M1 Region Research Articles

Motor Cortex Stimulation for Pain and Movement Disorders

Since initial reports in the early 1990s, stimulation of the M1 region of the cortex (MCS) has been used to treat chronic refractory pain conditions and a variety of movement disorders. A Medline search of literature between 1991 and 2007 revealed 512 cases using MCS. Although most of these relate to the treatment of pain (422), 84 of them involve movement disorders. More recently, several studies have specifically looked at treating Parkinson's disease (PD) with MCS. We report here several of our own cases using MCS to treat poststroke and non-poststroke pain syndromes and movement disorders (n = 8), PD (n = 4), ET (n = 2), and cortico-basal degeneration (n = 1). We also cover the essential history of this procedure and our current research using computational modeling to understand further the underlying mechanisms of MCS.

Identification of the domains of the influenza A virus M1 matrix protein required for NP binding, oligomerization and incorporation into virions

The matrix (M1) protein of influenza A virus is a multifunctional protein that plays essential structural and functional roles in the virus life cycle. It drives virus budding and is the major protein component of the virion, where it forms an intermediate layer between the viral envelope and integral membrane proteins and the genomic ribonucleoproteins (RNPs). It also helps to control the intracellular trafficking of RNPs. These roles are mediated primarily via protein–protein interactions with viral and possibly cellular proteins. Here, the regions of M1 involved in binding the viral RNPs and in mediating homo-oligomerization are identified. In vitro, by using recombinant proteins, it was found that the middle domain of M1 was responsible for binding NP and that this interaction did not require RNA. Similarly, only M1 polypeptides containing the middle domain were able to bind to RNP–M1 complexes isolated from purified virus. When M1 self-association was examined, all three domains of the protein participated in homo-oligomerization although, again, the middle domain was dominant and self-associated efficiently in the absence of the N- and C-terminal domains. However, when the individual fragments of M1 were tagged with green fluorescent protein and expressed in virus-infected cells, microscopy of filamentous particles showed that only full-length M1 was incorporated into budding virions. It is concluded that the middle domain of M1 is primarily responsible for binding NP and self-association, but that additional interactions are required for efficient incorporation of M1 into virus particles.

Apport de l’analyse spectrale à l’étude de la connectivité cérébrale en IRMf: Dissociation fonctionnelle de l’aire motrice supplémentaire

To validate, through functional magnetic resonance imaging (fMRI) from spectral analysis of time series during a visuomotor task, a model of functional connectivity mainly constituted by the pre-supplementary motor area (pre-SMA), the supplementary motor area proper (SMA-proper) and the primary motor cortex (M1).The paradigm that was tried out in young subjects (n=5) consisted of a preparation task of motor movement. We firstly proceeded with an estimate in the frequency domain of coherency coefficients and values of phase shift between these three areas. Secondly, the estimated coherency coefficients were integrated to a model of functional connectivity. Two interaction coefficients were calculated, one for the related M1 and pre-SMA regions, the other one for the related M1 and SMA-proper regions.Our results demonstrate hemodynamic activity that definitely occurred earlier in the pre-SMA area during the preparatory period of the task. In the same way, a more important interaction was found between M1 and pre-SMA areas, which corroborates the assumption of the prevalent role played by these two areas in the case of a preparation task of a motor movement. Thus, this study has allowed highlighting a functional dissociation between the two portions of the SMA.

Sub-chronic psychotomimetic phencyclidine induces deficits in reversal learning and alterations in parvalbumin-immunoreactive expression in the rat

Acute administration of the psychotomimetic phencyclidine (PCP) can mimic some features of schizophrenia, while a repeated treatment regimen of PCP may provide a more effective way to model in animals the enduring cognitive dysfunction observed in many schizophrenic patients. The present study aims to investigate behavioural and neuropathological effects of sub-chronic PCP administration. The cognitive deficit induced by sub-chronic PCP was examined using a previously established operant reversal-learning paradigm. Subsequently, the effect of sub-chronic PCP on parvalbumin-immunoreactive (parvalbumin-IR) neurons was assessed using immunohistochemical techniques. Rats were trained to respond for food in an operant reversal-learning paradigm for approximately 6 weeks, followed by sub-chronic administration of PCP (2mg/kg) or vehicle twice daily for 7 days followed 7 days later by behavioural testing. Six weeks post PCP, brains were analysed using immunohistochemical techniques to determine the size and density of parvalbumin-IR in the frontal cortex and hippocampus. Sub-chronic PCP significantly reduced (p <0.001) percentage correct responding in the reversal phase relative to the initial phase, an effect that persisted throughout the experimental period (4 weeks). The density of parvalbumin-IR neurons was reduced in the hippocampus, with significant reductions in the dentate gyrus and CA2/3 regions (p <0.001). There were significant changes in the frontal cortex, with a reduction (p <0.01) in the M1 (motor area 1) region and increases in the M2 (motor area 2) region and cingulate cortex (p <0.01-p <0.001). These results parallel findings of profound hippocampal and more subtle cortical deficits of parvalbumin-IR neurons in schizophrenia, and provide evidence to suggest that sub-chronic PCP can induce a lasting cognitive deficit, an effect that may be related to the observed neuronal deficits.

The channel-lining 6′ amino acid in the second membrane-spanning region of ionotropic GABA receptors has more profound effects on 4′-ethynyl-4-n-propylbicycloorthobenzoate binding than the 2′ amino acid

The noncompetitive antagonist of ionotropic gamma-aminobutyric acid (GABA) receptors 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) is a useful tool to probe the antagonist-binding site. In the present study, four mutants of the human GABA(A) receptor beta3 subunit were stably expressed in S2 cells and examined for their abilities to bind [(3)H]EBOB to identify the binding site of EBOB. The homo-oligomeric beta3 GABA receptor was used as a housefly GABA receptor model, as the beta3 subunit has a high sequence similarity with the housefly Rdl subunit in the second membrane-spanning (M2) region. The A274S mutation at the -1' position in the M2 region had no effect on [(3)H]EBOB binding. The A277S mutation at the 2' position led to a decrease in the affinity of EBOB for the GABA receptor. The T281V mutant at the 6' position and the A277S/T281V double mutant completely abolished the binding ability. A beta3 GABA receptor homology model predicts these interactions between the receptor and EBOB. These results suggest that EBOB interacts with threonine 281 and alanine 277, and that threonine 281 plays a more critical role in interacting with EBOB than alanine 277.

Malian Paenungulata (Mammalia: Placentalia): new African afrotheres from the early Eocene

ABSTRACT Fossils from early Tertiary phosphate deposits of northern Mali include a new diminutive proboscidean taxon intermediate in size between Daouitherium rebouli and Phosphatherium escuilliei. Although a fragmentary specimen, it has two dentary characters previously demonstrated to be diagnostic for basal proboscideans: a labially expanded mandibular corpus in the region of m2 and m3 and a mesiolabially positioned mandibular ramus. The locality that produced the fossils, Tamaguèlelt, has historically been hard to date even though it has produced many vertebrates. A section is presented that includes the locality and relates it to surrounding sediments. The locality is Eocene, lies very close to the Paleocene-Eocene boundary, and is most likely early Eocene (Ypresian). The depositional environment suggests substantial reworking of older sediments, and the fossils within them, including the proboscideans, may be even older than early Eocene. The Mali proboscidean is one of the oldest members of Afrotheria that has been found in Africa and this locality is the southernmost African early Eocene locality for mammals.

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.

Amygdala interconnections with the cingulate motor cortex in the rhesus monkey

Amygdala interconnections with the cingulate motor cortices were investigated in the rhesus monkey. Using multiple tracing approaches, we found a robust projection from the lateral basal nucleus of the amygdala to Layers II, IIIa, and V of the rostral cingulate motor cortex (M3). A smaller source of amygdala input arose from the accessory basal, cortical, and lateral nuclei, which targeted only the rostral region of M3. We also found a light projection from the lateral basal nucleus to the same layers of the caudal cingulate motor cortex (M4). Experiments examining this projection to cingulate somatotopy using combined neural tracing strategies and stereology to estimate the total number of terminal-like immunoreactive particles demonstrated that the amygdala projection terminates heavily in the face representation of M3 and moderately in its arm representation. Fewer terminal profiles were found in the leg representation of M3 and the face, arm, and leg representations of M4. Anterograde tracers placed directly into M3 and M4 revealed the amygdala connection to be reciprocal and documented corticofugal projections to the facial nucleus, surrounding pontine reticular formation, and spinal cord. Clinically, such pathways would be in a position to contribute to mediating movements in the face, neck, and upper extremity accompanying medial temporal lobe seizures that have historically characterized this syndrome. Alterations within or disruption of the amygdalo-cingulate projection to the rostral part of M3 may also have an adverse effect on facial expression in patients presenting with neurological or neuropsychiatric abnormalities of medial temporal lobe involvement. Finally, the prominent amygdala projection to the face region of M3 may significantly influence the outcome of higher-order facial expressions associated with social communication and emotional constructs such as fear, anger, happiness, and sadness.

Two novel missense and one novel nonsense CFTR mutations in Iranian males with congenital bilateral absence of the vas deferens

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia. Nearly 75% of men with CBAVD have at least one detectable common cystic fibrosis (CF) transmembrane conductance regulator (CFTR) mutation. To study the involvement of CFTR mutations in the Iranian population with presumed low CF frequency, we analysed 112 Iranian CBAVD males. Three Iranian CBAVD males with no clinical CF phenotype indicated by a normal karyotype, normal pancreatic function and sweat chloride concentration and no Y chromosome microdeletions were studied for CFTR mutations, IVS8-5T mutations and M470V exon 10 missense polymorphism. The entire coding sequence of each gene was analysed using a combination of the denaturing gradient-gel electrophoresis or by single-strand conformation analysis and direct DNA sequencing. Also, 52 fertile males were tested as controls to rule out polymorphism. This approach allowed us to detect one novel nonsense mutation (K536X) in the nucleotide-binding domain 1 (NBD1) region and two novel missense mutations (Y122H and T338A) in the M2 and M6 regions of CFTR gene in our studied population, which were not reported previously. Also, the conservation of changed nucleotide and amino acid in mutated regions was analysed by aligning with nine different species. K536X nonsense mutation (transversion) was found in the first NBD (NBF1), which plays an important regulatory role in CFTR function. It was, therefore, considered as a severe allele responsible for elevated sweat chloride levels and obstructive azoospermia. Because Y122H and T338A mutations were compound heterozygote with the IVS8-5T, it is difficult to judge the severity of these mutations and their role in the CBAVD phenotype.

Severe wear and tooth loss in wild ring-tailed lemurs ( Lemur catta): A function of feeding ecology, dental structure, and individual life history

The ring-tailed lemurs at Beza Mahafaly Special Reserve, Madagascar, exhibit a high frequency of severe wear and antemortem tooth loss. As part of a long-term study, we collected dental data on 83 living adult ring-tailed lemurs during 2003 and 2004. Among these individuals, 192 teeth were scored as absent. The most frequently missing tooth position is M1 (24%). As M1 is the first tooth to erupt, its high frequency of absence (primarily a result of wear) is not remarkable. However, the remaining pattern of tooth loss does not correlate with the sequence of eruption. We suggest that this pattern is a function of 1) feeding ecology, as hard, tough tamarind fruit is a key fallback food of ring-tailed lemurs living in gallery forests; 2) food processing, as tamarind fruit is primarily processed in the P3–M1 region of the mouth; and 3) tooth structure, as ring-tailed lemurs possess thin dental enamel. The incongruity between thin enamel and use of a hard, tough fallback food suggests that ring-tailed lemurs living in riverine gallery forests may rely on resources not used in the past. When comparing dental health in the same individuals ( n = 50) between 2003 and 2004, we found that individual tooth loss can show a rapid increase over the span of one year, increasing by as much as 20%. Despite this rapid loss, individuals are able to survive, sometimes benefiting from unintentional assistance from conspecifics, from which partially processed tamarind fruit is obtained. Although less frequent in this population, these longitudinal data also illustrate that ring-tailed lemurs lose teeth due to damage and disease, similar to other nonhuman primates. The relationship between tooth loss, feeding ecology, dental structure, and individual life history in this population has implications for interpreting behavior based on tooth loss in the hominid fossil record.

Characterization of H9N2 influenza A viruses isolated from chicken products imported into Japan from China

We characterized eleven H9N2 influenza A viruses isolated from chicken products imported from China. Genetically they were classified into six distinct genotypes, including five already known genotypes and one novel genotype. This suggested that such multiple genotypes of the H9N2 virus have possibly already become widespread and endemic in China. Two isolates have amino-acid substitutions that confer resistance to amantadine in the M2 region, and this supported the evidence that this mutation might be a result of the wide application of amantadine for avian influenza treatment in China. These findings emphasize the importance of surveillance for avian influenza virus in this region, and of quarantining imported chicken products as potential sources for the introduction of influenza virus.

Muscle representation in the macaque motor cortex: An anatomical perspective

How are the neurons that directly influence the motoneurons of a muscle distributed in the primary motor cortex (M1)? To answer this classical question we used retrograde transneuronal transport of rabies virus from single muscles of macaques. This enabled us to define cortico-motoneuronal (CM) cells that make monosynaptic connections with the motoneurons of the injected muscle. We examined the distribution of CM cells that project to motoneurons of three thumb and finger muscles. We found that the CM cells for these digit muscles are restricted to the caudal portion of M1, which is buried in the central sulcus. Within this region of M1, CM cells for one muscle display a remarkably widespread distribution and fill the entire mediolateral extent of the arm area. In fact, CM cells for digit muscles are found in regions of M1 that are known to contain the shoulder representation. The cortical territories occupied by CM cells for different muscles overlap extensively. Thus, we found no evidence for a focal representation of single muscles in M1. Instead, the overlap and intermingling among the different populations of CM cells may be the neural substrate to create a wide variety of muscle synergies. We found two additional surprising results. First, 15-16% of the CM cells originate from area 3a, a region of primary somatosensory cortex. Second, the size range of CM cells includes both "fast" and "slow" pyramidal tract neurons. These observations are likely to lead to dramatic changes in views about the function of the CM system.

Calmodulin Binding to Peptides Derived from the i3 Loop of Muscarinic Receptors

This study was conducted to identify and characterize the structural requirements of a calmodulin-binding motif identified in the third intracellular (i3) loop of muscarinic acetylcholine receptors (M1-M5), a region important for G protein coupling. GST fusion proteins and synthetic peptides derived from the hM1 i3 loop were tested for binding to CaM using a cross-linking gel shift assay and a dansyl-CaM fluorescence assay. Mutagenesis studies further characterized the structural requirements for the interaction and identified critical residues. 28-Mer peptides from the C terminus of i3, representing the putative calmodulin domains of M1, M2, and M3, were found capable of interacting with CaM. In addition, smaller peptides defined a 5-amino-acid sequence essential for calmodulin binding. Studies performed with M1 peptides derived from GST fusion proteins, representing larger portions of the i3 C terminus, suggested the presence of a second adjacent CaM binding site. Mutagenesis studies identified two mutants that are unable to bind CaM: a point mutation, E360A, and a deletion mutant, delta232-358. Calmodulin can bind to an M1 region implicated in G protein coupling. This indicates an important role for CaM in the regulation of muscarinic signal transduction.

Optical coherence tomography reveals in vivo cortical plasticity of adult mice in response to peripheral neuropathic pain

We examined neural plasticity in mice in vivo using optical coherence tomography (OCT) of primary somatosensory (S1) and motor (M1) cortices of mice under the influence of sciatic nerve chronic constriction injury (CCI), a model of neuropathic pain widely utilized in rats. The OCT system used in this study provided cross-sectional images of the cortical tissue of mice up to a depth of about 1 mm with longitudinal resolution up to 11 μm. This is the first study to evaluate neural plasticity in vivo using OCT. CCI mice exhibited cold allodynia and spontaneuos pain behaviors, which are signs of neuropathic pain, 30 days after sciatic nerve ligation, when OCT observation of S1 and M1 cortices was carried out. The scattering intensity of near-infrared light within the hind paw area of S1 and M1 regions in the contralateral hemisphere was significantly higher than in the ipsilateral hemisphere. These CCI-induced increases in scattering intensity within cortical regions associated with the hind paw probably reflect elevated neural activity associated with neuropathic pain. Synapses and mitochondria are believed to have high light scattering coefficients, since they contain remarkably high concentrations of proteins and complicated membrane structure. Number densities of mitochondria and synapses are known to increase in parallel with increases in neural activity. Our findings thus suggest that neuropathic pain gives rise to neural plasticity within the hind paw area of S1 and M1 contralateral to the ligated sciatic nerve.

Novel Seizure Phenotype and Sleep Disruptions in Knock-In Mice with Hypersensitive α4*Nicotinic Receptors

A leucine to alanine substitution (L9'A) was introduced in the M2 region of the mouse alpha4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, alpha4(L9'A)beta2 nAChRs were > or =30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9'A mutation and studied their cellular responses, seizure phenotype, and sleep-wake cycle. Seizure studies on alpha4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of alpha4or beta2 subunits. Thalamic cultures and synaptosomes from L9'A mice were hypersensitive to nicotine-induced ion flux. L9'A mice were approximately 15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9'A mice differed qualitatively from those in WT: L9'A seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9'A mice were partial, whereas WT seizures were generalized. When L9'A homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure approximately 20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9'A mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive alpha4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE.

Functional recovery and increased PSA-NCAM expression following delayed administration of an anti-MAG antibody post stroke in the rat

Stroke often renders its victims functionally impaired for long periods of time if not permanently. Inhibition of regeneration by molecules found in CNS myelin such as myelin-associated glycoprotein (MAG) and Nogo are thought to limit recovery post-stroke. We have previously demonstrated that an anti-MAG antibody, SB680949 administered 1 h post transient middle cerebral artery occlusion (tMCAO) in the rat results in improved function and neuroprotection 1. In this study we have investigated the potential time window for intervention using this antibody. In addition, we investigated expression of PSA-NCAM, a cell surface macromolecule whose expression is associated with neurogenesis and synaptic plasticity. tMCAO (90 mins) was induced in male Sprague-Dawley rats (300–350 g) as described previously 2. Two doses of SB680949 were administered intracerebroventricularly 24 hours apart, starting at 1, 6 or 24 hours following tMCAO. Control animals were dosed with IgG1 control antibody at 24 and 48 hours. Functional ability was assessed weekly using a 32 point composite neuroscore 3 and the cylinder test of forepaw placement 4. At 8 weeks, animals were deeply anaesthetised and perfused fixed with ice cold 4% paraformaldehyde and brains processed for histological assessment. Administration of SB680949 starting at 1 hour, but not at other time points, reduced lesion volume relative to animals injected with control antibody (12.3 2.1 versus 20.4 2.9 % of contralateral hemispheric volume P<0.05). Administration of SB680949 at 1 h improved function in the right (impaired) forepaw in the cylinder test at 1, 5 (P< 0.05), 7 (P< 0.01) and 8 weeks (P < 0.01). Administration of SB680949 starting at 24 h improved function in the right forepaw at 2, 5, 6 (P<0.05), 7 and 8 weeks (P<0.01). When SB680949 administration was started at 6 hours, no beneficial effect was seen in the cylinder test. SB680949 did not positively affect function as assessed by 32 point neuroscore. PSA-NCAM expression increased in the M1 region of the cortex in animals treated with SB680949 starting at 1 h(20.72.5 PSA-NCAM positive neurones in the non lesioned hemisphere of SB680949 treated animals versus 13.71.5 in the non lesioned hemisphere of control treated animals, P<0.05) and 24 hours (18.7 4.5 PSA-NCAM positive neurones in the non lesioned hemisphere of SB680949 treated animals versus 11.9 1.8 in the lesioned hemisphere of anti-MAG treated animals, P<0.05). These data confirm that early administration of SB680949 results in neuroprotection and improved function. Although when administered 24 hours post stroke anti-MAG treatment fails to offer neuroprotection, the degree of functional recovery is comparable. Increased expression of PSA-NCAM in the motor cortex in the groups displaying improved motor function supports the hypothesis that anti-MAG treatment enhances functional recovery through an increase in neurogenesis and enhanced plasticity.

Pharmacological Implications of Two Distinct Mechanisms of Interaction of Memantine withN-Methyl-d-aspartate-Gated Channels

Unlike other N-methyl-D-aspartate receptor (NMDAR) antagonists, clinical trials have shown that memantine is clinically tolerated and effective in the treatment of Alzheimer's disease. The mechanism for memantine tolerability, however, remains contentious but may be partly explained by its uncompetitive antagonism. The specific site of memantine block in the NMDAR channel interacts with magnesium and is assumed to be at or near a narrow constriction representing the channel selectivity filter. A second, very low-affinity site of memantine action has also been reported. Here, using mutational analysis and substituted cysteine accessibility methods on recombinant NR1/NR2A NMDARs expressed in Xenopus oocytes, we precisely localize both the specific and second memantine-blocking sites. Intriguingly, memantine interacts with its specific blocking site in the same fashion as intracellular rather than extracellular Mg(2+). Thus, the N-site asparagine (N) in the M2 region of the NR1 subunit represents the dominant site for uncompetitive antagonism by memantine. The N and N + 1 site asparagines in NR2A produce strong electrostatic interactions with memantine. In contrast, the second (superficial) memantine-blocking site, located at the extracellular vestibule of the channel, appears to be nonspecific and overlaps the site occupied by the nonspecific pore blocker hexamethonium. Residues in the post-M3 segment of the NR1 subunit are not directly involved in memantine binding. The distinct patterns of interaction and the relative degree of affinity of memantine for these two binding sites contribute to the drug's excellent pharmacological profile of clinical tolerability. In the future, these parameters should be considered in searching for improved neuroprotective agents in this class.

Absence of localized grey matter volume changes in the motor cortex following spinal cord injury

The consequences of spinal cord injury (SCI) have considerable effects on motor function, typically resulting in functional impairment. Pathological changes have been studied at the site of trauma, rostrocaudally within the cord, and in the periphery. Few studies, however, have investigated the consequences of SCI at the cortical level. Magnetic resonance imaging (MRI) was used to explore the morphological changes in the grey and white matter within the primary motor (M1) cortex of individuals with cervical SCI. The “precentral knob,” a landmark of M1 cortex dedicated to hand function, was selected for regionally specific measurements of change. Thirty-one hemispheres of SCI subjects and 28 hemispheres of control subjects were compared using a manual measurement after the images were segmented into grey matter, white matter, and cerebral spinal fluid (CSF). No significant differences in grey matter area measured at the precentral knob were found with the manual approach. An automated voxel-based morphometric analysis was also performed and demonstrated no significant differences in grey or white matter volume within an M1 region of interest. These data suggest that there is no gross anatomical change within M1 following cervical SCI. Our previously reported findings of reorganization of cortical motor output maps following SCI therefore likely result from changes in functional organization rather than anatomical changes.

A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site.

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta 19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Delta F/F) approximately 10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for Delta F agreed well with those for epibatidine-induced currents, but were shifted approximately 100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alpha gamma-binding site than to the alpha delta site and (ii) ACh binds with reverse-site selectivity, these data suggest that Delta F monitors an event linked to binding specifically at the alpha delta-subunit interface. In experiments with flash-applied agonists, the earliest detectable Delta F occurs within milliseconds, i.e., during activation. At low [ACh] (< or = 10 microM), a phase of Delta F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Delta F is complete before the slowest phase of recovery from desensitization (time constant approximately 250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alpha delta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes.

The M1 matrix protein controls the filamentous phenotype of influenza A virus

We show that most isolates of influenza A induce filamentous changes in infected cells in contrast to A/WSN/33 and A/PR8/34 strains which have undergone extensive laboratory passage and are mouse-adapted. Using reverse genetics, we created recombinant viruses in the naturally filamentous genetic background of A/Victoria/3/75 and established that this property is regulated by the M1 protein sequence, but that the phenotype is complex and several residues are involved. The filamentous phenotype was lost when the amino acid at position 41 was switched from A to V, at the same time, this recombinant virus also became insensitive to the antibody 14C2. On the other hand, the filamentous phenotype could be fully transferred to a virus containing RNA segment 7 of the A/WSN/33 virus by a combination of three mutations in both the amino and carboxy regions of the M1 protein. This observation suggests that an interaction among these regions of M1 may occur during assembly.