A Rapid Protocol for Intraoperative Assessment of Peripheral Nerve Myelinated Axon Count and Its Application to Cross-Facial Nerve Grafting.

BACKGROUND: Donor nerve myelinated axon counts correlate with functional outcomes in reanimation procedures; however, there exists no reliable means for their intraoperative quantification. In this article, the authors report a novel protocol for rapid quantification of myelinated axons from frozen sections, and demonstrate its applicability to surgical practice. METHODS: The impact of various fixation and FluoroMyelin Red staining strategies on resolved myelin sheath morphology from cryosections of rat and rabbit femoral and sciatic nerves was assessed. A protocol comprising fresh cryosection and rapid staining was developed, and histomorphometric results were compared against conventional osmium-postfixed, resin-embedded, toluidine blue-stained sections of rat sciatic nerve. The rapid protocol was applied for intraoperative quantification of donor nerve myelinated axon count in a cross-facial nerve grafting procedure. RESULTS: Resolution of myelinated axon morphology suitable for counting was realized within 10 minutes of tissue harvest. Although mean myelinated axon diameter appeared larger using the rapid fresh-frozen as compared to conventional nerve processing techniques (mean ± SD; rapid, 9.25 ± 0.62 µm; conventional, 6.05 ± 0.71 µm; p < 0.001), no difference in axon counts was observed on high-power fields (rapid, 429.42 ± 49.32; conventional, 460.32 ± 69.96; p = 0.277). Whole nerve myelinated axon counts using the rapid protocol herein (8435.12 ± 1329.72) were similar to prior reports using conventional osmium processing of rat sciatic nerve. CONCLUSIONS: A rapid protocol for quantification of myelinated axon counts from peripheral nerves using widely available equipment and techniques has been described, rendering possible intraoperative assessment of donor nerve suitability for reanimation.

Glial cell line-derived neurotrophic factor promotes increased phenotypic marker expression in femoral sensory and motor-derived Schwann cell cultures.

Schwann cells (SCs) secrete growth factors and extracellular matrix molecules that promote neuronal survival and help guide axons during regeneration. Transplantation of SCs is a promising strategy for enhancing peripheral nerve regeneration. However, we and others have shown that after long-term in vitro expansion, SCs revert to a de-differentiated state similar to the phenotype observed after injury. In vivo, glial cell-line derived neurotrophic factor (GDNF) may guide the differentiation of SCs to remyelinate regenerating axons. Therefore, we hypothesized that exogenous GDNF may guide the differentiation of SCs into their native phenotypes in vitro through stimulation of GDNF family receptor (GFR)α-1. When activated in SCs, GFRα-1 promotes phosphorylation of Fyn, a Src family tyrosine kinase responsible for mediating downstream signaling for differentiation and proliferation. In this study, SCs harvested from the sensory and motor branches of rat femoral nerve were expanded in vitro and then cultured with 50 or 100ng/mL of GDNF. The exogenous GDNF promoted differentiation of sensory and motor-derived SCs back to their native phenotypes, as demonstrated by decreased proliferation after 7days and increased expression of S100Βß and phenotype-specific markers. Furthermore, inhibiting Fyn with Src family kinase inhibitors, PP2 and SU6656, and siRNA-mediated knockdown of Fyn reduced GDNF-stimulated differentiation of sensory and motor-derived SCs. These results demonstrate that activating Fyn is necessary for GDNF-stimulated differentiation of femoral nerve-derived SCs into their native phenotypes in vitro. Therefore GDNF could be incorporated into SC-based therapies to promote differentiation of SCs into their native phenotype to improve functional nerve regeneration.

Schwann cells seeded in acellular nerve grafts improve functional recovery.

INTRODUCTION: This study evaluated whether Schwann cells (SCs) from different nerve sources transplanted into cold-preserved acellular nerve grafts (CP-ANGs) would improve functional regeneration compared with nerve isografts. METHODS: SCs isolated and expanded from motor and sensory branches of rat femoral and sciatic nerves were seeded into 14mm CP-ANGs. Growth factor expression, axonal regeneration, and functional recovery were evaluated in a 14-mm rat sciatic injury model and compared with isografts. RESULTS: At 14 days, motor or sensory-derived SCs increased expression of growth factors in CP-ANGs versus isografts. After 42 days, histomorphometric analysis found CP-ANGs with SCs and isografts had similar numbers of regenerating nerve fibers. At 84 days, muscle force generation was similar for CP-ANGs with SCs and isografts. SC source did not affect nerve fiber counts or muscle force generation. CONCLUSIONS: SCs transplanted into CP-ANGs increase functional regeneration to isograft levels; however SC nerve source did not have an effect.

PlexinA4 distribution in the adult rat spinal cord and dorsal root ganglia.

PlexinsA1-A4 participate in class 3 semaphorin signaling as co-receptors to neuropilin 1 and 2, PlexinA4 being the latest member of the PlexinA subfamily to be identified. Little is known about the cellular distribution of PlexinA4 in the spinal cord and dorsal root ganglion (DRG). Here, immunohistochemical studies using antibodies to PlexinA4 revealed immunolabeling in neurons in both dorsal and, to a greater extent, ventral horns of the spinal cord. Ventral horn PlexinA4 positive neurons exhibited morphology, size, and location consistent with both motor neurons and interneurons. Labeling was found in motor axons exiting through the ventral roots, and more widespread labeling was observed in ascending and descending white matter tracts. Within the DRG, immunostaining was observed in neuronal cell bodies as well as the central and peripheral processes of these cells. PlexinA4 is expressed in the peripheral nervous system where its expression is regulated upon nerve injury. This is the first detailed description of the cellular and subcellular distribution of PlexinA4 in the adult spinal cord and DRG, and it will set the basis for future studies on the potential role of PlexinA4 in regeneration and repair of the adult central and peripheral nervous system.

Differential gene expression in motor and sensory Schwann cells in the rat femoral nerve.

Phenotypic differences in Schwann cells (SCs) may help to guide axonal regeneration down motor or sensory specific pathways following peripheral nerve injury. The goal of this study was to identify phenotypic markers for SCs harvested from the cutaneous (sensory) and quadriceps (motor) branches of the rat femoral nerve and to study the effects of expansion culture on the expression patterns of these motor or sensory phenotypic markers. RNA was extracted from SCs harvested from the motor and sensory branches of the rat femoral nerve and analyzed using Affymetrix Gene Chips (Rat Genome 230 v2.0 Array A). Genes that were upregulated in motor SCs compared with the sensory SCs or vice versa were identified, and the results were verified for a subset of genes using quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of the "phenotype-specific" genes were then evaluated in SC expansion cultures at various time points over 30 days by qRT-PCR to determine the effect of expansion on SC phenotype. Expression levels of the phenotype-specific genes were significantly altered after expansion culture for both the motor and the sensory markers compared with fresh nerve tissue. These results indicate that both motor and sensory SC gene expression patterns are disrupted during expansion in vitro and may affect the ability of SCs to express phenotype-specific genes after transplantation.

Motoneuron survival after chronic and sequential peripheral nerve injuries in the rat.

OBJECT: Surgical repair of peripheral nerves following chronic nerve injury is associated with poor axonal regeneration and outcome. An underlying possibility is that chronic injuries may increase motoneuron cell death. The hypothesis that substantial motoneuron death follows chronic and sequential nerve injuries was tested in adult rats in this study. METHODS: Thirty adult male Lewis rats underwent bilateral multistage surgeries. At initial surgery, Fast Blue (FB) tracer was injected at a nerve-crush injury site in the right control femoral motor nerve. The left femoral motor nerve was transected at the same level and either capped to prevent regeneration (Group 1), or repaired to allow axonal regeneration and reinnervation of the target quadriceps muscle (Group 2) (15 rats in each group). After 8 weeks in 6 rats/group, the left femoral nerve was cut and exposed to FB just proximal to prior nerve capping or repair and the rats were evaluated for FB-labeled motoneuron counts bilaterally in the spinal cord (this was considered survival after initial injury). In the remaining 9 animals/group, the left nerve was recut (sequential injury), exposed to FB, and repaired to a fresh distal saphenous nerve stump to permit axonal regeneration. Following another 6 weeks, Fluoro-Gold, a second retrograde tracer, was applied to the cut distal saphenous nerve. This allowed us to evaluate the number of motoneurons that survived (maintained FB labeling) and the number of motoneurons that survived but that also regenerated axons (double labeled with FB and Fluoro-Gold). RESULTS: A mean number of 350 and 392 FB-labeled motoneurons were found after 8 weeks of nerve injury on the right and the left sides, respectively. This indicated no significant cell death due to initial nerve injury alone. A similar number (mean 390) of motoneurons were counted at final end point at 14 weeks, indicating no significant cell death after sequential and chronic nerve injury. However, only 50% (mean 180) of the surviving motoneurons were double labeled, indicating that only half of the population regenerated their axons. CONCLUSIONS: The hypothesis that significant motoneuron cell death occurs after chronic and or sequential nerve injury was rejected. Despite cell survival, only 50% of motoneurons are capable of exhibiting a regenerative response, consistent with our previous findings of reduced regeneration after chronic axotomy.

Conduction failures in rabbit saphenous nerve unmyelinated fibers.

Recent experimental and theoretical data indicate that the functional capabilities of axons with specialized structures are much more diverse than traditionally thought. However, few observations were concerned with the main axons without arborization. In the present study, electrical stimulation of the saphenous nerve at different frequencies (2, 5, 10, 20 Hz) was used to test the role of activity-dependent effects on the pattern of action potentials that propagate along individual unmyelinated fibers (C fibers) within the trunk of the saphenous nerve in rabbits. Three basic types of C fiber responses to repetitive stimulation were observed: type-1 fibers showed an entrained response without conduction failure; type-2 fibers discharged with intermittent conduction failures; while only sporadic conduction failures happened in type 3. The failure modality in type-2 and type-3 fibers is closely related to the conductive distance as well as the frequency and duration of stimuli which lead to a critical level of conduction velocity slowing. A novel fluctuation in interspike intervals was always observed immediately before the occurrence of the failures, implying that the fluctuation of conduction velocity is correlated with imminent failures. Both the 4-aminopyridine-sensitive potassium current and hyperpolarization-activated cation current were recognized to be involved in the regulation of conduction failure patterns. The results confirmed, at least in part, the existence of conduction failures in the main axon of C fibers, suggesting that axonal operations may also be determinants for adaptation phenomenon and information processing in peripheral nervous system.

Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression.

Brief electrical stimulation enhances the regenerative ability of axotomized motor [Nix, W.A., Hopf, H.C., 1983. Electrical stimulation of regenerating nerve and its effect on motor recovery. Brain Res. 272, 21-25; Al-Majed, A.A., Neumann, C.M., Brushart, T.M., Gordon, T., 2000. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J. Neurosci. 20, 2602-2608] and sensory [Brushart, T.M., Jari, R., Verge, V., Rohde, C., Gordon, T., 2005. Electrical stimulation restores the specificity of sensory axon regeneration. Exp. Neurol. 194, 221-229] neurons. Here we examined the parameter of duration of stimulation on regenerative capacity, including the intrinsic growth programs, of sensory neurons. The effect of 20 Hz continuous electrical stimulation on the number of DRG sensory neurons that regenerate their axons was evaluated following transection and surgical repair of the femoral nerve trunk. Stimulation was applied proximal to the repair site for 1 h, 3 h, 1 day, 7 days or 14 days at the time of nerve repair. Following a 21-day regeneration period, DRG neurons that regenerated axons into the muscle and cutaneous sensory nerve branches were retrogradely identified. Stimulation of 1 h led to a significant increase in DRG neurons regenerating into cutaneous and muscle branches when compared to 0 h (sham) stimulation or longer periods of stimulation. Stimulation for 1 h also significantly increased the numbers of neurons that regenerated axons beyond the repair site 4 days after lesion and was correlated with a significant increase in expression of growth-associated protein 43 (GAP-43) mRNA in the regenerating neurons at 2 days post-repair. An additional indicator of heightened plasticity following 1 h stimulation was elevated expression of brain-derived neurotrophic factor (BDNF). The effect of brief stimulation on enhancing sensory and motoneuron regeneration holds promise for inducing improved peripheral nerve repair in the clinical setting.

BDNF/TrkB signaling regulates HNK-1 carbohydrate expression in regenerating motor nerves and promotes functional recovery after peripheral nerve repair.

Functional recovery after peripheral nerve injury is often poor despite high regenerative capacity of peripheral neurons. In search for novel treatments, brief electrical stimulation of the acutely lesioned nerve has recently been identified as a clinically feasible approach increasing precision of axonal regrowth. The effects of this stimulation appear to be mediated by BDNF and its receptor, TrkB, but the down-stream effectors are unknown. A potential candidate is the HNK-1 carbohydrate known to be selectively reexpressed in motor but not sensory nerve branches of the mouse femoral nerve and to enhance growth of motor but not sensory axons in vitro. Here, we show that short-term low-frequency electrical stimulation (1 h, 20 Hz) of the lesioned and surgically repaired femoral nerve in wild-type mice causes a motor nerve-specific enhancement of HNK-1 expression correlating with previously reported acceleration of muscle reinnervation. Such enhanced HNK-1 expression was not observed after electrical stimulation in heterozygous BDNF or TrkB-deficient mice. Accordingly, the degree of proper reinnervation of the quadriceps muscle, as indicated by retrograde labeling of motoneurons, was reduced in TrkB+/- mice compared to wild-type littermates. Also, recovery of quadriceps muscle function, evaluated by a novel single-frame motion analysis approach, and axonal regrowth into the distal nerve stump, assessed morphologically, were considerably delayed in TrkB+/- mice. These findings indicate that BDNF/TrkB signaling is important for functional recovery after nerve repair and suggest that up-regulation of the HNK-1 glycan is linked to this phenomenon.

Specific expression of an HNK-1 carbohydrate epitope and NCAM on femoral nerve Schwann cells in mice.

Schwann cells are glial cells of the peripheral nervous system. There are two known subtypes of Schwann cells: those that are myelin-forming; and those that are non-myelin-forming. In this study, we looked at the expression of cell adhesion molecules in Schwann cells to determine whether other subtypes might exist. We used immunohistological analysis of femoral nerve segments containing sensory and motor fascicles, stained with anti-HNK-1, M6749 and anti-neural cell adhesion molecule (NCAM) monoclonal antibodies. Anti-HNK-1 and M6749 were positive in the motor fascicle, while anti-NCAM was positive in the sensory fascicle. Immunoblot analysis with the anti-HNK-1 and M6749 antibodies showed stronger immunoreactivity in the motor fraction than in the sensory fraction in the 100 kDa band. With the anti-NCAM antibody, the 140 and 120 kDa bands were seen in the sensory fascicle fraction, but not in the motor fascicle fraction. HNK-1-positive-cells were seen in motor fascicles 7 days after transection. However, the level of immunoreactivity diminished at 14 days, and no immunoreactivity was seen at 21 days. NCAM-positive cells were not observed 3 days after transection. In development, HNK-1-positive-cells and NCAM-positive cells were seen after P-21. These results suggest that the Schwann cells from the motor and the sensory fascicles have different subtypes. The motor and sensory Schwann cells may play different roles and function in a different way during peripheral nerve regeneration. In addition, there could be more stages of Schwann cell differentiation than previously thought; it is possible that myelin-forming Schwann cells differentiate into HNK-1-positive-cells (motor myelin-forming Schwann cells) and HNK-1-negative-cells (sensory myelin-forming Schwann cells), and non-myelin-forming Schwann cells differentiate into NCAM-positive cells (sensory non-myelin-forming Schwann cells) and NCAM-negative cells (autonomic non-myelin-forming Schwann cells).

Manipulations of the mouse femoral nerve influence the accuracy of pathway reinnervation by motor neurons.

Previous studies using the femoral nerve model in both mice and rats have shown that regenerating motor axons prefer to reinnervate the terminal nerve branch to muscle versus a terminal nerve branch to skin, a process that has been termed preferential motor reinnervation (PMR). If end organ contact with muscle and skin is prevented, this preferential motor reinnervation still occurs in the rat. To better understand the process of preferential motor reinnervation in the mouse, we examined motor neuron reinnervation of muscle and cutaneous pathways without any end organ contact as well as with only cutaneous end organ contact. Surprisingly, there was no preferential motor reinnervation: Motor neurons preferred the cutaneous pathway over the muscle pathway when all end organ contact was prevented and showed an even greater preference for the cutaneous pathway when it was attached to skin.

Chronic Schwann cell denervation and the presence of a sensory nerve reduce motor axonal regeneration.

Motor axonal regeneration is compromised by chronic distal nerve stump denervation, induced by delayed repair or prolonged regeneration distance, suggesting that the pathway for regeneration is progressively impaired with time and/or distance. In the present experiments, we tested the impacts of (i) chronic distal sensory nerve stump denervation on axonal regeneration and (ii) sensory or motor innervation of a nerve graft on the ability of motoneurons to regenerate their axons from the opposite end of the graft. Using the motor and sensory branches of rat femoral nerve and application of neuroanatomical tracers, we evaluated the numbers of regenerated femoral motoneurons and nerve fibers when motoneurons regenerated (i) into freshly cut and 2-month chronically denervated distal sensory nerve stump, (ii) alone into a 4-cm-long distally ligated sensory autograft (MGL) and, (iii) concurrently as sensory (MGS) or motor (MGM) nerves regenerated into the same autograft from the opposite end. We found that all (315 +/- 24: mean +/- SE) the femoral motoneurons regenerated into a freshly cut distal sensory nerve stump as compared to 254 +/- 20 after 2 months of chronic denervation. Under the MGL condition, 151 +/- 5 motoneurons regenerated, which was not significantly different from the MGM group (134 +/- 13) but was significantly reduced to 99 +/- 2 in the MGS group (P < 0.05). The number of regenerated nerve fibers was 1522 +/- 81 in the MGL group, 888 +/- 18 in the MGM group, and 516 +/- 44 in the MGS group, although the high number of nerve fibers in the MGL group was due partly to the elaboration of multiple sprouts. Nerve fiber number and myelination were reduced in the MGS group and increased in the MGM group. These results demonstrate that both chronic denervation and the presence of sensory nerve axons reduced desired motor axonal regeneration into sensory pathways. A common mechanism may involve reduced responsiveness of sensory Schwann cells within the nerve graft or chronically denervated distal nerve stump to regenerating motor axons. The findings confirm that motor regeneration is optimized by avoiding even short-term denervation. They also imply that repairing pure motor nerves (without their cutaneous sensory components) to distal nerve stumps should be considered clinically when motor recovery is the main desired outcome.

Electrical stimulation accelerates and increases expression of BDNF and trkB mRNA in regenerating rat femoral motoneurons.

Electrical stimulation promotes the speed and accuracy of motor axonal regeneration. The positive effects of stimulation are mediated at the cell body. Here we characterize the effect of electrical stimulation on motoneuronal expression of BDNF and its receptor, trkB, two genes whose expression levels in motoneurons correlate with regeneration and are regulated by electrical activity in a variety of neurons. We used semiquantitative in situ hybridization to measure expression of mRNA encoding BDNF and the full-length trkB receptor at intervals of 8 h, 2 days and 7 days after unilateral femoral nerve cut, suture, and stimulation. Expression in regenerating motoneurons was compared to that of contralateral intact motoneurons. BDNF and trkB signals were not significantly upregulated 8 h and 2 days after femoral nerve suture and sham stimulation. By 7 days, there was a 2-fold increase in both BDNF and trkB mRNA expression. In contrast, stimulation of cut and repaired nerves for only 1 h led to rapid upregulation of BDNF and trkB mRNA by 3-fold and 2-fold, respectively, within the first 8 h. The stimulation effect peaked at 2 days with 6-fold and 4-fold increases in the signals, respectively. Thereafter, the levels of BDNF and trkB mRNA expression declined to equal the 2-fold increase seen at 7 days after nerve repair and sham-stimulation. We conclude that brief electrical stimulation stimulates BDNF and trkB expression in regenerating motoneurons. Because electrical stimulation is known to accelerate axonal regeneration, we suggest that changes in the expression of BDNF and trkB correlate with acceleration of axonal regeneration.

Motor nerve topology reflects myocyte morphology in hamster retractor and epitrochlearis muscles.

Neuromuscular activation is a primary determinant of metabolic demand and oxygen transport. The m. retractor and m. epitrochlearis are model systems for studying metabolic control and oxygen transport; however, the organization of muscle fibers and motor nerves in these muscles is unknown. We tested whether the topology of motor innervation was related to the morphology of muscle fibers in m. retractor and m. epitrochlearis of male hamsters ( approximately 100 g). Respective muscles averaged 47 and 12 mm in length 100 and 35 mg in mass. Staining for acetylcholinesterase revealed neuromuscular junctions arranged in clusters throughout m. retractor and as a central band across m. epitrochlearis, suggesting differences in fiber morphology. For both muscles, complete cross-sections contained approximately 1,700 fibers. Fiber cross-sectional areas were distributed nearly normal in m. epitrochlearis (mean = 1,559 +/- 17 microm(2)) and skewed left (P < 0.05) in m. retractor (mean = 973 +/- 15 microm(2)). Single fiber length (Lf) spanned muscle length (Lm) in m. epitrochlearis, while fibers tapered to terminate within m. retractor (Lf/Lm = 0.43 +/- 0. 02). With myelin staining, a single branch of ulnar nerve projected axons across the midregion of m. epitrochlearis. For m. retractor, the spinal accessory nerve branched to give rise to proximal and distal regions of innervation, with intermingling of axons between nerve branches. Nerve bundle cross-sections stained for acetylcholinesterase indicate that each motor axon projects to an average of 65 muscle fibers in m. epitrochlearis and 100 in m. retractor. Differences in fiber morphology, innervation topology, and neuromuscular organization may contribute to the heterogeneity of metabolic demand and oxygen supply in skeletal muscle.

Comparison of heteronymous monosynaptic Ia facilitation in young and elderly subjects in supine and standing positions.

BACKGROUND: The control of posture and balance is a primary concern among the elderly. Postural instability has been identified as a contributor to the greater incidence of falling among this segment of the population. One important neuromuscular mechanism identified as important in the control of posture and balance is the segmental reflex system. The purpose of this study was to examine the role of presynaptic inhibition in modulating the reflex system in young and elderly subjects. METHODS: To estimate the influence of body position on presynaptic inhibition to the soleus motor pool between young and elderly subjects, 11 young (mean age=23.9 yrs.) and 9 elderly (mean age=72.1 yrs.) subjects were examined in two different body positions: supine and standing. This study utilized the heteronymous facilitation protocol, as described by Hultborn et al. (1987), to estimate presynaptic inhibition of the Ia afferent pathway onto the soleus alpha-motoneuron pool. Maximal soleus H-reflex (H-max) and motor response (M-max) amplitudes were determined prior to testing at each condition, and the H-max/M-max ratio at each body position was determined. To estimate presynaptic inhibition at each body position, subjects received 24 test soleus H-reflex stimuli ( approximately 15% M-max), and 24 soleus H-reflexes conditioned by stimulation of the ipsilateral femoral nerve. RESULTS: Results demonstrated a significant decrease in H-max/M-max ratio from supine (66.1%) to standing (56.8%) for the young subjects, whereas the elderly subjects demonstrated no changes in the H-max/M-max ratio between body positions (39.8% supine; 39.8% standing). The conditioning stimulus produced a significant change in the test reflex for the young subjects during supine testing (51.1% increase) but not standing (3.4% increase). The elderly subjects demonstrated no significant changes in the test reflex produced by the heteronymous conditioning at either condition (17.6% increase supine; 4.9% increase standing). CONCLUSIONS: These results demonstrate differential effects of both H-reflex modulation and heteronymous conditioning for elderly subjects when compared with young adults. These differences may be an adaptive phenomenon of the aging neuromuscular system, exemplified by a decreased ability to modulate the reflex system in the elderly group.

On the effect of chemically activated fine muscle afferents on interneurones mediating group I non-reciprocal inhibition of extensor ankle and knee muscles in humans.

In a previous paper it was shown that muscle nociceptive discharge depressed the activity of interneurones mediating group I non-reciprocal inhibition (or Ib interneurones) in humans [A. Rossi, B. Decchi, Changes in Ib heteronymous inhibition to soleus motoneurons during cutaneous and muscle nociceptive stimulation in humans, Brain Res. 774 (1997) 55-61.]. However, since nociceptive discharge depressed the size of the soleus H-reflex (by which Ib inhibition was tested) the question arises as to whether modification of motoneurone membrane conductance per se could depress the size of Ib inhibitory post-synaptic potentials. The results of the present study suggest that the contribution of motoneurone hyperpolarization to Ib disinhibition is negligible and that muscle nociceptive discharge actually depresses the activity of these pathways.

Pharmacologically evoked fictive motor patterns in the acutely spinalized marmoset monkey (Callithrix jacchus).

The existence of a spinal network capable of generating rhythmic alternating activity resembling locomotion still has not been firmly established in primates, including man, although evidence for one is accumulating. The present study investigated whether it is possible to activate such a network by administration of a variety of pharmacological agents to acutely spinalized marmoset monkeys (Callithrix jacchus) in the absence of phasic afferent input to the spinal cord. Fourteen marmoset monkeys were decerebrated, spinalized, and paralyzed. The nerves supplying both hindlimbs were cut and recorded from. In 5 monkeys the effect of electrical stimulation of the brainstem was investigated before spinalization. In 3 of these monkeys, rhythmic activity alternating between extensors and flexor nerves was seen. In the 2 other monkeys only synchronized activity was elicited. In acutely spinalized monkeys, administration of L-3,4-dihydroxyphenylalanine (L-dopa; 3-4 h after treatment with nialamide) failed to evoke any rhythmic alternating activity. In contrast, administration of clonidine elicited alternating activity in all of 8 monkeys tested. In 4 of these monkeys, the activity was restricted to alternation between ipsilateral and contralateral flexor nerves, whereas alternating activity between ipsilateral flexors and extensors was also seen in the other 4 monkeys. Administration of excitatory amino acids (NMDA or NMA) also elicited rhythmic alternating activity in 7 of 10 spinalized monkeys. In 4, rhythmic alternating activity was seen between extensors and flexors on one limb as well as between ipsilateral and contralateral flexors. In 3 monkeys NMDA/NMA produced alternation between extensors and flexors of one limb without alternation between the ipsilateral and contralateral sides. Administration of noradrenaline failed to elicit any rhythmic activity, but rather completely depressed already existing activity. Administration of serotonin (5-HT) was ineffective in facilitating alternating activity in 6 of 8 monkeys and was facilitatory to rhythmic activity in the other 2. We suggest that these data provide further evidence of a network capable of eliciting rhythmic alternating activity resembling locomotion in the primate spinal cord. The network, however, seems to be more difficult to activate pharmacologically in those conditions than in other mammals. This may especially be the case in higher primates, including man.

Sciatic and femoral nerve sensory neurones occupy different regions of the L4 dorsal root ganglion in the adult rat.

The topographical distribution of sciatic and femoral nerve sensory neuronal somata in the L4 dorsal root ganglion of the adult rat was mapped after retrograde tracing with one or two of the dyes Fast Blue, Fluoro-Gold, or Diamidino Yellow. The tracers were applied to the proximal transected end of either nerve alone, or from both nerves in the same animal using separate tracers. Three-dimensional reconstructions of the distribution of labelled neurones were made from serial sections of the L4 dorsal root ganglion which is the only ganglion that these two nerves share. The results showed that with little overlap, femoral nerve neurones distribute dorsally and rostrally whereas sciatic nerve neurones distribute medially and ventrally. This finding indicates the existence of a somatotopical organisation for the representation of different peripheral nerves in dorsal root ganglia of adult animals.

Physiologic studies of spinal inhibitory circuits in patients with stiff-person syndrome.

OBJECTIVE: To test inhibitory spinal circuits in patients with stiff-person syndrome (SPS). BACKGROUND: Patients with SPS have fluctuating muscle stiffness and spasms, and most have antibodies against GABAergic neurons. We predicted they would also have abnormalities of spinal GABAergic circuits. DESIGN/METHODS: Physiologic methods using H-reflexes were used to test reciprocal inhibition in the forearm and thigh, vibration-induced inhibition of flexor carpi radialis and soleus H-reflexes, recurrent inhibition, and nonreciprocal (1b) inhibition of soleus H-reflexes. RESULTS: Vibration-induced inhibition of H-reflexes was diminished in eight of nine patients tested, but the presynaptic period of reciprocal inhibition was normal in most patients. Both circuits are presumed to involve presynaptic inhibition and GABAergic interneurons. Presumed glycinergic circuits, including the first period of reciprocal inhibition and nonreciprocal (1b) inhibition, showed occasional abnormalities. Recurrent inhibition was normal in all five patients tested. CONCLUSION: Differences between the two presumptive GABAergic circuits may indicate that not all populations of GABAergic neurons are uniformly affected in SPS. The involvement of presumptive glycinergic circuits in some patients could point to impairment of nonGABAergic neurons, unrecognized involvement of GABAergic neurons in these inhibitory circuits, or, more likely, alterations of supraspinal systems that exert descending control over spinal circuits.

Organization of hindlimb nerve projections to the rat spinal cord: a choleragenoid horseradish peroxidase study.

The aim of the present study has been to investigate the projections of hindlimb muscle afferent fibers to the spinal cord with particular emphasis on the ventral horn and the column of Clarke. Following transections of the appropriate ventral roots, injections of the B-subunit of cholera toxin conjugated to horseradish peroxidase were made into the tibial, peroneal, hamstring, superior gluteal, femoral, and obturator nerves in one group of adult rats. In another group of rats, similar experiments were done with intact ventral roots in order to map the location in the ventral horn of the motoneuron cell columns supplying each investigated nerve. An extensive overlap was found for the different nerve projections to Rexed's laminae V-VII. A somatotopic organization of the nerve projections was seen in the lamina IX cell groups of the ventral horn as well as in the column of Clarke, even though an overlap existed. The densest primary afferent projection from each injected nerve was to its homonymous motoneurons. Only a small to moderate overlap between the projections of the tributary branches of the sciatic nerve was found in the ventral horn, whereas the obturator and femoral nerve projections showed more profound overlap. In the column of Clarke, hindlimb nerves innervating distal muscles projected medially, and nerves innervating proximal muscles projected laterally.