. in one instance, an r culture maintained for passages produced × npcs, which underwent normal neuronal development, indicating that extended periods in suspension culture may not interfere with neurogenic competence. differentiated cells express transcriptional, morphological and immunological markers of neurogenesis neuronal maturation was characterized between div 1– using the dendritic marker map the axonal marker map-tau and the pre-synaptic marker synapsin-. although map was uniformly present at div map-tau expression was not widely observed in the majority of neurons until div axonal arborization increased extensively between div 3– while dendritic extension occurred predominantly after div. weak synapsin puncta were widely dispersed along axons at div primarily in the absence of proximal dendrites, but as of div synapsin- staining accumulated at axodendritic interfaces. by div an extensive “lawn” of neurites was apparent, with robust axonal arborization and elongated dendrites appearing in close proximity to single or fasciculated axons. greater than 99% of surviving cells expressed neuron-specific markers at div and less than one gfap+ glial cell was observed per mm at div. glial cells that did survive were most often observed at regions of high neuron density, suggesting that a supportive microenvironment may be permissive for glial persistence in the absence of serum. a presumptive neuronal phenotype was further evaluated by expression profiling at div an average of ± nuclear mrna transcripts were detected at a single copy or higher, of which about were present in high abundance. overall, esns expressed a broad range of neurotypic genes, and 28% of the most abundant transcripts coded for neuron-specific proteins. gene expression was highly enriched for a neuronal phenotype and strongly associated with neuron-specific canonical pathways and functions. esns expressed high levels of glutamatergic markers, with low-to-moderate levels of gabaergic markers and virtually no markers of cholinergic, serotonergic, dopaminergic or motor neuron differentiation. a variety of neurotransmitter receptors were expressed, including those for glycine, gaba, acetylcholine and glutamate. esns also expressed transcripts essential for synaptic activity and electrochemical signal propagation, including subunits of the neuronal n- and p/q-type voltage-dependent ca2+ channels, snare proteins, synaptic vesicle-associated proteins, na+/k+ pumps, and a large number of voltage-gated na+, k+ and cl- channels. depolarizing stimuli evoke reversible ca2+ uptake the expression of gated ion channels and pumps important in maintaining and altering membrane polarity suggested that application of depolarizing stimuli might elicit ca2+ uptake. we altered the plasma membrane potential using indirect and direct methods and found that both methods of depolarization elicited reversible ca2+ influxes. the functional verification of ca2+ uptake in response to classical depolarizing stimuli confirms intracellular recordings and neurotransmitter release assays, and suggests that esns may be sensitive to neurotoxic stimuli whose mechanism of action involves dysregulation of electrochemical signal propagation. esns are sensitive to glutamatergic excitotoxicity glutamatergic excitotoxicity has been attributed to the pathologic internalization of ca2+ through post-synaptic nmda receptors, compounded by activation of vdccs in response to excitatory post-synaptic currents from ampa and ka receptors. in primary neuron cultures, neurotoxicity has been reported over a wide range of glutamate doses and exposure durations. esns express transcripts for nmda, ka and ampa receptor subunits and the nmda-associated protein grina at high levels, suggesting that esns may be functionally sensitive to glu treatment. treatment of esns with μm glu resulted in acute levels of ca2+ uptake similar to those caused by k+, whereas the inhibitory neurotransmitter gaba or vehicle controls had no effect. glu treatment resulted in significant time- and dose-dependent toxicity, further confirmed by morphological evidence of neurite degeneration h after a μm treatment. co-administration of the glur antagonists apv and cnqx afforded complete protection against toxicity after a h exposure to and μm glu, and 50% protection against μm glu. these results indicate that esns undergo a time- and dose-dependent glu toxicity that is mediated by ionotropic glurs. neuron viability and ca2+ influx following exposure to alpha-latrotoxin unlike glu exposure, which induces epscs in post-synaptic compartments, ltx forms ca2+-permissive pores in the presynaptic membrane that result in fulminant neurotransmitter release and activation of non-synaptic ca2+-sensitive intracellular pathways. esns express transcripts of known ltx receptors, and ltx treatment of esns evokes unregulated ca2+ influx, followed by morphological and biochemical indicators of neurotoxicity. to better characterize ltx toxicity, we evaluated dose-dependent relationships between ltx, onset and magnitude of ca2+ uptake and neuron cell death. the ec for ca2+ uptake at min was determined to be pm, with positive correlations between dose, onset and magnitude of ca2+ uptake. we also demonstrated a strong correlation between ltx dose and inhibition of metabolic activity after a min exposure. these findings illustrate the compatibility of esns with moderate-throughput screening approaches, confirm that esn sensitivity and response to ltx are similar to those of primary neurons, and demonstrate that esns offer a novel platform with which to screen for therapeutics that prevent ltx toxicity using multi-well formats. esns are a biologically relevant model of bont intoxication esns strongly express transcripts and protein for proteolytic targets of the seven bont serotypes and known protein receptors for presynaptic uptake ). we have shown that esns are sensitive to femtomolar concentrations of bont/a and /e. to determine whether esns are a suitable model for the remaining serotypes, we evaluated cleavage of the target snare proteins after a h exposure to bonts /b, /c, /d, /f and /g. esns exhibited similar or improved sensitivities to all bont serotypes tested compared to primary mouse spinal cord or cerebellar granule cell neurons, and several orders of magnitude improved sensitivity over neuroblastoma cells. bont/c is the only holotoxin that targets multiple snare proteins; interestingly, we found that intoxication of esns with bont/c resulted in cleavage of snap- as well as syntaxin- with roughly equivalent ec values. notes: primary neurons’ values are compiled from two sources. annotation by represents 50% inhibition of neurotransmitter release in rat cerebellar neuron cultures exposed to bonts for h, whereas annotation by represents cleavage of 50% of snap- protein in fetal mouse spinal cord cultures exposed to bont for h. sh-sy5y neuroblastomas’ values indicate inhibition of 50% of noradrenalin release in sh-sy5y human neuroblastomas cells after h incubation. esn values represent 50% cleavage of snare proteins after a h tonic intoxication. esn ec values for bont/b, /c, /d, /f and /g are from this manuscript, while bont/a and /e are from mcnutt et al.. nr, not reported. bont holotoxin binds to pre-synaptic receptors and is internalized via synaptic endocytosis. following cell entry, the proteolytically active light chain is released to the synaptic compartment through pores formed by the heavy chain in the endosome membrane. bont uptake and/or activation has been reported to be enhanced following intoxication under depolarizing conditions. we found that a min intoxication of esns by bont/a in depolarizing media increased snap- cleavage by five-fold after h as compared to two different basal media. discussion esc lines remain mitotically active, pluripotent and neurogenic following adaptation to feeder-independent suspension culture with the aim of developing a more facile, robust and efficient method for the production of homogenous neuronal cultures from mouse esc lines, we evaluated the neurogenic capacity of escs adapted to feeder-independent suspension culture and differentiated under rotary conditions. mitotic rates of suspension-adapted r d and c57bl/ esc lines were stabilized by five passages, and all esc lines remained mitotically active and expressed the pluripotency marker oct3/ in the absence of feeder cells. germline competence was not evaluated, so it is unclear whether these culture conditions restrict non-neurogenic developmental fates. adaptation to suspension culture streamlined and economized esc culture and neuronal differentiation by obviating the need for feeder cells and allowing the initiation of neuronal differentiation directly from suspension cultures every h. suspension-cultured escs remained neurogenic, and differentiated neurons exhibited a multitude of neurotypic characteristics when maintained in relatively inexpensive, commercially available media. we found that the simple addition of a rotary step during differentiation significantly increased neuron yield to levels sufficient for moderate-throughput screening techniques. these improvements resulted in the generation of sufficient npcs from one 10-cm dish to plate cm of tissue culture surface at cells per cm morphological and immunological characterization of neurogenesis the neurotypic character of esns was confirmed using morphology, transcriptional profiling, immunocytochemistry and functional characterization. temporal changes in map map-tau and synapsin- expression and localization demonstrated a characteristic progression through the first stages of neurogenesis, as described by dotti et al. in embryonic hippocampal neurons, culminating in a dense axodendritic arbor with punctate localization of synapsin- at axodendritic interfaces. as soon as div a majority of esns produced short map2+ processes that were indistinguishable from one another, characteristic of development stage neurons. between div 1– most esns underwent rapid extension of minor neurites and increased expression of axonal markers, characteristic of ds. axons then underwent rapid extension and arborization, accompanied by the onset of dendritic outgrowth and synaptogenesis at ds. the earliest appearance of synapsin accumulation at axodendritic interfaces appeared between div 7– consistent with previous demonstrations of characteristic pre- and post-synaptic architectures at div. it should be noted that the plating density is important in producing a neuronal population with few contaminating, non-neuronal cells. not only does serum withdrawal at div initiate the final stages of neuronal differentiation, but it also eliminates the β3-tubulin non-expressing cells, which are likely to be glial cells. glia that survive beyond div tend to be localized to regions of high neuron density. thus, if npcs are plated at high density, then increased numbers of glia survive serum-starvation. if unchecked, they can proliferate and eventually overwhelm the esn culture. we have found that glial contamination due to overplating can be suppressed without detectibly altering neuron viability or function by addition of 5-fluoro-deoxyuridine/uridine from div 8– during synaptogenesis, presynaptic proteins are bundled and transported along axons in mobile precursors of presynaptic sites known as cytoplasmic transport packets. because synapsin- has been identified as a component of ctps, the abundant distribution of weak synapsin- puncta along axons in the absence of proximal map2+ processes at div may represent the presence of ctps. interestingly, mobile puncta have been demonstrated to undergo synaptic vesicle recycling in the absence of post-synaptic partners, possibly accounting for the observation that esns are highly sensitive to bont/a at div prior to the formation of established synapses. transcriptional characterization of neurogenesis expression profiling and gene enrichment analyses of rna isolated from div esns were consistent with immunocytological evidence of neuronal phenotype. esns express a large set of neurotypic transcripts, including the post-mitotic nuclear marker neun, and abundant transcripts indicative of a vglut2+/vglut1- glutamatergic neuron subtype, with a smaller fraction expressing gabaergic genes. these findings corroborate previous reports indicating that ≫ 95% of esns are vglut2+ and release glutamate in ca2+-dependent fashion under depolarizing stimuli. consistent with evidence of synaptogenesis, transcripts of genes essential to synapse formation and function are also abundant. for example, snap- vamp and syntaxin 1a/b are all expressed in the top 2% of all transcripts, emphasizing the importance of neurotransmitter release to neuronal function. esns also express a large number of genes essential for neurotransmitter reception and intracellular electrochemical signal propagation, providing a mechanistic basis for previous demonstrations of ca2+-dependent, k+-evoked glutamate release and tetrodotoxin-sensitive measurements of action potentials at div. functional characterization of esn responses to neuron-specific stimuli a relevant neuron-based research platform must exhibit functional responses and sensitivities to neurotropic stimuli that are consistent with primary neurons. we first demonstrated that two classical methods of membrane depolarization rapidly induced reversible ca2+ uptake, confirming that esns functionally express a wide range of voltage-gated ion channels. esns exposed to glutamate or ltx exhibited acute ca2+ influx followed by cell death at doses and timescales similar to those reported in primary neuron populations. these data suggest that esns offer a scalable and genetically tractable neuron-based model system that is amenable to characterization of excitotoxic mechanisms and novel identification of therapeutic targets. furthermore, the ability to evaluate multiple metrics of toxicity in multi-well formats will facilitate moderate-throughput screening for antagonists of nmda receptor activity, antagonists of ltx pore formation/function or small molecules that mitigate acute or chronic aspects of excitotoxic progression. unlike the excitogenic neurotoxins, the bonts inhibit neurotransmitter release by cleaving snare proteins with exquisite sensitivity. since intoxication by bont holotoxins is highly specific and critically dependent on the function of a large set of neuron-specific proteins within the presynaptic compartment, the sensitivity of a cell population is therefore a reflection of presynaptic function. in combination with previous work, we have now shown that esns are sensitive to all seven classical bont serotypes, with ec values that are similar to those of primary neuron cultures and several orders of magnitude improved over neurogenic cell lines. to our knowledge, this is the first time that a cell line has been shown to be responsive to all serotypes, including the dual-specificity of serotype /c. these data indicate that esns functionally express the necessary receptors, co-factors and substrates for the internalization and activation of all of the bont serotypes, and suggest that bont is trafficked and processed in a fashion similar to primary neurons. although the general mode of action is understood for each of these neurotoxins, efforts to precisely characterize cellular mechanisms of action have been complicated by the lack of a neuromimetic model system that is amenable to modern molecular and biochemical techniques. for the first time, we demonstrate a single model system that is highly sensitive to these neurotoxins at physiologically meaningful concentrations, providing compelling evidence that esns are a biologically relevant, functional model for neurotoxicity research. given that no other neurogenic model has demonstrated comparable sensitivity and neuronal homogeneity to the botulinum neurotoxins, esns appear to be a novel and relevant model in which to conduct basic research, evaluate therapeutic candidates, determine potencies and diagnose the presence of multiple bont serotypes.  we demonstrate a variant of the 4/ neuronal differentiation process that results in high yields of enriched neurons, which respond to a variety of neurotropic stimuli in a sensitive and reproducible manner. the robust production of a highly enriched population that exhibits morphological and functional evidence of a neuronal phenotype resolves problems that have hindered moderate-throughput drug screening approaches based on primary neurons and neurogenic cells. esns do not suffer from allelic variability and are genetically tractable, allowing the interrogation of the role of specific genes in neurogenesis. repeated production of large quantities of esns is relatively simple and offers a highly reductionist model amenable to basic investigations of developmental and functional questions. unlike dissociated primary cultures derived from neonatal or postnatal cns tissues, esn production does not involve disruption of previously established neuronal interactions. neurogenesis is synchronized and neuron populations are highly enriched, reducing intra- and inter-experimental variability. we anticipate that the ability to recapitulate pathophysiological responses in a relatively homogeneous, scalable and genetically tractable neuron model system will facilitate new approaches to neurotoxicity and neurogenesis research, including detailed investigation into molecular mechanisms of action, “omics”-based discovery and diagnostic screening. although more work is needed to characterize neurogenesis and neuron maturation, the apparent similarities between esns and dissociated primary neuron cultures suggest that esns may also serve as a model system to explore mechanistic aspects of neuron development, synaptogenesis, neurotransmission and plasticity. the same features that make esns a productive model for neurotoxicity make them attractive platforms for mechanistic exploration of neurogenic mechanisms. 
recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. the functional responses of esns to neurotropic toxins with distinctly different targets and mechanisms of toxicity, such as glutamate, α-latrotoxin, and botulinum neurotoxin, were also evaluated. with the objective of developing a more facile, robust and efficient method to generate enriched glutamatergic neuronal cultures, we evaluated the neurogenic capacity of three mouse embryonic stem cell lines adapted to feeder-independent suspension culture. the functional characterization of pathophysiological responses to neurotropic toxins and the compatibility with multi-well plating formats were used to demonstrate the suitability of esns as a discovery platform for molecular mechanisms of action, moderate-throughput analytical approaches and diagnostic screening. these data providing compelling evidence that esns offer a neuromimetic platform suitable for the evaluation of molecular mechanisms of neurotoxicity. furthermore, for the first time we demonstrate a cell-based model that is sensitive to all seven bont serotypes with ec values comparable to those reported in primary neuron populations. however, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available. suspension-adapted escs expressed markers of pluripotency through at least passages, and differentiation produced 97× neural progenitor cells per 10-cm dish. expression profiling demonstrated the presence of transcripts necessary for neuronal function and confirmed that esn populations were predominantly glutamatergic. greater than 99% of embryonic stem cell-derived neurons expressed neuron-specific markers by h after plating and rapidly developed complex axodendritic arbors and appropriate compartmentalization of neurotypic proteins. derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. furthermore, esns were functionally receptive to all toxins with sensitivities and responses consistent with primary neurons.
 recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. however, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available. methods with the objective of developing a more facile, robust and efficient method to generate enriched glutamatergic neuronal cultures, we evaluated the neurogenic capacity of three mouse embryonic stem cell lines adapted to feeder-independent suspension culture. neurogenesis and neuronal maturation were characterized as a function of time in culture using immunological, genomic, morphological and functional metrics. the functional responses of esns to neurotropic toxins with distinctly different targets and mechanisms of toxicity, such as glutamate, α-latrotoxin, and botulinum neurotoxin, were also evaluated.  suspension-adapted escs expressed markers of pluripotency through at least passages, and differentiation produced 97× neural progenitor cells per 10-cm dish. greater than 99% of embryonic stem cell-derived neurons expressed neuron-specific markers by h after plating and rapidly developed complex axodendritic arbors and appropriate compartmentalization of neurotypic proteins. expression profiling demonstrated the presence of transcripts necessary for neuronal function and confirmed that esn populations were predominantly glutamatergic. furthermore, esns were functionally receptive to all toxins with sensitivities and responses consistent with primary neurons.  these findings demonstrate a cost-effective, scalable and flexible method to produce a highly enriched glutamatergic neuron population. the functional characterization of pathophysiological responses to neurotropic toxins and the compatibility with multi-well plating formats were used to demonstrate the suitability of esns as a discovery platform for molecular mechanisms of action, moderate-throughput analytical approaches and diagnostic screening. furthermore, for the first time we demonstrate a cell-based model that is sensitive to all seven bont serotypes with ec values comparable to those reported in primary neuron populations. these data providing compelling evidence that esns offer a neuromimetic platform suitable for the evaluation of molecular mechanisms of neurotoxicity. mouse embryonic stem cellsneurotoxicityin vitro modelingbontltxglutamate 