Boutons containing vesicular zinc define a subpopulation of synapses with low AMPAR content in rat hippocampus

Cortical regions of the brain stand out for their high content in synaptic zinc, which may thus be involved in synaptic function. The relative number, chemical nature and transmitter receptor profile of synapses that sequester vesicular zinc are largely unknown. To address this, we combined pre-embedding zinc histochemistry and post-embedding immunogold electron microscopy in rat hippocampus. All giant mossy fibre (MF) terminals in the CA3 region and approximately 45% of boutons making axospinous synapses in stratum radiatum in CA1 contained synaptic vesicles that stained for zinc. Both types of zinc-positive boutons selectively expressed the vesicular zinc transporter ZnT-3. Zinc-positive boutons further immunoreacted to the vesicular glutamate transporter VGLUT-1, but not to the transmitter gamma-aminobutyric acid. Most dendritic spines in CA1 immunoreacted to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunits GluR1-3 (approximately 80%) and to N-methyl-D-aspartate receptor (NMDAR) subunits NR1 + NR2A/B (approximately 90%). Synapses made by zinc-positive boutons contained 40% less AMPAR particles than those made by zinc-negative boutons, whereas NMDAR counts were similar. Further analysis indicated that this was due to the reduced synaptic expression of both GluR1 and GluR2 subunits. Hence, the levels of postsynaptic AMPARs may vary according to the presence of vesicular zinc in excitatory afferents to CA1. Zinc-positive and zinc-negative synapses may represent two glutamatergic subpopulations with distinct synaptic signalling.     

Spinal axonal injury transiently elevates the level of metabotropic glutamate receptor 5, but not 1, in cord-projection central neurons

In investigating the effect of spinal injury on cord-projection central neurons, we found that rat rubrospinal neurons retained glutamatergic afferents and, in general, ionotropic glutamate receptor expression following spinal axotomy. Since glutamate also acts on second-messenger-coupled metabotropic receptors, the expression of group I metabotropic glutamate receptors, mGluR1 and mGluR5, was examined following similar treatment. mGluR1 expression began to decline in the perikarya 2 days postlesion and a day later in the neuropil. The decline slowed down by the fifth day and recovered in both the perikarya and neuropil 1 week postlesion. However, expression in both the perikarya and neuropil declined again and persisted up to 2 years postlesion. Similarly, the mGluR5 displayed an early transient decrease and returned to normal levels by 7 days post-lesion. However, rather than progressing to a secondary decline, the expression of mGluR5 increased to levels dramatically higher than those of control nuclei at 2-4 weeks postlesion, subsiding again by 8 weeks, and remaining low up to 2 years postinjury. Although mGluR5 has been shown to save cultured neurons from excitotoxic cell death, its elevated expression in the present model corresponds in time to an increased input/output relationship and excitability of the injured neurons as well as a period of maximal somatic shrinkage and cell loss. In addition to the cell bodies and dendrites, axon-like profiles also contain mGluR1. Their decrease following rubrospinal axotomy suggests that axonal injury may also compromise the presynaptic regulation of afferent activities onto injured cord-projection central neurons.     

Painful nerve injury shortens the intracellular Ca2+ signal in axotomized sensory neurons of rats

BACKGROUND: Neuropathic pain is inadequately treated and poorly understood at the cellular level. Because intracellular Ca signaling critically regulates diverse neuronal functions, the authors examined effects of peripheral nerve injury on the Ca transient that follows neuronal activation. METHODS: Cytoplasmic Ca levels were recorded by digital microfluorometry from dissociated dorsal root ganglion neurons of hyperalgesic animals after ligation of the fifth lumbar spinal nerve and control animals. Neurons were activated by field stimulation or by K depolarization. RESULTS: Transients in presumptively nociceptive, small, capsaicin-sensitive neurons were diminished after axotomy, whereas transient amplitude increased in axotomized nonnociceptive neurons. Axotomy diminished the upward shift in resting calcium after transient recovery. In contrast, nociceptive neurons adjacent to axotomy acquired increased duration of the transient and greater baseline shift after K activation. Transients of nonnociceptive neurons adjacent to axotomy showed no changes after injury. In nociceptive neurons from injured rats that did not develop hyperalgesia, transient amplitude and baseline offset were large after axotomy, whereas transient duration in the adjacent neurons was shorter compared with neurons excised from hyperalgesic animals, which show normalization of these features. CONCLUSIONS: A diminished Ca signal in axotomized neurons may be in part due to loss of Ca influx through voltage-gated Ca channels. The upward shift in resting Ca level after activation, which is diminished after axotomy in presumed nociceptive neurons, is a previously unrecognized aspect of neuronal plasticity. These changes in the critical Ca signal may mediate various injury-related abnormalities in Ca-dependent neuronal.     

Propofol inhibits phosphorylation of N-methyl-D-aspartate receptor NR1 subunits in neurons

BACKGROUND: Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-D-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation. METHODS: The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons. RESULTS: Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist-induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits. CONCLUSIONS: Together, these data indicate an inhibitory effect of a general anesthetic propofol on NMDAR NR1 subunit phosphorylation in neurons. This inhibition was mediated through a signaling mechanism involving activation of protein phosphatase 2A.     

Sequence of cellular changes following localized axotomy to cortical neurons in glia-free culture

This study utilizes an in vitro model of localized physical injury to axons to examine the specific responses of neocortical neurons to trauma in isolation from glia cell types. The neuronal response to axotomy was closely linked with nerve cell maturity. Cultures grown for 14 days in vitro showed no accumulation of either neurofilaments or, the axonal sprouting marker, GAP43, within injured axons following injury. In older cultures (21 days in vitro), however, temporally distinct axonal changes were evident following transection of axonal bundles. At 12 h postinjury, these included extensive accumulation of neurofilaments into ring-like structures within the cut stumps and an increase in punctate GAP43 labelling throughout the damaged area. At 24 h postinjury, bulb-like accumulations of neurofilaments were also present within the transected axons. Finally at 3 days postinjury, distinct GAP43 and neurofilament immunolabeled axons, and GAP43 immunopositive growth cones, emanated from the cut stump. These results indicate that injured axons of mature neurons undergo a defined series of reactive changes, ultimately culminating in a sprouting response, which occur independently of the presence or effects of glial cell populations.     

Colocalization of glutamate ionotropic receptor subunits in the human temporal neocortex

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors represent major classes of glutamate receptors (GluR) which play fundamental roles in normal excitatory synaptic activity and, probably, in the etiology of several brain diseases. These receptors are composed of multiple receptor subunit proteins, and the differential expression of these subunits in cortical neurons is considered to be one of the substrates for the functional diversity of cortical excitatory circuitry. In the monkey neocortex, different subpopulations of neurons have been identified on the basis of immunocytochemical colocalization studies using subunit-specific antibodies, but no comparable investigations have been made in the human neocortex. The aim of the present study was to determine quantitatively GluR subunit combinations in the human temporal neocortex by double-labeling immunocyto- chemical experiments. We quantified the neuronal populations expressing different receptor subtypes with fluorescent tags visualizing them with confocal laser microscopy. We studied AMPA, kainate- and NMDA-receptor subunits, using antibodies against GluR1, GluR2, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1 subunits. A high degree of colocalization (93-100%) using combinations of antibodies against GluR2 with GluR2/3, GluR2/3 with GluR2/4, and GluR2 or GluR2/4 with NMDAR1 was found, whereas for other combinations the degree of colocalization varied between 38% and 88%. Some of the percentages reported here are similar to those found in the monkey cortex, whereas others differ considerably. These results emphasize the diversity of excitatory circuits in the human neocortex, and suggest species differences with regard to some of these GluR-mediated circuits.     

Propofol Inhibits Phosphorylation of N-methyl-d-aspartate Receptor NR1 Subunits in Neurons

Background: Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-d-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation.

Methods: The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons.

Results: Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist-induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits.     

Painful Nerve Injury Shortens the Intracellular Ca2+ Signal in Axotomized Sensory Neurons of Rats

Background: Neuropathic pain is inadequately treated and poorly understood at the cellular level. Because intracellular Ca2+ signaling critically regulates diverse neuronal functions, the authors examined effects of peripheral nerve injury on the Ca2+ transient that follows neuronal activation.

Methods: Cytoplasmic Ca2+ levels were recorded by digital microfluorometry from dissociated dorsal root ganglion neurons of hyperalgesic animals after ligation of the fifth lumbar spinal nerve and control animals. Neurons were activated by field stimulation or by K+ depolarization.

Results: Transients in presumptively nociceptive, small, capsaicin-sensitive neurons were diminished after axotomy, whereas transient amplitude increased in axotomized nonnociceptive neurons. Axotomy diminished the upward shift in resting calcium after transient recovery. In contrast, nociceptive neurons adjacent to axotomy acquired increased duration of the transient and greater baseline shift after K+ activation. Transients of nonnociceptive neurons adjacent to axotomy showed no changes after injury. In nociceptive neurons from injured rats that did not develop hyperalgesia, transient amplitude and baseline offset were large after axotomy, whereas transient duration in the adjacent neurons was shorter compared with neurons excised from hyperalgesic animals, which show normalization of these features.     

The effect of three inhaled anesthetics in mice harboring mutations in the GluR6 (kainate) receptor gene

Combinations of GluR5-GluR7, KA1, and KA2 subunits form kainate receptors, a subtype of excitatory ionotropic glutamate receptors. Isoflurane enhances the action of kainate receptors comprising GluR6 subunits expressed in oocytes. To test whether alterations of the GluR6 subunit gene affect the actions of inhaled anesthetics in vivo, we measured the minimum alveolar concentration of desflurane, isoflurane, and halothane in mice lacking the kainate receptor subunit GluR6 (GluR6 knockout mice) and mice with a dominant negative glutamine/arginine (Q/R) editing mutation in membrane domain 2 of the GluR6 receptor (GluR6 editing mutants), which increases the calcium permeability of kainate receptors containing GluR6Q. We also measured the capacity of isoflurane to interfere with Pavlovian fear conditioning to a tone and to context. Absence of the GluR6 subunit did not change the minimum alveolar concentration of isoflurane, desflurane, or halothane. Possibly, kainate receptors assembled from the remaining kainate receptor subunits compensate for the absent subunits and thereby produce a normal minimum alveolar concentration. A Q/R mutation that dominantly affects kainate receptors containing the GluR6 subunit in mice increased isoflurane minimum alveolar concentration (by 12%; P < 0.01), decreased desflurane minimum alveolar concentration (by 18%; P < 0.001), and did not change halothane minimum alveolar concentration (P = 0.25). These data may indicate that kainate receptors containing GluR6Q subunits differently modulate, directly or indirectly, the mechanism by which inhaled anesthetics cause immobility. The mutations of GluR6 that were studied did not affect the capacity of isoflurane to interfere with fear conditioning.     

Fate of the soma and dendrites of cord-projection central neurons after proximal and distal spinal axotomy: an intracellular dye injection study

We used rat rubrospinal neurons as a model to study the soma-dendritic morphology of cord-projection neurons following spinal axonal injury. We examined lumbar-projection neurons following both upper cervical and lower thoracic axotomy to find out whether changes were dependent on the proximity of the lesion to the cell body. Axotomized neurons were marked with retrograde tracer and studied 4 and 8 weeks later with intracellular dye injection technique. Axotomy resulted in prominent shrinkage of their soma and relatively minor reduction of their dendritic spreads. The degree of soma shrinkage depended on both the duration of survival and the proximity of lesion. In addition, dendritic modification peaked 4 weeks following proximal lesion, which was also achieved 8 weeks following distal axotomy. Tractotomy at upper cervical and lower thoracic levels also allowed us to compare the effect of distal axotomy on cervical and lumbar-projection neurons. Results show that although cervical-projection neurons responded more quickly than lumbar-projecting ones, they however showed a similar degree of alteration in both their soma and dendrites 8 weeks following distal axotomy. In summary, cord-projection neurons survived 8 weeks following either upper cervical or lower thoracic axotomy with relatively intact dendritic features. Taken together, our data thus far suggest that cord-projection central neurons continue to integrate inputs and control supraspinal targets following spinal axotomy. The minor dendritic shrinkage within two months of spinal axotomy rejuvenates hopes for functional recovery if regeneration of their spinal axons can be achieved at least within this time frame.     

Selective postsynaptic co-localization of MCT2 with AMPA receptor GluR2/3 subunits at excitatory synapses exhibiting AMPA receptor trafficking

MCT2 is the main neuronal monocarboxylate transporter needed by neurons if they are to use lactate as an additional energy substrate. Previous evidence suggested that some MCT2 could be located in postsynaptic elements of glutamatergic synapses. Using post-embedding electron microscopic immunocytochemistry, it is demonstrated that MCT2 is present at postsynaptic density of asymmetric synapses, in the stratum radiatum of both rat hippocampal CA1 and CA3 regions, as well as at parallel fibre-Purkinje cell synapses in mouse cerebellum. MCT2 levels were significantly lower at mossy fibre synapses on CA3 neurons, and MCT2 was almost absent from symmetric synapses on CA1 pyramidal cells. It could also be demonstrated using quantitative double-labeling immunogold cytochemistry that MCT2 and AMPA receptor GluR2/3 subunits have a similar postsynaptic distribution at asymmetric synapses with high levels expressed within the postsynaptic density. In addition, as for AMPA receptors, a significant proportion of MCT2 is located on vesicular membranes within the postsynaptic spine, forming an intracellular pool available for a putative postsynaptic endo/exocytotic trafficking at these excitatory synapses. Altogether, the data presented provide evidence for MCT2 expression in the postsynaptic density area at specific subsets of glutamatergic synapses, and also suggest that MCT2, like AMPA receptors, could undergo membrane trafficking.     

Mechanical loading modulates glutamate receptor subunit expression in bone

The cellular mechanisms coupling mechanical loading with bone remodeling remain unclear. In the CNS, the excitatory amino acid glutamate (Glu) serves as a potent neurotransmitter exerting its effects via various membrane Glu receptors (GluR). Nerves containing Glu exist close to bone cells expressing functional GluRs. Demonstration of a mechanically sensitive glutamate/aspartate transporter protein and the ability of glutamate to stimulate bone resorption in vitro suggest a role for glutamate linking mechanical load and bone remodeling. We used immunohistochemical techniques to identify the expression of N-methyl-d-aspartate acid (NMDA) and non-NMDA (AMPA or kainate) ionotropic GluR subunits on bone cells in vivo. In bone sections from young adult rats, osteoclasts expressed numerous GluR subunits including AMPA (GluR2/3 and GluR4), kainic acid (GluR567) and NMDA (NMDAR2A, NMDAR2B and NMDAR2C) receptor subtypes. Bone lining cells demonstrated immunoexpression for NMDAR2A, NMDAR2B, NMDAR2C, GluR567, GluR23, GluR2 and GluR4 subunits. Immunoexpression was not evident on osteocytes, chondrocytes or vascular channels. To investigate the effects of mechanical loading on GluR expression, we used a Materials Testing System (MTS) to apply 10 N sinusoidal axial compressive loads percutaneously to the right limbs (radius/ulna, tibia/fibula) of rats. Each limb underwent 300-load cycles/day (cycle rate, 1 Hz) for 4 consecutive days. Contralateral, non-loaded limbs served as controls. Mechanically loaded limbs revealed a load-induced loss of immunoexpression for GluR2/3, GluR4, GluR567 and NMDAR2A on osteoclasts and NMDAR2A, NMDAR2B, GluR2/3 and GluR4 on bone lining cells. Both neonatal rabbit and rat osteoclasts were cultured on bone slices to investigate the effect of the NMDA receptor antagonist, MK801, and the AMPA/kainic acid receptor antagonist, NBQX, on osteoclast resorptive activity in vitro. The inhibition of resorptive function seen suggested that both NMDAR and kainic acid receptor function are required for normal osteoclast function. While the exact role of ionotropic GluRs in skeletal tissue remains unclear, the modulation of GluR subunit expression by mechanical loading lends further support for participation of Glu as a mechanical loading effector. These ionotropic receptors appear to be functionally relevant to normal osteoclast resorptive activity.     

Modulation of AMPA receptor GluR1 subunit phosphorylation in neurons by the intravenous anaesthetic propofol

Background: The ionotropic glutamate receptor is a potential molecular sitein the central nervous system that general anaesthetics mayinteract with to produce some of their biological actions. Proteinphosphorylation has been well documented to occur in the intracellularC-terminal domain of -amino-3-hydroxy-5-methylisoxazole-4-propionicacid (AMPA) subtype of glutamate receptors, which representsa pivotal mechanism for the post-translational modulation ofAMPA receptor functions. In this study, we investigated a possibleinfluence of an i.v. anaesthetic agent propofol on the phosphorylationof AMPA receptor GluR1 subunits in cultured neurons. Methods: The effect of propofol on phosphorylation of GluR1 subunitsat serine 831 and 845 was assayed in cultured rat striatal andcortical neurons by western blot with phospho- and site-specificantibodies. Results: Propofol consistently elevated phosphorylation of GluR1 subunitsat the C-terminal serine 845 site in both striatal and corticalneurons. The elevation in phosphorylation was concentration-dependentand started at a low concentration (3 µM). This increasein serine 845 phosphorylation was rapid and sustained duringthe entire course of propofol exposure. In contrast to serine845, phosphorylation of GluR1 at serine 831 was not alteredby propofol in striatal and cortical neurons. Total GluR1 abundanceremained unchanged in response to propofol incubation. Conclusions: These data indicate that propofol possesses the ability to upregulateAMPA receptor GluR1 subunit phosphorylation at a specific serine845 site in neurons and provide evidence supporting the AMPAreceptor as a molecular target for general anaesthetics.     

Inhibition of NR2B phosphorylation restores alterations in NMDA receptor expression and improves functional recovery following traumatic brain injury in mice

Traumatic brain injury (TBI) triggers a massive glutamate efflux, hyperactivation of N-methyl-D-aspartate receptors (NMDARs) and neuronal cell death. Previously it was demonstrated that, 15 min following experimentally induced closed head injury (CHI), the density of activated NMDARs increases in the hippocampus, and decreases in the cortex at the impact site. Here we show that CHI-induced alterations in activated NMDARs correlate with changes in the expression levels of the major NMDARs subunits. In the hippocampus, the expression of NR1, NR2A, and NR2B subunits as well as the GluR1 subunit of the AMPA receptor (AMPAR) were increased, while in the cortex at the impact site, we found a decrease in the expression of these subunits. We demonstrate that CHI-induced increase in the expression of NMDAR subunits and GluR1 in the hippocampus, but not in the cortex, is associated with an increase in NR2B tyrosine phosphorylation. Furthermore, inhibition of NR2B-phosphorylation by the tyrosine kinase inhibitor PP2 restores the expression of this subunit to its normal levels. Finally, a single injection of PP2, prior to the induction of CHI, resulted in a significant improvement in long-term recovery of motor functions observed in CHI mice. These results provide a new mechanism by which acute trauma contributes to the development of secondary damage and functional deficits in the brain, and suggests a possible role for Src tyrosine kinase inhibitors as preoperative therapy for planned neurosurgical procedures.     

MK-801 upregulates NR2A protein levels and induces functional recovery of the ipsilateral hemidiaphragm following acute C2 hemisection in adult rats

BACKGROUND: C2 hemisection results in paralysis of the ipsilateral hemidiaphragm. Recent data indicate that an upregulation of the N-methyl-D-aspartate (NMDA) receptor 2A subunit following chronic C2 hemisection is associated with spontaneous hemidiaphragmatic recovery following injury. MK-801, an antagonist of the NMDA receptor, upregulates the NR2A subunit in neonatal rats. HYPOTHESIS: We hypothesized that administration of MK-801 to adult, acute C2-hemisected rats would result in an increase of NR2A in the spinal cord. Furthermore, we hypothesized that upregulation of NR2A would be associated with recovery of the ipsilateral hemidiaphragm as in the chronic studies. DESIGN: To develop a dose-response curve, adult rats were treated with varying doses of MK-801 and their spinal cords harvested and assessed for NR2A as well as AMPA GluR1 and GluR2 subunit protein levels. In the second part of this study, C2-hemisected animals received MK-801. Following treatment, the animals were assessed for recovery of the hemidiaphragm through electromyographic recordings and their spinal cords assessed for NR2A, GluR1, and GluR2. RESULTS: Treatment with MK-801 leads to an increase of the NR2A subunit in the spinal cords of adult noninjured rats. There were no changes in the expression of GluR1 and GluR2 in these animals. Administration of MK-801 to C2-hemisected rats resulted in recovery of the ipsilateral hemidiaphragm, an increase of NR2A, and a decrease of GluR2. CONCLUSION: Our findings strengthen the evidence that the NR2A subunit plays a substantial role in mediating recovery of the paralyzed hemidiaphragm following C2 spinal cord hemisection.     

Interneurons are the source and the targets of the first synapses formed in the rat developing hippocampal circuit

In hippocampal CA1 pyramidal neurons, GABAergic synapses are established before glutamatergic synapses. GABAergic interneurons should therefore develop and acquire synapses at an earlier stage to provide the source for GABAergic synapses. We now report that this is indeed the case. At birth and in utero, when nearly all pyramidal neurons are not yet functional, most interneurons have already either GABAergic only or GABAergic and glutamatergic postsynaptic currents. At birth, the morphological maturation of interneurons parallels their individual functional responses. In addition, the formation of functional interneurons types appears to be a sequential process. Interneurons that innervate other interneurons acquire GABA(A) synapses before peridendritic interneurons, but also before perisomatic interneurons that are not yet functional at birth. Therefore, interneurons are the source and the targets of the first synapses formed in the developing circuit. Since GABA was shown to be excitatory in utero, interneurons provide all the excitatory drive at a time when the principal cells are silent. They could therefore play a central role in the formation of the cortical circuit at early developmental stages.     

Peripheral nerve regeneration.

The success of peripheral nerve regeneration is dependent on the survival of axotomized neurons, the efficacy of axonal outgrowth from those neurons, and the specificity of reinnervation of peripheral targets by those neurons. Experimental evidence indicates that following peripheral injury, primary sensory (DRG) neurons and in some cases, motoneurons are lost. This cell death, which can involve one third or more of the axotomized neurons, suggests that some neurons in the adult are dependent on nerve or target-derived neurotrophic factors. One of these factors, NGF, when supplied to the cut proximal stump of the sciatic nerve, can save 100% of the DRG neurons that would normally succumb to axonal injury. But not all neurons are NGF-dependent, and other factors, including gonadal hormones, may be important to their survival following axotomy. Axonal elongation following peripheral nerve injury is dependent upon molecules in the extracellular matrix as well as secreted molecules from nonneuronal cells within the distal stump of the nerve. Extracellular matrix molecules such as laminin provide an adhesive substrate for axonal growth; but Schwann cells in the distal stump, which have been shown to synthesize increased amounts of NGF following peripheral nerve injury, appear to be essential for axonal elongation. Although neuronal survival and the efficacy of axonal elongation are important to peripheral nerve regeneration, the most important determinant of the success of peripheral nerve regeneration is the specificity of reinnervation. There remains some debate over whether regenerating axons are physically guided to the appropriate targets by mechanical guides in the form of basal laminar tubes, or whether they are lured by neurotropic factors derived from the distal nerve stump and targets. There is evidence that both factors are operative in the adult PNS. However, although recent data suggest that neurotropic factors within the adult nerve can influence the sorting of regenerating axons, clinical and experimental data indicate that physical constraints of nerve cytoarchitecture can override those tropic factors. Finally, although some degree of specificity of reinnervation of peripheral targets has been demonstrated, particularly for sensory receptors in skin and muscle, there are typically perturbations of sensation and movement due to axonal misrouting and aberrant reinnervation. Further laboratory research is needed to understand how neuron-target specificity is established during development of the PNS and to determine how the developmental mechanisms can be exploited to reestablish that specificity following peripheral nerve injury.     

Regeneration of Auditory Nerve Following Complete Sectioning and Intrathecal Application of the IN-1 Antibody

Summary.  The cochlear nerve of adult Lewis rats was following microsurgical exposure in the cerebellopontine angle (CPA). The lesions completely interrupted the auditory nerve axons at the lesion site producing ipsilateral deafness in all animals. The rats were then treated with a recombinant Fab fragment of the antibody IN-1 against nerve growth inhibitory proteins for one to two weeks. An age-matched control group of rats was treated with unspecific mouse IgG antibody. Because the cochlear nerve lesions resulted in significant neuronal apoptosis of spiral ganglion cells, neurotrophin-3 (NT-3) was applied to the lesion site immediately post-injury in some rats. Electrophysiological studies were carried out by recording the brainstem auditory evoked potentials (BAEP) before and immediately after the lesion, and at regular intervals up to 2 months after injury. Cochlear nerve fibres were anterogradely traced by horseradish peroxidase (HRP) or biotinylated dextran amine (BDA) injected into the spiral ganglion. The results achieved in this study were consistent with the following conclusions: 1) transection of the adult rat cochlear nerve at the CPA results in functional deafness, disappearance of BAEP, apoptosis of parent axotomized neurons of the spiral ganglion, and interruption of labelled axons close to the lesion site; 2) NT-3 is able to partially rescue axotomized neurons of the spiral ganglion; 3) injured cochlear nerve fibres show a limited spontaneous sprouting and regrowth response which does not lead to BAEP recovery; 4) intrathecal treatment with IN-1 directed against myelin-associated neurite growth inhibitory proteins promotes significant elongation of the injured fibres; and 5) the regenerating fibres seem to navigate to correct targets, and be able to establish synaptic connections for functional recovery as depicted by BAEP examinations.     

The response of rubrospinal neurons to axotomy at different stages of development in the North American opossum.

Rubral axons can grow around a lesion of their pathway in the thoracic spinal cord of developing opossums and a critical period exists for that plasticity. The critical period probably begins when rubral axons first grow into the thoracic cord, and it extends until approximately postnatal day 30. We previously noted that most rubrospinal neurons die after transection of their axon during the critical period, suggesting that plasticity results primarily from growth of axons not damaged by the lesion (Xu and Martin, J. Comp. Neurol. 279, 368-381, 1989). That observation led us to study the response of rubrospinal neurons to axotomy in more detail and at additional stages of development, using a prelabeling paradigm. We first injected fast blue (FB) into the caudal thoracic or rostral lumbar spinal cord in animals ranging from estimated postnatal day 9 to 50 and, about 4 days later, lesioned the rubrospinal tract several segments rostral to the injection. Approximately 30 days later, the animals were killed so that the red nucleus could be searched for labeled neurons. During the critical period for plasticity, rubrospinal neurons showed signs of degeneration 1 week after their axon was cut. When animals were killed 2-3 weeks after lesioning, there was an obvious decrease in axotomized neurons within the red nucleus, and by 4 weeks, more than 75% of them had degenerated. The marked susceptibility of rubrospinal neurons to axotomy during the critical period for plasticity is consistent with the hypothesis that developmental plasticity of the rubrospinal tract results primarily from growth of axons that were not damaged by the lesion. Our results also suggest that survival of axotomized rubrospinal neurons increases with age.