Elevating spinal levels of neurotrophin NT-3 (NT3) while increasing expression of the NR2D subuni... more 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.
To encourage re-establishment of functional innervation of ipsilateral lumbar motoneurons by desc... more 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.
Elevating spinal levels of neurotrophin NT-3 (NT3) while increasing expression of the NR2D subuni... more 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.
PDF] [Full Text] [Abstract] , February 1, 2005; 312 (2): 669-677. J. Pharmacol. Exp. Ther. V. L. ... more PDF] [Full Text] [Abstract] , February 1, 2005; 312 (2): 669-677. J. Pharmacol. Exp. Ther. V. L. Arvanian, V. Motin and L. M. Mendell Inputs to Motoneurons in Neonatal Rat Spinal Cord Comparison of Metabotropic Glutamate Receptor Responses at Segmental and Descending [PDF] [Full Text] [Abstract] , September 1, 2005; 94 (3): 1798-1804. J Neurophysiol . Viral delivery of NR2D subunits reduces Mg 2ϩ block of NMDA receptor and restores NT-3induced potentiation of AMPA/kainate responses in maturing rat motoneurons.
We investigated the acute effects of bath applied BDNF on synaptic input to motoneurons in the he... more We investigated the acute effects of bath applied BDNF on synaptic input to motoneurons in the hemisected spinal cord of the neonatal rat. Motoneurons were recorded intracellularly, and BDNF-induced modulation of the synaptic response to stimulation of the homologous dorsal root (DR) and the ventrolateral funiculus (VLF) was examined. All motoneurons exhibited long-lasting (up to several hours) depression of the DR-activated monosynaptic AMPA/kainate-receptor mediated EPSP in response to BDNF but in about half of the motoneurons this was preceded by facilitation. VLF-evoked AMPA/kainate EPSPs in the same motoneurons were unaffected. BDNF effects were blocked by K252a and were not observed in neonates older than 1 week. Bath applied NMDA antagonists APV and MK-801 abolished both facilitatory and inhibitory actions of BDNF on the AMPA/kainate responses indicating the requirement for functional NMDA receptors. The pharmacologically isolated, DR-evoked, NMDA receptor-mediated response exhibited the same pattern of changes after BDNF superfusion. When introduced into the motoneuron through the recording microelectrode, MK-801 selectively blocked the facilitatory action of BDNF. Furthermore, BDNF enhanced NMDAinduced depolarization of the motoneuron in the presence of tetrodotoxin (TTX), thus, con®rming its facilitatory effect on motoneuron NMDA receptors. Bath application of either BDNF or NMDA depressed the monosynaptic EPSP after selective blockade of postsynaptic NMDA receptors indicating a role for presynaptic NMDA receptors in BDNF-induced inhibitory action. Thus, BDNF-induced facilitation of monosynaptic EPSPs in neonatal rats is mediated by direct effects on postsynaptic NMDA receptors, while its inhibitory action occurs presynaptically.
The pathway mediating the monosynaptic stretch reflex has served as an important model system for... more The pathway mediating the monosynaptic stretch reflex has served as an important model system for studies of plasticity in the spinal cord. Its usefulness is extended by evidence that neurotrophins, particularly neurotrophin-3 (NT-3), which has been shown to promote spinal axon elongation, can modulate the efficacy of the muscle spindle-motoneurone connection both after peripheral nerve injury and during development. The findings summarized here emphasize the potential for neurotrophins to modify function of both damaged and undamaged neurones. It is important to recognize that these effects may be functionally detrimental as well as beneficial.
at some nodes of Ranvier. These results identify a novel acute action of CSPGs on axonal conducti... more 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.
The expression of neurotrophins and their receptors in the adult spinal cord indicates that they ... more The expression of neurotrophins and their receptors in the adult spinal cord indicates that they have postnatal actions in addition to their well-known prenatal ones on axonal growth and cell survival. In this review we summarize evidence in support of mechanisms by which neurotrophins acutely modulate the response both of sensory neurons and of synapses within the spinal cord. The selective action of neurotrophins is achieved via restricted expression of high affinity trk receptors through which the neurotrophins act. Activation of trk receptors enhances the response of the vanilloid VR-1 receptor in nociceptive neurons leading to peripheral sensitization of the response to capsaicin or noxious heat. At synapses on motoneurons trk receptor activation enhances the response of NMDA receptors that in turn can increase the response of AMPA/kainate receptors on the same cell. Both of these sensitizing actions have a very rapid onset that is contrasted with slower neurotrophin effects on growth of axotomized afferents. It is likely that these different functional effects of neurotrophins reflect activation of different intracellular signaling pathways. These studies suggest mechanisms by which neurotrophins might be used to improve function of the damaged spinal cord.
Lumbar motoneurons can be activated monosynaptically by two glutamatergic synaptic inputs: segmen... more Lumbar motoneurons can be activated monosynaptically by two glutamatergic synaptic inputs: segmental dorsal root (DR) and descending ventrolateral funiculus (VLF). To determine if their Nmethyl-D-aspartate (NMDA) receptors are independent, we used (5R,10S)-(+)-5-methyl-10,11dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK-801), known to induce a use-dependent irreversible block of NMDA receptors (NMDAR). In the presence of MK-801 (in bath) and non-NMDA antagonists (in bath, to isolate NMDA receptors pharmacologically) we first stimulated DR. After MK-801 blockade of DR synaptic input, the VLF was stimulated. Its response was found to be not significantly different than its control value suggesting that the DR stimulus activated very few if any receptors also activated by VLF stimulation. Similar findings were made if the stimulation order was reversed. Both inputs also elicited a polysynaptic NMDA receptor-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 DR and VLF responses could recover although the recovery of the DR monosynaptic and polysynaptic responses was reliably greater than those associated with 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 CNS white matter inputs at this developmental stage.
We explored functional recovery in two spinal cord injury models following a novel combination tr... more 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 3 rd 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.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 24, 2003
We report that neurotrophin-3 (NT-3), delivered chronically via fibroblasts implanted intrathecal... more 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...
Arvanian, Victor L. and Lorne M. Mendell. Removal of NMDA receptor Mg 2ϩ block extends the action... more Arvanian, Victor L. and Lorne M. Mendell. Removal of NMDA receptor Mg 2ϩ block extends the action of NT-3 on synaptic transmission in neonatal rat motoneurons. J Neurophysiol 86: [123][124][125][126][127][128][129] 2001. NT-3 has previously been reported to enhance AMPA/kainate receptor-mediated synaptic responses in motoneurons via an effect on the N-methyl-D-aspartate (NMDA) receptor. To investigate neurotrophin-3 (NT-3) action further, we measured the NMDA receptor (NMDAR)-mediated synaptic response directly by intracellular recording in motoneurons after blocking AMPA/kainate, GABA A , GABA B and glycine receptor-mediated responses pharmacologically. Two pathways were stimulated, the segmental dorsal root (DR) and the descending ventrolateral fasciculus (VLF). The DR-evoked NMDAR-mediated response in motoneurons of rats younger than 1 wk has two components, the initial one of which is generated monosynaptically. NT-3 strongly potentiated both NMDA components in a rapidly reversible manner. No NMDAR-mediated responses were present at VLF connections and at DR connections in older (1-to 2-wk-old) neonates. Bath-applied NT-3-induced potentiation of the AMPA/kainate receptor-mediated response occurred only at connections that exhibit a synaptic NMDA receptor-mediated response. Reducing Mg 2ϩ concentration in the bathing solution restored the NMDAR-mediated response elicited by DR stimulation in older neonates and by VLF throughout the neonatal period (0 -2 wk). In low-Mg 2ϩ , NT-3 enhanced AMPA/kainate receptor-mediated responses elicited by inputs normally not influenced by NT-3. Thus a major reason for the loss of NT-3 action on AMPA/kainate synaptic responses is the reduced activity of the NMDA receptor due to developing Mg 2ϩ block of NMDA receptor-channel complex as the animal matures, and both can be re-established by reducing Mg 2ϩ concentration in fluid bathing the spinal cord.
Viral delivery of NR2D subunits reduces Mg 2ϩ block of NMDA receptor and restores NT-3induced pot... more Viral delivery of NR2D subunits reduces Mg 2ϩ block of NMDA receptor and restores NT-3induced 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 Mg 2ϩ 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 Mg 2ϩ 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 Mg 2ϩ blockade. Viruses carrying the -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.
Although most spinal cord injuries are anatomically incomplete, only limited functional recovery ... more 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.
We investigated whether administration of neurotrophin-3 (NT-3) and NMDA-2D-expressing units, fou... more 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 NR2Dexpressing HSV-1 amplicons (HSVnr2d) were implanted above the lesion. Control animals were treated with an amplicon-expressing hgalactosidase (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. D
We compared the contribution of metabotropic glutamate receptors (mGluRs) to the generation and m... more 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.
Elevating spinal levels of neurotrophin NT-3 (NT3) while increasing expression of the NR2D subuni... more 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.
To encourage re-establishment of functional innervation of ipsilateral lumbar motoneurons by desc... more 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.
Elevating spinal levels of neurotrophin NT-3 (NT3) while increasing expression of the NR2D subuni... more 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.
PDF] [Full Text] [Abstract] , February 1, 2005; 312 (2): 669-677. J. Pharmacol. Exp. Ther. V. L. ... more PDF] [Full Text] [Abstract] , February 1, 2005; 312 (2): 669-677. J. Pharmacol. Exp. Ther. V. L. Arvanian, V. Motin and L. M. Mendell Inputs to Motoneurons in Neonatal Rat Spinal Cord Comparison of Metabotropic Glutamate Receptor Responses at Segmental and Descending [PDF] [Full Text] [Abstract] , September 1, 2005; 94 (3): 1798-1804. J Neurophysiol . Viral delivery of NR2D subunits reduces Mg 2ϩ block of NMDA receptor and restores NT-3induced potentiation of AMPA/kainate responses in maturing rat motoneurons.
We investigated the acute effects of bath applied BDNF on synaptic input to motoneurons in the he... more We investigated the acute effects of bath applied BDNF on synaptic input to motoneurons in the hemisected spinal cord of the neonatal rat. Motoneurons were recorded intracellularly, and BDNF-induced modulation of the synaptic response to stimulation of the homologous dorsal root (DR) and the ventrolateral funiculus (VLF) was examined. All motoneurons exhibited long-lasting (up to several hours) depression of the DR-activated monosynaptic AMPA/kainate-receptor mediated EPSP in response to BDNF but in about half of the motoneurons this was preceded by facilitation. VLF-evoked AMPA/kainate EPSPs in the same motoneurons were unaffected. BDNF effects were blocked by K252a and were not observed in neonates older than 1 week. Bath applied NMDA antagonists APV and MK-801 abolished both facilitatory and inhibitory actions of BDNF on the AMPA/kainate responses indicating the requirement for functional NMDA receptors. The pharmacologically isolated, DR-evoked, NMDA receptor-mediated response exhibited the same pattern of changes after BDNF superfusion. When introduced into the motoneuron through the recording microelectrode, MK-801 selectively blocked the facilitatory action of BDNF. Furthermore, BDNF enhanced NMDAinduced depolarization of the motoneuron in the presence of tetrodotoxin (TTX), thus, con®rming its facilitatory effect on motoneuron NMDA receptors. Bath application of either BDNF or NMDA depressed the monosynaptic EPSP after selective blockade of postsynaptic NMDA receptors indicating a role for presynaptic NMDA receptors in BDNF-induced inhibitory action. Thus, BDNF-induced facilitation of monosynaptic EPSPs in neonatal rats is mediated by direct effects on postsynaptic NMDA receptors, while its inhibitory action occurs presynaptically.
The pathway mediating the monosynaptic stretch reflex has served as an important model system for... more The pathway mediating the monosynaptic stretch reflex has served as an important model system for studies of plasticity in the spinal cord. Its usefulness is extended by evidence that neurotrophins, particularly neurotrophin-3 (NT-3), which has been shown to promote spinal axon elongation, can modulate the efficacy of the muscle spindle-motoneurone connection both after peripheral nerve injury and during development. The findings summarized here emphasize the potential for neurotrophins to modify function of both damaged and undamaged neurones. It is important to recognize that these effects may be functionally detrimental as well as beneficial.
at some nodes of Ranvier. These results identify a novel acute action of CSPGs on axonal conducti... more 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.
The expression of neurotrophins and their receptors in the adult spinal cord indicates that they ... more The expression of neurotrophins and their receptors in the adult spinal cord indicates that they have postnatal actions in addition to their well-known prenatal ones on axonal growth and cell survival. In this review we summarize evidence in support of mechanisms by which neurotrophins acutely modulate the response both of sensory neurons and of synapses within the spinal cord. The selective action of neurotrophins is achieved via restricted expression of high affinity trk receptors through which the neurotrophins act. Activation of trk receptors enhances the response of the vanilloid VR-1 receptor in nociceptive neurons leading to peripheral sensitization of the response to capsaicin or noxious heat. At synapses on motoneurons trk receptor activation enhances the response of NMDA receptors that in turn can increase the response of AMPA/kainate receptors on the same cell. Both of these sensitizing actions have a very rapid onset that is contrasted with slower neurotrophin effects on growth of axotomized afferents. It is likely that these different functional effects of neurotrophins reflect activation of different intracellular signaling pathways. These studies suggest mechanisms by which neurotrophins might be used to improve function of the damaged spinal cord.
Lumbar motoneurons can be activated monosynaptically by two glutamatergic synaptic inputs: segmen... more Lumbar motoneurons can be activated monosynaptically by two glutamatergic synaptic inputs: segmental dorsal root (DR) and descending ventrolateral funiculus (VLF). To determine if their Nmethyl-D-aspartate (NMDA) receptors are independent, we used (5R,10S)-(+)-5-methyl-10,11dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK-801), known to induce a use-dependent irreversible block of NMDA receptors (NMDAR). In the presence of MK-801 (in bath) and non-NMDA antagonists (in bath, to isolate NMDA receptors pharmacologically) we first stimulated DR. After MK-801 blockade of DR synaptic input, the VLF was stimulated. Its response was found to be not significantly different than its control value suggesting that the DR stimulus activated very few if any receptors also activated by VLF stimulation. Similar findings were made if the stimulation order was reversed. Both inputs also elicited a polysynaptic NMDA receptor-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 DR and VLF responses could recover although the recovery of the DR monosynaptic and polysynaptic responses was reliably greater than those associated with 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 CNS white matter inputs at this developmental stage.
We explored functional recovery in two spinal cord injury models following a novel combination tr... more 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 3 rd 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.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 24, 2003
We report that neurotrophin-3 (NT-3), delivered chronically via fibroblasts implanted intrathecal... more 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...
Arvanian, Victor L. and Lorne M. Mendell. Removal of NMDA receptor Mg 2ϩ block extends the action... more Arvanian, Victor L. and Lorne M. Mendell. Removal of NMDA receptor Mg 2ϩ block extends the action of NT-3 on synaptic transmission in neonatal rat motoneurons. J Neurophysiol 86: [123][124][125][126][127][128][129] 2001. NT-3 has previously been reported to enhance AMPA/kainate receptor-mediated synaptic responses in motoneurons via an effect on the N-methyl-D-aspartate (NMDA) receptor. To investigate neurotrophin-3 (NT-3) action further, we measured the NMDA receptor (NMDAR)-mediated synaptic response directly by intracellular recording in motoneurons after blocking AMPA/kainate, GABA A , GABA B and glycine receptor-mediated responses pharmacologically. Two pathways were stimulated, the segmental dorsal root (DR) and the descending ventrolateral fasciculus (VLF). The DR-evoked NMDAR-mediated response in motoneurons of rats younger than 1 wk has two components, the initial one of which is generated monosynaptically. NT-3 strongly potentiated both NMDA components in a rapidly reversible manner. No NMDAR-mediated responses were present at VLF connections and at DR connections in older (1-to 2-wk-old) neonates. Bath-applied NT-3-induced potentiation of the AMPA/kainate receptor-mediated response occurred only at connections that exhibit a synaptic NMDA receptor-mediated response. Reducing Mg 2ϩ concentration in the bathing solution restored the NMDAR-mediated response elicited by DR stimulation in older neonates and by VLF throughout the neonatal period (0 -2 wk). In low-Mg 2ϩ , NT-3 enhanced AMPA/kainate receptor-mediated responses elicited by inputs normally not influenced by NT-3. Thus a major reason for the loss of NT-3 action on AMPA/kainate synaptic responses is the reduced activity of the NMDA receptor due to developing Mg 2ϩ block of NMDA receptor-channel complex as the animal matures, and both can be re-established by reducing Mg 2ϩ concentration in fluid bathing the spinal cord.
Viral delivery of NR2D subunits reduces Mg 2ϩ block of NMDA receptor and restores NT-3induced pot... more Viral delivery of NR2D subunits reduces Mg 2ϩ block of NMDA receptor and restores NT-3induced 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 Mg 2ϩ 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 Mg 2ϩ 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 Mg 2ϩ blockade. Viruses carrying the -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.
Although most spinal cord injuries are anatomically incomplete, only limited functional recovery ... more 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.
We investigated whether administration of neurotrophin-3 (NT-3) and NMDA-2D-expressing units, fou... more 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 NR2Dexpressing HSV-1 amplicons (HSVnr2d) were implanted above the lesion. Control animals were treated with an amplicon-expressing hgalactosidase (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. D
We compared the contribution of metabotropic glutamate receptors (mGluRs) to the generation and m... more 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.
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Papers by Victor Arvanian