Acetylcholine receptors from human muscle as pharmacological targets for ALS therapy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons that leads to progressive paralysis of skeletal muscle. Studies of ALS have revealed defects in expression of acetylcholine receptors (AChRs) in skeletal muscle that occur even in the absence of motor neuron anomalies. The endocannabinoid palmitoylethanolamide (PEA) modified the clinical conditions in one ALS patient, improving muscle force and respiratory efficacy. By microtransplanting muscle membranes from selected ALS patients into Xenopus oocytes, we show that PEA reduces the desensitization of acetylcholine-evoked currents after repetitive neurotransmitter application (i.e., rundown). The same effect was observed using muscle samples from denervated (non-ALS) control patients. The expression of human recombinant α1ß1γδ (γ-AChRs) and α1ß1εδ AChRs (ε-AChRs) in Xenopus oocytes revealed that PEA selectively affected the rundown of ACh currents in ε-AChRs. A clear up-regulation of the α1 subunit in muscle from ALS patients compared with that from non-ALS patients was found by quantitative PCR, but no differential expression was found for other subunits. Clinically, ALS patients treated with PEA showed a lower decrease in their forced vital capacity (FVC) over time as compared with untreated ALS patients, suggesting that PEA can enhance pulmonary function in ALS. In the present work, data were collected from a cohort of 76 ALS patients and 17 denervated patients. Our results strengthen the evidence for the role of skeletal muscle in ALS pathogenesis and pave the way for the development of new drugs to hamper the clinical effects of the disease.

Nicotinic and muscarinic agonists and acetylcholinesterase inhibitors stimulate a common pathway to enhance GluN2B-NMDAR responses.

Nicotinic and muscarinic ACh receptor agonists and acetylcholinesterase inhibitors (AChEIs) can enhance cognitive function. However, it is unknown whether a common signaling pathway is involved in the effect. Here, we show that in vivo administration of nicotine, AChEIs, and an m1 muscarinic (m1) agonist increase glutamate receptor, ionotropic, N-methyl D-aspartate 2B (GluN2B)-containing NMDA receptor (NR2B-NMDAR) responses, a necessary component in memory formation, in hippocampal CA1 pyramidal cells, and that coadministration of the m1 antagonist pirenzepine prevents the effect of cholinergic drugs. These observations suggest that the effect of nicotine is secondary to increased release of ACh via the activation of nicotinic ACh receptors (nAChRs) and involves m1 receptor activation through ACh. In vitro activation of m1 receptors causes the selective enhancement of NR2B-NMDAR responses in CA1 pyramidal cells, and in vivo exposure to cholinergic drugs occludes the in vitro effect. Furthermore, in vivo exposure to cholinergic drugs suppresses the potentiating effect of Src on NMDAR responses in vitro. These results suggest that exposure to cholinergic drugs maximally stimulates the m1/guanine nucleotide-binding protein subunit alpha q/PKC/proline-rich tyrosine kinase 2/Src signaling pathway for the potentiation of NMDAR responses in vivo, occluding the in vitro effects of m1 activation and Src. Thus, our results indicate not only that nAChRs, ACh, and m1 receptors are on the same pathway involving Src signaling but also that NR2B-NMDARs are a point of convergence of cholinergic and glutamatergic pathways involved in learning and memory.

GABAρ subunits confer a bicuculline-insensitive component to GFAP+ cells of cerebellum.

GABA-A receptors mediating synaptic or extrasynaptic transmission are molecularly and functionally distinct, and glial cells are known to express a plethora of GABA-A subunits. Here we demonstrate that GFAP(+) cells of the granular layer of cerebellum express GABAρ subunits during early postnatal development, thereby conferring peculiar pharmacologic characteristics to GABA responses. Electron microscopy revealed the presence of GABAρ in the plasma membrane of GFAP(+) cells. In contrast, expression in the adult was restricted to Purkinje neurons and a subset of ependymal cells. Electrophysiological studies in vitro revealed that astrocytes express functional receptors with an EC50 of 52.2 ± 11.8 µM for GABA. The evoked currents were inhibited by bicuculline (100 µM) and TPMPA (IC50, 5.9 ± 0.6 µM), indicating the presence of a GABAρ component. Coimmunoprecipitation demonstrated protein-protein interactions between GABAρ1 and GABAα1, and double immunofluorescence showed that these subunits colocalize in the plasma membrane. Three populations of GABA-A receptors in astrocytes were identified: classic GABA-A, bicuculline-insensitive GABAρ, and GABA-A-GABAρ hybrids. Clusters of GABA-A receptors were distributed in the perinuclear space and along the processes of GFAP(+) cells. Time-lapse microscopy showed GABAρ2-GFP accumulation in clusters located in the soma and along the processes. The clusters were relatively immobile, with mean displacement of 9.4 ± 0.9 µm and a net distance traveled of 1-2 µm, owing mainly to directional movement or simple diffusion. Modulation of GABAρ dynamics may be a novel mechanism of extrasynaptic transmission regulating GABAergic control of GFAP(+) cells during early postnatal development.

Dipicrylamine Modulates GABAρ1 Receptors through Interactions with Residues in the TM4 and Cys-Loop Domains.

Dipicrylamine (DPA) is a commonly used acceptor agent in Förster resonance energy transfer experiments that allows the study of high-frequency neuronal activity in the optical monitoring of voltage in living cells. However, DPA potently antagonizes GABAA receptors that contain α1 and ß2 subunits by a mechanism which is not clearly understood. In this work, we aimed to determine whether DPA modulation is a general phenomenon of Cys-loop ligand-gated ion channels (LGICs), and whether this modulation depends on particular amino acid residues. For this, we studied the effects of DPA on human homomeric GABAρ1, α7 nicotinic, and 5-HT3A serotonin receptors expressed in Xenopus oocytes. Our results indicate that DPA is an allosteric modulator of GABAρ1 receptors with an IC50 of 1.6 µM, an enhancer of α7 nicotinic receptors at relatively high concentrations of DPA, and has little, if any, effect on 5-HT3A receptors. DPA antagonism of GABAρ1 was strongly enhanced by preincubation, was slightly voltage-dependent, and its washout was accelerated by bovine serum albumin. These results indicate that DPA modulation is not a general phenomenon of LGICs, and structural differences between receptors may account for disparities in DPA effects. In silico modeling of DPA docking to GABAρ1, α7 nicotinic, and 5-HT3A receptors suggests that a hydrophobic pocket within the Cys-loop and the M4 segment in GABAρ1, located at the extracellular/membrane interface, facilitates the interaction with DPA that leads to inhibition of the receptor. Functional examinations of mutant receptors support the involvement of the M4 segment in the allosteric modulation of GABAρ1 by DPA.

Anorexia Reduces GFAP+ Cell Density in the Rat Hippocampus.

Anorexia nervosa is an eating disorder observed primarily in young women. The neurobiology of the disorder is unknown but recently magnetic resonance imaging showed a volume reduction of the hippocampus in anorexic patients. Dehydration-induced anorexia (DIA) is a murine model that mimics core features of this disorder, including severe weight loss due to voluntary reduction in food intake. The energy supply to the brain is mediated by astrocytes, but whether their density is compromised by anorexia is unknown. Thus, the aim of this study was to estimate GFAP+ cell density in the main regions of the hippocampus (CA1, CA2, CA3, and dentate gyrus) in the DIA model. Our results showed that GFAP+ cell density was significantly reduced (~20%) in all regions of the hippocampus, except in CA1. Interestingly, DIA significantly reduced the GFAP+ cells/nuclei ratio in CA2 (-23%) and dentate gyrus (-48%). The reduction of GFAP+ cell density was in agreement with a lower expression of GFAP protein. Additionally, anorexia increased the expression of the intermediate filaments vimentin and nestin. Accordingly, anorexia increased the number of reactive astrocytes in CA2 and dentate gyrus more than twofold. We conclude that anorexia reduces the hippocampal GFAP+ cell density and increases vimentin and nestin expression.

Loss of functional GABA(A) receptors in the Alzheimer diseased brain.

The cholinergic and glutamatergic neurotransmission systems are known to be severely disrupted in Alzheimer's disease (AD). GABAergic neurotransmission, in contrast, is generally thought to be well preserved. Evidence from animal models and human postmortem tissue suggest GABAergic remodeling in the AD brain. Nevertheless, there is no information on changes, if any, in the electrophysiological properties of human native GABA receptors as a consequence of AD. To gain such information, we have microtransplanted cell membranes, isolated from temporal cortices of control and AD brains, into Xenopus oocytes, and recorded the electrophysiological activity of the transplanted GABA receptors. We found an age-dependent reduction of GABA currents in the AD brain. This reduction was larger when the AD membranes were obtained from younger subjects. We also found that GABA currents from AD brains have a faster rate of desensitization than those from non-AD brains. Furthermore, GABA receptors from AD brains were slightly, but significantly, less sensitive to GABA than receptors from non-AD brains. The reduction of GABA currents in AD was associated with reductions of mRNA and protein of the principal GABA receptor subunits normally present in the temporal cortex. Pairwise analysis of the transcripts within control and AD groups and analyses of the proportion of GABA receptor subunits revealed down-regulation of α1 and γ2 subunits in AD. In contrast, the proportions of α2, ß1, and γ1 transcripts were up-regulated in the AD brains. Our data support a functional remodeling of GABAergic neurotransmission in the human AD brain.

Dehydration-Induced Anorexia Reduces Astrocyte Density in the Rat Corpus Callosum.

Anorexia nervosa is an eating disorder associated with severe weight loss as a consequence of voluntary food intake avoidance. Animal models such as dehydration-induced anorexia (DIA) mimic core features of the disorder, including voluntary reduction in food intake, which compromises the supply of energy to the brain. Glial cells, the major population of nerve cells in the central nervous system, play a crucial role in supplying energy to the neurons. The corpus callosum (CC) is the largest white matter tract in mammals, and more than 99% of the cell somata correspond to glial cells in rodents. Whether glial cell density is altered in anorexia is unknown. Thus, the aim of this study was to estimate glial cell density in the three main regions of the CC (genu, body, and splenium) in a murine model of DIA. The astrocyte density was significantly reduced (~34%) for the DIA group in the body of the CC, whereas in the genu and the splenium no significant changes were observed. DIA and forced food restriction (FFR) also reduced the ratio of astrocytes to glial cells by 57.5% and 22%, respectively, in the body of CC. Thus, we conclude that DIA reduces astrocyte density only in the body of the rat CC.

Characterization of an outward rectifying chloride current of Xenopus tropicalis oocytes.

Here, we describe an outward rectifying current in Xenopus tropicalis oocytes that we have called xtClC-or. The current has two components; the major component is voltage activated and independent of intracellular or extracellular Ca(2+), whereas the second is a smaller component that is Ca(2+) dependent. The properties of the Ca(2+)-independent current, such as voltage dependence and outward rectification, resemble those of ClC anion channels/transporters. This current is sensitive to NPPB and NFA, insensitive to 9AC and DIDS, and showed a whole-cell conductance sequence of SCN(-)>I(-)>Br(-)>CI(-). RT-PCR revealed the expression in oocytes of ClC-2 to ClC-7, and major reductions of current amplitudes were observed when a ClC-5 antisense oligonucleotide was injected into oocytes. The Ca(2+)-dependent component was abated after injection of 10mM BAPTA or EGTA, whereas 10mMMg(2+) inhibited the current to 26±3.1%. This component was blocked by 9-AC, NFA, and NPPB, whereas DIDS did not elicit any evident effect. The ion sequence selectivity was SCN=I(-)>Br(-)>Cl(-). To try to determine the molecular identity that gives rise to this component we assessed by RT-PCR the expression of the Ca(2+)-dependent Cl(-) channel TMEM16A, which was found to be present in the oocytes. However, injection of antisense TMEM16A oligonucleotides did not inhibit the transient outward current. This result fits well with the electrophysiological data. Together, these results suggest that ClC-5 is a major, but not the sole channel responsible for this outwardly rectifying Cl(-) current.

Modulation of GABA-A receptors of astrocytes and STC-1 cells by taurine structural analogs.

Taurine activates and modulates GABA receptors in vivo as well as those expressed in heterologous systems. This study aimed to determine whether the structural analogs of taurine: homotaurine and hypotaurine, have the ability to activate GABA-A receptors that include GABAρ subunits. The expression of GABA-A receptors containing GABAρ has been reported in the STC-1 cells and astrocytes. In both cell types, taurine, homo-, and hypotaurine gated with low efficiency a picrotoxin-sensitive GABA-A receptor. The known bimodal modulatory effect of taurine on GABAρ receptors was not observed; however, differences between the activation and deactivation rates were detected when they were perfused together with GABA. In silico docking simulations suggested that taurine, hypo-, and homotaurine do not form a cation-π interaction such as that generated by GABA in the agonist-binding site of GABAρ. This observation complements the electrophysiological data suggesting that taurine and its analogs act as partial agonists of GABA-A receptors. All the observations above suggest that the structural analogs of taurine are partial agonists of GABA-A receptors that occupy the agonist-binding site, but their structures do not allow the proper interaction with the receptor to fully gate its Cl(-) channel.

Physiological characterization of human muscle acetylcholine receptors from ALS patients.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: "microtransplantation" of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease.

Identification of the minimal promoter for specific expression of the GABAρ1 receptor in retinal bipolar cells.

γ-aminobutyric acid (GABA)ρ receptors regulate rapid synaptic ion currents in the axon end of retinal ON bipolar neurons, acting as a point of control along the visual pathway. In the GABAρ1 subunit knock out mouse, inhibition mediated by this receptor is totally eliminated, showing its role in neural transmission in retina. GABAρ1 mRNA is expressed in mouse retina after post-natal day 7, but little is known about its transcriptional regulation. To identify the GABAρ1 promoter, in silico analyses were performed and indicated that a 0.290-kb fragment, flanking the 5'-end of the GABAρ1 gene, includes putative transcription factor-binding sites, two Inr elements, and lacks a TATA-box. A rapid amplification of cDNA ends (RACE) assay showed three transcription start sites (TSS) clustered in the first exon. Luciferase reporter assays indicated that a 0.232-kb fragment upstream from the ATG is the minimal promoter in transfected cell lines and in vitro electroporated retinae. The second Inr and AP1 site are important to activate transcription in secretin tumor cells (STC-1) and retina. Finally, the 0.232-kb fragment drives green fluorescent protein (GFP) expression to the inner nuclear layer, where bipolar cells are present. This first work paves the way for further studies of molecular elements that control GABAρ1 transcription and regulate its expression during retinal development.

γ-Aminobutyric acid-ρ expression in ependymal glial cells of the mouse cerebellum.

The ependymal glial cells (EGCs) from the periventricular zone of the cerebellum were studied to determine their distribution and the functional properties of their γ-aminobutyric acid type A (GABA(A) ) receptors. EGCs were identified by the presence of ciliated structures on their ventricular surface and their expression of glial fibrillary acidic protein (GFAP). Interestingly, diverse cell types, including neurons, astrocytes, and other types of glia, were identified in the subventricular zone by their current profiles. Electron microscopy showed ciliated cells and myelinated axons in this zone, but we found no collateral connections to suggest the presence of functional synapses. GABA-mediated currents were recorded from EGCs in cerebellar slices from postnatal days 13 to 35 (PN13-PN35). These currents were blocked by TPMPA (a highly specific GABA(A) ρ subunit antagonist) and bicuculline (a selective antagonist for classic GABA(A) receptors). Pentobarbital failed to modulate GABA(A)-mediated currents despite the expression of GABAα1 and GABAγ2 subunits. In situ hybridization, RT-PCR, and immunofluorescence studies confirmed GABAρ1 expression in EGCs of the cerebellum. We conclude that cerebellar EGCs express GABAρ1, which is functionally involved in GABA(A) receptor-mediated responses that are unique among glial cells of the brain.

Structure-function study of the fourth transmembrane segment of the GABAρ1 receptor.

The Cys-loop family of receptors mediates synaptic neurotransmission in the central nervous system of vertebrates. These receptors share several structural characteristics and assemble in the plasma membrane as multimers with fivefold symmetry. Of these, the ionotropic GABA receptors are key players in the pathogenesis of diseases like epilepsy, anxiety, and schizophrenia. Different experimental approaches have shed some light on the mechanisms behind the function of these receptors; but little is known about their structure at high resolution. Sequence homology with the nicotinic acetylcholine receptor predicts that ionotropic GABA receptors possess four transmembrane segments (TM1-4) and that TM2 forms the wall of the ion channel. However, the role of the other three segments is unclear. The GABAρ1 receptor plays a fundamental role in the regulation of neurotransmission along the visual pathway, is highly sensitive to GABA, and exhibits little desensitization. In our recent investigations of the role of TM4 in receptor function, a key residue in this domain (W475) was found to be involved in activation of the receptor. Here we have generated a structural model of the GABAρ1 receptor in silico and assessed its validity by electrophysiologically testing nine amino acid substitutions of W475 and deletions of the neighboring residues (Y474 and S476). The results identify a critical linkage between the ligand-binding domain and the TM4 domain and provide a framework for more detailed structure-function analyses of ionotropic GABA receptors.

Enhancement of GABA(A)-current run-down in the hippocampus occurs at the first spontaneous seizure in a model of temporal lobe epilepsy.

Refractory temporal lobe epilepsy (TLE) is associated with a dysfunction of inhibitory signaling mediated by GABA(A) receptors. In particular, the use-dependent decrease (run-down) of the currents (I(GABA)) evoked by the repetitive activation of GABA(A) receptors is markedly enhanced in hippocampal and cortical neurons of TLE patients. Understanding the role of I(GABA) run-down in the disease, and its mechanisms, may allow development of medical alternatives to surgical resection, but such mechanistic insights are difficult to pursue in surgical human tissue. Therefore, we have used an animal model (pilocarpine-treated rats) to identify when and where the increase in I(GABA) run-down occurs in the natural history of epilepsy. We found: (i) that the increased run-down occurs in the hippocampus at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made), and then extends to the neocortex and remains constant in the course of the disease; (ii) that the phenomenon is strictly correlated with the occurrence of spontaneous seizures, because it is not observed in animals that do not become epileptic. Furthermore, initial exploration of the molecular mechanism disclosed a relative increase in alpha4-, relative to alpha1-containing GABA(A) receptors, occurring at the same time when the increased run-down appears, suggesting that alterations in the molecular composition of the GABA receptors may be responsible for the occurrence of the increased run-down. These observations disclose research opportunities in the field of epileptogenesis that may lead to a better understanding of the mechanism whereby a previously normal tissue becomes epileptic.

Expression of GABAρ receptors in the neostriatum: localization in aspiny, medium spiny neurons and GFAP-positive cells.

GABAergic transmission in the neostriatum plays a central role in motor coordination, in which a plethora of GABA-A receptor subunits combine to modulate neural inhibition. GABAρ receptors were originally described in the mammalian retina. These receptors possess special electrophysiological and pharmacological properties, forming a characteristic class of ionotropic receptors. In previous studies, we suggested that GABAρ receptors are expressed in the neostriatum, and in this report we show that they are indeed present in all the calretinin-positive interneurons of the neostriatum. In addition, they are located in calbindin-positive interneurons and projection neurons that express the dopamine D(2) receptor. GABAρ receptors were also located in 30% of the glial fibrillary acidic protein-positive cells, and may therefore also contribute to gliotransmission. Quantitative reverse transcription-PCR suggested that the mRNAs of this receptor do not express as much as in the retina, and that GABAρ2 is more abundant than GABAρ1. Electrophysiological recordings in brain slices provided evidence of neurons expressing a cis-4-aminocrotonic acid-activated, 1,2,5,6-tetrahydropyridine-4-yl methylphosphinic acid-sensitive ionotropic GABA receptor, indicating the presence of functional GABAρ receptors in the neostriatum. Finally, electron-microscopy and immunogold located the receptors mainly in perisynaptic as well as in extrasynaptic sites. All these observations reinforce the importance of GABAρ receptors in the neostriatum and contribute to the diversity of inhibitory regulation in this area.

Serotonin receptors in hippocampus.

Serotonin is an ancient molecular signal and a recognized neurotransmitter brainwide distributed with particular presence in hippocampus. Almost all serotonin receptor subtypes are expressed in hippocampus, which implicates an intricate modulating system, considering that they can be localized as autosynaptic, presynaptic, and postsynaptic receptors, even colocalized within the same cell and being target of homo- and heterodimerization. Neurons and glia, including immune cells, integrate a functional network that uses several serotonin receptors to regulate their roles in this particular part of the limbic system.

Microtransplantation of acetylcholine receptors from normal or denervated rat skeletal muscles to frog oocytes.

Cell membranes, carrying neurotransmitter receptors and ion channels, can be 'microtransplanted' into frog oocytes. This technique allows a direct functional characterization of the original membrane proteins, together with any associated molecules they may have, still embedded in their natural lipid environment. This approach has been previously demonstrated to be very useful to study neurotransmitter receptors and ion channels contained in cell membranes isolated from human brains. Here, we examined the possibility of using the microtransplantation method to study acetylcholine receptors from normal and denervated rat skeletal muscles. We found that the muscle membranes, carrying their fetal or adult acetylcholine receptor isoforms, could be efficiently microtransplanted to the oocyte membrane, making the oocytes become sensitive to acetylcholine. These results show that oocytes injected with skeletal muscle membranes efficiently incorporate functional acetylcholine receptors, thus making the microtransplantation approach a valuable tool to further investigate receptors and ion channels of human muscle diseases.

Functional impact of serial deletions at the C-terminus of the human GABArho1 receptor.

GABArho1 receptors are formed by homopentameric assemblies that gate a chloride ion-channel upon activation by the neurotransmitter. Very little is known about the structural and functional roles played by the different domains that form each subunit; but one of them, the fourth transmembrane segment (TM4), is known to form a hydrophobic bundle together with three other TM segments that are necessary to stabilize the structure of the receptor. In this study we progressively removed amino acid residues from the C-terminus of the human GABArho1 and studied the functional properties of the receptor mutants expressed in X. laevis oocytes. We found that deletions of up to the last four residues gave rise to receptors that were still functional, generating currents of 3.92 microA for the wt, 5.75 microA for S479X, 1.82 microA for F478X, 0.52 microA for I477X and 0.27 microA for S476X when exposed to 5 microM GABA; surprisingly, the mutant with one residue removed resulted more sensitive to the agonists. Further deletions, up to residue W475, resulted in receptors that did not gate an ion-channel. In addition, deleting the signal sequence, from R2-A15, in the N-terminus produced non-functional receptors. This study reveals that GABArho1 can tolerate removal of several residues that form the fourth transmembrane segment up to a critical point, signaled by W475, beyond which the mutant protein is translated but does not form functional receptors. A comparative study is presented of some electrophysiological and pharmacological properties of the deletion mutants that were able to generate GABA currents.

The metal-coordinated Casiopeína IIIEa induces the petite-like phenotype in Saccharomyces cerevisiae.

The Casiopeínas® are mixed chelate copper (II) complexes and promising antineoplastics agents against cancer cells and tumors in vitro and in vivo. However, the action mode of these compounds is poorly characterized. In this work the effect of the antineoplastic Casiopeína IIIEa on the metabolism and ultrastructure of the yeast Saccharomyces cerevisiae was investigated. Exposure of cells growing in rich or in low-iron medium to 5 µM of the compound decreased duplication time and reduced oxygen consumption. Those cells formed smaller colonies when growing in a non-fermentable carbon source and low-iron medium, and under the light microscope, multiple folds were observed along the plasma membrane accompanied with a reduction in the diameter of the yeast. These observations were confirmed under the electron microscope, which also revealed a slight reduction of the mitochondrial size. A correlation was found with smaller colonies exhibiting lower rates of oxygen consumption, and yeast labelled with fluorescent MitoTracker(TM) consistently exhibited reduced mitochondrial activity. It appears that Casiopeína IIIEa gives rise to smaller yeast and petite-like colonies by reducing the mitochondrial respiratory activity without significantly affecting the mitochondrial structure.

Microtransplantation of neurotransmitter receptors from postmortem autistic brains to Xenopus oocytes.

Autism is a complex disorder that arises from the pervasive action of genetic and epigenetic factors that alter synaptic connectivity of the brain. Although GABA and glutamate receptors seem to be two of those factors, very little is known about the functional properties of the autistic receptors. Autistic tissue samples stored in brain banks usually have relatively long postmortem times, and it is highly desirable to know whether neurotransmitter receptors in such tissues are still functional. Here we demonstrate that native receptors microtransplanted from autistic brains, as well as de novo mRNA-expressed receptors, are still functional and susceptible to detailed electrophysiological characterization even after long postmortem intervals. The opportunity to study the properties of human receptors present in diseased brains not only opens new avenues toward understanding autism and other neurological disorders, but it also makes the microtransplantation method a useful translational system to evaluate and develop novel medicinal drugs.