Consequences of zygote injection and germline transfer of mutant human mitochondrial DNA in mice.

Considerable evidence supports mutations in mitochondrial genes as the cause of maternally inherited diseases affecting tissues that rely primarily on oxidative energy metabolism, usually the nervous system, the heart, and skeletal muscles. Mitochondrial diseases are diverse, and animal models currently are limited. Here we introduced a mutant human mitochondrial gene responsible for Leber hereditary optic neuropathy (LHON) into the mouse germ line using fluorescence imaging for tissue-specific enrichment in the target retinal ganglion cells. A mitochondria-targeted adeno-associated virus (MTS-AAV) containing the mutant human NADH ubiquinone oxidoreductase subunit 4 (ND4) gene followed by mitochondrial-encoded mCherry was microinjected into zygotes. Female founders with mCherry fluorescence on ophthalmoscopy were backcrossed with normal males for eight generations. Mutant human ND4 DNA was 20% of mouse ND4 and did not integrate into the host genome. Translated human ND4 protein assembled into host respiratory complexes, decreasing respiratory chain function and increasing oxidative stress. Swelling of the optic nerve head was followed by progressive demise of ganglion cells and their axons, the hallmarks of human LHON. Early visual loss that began at 3 mo and progressed to blindness 8 mo after birth was reversed by intraocular injection of MTS-AAV expressing wild-type human ND4. The technology of introducing human mitochondrial genes into the mouse germ line has never been described, to our knowledge, and has implications not only for creating animal models recapitulating the counterpart human disorder but more importantly for reversing the adverse effects of the mutant gene using gene therapy to deliver the wild-type allele.

Fluorescent reporters of monoamine transporter distribution and function.

Serotonin is a monoamine serving as a chemical messenger in diverse brain regions, as well as in blood and various other organs. We synthesized six ethylamine functionalized fluorophores as fluorescent probes for serotonin. The one with best spectral properties and aqueous solubility, 6-amino-2-(2-aminoethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione, was studied in detail both in vivo and in vitro. It was shown to act as a ligand for serotonin transporter (SERT) without acute cerebral or cardiovascular toxicity or adverse effects. Fluorescent serotonin analogs can be used for direct visualization of SERT distribution and activity in live tissue.

Brainstem-Evoked Transcription of Defensive Genes After Spinal Cord Injury.

The spinal cord after injury shows altered transcription in numerous genes. We tested in a pilot study whether the nucleus raphé magnus, a descending serotonergic brainstem region whose stimulation improves recovery after incomplete spinal cord injury (SCI), can influence these transcriptional changes. Rats received 2 h of low-frequency electrical stimulation in the raphé magnus 3 days after an impact contusion at segment T8. Comparison groups lacked injuries or activated stimulators or both. Immediately following stimulation, spinal cords were extracted, their RNA transcriptome sequenced, and differential gene expression quantified. Confirming many previous studies, injury primarily increased inflammatory and immune transcripts and decreased those related to lipid and cholesterol synthesis and neuronal signaling. Stimulation plus injury, contrasted with injury alone, caused significant changes in 43 transcripts (39 increases, 4 decreases), all protein-coding. Injury itself decreased only four of these 43 transcripts, all reversed by stimulation, and increased none of them. The non-specific 5-HT7 receptor antagonist pimozide reversed 25 of the 43 changes. Stimulation in intact rats principally caused decreases in transcripts related to oxidative phosphorylation, none of which were altered by stimulation in injury. Gene ontology (biological process) annotations comparing stimulation with either no stimulation or pimozide treatment in injured rats highlighted defense responses to lipopolysaccharides and microorganisms, and also erythrocyte development and oxygen transport (possibly yielding cellular oxidant detoxification). Connectivity maps of human orthologous genes generated in the CLUE database of perturbagen-response transcriptional signatures showed that drug classes whose effects in injured rats most closely resembled stimulation without pimozide include peroxisome proliferator-activated receptor agonists and angiotensin receptor blockers, which are reportedly beneficial in SCI. Thus the initial transcriptional response of the injured spinal cord to raphé magnus stimulation is upregulation of genes that in various ways are mostly protective, some probably located in recently arrived myeloid cells.

Cellular Changes in Injured Rat Spinal Cord Following Electrical Brainstem Stimulation.

Spinal cord injury (SCI) is a major cause of disability and pain, but little progress has been made in its clinical management. Low-frequency electrical stimulation (LFS) of various anti-nociceptive targets improves outcomes after SCI, including motor recovery and mechanical allodynia. However, the mechanisms of these beneficial effects are incompletely delineated and probably multiple. Our aim was to explore near-term effects of LFS in the hindbrain's nucleus raphe magnus (NRM) on cellular proliferation in a rat SCI model. Starting 24 h after incomplete contusional SCI at C5, intermittent LFS at 8 Hz was delivered wirelessly to NRM. Controls were given inactive stimulators. At 48 h, 5-bromodeoxyuridine (BrdU) was administered and, at 72 h, spinal cords were extracted and immunostained for various immune and neuroglial progenitor markers and BrdU at the level of the lesion and proximally and distally. LFS altered cell marker counts predominantly at the dorsal injury site. BrdU cell counts were decreased. Individually and in combination with BrdU, there were reductions in CD68 (monocytes) and Sox2 (immature neural precursors) and increases in Blbp (radial glia) expression. CD68-positive cells showed increased co-staining with iNOS. No differences in the expression of GFAP (glia) and NG2 (oligodendrocytes) or in GFAP cell morphology were found. In conclusion, our work shows that LFS of NRM in subacute SCI influences the proliferation of cell types implicated in inflammation and repair, thus providing mechanistic insight into deep brain stimulation as a neuromodulatory treatment for this devastating pathology.

Some Autonomic Deficits of Acute or Chronic Cervical Spinal Contusion Reversed by Interim Brainstem Stimulation.

Prolonged electrical stimulation of the hindbrain's nucleus raphe magnus (NRM) or of its major midbrain input region, the periaqueductal gray (PAG), was previously found in rats to promote recovery from sensory-motor and histological deficits of acute thoracic spinal cord injury (SCI). Here, some visceral deficits of acute and chronic midline cervical (C5) contusion are similarly examined. Cranially implanted wireless stimulators delivered intermittent 8 Hz, 30-70 µA cathodal pulse trains to a brainstem microelectrode. Injured controls were given inactive stimulators; rats without injuries or implants were also compared. Rectal distension or squeezing of the forepaws caused an exaggerated rise in mean arterial pressure in injured, untreated rats under anesthesia on post-injury week 6, probably reflecting autonomic dysreflexia (AD). These pressor responses became normal when 7 days of unilateral PAG stimulation was started on the injury day. Older untreated injuries (weeks 18-19) showed normal pressor responses, but unexpectedly had significant resting and nociceptive bradycardia, which was reversed by 3 weeks of PAG stimulation started on weeks 7 or 12. Subsequent chronic studies examined gastric emptying (GE), as indicated by intestinal transit of gavaged dye, and serum chemistry. GE and fasting serum insulin were reduced on injury weeks 14-15, and were both normalized by ∼5 weeks of PAG stimulation begun in weeks 7-8. Increases in calcitonin gene-related peptide, a prominent visceral afferent neurotransmitter, measured near untreated injuries (first thoracic segment) in superficial dorsal laminae were reversed by acutely or chronically initiated PAG stimulation. The NRM, given 2-3 weeks of stimulation beginning 2 days after SCI, prevented abnormalities in both pressor responses and GE on post-injury week 9, consistent with its relaying of repair commands from the PAG. The descending PAG-NRM axis thus exhibits broadly restorative influences on visceral as well as sensory-motor deficits, improving chronic as well as acute signs of injury.

Inhibition by the chromaffin cell-derived peptide serine-histogranin in the rat's dorsal horn.

The heptadecapeptide histogranin, synthesized by adrenal chromaffin cells, is implicated in the analgesia produced by transplanting chromaffin cells into the spinal cord, including block of hyperalgesia mediated by NMDA-subtype glutamate receptors. To examine the neurophysiological basis for this analgesia, we applied the stable analog [Ser(1)]-histogranin (SHG) by iontophoresis near extracellularly recorded wide-dynamic range (WDR) neurons in anesthetized rats. When SHG was applied during peripheral electrical stimulation of A and C fibers at 0.1Hz, the C-fiber response was significantly inhibited but the A-fiber response was unaffected. SHG also opposed the NMDA-receptor-dependent post-tetanic facilitation (wind-up) of C-fiber responses produced by increasing the rate of peripheral afferent stimulation to 1Hz for 20s. To test whether block of NMDA-subtype receptors could be wholly or partially responsible for this suppression, SHG was applied during sequential pulsed iontophoresis of three agonists targeting distinct excitatory synaptic receptors: NMDA, kainate and substance P. All three excitatory effects were reversed by SHG; this reversal outlasted the 10-30min observation period when higher SHG doses were applied (>60nA). Histogranin therefore probably produces prolonged spinal analgesia by opposing the basal and potentiating synaptic effects of C-fibers on dorsal horn neurons. Actions besides or in addition to NMDA-receptor antagonism (e.g., agonism at inhibitory postsynaptic receptors or block of voltage-gated cation channels on C-fibers) are implied by the diversity of excitatory transmitters opposed by SHG.

Surgical Neurostimulation for Spinal Cord Injury.

Traumatic spinal cord injury (SCI) is a devastating neurological condition characterized by a constellation of symptoms including paralysis, paraesthesia, pain, cardiovascular, bladder, bowel and sexual dysfunction. Current treatment for SCI involves acute resuscitation, aggressive rehabilitation and symptomatic treatment for complications. Despite the progress in scientific understanding, regenerative therapies are lacking. In this review, we outline the current state and future potential of invasive and non-invasive neuromodulation strategies including deep brain stimulation (DBS), spinal cord stimulation (SCS), motor cortex stimulation (MCS), transcutaneous direct current stimulation (tDCS) and repetitive transcranial magnetic stimulation (rTMS) in the context of SCI. We consider the ability of these therapies to address pain, sensorimotor symptoms and autonomic dysregulation associated with SCI. In addition to the potential to make important contributions to SCI treatment, neuromodulation has the added ability to contribute to our understanding of spinal cord neurobiology and the pathophysiology of SCI.

Monoamine Release in the Cat Lumbar Spinal Cord during Fictive Locomotion Evoked by the Mesencephalic Locomotor Region.

Spinal cord neurons active during locomotion are innervated by descending axons that release the monoamines serotonin (5-HT) and norepinephrine (NE) and these neurons express monoaminergic receptor subtypes implicated in the control of locomotion. The timing, level and spinal locations of release of these two substances during centrally-generated locomotor activity should therefore be critical to this control. These variables were measured in real time by fast-cyclic voltammetry in the decerebrate cat's lumbar spinal cord during fictive locomotion, which was evoked by electrical stimulation of the mesencephalic locomotor region (MLR) and registered as integrated activity in bilateral peripheral nerves to hindlimb muscles. Monoamine release was observed in dorsal horn (DH), intermediate zone/ventral horn (IZ/VH) and adjacent white matter (WM) during evoked locomotion. Extracellular peak levels (all sites) increased above baseline by 138 ± 232.5 nM and 35.6 ± 94.4 nM (mean ± SD) for NE and 5-HT, respectively. For both substances, release usually began prior to the onset of locomotion typically earliest in the IZ/VH and peaks were positively correlated with net activity in peripheral nerves. Monoamine levels gradually returned to baseline levels or below at the end of stimulation in most trials. Monoamine oxidase and uptake inhibitors increased the release magnitude, time-to-peak (TTP) and decline-to-baseline. These results demonstrate that spinal monoamine release is modulated on a timescale of seconds, in tandem with centrally-generated locomotion and indicate that MLR-evoked locomotor activity involves concurrent activation of descending monoaminergic and reticulospinal pathways. These gradual changes in space and time of monoamine concentrations high enough to strongly activate various receptors subtypes on locomotor activated neurons further suggest that during MLR-evoked locomotion, monoamine action is, in part, mediated by extrasynaptic neurotransmission in the spinal cord.

Prolonged stimulation of a brainstem raphe region attenuates experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS), a neuroinflammatory disease, has few treatment options, none entirely adequate. We studied whether prolonged electrical microstimulation of a hindbrain region (the nucleus raphe magnus) can attenuate experimental autoimmune encephalomyelitis, a murine model of MS induced by MOG35-55 injection. Eight days after symptoms emerged, a wireless electrical stimulator with an attached microelectrode was implanted cranially, and daily intermittent stimulation was begun in awake, unrestrained mice. The thoracic spinal cord was analyzed for changes in histology (on day 29) and gene expression (on day 37), with a focus on myelination and cytokine production. Controls, with inactive implants, showed a phase of disease exacerbation on days 19-25 that stimulation for >16days eliminated. Prolonged stimulation also reduced numbers of infiltrating immune cells and increased numbers of myelinated axons. It additionally lowered genetic expression of some pro-inflammatory cytokines (interferon gamma and tumor necrosis factor) and platelet-derived growth factor receptor alpha, a marker of oligodendrocyte precursors, while raising expression of myelin basic protein. Studies of restorative treatments for MS might profitably consider ways to stimulate the raphe magnus, directly or via its inputs, or to emulate its serotonergic and peptidergic output.

Deep Brain Stimulation Improves the Symptoms and Sensory Signs of Persistent Central Neuropathic Pain from Spinal Cord Injury: A Case Report.

Central neuropathic pain (CNP) is a significant problem after spinal cord injury (SCI). Pharmacological and non-pharmacological approaches may reduce the severity, but relief is rarely substantial. While deep brain stimulation (DBS) has been used to treat various chronic pain types, the technique has rarely been used to attenuate CNP after SCI. Here we present the case of a 54-year-old female with incomplete paraplegia who had severe CNP in the lower limbs and buttock areas since her injury 30 years prior. She was treated with bilateral DBS of the midbrain periaqueductal gray (PAG). The effects of this stimulation on CNP characteristics, severity and pain-related sensory function were evaluated using the International SCI Pain Basic Data Set (ISCIPBDS), Neuropathic Pain Symptom Inventory (NPSI), Multidimensional Pain Inventory and Quantitative Sensory Testing before and periodically after initiation of DBS. After starting DBS treatment, weekly CNP severity ratings rapidly decreased from severe to minimal, paralleled by a substantial reduction in size of the painful area, reduced pain impact and reversal of pain-related neurological abnormalities, i.e., dynamic-mechanical and cold allodynia. She discontinued pain medication on study week 24. The improvement has been consistent. The present study expands on previous findings by providing in-depth assessments of symptoms and signs associated with CNP. The results of this study suggest that activation of endogenous pain inhibitory systems linked to the PAG can eliminate CNP in some people with SCI. More research is needed to better-select appropriate candidates for this type of therapy. We discuss the implications of these findings for understanding the brainstem's control of chronic pain and for future progress in using analgesic DBS in the central gray.

The midbrain central gray best suppresses chronic pain with electrical stimulation at very low pulse rates in two human cases.

Deep brain stimulation in the midbrain׳s central gray can relieve neuropathic pain in man, but for unclear reasons sometimes fails intraoperatively or in early weeks. Here we describe continuous bilateral stimulation in the central gray of two subjects with longstanding, severe neuropathic pain from spinal cord injury. Stimulation parameters were recursively adjusted over many weeks to optimize analgesia while minimizing adverse effects. In early weeks, adjustments were made in periodic office visits; subjects later selected ad libitum at home among several blinded choices while rating pain twice daily. Both subjects received significantly better pain relief when stimulus pulse rates were low. The best relief occurred with 2 Hz cycled on for 1s and off for 2s. After inferior parameters were set, pain typically climbed slowly over 1-2 days; superior parameters led to both slow and fast improvements. Over many weeks of stimulation at low pulse rates, both subjects experienced significantly less interference from pain with sleep. One subject, with major pain relief, also showed less interference with social/recreational ability and mood; the other subject, despite minor pain relief, experienced a significantly positive global impression of change. Oscillopsia, the only observed complication of stimulation, disappeared at low mean pulse rates (≤ 3/s). These subjects׳ responses are not likely to be unique even if they are uncommon. Thus daily or more frequent pain assessment, combined with slower periodic adjustment of stimulation parameters that incorporate mean pulse rates about one per second, will likely improve success with this treatment.

Analgesic effects of dietary caloric restriction in adult mice.

Nociception was studied in male mice, mostly of the C57BL/6 strain, during continuous or prolonged restriction of caloric intake (60% of ad-libitum) from midlife to senescence (up to 105 weeks). Restricted mice showed fewer licking or biting responses 20-60 min after hind paw injection of 5% formalin at 46 and 70 weeks, but not at 93 weeks. Also, they showed longer response latencies around 46 weeks of age in the 52 degrees C hot-plate test, which partial tail amputation failed to affect, although it did produce at least 2 weeks of chronic neuropathic hypersensitivity in ad libitum controls. Injection of collagen subcutaneously at 36-42 weeks led to chronic hyperalgesia in the DBA/1 but not the C57BL/6 strain, measured weekly by the barely nociceptive 50 degrees C hot-plate test to minimize damage. This collagen-induced arthritic hyperalgesia was then gradually and reversibly blocked during 9-15 weeks of caloric restriction starting at 53-58 weeks. In longitudinal trials on normal mice, performed every 2-4 weeks between 42 and 105 weeks with the 50 degrees C hot-plate, caloric restriction led to altered latencies (higher relative to controls) only in the last 10-20 weeks, perhaps because it delayed the onset of age-related peripheral neuropathies. In conclusion, long-term caloric restriction leads to significant hypoalgesia in pre-senescent mice subjected to above-threshold pain of widely different durations, the effect disappearing at later ages unless spontaneous neuropathies become influential. A reduction in cumulative food intake thus appears to generate antinociceptive signals in adult male mice, perhaps serving specifically to promote riskier behavior during prolonged food shortages.

Detection of abnormal cerebral excitability by coincident stimulation and recording.

OBJECTIVE: A method for mapping brain excitability and detecting abnormalities, by concurrently stimulating and recording 'focal' compound responses through one microelectrode, was evaluated in three rat epilepsy models in comparison with distal stimulation of perforant path afferents. METHODS: A fixed trajectory from neocortex to dentate gyrus was mapped under halothane anesthesia. Several weeks earlier, tetanus toxin or vehicle was microinjected into the dentate polymorphic layer, or else rats were genetically epilepsy-prone (GEPR-9) or epilepsy-resistant (GERR-0). Other (unmapped) rats received acute penicillin microinjections within the dentate granular layer. RESULTS: Focal responses, although widespread, proved largest in the dentate (>+/-0.5 mV). Tetanus toxin diminished focal responses near the microinjection site versus vehicle-microinjected (66%) or contralateral controls (55%), but enhanced them elsewhere in the dentate. It enhanced distal responses at all hippocampal locations. Focal but not distal responses were higher in GEPR-9 than in GERR-0 rats at widespread forebrain locations (mean 233%). Penicillin facilitated both focal and distal dentate responses, but the focal facilitation peaked sooner (about 75 versus 180 min). CONCLUSIONS: Focal responses better uncover pervasive or discrete excitability differences. SIGNIFICANCE: Focal mapping may aid in diagnostic imaging and intraoperative targeting, offering high resolution, rapid performance, low stimulus currents and minimal invasion.

Hindbrain raphe stimulation boosts cyclic adenosine monophosphate and signaling proteins in the injured spinal cord.

Early recovery from incomplete spinal cord contusion is improved by prolonged stimulation of the hindbrain's serotonergic nucleus raphe magnus (NRM). Here we examine whether increases in cyclic adenosine monophosphate (cAMP), an intracellular signaling molecule with several known restorative actions on damaged neural tissue, could play a role. Subsequent changes in cAMP-dependent phosphorylation of protein kinase A (PKA) and PKA-dependent phosphorylation of the transcription factor "cAMP response element-binding protein" (CREB) are also analyzed. Rats with moderate weight-drop injury at segment T8 received 2h of NRM stimulation beginning three days after injury, followed immediately by separate extraction of cervical, thoracic and lumbar spinal cord for immunochemical assay. Controls lacked injury, stimulation or both. Injury reduced cAMP levels to under half of normal in all three spinal regions. NRM stimulation completely restored these levels, while producing no significant change in non-injured rats. Pretreatment with the 5-HT7 receptor antagonist pimozide (1 mg/kg, intraperitoneal) lowered cAMP in non-injured rats to injury amounts, which were unchanged by NRM stimulation. The phosphorylated fraction of PKA (pPKA) and CREB (pCREB) was reduced significantly in all three regions after SCI and restored by NRM stimulation, except for pCREB in lumbar segments. In conclusion, SCI produces spreading deficits in cAMP, pPKA and pCREB that are reversible by Gs protein-coupled 5-HT receptors responding to raphe-spinal activity, although these signaling molecules are not reactive to NRM stimulation in normal tissue. These findings can partly explain the benefits of NRM stimulation after SCI.

A long-lasting wireless stimulator for small mammals.

The chronic effects of electrical stimulation in unrestrained awake rodents are best studied with a wireless neural stimulator that can operate unsupervised for several weeks or more. A robust, inexpensive, easily built, cranially implantable stimulator was developed to explore the restorative effects of brainstem stimulation after neurotrauma. Its connectorless electrodes directly protrude from a cuboid epoxy capsule containing all circuitry and power sources. This physical arrangement prevents fluid leaks or wire breakage and also simplifies and speeds implantation. Constant-current pulses of high compliance (34 volts) are delivered from a step-up voltage regulator under microprocessor control. A slowly pulsed magnetic field controls activation state and stimulation parameters. Program status is signaled to a remote reader by interval-modulated infrared pulses. Capsule size is limited by the two batteries. Silver oxide batteries rated at 8 mA-h were used routinely in 8 mm wide, 15 mm long and 4 mm high capsules. Devices of smaller contact area (5 by 12 mm) but taller (6 mm) were created for mice. Microstimulation of the rat's raphe nuclei with intermittent 5-min (50% duty cycle) trains of 30 µA, 1 ms pulses at 8 or 24 Hz frequency during 12 daylight hours lasted 21.1 days ±0.8 (mean ± standard error, Kaplan-Meir censored estimate, n = 128). Extended lifetimes (>6 weeks, no failures, n = 16) were achieved with larger batteries (44 mA-h) in longer (18 mm), taller (6 mm) capsules. The circuit and electrode design are versatile; simple modifications allowed durable constant-voltage stimulation of the rat's sciatic nerve through a cylindrical cathode from a subcutaneous pelvic capsule. Devices with these general features can address in small mammals many of the biological and technical questions arising neurosurgically with prolonged peripheral or deep brain stimulation.

Midbrain raphe stimulation improves behavioral and anatomical recovery from fluid-percussion brain injury.

The midbrain median raphe (MR) and dorsal raphe (DR) nuclei were tested for their capacity to regulate recovery from traumatic brain injury (TBI). An implanted, wireless self-powered stimulator delivered intermittent 8-Hz pulse trains for 7 days to the rat's MR or DR, beginning 4-6 h after a moderate parasagittal (right) fluid-percussion injury. MR stimulation was also examined with a higher frequency (24 Hz) or a delayed start (7 days after injury). Controls had sham injuries, inactive stimulators, or both. The stimulation caused no apparent acute responses or adverse long-term changes. In water-maze trials conducted 5 weeks post-injury, early 8-Hz MR and DR stimulation restored the rate of acquisition of reference memory for a hidden platform of fixed location. Short-term spatial working memory, for a variably located hidden platform, was restored only by early 8-Hz MR stimulation. All stimulation protocols reversed injury-induced asymmetry of spontaneous forelimb reaching movements tested 6 weeks post-injury. Post-mortem histological measurement at 8 weeks post-injury revealed volume losses in parietal-occipital cortex and decussating white matter (corpus callosum plus external capsule), but not hippocampus. The cortical losses were significantly reversed by early 8-Hz MR and DR stimulation, the white matter losses by all forms of MR stimulation. The generally most effective protocol, 8-Hz MR stimulation, was tested 3 days post-injury for its acute effect on forebrain cyclic adenosine monophosphate (cAMP), a key trophic signaling molecule. This procedure reversed injury-induced declines of cAMP levels in both cortex and hippocampus. In conclusion, midbrain raphe nuclei can enduringly enhance recovery from early disseminated TBI, possibly in part through increased signaling by cAMP in efferent targets. A neurosurgical treatment for TBI using interim electrical stimulation in raphe repair centers is suggested.

Promotion of recovery from thoracic spinal cord contusion in rats by stimulation of medullary raphe or its midbrain input.

BACKGROUND: . Brainstem regions with descending axons could influence recovery after spinal cord injury, thus presenting potential targets for treatment. Neurons in the hindbrain raphe magnus respond to sensory and chemical concomitants of trauma (eg, pain, circulating cytokines) and release trophic substances (serotonin, neuropeptides) in widespread spinal regions. OBJECTIVES: . The authors tested in rats whether intermittent stimulation of the raphe magnus or its major midbrain input, the periaqueductal gray, influences recovery from incomplete thoracic (T8) injury. METHODS: . After a moderate weight-drop injury, an implanted wireless stimulator intermittently delivered cathodal pulses (8 Hz, 30 µA) through a microelectrode for 12 hours daily over multiple days. Stimulators in controls were inactive or not implanted. RESULTS: . Raphe magnus stimulation, started 1 to 2 hours after injury, enduringly improved open-field motor performance (measured weekly for 8 weeks) and footprint and gridwalk performance (measured in the 9th week). These improvements increased with days of stimulation (1-7). Myelinated axons in perilesional white matter and serotonin-containing terminals in gray matter, quantified 14 weeks postinjury, also increased. In separate parametric studies that examined open-field behavior following 14 days of stimulation, starting 2 days after injury was better than 7 days or 2 hours; starting at 8 weeks appeared ineffective. The periaqueductal gray, an established, safe stimulation target in man, similarly facilitated recovery of motor performance and myelination (but not serotonergic terminals) when stimulated for 4 to 7 days. CONCLUSIONS: . Raphe magnus neurons mediate restorative feedback in acute spinal cord injury. Their interim activation, direct or indirect (via periaqueductal gray), offers a basis for possible treatments in patients.

Intraspinal transplantation of GABAergic neural progenitors attenuates neuropathic pain in rats: a pharmacologic and neurophysiological evaluation.

Dysfunctional γ-aminobutyric acid (GABA)-ergic inhibitory neurotransmission is hypothesized to underlie chronic neuropathic pain. Intraspinal transplantation of GABAergic neural progenitor cells (NPCs) may reduce neuropathic pain by restoring dorsal horn inhibition. Rat NPCs pre-differentiated to a GABAergic phenotype were transplanted into the dorsal horn of rats with unilateral chronic constriction injury (CCI) of the sciatic nerve. GABA signaling in antinociceptive effects of NPC grafts was tested with the GABA(A) receptor antagonist bicuculline (BIC), GABA(B) receptor antagonist CGP35348 (CGP) and GABA reuptake inhibitor SKF 89976A (SKF). NPC-treated animals showed decreased hyperalgesia and allodynia 1-3week post-transplantation; vehicle-injected CCI rats continued displaying pain behaviors. Intrathecal application of BIC or CGP attenuated the antinociceptive effects of the NPC transplants while SKF injection induced analgesia in control rats. Electrophysiological recordings in NPC treated rats showed reduced responses of wide dynamic range (WDR) neurons to peripheral stimulation compared to controls. A spinal application of BIC or CGP increased wind-up response and post-discharges of WDR neurons in NPC treated animals. Results suggest that transplantation of GABAergic NPCs attenuate pain behaviors and reduce exaggerated dorsal horn neuronal firing induced by CCI. The effects of GABA receptor inhibitors suggest participation of continuously released GABA in the grafted animals.

Electrical activity suppresses axon growth through Ca(v)1.2 channels in adult primary sensory neurons.

BACKGROUND: Primary sensory neurons of the dorsal root ganglia (DRG) regenerate their spinal cord axon if the peripheral nerve axon has previously been cut. This conditioning lesion confers axon growth competence to the neurons. However, the signal that is sensed by the cell upon peripheral lesion to initiate the regenerative response remains elusive. RESULTS: We show here that loss of electrical activity following peripheral deafferentiation is an important signal to trigger axon regrowth. We first verified that firing in sensory fibers, as recorded from dorsal roots in vivo, declined after peripheral lesioning but was not altered after central lesioning. We found that electrical activity strongly inhibited axon outgrowth in cultured adult sensory neurons. The inhibitory effect depended on the L-type voltage-gated Ca(2+) channel current and involved transcriptional changes. After a peripheral lesion, the L-type current was consistently diminished and the L-type pore-forming subunit, Ca(v)1.2, was downregulated. Genetic ablation of Ca(v)1.2 in the nervous system caused an increase in axon outgrowth from dissociated DRG neurons and enhanced peripheral nerve regeneration in vivo. CONCLUSIONS: Our data indicate that cessation of electrical activity after peripheral lesion contributes to the regenerative response observed upon conditioning and might be necessary to promote regeneration after central nervous system injury.

Restorative effects of stimulating medullary raphe after spinal cord injury.

Serotonin in the spinal cord acutely modulates nociceptive transmission and motor reflexes and may also assist functional restoration after spinal cord injury (SCI). It is released there mainly by descending axons of the medulla's nucleus raphe magnus (NRM). We examined whether mechanical allodynia (cutaneous hypersensitivity) after incomplete SCI is sustainably reversed by prolonged, intermittent electrical stimulation of the NRM and whether altered NRM activity accounts for the allodynia. NRM stimulation was given to rats over several days (average 3.2), beginning about 1 hour after moderate thoracic contusion injury. This stimulation reduced mechanical allodynia in forepaws but not hindpaws at 6 weeks after injury (vs nonstimulated controls). Histologically, the stimulation augmented white matter and reduced astrocytosis (glial fibrillary acidic protein immunostaining) in injured segments at 15 weeks. Cavity volume and perilesion neuron numbers were unchanged. Single-cell extracellular recording 12 to 14 weeks after thoracic contusion injury revealed generally higher spontaneous firing and weaker responses to above-injury noxious stimulation in both inhibited and excited NRM neurons; unresponsive neurons were fewer. Neurons inhibited from dermatomes above the injury were excited from below. Altered NRM activity is unlikely to cause SCI allodynia, since inhibited and excited classes are believed to oppositely modulate nociception. Prolonged, early NRM stimulation probably reverses above-injury allodynia by facilitating qualitative recovery of remaining tissue.