Dynamic balance of metabotropic inputs causes dorsal horn neurons to switch functional states

Sensory relay structures in the spinal cord dorsal horn are now thought to be active processing structures that function before supraspinal sensory integration. Dorsal horn neurons directly receive nociceptive (pain) signals from the periphery, express a high degree of functional plasticity and are involved in long-term sensitization and chronic pain. We show here that deep dorsal horn neurons (DHNs) in Wistar rats can switch their intrinsic firing properties from tonic to plateau or endogenous bursting patterns, depending upon the balance of control by metabotropic glutamate (mGlu) and GABA(B) receptors. We further show that this modulation acts on at least one common target, the inwardly rectifying potassium channel (Kir3). Finally, we found that these firing modes correspond to specific functional states of information transfer in which dorsal horn neurons can faithfully transmit, greatly enhance or block the transfer of nociceptive information.     

Sodium nitroprusside and cGMP decrease Ca2+ channel availability in basilar artery smooth muscle cells

 We studied the effect of the nitric oxide (NO) donor, sodium nitroprusside (SNP), on the macroscopic and single-channel currents due to the 22-pS Ca²⁺ channel in smooth muscle cells from guinea pig basilar artery. In nystatin-perforated whole-cell recordings, 50 nM SNP decreased the macroscopic current to 63±12% of control values, without changing the voltage dependence of the current. In cell-attached patches with BAY-K8644 in the pipette, SNP caused a comparable decrease in single-channel availability (n ·P _o) that was dose dependent over the range of 10 nM to 10 μM SNP. SNP had no effect on single-channel properties, including slope conductance, voltage dependence of activation, the number of open states, the time constants of the open states, and the proportion of time spent in each open state. The effect of SNP (50 nM) on single Ca²⁺ channel openings was reproduced by 8-Br-cGMP (100 μM), which also reduced channel availability without altering channel properties. The protein kinase inhibitor H-8 (1.5 μM), which exhibits relative specificity for cGMP-dependent protein kinase, completely inhibited the decrease in single-channel availability expected with SNP. The dose-dependent decrease in Ca²⁺ channel availability caused by SNP was not altered by prior application of 8-Br-cAMP or forskolin, both of which cause an increase in Ca²⁺ channel availability in these cells. Our findings suggest that NO decreases openings of Ca²⁺ channels in basilar artery smooth muscle cells without altering channel properties, and that it does so by a mechanism likely to involve cGMP-dependent protein kinase. Received: 2 July 1996 / Received after revision: 30 September 1996 / Accepted: 2 October 1996     

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3:5-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g_j) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas g_j was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g_j in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (_j) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the _j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [³²P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [³²P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g_j, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.     

Control of established experimental allergic encephalomyelitis by inhibition of tumor necrosis factor (TNF) activity within the central nervous system using monoclonal antibodies and TNF receptor-immunoglobulin fusion proteins

Tumor necrosis factor (TNF) activity was inhibited during the development of actively-induced, chronic relapsing experimental allergic encephalomyelitis (CREAE) in Biozzi AB/H mice, using a mouse TNF-specific (TN3.19.12) antibody and bivalent human p55 and p75 TNF receptor-immunoglobulin (TNFR-Ig) fusion proteins. The development of disease could be inhibited when repeated doses of antibody were administered prior to the anticipated onset. It has now also been shown that a therapeutic effect is evident even when antibody is administered after the onset of clinical signs, further indicating an important role for TNF in pathogenic effector mechanisms in CREAE. Although biologically-active TNF was not detected in the circulation, TNF-α was detected in lesions within the central nervous system (CNS). This suggested that the CNS may be the main site for TNF-specific immunomodulation and was supported by the observation that intracranial injection was significantly more potent than that administered systemically, for both antibody and TNFR-Ig fusion proteins. The fusion proteins were as effective as antibody at doses 10—100-fold lower than that used for antibody, reflecting their higher neutralizing capacity in vitro. Although treatment was not curative and relapse inevitably occurred in this model if treatment was not sustained, the data indicate that anti-TNF immunotherapy, especially within the CNS, can inhibit CREAE and may, therefore, be useful in the control of human neuroimmunological diseases.     

Interleukin-1β and anaphylatoxins exert a synergistic effect on NGF expression by astrocytes

C3a and C5a anaphylatoxins are proinflammatory polypeptides released during complement activation. They exert their biological activities through interaction with two G protein-coupled receptors named C3aR and C5aR, respectively. In the brain, these receptors are expressed on glial cells, and some recent data have suggested that anaphylatoxins could mediate neuroprotection. In this study, we used RT-PCR and ribonuclease protection assays (RPA) to investigate the role of anaphylatoxins on neurotrophin expression by the human glioblastoma cell line T98G and by rat astrocytes. Our data show that for both cell types, anaphylatoxins upregulate expression of NGF mRNA. This response depended on a G protein-coupled pathway since pre-treatment of cells with pertussis toxin (PTX) completely blocked NGF mRNA increases. This effect was anaphylatoxin-specific since pre-incubation with anti-C3a or anti-C5aR antibodies abolished the effects of C3a and C5a, respectively. The regulation of NGF mRNA by anaphylatoxins was not accompanied by translation into protein expression, but there was a significant synergic effect of anaphylatoxins/IL-1b costimulation. Our demonstration of involvement of anaphylatoxins in the NGF release process by astrocytes suggests that C3a and C5a could modulate neuronal survival in the CNS.     

Molecular genetic analysis of familial early-onset Alzheimer's disease linked to chromosome 14q24.3

Genetic linkage studies have indicated that chromosome 14q24.3harbours a major locus for early-onset (onset age <65 years)Alzheimer's disease (AD3). Positional cloning efforts have identifieda novel gene S182 or presenilin 1 as the AD3 gene. We have mappedS182 in the AD3 candidate region between D14S277 and D14S284defined by genetic linkage studies in the two chromosome 14linked, early-onset AD families AD/A and AD/B. We have shownthat S182 is expressed in lymphoblasts and have determined thecomplete cDNA in both brain and lymphoblasts by RT-PCR sequencing.S182 is alternatively spliced in both brain and lymphoblastswithin a putative phosphorylation site located 5' in the codingregion. We identified two novel mutations, Ile143Thr and Gly384Alalocated in, respectively, the second transmembrane domain andin the sixth hydrophilic loop of the putative transmembranestructure of S182. As families AD/A and AD/B have a very similarAD phenotype our observation of two mutations in functionallydifferent domains suggest that onset age and severity of ADmay not be very helpful predictors of the location of putativeS182 mutations.     

Refined genetic mapping of autosomal recessive chronic distal spinal muscular atrophy to chromosome 11q13.3 and evidence of linkage disequilibrium in European families

Chronic distal spinal muscular atrophy (Chronic DSMA, MIM (*)607088) is a rare autosomal recessive disorder characterized by a progressive motor weakness and muscular atrophy, predominating in the distal parts of the limbs. A form of Chronic DSMA gene has been previously mapped to chromosome 11q13 in the 10.3 cM interval defined by loci D11S1889 and D11S1321. By linkage analysis in 12 European Chronic DSMA families, we showed that a disease gene maps to chromosome 11q13.3 (Z(max)=6.66 at theta=0.00 at the DSM4 locus) and suggested that this condition is genetically homogeneous. Recombination events allowed us to reduce the genetic interval to a 2.6 cM region, telomeric to the IGHMBP2 gene, excluding this gene as the disease causing gene in Chronic DSMA. Moreover, partial linkage disequilibrium was found between three rare alleles at loci D11S1369, DSM4 and D11S4184 and the mutant chromosome in European patients. Analysis of the markers at these loci strongly suggests that most Chronic DSMA chromosomes are derived from a single ancestor. Refinement of the Chronic DSMA locus will hopefully allow to test candidate genes and lead to identification of the disease-causing mutations.     

Force enhancement by PEG during ramp stretches of skeletal muscle

We have investigated the effects of a stronger actomyosin bond on force (Ps) during rapid stretch of active permeabilized rabbit psoas muscle fibers as a function of temperature from 5 to 30 degrees C. The strength of the actomyosin bond is enhanced by addition of polyethylene glycol (PEG), especially in pre-powerstroke states [Chinn et al. (2000) Biophys J 49: 437-451]. We have hypothesized that such states produce much of the force when activated muscles are stretched [Getz et al. (1998) Biophys J 75: 2971-2983]. Addition of PEG to activated fibers produced a small increase in isometric tension, Po (50-90 kN/m2), which was approximately independent of temperature. In contrast PEG produced a dramatic increase in Ps at low temperatures (200-300 kN/m2), but a modest increase at higher temperatures (70-90 kN/m2). We also measured Ps and Po in solutions containing the phosphate analog aluminum fluoride (AlF4) with and without PEG. In the absence of PEG, AlF4 reduced Po much more than Ps. Addition of PEG did not enhance Po, but enhanced Ps significantly. The contrasting effects of PEG on Ps and Po, and the effect of temperature can be explained by a model in which stretch force is produced by pre-powerstroke cross-bridges whose maximum distension is increased by PEG, and isometric force is produced by strongly bound cross-bridges whose bond strength is also enhanced by PEG, but to a lesser extent.