Acetyl-L-carnitine treatment following spinal cord injury improves mitochondrial function correlated with remarkable tissue sparing and functional recovery.

We have recently documented that treatment with the alternative biofuel, acetyl-L-carnitine (ALC, 300 mg/kg), as late as 1 h after T10 contusion spinal cord injury (SCI), significantly maintained mitochondrial function 24 h after injury. Here we report that after more severe contusion SCI centered on the L1/L2 segments that are postulated to contain lamina X neurons critical for locomotion (the "central pattern generator"), ALC treatment resulted in significant improvements in acute mitochondrial bioenergetics and long-term hind limb function. Although control-injured rats were only able to achieve slight movements of hind limb joints, ALC-treated animals produced consistent weight-supported plantar steps 1 month after injury. Such landmark behavioral improvements were significantly correlated with increased tissue sparing of both gray and white matter proximal to the injury, as well as preservation of choline acetyltransferase (ChAT)-positive neurons in lamina X rostral to the injury site. These findings signify that functional improvements with ALC treatment are mediated, in part, by preserved locomotor circuitry rostral to upper lumbar contusion SCI. Based on beneficial effects of ALC on mitochondrial bioenergetics after injury, our collective evidence demonstrate that preventing mitochondrial dysfunction acutely "promotes" neuroprotection that may be associated with the milestone recovery of plantar, weight-supported stepping.

Task-specificity vs. ceiling effect: step-training in shallow water after spinal cord injury.

While activity-based rehabilitation is one of the most promising therapeutic approaches for spinal cord injury, the necessary components for optimal locomotor retraining have not yet been determined. Currently, a number of different activity-based approaches are being investigated including body weight-supported treadmill training (with and without manual assistance), robotically-assisted treadmill training, bicycling and swimming, among others. We recently showed, in the adult rat, that intensive rehabilitation based on swimming brought about significant improvements in hindlimb performance during swimming but did not alter the normal course of recovery of over-ground walking (Smith et al., 2006a,b, 2009). However, swimming lacks the phasic limb-loading and plantar cutaneous feedback thought to be important for weight-supported step training. So, we are investigating an innovative approach based on walking in shallow water where buoyancy provides some body weight support and balance while still allowing for limb-loading and appropriate cutaneous afferent feedback during retraining. Thus, the aim of this study is to determine if spinal cord injured animals show improved overground locomotion following intensive body weight-supported locomotor training in shallow water. The results show that training in shallow water successfully improved stepping in shallow water, but was not able to bring about significant improvements in overground locomotion despite the fact that the shallow water provides sufficient body weight support to allow acutely injured rats to generate frequent plantar stepping. These observations support previous suggestions that incompletely injured animals retrain themselves while moving about in their cages and that daily training regimes are not able to improve upon this already substantial functional improvement due to a ceiling effect, rather than task-specificity, per se. These results also support the concept that moderately-severe thoracic contusion injuries decrease the capacity for body weight support, but do not decrease the capacity for pattern generation. In contrast, animals with severe contusion injuries could not support their body weight nor could they generate a locomotor pattern when provided with body weight support via buoyancy.

Reticulospinal pathways in the ventrolateral funiculus with terminations in the cervical and lumbar enlargements of the adult rat spinal cord.

In the mammalian spinal cord, the ventrolateral funiculus (VLF) has been identified as critical to postural control and locomotor function, in part due to the reticulospinal pathways it contains. The primary purpose of this descriptive study was to investigate the distribution of neurons in the medulla labeled retrogradely from the VLF and the intermediate gray matter of specific lumbar and cervical spinal cord segments in the adult rat. We made discrete injections of Fluoro-Ruby (FR) into the intermediate gray matter at the cervical (C) 5/6, 7/8 or lumbar (L) 2 segmental levels followed by a single injection of Fluoro-Gold (FG) into the right VLF at T9. Double-labeled medullary neurons were found primarily in the gigantocellular group of nuclei (Gi), distributed both ipsilaterally and contralaterally following cervical or lumbar FR injections. In addition, a substantial population of neurons contained within the vestibular group of nuclei was double labeled both ipsilaterally and contralaterally. We also identified a substantial population of Gi-related neurons located ipsilateral to the VLF injections that were double labeled following left unilateral FR injections at C5/6, C7/8 or L2. These results describe a substantial population of ipsilateral and commissural medullary neurons that project to both cervical and thoracolumbar segments. Two different populations of commissural neurons are described, one with axons that cross the midline rostral to T9, and one with axons that cross the midline caudal to T9. These observations provide strong additional evidence for a pattern of reticulo- and vestibulospinal projections that include substantial numbers of commissural neurons and project to multiple cervical and thoracolumbar levels.

Inter-enlargement pathways in the ventrolateral funiculus of the adult rat spinal cord.

The ventrolateral funiculus (VLF) in the spinal cord contains important ascending and descending pathways related to locomotion and interlimb coordination. The primary purpose of this descriptive study was to investigate the distribution of inter-enlargement pathways in the adult rat spinal cord with an emphasis on the VLF. We made discrete unilateral injections of Fluoro-Gold (FG) into the right VLF at thoracic segment (T) 9, and either unilateral or bilateral injections of Fluoro-Ruby (FR) into the intermediate gray matter at the cervical (C) 5-6, C7-8, or lumbar (L) 2 segmental levels. Inter-enlargement neurons with ascending axons in the right VLF were found bilaterally in laminae VII and VIII throughout the rostral lumbar spinal cord (L1-L3) and predominantly contralaterally in the caudal lumbosacral (L4-S1) spinal cord. Following left unilateral FR injections at C5-6 or C7-8 and right unilateral VLF injections of FG at T9, very few double-labeled neurons could be found anywhere in the lumbar spinal cord. Similar injections of FR at L2 revealed an almost symmetrical bilateral distribution of double-labeled neurons throughout the cervical spinal cord (C1-8). These results describe ascending and descending pathways within the spinal cord that interconnect the two enlargements and involve both commissural and ipsilateral interneurons. The majority of inter-enlargement neurons had axons within the VLF at T9. These observations support the hypothesis that the VLF contains long ascending and descending axons with propriospinal inter-enlargement, commissural and ipsilateral connections that are anatomically well-suited to mediate interlimb coordination.

Embryonic brain precursors transplanted into kainate lesioned rat spinal cord.

Embryonic day 14 rat cerebral cortex-derived precursors were expanded with FGF2 and labeled with BrdU prior to being transplanted into the kainic acid-lesioned adult rat spinal cord. While these precursors give rise to cells with neuronal, astrocytic and oligodendroglial phenotypes vitro, they remained largely undifferentiated up to 12 weeks in vivo. Numerous BrdU-labeled cells were found in injured gray matter, and also lining the dilated central canal that sometimes accompanies these lesions. BrdU-labeled cells never co-expressed Map2ab, rarely co-expressed GFAP but often co-expressed nestin, even after 12 weeks in vivo. These observations suggest that the environment of the kainic acid-injured spinal cord is not hostile to transplanted embryonic cerebral cortex-derived precursors, but also is not conducive to their neuronal differentation.

Lasting paraplegia caused by loss of lumbar spinal cord interneurons in rats: no direct correlation with motor neuron loss.

OBJECT: The aims of this study were to investigate further the role played by lumbar spinal cord interneurons in the generation of locomotor activity and to develop a model of spinal cord injury suitable for testing neuron replacement strategies. METHODS: Adult rats received intraspinal injections of kainic acid (KA). Locomotion was assessed weekly for 4 weeks by using the Basso, Beattie, and Bresnahan (BBB) 21-point locomotor scale, and transcranial magnetic motor evoked potentials (MMEPs) were recorded in gastrocnemius and quadriceps muscles at 1 and 4 weeks. No changes in transcranial MMEP latency were noted following KA injection, indicating that the descending motor pathways responsible for these responses, including the alpha motor neurons, were not compromised. Rats in which KA injections included much of the L-2 segment (10 animals) showed severe locomotor deficits, with a mean BBB score of 4.5 +/- 3.6 (+/- standard deviation). Rats that received lesions rostral to the L-2 segment (four animals) were able to locomote and had a mean BBB score of 14.6 +/- 2.6. Three rats that received only one injection bilaterally centered at L-2 (three animals) had a mean BBB score of 3.2 +/- 2. Histological examination revealed variable loss of motor neurons limited to the injection site. There was no correlation between motor neuron loss and BBB score. CONCLUSIONS: Interneuron loss centered on the L-2 segment induces lasting paraplegia independent of motor neuron loss and white matter damage, supporting earlier suggestions that circuitry critical to the generator of locomotor activity (the central pattern generator) resides in this area. This injury model may prove ideal for studies of neuron replacement strategies.

Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations.

The effects of specific mitogens and substrates on the proliferative capacity and the differentiated phenotypic plasticity of neural precursor cell populations isolated from the adult rat subventricular zone (SVZ) were examined. SVZ cells were grown on uncoated tissue culture plastic, extracellular matrix, or poly-D-ornithine with either laminin or fibronectin. SVZ neural precursor cells could not be generated with platelet-derived growth factor (PDGF), granulocyte macrophage colony stimulating factor, stem cell factor, heparin-binding epidermal growth factor (HB-EGF), granulocyte colony stimulating factor, or ciliary neurotrophic factor (CNTF), but could be with EGF, fibroblast growth factor 2 (FGF2), and FGF2 plus heparin. Varying combinations of substrate and mitogen resulted in very different expansion rates and/or lineage potential. Neurons, oligodendrocytes, and astrocytes differentiated from all cultures, but EGF-generated neural precursor cells were more restricted to an astrocytic lineage and FGF2-generated neural precursor cells had a greater capacity for neuronal differentiation. In both EGF- and FGF2-generated cell populations, CNTF increased the number of differentiated astrocytes, triiodothyronine oligodendrocytes, PDGF neurons, and brain-derived neurotrophic factor neurons only from EGF cells. Electrophysiological analysis of differentiated cells showed three distinct phenotypes, glial, neuronal, and presumed precursor cells, although the neuronal properties were immature. Collectively, these data indicate that CNS neural precursor cell populations isolated with different mitogens and substrates are intrinsically different and their characteristics cannot be directly compared.

Electrophysiological properties of mitogen-expanded adult rat spinal cord and subventricular zone neural precursor cells.

Growth factor-expanded neural precursor cells isolated from the mammalian central nervous system can differentiate into neurons and glia. Although the morphological and neurochemical development of these neural precursor cells has been investigated, little attention has been paid to their electrophysiology. This study examined the electrophysiological properties of neurons and glia derived from neural precursor cells isolated from the adult rat spinal cord (SC) and subventricular zone (SVZ). Cells were cultured in medium containing epidermal growth factor and/or fibroblast growth factor-2. After at least two passages, spheres of neural precursor cells were plated on coated coverslips and maintained in culture for up to 6 weeks. Whole-cell patch recordings were made using standard current clamp techniques. Immature action potentials were observed within hours of plating for both SC and SVZ cells. Input resistance and time constants decreased over the first week after plating and no further changes were found at later times. At similar times following plating, however, SVZ cells had a lower input resistance and shorter time constant compared to SC cells. SVZ cells also had higher resting membrane potentials and smaller after hyperpolarizations than those of SC cells, despite no significant difference in the amplitude of action potentials. Neither the SC nor the SVZ cells were capable of eliciting more than a single action potential in response to injected current. While all SC cells tested were depolarized by glutamate, the response of SVZ cells to glutamate varied considerably. This study revealed that neural precursor cells from SC and SVZ differ in both active and passive membrane properties. It appears also that the electrophysiological development of SC and SVZ precursor-derived neurons is incomplete under the conditions used. These observations suggest that the neural precursor cells from different anatomical locations may be physiologically diverse and may exhibit some differences in commitment toward neuronal or glial phenotypes.

Comparing deficits following excitotoxic and contusion injuries in the thoracic and lumbar spinal cord of the adult rat.

The majority of human spinal cord injuries involve gray matter loss from the cervical or lumbar enlargements. However, the deficits that arise from gray matter damage are largely masked by the severe deficits due to associated white matter damage. We have developed a model to examine gray matter-specific deficits and therapeutic strategies that uses intraspinal injections of the excitotoxin kainic acid into the T9 and L2 regions of the spinal cord. The resulting deficits have been compared to those from standard contusion injuries at the same levels. Injuries were assessed histologically and functional deficits were determined using the Basso, Beattie, and Bresnahan (BBB) 21-point open field locomotor scale and transcranial magnetic motor evoked potentials (tcMMEPs). Kainic acid injections into T9 resulted in substantial gray matter damage; however, BBB scores and tcMMEP response latencies were not different from those of controls. In contrast, kainic acid injections into L2 resulted in paraplegia with BBB scores similar to those following contusion injuries at either T9 or L2, without affecting tcMMEP response latencies. These observations demonstrate that gray matter loss can result in significant functional deficits, including paraplegia, in the absence of a disruption of major descending pathways.

Neuronal excitatory properties of human immunodeficiency virus type 1 Tat protein.

Neuronal dysfunction and cell death in patients with human immunodeficiency virus type-1 (HIV-1) infection may be mediated by HIV-1 proteins and products released from infected cells. Two HIV-1 proteins, the envelope glycoprotein gp120 and nonstructural protein Tat, are neurotoxic. We have determined the neuroexcitatory properties of HIV-1 tat protein using patch-clamp recording techniques. When fmoles of Tat were applied extracellularly, it elicited dose-dependent depolarizations of human fetal neurons in culture and rat CA1 neurons in slices, both in the absence and presence of tetrodotoxin. These responses were voltage-dependent, reversed at approximately 0 mV, and were significantly increased by repetitive applications with no evidence of desensitization. That these responses to Tat were due to direct actions on neurons was supported by observations that Tat dose-dependently depolarized outside-out patches excised from cultured human neurons. Removal of extracellular Ca2+ decreased the responses both in neurons and membrane patches. This is the first demonstration that an HIV-1 protein can, in the absence of accessory cells, directly excite neurons and leads us to speculate that Tat may be a causative agent in HIV-1 neurotoxicity.

Locomotor rhythm evoked by ventrolateral funiculus stimulation in the neonatal rat spinal cord in vitro.

Spinal cords from 2- to 8-day-old rats, maintained in vitro, were used to investigate the effects of discrete electrical stimuli applied to the ventrolateral funiculus (VLF) on motor neuron activity recorded from the lumbar ventral roots. Short trains of stimuli (1-3 s) delivered to one VLF in the low cervical region elicited rhythmic activity that persisted for up to 30 s. Responses consisted of short periods of activity (1-5 s) occurring simultaneously in the ipsilateral L5 and contralateral L3 ventral roots that alternated with similar bursts of activity in the ipsilateral L3 ventral root, a pattern consistent with locomotion. The rhythmicity of the ventral root responses to VLF stimulation was not affected by midsagittal sectioning of the preparation rostral to T10 and/or caudal to L4. Midsagittal sectioning of the lower thoracic or upper lumbar segments, however, disrupted the rhythmicity of the ventral root responses, leaving only long-duration simultaneous activation of the ipsilateral roots following VLF stimulus trains. The minimum lesion that effectively abolished the rhythmicity was one that divided only the L2 and L3 segments. In preparations rendered arrhythmic to VLF stimulation by an L2/L3 midsagittal lesion, rhythmicity could still be induced by N-methyl-D-aspartate (NMDA; 2-5 microM) and serotonin (5-HT; 20-50 microM), a drug combination commonly used to induce locomotor-like rhythmicity and air-stepping in vitro. Field potentials recorded following single stimuli delivered to the VLF revealed short-latency, large-amplitude responses in the ventral horn and intermediate gray both ipsilateral and contralateral to the stimulus site at T12 and L2. These observations suggest that 1) the discrete pathway under study may be an important descending locomotor command pathway and 2) this pathway has a strong bilateral projection in the lower thoracic and upper lumbar segments that is crucial for the initiation of VLF-induced rhythmic motor output. The induction of rhythmicity by NMDA/5-HT in an L2/L3-lesioned preparation suggests that these two rhythmogenic mechanisms may represent different levels within the circuitry that comprises the central pattern generator for locomotion. The rhythmic activity resulting from VLF stimulation is dependent on a bilateral projection that can be bypassed by the generalized excitation and subsequent rhythmicity that results from bath application of the NMDA/5-HT combination.

Identification of a human immunodeficiency virus type 1 Tat epitope that is neuroexcitatory and neurotoxic.

Tat is an 86- to 104-amino-acid viral protein that activates human immunodeficiency virus type 1 expression, modifies several cellular functions, and causes neurotoxicity. Here, we determined the extent to which peptide fragments of human immunodeficiency virus type 1 BRU Tat1-86 produced neurotoxicity, increased levels of intracellular calcium ([Ca2+]i), and affected neuronal excitability. Tat31-61 but not Tat48-85 dose dependently increased cytotoxicity and levels of [Ca2+]i in cultured human fetal brain cells. Similarly, Tat31-61 but not Tat48-85 depolarized rat hippocampal CA1 neurons in slices of rat brain. The neurotoxicity and increases in [Ca2+]i could be significantly inhibited by non-N-methyl-D-aspartate excitatory amino acid receptor antagonists. Shorter 15-mer peptides which overlapped by 10 amino acids each and which represented the entire sequence of Tat1-86 failed to produce any measurable neurotoxicity. Although it remains to be determined if Tat acts directly on neurons and/or indirectly via glial cells, these findings do suggest that Tat neurotoxicity is conformationally dependent, that the active site resides within the first exon of Tat between residues 31 to 61, and that these effects are mediated at least in part by excitatory amino acid receptors.

Neurons derived from P19 embryonal carcinoma cells develop responses to excitatory and inhibitory neurotransmitters.

Cells of the P19 line of embryonal carcinoma cells differentiate into neurons, astrocytes and oligodendrocytes following treatment with retinoic acid. The neurons from these differentiating P19 cultures synthesize a pattern of neurotransmitters that resembles that of neurons of the forebrain. We treated P19 cells with retinoic acid and then implanted them into the striatum of adult rats. After times ranging from 1 to 15 weeks post-implantation, brain slices containing the implanted tissue were prepared and used for intracellular recording of electrical activity and responsiveness to application of neurotransmitters. Within 2 weeks of implantation, the P19-derived neurons had developed responsiveness to the excitatory neurotransmitter glutamate and the inhibitory transmitters gamma-aminobutyric acid and glycine. These neurons also exhibited spontaneous synaptic potentials. The responses to glutamate appear to be mediated by N-methyl-D-aspartic acid as well as non-N-methyl-D-aspartic acid receptor subtypes. Gamma-aminobutyric acid evoked bicuculline-sensitive depolarizing responses in the younger grafts and biphasic depolarizing/hyperpolarizing responses in older ones. Responses to glycine were strychnine sensitive and also showed age-related changes from depolarizing to biphasic character. Synaptic potentials in the younger grafts were exclusively depolarizing, but in older ones both depolarizing and hyperpolarizing events were observed. The synaptic potentials appear to arise from synaptic connections between P19-derived neurons within the grafts. Many of the features of P19-derived neurons are similar to those of neurons in the developing forebrain.

Lamina VII neurons are rhythmically active during locomotor-like activity in the neonatal rat spinal cord.

The midsagittally-sectioned lumbar spinal cord with thoracic segments intact retains the capacity for locomotor-like activity. Intracellular recordings were used to characterize the activity and concurrently label lumbar neurons in lamina VII, an area previously implicated in the generation of locomotion. Sharp electrodes were shown to preferentially impale larger neurons. These neurons undergo rhythmic voltage oscillations, presumably synaptically driven, during locomotor-like activity induced by bath application of N-methyl-D-aspartate and 5-hydroxytryptamine. This supports the hypothesis that synaptic activity recruits neurons in lamina VII that are associated with locomotor behavior.

Long-duration, frequency-dependent motor responses evoked by ventrolateral funiculus stimulation in the neonatal rat spinal cord.

Three variations of the in vitro neonatal rat spinal cord preparation were used to investigate motor responses to stimulation of the ventrolateral funiculus (VLF). In a partially hemisected spinal cord preparation, stimuli elicited frequency-dependent activity in lumbar ventral roots that outlasted the stimulus train by up to 30 s. In a spinal cord-hindlimb preparation, trains of VLF stimuli elicited slow, step-like flexor-extensor hindlimb movement that also persisted for up to 30 s beyond the stimulus. Finally, in a partially hemisected spinal cord preparation where 5-hydroxytryptamine/N-methyl-D-aspartate was used to induce locomotor-like rhythmic activity, short trains of VLF stimuli were capable of perturbing the locomotor rhythm, transiently altering its frequency. Application of pharmacological antagonists suggests that these responses may be the result of stimulation of a descending pathway that includes glutamatergic and catecholaminergic fibres comprising part of a descending locomotor command path.

Human immunodeficiency virus type 1 tat activates non-N-methyl-D-aspartate excitatory amino acid receptors and causes neurotoxicity.

The human immunodeficiency virus type 1 (HIV-1) protein Tat is known to be released from HIV-1-infected cells. We show that micromolar concentrations of Tat depolarized young rat and adult human neurons. In addition, Tat, at similar concentrations, was toxic to human fetal neurons in culture. Tat-induced responses were insensitive to the Na+ channel blocker tetrodotoxin, suggesting a direct effect of Tat on neurons. Tat-induced depolarizations and cytotoxicity were blocked by the excitatory amino acid antagonist kynurenate. The N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate had little effect on Tat-induced depolarizations but did provide protection from Tat neurotoxicity. These results suggest that Tat, released from HIV-1-infected cells, may be an important mediator of neurotoxicity observed in HIV-1 encephalopathy.

In vivo electrophysiological maturation of neurons derived from a multipotent precursor (embryonal carcinoma) cell line.

The multipotent embryonal carcinoma (EC) P19 cell line differentiates into neurons, glia and smooth muscle following exposure to retinoic acid (RA). RA-induced differentiation is irreversible and the neurons that develop are abundant, post-mitotic, and survive for prolonged periods in culture or when grafted into the CNS of adult rats. Striatal slices containing grafted P19 cells were studied with intracellular recording and labelling techniques to examine the development of electrophysiological and morphological properties of P19-derived neurons over a period of 6 to 120 days after grafting into ibotenic acid lesioned striatum. Cells from 1-week-old grafts had a range of immature electrophysiological characteristics including unstable resting membrane potentials (RMP's) and very high membrane input resistances (Rin's). Many were not able to produce action potentials (AP's). In contrast, the majority of cells recorded from 2- and 3-week-old grafts had stable RMP's, moderate Rin's, and were able to produce regenerative AP's. In grafts over 4 weeks of age, the majority of P19-derived neurons had mature neuronal electrophysiological characteristics including RMP's of -60 mV, Rin's of 100-300 M omega, and overshooting AP's. Morphologically, P19 derived neurons increase in soma size from 12-15 mu in diameter in 7-14-day-old grafts, to 25-35 mu in diameter in grafts 50-120 days old. Developing neurons exhibited a variety of morphotypes with increasingly complex processes and lengths of process extension. Our results demonstrate a developmental progression of the electrophysiology of P19-derived neurons, culminating in mature characteristics closely resembling those of adult rodent hippocampal or cortical pyramidal neurons. The ability to easily alter these cells genetically provides a powerful model for addressing issues specific to neuronal development.

Murine embryonal carcinoma-derived neurons survive and mature following transplantation into adult rat striatum.

P19 embryonal carcinoma cells are pluripotent and can be efficiently induced to differentiate in culture into neurons and astroglia by brief treatment with retinoic acid. Retinoic acid-treated P19 cells survive after grafting into the adult rat striatum and differentiate into neurons and glia within the transplantation site. No tumours develop from the grafted cells which continue to express foreign genes that had been transfected into the parental P19 cells. The neurons in these grafts express a variety of neurotransmitters similar to those formed in retinoic acid-treated P19 cell cultures and they mature to acquire the electrophysiological properties expected of fully developed neurons. These results suggest that P19 cells may be used for studies related to neuronal cell development and maturation and that P19 cells may be considered for cell replacement strategies in neurodegenerative disorders of the central nervous system.

Electrophysiological changes accompanying DSP-4 lesions of rat locus coeruleus neurons.

The electrophysiological characteristics of intracellularly recorded locus coeruleus (LC) neurons in brain stem slices from DSP-4 treated animals have been compared to those from untreated controls. LC neurons from DSP-4 treated animals had action potentials and Ca2+ spikes (elicited in the presence of TTX) of significantly reduced duration compared to controls. These observations suggest that chemical axotomy with DSP-4 reduces Ca2+ conductance in neurons of the locus coeruleus.