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.)

Expression and Function of GABA Receptors in Myelinating Cells

Myelin facilitates the fast transmission of nerve impulses and provides metabolic support to axons. Differentiation of oligodendrocyte progenitor cells (OPCs) and Schwann cell (SC) precursors is critical for myelination during development and myelin repair in demyelinating disorders. Myelination is tightly controlled by neuron-glia communication and requires the participation of a wide repertoire of signals, including neurotransmitters such as glutamate, ATP, adenosine, or gamma-aminobutyric acid (GABA). GABA is the main inhibitory neurotransmitter in the central nervous system (CNS) and it is also present in the peripheral nervous system (PNS). The composition and function of GABA receptors (GABARs) are well studied in neurons, while their nature and role in glial cells are still incipient. Recent studies demonstrate that GABA-mediated signaling mechanisms play relevant roles in OPC and SC precursor development and function, and stand out the implication of GABARs in oligodendrocyte (OL) and SC maturation and myelination. In this review, we highlight the evidence supporting the novel role of GABA with an emphasis on the molecular identity of the receptors expressed in these glial cells and the possible signaling pathways involved in their actions. GABAergic signaling in myelinating cells may have potential implications for developing novel reparative therapies in demyelinating diseases.This work was supported by CIBERNED (CB06/05/0076; CM) and by grants from the Ministry of Economy and Competitiveness, Government of Spain (SAF2016-75292-R and PID2019-109724RB-I00; CM), Basque Government (IT1203-19; CM), CONACYT-Mexico (No. 252121; RA), PAPIIT-UNAM-Mexico (IN203519; RA) and NIH (R21AG053740 and R21MH113177; AL). MS-R was hired thanks to the Gangoiti Foundation (Bilbao). LB-C and RO hold fellowships from Basque Government and CONACYT-Mexico, respectively

Synaptic Disruption by Soluble Oligomers in Patients with Alzheimer’s and Parkinson’s Disease

Neurodegenerative diseases are the result of progressive dysfunction of the neuronal activity and subsequent neuronal death. Currently, the most prevalent neurodegenerative diseases are by far Alzheimer’s (AD) and Parkinson’s (PD) disease, affecting millions of people worldwide. Although amyloid plaques and neurofibrillary tangles are the neuropathological hallmarks for AD and Lewy bodies (LB) are the hallmark for PD, current evidence strongly suggests that oligomers seeding the neuropathological hallmarks are more toxic and disease-relevant in both pathologies. The presence of small soluble oligomers is the common bond between AD and PD: amyloid β oligomers (AβOs) and Tau oligomers (TauOs) in AD and α-synuclein oligomers (αSynOs) in PD. Such oligomers appear to be particularly increased during the early pathological stages, targeting synapses at vulnerable brain regions leading to synaptic plasticity disruption, synapse loss, inflammation, excitation to inhibition imbalance and cognitive impairment. Absence of TauOs at synapses in individuals with strong AD disease pathology but preserved cognition suggests that mechanisms of resilience may be dependent on the interactions between soluble oligomers and their synaptic targets. In this review, we will discuss the current knowledge about the interactions between soluble oligomers and synaptic dysfunction in patients diagnosed with AD and PD, how it affects excitatory and inhibitory synaptic transmission, and the potential mechanisms of synaptic resilience in humans

Regional transcriptome analysis of AMPA and GABAA receptor subunit expression generates E/I signatures of the human brain

Abstract Theoretical and experimental work has demonstrated that excitatory (E) and inhibitory (I) currents within cortical circuits stabilize to a balanced state. This E/I balance, observed from single neuron to network levels, has a fundamental role in proper brain function and its impairment has been linked to numerous brain disorders. Over recent years, large amount of microarray and RNA-Sequencing datasets have been collected, however few studies have made use of these resources for exploring the balance of global gene expression levels between excitatory AMPA receptors (AMPARs) and inhibitory GABAA receptors. Here, we analyzed the relative relationships between these receptors to generate a basic transcriptional marker of E/I ratio. Using publicly available data from the Allen Brain Institute, we generated whole brain and regional signatures of AMPAR subunit gene expression in healthy human brains as well as the transcriptional E/I (tE/I) ratio. Then we refined the tE/I ratio to cell-type signatures in the mouse brain using data from the Gene Expression Omnibus. Lastly, we applied our workflow to developmental data from the Allen Brain Institute and revealed spatially and temporally controlled changes in the tE/I ratio during the embryonic and early postnatal stages that ultimately lead to the tE/I balance in adults

Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis

Metabotropic glutamate receptors (mGluRs) are membrane receptors that play a central role in the modulation of synaptic transmission and neuronal excitability and whose dysregulation is implicated in diverse neurological disorders. Most current understanding about the electrophysiological properties of such receptors has been determined using recombinant proteins. However, recombinant receptors do not necessarily recapitulate the properties of native receptors due to the lack of obligated accessory proteins, some of which are differentially expressed as function of developmental stage and brain region. To overcome this limitation, we sought to microtransplant entire synaptosome membranes from frozen rat cortex into Xenopus oocytes, and directly analyze the responses elicited by native mGluRs. We recorded ion currents elicited by 1 mM glutamate using two electrodes voltage clamp. Glutamate produced a fast ionotropic response (6 ± 0.3 nA) in all microtransplanted oocytes (n = 218 oocytes) and a delayed oscillatory response (52 ± 7 nA) in 73% of them. The participation of Group 1 mGluRs was confirmed by the presence of metabotropic oscillations during the administration of (±)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD; Group 1 mGluR agonist), and the absence of oscillations during co-administration of N-(1-adamantyl)quinoxaline-2-carboxamide (NPS 2390; Group 1 mGluR antagonist). Since both mGluR1 and mGluR5 belong to Group 1 mGluRs, further investigation revealed that mGluR1 antagonism with LY 456236 has little effect on metabotropic oscillations, while mGluR5 antagonism with 100 µM AZD 9272 has significant reduction of metabotropic currents elicited by ACPD and glutamate. We confirmed the expression of mGluR1 and mGluR5 in native synaptosomes by immunoblots, both of which are enhanced when compared to their counterpart proteins in rat cortex tissue lysates. Finally, these results demonstrate the merit of using microtransplantation of native synaptosomes for the study of mGluRs and the contribution of mGluR5 to the metabotropic glutamate signaling, providing a better tool for the understanding of the role of these receptors in neurological disorders

Dipicrylamine Modulates GABAr1 Receptors through Interactions with Residues in the TM4 and Cys-Loop Domains s

ABSTRACT 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 GABA A receptors that contain a1 and b2 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 GABAr1, a7 nicotinic, and 5-HT 3A serotonin receptors expressed in Xenopus oocytes. Our results indicate that DPA is an allosteric modulator of GABAr1 receptors with an IC 50 of 1.6 mM, an enhancer of a7 nicotinic receptors at relatively high concentrations of DPA, and has little, if any, effect on 5-HT 3A receptors. DPA antagonism of GABAr1 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 GABAr1, a7 nicotinic, and 5-HT 3A receptors suggests that a hydrophobic pocket within the Cys-loop and the M4 segment in GABAr1, 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 GABAr1 by DPA

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 a1 and b2 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 GABAr1, a7 nicotinic, and 5-HT3A serotonin receptors expressed in Xenopus oocytes. Our results indicate that DPA is an allosteric modulator of GABAr1 receptors with an IC50 of 1.6 mM, an enhancer of a7 nicotinic receptors at relatively high concentrations of DPA, and has little, if any, effect on 5-HT3A receptors. DPA antagonism of GABAr1 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 GABAr1, a7 nicotinic, and 5-HT3A receptors suggests that a hydrophobic pocket within the Cys-loop and the M4 segment in GABAr1, 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 GABAr1 by DPA