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TMEM16A is a Ca 2+ -activated Cl - channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 agonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation-transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the lack of muscle electrical activity, as judged by partial reversal upon field electrical stimulation of in vitro denervated skeletal muscle fibers., (© 2022 Volpe et al.)

Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts.

It has long been known that regular physical exercise induces short and long term benefits reducing the risk of cardiovascular disease, diabetes, osteoporosis, cancer and improves sleep quality, cognitive level, mobility, autonomy in enderly. More recent is the evidence on the endocrine role of the contracting skeletal muscle. Exercise triggers the release of miokines, which act in autocrine, paracrine and endocrine ways controlling the activity of muscles but also of other tissues and organs such as adipose tissue, liver, pancreas, bones, and brain. The mechanism of release is still unclear. Neuromuscular electrical stimulation reproduces the beneficial effects of physical activity producing physiological metabolic, cardiovascular, aerobic responses consistent with those induced by exercise. In vitro, Electrical Pulse Stimulations (EPS) of muscle cells elicit cell contraction and mimic miokine release in the external medium. Here we show that, in cultured mouse myotubes, EPS induce contractile activity and the release of the myokine IL-6. Gadolinium highly reduces EPS-induced IL-6 release, suggesting the involvement of mechanical activated ion channels. The chemical activation of mechanosensitive Piezo1 channels with the specific agonist Yoda1 stimulates IL-6 release similarly to EPS, suggesting the involvement of Piezo1 channels in the control of the myokine release. The expression of Piezo1 protein in myotubes was confirmed by the Western blot analysis. To the best of our knowledge, this is the first evidence of a Piezo1-mediated effect in myokine release and suggests a potential translational use of specific Piezo1 agonists for innovative therapeutic treatments reproducing/enhancing the benefits of exercise mediated by myokines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Piezo1 channels are highly mechanically-activated cation channels that can sense and transduce the mechanical stimuli into physiological signals in different tissues including skeletal muscle. In this focused review, we summarize the emerging evidence of Piezo1 channel-mediated effects in the physiology of skeletal muscle, with a particular focus on the role of Piezo1 in controlling myogenic precursor activity and skeletal muscle regeneration and vascularization. The disclosed effects reported by pharmacological activation of Piezo1 channels with the selective agonist Yoda1 indicate a potential impact of Piezo1 channel activity in skeletal muscle regeneration, which is disrupted in various muscular pathological states. All findings reported so far agree with the idea that Piezo1 channels represent a novel, powerful molecular target to develop new therapeutic strategies for preventing or ameliorating skeletal muscle disorders characterized by an impairment of tissue regenerative potential.

Aim: Mechanosensitive Piezo1 ion channels emerged recently as important contributors to various vital functions including modulation of the blood supply to skeletal muscles. The specific Piezo1 channel agonist Yoda1 was shown to regulate the tone of blood vessels similarly to physical exercise. However, the direct role of Piezo1 channels in muscle function has been little studied so far. We therefore investigated the action of Yoda1 on the functional state of skeletal muscle precursors (satellite cells and myotubes) and on adult muscle fibres., Methods: Immunostaining, electrophysiological intracellular recordings and Ca 2+ imaging experiments were performed to localize and assess the effect of the chemical activation of Piezo1 channels with Yoda1, on myogenic precursors, adult myofibres and at the adult neuromuscular junction., Results: Piezo1 channels were detected by immunostaining in satellite cells (SCs) and myotubes as well as in adult myofibres. In the skeletal muscle precursors, Yoda1 treatment stimulated the differentiation and cell fusion rather than the proliferation of SCs. Moreover, in myotubes, Yoda1 induced significant [Ca 2+ ] i transients, without detectable [Ca 2+ ] i response in adult myofibres. Furthermore, although expression of Piezo1 channels was detected around the muscle endplate region, Yoda1 application did not alter either the nerve-evoked or spontaneous synaptic activity or muscle contractions in adult myofibres., Conclusion: Our data indicate that the chemical activation of Piezo1 channels specifically enhances the differentiation of skeletal muscle precursors, suggesting a possible new strategy to promote muscle regeneration., (© 2021 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Homer represents a diversified family of scaffold and transduction proteins made up of several isoforms. Here, we present preliminary observations on skeletal muscle adaptation and plasticity in a transgenic model of Homer 2 -/- mouse using a multifaceted approach entailing morphometry, quantitative RT-PCR (Reverse Transcription PCR), confocal immunofluorescence, and electrophysiology. Morphometry shows that Soleus muscle (SOL), at variance with Extensor digitorum longus muscle (EDL) and Flexor digitorum brevis muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts. In SOL of Homer 2 -/- mice, quantitative RT-PCR indicated the upregulation of Atrogin-1 and Muscle ring finger protein 1 (MuRF1) genes, and confocal immunofluorescence showed the decrease of neuromuscular junction (NMJ) Homer content. Electrophysiological measurements of isolated FDB fibres from Homer 2 -/- mice detected the exclusive presence of the adult ε-nAChR isoform excluding denervation. As for NMJ morphology, data were not conclusive, and further work is needed to ascertain whether the null Homer 2 phenotype induces any endplate remodelling. Within the context of adaptation and plasticity, the present data show that Homer 2 is a co-regulator of the normotrophic status in a muscle specific fashion.

Since the pioneering works of Ricardo Miledi, the neuromuscular junction represents the best example of a synapse where ACh is the neurotransmitter acting on nicotinic ACh receptors. ATP, co-released with ACh, is promptly degraded to Ado, which acts as a modulator of the cholinergic synaptic activity. Consequently, both ACh and adenosine play a crucial role in controlling the nerve-muscle communication. Apart from their role in the context of synaptic transmission, ACh and adenosine are autocrinally released by skeletal muscle cells, suggesting also a non nerve-driven function of these molecules. Indeed, the existence of cholinergic and adenosinergic systems has been widely described in many other non neuronal cell types. In this review, we will describe the two systems and their interplay in non-innervated differentiating skeletal muscle cells, and in innervated adult skeletal muscle fibers. We believe that the better comprehension of the interactions between the activity of nAChRs and adenosine could help the knowledge of skeletal muscle physiology. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries., Competing Interests: Declarations of interest None., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

An in vitro system of electrical stimulation was used to explore whether an innovative "noisy" stimulation protocol derived from human electromyographic recordings (EMGstim) could promote muscle regeneration. EMGstim was delivered to cultured mouse myofibers isolated from Flexor Digitorum Brevis, preserving their satellite cells. In response to EMGstim, immunostaining for the myogenic regulatory factor myogenin, revealed an increased percentage of elongated myogenin-positive cells surrounding the myofibers. Conditioned medium collected from EMGstim-treated cell cultures, promoted satellite cells differentiation in unstimulated myofiber cell cultures, suggesting that extracellular soluble factors could mediate the process. Interestingly, the myogenic effect of EMGstim was mimicked by exogenously applied ATP (0.1 μM), reduced by the ATP diphosphohydrolase apyrase and prevented by blocking endogenous ATP release with carbenoxolone. In conclusion, our results show that "noisy" electrical stimulations favor muscle progenitor cell differentiation most likely via the release of endogenous ATP from contracting myofibres. Our data also suggest that "noisy" stimulation protocols could be potentially more efficient than regular stimulations to promote in vivo muscle regeneration after traumatic injury or in neuropathological diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The biochemical properties of muscle extracellular matrix are essential for stem cell adhesion, motility, proliferation and myogenic development. Recombinant elastin-like polypeptides are synthetic polypeptides that, besides maintaining some properties of the native protein, can be tailored by fusing bioactive sequences to their C-terminal. Our laboratory synthesized several Human Elastin-Like Polypeptides (HELP) derived from the sequence of human tropoelastin. Here, we developed a novel HELP family member by fusing the elastin-like backbone to the sequence of human Epidermal Growth Factor. We employed this synthetic protein, named HEGF, either alone or in combination with other proteins of the HELP family carrying RGD-integrin binding sites, as adhesion substrate for C2C12 myoblasts and satellite cells primary cultures. Adhesion of myoblasts to HEGF-based substrates induced scattering, decreased adhesion and cytoskeleton assembly; the concomitant presence of the RGD motifs potentiated all these effects. Recombinant substrates induced myoblasts proliferation, differentiation and the development of multinucleated myotubes, thus favoring myoblasts expansion and preserving their myogenic potential. The effects induced by adhesion substrates were inhibited by AG82 (Tyrphostin 25) and herbimycin A, indicating their dependence on the activation of both the EGF receptor and the tyrosine kinase c-src. Finally, HEGF increased the number of muscle stem cells (satellite cells) derived from isolated muscle fibers in culture, thus highlighting its potential as a novel substrate for skeletal muscle regeneration strategies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Adenosine is a powerful modulator of skeletal neuromuscular transmission, operating via inhibitory or facilitatory purinergic-type P1 receptors. To date, studies have been focused mainly on the effect of adenosine on presynaptic P1 receptors controlling transmitter release. In this study, using two-microelectrode voltage-clamp and single-channel patch-clamp recording techniques, we have explored potential postsynaptic targets of adenosine and their modulatory effect on nicotinic acetylcholine receptor (nAChR)-mediated synaptic responses in adult mouse skeletal muscle fibers in vitro. In the whole-mount neuromuscular junction (NMJ) preparation, adenosine (100 μM) significantly reduced the frequency of the miniature endplate currents (MEPCs) and slowed their rising and decay time. Consistent with a postsynaptic site of action, adenosine and the potent P1 receptor agonist NECA significantly increased the open probability, the frequency and the open time of single nAChR channels, recorded at the endplate region. Using specific ligands for the P1 receptor subtypes, we found that the low-affinity P1 receptor subtype A 2B was responsible for mediating the effects of adenosine on the nAChR channel openings. Our data suggest that at the adult mammalian NMJ, adenosine acts not only presynaptically to modulate acetylcholine transmitter release, but also at the postsynaptic level, to enhance the activity of nAChRs. Our findings open a new scenario in understanding of purinergic regulation of nAChR activity at the mammalian endplate region., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Acetylcholinesterase (AChE) and agrin, a heparan-sulfate proteoglycan, reside in the basal lamina of the neuromuscular junction (NMJ) and play key roles in cholinergic transmission and synaptogenesis. Unlike most NMJ components, AChE and agrin are expressed in skeletal muscle and α-motor neurons. AChE and agrin are also expressed in various other types of cells, where they have important alternative functions that are not related to their classical roles in NMJ. In this review, we first focus on co-cultures of embryonic rat spinal cord explants with human skeletal muscle cells as an experimental model to study functional innervation in vitro. We describe how this heterologous rat-human model, which enables experimentation on highly developed contracting human myotubes, offers unique opportunities for AChE and agrin research. We then highlight innovative approaches that were used to address salient questions regarding expression and alternative functions of AChE and agrin in developing human skeletal muscle. Results obtained in co-cultures are compared with those obtained in other models in the context of general advances in the field of AChE and agrin neurobiology., Competing Interests: The authors declare no conflict of interest.

Electrical stimulation (ES) of skeletal muscle partially mimics the benefits of physical activity. However, the stimulation protocols applied clinically to date, often cause unpleasant symptoms and muscle fatigue. Here, we compared the efficiency of a "noisy" stimulus waveform derived from human electromyographic (EMG) muscle patterns, with stereotyped 45 and 1 Hz electrical stimulations applied to mouse myotubes in vitro. Human gastrocnemius medialis electromyograms recorded from volunteers during real locomotor activity were used as a template for a noisy stimulation, called EMGstim. The stimulus-induced electrical activity, intracellular Ca(2+) dynamics and mechanical twitches in the myotubes were assessed using whole-cell perforated patch-clamp, Ca(2+) imaging and optical visualization techniques. EMGstim was more efficient in inducing myotube cell firing, [Ca(2+)]i changes and contractions compared with more conventional electrical stimulation. Its stimulation strength was also much lower than the minimum required to induce contractions via stereotyped stimulation protocols. We conclude that muscle cells in vitro can be more efficiently depolarized using the "noisy" stochastic stimulation pattern, EMGstim, a finding that suggests a way to favor a higher level of electrical activity in a larger number of cells.

Aims: The autocrine activity of the embryonic isoform of the nicotinic acetylcholine receptor is crucial for the correct differentiation and trophism of skeletal muscle cells before innervation. The functional activity of extracellular adenosine and adenosine receptor subtypes expressed in differentiating myotubes is still unknown. In this study, we performed a detailed analysis of the role of adenosine receptor-mediated effects on the autocrine-mediated nicotinic acetylcholine receptor channel openings and the associated spontaneous intracellular calcium 'spikes' generated in differentiating mouse myotubes in vitro., Methods: Cell-attached patch-clamp recordings and intracellular calcium imaging experiments were performed in contracting myotubes derived from mouse satellite cells., Results: The endogenous extracellular adenosine and the adenosine receptor-mediated activity modulated the properties of the embryonic isoform of the nicotinic acetylcholine receptor in myotubes in vitro, by increasing the mean open time and the open probability of the ion channel, and sustaining nicotinic acetylcholine receptor-driven intracellular [Ca(2+) ]i 'spikes'. The pharmacological characterization of the adenosine receptor-mediated effects suggested a prevalent involvement of the A2B adenosine receptor subtype., Conclusion: We propose that the interplay between endogenous adenosine and nicotinic acetylcholine receptors represents a potential novel strategy to improve differentiation/regeneration of skeletal muscle., (© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Rett Syndrome (RTT) is a neurodevelopmental disorder associated with intellectual disability, mainly caused by loss-of-function mutations in the MECP2 gene. RTT brains display decreased neuronal size and dendritic arborization possibly caused by either a developmental failure or a deficit in the maintenance of dendritic arbor structure. To distinguish between these two hypotheses, the development of Mecp2-knockout mouse hippocampal neurons was analyzed in vitro. Since a staging system for the in vitro development of mouse neurons was lacking, mouse and rat hippocampal neurons development was compared between 1-15 days in vitro (DIV) leading to a 6-stage model for both species. Mecp2-knockout hippocampal neurons displayed reduced growth of dendritic branches from stage 4 (DIV4) onwards. At stages 5-6 (DIV9-15), synapse number was lowered in Mecp2-knockout neurons, suggesting increased synapse elimination. These results point to both a developmental and a maintenance setback affecting the final shape and function of neurons in RTT.

Palytoxin (PLTX) is a highly toxic hydrophilic polyether detected in several edible marine organisms from intra-tropical areas, where seafood poisoning were reported. Symptoms usually start with gastro-intestinal malaise, often accompanied by myalgia, muscular cramps, dyspnea and, sometimes, arrhythmias. Monitoring programs in the Mediterranean Sea have detected PLTX-like molecules in edible mollusks and echinoderms. Despite the potential exposure of the human population and its high toxic potential, the toxicological profile of the molecule is still an issue. Thus, the effects of repeated oral administration of PLTX in mice were investigated. Seven days of PLTX administration caused lethality and toxic effects at doses ≥ 30 μg/kg/day. A NOAEL was estimated equal to 3 μg/kg/day, indicating a quite steep dose-response curve. This value, due to the limited number of animal tested, is provisional, although represents a sound basis for further testing. Macroscopic alterations at gastrointestinal level (gastric ulcers and intestinal fluid accumulation) were observed in mice dead during the treatment period. Histological analysis highlighted severe inflammation, locally associated with necrosis, at pulmonary level, as well as hyper-eosinophilia and fiber separation in myocardium. A cardiac damage was supported by the in vitro effect of the toxin on cardiomyocytes, indicating a severe and irreversible impairment of their electrical properties: electrophysiological recordings detected a progressive cell depolarization, arrest of action potentials and beating., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Long-term potentiation (LTP) is accompanied by increased spine density and dimensions triggered by signaling cascades involving activation of the neurotrophin brain-derived neurotrophic factor (BDNF) and cytoskeleton remodeling. Chemically-induced long-term potentiation (c-LTP) is a widely used cellular model of plasticity, whose effects on spines have been poorly investigated. We induced c-LTP by bath-application of the N-methyl-d-aspartate receptor (NMDAR) coagonist glycine or by the K(+) channel blocker tetraethylammonium (TEA) chloride in cultured hippocampal neurons and compared the changes in dendritic spines induced by the two models of c-LTP and determined if they depend on BDNF/TrkB signaling. We found that both TEA and glycine induced a significant increase in stubby spine density in primary and secondary apical dendrites, whereas a specific increase in mushroom spine density was observed upon TEA application only in primary dendrites. Both TEA and glycine increased BDNF levels and the blockade of tropomyosin-receptor-kinase receptors (TrkRs) by the nonselective tyrosine kinase inhibitor K-252a or the selective allosteric TrkB receptor (TrkBR) inhibitor ANA-12, abolished the c-LTP-induced increase in spine density. Surprisingly, a blockade of TrkBRs did not change basal spontaneous glutamatergic transmission but completely changed the synaptic plasticity induced by c-LTP, provoking a shift from a long-term increase to a long-term depression (LTD) in miniature excitatory postsynaptic current (mEPSC) frequency. In conclusion, these results suggest that BDNF/TrkB signaling is necessary for c-LTP-induced plasticity in hippocampal neurons and its blockade leads to a switch of c-LTP into chemical-LTD (c-LTD)., (Copyright © 2013 Wiley Periodicals, Inc.)

Acetylcholinesterase (AChE) and agrin play unique functional roles in the neuromuscular junction (NMJ). AChE is a cholinergic and agrin a synaptogenetic component. In spite of their different functions, they share several common features: their targeting is determined by alternative splicing; unlike most other NMJ components they are expressed in both, muscle and motor neuron and both reside on the synaptic basal lamina of the NMJ. Also, both were reported to play various nonjunctional roles. However, while the origin of basal lamina bound agrin is undoubtedly neural, the neural origin of AChE, which is anchored to the basal lamina with collagenic tail ColQ, is elusive. Hypothesizing that motor neuron proteins targeted to the NMJ basal lamina share common temporal pattern of expression, which is coordinated with the formation of basal lamina, we compared expression of agrin isoforms with the expression of AChE-T and ColQ in the developing rat spinal cord at the stages before and after the formation of NMJ basal lamina. Cellular origin of AChE-T and agrin was determined by in situ hybridization and their quantitative levels by RT PCR. We found parallel increase in expression of the synaptogenetic (agrin 8) isoform of agrin and ColQ after the formation of basal lamina supporting the view that ColQ bound AChE and agrin 8 isoform are destined to the basal lamina. Catalytic AChE-T subunit and agrin isoforms 19 and 0 followed different expression patterns. In accordance with the reports of other authors, our investigations also revealed various alternative functions for AChE and agrin. We have already demonstrated participation of AChE in myoblast apoptosis; here we present the evidence that agrin promotes the maturation of heavy myosin chains and the excitation-contraction coupling. These results show that common features of AChE and agrin extend to their capacity to play multiple roles in muscle development., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Reactive oxygen species (ROS) and extracellular adenosine 5'-triphosphate (ATP) participate in autocrine and paracrine regulation in skeletal muscle. However, the link between these two signaling systems is not well established. Here, we studied cell proliferation as a possible consequence of the trophic effect of ATP in cultured skeletal mouse myoblasts and we tested the possibility that low concentrations of ROS represent the intermediate signaling molecule mediating this effect. Exposure to 10 μM ATP increased proliferation of mouse myoblasts by ~20%. ATP also induced intracellular Ca(2+) oscillations, which were independent of extracellular Ca(2+). Both effects of ATP were prevented by suramin, a broad-spectrum purinergic P2 receptor antagonist. In contrast, the adenosine receptor blocker CGS-15943 did not modify the ATP-mediated effects. Consistent with this, adenosine per se did not change myoblast growth, indicating the direct action of ATP via P2 receptor activation. The proliferative effect of ATP was prevented after depletion of hydrogen peroxide (H(2)O(2)) by the peroxidase enzyme catalase. Low-micromolar concentrations of exogenous H(2)O(2) mimicked the stimulatory effect of ATP on myoblast growth. DCF imaging revealed ATP-induced catalase and DPI-sensitive ROS production in myoblasts. In conclusion, our results indicate that extracellular ATP controls mouse myoblast proliferation via induction of ROS generation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Brain-derived neurotrophic factor (BDNF) is encoded by multiple BDNF transcripts, whose function is unclear. We recently showed that a subset of BDNF transcripts can traffic into distal dendrites in response to electrical activity, while others are segregated into the somatoproximal domains. Physical exercise and antidepressant treatments exert their beneficial effects through upregulation of BDNF, which is required to support survival and differentiation of newborn dentate gyrus (DG) neurons. While these DG processes are required for the antidepressant effect, a role for CA1 in antidepressant action has been excluded, and the effect on CA3 neurons remains unclear. Here, we show for the first time that physical exercise and antidepressants induce local increase of BDNF in CA3. Voluntary physical exercise for 28 consecutive days, or 2-week treatment with 10 mg/kg per day fluoxetine or reboxetine, produced a global increase of BDNF mRNA and protein in the neuronal somata of the whole hippocampus and a specific increase of BDNF in dendrites of CA3 neurons. This increase was accounted for by BDNF exon 6 variant. In cultured hippocampal neurons, application of serotonin or norepinephrine (10-50 μM) induced increase in synaptic transmission and targeting of BDNF mRNA in dendrites. The increased expression of BDNF in CA3 dendrites following antidepressants or exercise further supports the neurotrophin hypothesis of antidepressants action and confirms that the differential subcellular localization of BDNF mRNA splice variants provides a spatial code for a selective expression of BDNF in specific subcellular districts. This selective expression may be exploited to design more specific antidepressants.

Background and Objectives: The few existing studies of sexual dysfunction in women on hemodialysis are limited by small sample size. This large, cross-sectional study evaluated the prevalence and correlates of female sexual dysfunction in advanced kidney disease. DESIGN, SETTING, PARTICIPANTS, METHODS: A total of 1472 women with ESRD undergoing hemodialysis were recruited to a multinational, cross-sectional study conducted within a collaborative dialysis network in Europe and South America. Sexual dysfunction was identified by the Female Sexual Function Index. Correlates of self-reported sexual dysfunction were identified by regression analyses., Results: Of the 1472 women, 659 completed questionnaires (45%). More than half (362 of 659 [55%]) lived with a partner, and 232 of 659 (35%) reported being sexually active. Of these 659 respondents, 555 (84%) reported sexual dysfunction. Women with a partner (282 of 362 [78%]) were less likely to report sexual dysfunction than those without a partner (273 of 297 [92%]) (P<0.001). Sexual dysfunction was independently associated with age, depressive symptoms, less education, menopause, diabetes, and diuretic therapy. Nearly all women who were not wait-listed for a kidney transplant and were living without a partner (249 of 260 [96%]) reported sexual dysfunction. More than half (128 of 232 [55%]) of sexually active women reported sexual dysfunction, associated with age, depressive symptoms, menopause, low serum albumin, and diuretic therapy., Conclusions: This descriptive study suggests most women on hemodialysis experience sexual problems. Additional research on the relevance of sexual dysfunction to symptom burden and quality of life in these women is needed.

Background: Factors associated with erectile dysfunction in men on haemodialysis are incompletely identified due to suboptimal existing studies. We determined the prevalence and correlates of erectile dysfunction and identified combinations of clinical characteristics associated with a higher risk of erectile dysfunction using recursive partitioning and amalgamation (REPCAM) analysis., Methods: We conducted a multinational cross-sectional study in men on haemodialysis within a collaborative network. Erectile dysfunction and depressive symptoms were evaluated using the erectile function domain of the International Index of Erectile Function questionnaire and the Center for Epidemiological Studies-Depression Scale, respectively., Results: Nine hundred and forty-six (59%) of 1611 eligible men provided complete data for erectile dysfunction. Eighty-three per cent reported erectile dysfunction and 47% reported severe erectile dysfunction. Four per cent of those with erectile dysfunction were receiving pharmacological treatment. Depressive symptoms were the strongest correlate of erectile dysfunction [adjusted odds ratio 2.41 (95% confidence interval (CI) 1.57-3.71)]. Erectile dysfunction was also associated with age (1.06, 1.05-1.08), being unemployed (1.80, 1.17-2.79) or receiving a pension (2.05, 1.14-3.69) and interdialytic weight gain (1.9-2.87 kg, 1.92 [CI 1.19-3.09]; >2.87 kg, 1.57 [CI 1.00-2.45]). Married men had a lower risk of erectile dysfunction (0.49, 0.31-0.76). The prevalence of erectile dysfunction was highest (94%) in unmarried and unemployed or retired men who have depressive symptoms., Conclusions: Most men on haemodialysis experience erectile dysfunction and are untreated. Given the prevalence of this condition and the relative lack of efficacy data for pharmacological agents, we suggest that large trials of pharmacological and non-pharmacological interventions for erectile dysfunction and depression are needed.

Adult spinal cord has little regenerative potential, thus limiting patient recovery following injury. In this study, we describe a new population of cells resident in the adult rat spinal cord meninges that express the neural stem/precursor markers nestin and doublecortin. Furthermore, from dissociated meningeal tissue a neural stem cell population was cultured in vitro and subsequently shown to differentiate into functional neurons or mature oligodendrocytes. Proliferation rate and number of nestin- and doublecortin-positive cells increased in vivo in meninges following spinal cord injury. By using a lentivirus-labeling approach, we show that meningeal cells, including nestin- and doublecortin-positive cells, migrate in the spinal cord parenchyma and contribute to the glial scar formation. Our data emphasize the multiple roles of meninges in the reaction of the parenchyma to trauma and indicate for the first time that spinal cord meninges are potential niches harboring stem/precursor cells that can be activated by injury. Meninges may be considered as a new source of adult stem/precursor cells to be further tested for use in regenerative medicine applied to neurological disorders, including repair from spinal cord injury., (Copyright © 2011 AlphaMed Press.)

Reactive oxygen species (ROS), normally generated in skeletal muscles, could control excitability of muscle fibers through redox modulation of membrane ion channels. However, the mechanisms of ROS action remain largely unknown. To investigate the action of ROS on electrical properties of muscle cells, patch-clamp recordings were performed after application of hydrogen peroxide (H₂O₂) to skeletal myotubes. H₂O₂ facilitated sodium spikes after a hyperpolarizing current pulse, by decreasing the latency for spike initiation. Importantly, the antioxidant N-acetylcysteine induced the opposite effect, suggesting the redox control of muscle excitability. The effect of H₂O₂ was abolished in the presence of catalase. The kinetics of sodium channels were not affected by H₂O₂. However, the fast inward rectifier K(+) (K(IR)) currents, activated by hyperpolarization, were reduced by H₂O₂, similar to the action of the potassium channel blockers Ba(2+) and Cs(+). The block of the outward tail current contributing to K(IR) deactivation can explain the shorter latency for spike initiation. We propose that the K(IR) current is an important target for ROS action in myotubes. Our data would thus suggest that ROS are involved in the control of the excitability of myotubes and, possibly, in the oscillatory behavior critical for the plasticity of developing muscle cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.

Recent investigations suggest that the effects of neural agrin might not be limited to neuromuscular junction formation and maintenance and that other aspects of muscle development might be promoted by agrin. Here we tested the hypothesis that agrin induces a change in the excitability properties in primary cultures of non-innervated human myotubes. Electrical membrane properties of human myotubes were recorded using the whole-cell patch-clamp technique. Cell incubation with recombinant chick neural agrin (1 nM) led to a more negative membrane resting potential. Addition of strophanthidin, a blocker of the Na(+)/K(+) ATPase, depolarized agrin-treated myotubes stronger than control, indicating, in the presence of agrin, a higher contribution of the Na(+)/K(+) ATPase in establishing the resting membrane potential. Indeed, larger amounts of both the alpha1 and the alpha2 isoforms of the Na(+)/K(+) ATPase protein were expressed in agrin-treated cells. A slight but significant down-regulation of functional apamin-sensitive K(+) channels was observed after agrin treatment. These results indicate that neural agrin might act as a trophic factor promoting the maturation of membrane electrical properties during differentiation, confirming the role of agrin as a general promoter of muscle development.

Oral administration of yessotoxin (YTX) has been reported to induce ultrastructural alterations in rodent cardiac muscle. To study its effects on various fundamental aspects of cardiac muscle cells activity, that is, cell beating, Ca(2+) and cyclic adenosine 3',5'-monophosphate (cAMP) levels, as well as cell vitality, a primary culture of rat cardiomyocytes was used. Patch-clamp recordings, Ca(2+) imaging, and cAMP assays were performed on cultured cardiomyocytes to characterize YTX effects on the cell beating frequency. 3-(4,5-Dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) and sulforhodamine B (SRB) tests were carried out to determine its effect on cardiomyocytes viability. Videoimaging techniques showed a time- and concentration-dependent reduction in the beating frequency after 1, 5, and 24 h incubation with YTX (0.1-1 microM). This effect was neither associated to the uncoupling between the membrane electrical activity and Ca(2+) release from intracellular stores nor to the impairment of the mechanisms controlling the Ca(2+) homeostasis. In addition, 1 microM YTX did not modify basal cAMP levels in cardiomyocytes. MTT and SRB assays revealed that incubation of cardiomyocytes with YTX (0.01-1 microM; 24, 48, and 72 h) caused a decrease in cell viability in a concentration- and time-dependent way. This effect was still evident in cardiomyocytes exposed to YTX for 1, 5, and 24 h and cultured up to 72 h in YTX-free medium. Our results demonstrate that, at nanomolar concentrations, a short incubation with YTX causes an inhibition of the beating activity and an irreversible reduction of viability of cardiac cells in vitro.

The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca(2+) imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca(2+) channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca(2+) channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.

Two copies of native trnK (UUU) gene are encoded on the sunflower mitochondrial DNA. They lie within two 12-kb direct repeats, presumably generated by a duplication event. During an investigation aimed at detecting DNA regions activating the trnK1 and trnK2 genes, three distinct promoters have been identified. Their locations were deduced using standard procedures (RT-PCR, RNA capping and 5'RACE) usually employed for the detection of transcription initiation sites (TISs). Promoters P3 and P2 control two independent partially overlapping transcription units containing the trnK2 and ccb206 genes, respectively. Promoter P1 has been mapped about 5200 bp upstream of the trnK1 gene which is part of a transcription unit also containing exons c, d and e of the nad2 gene, 5' to the tRNA gene. Most probably this promoter is not alone in controlling this transcription unit because this DNA region could be cotranscribed, at least partially, starting from other two promoters located upstream of the trnC and trnN genes, respectively. These genes have been previously mapped in a 5' region adjacent to the cluster containing nad2 exons c, d and e and the trnK1 gene. The comparative analysis of promoters P3 and P1 suggests that the difference between them could be related to the duplication event generating the second copy of trnK gene. The availability of a high number of new promoters belonging to dicot mitochondrial genomes makes possible to note some of their specific features.

Three proteinase inhibitor genes have been identified in the rapeseed (Brassica napus) genome. They are highly homologous to other genes of the mustard inhibitor (MSI) family of proteinase inhibitors characteristic of Cruciferae. In germinating seeds, only the transcript of one gene, coding for a trypsin inhibitor, is detectable by Northern analysis. The other two genes are transcribed at basal levels detectable only by reverse transcription PCR. One of the other two genes (rti-2) encodes a polypeptide with a glutamic residue in the P1 position, characteristic of glutamyl proteinase inhibitors. The recombinant RTI-2 protein strongly inhibits (Ki=44 nM) a glutamyl proteinase from Streptomyces griseus.

Mouse skeletal myotubes differentiated in vitro exhibited spontaneous contractions associated with electrical activity. The ionic conductances responsible for the origin and modulation of the spontaneous activity were examined using the whole-cell patch-clamp technique and measuring [Ca(2+)](i) transients with the Ca(2+) indicator, fura 2-AM. Regular spontaneous activity was characterized by single TTX-sensitive action potentials, followed by transient increases in [Ca(2+)](i). Since the bath-application of Cd(2+) (300 microM) or Ni(2+) (50 muM) abolished the cell firing, T-type (I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents were investigated in spontaneously contracting myotubes. The low activation threshold (around -60 mV) and the high density of I(Ca,T) observed in contracting myotubes suggested that I(Ca,T) initiated action potential firing, by bringing cells to the firing threshold. The results also suggested that the activity of I(Ca,L) could sustain the [Ca(2+)](i) transients associated with the action potential, leading to the activation of apamin-sensitive SK-type Ca(2+)-activated K(+) channels and the afterhyperpolarization (AHP) following single spikes. In conclusion, an interplay between voltage-dependent inward (Na(+) and Ca(2+)) and outward (SK) conductances is proposed to mediate the spontaneous pacemaker activity in cultured muscle myotubes during the process of myogenesis.

Na(+) currents were measured in myocytes from a fetus with congenital myotonic dystrophy type 1 (DM1) using the patch-clamp whole-cell technique. Steady-state activation and inactivation properties of Na(+) channels were not substantially different between these cells and age-matched control cells. However, a decrease in Na(+) channel density and a faster rate of recovery from inactivation were found in myocytes from congenital DM1 suggesting that changes in functional Na(+) channels may affect cell excitability of muscle cells of patients with this disorder.

The RT-PCR approach was used to estimate the expression of gamma-aminobutyric acid (GABA)(A) rho receptor subunits in the hippocampus of neonatal and adult rats. All three rho subunits were detected at postnatal day (P) 2, the rho3 subunit being expressed at an extremely low level. The rho1 and rho2 products appeared to be developmentally regulated; they were found to be more pronounced in adulthood. In another set of experiments, to correlate gene expression with receptor function, GABA(A) rho subunit mRNAs were detected with single-cell RT-PCR in CA3 pyramidal cells (from P3-P4 hippocampal slices), previously characterized with electrophysiological experiments for their bicuculline-sensitive or -insensitive responses to GABA. In 6 of 19 cells (31%), pressure application of GABA evoked at -70 mV inward currents that persisted in the presence of 100 microM bicuculline (314 plus minus 129 pA). RT-PCR performed in two of these neurons revealed the presence of rho1 and rho2 subunits, the latter being present with the alpha2 subunit. A rho2 subunit was also found in 1 neuron (among 9) exhibiting a response to GABA, which was completely abolished by bicuculline. This might be due to the lack of putative accessory GABA(A) subunits that can coassemble with rho2 to make functional receptors. Similar experiments from 10 P15 CA3 pyramidal cells failed to reveal any rho1-3 transcripts. However, these neurons abundantly express alpha3 subunits. It is likely that in CA3 pyramidal cells of neonatal and adult hippocampus GABA(A) rho subunits are present but at very low levels of expression.

Stratum (st.) radiatum interneurons represent a heterogeneous class of hippocampal cells with as yet poorly characterized physiological properties. Intracellular staining with biocytin, in situ hybridization, and patch-clamp recording have been combined to investigate the morphological and electrophysiological properties of these cells in the CA3 hippocampal region in young rats [postnatal days 10 to 21 (P10-21)]. Labeled cells presented a heterogeneous morphology with various soma shapes, often found multipolar, and dendritic arborizations confined to st. radiatum. The passive membrane properties of these st. radiatum interneurons showed instead no significant differences between P10 and P21. Low resting potential, high-input resistance, and short time constants characterized CA3 st. radiatum interneurons, which were silent at rest. Action potentials, elicited by brief current pulses, were lower and shorter than in pyramidal cells and followed by a Ca(2+)-dependent medium-duration afterhyperpolarizing potential (mAHP). Prolonged depolarizing current injection generated trains of action potentials that fired at constant frequency after a slight accommodation. The maximum steady-state firing rate was 31 +/- 4 (SD) Hz. Hyperpolarizing current pulses revealed a prominent inward rectification characterized by a "sag," followed by a depolarizing rebound that triggered action potentials. Sag and anodal brake excitation were blocked by Cs(+), suggesting that they were mediated by a hyperpolarization-activated cation conductance (I(h)). In the presence of tetrodotoxin and tetraethylammonium, biphasic tail currents were elicited in voltage clamp after a depolarizing step inducing Ca(2+) influx. Tail currents presented a fast Ca(2+)-activated and apamin-sensitive component (I(AHP)) and were further reduced by carbachol. The presence of I(AHP) was consistent with the high expression level of the apamin-sensitive SK2 subunit transcript in CA3 st. radiatum interneurons as detected by in situ hybridization. Different pharmacological agents were shown to affect the afterhyperpolarizing potential as well as the firing properties of st. radiatum interneurons. Exposure to Ca(2+)-free solutions mainly affected the late phase of repolarization and strongly reduced the mAHP. The mAHP was also attenuated by carbachol and by apamin, suggesting it to be partly mediated by I(AHP). Reduction of the mAHP increased the interneuron firing frequency. In conclusion, st. radiatum interneurons of CA3 hippocampal region represent a class of nonpyramidal cells with action potentials followed by an AHP of relatively short duration, partially generated by apamin and carbachol-sensitive conductances involved in the regulation of the cell firing rate.

Whole cell patch-clamp recording and intracellular staining with biocytin allowed the morphological and electrophysiological characterization of "giant" cells, studied in stratum (st.) radiatum of the CA3 region in 17- to 21-day-old rat hippocampal slices. These neurons had extensive dendritic arborization, a triangular soma, and a bipolar vertical orientation with axons directed to the pyramidal layer or extended into the st. oriens. Giant cells had significantly higher input resistance and shorter action potentials compared with CA3 pyramidal cells. Evoked action potentials were typically followed by an afterdepolarizing potential (ADP). During depolarizing current injection, most (80%) of recorded giant cells displayed a regular firing pattern (maximum steady-state firing rate, approximately 30 Hz) characterized by a modest early accommodation, whereas irregular firing was observed in the remaining 20% of giant cells. Hyperpolarizing current pulses induced a slow inward rectification of the electrotonic voltage responses, blocked by 2 mM external Cs(+). N-methyl-D-aspartate (NMDA) and non-NMDA-mediated excitatory postsynaptic currents (EPSCs) measured under voltage clamp were distinguished on the basis of their voltage dependence and sensitivity to specific NMDA and non-NMDA glutamate receptor blockers. Non-NMDA EPSCs possessed a linear current-voltage relationship. EPSCs elicited by st. lucidum stimulation were reversibly reduced (mean, 23%) by the group II metabotropic glutamate receptor agonist (2S, 1'R, 2'R, 3'R)-2-(2,3-dicarboxyl-cyclopropyl)-glycine (DCG-IV, 1 microM). GABA(A)-mediated postsynaptic currents were subject to paired-pulse depression that was inhibited by the GABA(B) antagonist CGP 55845A (5 microM). We conclude that CA3 giant cells represent a particular class of hippocampal neuron located in st. radiatum that shares only some morphological and physiological properties with principal cells.

The properties of inward-rectifying membrane currents in immature rat olfactory cortex neurones (postnatal day (P) 10-22) were analysed using whole-cell patch-clamp recordings. In 78% of cells (40/51), injection of hyperpolarizing current pulses elicited graded electrotonic potentials showing a slowly developing sag in the membrane potential. Under voltage clamp, negative commands from -50 mV activated slow inward current (ISlow) relaxations whose amplitude and exponential rate of onset increased with increasing hyperpolarization (n=40); the ISlow activation time constant (tauon) ranged from 650+/-116 (mean+/-S.E.M.) ms at -70 mV to 177+/-18 ms at -120 mV; n=34). By contrast, in 11/51 neurones, similar negative commands revealed only fast-type inward rectification (IIR) with either rapid (n=9) or 'instantaneous' onset kinetics (n=2). ISlow activation threshold was at approximately -60 mV, with full activation at -120 mV; the half-maximal voltage (V0.5) and slope factor (k) of activation were: -85+/-0.4 mV and 11+/-0.5, respectively (n=13). The estimated reversal potential for ISlow was -28+/-2 mV (n=5). No obvious age-dependent changes in maximal ISlow current amplitude or density (at -120 mV) or in the proportion of cells showing IIR were found between P10 and P22. Islow was blocked by Cs+ (5 mM, n=6) or the specific h-current blocker ZD 7288 (50 microM, n=11) but not Ba2+ (500 microM, n=7); in contrast, IIR was blocked by Cs+ or Ba2+ but not ZD 7288. It is concluded that unlike adult olfactory cortical cells, immature olfactory neurones can exhibit both slow and fast-types of inward rectification: the more predominant ISlow component, resembled the h-current (Ih) previously identified in other central neurones., (Copyright 1998 Elsevier Science B.V.)

The whole-cell configuration of the patch clamp technique was used to record miniature gamma-aminobutyric acidA (GABAA) receptor-mediated currents (in tetrodotoxin, 1 microM and kynurenic acid 1 mM) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6-9) old rats. Switching from a Ca2+-containing to a nominally Ca2+-free medium (in which Ca2+ was substituted with Mg2+, in the presence or in the absence of 100 microM EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration-dependent increase in frequency but not in amplitude of tetrodotoxin-resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 microM over control) was 1.8+/-0.5, (n = 9). In nominally Ca2+-free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mM), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mM) reversibly reduced the amplitude of the current induced by GABA (100 microM) indicating a clear postsynaptic effect on GABAA receptor. Superfusion of ryanodine (30 microM), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine-ryanodine-sensitive stores facilitates GABA release.

The whole cell configuration of the patch-clamp technique was used to study the modulation gamma-aminobutyric acid (GABA)-mediated postsynaptic currents by ruthenium red in CA3 hippocampal neurons in slices obtained from postnatal (P) days P6-P10 old rats. In the presence of kynurenic acid (1 mM), ruthenium red (100 microM) completely blocked stimulus-elicited GABA-mediated postsynaptic currents and reduced by 50% the amplitude of the spontaneous ones. Ruthenium red (100 microM) increased the frequency but not the amplitude of miniature GABAergic currents recorded in the presence of tetrodotoxin (1 microM) and kynurenic acid (1 mM), an effect that was prevented by heparin (100 microM). Ruthenium red did not modify the kinetics of miniature postsynaptic currents and the currents induced by exogenous application of GABA (10 microM) in the presence of tetrodotoxin, suggesting that its action was presynaptic in origin. The effects of ruthenium red on quantal GABA release was independent of external calcium. In a nominally Ca2+-free solution the potentiating effect induced by this polyvalent cation on miniature postsynaptic currents was still present. Intracellular calcium stores were not involved in ruthenium red action, because this polyvalent cation was able to facilitate miniature currents also in the presence of thapsigargin (10-20 microM). These results indicate that ruthenium red has a dual action on GABA release from GABAergic interneurons: it reduces the amplitude of spontaneous events and increases the frequency of miniature currents. The former effect is calcium-dependent, whereas the latter is calcium independent.

Intracellular and patch clamp recording techniques were used to investigate the role of GABA in immature CA3 hippocampal neurons. During the first postnatal week spontaneous GABA release was detected as spontaneous ongoing synaptic potentials (SPSPs) or giant depolarizing potentials (GDPs). GDPs were generated at regular intervals and regulated by ionotropic glutamate receptors (GluRs), whereas SPSPs occurred randomly and were unaffected by ionotropic GluRs. Both GDPs and SPSPs were positively modulated by metabotropic GluRs through cyclic AMP-dependent protein kinase. Moreover GABA controlled its own release through GABAA and GABAB receptors, probably localized on GABAergic nerve terminals. At this developmental stage, GABA depolarized CA3 pyramidal cells through two distinct classes of chloride-permeable receptors: bicuculline sensitive and insensitive, respectively. The bicuculline-insensitive responses were blocked by picrotoxin in a noncompetitive way. Whole-cell GABA currents, recorded in the presence of bicuculline, had a slower desensitization rate and faster recovery from desensitization. In excised outside-out patches, in the presence of bicuculline, GABA activated single-channel currents with conductances of 14, 22, and 31 pS. These values were similar to those obtained when GABA was applied in the absence of bicuculline. Interestingly, GABA responses obtained in the absence of bicuculline, were sensitive to the blocking effect of zinc, whereas bicuculline-resistant responses were almost unaffected by this divalent cation. Expression of different subunits in native receptors (particularly of the alpha and rho type) may account for the functional differences observed in the present experiments. Activation of bicuculline-insensitive receptors would strengthen and prolong the depolarizing action of GABA, thus favoring the entry of calcium through voltage-dependent calcium channels. This calcium signal may be essential in promoting stabilization of synaptic contacts during a critical period of postnatal development.

Whole-cell recordings were made from neurones in slices of rat olfactory cortex (10-19 days old), using potassium methylsulphate-filled pipettes. Positive commands applied from -60 mV in the presence of oxotremorine-M (10-20 microgramM, 20 of 30 cells) or trans-ACPD (10-50 microgramM, 4 of 9 cells) evoked a slow inward tail current similar to the K+ -mediated tail current (I K,ADP) recorded with sharp electrodes. I ADP was reduced by hyperpolarization, showed a 27% decrease in input conductance at its peak and was depressed by Cd(2+), 4AP or high K+ but unaffected by Cs+, Ba(2+) or TEA (5 mM). IADP was significantly larger in cells between postnatal days 13 and 15 than in younger neurones (10-12 days). These data show that stable whole-cell recordings of I ADP can be made from olfactory neurones in cortical slices, and support our hypothesis that IADP is a slowly reactivating, Ca(2+) -sensitive K+ conductance.

1. Intracellular recordings were used to study the role of metabotropic glutamate receptors (mGluRs) in modulating GABA-mediated giant depolarizing potentials (GDPs) in immature rat hippocampal CA3 neurones. 2. The mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM) reduced the frequency of GDPs. The broad-spectrum ionotropic glutamate receptor antagonist kynurenic acid (1 mM) blocked GDPs. 3. In the presence of kynurenic acid, both tetanic stimulation of the hilus or bath application of quisqualic acid (1 microM) and trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 20 microM) induced the appearance of GDPs. These effects were antagonized by MCPG (1 mM) or L(+)-2-amino-3-phosphonopropionic acid (L-AP3) and blocked by bicuculline (10 microM). 4. 8-Bromo-cAMP (8-Br-cAMP, 0.3 mM), 3-isobutyl-1-methylxanthine (IBMX, 200 microM) or forskolin (30 microM) mimicked the effects of mGluR agonists on GDPs. The forskolin analogue 1,9-dideoxyforskolin (30 microM), which does not activate adenylate cyclase, was ineffective. 5. Incubation of slices in the presence of the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) (500 microM) or superfusion of Rp-cAMPS (20 microM) prevented the effects of forskolin or t-ACPD on GDPs. In the presence of kynurenic acid, the protein kinase C activator, phorbol 12,13-diacetate (2 microM) induced the appearance of GDPs. This effect was prevented by staurosporine (1 microM). However, staurosporine (1-3 microM) did not modify the effects of t-ACPD on GDPs. 6. It is suggested that, during development, mGluRs enhance the synchronous release of GABA, responsible for GDPs, through cAMP-dependent protein kinase.