Francesca Grassi

Tumor necrosis factor alters synaptic transmission in rat hippocampal slices

The effects of human recombinant tumor necrosis factor (TNF-alpha) on the synaptic transmission were studied in rat hippocampal slices by using extracellular field potential recordings. Population spikes and/or excitatory postsynaptic potentials were extracellularly recorded in hippocampus CA1 region from stratum pyramidale and stratum radiatum, respectively, and synaptic transmission was examined in the Schaffer collateral/commissural-CA1 pathway. Basal neurotransmission slightly and promptly increased in slices acutely exposed to TNF-alpha (1-100 nM). Examination of the long-term potentiation (LTP) revealed that a brief treatment with the cytokine did not influence LTP, while a long-lasting application of TNF-alpha (50 min or more) inhibited LTP in a dose-dependent way in the range of 1-100 nM. A role for TNF-alpha as a peptide of immunological significance belonging to the family of brain neuromodulators is discussed.

Neuromuscular junction disassembly and muscle fatigue in mice lacking neurotrophin-4.

Neurotrophin-4 (NT-4) is produced by slow muscle fibers in an activity-dependent manner and promotes growth and remodeling of adult motorneuron innervation. However, both muscle fibers and motor neurons express NT-4 receptors, suggesting bidirectional NT-4 signaling at the neuromuscular junction. Mice lacking NT-4 displayed enlarged and fragmented neuromuscular junctions with disassembled postsynaptic acetylcholine receptor (AChR) clusters, reduced AChR binding, and acetylcholinesterase activity. Electromyographic responses, posttetanic potentiation, and action potential amplitude were also significantly reduced in muscle fibers from NT-4 knock-out mice. Slow-twitch soleus muscles from these mice fatigued twice as rapidly as those from wild-type mice during repeated tetanic stimulation. Thus, muscle-derived NT-4 is required for maintenance of postsynaptic AChR regions, normal muscular electrophysiological responses, and resistance to muscle fatigue. This neurotrophin may therefore be a key component of an activity-dependent feedback mechanism regulating maintenance of neuromuscular connections and muscular performance.

Acetylcholine May Regulate its Own Nicotinic Receptor-Channel through the C-Kinase System

Acetylcholine (ACh)-activated channel properties were examined on an aneural culture of chick embryo myotubes by using patch-clamp techniques. Changes in conductance, open time and closed time were induced by the selective activator of the calcium- and phospholipid-dependent C-kinase (PKc), 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of TPA was mimicked by exogenous phospholipase C and was blocked by the PKc inhibitor, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine. In addition to its gating action, ACh was shown to stimulate phosphoinositide turnover and to translocate PKc from the cytosol to the cell membrane. Both these ACh-induced effects were inhibited by curare and not substantially affected by atropine. Bath-applied ACh outside the patch-pipette in the cell-attached patch-clamp mode, had a strong effect on the ACh-activated channels in the patch membrane, in a way that resembled the action of TPA . These findings raise the possibility that ACh regulates its own nicotinic receptors through the C-kinase system.

TNF-α increases the frequency of spontaneous miniature synaptic currents in cultured rat hippocampal neurons

Abstract Tumor necrosis factor-α (TNF-α) is a cytokine secreted by activated astrocytes and is known to alter evoked synaptic activity in slices of adult rat hippocampus. In this paper we show that TNF-α increases the frequency of spontaneous miniature synaptic currents in cultured hippocampal neurons, acting at nanomolar concentrations. In addition, we show that the mRNA for the 55 kDa TNF-α receptor (TNF-R1) is detected in embryonic rat hippocampal cultures, as well as in acutely dissected embryonic and adult rat hippocampi. Possible transduction pathways mediating the TNF-α effect are discussed.

Interferon inhibits synaptic potentiation in rat hippocampus

The effects of rat interferon (IFN) on the electrically-induced potentiation of the synaptic transmission were studied in rat hippocampal slices by using extracellular field potential recordings. The treatment with rat IFN (120 U/ml) reduced the size of short-term potentiation (STP) and suppressed long-term potentiation (LTP). These IFN-induced effects were dose-dependent in the range of 50-500 U/ml. In addition, IFN slightly attenuated the potentiation when applied during the maintenance of LTP. Basal synaptic transmission was affected by IFN at concentrations greater than or equal to 250 U/ml. Following an acute exposure to IFN (500-200 U/ml), cultured embryonic neurones from rat hippocampus often exhibited an attenuation of N-methyl-D-aspartate-induced currents and a variation (increase or decrease) of voltage-activated Ca2+ current amplitude. A possible role of IFN as neuromodulator in mammalian brain during immune responses is discussed.

α5 Subunit forms functional α3β4α5 nAChRs in transfected human cells

NACHRS heterologously expressed in human cells after transient transfection with α3β4α5 or α3β4 subunit cDNAs exhibited similar sensitivities to antagonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose—current response curves after co-expression of α5, α3 and β4 subunits. One component was comparable to that obtained in α3β4-transfected cells, while the additional component, putatively attributed to an α3β4α5 nAChR population, showed a Hill coefficient > 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the α5 subunit participates in the assembly of α3β4α5 nAChRs complexes in human cells, adding a new member to the family of neuronal nicotinic receptors.

Acetylcholine receptor channels are present in undifferentiated satellite cells but not in embryonic myoblasts in culture

The expression and the physiological properties of acetylcholine receptors (AChRs) of mononucleated myogenic cells, isolated from either embryonic or adult muscle of the mouse, have been investigated using the gigaohm seal patch-clamp technique in combination with immunocytochemistry (with an anti-myosin antibody) and alpha-bungarotoxin binding techniques. Undifferentiated (myosin-negative) embryonic myoblasts, grown either in mass culture or under clonal conditions, were found to be unresponsive to ACh and did not bind alpha-bungarotoxin. On the contrary, undifferentiated satellite cells (from adult muscle) exhibited channels activated by ACh and alpha-bungarotoxin binding sites similar to those observed in differentiated (myosin-positive) embryonic myoblasts and myotubes. Two classes of ACh-activated channels with different opening frequencies were identified. The major class of channels had a conductance of about 42 pS and mean open time of 3.1-8.2 msec. The minor class of channels had smaller conductance (about 17 pS) and similar open time. During differentiation, the conductance of the two channels did not change significantly, while channel lifetime became shorter in myotubes derived from satellite cells but not in myotubes derived from embryonic myoblasts. The relative proportion of small over large channels was significantly larger in embryonic than in adult myogenic cells.

The human adult subtype ACh receptor channel has high Ca2+ permeability and predisposes to endplate Ca2+ overloading

Slow‐channel congenital myasthenic syndrome, caused by mutations in subunits of the endplate ACh receptor (AChR), results in prolonged synaptic currents and excitotoxic injury of the postsynaptic region by Ca2+ overloading. The Ca2+ overloading could be due entirely to the prolonged openings of the AChR channel or could be abetted by enhanced Ca2+ permeability of the mutant channels. We therefore measured the fractional Ca2+ current, defined as the percentage of the total ACh‐evoked current carried by Ca2+ ions (Pf), for AChRs harbouring the αG153S or the αV249F slow‐channel mutation, and for wild‐type human AChRs in which Pf has not yet been determined. Experiments were performed in transiently transfected GH4C1 cells and human myotubes with simultaneous recording of ACh‐evoked whole‐cell currents and fura‐2 fluorescence signals. We found that the Pf of the wild‐type human endplate AChR was unexpectedly high (Pf∼7%), but neither the αV249F nor the αG153S mutation altered Pf. Fetal human AChRs containing either the wild‐type or the mutated α subunit had a much lower Pf (2–3%). We conclude that the Ca2+ permeability of human endplate AChRs is higher than that reported for any other human nicotinic AChR, with the exception of α7‐containing AChRs (Pf > 10%); and that neither the αG153S nor the αV249F mutations affect the Pf of fetal or adult endplate AChRs. However, the intrinsically high Ca2+ permeability of human AChRs probably predisposes to development of the endplate myopathy when opening events of the AChR channel are prolonged by altered AChR‐channel kinetics.

Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)‐induced single‐channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat α3β4, chick α<3/β2, α4β2, α4β4, α7and α8, expression levels were high enough to allow measurements of acetylcholine‐evoked whole‐cell currents and nicotine‐elicited Ca2+ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine‐evoked events of α8 nAChR had a slope conductance of 23 pS, whereas two conductance classes (19–23 and 32–45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric α7 and α8 nAChRs (6–7 ms) than for heteromeric nAChRs (1–3 ms), with the exception of α3α4nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (α3α4and α3α2) exhibited single‐channel characteristics similar to those reported for native receptors. The variety of nAChRs channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.

Rare Missense Variants of Neuronal Nicotinic Acetylcholine Receptor Altering Receptor Function Are Associated with Sporadic Amyotrophic Lateral Sclerosis

Sporadic amyotrophic lateral sclerosis (SALS) is a motor neuron degenerative disease of unknown etiology. Current thinking on SALS is that multiple genetic and environmental factors contribute to disease liability. Since neuronal acetylcholine receptors (nAChRs) are part of the glutamatergic pathway, we searched for sequence variants in CHRNA3, CHRNA4 and CHRNB4 genes, encoding neuronal nicotinic AChR subunits, in 245 SALS patients and in 450 controls. We characterized missense variants by in vitro mutagenesis, cell transfection and electrophysiology. Sequencing the regions encoding the intracellular loop of AChRs subunits disclosed 15 missense variants (6.1%) in 14 patients compared with only six variants (1.3%) in controls (P = 0.001; OR 4.48, 95% CI 1.7-11.8). The frequency of variants in exons encoding extracellular and transmembrane domains and in intronic regions did not differ. NAChRs formed by mutant alpha3 and alpha4 and wild-type (WT) beta4 subunits exhibited altered affinity for nicotine (Nic), reduced use-dependent rundown of Nic-activated currents (I(Nic)) and reduced desensitization leading to sustained intracellular Ca(2+) concentration, in comparison with WT-nAChR. The cellular loop has a crucial importance for receptor trafficking and regulating ion channel properties. Missense variants in this domain are significantly over-represented in SALS patients and alter functional properties of nAChR in vitro, resulting in increased Ca(2+) entry into the cells. We suggest that these gain-of-function variants might contribute to disease liability in a subset of SALS because Ca(2+) signals mediate nAChRs neuromodulatory effects, including regulation of glutamate release and control of cell survival.