Enzyme histochemical profile of immunohistochemically identified Renshaw cells in rat lumbar spinal cord.

Activity levels of cytochrome oxidase, acid phosphatase, and NADPH diaphorase were examined in the perikarya of immunohistochemically identified Renshaw cells from sections of rat lumbar spinal cord. Renshaw cell profiles were identified on the basis of their characteristic anti-gephyrin-immunofluorescent labelling. Intrasomatic densities of enzyme histochemical reaction product were employed as indicators of relative mitochondrial activity (cytochrome oxidase), intracytoplasmic digestion (acid phosphatase), or putative nitrergic signalling (NAPDH-diaphorase). Approximately half of the Renshaw cell somata examined displayed moderate levels of cytochrome oxidase reaction product (142 of 262 Renshaw cells) or low levels of acid phosphatase activity (156 of 243 Renshaw cells). A majority (160 of 202 cells) of Renshaw cells contained low intrasomatic levels of NADPH-diaphorase activity but most of these cells were closely apposed by at least one NADPH-diaphorase reactive axonal varicosity. Our findings suggest that moderate levels of perikaryal oxidative metabolism and low levels of intracytoplasmic digestion are sufficient for, and support, the unique physiological capabilities of Renshaw cells. The presence of NADPH-diaphorase containing somatic close contacts indicate that nitric oxide may have at least a minor role in the regulation of Renshaw cell activity. These results are complementary and consistent with previous morphological and pharmacological demonstrations of Renshaw cell heterogeneity.

Differential distribution of metabotropic glutamate receptors 1a, 1b, and 5 in the rat spinal cord.

Metabotropic glutamate receptors (mGluRs) modulate somatosensory, autonomic, and motor functions at spinal levels. mGluR postsynaptic actions over spinal neurons display the pharmacologic characteristics of type I mGluRs; however, the spinal distribution of type I mGluR isoforms remains poorly defined. In this study, the authors describe a differential distribution of immunoreactivity to various type I mGluR isoforms (mGluR1a, mGluR5a,b, and mGluR1b) that suggests a correlation between specific isoforms and particular aspects of spinal cord function. Two different antisera raised against mGluR5a,b detected intense immunoreactivity within nociceptive afferent terminal fields (laminae I and II) and also in autonomic regions (parasympathetic and sympathetic). In contrast, two of three anti-mGluR1a antibodies did not immunostain lamina I or II. Laminae I and II immunostaining by a third anti-mGluR1a antibody was competed by a peptide sequence obtained from a homologous region in mGluR5, suggesting possible cross reactivity in fixed tissue. Autonomic neurons did not express mGluR1a immunoreactivity. All anti-mGluR1a antibodies strongly and specifically immunolabeled dendritic and somatic membranes of neurons in the deep dorsal horn (lamina III-V) and the ventral horn (lamina VI-IX). Somatic motoneurons expressed mGluR1a immunoreactivity but little or no mGluR5 immunoreactivity. Phrenic and pudendal motoneurons expressed the highest level of mGluR1a immunoreactivity in the spinal cord. Intense mGluR1b immunoreactivity was restricted to a few scattered neurons and a prominent group of neurons in lamina X. Lamina II neurons expressed low levels of mGluR1b immunoreactivity. Ultrastructurally, type I mGluR immunoreactivity was found mostly at extrasynaptic sites on the plasma membrane, but it was also found perisynaptically, in the body of the postsynaptic regions or in relation to intracytoplasmic structures.

Spinal cholinergic neurons activated during locomotion: localization and electrophysiological characterization.

The objective of the present study was to determine the location of the cholinergic neurons activated in the spinal cord of decerebrate cats during fictive locomotion. Locomotion was induced by stimulation of the mesencephalic locomotor region (MLR). After bouts of locomotion during a 7-9 h period, the animals were perfused and the L(3)-S(1) spinal cord segments removed. Cats in the control group were subjected to the same surgical procedures but no locomotor task. The tissues were sectioned and then stained by immunohistochemical methods for detection of the c-fos protein and choline acetyltransferase (ChAT) enzyme. The resultant c-fos labeling in the lumbar spinal cord was similar to that induced by fictive locomotion in the cat. ChAT-positive cells also clearly exhibited fictive locomotion induced c-fos labeling. Double labeling with c-fos and ChAT was observed in cells within ventral lamina VII, VIII, and possibly IX. Most of them were concentrated in the medial portion of lamina VII close to lamina X, similar in location to the partition and central canal cells found by Barber and collaborators. The number of ChAT and c-fos-labeled neurons was increased following fictive locomotion and was greatest in the intermediate gray, compared with dorsal and ventral regions. The results are consistent with the suggestion that cholinergic interneurons in the lumbar spinal cord are involved in the production of fictive locomotion. Cells in the regions positive for double-labeled cells were targeted for electrophysiological study during locomotion, intracellular filling, and subsequent processing for ChAT immunohistochemistry. Three cells identified in this way were vigorously active during locomotion in phase with ipsilateral extension, and they projected to the contralateral side of the spinal cord. Thus a new population of spinal cord cells can be defined: cholinergic partition cells with commissural projections that are active during the extension phase of locomotion.

Peptidergic input to immunohistochemically-identified Renshaw cells.

Peptidergic influences on Renshaw cells were assessed in rat using gephyrin-immunoreactivity, as a Renshaw cell specific marker, in combination with substance P, calcitonin gene-related peptide- and nicotinic acetylcholine receptor-immunolabelling. An average of 3.9 substance P-, and 8.1 calcitonin gene-related peptide-, and 16.3 nicotinic acetylcholine receptor-immunoreactive close contacts or puncta were observed per Renshaw cell. Most appositions were somatic. These results provide neuroanatomical support for the peptidergic modulation of Renshaw cells.

Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp41 coiled-coil pocket.

The HIV-1 gp41 protein promotes viral entry by mediating the fusion of viral and cellular membranes. A prominent pocket on the surface of a central trimeric coiled coil within gp41 was previously identified as a potential target for drugs that inhibit HIV-1 entry. We designed a peptide, IQN17, which properly presents this pocket. Utilizing IQN17 and mirror-image phage display, we identified cyclic, D-peptide inhibitors of HIV-1 infection that share a sequence motif. A 1.5 A cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these inhibitors make intimate contact with the gp41 pocket. Our studies validate the pocket per se as a target for drug development. IQN17 and these D-peptide inhibitors are likely to be useful for development and identification of a new class of orally bioavailable anti-HIV drugs.

Distribution of 5-hydroxytryptamine-immunoreactive boutons on immunohistochemically-identified Renshaw cells in cat and rat lumbar spinal cord.

A combination of anti-gephyrin- and anti-calbindin D28k-immunoreactivity was used to identify 129 and 171 Renshaw cells and their dendrites in cat and rat lumbar spinal cord, respectively. Using anti-5-hydroxytryptamine-immunoreactivity to label serotoninergic fibers and boutons, 1048 serotoninergic boutons were observed in close contact with the immunolabeled Renshaw cells, with an average of 4.4 and 2.8 close contacts on cat and rat Renshaw cells, respectively. Two-thirds of the observed appositions were formed on the somatic membrane.

Temperature dependence of intramolecular dynamics of the basic leucine zipper of GCN4: implications for the entropy of association with DNA.

The basic leucine zipper domain of the yeast transcription factor GCN4 consists of a C-terminal leucine zipper and an N-terminal basic DNA-binding region that achieves a stable structure only after association with DNA. Backbone dynamics of a peptide encompassing the basic and leucine zipper bZip domain (residues 226-281) are described using NMR spectroscopy. The 15N longitudinal relaxation rates, 15N transverse relaxation rates, and {1H}-15N nuclear Overhauser effects were measured for the backbone amide nitrogen atoms at 290 K, 300 K, and 310 K. The relaxation data were interpreted using reduced spectral density mapping to determine values of the spectral density function, J(omega), at the frequencies 0, omegaN, and 0.87omegaH to characterize overall and intramolecular motions on picosecond-nanosecond timescales. To account for the temperature dependence of overall rotational diffusion, the J(0) values were normalized using Stoke's Law. At 310 K, the 13Calpha and 13CO chemical shifts in conjunction with the spectral density values indicate that the leucine zipper sequence forms a highly ordered alpha-helix, while the basic region populates an ensemble of highly dynamic transient structures with substantial helical character. The normalized values of J(0) and the values of J(0.87omegaH) for residues in the leucine zipper dimerization domain are independent of temperature. In contrast, residues in the basic region exhibit pronounced increases in the normalized J(0) and decreases in J(0.87omegaH) as temperature is decreased. A strong correlation exists between the temperature dependence of 13CO chemical shifts and of J(0.87omegaH). These results suggest that, for the basic region, lowering the temperature increases the population of transient helical conformations, and concomitantly reduces the amplitude or timescale of conformational fluctuations on picosecond-nanosecond timescales. Changes in the conformational dynamics of the peptide backbone of the basic region that accompany DNA binding contribute to the overall thermodynamics of complex formation. The change in backbone conformational entropy derived from NMR spin-relaxation data agrees well with the result calculated from calorimetric measurements. Restriction of the conformational space accessible to the basic region may significantly reduce the entropic cost associated with formation of the basic region helices consequent to DNA binding.

Calbindin D28k expression in immunohistochemically identified Renshaw cells.

Double immunofluorescence was utilized to determine whether Renshaw cells contain calbindin D28k immunoreactivity. Renshaw cells were identified by their characteristic expression patterns of gephyrin immunoreactivity in sections of rat and cat lumbar spinal cord. In the rat, all neurons classified as Renshaw cells (n = 487) also contained calbindin D28k-immunoreactivity, and all calbindin D28k-immunoreactive cells located in the ventral-most region of lamina VII expressed the characteristic gephyrin labeling and morphology of Renshaw cells. In the cat, fewer than half of the Renshaw cells (47%; n = 128) were double-labeled. In both species, occasional calbindin D28k-immunoreactive Renshaw cells were identified within motor nuclei in lamina IX. The distinctive immunolabeling of Renshaw cells allowed us to estimate that there are about 250 Renshaw cells in each ventral horn of the fourth lumbar segment of rat spinal cord, and about 750 cells per ventral horn in the L6 segment of the cat. We conclude that the functional properties of Renshaw cells, including their ability to fire action potentials at high rates, likely require specific homeostatic mechanisms including strong intracellular calcium buffering, the precise mechanisms of which may vary between species.

3D accordion spectroscopy for measuring 15N and 13CO relaxation rates in poorly resolved NMR spectra.

An experimental approach for the measurement of nuclear magnetic spin relaxation rate constants that combines triple-resonance techniques and accordion spectroscopy is described. Pulse sequences are discussed for the measurement of backbone 15N and 13CO R1 relaxation rate constants. The three-dimensional HNCO triple-resonance technique is employed to gain improved spectral resolution over conventional two-dimensional methods by frequency labeling both the 15N and 13CO spins. Accordion spectroscopy is used to reduce the dimensionality of the relaxation experiment. The "negative-time accordion" approach (A. M. Mandel and A. G. Palmer (1994), J. Magn. Reson. A 110, 62-72) is used for extracting rate constants from the t1 interferograms. The experiments are demonstrated using a 13C/15N isotopically enriched sample of the third fibronectin type III domain of human tenascin.

Calcitonin gene-related peptide: distribution and effects on spontaneous rhythmic activity in embryonic chick spinal cord.

Immunohistochemical and in vitro electrophysiological techniques were utilized to examine the distribution and possible role of calcitonin gene-related peptide (CGRP) in the spinal cord of the developing chick. CGRP-like immunoreactivity first appeared in the lateral motor column of the lumbosacral spinal cord at embryonic day 6 followed by the emergence of fiber immunoreactivity in the dorsal horn at embryonic day 11. A rostrocaudal survey of the cervical to lumbosacral spinal cord in embryonic day 18 chick demonstrated robust CGRP-like immunoreactivity at all levels in both putative motor neurons and dorsal horn fibers. Additionally, small immunoreactive lamina VII neurons were observed in sections of lumbosacral cord. In the embryonic day 10 (E10) in vitro reduced spinal cord preparation, bath application of the calcitonin gene-related peptide antagonist human alpha-CGRP fragment 8-37 decreased the frequency and increased the duration of episodes of spontaneously occurring rhythmic activity. Conversely, application of alpha or beta forms of calcitonin gene-related peptide increased the frequency of the rhythmic episodes. The electrophysiological results suggest there is a constitutive release of calcitonin gene-related peptide contributing to the spontaneous rhythmic activity. Immunohistochemical results from E10 animals suggest that CGRP-like immunoreactive putative motoneurons may be the source of the released CGRP.

Effect of sciatic nerve transection or TTX application on enzyme activity in rat spinal cord.

To clarify the differential effects on spinal circuitry caused by physical vs functional disconnection from the periphery, we compared changes produced by 3-, 7- or 14-day unilateral sciatic axotomy or tetrodotoxin (TTX) nerve blockade on the abundance or activity of NADPH diaphorase (NDP), cytochrome oxidase (CO) and acid phosphatase (AP) in the spinal cord. Following axotomy, AP and NDP were decreased in the dorsal horn and increased in large cells in the dorsolateral motor nuclei while CO was decreased in ventral horn neuropil. TTX induced a decrease of CO in the ventral horn and NDP in the dorsal horn. This suggests that physical vs functional disconnection causes modulation of distinct intracellular pathways in sensory afferents, dorsal horn neurons and motoneurons.

Backbone dynamics of homologous fibronectin type III cell adhesion domains from fibronectin and tenascin.

BACKGROUND: Fibronectin type III domains are found as autonomously-folded domains in a large variety of multidomain proteins, including extracellular matrix proteins. A subset of these domains employ an Arg-Gly-Asp (RGD) tripeptide motif to mediate contact with cell-surface receptors (integrins). This motif mediates protein-protein interactions in a diverse range of biological processes, such as in tissue development, would healing and metastasis. The molecular basis for affinity and specificity of cell adhesion via type III domains has not been clearly established. The tenth type III domain from fibronectin (FNfn10) and the third type III domain from tenascin-C (TNfn3) have 27% sequence identity and share the same overall protein fold, but present the RGD motifs in different structural contexts. The dynamical properties of the RGD motifs may affect the specificity and affinity of the FNfn10 and TNfn3 domains. Structure-dynamics correlations for these structurally homologous proteins may reveal common molecular features which are important to the dynamical properties of proteins. RESULTS: The intramolecular dynamics of the protein backbones of FNfn10 and TNfn3 have been studied by 15N nuclear spin relaxation. The FG loop in FNfn10, which contains the RGD motif, exhibits extensive flexibility on picosecond to nanosecond timescales, but motions on microsecond to millisecond timescales are not observed. The equivalent region in TNfn3 is as rigid as regular elements of secondary structure. The CC' loop also is more flexible on picosecond-nanosecond timescales in FNfn10 than in TNfn3. Conformational exchange, reflecting flexibility on microsecond-millisecond timescales, is observed in beta strands A and B of both FNfn10 and TNfn3. CONCLUSIONS: Comparison of the structures of the FNfn10 and TNfn3 reveals several features related to their different dynamical properties. The larger amplitude motions of loops in FNfn10 are consistent with the hypothesis that flexibility of these regions facilitates induced-fit recognition of fibronectin by multiple receptors. Similarly, the more rigid loops of TNfn3 may reflect greater specificity for particular integrins. The correlations observed between structural features and dynamical properties of the homologous type III domains indicate the influence of hydrogen bonding and hydrophobic packing on dynamical fluctuations in proteins.

Downregulation of metabotropic glutamate receptor 1a in motoneurons after axotomy.

Axotomized motoneurons display drastic modifications in synaptic structure and function related to their disconnection from the periphery and establishment of a regenerative metabolic functional mode. The molecular basis of these modifications is not fully understood. Here we describe changes in metabotropic glutamate receptor 1a (mGluR1a)-immunoreactivity 3, 7 or 14 days after unilateral aciatic transection. mGluR1a-immunoreactivity was distributed throughout the somatic cytoplasm and somatodendritic membrane of uninjured motoneurons and was significantly reduced in axotomized motoneurons. This reduction was observed at 3 days and grew progressively over 2 weeks. These findings suggest that downregulation of mGluR1a could contribute to reduced excitatory neurotransmission in axotomized motoneurons.

A descriptive study of childhood injuries in Kingston, Ontario, using data from a computerized injury surveillance system.

This report uses data from the Kingston and Region Injury Surveillance Program (KRISP), a subset of the Canadian Hospitals Injury Reporting and Prevention Program (CHIRPP), to describe rates and identify patterns of injury among children aged 0-19 years in Kingston and area. During 1994, there were 7572 reported injury events, resulting in an overall rate of 173.6 injuries per 1000 children per year (males: 202 per 1000; females: 143.7 per 1000). Four major patterns of injury were identified as priorities for intervention: 1) household injuries among children aged 0-4 years; 2) injuries occurring on playgrounds to children aged 5-14 years; 3) sports injuries among 10-19-year-old children and youth; and 4) bicycle-related injuries among children 5-14 years of age. Discussion focuses on the use of the surveillance system in prioritizing interventions and evaluating injury prevention programs for this population.

Cytochemical characteristics of cat spinal neurons activated during fictive locomotion.

Using standard immunohistochemical and histochemical techniques, we have examined the neurochemical characteristics of a subpopulation of locomotor-related neurons as labeled by the activity-dependent marker c-fos. Results were compared to those obtained from a small sample of intracellularly labeled locomotor-related neurons. In the paralyzed, decerebrate cat, fictive locomotion was evoked by electrical stimulation of the mesencephalic locomotor region. Most c-fos-immunoreactive neurons were distributed in medial lamina VI and VII and in lamina VIII and X. Double labeling of c-fos with various cytochemical markers revealed that about one-third of the c-fos-immunoreactive neurons were choline acetyltransferase immunoreactive, about one-third were glutamate immunoreactive, and about one-third were aspartate immunoreactive. In addition, approximately 15% of the c-fos-labeled neurons contained NADPH-diaphrorase reaction product, while almost 40% appeared to receive close contacts from calcitonin gene-related peptide-immunoreactive fibers and boutons. Choline acetyltransferase- or aspartate immunoreactivity was observed in some intracellularly labeled neurons. These findings have implications regarding the putative neurotransmitters utilized by subpopulations of locomotor-related neurons in the cat spinal cord.