Mammalian Spinal Neurons Research Articles

Single-cell transcriptomic analysis reveals diversity within mammalian spinal motor neurons

Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate movements. In vertebrates, the molecular identities of the cardinal MN types such as those innervating limb versus trunk muscles are well elucidated. Yet the identities of finer subtypes within these cell populations that innervate individual muscle groups remain enigmatic. Here we investigate heterogeneity in mouse MNs using single-cell transcriptomics. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. Additionally, we uncover that axial MNs are subdivided into three molecularly distinct subtypes, defined by mediolaterally-biased Satb2, Nr2f2 or Bcl11b expression patterns with different axon guidance signatures. These three subtypes are present in chicken and human embryos, suggesting a conserved axial MN expression pattern across higher vertebrates. Overall, our study provides a molecular resource of spinal MN types and paves the way towards deciphering how neuronal subtypes evolved to accommodate vertebrate motor behaviors.

The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs PR50 and GR50 are highly toxic when expressed in Caenorhabditis elegans, and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNA interference (RNAi) screen for suppressors of PR50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by PR50 expression. All of these genes have vertebrate homologs, and 7 of 12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for GR50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that knockdown of bromodomain proteins in both C. elegans and mammalian neurons, which are known SPOP ubiquitination targets, suppresses the protective effect of SPOP inhibition. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent an entry point for therapeutic intervention to treat C9orf72 FTD/ALS.

Stimulation-induced Ca(2+) influx at nodes of Ranvier in mouse peripheral motor axons.

In peripheral myelinated axons of mammalian spinal motor neurons, Ca(2+) influx was thought to occur only in pathological conditions such as ischaemia. Using Ca(2+) imaging in mouse large motor axons, we find that physiological stimulation with trains of action potentials transiently elevates axoplasmic [C(2+)] around nodes of Ranvier. These stimulation-induced [Ca(2+)] elevations require Ca(2+) influx, and are partially reduced by blocking T-type Ca(2+) channels (e.g. mibefradil) and by blocking the Na(+)/Ca(2+) exchanger (NCX), suggesting an important contribution of Ca(2+) influx via reverse-mode NCX activity. Acute disruption of paranodal myelin dramatically increases stimulation-induced [Ca(2+)] elevations around nodes by allowing activation of sub-myelin L-type (nimodipine-sensitive) Ca(2+) channels. The Ca(2+) that enters myelinated motor axons during normal activity is likely to contribute to several signalling pathways; the larger Ca(2+) influx that occurs following demyelination may contribute to the axonal degeneration that occurs in peripheral demyelinating diseases. Activity-dependent Ca(2+) signalling is well established for somata and terminals of mammalian spinal motor neurons, but not for their axons. Imaging of an intra-axonally injected fluorescent [Ca(2+)] indicator revealed that during repetitive action potential stimulation, [Ca(2+)] elevations localized to nodal regions occurred in mouse motor axons from ventral roots, phrenic nerve and intramuscular branches. These [Ca(2+)] elevations (∼ 0.1 μm with stimulation at 50 Hz, 10 s) were blocked by removal of Ca(2+) from the extracellular solution. Effects of pharmacological blockers indicated contributions from both T-type Ca(2+) channels and reverse mode Na(+)/Ca(2+) exchange (NCX). Acute disruption of paranodal myelin (by stretch or lysophosphatidylcholine) increased the stimulation-induced [Ca(2+)] elevations, which now included a prominent contribution from L-type Ca(2+) channels. These results suggest that the peri-nodal axolemma of motor axons includes multiple pathways for stimulation-induced Ca(2+) influx, some active in normally-myelinated axons (T-type channels, NCX), others active only when exposed by myelin disruption (L-type channels). The modest axoplasmic peri-nodal [Ca(2+)] elevations measured in intact motor axons might mediate local responses to axonal activation. The larger [Ca(2+) ] elevations measured after myelin disruption might, over time, contribute to the axonal degeneration observed in peripheral demyelinating neuropathies.

Replication of association of CHRNA4 rare variants with sporadic amyotrophic lateral sclerosis: The Italian multicentre study

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels widely expressed throughout the mammalian brain, including bulbar and spinal motor neurons. They are involved in neuroprotection and in control of release of many neurotransmitters, including glutamate. Previous data raised the hypothesis that rare variants in the region coding the intracellular loop subunits of nAChRs might represent one of several genetic risk factors for SALS. The aim of present study was to replicate the study in an independent cohort of ALS patients. We analysed 718 sporadic ALS patients from five Italian ALS centres and 1300 ethnically matched controls. We focused primarily on CHRNA4, encoding α4 subunit, since most mutations were previously detected in this gene. We observed a significant association between CHRNA4 mutations and ALS (OR 2.91; 95% CI 1.4080-6.0453; p = 0.0056). Most mutations detected in patients were not present in the dbSNP134 and in 3500 ethnically matched control chromosomes and affected evolutionary conserved amino acid residues. In conclusion, the present data confirm that CHRNA4 variants are overrepresented in SALS strengthening the hypothesis can they act as predisposing genetic factors for SALS.

Apoptosis of limb innervating motor neurons and erosion of motor pool identity upon lineage specific Dicer inactivation

Diversification of mammalian spinal motor neurons into hundreds of subtypes is critical for the maintenance of body posture and coordination of complex movements. Motor neuron differentiation is controlled by extrinsic signals that regulate intrinsic genetic programs specifying and consolidating motor neuron subtype identity. While transcription factors have been recognized as principal regulators of the intrinsic program, the role of posttranscriptional regulations has not been systematically tested. MicroRNAs produced by Dicer mediated cleavage of RNA hairpins contribute to gene regulation by posttranscriptional silencing. Here we used Olig2-cre conditional deletion of Dicer gene in motor neuron progenitors to examine effects of miRNA biogenesis disruption on postmitotic spinal motor neurons. We report that despite the initial increase in the number of motor neuron progenitors, disruption of Dicer function results in a loss of many limb- and sympathetic ganglia-innervating spinal motor neurons. Furthermore, it leads to defects in motor pool identity specification. Thus, our results indicate that miRNAs are an integral part of the genetic program controlling motor neuron survival and acquisition of subtype specific properties.

Adult rat spinal cord culture on an organosilane surface in a novel serum-free medium

In this study, we have documented by morphological analysis, immunocytochemistry, and electrophysiology, the development of a culture system that promotes the growth and long-term survival of dissociated adult rat spinal cord neurons. This system comprises a patternable, nonbiological, cell growth-promoting organosilane substrate coated on a glass surface and an empirically derived novel serum-free medium, supplemented with specific growth factors (acidic fibroblast growth factor, heparin sulfate, neurotrophin-3, brain-derived neurotrophic factor, glial-derived neurotrophic factor, cardiotrophin-1, and vitronectin). Neurons were characterized by immunoreactivity for neurofilament 150, neuron-specific enolase, Islet-1 antibodies, electrophysiology, and the cultures were maintained for 4-6 wk. This culture system could be a useful tool for the study of adult mammalian spinal neurons in a functional in vitro system.

Reduction of NaCl increases survival of mammalian spinal neurons subjected to dendrite transection injury

Neurites were transected from spinal neurons in media with normal (125 mM) or reduced NaCI (sucrose substitution). After 12 h the normal ionic environment (conditioned medium with serum) was restored. A one-factor ANOVA comparison found a significant difference in 48 h survival (P = 0.0001). Survival was highest when NaCl was reduced 50% (74% ± 19 vs. 22% ± 19 in normal NaCI). Na+ - and Cl−-mediated deterioration may contribute to both gray and white matter injury in CNS trauma.

Reduction of NaCl increases survival of mammalian spinal neurons subjected to dendrite transection injury

Neurites were transected from spinal neurons in media with normal (125 mM) or reduced NaCI (sucrose substitution). After 12 h the normal ionic environment (conditioned medium with serum) was restored. A one-factor ANOVA comparison found a significant difference in 48 h survival (P = 0.0001). Survival was highest when NaCl was reduced 50% (74% ± 19 vs. 22% ± 19 in normal NaCI). Na+ - and Cl−-mediated deterioration may contribute to both gray and white matter injury in CNS trauma.

Effects of methylprednisolone on lesioned and uninjured mammalian spinal neurons: viability, ultrastructure, and network electrophysiology.

An in vitro investigation was undertaken to provide information regarding the effectiveness of methylprednisolone sodium succinate (MPSS) as a treatment for the primary mechanical injury of spinal cord (SC) trauma. Exposure of uninjured mouse SC cells to MPSS for 24 h caused neuronal stress when the concentration exceeded 150 micrograms/mL; neuronal death occurred at concentrations above 600 micrograms/mL. The concentration range for MPSS protection of SC neurons subjected to a defined physical injury (laser microbeam transection of a primary dendrite 100 microns from the perikaryon) was very narrow: survival in the 30 micrograms/mL group differed significantly from the untreated control group (68.5% +/- 14.1 vs. 47.1% +/- 14.1), treatment with 20 or 60 micrograms/mL MPSS did not increase survival, and treatment with 100 micrograms/mL MPSS accelerated ultrastructural deterioration and increased the likelihood of death. Enhanced survival of lesioned neurons was observed when 30 micrograms/mL MPSS was applied within 15 min of dendrotomy but not when MPSS was administered 2 h after lesioning. Multimicroelectrode plate (MMEP) studies of SC network electrical activity indicated that MPSS associated readily with neuronal membranes. This finding was consistent with the hypothesis that MPSS may protect lesioned neurons by stabilizing damaged membranes, enhancing lesion resealing, and limiting the spread of ion-mediated damage. However, comparisons of neurite die-back 24 h after dendrotomy found no significant difference between MPSS-treated and control neurons. Application of 30 or 100 micrograms/mL MPSS increased the spontaneous burst activity of SC networks grown on MMEPs, however, there was no evidence that the increased excitability at these concentrations was the result of specific actions of MPSS on GABA or NMDA synapses.

S32-5 - Presynaptic and postsynaptic actions of serotonin on the mammalian spinal neurons in slice

S32-5 - Presynaptic and postsynaptic actions of serotonin on the mammalian spinal neurons in slice

In vitro investigations of the effects of nonfreezing low temperatures on lesioned and uninjured mammalian spinal neurons.

This two-part investigation explored the parameters and mechanisms of: (1) injury to spinal cord (SC) neurons by nonfreezing low temperatures, and (2) hypothermic protection of SC neurons subjected to a defined, physical injury (dendrite transection). Conclusions from the studies of hypothermic injury were: (1) morphologic and ultrastructural signs of stress developed in SC neurons as the temperature was decreased below 17 degrees C; (2) most neurons showing stress during cooling died upon rewarming to 37 degrees C; (3) spontaneous SC network activity was not significantly changed by cooling to 17 degrees C for 2 hours and rewarming, but cooling to 10 degrees C for 1 hour caused a reduction of burst frequency after rewarming, and cooling to 10 degrees C for 2 hours resulted in electrical silence after rewarming; and (4) application of N-methyl-D-aspartate (NMDA) antagonists before cooling prevented neuronal death, ultrastructural damage, and loss of activity upon rewarming, but application after cooling (before rewarming) was not protective. Conclusions from the studies of hypothermic protection were: (1) cooling at 17 degrees C for 2 hours followed by rewarming to 37 degrees C significantly increased lesioned neuron survival, but protection was lost when the period at 17 degrees C was increased to 6 hours; (2) NMDA blockade under normothermic (37 degrees C) or hypothermic (17 degrees C or 10 degrees C for 2 hours) conditions was not more protective of lesioned neurons than cooling to 17 degrees C (no NMDA antagonist); and (3) 200 microM thiopental or 100 microM pentobarbital increased lesioned neuron survival to a degree comparable to cooling for 2 hours at 17 degrees C.

An in vitro study of the effects of methylprednisolone on lesioned and uninjured mammalian spinal neurons

Cultured spinal neurons were subjected to dendrite amputation 100 μm from the perikaryon and treated with methylprednisolone (MP). Survival was significantly increased by 30 μg/ml MP but not by 10, 20 or 60 μg/ml. Survival was reduced by 100 μg/ml MP. These results suggest: (1) MP protects neurons subjected to physical trauma, and (2) the effective dose range is very narrow. These findings may have implications for MP's observed bimodal effects in spinal cord injury.

Progressive morphological abnormalities observed in rat spinal motor neurons at extended intervals after axonal regeneration.

It is generally accepted that mammalian spinal motor neurons return to normal after axotomy if their regenerated axons successfully reinnervate appropriate peripheral targets. However, morphological abnormalities, recently observed in spinal motor neurons examined 1 year after nerve crush injury, raise the possibility that delayed perikaryal changes occur after regeneration is complete. In order to distinguish between chronic and progressive alterations in neurons with long-term regenerated axons, rat spinal motor neurons and dorsal root ganglion cells were examined at 5 and 10 months following unilateral sciatic nerve crush. Neurons with regenerated axons were identified by retrograde labelling with horseradish peroxidase. The structural properties of neurons ipsilateral to nerve injury were compared to those of neurons from the spinal cord and dorsal root ganglia on the contralateral side and from age-matched control rats. At 5 months postcrush, the morphology of motor and sensory neurons ipsilateral to injury was comparable to that of control cells. However, several features of the motor neurons with regenerated axons distinguished them from control motor neurons at 10 months postcrush. Mean perikaryal area of ipsilateral spinal motor neurons was larger than the means for control motor neurons (p less than .001). Ipsilateral spinal motor neurons also appeared clustered within the spinal cord and had thicker dendrites. Dorsal root ganglion cells with regenerated axons were slightly larger than control cells at 10 months postcrush but they exhibited no other morphological changes. The present findings indicate that spinal motor neurons are progressively altered after their regenerated axons have reestablished functional synapses with their peripheral targets.

The actions of a conformationally restricted analogue of aspartic acid on mammalian spinal neurones

The effects of the 4 stereoisomers of 1-aminocyclopentane-1,2-dicarboxylic acid (CPA), a conformationally restricted analogue of aspartic acid, have been examined on spinal dorsal horn neurones of the rat in vivo. Unlike the corresponding 1,3-dicarboxylate compounds which are glutamate analogues and markedly excitatory, the CPA isomers had no evident excitatory actions of their own. Three were weak and non-specific antagonists of other amino acid-induced excitations, while the (+)- trans isomer had a slight potentiating effect.

Paradoxical effect of hypothermia on survival of lesioned and uninjured mammalian spinal neurons

Survival of cultured spinal neurons 2 h after dendrotomy increased significantly when the temperature was reduced 17 °C or 17 °C. Survival of lesioned neurons maintained at 17 °C for 2 h and then at 37 °C for 22 h was still significantly higher than survival of neurons maintained at 37 °C for 24 h. Below 17 °C both lesioned and uninjured neurons swelled. Upon rewarming to 37 °C, many swollen neurons died. Ketamine prevented hypothermic swelling and neuronal death.

NMDA Antagonists Prevent Hypothermic Injury and Death of Mammalian Spinal Neurons

Prolonged (2-6 h) cooling of monolayer cultures of dissociated murine spinal cord at temperatures below 17 degrees C caused pronounced swelling of neuronal perikarya and dendrites. The numbers of swollen neurons in a culture increased as the temperature was reduced, and at 7 degrees C-10 degrees C all of the neurons were swollen. On rewarming the cultures to 37 degrees C, the majority of the swollen neurons died (up to 74% at 10 degrees C). Glial cells were not affected. Addition of the NMDA antagonists D-2-amino-5-phosphonovalerate (DAPV, 100 microM), ketamine (100 microM), and dibenzocyclohepteneimine (MK801, 10 microM) to spinal cord cultures before lowering the temperature to 10 degrees C minimized the dendrosomatic swelling and reduced neuronal mortality from 74% to 10%. These data show a surprising sensitivity of some neurons to nonfreezing low temperatures and suggest direct involvement of the NMDA receptor in hypothermia-related neuronal death.

Neuronal Survival and Dynamics of Ultrastructural Damage After Dendrotomy in Low Calcium

To determine the contributions of calcium to development of ultrastructural damage and neuronal death after mechanical injury, we amputated primary dendrites from over 300 cultured mammalian spinal neurons under normal (1.8 mM) or low (less than or equal to 30 microM) calcium conditions. Two general categories of early ultrastructural change were seen in both normal and low calcium: (1) a lesion-dependent gradient of damage that moved centripetally through the proximal segment and penetrated the soma within 15 min and (2) dilation of the somal Golgi/smooth endoplasmic reticulum (SER), which preceded the wave of deterioration from the lesion. Although the somal Golgi/SER changes were similar in both normal and low calcium, the damage gradient in low calcium differed from the damage gradient in normal calcium. (1) Microtubules and neurofilaments were preserved, (2) mitochondria became more electron dense but did not develop electronlucent foci or high amplitude swelling, and (3) an extensive vesicular gradient formed consisting of rows of swollen SER vesicles. Sodium ionophores have been reported to cause similar changes. Survival studies showed that calcium reduction significantly delayed neuronal death. Survival was 63 +/- 16% vs 35 +/- 8% (p less than 0.003) at 2 h and 30 +/- 7% vs 23 +/- 8% at 6 h in low and normal calcium, respectively. Dead neurons that had been lesioned in low calcium also showed greater ultrastructural preservation than neurons that died after dendrotomy in normal calcium. We hypothesize that under low calcium conditions, the large sodium injury current plays an important role in neuronal deterioration and death after mechanical trauma.

Electrophysiological effects of FLFQPQRF amide, an endogenous brain morphine modulating peptide, on cultured mouse spinal-cord neurons

Intracellular recordings were made from dissociated fetal mouse spinal cord neurones in primary culture. Micropressure application of FLFQPQRFamide (10 −5 M in the delivery pipette), an endogenous mammalian brain morphine modulating peptide, onto the surface of spinal cord neurones induced, in a dose dependent manner, a transitory hyperpolarization followed by a long lasting depolarization of the membrane potential (n = 37). In contrast, no response was observed when the peptide was applied on dorsal root ganglia neurones (n = 30). The depolarizing phase of this response was underlied by an increase of the input resistance. Extrapolated reversal potential for the depolarizing phase was close to −80 mV while it was close to −40 mV for the hyperpolarizing phase. Increasing extracellular K + concentration raised the reversal potential value of depolarizing phases to more positive values. The amplitude of the depolarizing phase was reduced by application of tetraethylammonium (50 mM) while it was enhanced by application of 4-aminopyridine (3 mM). CaCl 2 application (3 mM) reversibly blocked the hyperpolarization and decreased the subsequent depolarization. In presence of Ba 2+ the extrapollated reversal potential of the hyperpolarizing phase was dramatically shifted to a more positive value. Finally FLFQPQRFamide induced response can be partially mimicked by FMRFamide application. Our observations indicate that FLFQPQRFamide can have multiple effects on membrane conductance of mammalian spinal cord neurones by acting on a single class of receptor. These effects of FLFQPQRFamide were found to be mainly excitatory.

Interaction of pyrethroids with mammalian spinal neurons

The effects of intravenous administration of non-cyano ( cis-permethrin) and cyano-substituted (deltamethrin) pyrethroids were studied on spontaneous and evoked ventral root activity in rat spinal cord and on spontaneous firing of ventral horn interneurons in the cat. Both pyrethroids had dramatic facilitatory effects on spontaneous firing rates of ventral roots and spinal interneurons and increased the amplitude of mono- and polysynaptically mediated ventral root responses to dorsal root stimulation. Spontaneous and evoked afferent sensory activity was slightly enhanced by cis-permethrin, but not by deltamethrin. In the cat diazepam (0.5 mg/kg, iv) was equally effective in antagonizing the facilitation of interneuronal firing resulting from either deltamethrin or cis-permethrin. These effects of pyrethroids on spinal neurons may underly the production of tremor and choreoathetosis-salivation toxicity symptoms in mammals.

Transparent indium-tin oxide electrode patterns for extracellular, multisite recording in neuronal cultures

Glass plates coated with transparent thin film conductors of indium-tin oxide (ITO), 100 nm thick and 10 μm wide, have been successfully used to record spike potentials from neuronal monolayer cultures. The material is non-toxic to mammalian spinal neurons and is stable under warm culture medium. Laser-deinsulated recording craters that expose 100 μm 2 of ITO yield impedances of 8–10 MΩ at 1 kHz with noise levels of 40 μV. Conventional gold plating of the craters reduces these impedances to below 3 MΩ. The material is easily etchable and sputtered glass plates of high quality are commercially available at relatively low cost. The high light transmittance of ITO makes the conductors essentially invisible and allows unobstructed observation of circuit components in monolayer cultures. The introduction of ITO as a thin film microelectrode material should accelerate the construction of high density recording patterns that could exceed 400 microelectrodes per mm 2.