Spinal Inhibitory Interneurons: Gatekeepers of Sensorimotor Pathways.

The ability to sense and move within an environment are complex functions necessary for the survival of nearly all species. The spinal cord is both the initial entry site for peripheral information and the final output site for motor response, placing spinal circuits as paramount in mediating sensory responses and coordinating movement. This is partly accomplished through the activation of complex spinal microcircuits that gate afferent signals to filter extraneous stimuli from various sensory modalities and determine which signals are transmitted to higher order structures in the CNS and to spinal motor pathways. A mechanistic understanding of how inhibitory interneurons are organized and employed within the spinal cord will provide potential access points for therapeutics targeting inhibitory deficits underlying various pathologies including sensory and movement disorders. Recent studies using transgenic manipulations, neurochemical profiling, and single-cell transcriptomics have identified distinct populations of inhibitory interneurons which express an array of genetic and/or neurochemical markers that constitute functional microcircuits. In this review, we provide an overview of identified neural components that make up inhibitory microcircuits within the dorsal and ventral spinal cord and highlight the importance of inhibitory control of sensorimotor pathways at the spinal level.

Functional Divergence of Delta and Mu Opioid Receptor Organization in CNS Pain Circuits.

Cellular interactions between delta and mu opioid receptors (DORs and MORs), including heteromerization, are thought to regulate opioid analgesia. However, the identity of the nociceptive neurons in which such interactions could occur in vivo remains elusive. Here we show that DOR-MOR co-expression is limited to small populations of excitatory interneurons and projection neurons in the spinal cord dorsal horn and unexpectedly predominates in ventral horn motor circuits. Similarly, DOR-MOR co-expression is rare in parabrachial, amygdalar, and cortical brain regions processing nociceptive information. We further demonstrate that in the discrete DOR-MOR co-expressing nociceptive neurons, the two receptors internalize and function independently. Finally, conditional knockout experiments revealed that DORs selectively regulate mechanical pain by controlling the excitability of somatostatin-positive dorsal horn interneurons. Collectively, our results illuminate the functional organization of DORs and MORs in CNS pain circuits and reappraise the importance of DOR-MOR cellular interactions for developing novel opioid analgesics.

An immunohistochemical study on a tetanus fatal case using toxin fragment C (TTC). Should it be a useful diagnostic tool?

A 65-year-old man fell in his garden and sustained a right pre-radial cutaneous laceration associated with a Colles' fracture. His status for tetanus immunization was uncertain; so a course of antitetanus treatment was immediately started. Two days after admission the man suddenly developed severe nucal pain, rigidity and dysphagia. A brain CT scan was negative. His condition progressively worsened and then he developed trismus. Cultures from the wound were negative for Clostridium tetani; the CSF analysis was negative. On the 9th day after admission, the man died. A presumptive clinical diagnosis of tetanus was made. Autopsy was performed 24 h after death. An immunohistochemical study was conducted with an antibody directed against tetanus toxin fragment C (TTC). By immunohistochemical evaluation, large motor neurons in the ventral horn were immunopositive for TTC. High power magnification of the ventral horn of spinal cord gray matter samples showed TTC immunoreactivity in motor neuron axons and cell bodies, using a confocal laser scanning microscope. The correct diagnosis could be established on the basis of pathological examination with TTC immunostaining.

Fibroblast growth factor homologous factor 2B: association with Nav1.6 and selective colocalization at nodes of Ranvier of dorsal root axons.

Voltage-gated sodium channels interact with cytosolic proteins that regulate channel trafficking and/or modulate the biophysical properties of the channels. Na(v)1.6 is heavily expressed at the nodes of Ranvier along adult CNS and PNS axons and along unmyelinated fibers in the PNS. In an initial yeast two-hybrid screen using the C terminus of Na(v)1.6 as a bait, we identified FHF2B, a member of the FGF homologous factor (FHF) subfamily, as an interacting partner of Na(v)1.6. Members of the FHF subfamily share approximately 70% sequence identity, and individual members demonstrate a cell- and tissue-specific expression pattern. FHF2 is abundantly expressed in the hippocampus and DRG neurons and colocalizes with Na(v)1.6 at mature nodes of Ranvier in myelinated sensory fibers in the dorsal root of the sciatic nerve. However, retinal ganglion cells and spinal ventral horn motor neurons show very low levels of FHF2 expression, and their axons exhibit no nodal FHF2 staining within the optic nerve and ventral root, respectively. Thus, FHF2 is selectively localized at nodes of dorsal root sensory but not ventral root motor axons. The coexpression of FHF2B and Na(v)1.6 in the DRG-derived cell line ND7/23 significantly increases the peak current amplitude and causes a 4 mV depolarizing shift of voltage-dependent inactivation of the channel. The preferential expression of FHF2B in sensory neurons may provide a basis for physiological differences in sodium currents that have been reported at the nodes of Ranvier in sensory versus motor axons.

Evidence for neuronal localisation of enteroviral sequences in motor neurone disease/amyotrophic lateral sclerosis by in situ hybridization.

Sequences resembling those of human enterovirus type B sequences have been associated with motor neurone disease/amyotrophic lateral sclerosis. In a previous study we detected enteroviral sequences in spinal cord/brain stem from cases of motor neurone disease/amyotrophic lateral sclerosis, but not controls. Adjacent tissue sections to two of those strongly positive for these sequences by reverse-transcriptase polymerase chain reaction were analyzed by in situ hybridization with digoxigenin-labelled virus-specific antisense riboprobes. In one case, a female aged 83 showing 12 month rapid progressive disease, signal was specifically localized to cells identifiable as motor neurones of the anterior horn. In another case, a male aged 63 with a 60-month history of progressive muscle weakness, dysarthia, dyspnoea and increased tendon reflexes, signal was located to neurones in the gracile/cuneate nuclei of the brain stem tissue block that had been analyzed. This case showed loss of neurones in the anterior horn of the spinal cord by histopathologic examination which would account for clinical signs of motor neurone disease/amyotrophic lateral sclerosis. Dysfunction of the gracile/cuneate nuclei might have been masked by the paralytic disease. These structures are adjacent to the hypoglossal nuclei, and suggest either localised dissemination from hypoglossal nuclei or a possible route of dissemination of infection through the brainstem to the hypoglossal nuclei. These findings provide further evidence for the possible involvement of enteroviruses in motor neurone disease/amyotrophic lateral sclerosis.

ISSLS prize winner: Erythropoietin inhibits spinal neuronal apoptosis and pain following nerve root crush.

STUDY DESIGN: The authors investigated the association of L5 proximal nerve root injury with spinal cord neuronal apoptosis (histologic) and whether exogenous erythropoietin therapy might reduce apoptosis/or pain (behavioral). OBJECTIVES: The first objective was to determine whether nerve root crush induces specific programmed cell death of spinal neurons in the dorsal and ventral horn and whether this is correlated with pain behaviors. The second objective was to determine if exogenous erythropoietin might reduce apoptosis and/or pain. SUMMARY OF BACKGROUND DATA: Whether spinal neuronal apoptosis is correlated with pain behaviors following nerve root injury remains unknown. It has been hypothesized that neuroprotective factors may alleviate pain behaviors by protecting neurons from death. Erythropoietin is a hematopoietic growth factor that recently has been demonstrated as a potent neuroprotective factor against ischemic damage in the brain. The effects of erythropoietin on pain and spinal cord neurons have not been examined. METHODS: Sprague-Dawley rats received a L5 proximal nerve root crush injury or sham operation and were separated into two treatment groups for subcutaneous injection: 1) vehicle; 2) recombinant human erythropoietin, 2680 U/kg. The rats were sacrificed, and spinal cords were removed for apoptotic and immunohistochemical analysis at 0, 1, and 3 days after surgery. To determine whether recombinant human erythropoietin prevented mechanical allodynia in animals with nerve root crushes (n = 12/group), both treatment groups were tested for pain behaviors using the von Frey test at -1, -2, -3, 1, 3, 7, 11, and 14 days after surgery. RESULTS: After nerve root injury, apoptotic neurons increased by 80% in the ipsilateral spinal cord and moderately in contralateral spinal cord in vehicle-treated animals compared to uninjured controls. Recombinant human erythropoietin reduced (P < 0.05) neuronal apoptosis in both superficial dorsal and ventral horns of the spinal cord. This corresponded with identification of erythropoietin and its receptors on spinal neurons and reductions in TNF-alpha colocalization in ventral horn neurons. Mechanical allodynia developed in the corresponding ipsilateral hind paw within 1 day and was sustained until day 14. Recombinant human erythropoietin-treated animals demonstrated faster recovery from mechanical allodynia compared with vehicle-treated controls (P < 0.05). CONCLUSIONS: Our findings indicated that L5 proximal nerve root crush increased neuronal apoptosis in the superficial dorsal and ventral horn that correlated with mechanical allodynia. Exogenous recombinant human erythropoietin facilitated receptor-mediated neuroprotection of spinal cord neurons and faster recovery from mechanical allodynia. Erythropoietin may be a potential therapeutic factor for patients with low back pain by providing pain relief and neuroprotection.

Uncoupling protein 2 in primary pain and temperature afferents of the spinal cord.

Uncoupling protein 2 (UCP2) is a mitochondrial protonophore that regulates cellular energy homeostasis. In this study, we explored the expression of UCP2 in the spinal cord. UCP2 was expressed in the substantia gelatinosa and ventral horn of the rodent and primate spinal cord. In all of these areas, UCP2 expression was associated with axons and axon terminals and direct appositions between UCP2-immunoreactive fibers and NMDA glutamate receptors-containing perikarya were frequently detected. All of the UCP2-labeled processes were also immunoreactive for substance P. The expression of UCP2 in primary sensory afferents of the spinal cord suggests that this mitochondrial uncoupler is involved in the mechanism of pain and temperature sensation.

Synaptic relationships between hair follicle afferents and neurones expressing GABA and glycine-like immunoreactivity in the spinal cord of the rat.

gamma-Aminobutyric acid (GABA) and glycine have been implicated in the inhibition of sensory pathways in the dorsal horn of the spinal cord. The object of this study is to investigate the interactions between neurones immunoreactive for GABA and/or glycine and hair follicle afferent terminals labelled by intracellular injection with neurobiotin. GABA and glycine-like immunoreactivity in axons and dendrites in synaptic contact with the afferent terminals was demonstrated by using a postembedding immunogold method, and serial section reconstruction was used to show the distribution and nature of these interactions in lamina III of the dorsal horn. Most afferent boutons (94%) were postsynaptic at axo-axonic synapses: 67% of presynaptic boutons presynaptic to the afferent terminals were immunoreactive for GABA and glycine, 24% for GABA alone, and 7% for glycine alone. Only a small percentage of dendrites postsynaptic to afferent boutons appeared to belong to inhibitory interneurones: 3% were immunoreactive for GABA and glycine, 10% for glycine alone, but 87% were immunoreactive for neither antibody. Many afferent boutons were the central terminals of what appeared to be type IIb glomeruli and were involved triadic synaptic arrangements at which boutons presynaptic to an afferent terminal also made axodendritic contacts with dendrites postsynaptic to the afferent. Many of the presynaptic boutons involved in the triads were immunoreactive for GABA and glycine. Because afferent terminals do not themselves express glycine receptors (Mitchell et al. [1993] J. Neurosci. 13:2371-2381), glycine may therefore act on dendrites postsynaptic to hair follicle afferent terminals at these triads.

A quantitative histochemical assay for activities of mitochondrial electron transport chain complexes in mouse spinal cord sections.

Mitochondrial dysfunction and degeneration are associated with neurodegenerative disorders. A dysfunctional mitochondrial electron transport chain (ETC) impairs ATP production and accelerates the generation of free radicals. To quantify ETC activity, solution-spectrophotometric assays and histochemical reactions on blue native polyacrylamide gel electrophoresis (BN-PAGE) gels have been used. These methods, however, do not provide information regarding mitochondrial ETC activities associated with specific regions in the central nervous system (CNS). Because neurodegenerative diseases often strike a specific subset of neurons within specific regions in the CNS, reliable methods for quantifying mitochondrial ETC activities in selected CNS regions are needed. We have studied the quantitative range of in situ histochemical assays for ETC complex I, II and IV and determined the optimal conditions for quantification of these ETC complex activities. We also demonstrate that these assays can detect a decrease in mitochondrial ETC activities in the ventral horn of spinal cords isolated from a transgenic mouse model for amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease.

Immune reactivity in a mouse model of familial ALS correlates with disease progression.

OBJECTIVE: The cause of motor neuron death in ALS is incompletely understood. This study aims to define the potential involvement of nonneuronal immune-inflammatory factors in the destruction of motor neurons in mutant superoxide dismutase-1 (SOD1) transgenic mice as a model of ALS. BACKGROUND: The presence of activated microglia, IgG and its receptor for Fc portion (FcgammaRI), and T lymphocytes in the spinal cord of both patients with ALS and experimental animal models of motor neuron disease strongly suggests that immune-inflammatory factors may be actively involved in the disease process. METHODS: The expression of immune-inflammatory factors was followed in both human mutant (G93A) SOD1 transgenic mice and human wild-type SOD1 transgenic mice, at different ages (40, 80, and 120 days). Fixed, frozen, free-floating sections of the lumbar spinal cord were stained with antibodies against CD11b, IgG, FcgammaRI, intercellular adhesion molecule-1 (ICAM-1), CD3, and glial fibrillary acidic protein. RESULTS: The earliest change observed was the upregulation of ICAM-1 in the ventral lumbar spinal cord of 40-day-old mutant SOD1 mice. IgG and FcgammaRI reactivities were detected on motor neurons as early as 40 days and on microglial cells at later stages. Microglial activation was first evident in the ventral horn at 80 days, whereas reactive astrocytes and T cells became most prominent in 120-day-old mutant SOD1 mice. CONCLUSION: The upregulation of proinflammatory factors during early presymptomatic stages as well as the expansion of immune activation as disease progresses in mutant SOD1 transgenic mice suggest that immune-inflammatory mechanisms could contribute to disease progression.

Ubiquitin immunoreactivity in presumed spinal interneurones in motor neurone disease.

Previous studies have demonstrated the presence of ubiquitin-immunoreactivity (Ub-IR) as inclusions and skeins in motor neurones of both the familial and sporadic forms of motor neurone disease (MND). There is evidence that interneurones also degenerate in MND, but Ub-IR in ventral horn spinal interneurones has not been studied previously. Here, Ub-IR was investigated in 1445 presumed interneurones and 1086 presumed motor neurones counted in three random 20-microm sections of the ventral horn of the third lumbar segment of the spinal cord of each of seven controls and seven patients with MND. The ventral horn was divided into four quadrants; the dorsomedial quadrant contains almost exclusively interneurones and the ventrolateral quadrant largely motor neurones. The neurones were also classified by morphological and size criteria into presumed interneurones (< 25 microm) and presumed motor neurones (>or= 25 microm). Ub-IR was classified as inclusions, skeins and dispersed cytoplasmic and nuclear staining. Ub-IR inclusions or skeins were not observed in the controls but 6.6% of neurones (motor neurones and interneurones) showed the presence of dispersed cytoplasm staining and nuclear staining. The incidence of Ub-IR cytoplasmic and nuclear staining was significantly greater in both motor neurones and interneurones of MND patients than controls. Ub-IR was less frequent in MND cases in which a great loss of neurones was observed. Ub-IR was significantly more frequent in motor neurones than interneurones, both in patients and controls. Ub-IR inclusions and skeins were only observed in motor neurones from MND patients. Ub-IR inclusions were not observed in presumed spinal interneurones, while skeins were only seen in three out of 565 of these cells (two of them in the dorsomedial quadrant) in two out of seven patients. Thus, although presumed spinal interneurones occasionally revealed Ub-IR features similar to motor neurones, the rare staining of Ub-IR skeins and the lack of Ub-IR inclusions in interneurones in MND suggests that these neurones only occasionally form ubiquitin-protein conjugates. Neuronal size, rather than type, may be important in determining whether ubiquitin-protein conjugates form in the ventral horn neurones in MND.

Persistent c-fos expression and NADPH-d reactivity in the medulla and the lumbar spinal cord in rat with short-term peripheral anosmia.

Here we examine hypothesis that short-term peripheral ZnSO(4)-induced anosmia can produce effects on c-fos expression within spinal cord and caudal medulla in male Wistar rats (n=4). Fos-like-immunoreactive cells revealed by avidin-biotin-peroxidase method show a significant bilateral increase in the nucleus proprius (layers 3 and 4) and medial part of layers 5 and 6. In substantia gelatinosa (layer 2(i)) and area 10 Fos-positive neurons were intermixed together with nicotin-amide adenine dineucleotide phosphate-diaphorase (NADPH-d)-reactive cells. Short-term anosmia enhanced c-fos expression in ventral horn (layers 7 and 8), ventrolateral segment and dorsal part of the spinal trigeminal nuclei. In anosmic rats varicose fibres and numerous NADPH-d-stained neurons were present in the gelatinous layer of the spinal trigeminal nucleus caudalis, and a separate population of Fos-positive cells was detected within this layer. Nucleus tractus solitaris also contained a few NADPH-d-reactive, medium sized neurons intermixed with Fos-immunoreactive cells.

Sustained induction of heme oxygenase-1 in the traumatized spinal cord.

Oxidative stress contributes to secondary injury after spinal cord trauma. Among the consequences of oxidative stress is the induction of heme oxygenase-1 (HO-1), an inducible isozyme that metabolizes heme to iron, biliverdin, and carbon monoxide. Here we examine the induction of HO-1 in the hemisected spinal cord, a model that results in reproducible degeneration in the ipsilateral white matter. HO-1 was induced in microglia and macrophages from 24 h to at least 42 days after injury. Within the first week after injury, HO-1 was induced in both the gray and the white matter. Thereafter, HO-1 expression was limited to degenerating fiber tracts. HSP70, a heat shock protein induced mainly by the presence of denatured proteins, was consistently colocalized with HO-1 in the microglia and macrophages. This study to demonstrates long-term induction of HO-1 and HSP70 in microglia and macrophages after traumatic injury and an association between induction of HO-1 and Wallerian degeneration. White matter degeneration is characterized by phagocytosis of cellular debris and remodeling of surviving tissue. This results in the metabolism, synthesis, and turnover of heme and heme proteins. Thus, sustained induction of HO-1 and HSP70 in microglia and macrophages suggests that tissue degeneration is an ongoing process, lasting 6 weeks and perhaps even longer.

Plasticity in the rat spinal cord seen in response to lesions to the motor cortex during development but not to lesions in maturity.

Motor cortical inputs and proprioreceptive muscle afferents largely target the same spinal cord region. This study explored the idea that during development the two inputs interact via an activity-dependent mechanism to produce mature patterns of innervation. In rats, the forelimb motor cortex was ablated unilaterally at either postnatal day 7 (P7), the beginning of corticospinal synaptogenesis in the cervical cord, or at P50. Comparisons were made with sham-operated animals. At P70, muscle afferents from the extensor digitorum communis muscle, contralateral to the lesion, were transganglionically labeled with cholera toxin B-subunit. Lower cervical spinal cord sections were immunostained for cholera toxin B, parvalbumin, and cJun. Our small lesions had no obvious effects upon forelimb function. However, developmental lesions, but not adult lesions, were shown to significantly increase the number of muscle afferent boutons present in the contralateral ventral horn, compared with sham-operated controls. Also, the ratio of parvalbumin-positive neurons contralateral/ipsilateral to the developmental lesion (but not adult lesions) was decreased and the ratio of cJun-positive motoneurons increased. Thus, an early motor cortex lesion resulted in retention of a proportion of muscle afferent synapses to the ventral horn that are known to be lost during normal development. Parvalbumin and cJun are markers of neuronal activity suggesting that spinal circuitry develops permanently altered activity patterns in response to an early cortical lesion, although this plasticity is lost in the mature animal.

Zinc-enriched (ZEN) terminals in mouse spinal cord: immunohistochemistry and autometallography.

The general distribution of zinc-enriched (ZEN) terminals in mouse spinal cord was investigated at light microscopic level by means of zinc transporter-3 immunohistochemistry (ZnT3(IHC)) and zinc selenium autometallography (ZnSe(AMG)). Staining for ZnT3(IHC) corresponded closely to the ZnSe(AMG) staining. Both appeared as dense grains of variable sizes and densities in the gray matter with a characteristic segmental laminar pattern. The white matter was unstained but contained rows of stained terminals radiating from the gray matter. In the dorsal horn, laminae I, III and IV were heavily stained, whereas lamina II appeared as the least stained area in the gray matter. Moderate staining was seen in laminae V and VI. In the ventral horn, large ZnT3(IHC) and ZnSe(AMG) grains, known from previous papers to represent ZEN terminals, were observed related in particular to motor neuronal somata and big dendrites. These ZEN terminals in the ventral horn were in general larger than those in the dorsal horn. This is the first description of the pattern of ZEN terminals in mouse spinal cord.

Urocortin expression in the human central nervous system.

OBJECTIVE AND STUDY DESIGN: Urocortin is a recently identified neuropeptide of the corticotrophin-releasing factor (CRF) family in the mammalian brain and has been demonstrated to stimulate ACTH secretion from pituitary cells, but its expression in human brain tissue including the hypothalamus has not been examined. In this study, we first examined urocortin expression in the hypothalamus (20 cases) and pituitary stalks (17 cases) of human brain obtained from autopsy using immunohistochemistry and mRNA in situ hybridization. RESULTS: Neither urocortin immunoreactivity nor mRNA hybridization signals were detected in the hypothalami and pituitary stalks while CRF immunoreactivity was detected in the paraventricular nuclei of the hypothalami in 10/20 cases and in nerve fibres of the stalks in 17/17 cases. These results indicate that urocortin does not act on the hypothalamo-pituitary-adrenal axis, at least not in the same manner as CRF in humans. We then examined urocortin expression in various portions of the brain in 7 cases. Both urocortin immunoreactivity and mRNA hybridization were detected in Purkinje cells of the cerebellum and anterior horn cells of the spinal cord in specimens examined. Urocortin expression was, however, variably seen in superior olivary nuclei (two out of six cases examined) and in the Edingar-Westphal nuclei (one out of three cases examined). CONCLUSIONS: The distribution of urocortin in the human central nervous system suggests that urocortin may work as a neurotransmitter like other neuropeptides in the human.

Developmental changes in the expression of alpha-, beta- and gamma-subspecies of protein kinase C at synapses in the ventral horn of the embryonic and postnatal rat spinal cord.

Developmental changes in expression of alpha-, beta- and gamma-subspecies of protein kinase C (PKC) at synapses in the ventral horn of the rat spinal cord were immunocytochemically investigated. On embryonic day 15, a few synapses were found in the ventral horn, and they gradually increased in number until postnatal day 21 or 28. During the embryonic period, immunoreactivity (IR) for all three subspecies was demonstrated in both the pre- and postsynaptic regions. In the former, IR was detected mainly along the outer surface of the synaptic vesicles, and in the latter, along the postsynaptic membranes. At these stages, synapses were morphologically immature, having a faint postsynaptic density and a few round synaptic vesicles. After birth, IR for PKCs at the postsynaptic densities became stronger, but gradually disappeared in most of the presynaptic regions. In adult, IR for PKCs was detected only at the postsynaptic densities. At the later postnatal stages, the synapses were fully mature, having a thick postsynaptic density, a great number of synaptic vesicles and a distinct synaptic cleft as those in adult animals. In addition, the developmental changes in expression of these subspecies of PKC in the presynaptic regions were quite different. These findings suggest that the increase in expression of PKC at postsynaptic densities might be closely related with the development of synaptic functions, and also that each subspecies of PKC may take part in different aspects of synaptogenesis.

The fate of motoneurons in the spinal cord after peripheral nerve repair: a quantitative study using the neural tracer horseradish peroxidase.

This study assessed the changes that occurred in the spinal motoneuron pool after the repair of a specific peripheral nerve by means of several clinically appropriate surgical techniques: nerve graft, muscle graft, and epineurial suture. The motoneuron pool relating to a single muscle was assessed at 50, 100, 200, and 300 days after repair via retrograde axonal transport of the neural tracer horseradish peroxidase. The results indicate that although a small portion of the motoneuron population dies following peripheral nerve surgery, this is not a significant number. The majority of the anterior horn cells appear to have the ability to both survive nerve transection and form new functional connections with the regenerated nerve after repair. The degree of cell loss is influenced by the nature of the injury and the method of repair implemented. Injuries involving neurotmesis result in the loss of a greater proportion of the cell population than less severe injuries involving axonotmesis. A greater proportion of the motoneuron population is preserved when the severed nerve has been repaired using a direct epineurial suture than when repair is achieved by means of a graft. The two methods of grafting produced comparable results, although the muscle graft tended to result in the preservation of a greater number of cells than the nerve graft, making it an acceptable alternative method for the surgical repair of short gaps in peripheral nerves.

Dexamethasone increases adrenalectomy-depressed Na+,K(+)-ATPase mRNA and ouabain binding in spinal cord ventral horn.

The Na+,K(+)-ATPase plays a key role in the regulation of ion fluxes and membrane repolarization in the CNS. We have studied glucocorticoid effects on biosynthesis of the Na+,K(+)-ATPase and on ouabain binding in the ventral horn of the spinal cord using intact rats, adrenalectomized (ADX) rats, and ADX rats receiving dexamethasone (ADX+DEX) during 4 days. Cryostat sections from spinal cords were incubated with a 35S-oligonucleotide coding for the alpha 3-subunit or a 3H-cDNA coding for the beta 1-subunit of the Na+,K(+)-ATPase using in situ hybridization techniques. In ventral horn motoneurons, grain density per cell and grain density per area of soma for both probes were slightly reduced in ADX rats but significantly increased in the ADX+DEX group, using ANOVA and the Bonferroni's test. Statistical analysis of frequency histograms of neuronal densities further indicated a significant shift to the right for intact rats compared with ADX rats for both probes. Concomitantly, [3H]ouabain binding to membrane preparations from ventral horns was reduced in ADX rats and restored to normal by DEX administration. No effect of adrenalectomy or DEX treatment was obtained in the dorsal horn. In conclusion, glucocorticoids positively modulate the mRNA for the alpha 3-subunit and the beta 1-subunit of the Na+,K(+)-ATPase and recover ouabain binding to normal values. The increments of the synthesis and activity of an enzyme affecting membrane repolarization and synaptic neurotransmission are consistent with the alleged stimulatory effect of glucocorticoids on spinal cord function.

Synaptic pathology of spinal anterior horn cells in amyotrophic lateral sclerosis: an immunohistochemical study.

We have applied immunohistochemical techniques to study synaptic alterations of the spinal anterior horn in amyotrophic lateral sclerosis (ALS), and other disorders involving upper or lower motor neurons. A monoclonal antibody to synaptophysin was used. Spinal cord tissues from normal individuals served as controls. As compared to these, a decrease in synaptophysin immunoreactivity was evident in the neuropil in the spinal anterior horn of ALS patients. However, synaptophysin expression in the perikarya and dendrites of remaining normal-appearing neurons in these patients was not decreased and occasionally it was even higher than in control neurons. Similar results were obtained with specimens from patients with lower motor neuron disease. Synaptophysin immunoreactivity in the neuropil and perikarya of the cases with focal spinal cord lesions with bilateral descending tract degeneration was similar to normal controls. Our data suggest that the alterations in synaptophysin expression occurring in ALS are mainly associated with the loss of lower motor neurons, and that the occasional increased perikaryal expression may be due to the neuronal atrophy, compensatory accumulation or abnormal synaptic vesicle degradation.