AAV Vector Mediated Delivery of NG2 Function Neutralizing Antibody and Neurotrophin NT-3 Improves Synaptic Transmission, Locomotion, and Urinary Tract Function after Spinal Cord Contusion Injury in Adult Rats.

NG2 is a structurally unique transmembrane chondroitin sulfate proteoglycan (CSPG). Its role in damaged spinal cord is dual. NG2 is considered one of key inhibitory factors restricting axonal growth following spinal injury. Additionally, we have recently detected its novel function as a blocker of axonal conduction. Some studies, however, indicate the importance of NG2 presence in the formation of synaptic contacts. We hypothesized that the optimal treatment would be neutralization of inhibitory functions of NG2 without its physical removal. Acute intraspinal injections of anti-NG2 monoclonal antibodies reportedly prevented an acute block of axonal conduction by exogenous NG2. For prolonged delivery of NG2 function neutralizing antibody, we have developed a novel gene therapy: adeno-associated vector (AAV) construct expressing recombinant single-chain variable fragment anti-NG2 antibody (AAV-NG2Ab). We examined effects of AAV-NG2Ab alone or in combination with neurotrophin NT-3 in adult female rats with thoracic T10 contusion injuries. A battery of behavioral tests was used to evaluate locomotor function. In vivo single-cell electrophysiology was used to evaluate synaptic transmission. Lower urinary tract function was assessed during the survival period using metabolic chambers. Terminal cystometry, with acquisition of external urethral sphincter activity and bladder pressure, was used to evaluate bladder function. Both the AAV-NG2Ab and AAV-NG2Ab combined with AAV-NT3 treatment groups demonstrated significant improvements in transmission, locomotion, and bladder function compared with the control (AAV-GFP) group. These functional improvements associated with improved remyelination and plasticity of 5-HT fibers. The best results were observed in the group that received combinational AAV-NG2Ab+AAV-NT3 treatment.SIGNIFICANCE STATEMENT We recently demonstrated beneficial, but transient, effects of neutralization of the NG2 proteoglycan using monoclonal antibodies delivered intrathecally via osmotic mini-pumps after spinal cord injury. Currently, we have developed a novel gene therapy tool for prolonged and clinically relevant delivery of a recombinant single-chain variable fragment anti-NG2 antibody: AAV-rh10 serotype expressing scFv-NG2 (AAV-NG2Ab). Here, we examined effects of AAV-NG2Ab combined with transgene delivery of Neurotrophin-3 (AAV-NT3) in adult rats with thoracic contusion injuries. The AAV-NG2Ab and AAV-NG2Ab+AAV-NT3 treatment groups demonstrated significant improvements of locomotor function and lower urinary tract function. Beneficial effects of this novel gene therapy on locomotion and bladder function associated with improved transmission to motoneurons and plasticity of axons in damaged spinal cord.

Buprenorphine, a partial opioid agonist, prevents modulation of H-reflex induced by pulsed electromagnetic stimulation in spinal cord injured rats.

Our recent study revealed that spinal electromagnetic stimulation (sEMS) applied at low (0.2 Hz) frequencies may improve diminished transmission in damaged spinal cord in spinal cord injured (SCI) rats. We have recently begun a pilot study investigating the effects of sEMS in non-injured and SCI humans. One unexpected result was the reduction of chronic low back pain (CLBP), reported by some patients following sEMS treatment. Chronic low back pain is one of the main causes of disability affecting the general population. Opioids are the most common drugs prescribed to US adults with CLBP. To optimize parameters for sEMS for pain treatment, in this study we used the SCI animal model and examined effects of sEMS applied at lumbosacral level on parameters and frequency-dependent depression (FDD) of Hoffmann H-reflex responses, known as common neurophysiological measures for evaluation of sensorimotor condition and plasticity in humans. We have also examined the interactive effects of sEMS and the opiate partial agonist Buprenorphine on the parameters of H-reflex in naïve and SCI rats. Consistent with previous reports, chronic SCI resulted in a marked decrease of threshold intensity required to evoke H-reflex and a lesser rate of FDD of the H-response in adult rats. Our current study revealed the optimum parameters of spinal EMS for best recovery of the properties of the H-reflex in chronic SCI animals. Here we demonstrate that electro-magnetic stimulation applied at spinal L4-L5 level with a pulsed mode (pulse at 20 Hz frequency for 5 sec with 25 sec break between pulses, total 40 trains for 20 min; PSEMS) reversed effects of SCI on key parameters of H-reflex: i.e. (1) restored the threshold intensity of electric current applied at tibial nerve to evoke the H-reflex and (2) recovered FDD properties of the H-reflex in SCI rats. Importantly, subcutaneous injections of Buprenorphine, prior to PSEMS administration, abolished the ability of PSEMS to recover both threshold intensity and FDD of the H-reflex in chronic SCI animals. These results suggest that a semi-synthetic opioid Buprenorphine and PSEMS might share common sites of action. We thus conclude that PSEMS might carry potential as a non-invasive treatment approach for chronic low back pain.

Modulation of H-reflex responses and frequency-dependent depression by repetitive spinal electromagnetic stimulation: From rats to humans and back to chronic spinal cord injured rats.

The lack of propagation of signals through survived fibers is among the major reasons for functional loss after incomplete spinal cord injury (SCI). Our recent results of animal studies demonstrate that spinal electromagnetic stimulation (SEMS) can enhance transmission in damaged spinal cord, and this type of modulation depends on the function of NMDA receptors at the neuronal networks below the injury level. Here, our pilot human study revealed that administration of repetitive SEMS induced long-lasting modulation of H-responses in both healthy and participants with chronic SCI. In order to understand the mechanisms underlying these effects, we have used an animal model and examined effects of SEMS on H-responses. Effects of SEMS on H-responses, frequency-dependent depression (FDD) of H-reflex, and possible underlying mechanisms have been examined in both naïve and rats with SCI. Our results demonstrate that consistent with the effects of SEMS on H-reflex seen in humans, repetitive SEMS induced similar modulation in excitability of peripheral nerve responses in both non-injured and rats with SCI. Importantly, our results confirmed the reduced FDD of H-reflex in SCI animals and revealed that SEMS was able to recover FDD in rats with chronic SCI. Using intraspinal injections of the NMDA receptor blocker MK-801, we have identified NMDA receptors as an important contributor to these SEMS-induced effects in rats with SCI. These results identify SEMS as a novel non-invasive technique for modulation of neuro-muscular circuits and, importantly, modulation of spinal networks after chronic SCI.

Transcranial magnetic stimulation (TMS) responses elicited in hindlimb muscles as an assessment of synaptic plasticity in spino-muscular circuitry after chronic spinal cord injury.

Electromagnetic stimulation applied at the cranial level, i.e. transcranial magnetic stimulation (TMS), is a technique for stimulation and neuromodulation used for diagnostic and therapeutic applications in clinical and research settings. Although recordings of TMS elicited motor-evoked potentials (MEP) are an essential diagnostic tool for spinal cord injured (SCI) patients, they are reliably recorded from arm, and not leg muscles. Mid-thoracic contusion is a common SCI that results in locomotor impairments predominantly in legs. In this study, we used a chronic T10 contusion SCI rat model and examined whether (i) TMS-responses in hindlimb muscles can be used for evaluation of conduction deficits in cortico-spinal circuitry and (ii) if plastic changes at spinal levels will affect these responses. In this study, plastic changes of transmission in damaged spinal cord were achieved by repetitive electro-magnetic stimulation applied over the spinal level (rSEMS). Spinal electro-magnetic stimulation was previously shown to activate spinal nerves and is gaining large acceptance as a non-invasive alternative to direct current and/or epidural electric stimulation. Results demonstrate that TMS fails to induce measurable MEPs in hindlimbs of chronically SCI animals. After facilitation of synaptic transmission in damaged spinal cord was achieved with rSEMS, however, MEPs were recorded from hindlimb muscles in response to single pulse TMS stimulation. These results provide additional evidence demonstrating beneficial effects of TMS as a diagnostic technique for descending motor pathways in uninjured CNS and after SCI. This study confirms the ability of TMS to assess plastic changes of transmission occurring at the spinal level.

Spinal electro-magnetic stimulation combined with transgene delivery of neurotrophin NT-3 and exercise: novel combination therapy for spinal contusion injury.

Our recent terminal experiments revealed that administration of a single train of repetitive spinal electromagnetic stimulation (sEMS; 35 min) enhanced synaptic plasticity in spinal circuitry following lateral hemisection spinal cord injury. In the current study, we have examined effects of repetitive sEMS applied as a single train and chronically (5 wk, every other day) following thoracic T10 contusion. Chronic studies involved examination of systematic sEMS administration alone and combined with exercise training and transgene delivery of neurotrophin [adeno-associated virus 10-neurotrophin 3 (AAV10-NT3)]. Electrophysiological intracellular/extracellular recordings, immunohistochemistry, behavioral testing, and anatomical tracing were performed to assess effects of treatments. We found that administration of a single sEMS train induced transient facilitation of transmission through preserved lateral white matter to motoneurons and hindlimb muscles in chronically contused rats with effects lasting for at least 2 h. These physiological changes associated with increased immunoreactivity of GluR1 and GluR2/3 glutamate receptors in lumbar neurons. Systematic administration of sEMS alone for 5 wk, however, was unable to induce cumulative improvements of transmission in spinomuscular circuitry or improve impaired motor function following thoracic contusion. Encouragingly, chronic administration of sEMS, followed by exercise training (running in an exercise ball and swimming), induced the following: 1) sustained strengthening of transmission to lumbar motoneurons and hindlimb muscles, 2) better retrograde transport of anatomical tracer, and 3) improved locomotor function. Greatest improvements were seen in the group that received exercise combined with sEMS and AAV-NT3.

Combination of chondroitinase ABC and AAV-NT3 promotes neural plasticity at descending spinal pathways after thoracic contusion in rats.

Transmission through descending pathways to lumbar motoneurons, although important for voluntary walking in humans and rats, has not been fully understood at the cellular level in contusion models. Major descending pathways innervating lumbar motoneurons include those at corticospinal tract (CST) and ventrolateral funiculus (VLF). We examined transmission and plasticity at synaptic pathways from dorsal (d)CST and VLF to individual motoneurons located in ventral horn and interneurons located in dorsomedial gray matter at lumbar segments after thoracic chronic contusion in adult anesthetized rats. To accomplish this, we used intracellular electrophysiological recordings and performed acute focal spinal lesions during the recordings. We directly demonstrate that after thoracic T10 chronic contusion the disrupted dCST axons spontaneously form new synaptic contacts with individual motoneurons, extending around the contusion cavity, through spared ventrolateral white matter. These detour synaptic connections are very weak, and strengthening these connections in order to improve function may be a target for therapeutic interventions after spinal cord injury (SCI). We found that degradation of scar-related chondroitin sulfate proteoglycans with the enzyme chondroitinase ABC (ChABC) combined with adeno-associated viral (AAV) vector-mediated prolonged delivery of neurotrophin NT-3 (AAV-NT3) strengthened these spontaneously formed connections in contused spinal cord. Moreover, ChABC/AAV-NT3 treatment induced the appearance of additional detour synaptic pathways innervating dorsomedial interneurons. Improved transmission in ChABC/AAV-NT3-treated animals was associated with increased immunoreactivity of 5-HT-positive fibers in lumbar dorsal and ventral horns. Improved locomotor function assessed with automated CatWalk highlights the physiological significance of these novel connections.

Neutralization of inhibitory molecule NG2 improves synaptic transmission, retrograde transport, and locomotor function after spinal cord injury in adult rats.

NG2 belongs to the family of chondroitin sulfate proteoglycans that are upregulated after spinal cord injury (SCI) and are major inhibitory factors restricting the growth of fibers after SCI. Neutralization of NG2's inhibitory effect on axon growth by anti-NG2 monoclonal antibodies (NG2-Ab) has been reported. In addition, recent studies show that exogenous NG2 induces a block of axonal conduction. In this study, we demonstrate that acute intraspinal injections of NG2-Ab prevented an acute block of conduction by NG2. Chronic intrathecal infusion of NG2-Ab improved the following deficits induced by chronic midthoracic lateral hemisection (HX) injury: (1) synaptic transmission to lumbar motoneurons, (2) retrograde transport of fluororuby anatomical tracer from L5 to L1, and (3) locomotor function assessed by automated CatWalk gait analysis. We collected data in an attempt to understand the cellular and molecular mechanisms underlying the NG2-Ab-induced improvement of synaptic transmission in HX-injured spinal cord. These data showed the following: (1) that chronic NG2-Ab infusion improved conduction and axonal excitability in chronically HX-injured rats, (2) that antibody treatment increased the density of serotonergic axons with ventral regions of spinal segments L1-L5, (3) and that NG2-positive processes contact nodes of Ranvier within the nodal gap at the location of nodal Na(+) channels, which are known to be critical for propagation of action potentials along axons. Together, these results demonstrate that treatment with NG2-Ab partially improves both synaptic and anatomical plasticity in damaged spinal cord and promotes functional recovery after HX SCI. Neutralizing antibodies against NG2 may be an excellent way to promote axonal conduction after SCI.

Repetitive spinal electromagnetic stimulation opens a window of synaptic plasticity in damaged spinal cord: role of NMDA receptors.

As we reported previously, propagation of action potentials through surviving axons is impaired dramatically, resulting in reduced transmission to lumbar motoneurons after midthoracic lateral hemisection (HX) in rats. The aim of the present study was to evoke action potentials through the spared fibers using noninvasive electromagnetic stimulation (EMS) over intact T2 vertebrae in an attempt to activate synaptic inputs to lumbar motoneurons and thus to enhance plasticity of spinal neural circuits after HX. We found that EMS was able to activate synaptic inputs to lumbar motoneurons and motor-evoked potentials (MEP) in hindlimb muscles in adult anesthetized rats. Amplitude of MEP was attenuated in parallel with the decline of responses recorded from the motoneuron pool after HX. Repetitive EMS (50 min, 0.2 Hz) facilitated the amplitudes of responses elicited by electric stimulation of lateral white matter or dorsal corticospinal tracts in HX rats. Facilitation sustained for at least 1.5 h after termination of EMS. The N-methyl-d-aspartate (NMDA) receptor blocker MK-801, injected intraspinally close to the recording electrode prior to EMS, did not alter these responses but blocked the EMS-induced facilitation, suggesting that activation of NMDA receptors is required to initiate an EMS-evoked increase. When MK-801 was administered after EMS-induced facilitation was established, it induced depression of these elevated responses. Results suggest that repetitive EMS over intact vertebrae could be used as a therapeutic approach to open a window of synaptic plasticity after incomplete midthoracic injuries, i.e., to activate NMDA receptors in the lumbar motoneuron pool at synaptic inputs and to strengthen transmission in damaged spinal cord.

Chondroitinase ABC combined with neurotrophin NT-3 secretion and NR2D expression promotes axonal plasticity and functional recovery in rats with lateral hemisection of the spinal cord.

Elevating spinal levels of neurotrophin NT-3 (NT3) while increasing expression of the NR2D subunit of the NMDA receptor using a HSV viral construct promotes formation of novel multisynaptic projections from lateral white matter (LWM) axons to motoneurons in neonates. However, this treatment is ineffective after postnatal day 10. Because chondroitinase ABC (ChABC) treatment restores plasticity in the adult CNS, we have added ChABC to this treatment and applied the combination to adult rats receiving a left lateral hemisection (Hx) at T8. All hemisected animals initially dragged the ipsilateral hindpaw and displayed abnormal gait. Rats treated with ChABC or NT3/HSV-NR2D recovered partial hindlimb locomotor function, but animals receiving combined therapy displayed the most improved body stability and interlimb coordination [Basso-Beattie-Bresnahan (BBB) locomotor scale and gait analysis]. Electrical stimulation of the left LWM at T6 did not evoke any synaptic response in ipsilateral L5 motoneurons of control hemisected animals, indicating interruption of the white matter. Only animals with the full combination treatment recovered consistent multisynaptic responses in these motoneurons indicating formation of a detour pathway around the Hx. These physiological findings were supported by the observation of increased branching of both cut and intact LWM axons into the gray matter near the injury. ChABC-treated animals displayed more sprouting than control animals and those receiving NT3/HSV-NR2D; animals receiving the combination of all three treatments showed the most sprouting. Our results indicate that therapies aimed at increasing plasticity, promoting axon growth and modulating synaptic function have synergistic effects and promote better functional recovery than if applied individually.

Combined delivery of Nogo-A antibody, neurotrophin-3 and the NMDA-NR2d subunit establishes a functional 'detour' in the hemisected spinal cord.

To encourage re-establishment of functional innervation of ipsilateral lumbar motoneurons by descending fibers after an intervening lateral thoracic (T10) hemisection (Hx), we treated adult rats with the following agents: (i) anti-Nogo-A antibodies to neutralize the growth-inhibitor Nogo-A; (ii) neurotrophin-3 (NT-3) via engineered fibroblasts to promote neuron survival and plasticity; and (iii) the NMDA-receptor 2d (NR2d) subunit via an HSV-1 amplicon vector to elevate NMDA receptor function by reversing the Mg(2+) block, thereby enhancing synaptic plasticity and promoting the effects of NT-3. Synaptic responses evoked by stimulation of the ventrolateral funiculus ipsilateral and rostral to the Hx were recorded intracellularly from ipsilateral lumbar motoneurons. In uninjured adult rats short-latency (1.7-ms) monosynaptic responses were observed. After Hx these monosynaptic responses were abolished. In the Nogo-Ab + NT-3 + NR2d group, long-latency (approximately 10 ms), probably polysynaptic, responses were recorded and these were not abolished by re-transection of the spinal cord through the Hx area. This suggests that these novel responses resulted from new connections established around the Hx. Anterograde anatomical tracing from the cervical grey matter ipsilateral to the Hx revealed increased numbers of axons re-crossing the midline below the lesion in the Nogo-Ab + NT-3 + NR2d group. The combined treatment resulted in slightly better motor function in the absence of adverse effects (e.g. pain). Together, these results suggest that the combination treatment with Nogo-Ab + NT-3 + NR2d can produce a functional 'detour' around the lesion in a laterally hemisected spinal cord. This novel combination treatment may help to improve function of the damaged spinal cord.

Alterations of action potentials and the localization of Nav1.6 sodium channels in spared axons after hemisection injury of the spinal cord in adult rats.

Previously, we reported a pronounced reduction in transmission through surviving axons contralateral to chronic hemisection (HX) of adult rat spinal cord. To examine the cellular and molecular mechanisms responsible for this diminished transmission, we recorded intracellularly from lumbar lateral white matter axons in deeply anesthetized adult rats in vivo and measured the propagation of action potentials (APs) through rubrospinal/reticulospinal tract (RST/RtST) axons contralateral to chronic HX at T10. We found decreased excitability in these axons, manifested by an increased rheobase to trigger APs and longer latency for AP propagation passing the injury level, without significant differences in axonal resting membrane potential and input resistance. These electrophysiological changes were associated with altered spatial localization of Nav1.6 sodium channels along axons: a subset of axons contralateral to the injury exhibited a diffuse localization (>10 µm spread) of Nav1.6 channels, a pattern characteristic of demyelinated axons (Craner MJ, Newcombe J, Black JA, Hartle C, Cuzner ML, Waxman SG. Proc Natl Acad Sci USA 101: 8168-8173, 2004b). This result was substantiated by ultrastructural changes seen with electron microscopy, in which an increased number of large-caliber, demyelinated RST axons were found contralateral to the chronic HX. Therefore, an increased rheobase, pathological changes in the distribution of Nav1.6 sodium channels, and the demyelination of contralateral RST axons are likely responsible for their decreased conduction chronically after HX and thus may provide novel targets for strategies to improve function following incomplete spinal cord injury.

Role of chondroitin sulfate proteoglycans in axonal conduction in Mammalian spinal cord.

Chronic unilateral hemisection (HX) of the adult rat spinal cord diminishes conduction through intact fibers in the ventrolateral funiculus (VLF) contralateral to HX. This is associated with a partial loss of myelination from fibers in the VLF (Arvanian et al., 2009). Here, we again measured conduction through the VLF using electrical stimulation while recording the resulting volley and synaptic potentials in target motoneurons. We found that intraspinal injection of chondroitinase-ABC, known to digest chondroitin sulfate proteoglycans (CSPGs), prevented the decline of axonal conduction through intact VLF fibers across from chronic T10 HX. Chondroitinase treatment was also associated with behavior suggestive of an improvement of locomotor function after chronic HX. To further study the role of CSPGs in axonal conduction, we injected three purified CSPGs, NG2 and neurocan, which increase in the vicinity of a spinal injury, and aggrecan, which decreases, into the lateral column of the uninjured cord at T10 in separate experiments. Intraspinal injection of NG2 acutely depressed axonal conduction through the injected region in a dose-dependent manner. Similar injections of saline, aggrecan, or neurocan had no significant effect. Immunofluorescence staining experiments revealed the presence of endogenous and exogenous NG2 at some nodes of Ranvier. These results identify a novel acute action of CSPGs on axonal conduction in the spinal cord and suggest that antagonism of proteoglycans reverses or prevents the decline of axonal conduction, in addition to stimulating axonal growth.

Input-specific plasticity of N-methyl-D-aspartate receptor-mediated synaptic responses in neonatal rat motoneurons.

Lumbar motoneurons can be activated monosynaptically by two glutamatergic synaptic inputs: the segmental dorsal root (DR) and the descending ventrolateral funiculus (VLF). To determine whether their N-methyl-d-aspartate (NMDA) receptors are independent, we used (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK-801), known to induce a use-dependent irreversible block of NMDA receptors (NMDARs). In the presence of MK-801 (in bath) and non-NMDA antagonists (in bath, to isolate NMDARs pharmacologically), we first stimulated the DR. After MK-801 blockade of DR synaptic input, the VLF was stimulated. Its response was found to be not significantly different from its control value, suggesting that the DR stimulus activated very few, if any, receptors also activated by VLF stimulation. Similar findings were obtained if the stimulation order was reversed. Both inputs also elicited a polysynaptic NMDAR-mediated response. Evoking the DR polysynaptic response in the presence of MK-801 eliminated the corresponding VLF response; the reverse did not occur. Surprisingly, when MK-801 was washed from the bath, both the DR and the VLF responses could recover, although the recovery of the DR monosynaptic and polysynaptic responses was reliably greater than those associated with the VLF. Recovery was prevented if extrasynaptic receptors were activated by bath-applied NMDA in the presence of MK-801, consistent with the possibility that recovery was due to movement of extrasynaptic receptors into parts of the membrane accessible to transmitter released by DR and VLF stimulation. These novel findings suggest that segmental glutamatergic inputs to motoneurons are more susceptible to plastic changes than those from central nervous system white matter inputs at this developmental stage.

Chronic spinal hemisection in rats induces a progressive decline in transmission in uninjured fibers to motoneurons.

Although most spinal cord injuries are anatomically incomplete, only limited functional recovery has been observed in people and rats with partial lesions. To address why surviving fibers cannot mediate more complete recovery, we evaluated the physiological and anatomical status of spared fibers after unilateral hemisection (HX) of thoracic spinal cord in adult rats. We made intracellular and extracellular recordings at L5 (below HX) in response to electrical stimulation of contralateral white matter above (T6) and below (L1) HX. Responses from T6 displayed reduced amplitude, increased latency and elevated stimulus threshold in the fibers across from HX, beginning 1-2 weeks after HX. Ultrastructural analysis revealed demyelination of intact axons contralateral to the HX, with a time course similar to the conduction changes. Behavioral studies indicated partial recovery which arrested when conduction deficits began. In conclusion, this study is the first demonstration of the delayed decline of transmission through surviving axons to individual lumbar motoneurons during chronic stage of incomplete spinal cord injury in adult rats. These findings suggest a chronic pathological state in intact fibers and necessity for prompt treatment to minimize it.

Combined treatment with neurotrophin-3 and LSD facilitates behavioral recovery from double-hemisection spinal injury in neonatal rats.

We explored functional recovery in two spinal cord injury models following a novel combination treatment (NT-3 + LSD). One group of rats received a staggered double hemisection (DH) at postnatal day 2 (P2) of the left hemicord at T11 and the right hemicord at T12. Another group received complete transection (CT) at T11 on P2. A third group was sham operated. Each of these groups was also treated with the drug combination. Drugs were administered intrathecally above the lesion during surgery, and again s.c. at P4, P6, P8, and P10. Intracellular recording in an in vitro spinal cord preparation at P10-P12 in DH rats revealed weak polysynaptic connections to lumbar motoneurons through the injury region, but only in those receiving NT-3 + LSD; NT-3 or LSD alone had no effect. In behavioral experiments, the frequency of rearing in an open field and hindlimb kicks during swimming was assessed every 3-4 days from P9 to P58. Both CT and DH injury severely impaired rearing and hindlimb kicking during swimming. DH rats treated with NT-3 + LSD showed significantly more kicks during swimming than untreated DH or CT rats and treated CT rats beginning as early as P9 and lasting through the duration of testing. Rearing behavior was also improved by treatment but beginning only in the 3rd postnatal week, the time at which it normally develops. Rearing frequency reached sham control levels by P40. Our results suggest this combination treatment may be a promising new strategy for facilitating recovery from moderate spinal cord injury.

Combined delivery of neurotrophin-3 and NMDA receptors 2D subunit strengthens synaptic transmission in contused and staggered double hemisected spinal cord of neonatal rat.

We investigated whether administration of neurotrophin-3 (NT-3) and NMDA-2D-expressing units, found previously to enhance transmission in neonatal rat spinal cord, strengthens synaptic connections in the injured neonatal cord. We employed electrophysiological methods to evaluate the strength of synaptic transmission to individual motoneurons in the contusion and staggered double hemisection spinal cord injury (SCI) models. SCI at caudal thoracic levels (T11-T12) was carried out at postnatal day 2 (P2). Plugs containing NT-3- secreting fibroblasts and NR2D-expressing HSV-1 amplicons (HSVnr2d) were implanted above the lesion. Control animals were treated with an amplicon-expressing beta-galactosidase (HSVlac). After 8-10 days of treatment, the rats were sacrificed and spinal cords were removed for intracellular recording. Untreated contused cords preserved a fraction of white matter and weak monosynaptic responses were observed through the injury region. However, no synaptic connections were observed in control cords receiving double hemisection injury. Combined treatment with NT-3 and HSVnr2d strengthened monosynaptic connections in contused cords and induced the appearance of weak but functional multisynaptic connections in double hemisected cords. In contrast, treatment with either NT-3 or HSVnr2d alone failed to induce appearance of synaptic responses through the hemisected region. These results suggest that chronic treatment with NT-3 secreting fibroblasts combined with facilitated function of NMDA receptors by HSVnr2d treatment strengthens connections that survive incomplete SCI and therefore that such combined treatment might facilitate recovery of function following SCI.

Comparison of metabotropic glutamate receptor responses at segmental and descending inputs to motoneurons in neonatal rat spinal cord.

We compared the contribution of metabotropic glutamate receptors (mGluRs) to the generation and modulation of synaptic responses elicited in intracellularly recorded L5 motoneurons from neonatal rats by segmental and descending fibers. Dorsal root (DR) stimulation at high intensity (C-fiber strength) evoked long latency (2-5-s) depolarization in addition to early monosynaptic and polysynaptic responses. Stimulation of the descending ventrolateral funiculus (VLF) failed to evoke a late response in the same motoneuron. The mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG; 0.4 mM) selectively blocked the long latency DR response. This mGluR-mediated response persisted in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate or N-methyl-d-aspartate (NMDA) antagonists, but not both, suggesting that glutamate transmission (either AMPA/kainate or NMDA) is required for mGluR-mediated inputs from small diameter sensory afferents to affect the motoneuron. Although MCPG inhibited the long latency DR response, it induced moderate facilitation of monosynaptic DR and VLF responses. The mGluR agonist 1s3r-ACPD induced motoneuron depolarization and depressed the monosynaptic DR and VLF responses. MCPG also facilitated the neurotrophin-3 and brain-derived neurotrophic factor induced strengthening of the monosynaptic DR responses (but only before P6, since neurotrophins are ineffective later at DR synapses and never at VLF synapses after birth). Our results suggest that mGluRs are involved in synaptic pathways to motoneurons made by DR but not VLF fibers. MCPG-induced facilitation of monosynaptic AMPA/kainate DR and VLF responses suggests the possibility of tonic mGluR-mediated inhibition of DR and VLF responses. We speculate that MCPG facilitates neurotrophin-induced strengthening of monosynaptic DR responses by reducing this tonic inhibition.

Viral delivery of NR2D subunits reduces Mg2+ block of NMDA receptor and restores NT-3-induced potentiation of AMPA-kainate responses in maturing rat motoneurons.

N-methyl-D-aspartate (NMDA) responsiveness of motoneurons declines during the initial 2 postnatal weeks due to increasing Mg2+ block of NMDA receptors. Using gene chip analyses, RT-PCR, and immunochemistry, we have shown that the NR2D subunit of the NMDA receptor (NMDAR), known to confer resistance to Mg2+ block, also declines in motoneurons during this period. We injected a viral construct (HSVnr2d) into the lumbar spinal cord on postnatal day 2 in an attempt to restore NMDAR function in motoneurons during the second postnatal week. Following HSVnr2d injection, we detected elevated levels of NR2D mRNA in spinal cord samples and NR2D protein specifically in motoneurons. These molecular changes were associated with marked functional alterations whereby NMDAR-mediated responses in motoneurons associated with both dorsal root (DR) and ventrolateral funiculus (VLF) inputs returned to values observed at E18 due to decreased Mg2+ blockade. Viruses carrying the beta-galactosidase gene did not induce these effects. NT-3 is known to potentiate AMPA-kainate responses in motoneurons if the response has an NMDAR-mediated component and thus is normally ineffective during the second postnatal week. Restoration of NMDAR-mediated responsiveness in the second postnatal week was accompanied by a return of the ability of neurotrophin-3 (NT-3) to potentiate the AMPA-kainate responses produced by both DR and VLF synaptic inputs. We conclude that delivery of the gene for a specific NMDA subunit can restore properties characteristic of younger animals to spinal cord motoneurons. This approach might be useful for enhancing the function of fibers surviving in the damaged spinal cord.

Chronic neurotrophin-3 strengthens synaptic connections to motoneurons in the neonatal rat.

We report that neurotrophin-3 (NT-3), delivered chronically via fibroblasts implanted intrathecally into neonatal rats, can facilitate synaptic transmission in the spinal cord. A small collagen plug containing NT-3-secreting fibroblasts was placed on the exposed dorsal surface of the spinal cord (L1) of 2-d-old rats; controls received beta-galactosidase-secreting fibroblasts. After 6 hr to 12 d of survival, synaptic potentials (EPSP) elicited by two synaptic inputs, L5 dorsal root and ventrolateral funiculus (VLF), were recorded intracellularly in L5 motoneurons in vitro. Preparations treated with NT-3 implants exhibited enhanced monosynaptic synaptic transmission from both inputs, which persisted over the entire testing period. Unlike acute enhancement of transmission by NT-3 (Arvanian and Mendell, 2001a), the chronic effect could occur at connections not normally eliciting an NMDA receptor-mediated response at the time of NT-3 exposure. Using susceptibility to blockade of the NMDA receptor by Mg2+ and APV, we confirmed that chronic treatment with NT-3 did not enhance NMDA receptor activity at these connections. Cords treated with chronic NT-3 also transiently displayed polysynaptic components activated by VLF that were blocked by the NMDA receptor antagonist APV. These novel NMDA receptor-mediated potentials may reflect changes in interneurons near the site of fibroblast implantation. We conclude that chronic NT-3 enhances the potency of segmental and descending projections via mechanisms different from those underlying acute changes.