Microtransplantation of cellular membranes from squid stellate ganglion reveals ionotropic GABA receptors

Author Posting. © Marine Biological Laboratory, 2013. This article is posted here by permission of Marine Biological Laboratory for personal use, not for redistribution. The definitive version was published in Biological Bulletin 224 (2013): 47-52.The squid has been the most studied cephalopod, and it has served as a very useful model for investigating the events associated with nerve impulse generation and synaptic transmission. While the physiology of squid giant axons has been extensively studied, very little is known about the distribution and function of the neurotransmitters and receptors that mediate inhibitory transmission at the synapses. In this study we investigated whether γ-aminobutyric acid (GABA) activates neurotransmitter receptors in stellate ganglia membranes. To overcome the low abundance of GABA-like mRNAs in invertebrates and the low expression of GABA in cephalopods, we used a two-electrode voltage clamp technique to determine if Xenopus laevis oocytes injected with cell membranes from squid stellate ganglia responded to GABA. Using this method, membrane patches containing proteins and ion channels from the squid's stellate ganglion were incorporated into the surface of oocytes. We demonstrated that GABA activates membrane receptors in cellular membranes isolated from squid stellate ganglia. Using the same approach, we were able to record native glutamate-evoked currents. The squid's GABA receptors showed an EC50 of 98 μmol l–1 to GABA and were inhibited by zinc (IC50 = 356 μmol l–1). Interestingly, GABA receptors from the squid were only partially blocked by bicuculline. These results indicate that the microtransplantation of native cell membranes is useful to identify and characterize scarce membrane proteins. Moreover, our data also support the role of GABA as an ionotropic neurotransmitter in cephalopods, acting through chloride-permeable membrane receptors.Grass Foundation Fellowships to L.C. and A.L. (www.grassfoundation.org). L.C. was additionally supported by the Ph.D. in Neurophysiology program of the University of Rome “La Sapienza.” All authors were Grass Fellows. This work was supported by Ministero della Sanita` Antidoping and PRIN project 2009 (to E.P.)

Dissecting the molecular determinants of GABAA receptors current rundown, a hallmark of refractory human epilepsy

GABAA receptors-(Rs) are fundamental for the maintenance of an efficient inhibitory function in the central nervous system (CNS). Their dysfunction is associated with a wide range of CNS disorders, many of which characterized by seizures and epilepsy. Recently, an increased use-dependent desensitization due to a repetitive GABA stimulation (GABAA current rundown) of GABAARs has been associated with drug-resistant temporal lobe epilepsy (TLE). Here, we aimed to investigate the molecular determinants of GABAA current rundown with two different heterologous expression systems (Xenopus oocytes and human embryonic kidney cells; HEK) which allowed us to manipulate receptor stoichiometry and to study the GABAA current rundown on different GABAAR configurations. To this purpose, we performed electrophysiology experiments using two-electrode voltage clamp in oocytes and confirming part of our results in HEK. We found that different degrees of GABAA current rundown can be associated with the expression of different GABAAR beta-subunits reaching the maximum current decrease when functional alpha1beta2 receptors are expressed. Furthermore, the blockade of phosphatases can prevent the current rundown observed in alpha1beta2 GABAARs. Since GABAAR represents one important therapeutic target in the treatment of human epilepsy, our results could open new perspectives on the therapeutic management of drug-resistant patients showing a GABAergic impairment

Priming stress patterns in word recognition

This study addresses the lexical representation of stress in a series of five intra-modal and cross-modal priming experiments in the Greek language using lexical decision tasks with auditory and visual targets. Three-syllable primes and targets were matched in first syllable segments, length, and other variables, and differed segmentally in the second and third syllable. Primes matched or mismatched targets in stress, which was placed on the penultimate or antepenultimate syllable. There was no evidence for stress priming in either accuracy or latency of responses to either words or pseudowords in any of these experiments, either intra-modally or cross-modally. In contrast, a control fragment priming experiment using only the first two syllables of the primes produced a significant effect of stress congruence for words but not for pseudowords. The results are interpreted in the context of previous findings in the literature as arising from lexical activation rather than from matching stress patterns. Overall, findings are consistent with lexical representations including stress information that is inseparable from segmental specification, rather than with abstract representations of metrical templates

Sonohysterography and liquid-based cytology in menopausal patients with abnormal endometrium

n/

Visuospatial attention lateralization in volleyball players and in rowers.

In the present study, differences in visuospatial attention lateralization were evaluated in athletes engaged in open-compared to closed-skill sports and sedentary nonathletes. 23 volleyball players (open skill; Italian national level and regional level), 10 rowers (closed skill, Italian national level), and 23 sedentary participants responded to a computerized line-length judgment task. Five lines, differing in the length of their right and left segments, were randomly presented; the respondent made a forced-choice decision about the respective length of the two segments. Volleyball players responded significantly faster; those at the higher competitive level were also more accurate, making a statistically significantly lower number of leftward errors as compared with rowers and controls. If such responses are due to training rather than self-selection of ability, then the results may suggest the possibility of changing the distribution of visuospatial attention by training in open-skill sports

Neuromuscular magnetic stimulation counteracts muscle decline in ALS patients: results of a randomized, double-blind, controlled study

The aim of the study was to verify whether neuromuscular magnetic stimulation (NMMS) improves muscle function in spinal-onset amyotrophic lateral sclerosis (ALS) patients. Twenty-two ALS patients were randomized in two groups to receive, daily for two weeks, NMMS in right or left arm (referred to as real-NMMS, rNMMS), and sham NMMS (sNMMS) in the opposite arm. All the patients underwent a median nerve conduction (compound muscle action potential, CMAP) study and a clinical examination that included a handgrip strength test and an evaluation of upper limb muscle strength by means of the Medical Research Council Muscle Scale (MRC). Muscle biopsy was then performed bilaterally on the flexor carpi radialis muscle to monitor morpho-functional parameters and molecular changes. Patients and physicians who performed examinations were blinded to the side of real intervention. The primary outcome was the change in the muscle strength in upper arms. The secondary outcomes were the change from baseline in the CMAP amplitudes, in the nicotinic ACh currents, in the expression levels of a selected panel of genes involved in muscle growth and atrophy, and in histomorphometric parameters of ALS muscle fibers. The Repeated Measures (RM) ANOVA with a Greenhouse-Geisser correction (sphericity not assumed) showed a significant effect [F(3, 63) = 5.907, p < 0.01] of rNMMS on MRC scale at the flexor carpi radialis muscle, thus demonstrating that the rNMMS significantly improves muscle strength in flexor muscles in the forearm. Secondary outcomes showed that the improvement observed in rNMMS-treated muscles was associated to counteracting muscle atrophy, down-modulating the proteolysis, and increasing the efficacy of nicotinic ACh receptors (AChRs). We did not observe any significant difference in pre- and post-stimulation CMAP amplitudes, evoked by median nerve stimulation. This suggests that the improvement in muscle strength observed in the stimulated arm is unlikely related to reinnervation. The real and sham treatments were well tolerated without evident side effects. Although promising, this is a proof of concept study, without an immediate clinical translation, that requires further clinical validation

GABA(A) receptor function is enhanced by Interleukin-10 in human epileptogenic gangliogliomas and its effect is counteracted by Interleukin-1 beta

Gangliogliomas (GGs) are low-grade brain tumours that cause intractable focal epilepsy in children and adults. In GG, as in epileptogenic focal malformations (i.e., tuberous sclerosis complex, TSC), there is evidence of sustained neuroinflammation with involvement of the pro-inflammatory cytokine IL-1β. On the other hand, anti-inflammatory mediators are less studied but bear relevance for understanding seizure mechanisms. Therefore, we investigated the effect of the key anti-inflammatory cytokine IL-10 on GABAergic neurotransmission in GG. We assessed the IL-10 dependent signaling by transcriptomic analysis, immunohistochemistry and performed voltage-clamp recordings on Xenopus oocytes microtransplanted with cell membranes from brain specimens, to overcome the limited availability of acute GG slices. We report that IL-10-related mRNAs were up-regulated in GG and slightly in TSC. Moreover, we found IL-10 receptors are expressed by neurons and astroglia. Furthermore, GABA currents were potentiated significantly by IL-10 in GG. This effect was time and dose-dependent and inhibited by blockade of IL-10 signaling. Notably, in the same tissue, IL-1β reduced GABA current amplitude and prevented the IL-10 effect. These results suggest that in epileptogenic tissue, pro-inflammatory mechanisms of hyperexcitability prevail over key anti-inflammatory pathways enhancing GABAergic inhibition. Hence, boosting the effects of specific anti-inflammatory molecules could resolve inflammation and reduce intractable seizures

Impaired duodenal Palmitoylethanolamide release underlies acid-induced mast cells activation in Functional Dyspepsia

Acid hypersensitivity is claimed to be a symptomatic trigger in functional dyspepsia (FD); however, the neuroimmune pathway(s) and the mediators involved in this process have not been systematically investigated. Palmitoylethanolamide (PEA) is an endogenous compound, able to modulate nociception and inflammation, but its role in FD has never been assessed

SPECIFIC DYSPEPTIC SYMPTOMS ARE ASSOCIATED WITH POOR RESPONSE TO THERAPY IN PATIENTS WITH GASTROESOPHAGEAL REFLUX DISEASE

Background: In gastroesophageal reflux disease (GORD) patients, coexistence of functional dyspepsia (FD) is known to be associated with poor response to proton pump inhibitors (PPIs), but the contribution of specific dyspepsia symptoms has not been systematically investigated yet. Objective: To characterize the impact of dyspepsia symptoms on PPIs response in GORD patients. Methods:. The enrolled subjects were 100 patients with diagnosis of GORD. All patients underwent a 24 hour pH-impedance test, while on PPIs-therapy. Patients were divided into two groups, refractory and responders, according to the persistence of GORD symptoms. A standardized questionnaire for FD was also administered to assess presence of dyspepsia symptoms. Results: In the subgroup of refractory patients FD was more prevalent than in responder ones, with postprandial fullness, nausea, vomiting, early satiation and epigastric pain being significantly prevalent in refractory GORD-patients. In the multivariate analysis only early satiation and vomiting were significantly associated with poor response to PPIs Conclusion: Coexistence of FD is associated with refractory-GORD. We showed that only early satiation and vomiting are risk factors for poor response to PPIs therapy. Our findings suggest that symptoms of early satiation and vomiting would help to identify the subset of PPIs-refractory GORD patients

I cattolici nella fabbrica del cinema e dei media: produzione, opere, protagonisti (1940-1970)

Il volume contiene alcuni esiti delle ricerche condotte nell’ambito del progetto PRIN 2012 I cattolici e il cinema in Italia tra gli anni ’40 e gli anni ’70, coordinato dall’Università degli Studi di Milano, capitalizzando riflessioni emerse nel corso di una serie di convegni che si sono succeduti dal 2014 al 2016.The issue contains some outcomes of the research project PRIN 2012 The Catholics and Cinema in Italy between the 40s and the 70s, coordinated by University of Milan, capitalizing reflections shared during a series of congresses between 2014 and 2016