Publications by authors named "Rafael Luján"

134 Publications

Age-Dependent Shift of AMPA Receptors From Synapses to Intracellular Compartments in Alzheimer's Disease: Immunocytochemical Analysis of the CA1 Hippocampal Region in APP/PS1 Transgenic Mouse Model.

Front Aging Neurosci 2020 6;12:577996. Epub 2020 Oct 6.

Synaptic Structure Laboratory, Departamento de Ciencias Médicas, Facultad de Medicina, Instituto de Investigación en Discapacidades Neurológicas (IDINE), Universidad de Castilla-La Mancha, Albacete, Spain.

Synapse loss occurs early in Alzheimer's disease (AD) patients and animal models. Alterations at synaptic level are a major morphological correlate of the memory deficits and related symptoms of AD. Given the predominant roles of synaptic AMPA receptors (AMPARs) in excitatory synaptic transmission in the brain, changes in their dynamic regulation are also implicated in the pathophysiology of AD. Here, we used immunolocalization techniques to analyze the expression and subcellular distribution of AMPARs in the hippocampal region of APP/PS1 mouse model of AD. Immunoblots and histoblots revealed that the total amount of AMPARs and their regional expression pattern in the hippocampus was similar in APP/PS1 mice and in age-matched wild type mice. At the ultrastructural level, two synapse populations were examined using SDS-digested freeze-fracture replica labeling in the in mice: (i) on spines of CA1 pyramidal cells; and (ii) on randomly found dendritic shafts of CA1 interneurons. While 1- and 6-months-old APP/PS1 mice exhibited no change, we observed a significant reduction at 12 months in AMPAR density at synapses in both pyramidal cells and interneurons, compared to wild-type. This reduction of AMPARs in dendritic spines was accompanied by a significant increase in AMPAR subunit proteins identified in intracellular compartments. Our data demonstrate an age-dependent reduction of synaptic AMPARs in APP/PS1 mice, which may contribute to impaired learning and memory at later stages of AD.
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http://dx.doi.org/10.3389/fnagi.2020.577996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572859PMC
October 2020

β-Adrenergic Receptors/Epac Signaling Increases the Size of the Readily Releasable Pool of Synaptic Vesicles Required for Parallel Fiber LTP.

J Neurosci 2020 11 12;40(45):8604-8617. Epub 2020 Oct 12.

Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense, 28040 Madrid, Spain

The second messenger cAMP is an important determinant of synaptic plasticity that is associated with enhanced neurotransmitter release. Long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell (PC) synapses depends on a Ca-induced increase in presynaptic cAMP that is mediated by Ca-sensitive adenylyl cyclases. However, the upstream signaling and the downstream targets of cAMP involved in these events remain poorly understood. It is unclear whether cAMP generated by β-adrenergic receptors (βARs) is required for PF-PC LTP, although noradrenergic varicosities are apposed in PF-PC contacts. Guanine nucleotide exchange proteins directly activated by cAMP [Epac proteins (Epac 1-2)] are alternative cAMP targets to protein kinase A (PKA) and Epac2 is abundant in the cerebellum. However, whether Epac proteins participate in PF-PC LTP is not known. Immunoelectron microscopy demonstrated that βARs are expressed in PF boutons. Moreover, activation of these receptors through their agonist isoproterenol potentiated synaptic transmission in cerebellar slices from mice of either sex, an effect that was insensitive to the PKA inhibitors (H-89, KT270) but that was blocked by the Epac inhibitor ESI 05. Interestingly, prior activation of these βARs occluded PF-PC LTP, while the β1AR antagonist metoprolol blocked PF-PC LTP, which was also absent in mice. PF-PC LTP is associated with an increase in the size of the readily releasable pool (RRP) of synaptic vesicles, consistent with the isoproterenol-induced increase in vesicle docking in cerebellar slices. Thus, the βAR-mediated modulation of the release machinery and the subsequent increase in the size of the RRP contributes to PF-PC LTP. G-protein-coupled receptors modulate the release machinery, causing long-lasting changes in synaptic transmission that influence synaptic plasticity. Nevertheless, the mechanisms underlying synaptic responses to β-adrenergic receptor (βAR) activation remain poorly understood. An increase in the number of synaptic vesicles primed for exocytosis accounts for the potentiation of neurotransmitter release driven by βARs. This effect is not mediated by the canonical protein kinase A pathway but rather, through direct activation of the guanine nucleotide exchange protein Epac by cAMP. Interestingly, this βAR signaling via Epac is involved in long term potentiation at cerebellar granule cell-to-Purkinje cell synapses. Thus, the pharmacological activation of βARs modulates synaptic plasticity and opens therapeutic opportunities to control this phenomenon.
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http://dx.doi.org/10.1523/JNEUROSCI.0716-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643300PMC
November 2020

Cellular and Subcellular Localisation of Kv4-Associated KChIP Proteins in the Rat Cerebellum.

Int J Mol Sci 2020 Sep 3;21(17). Epub 2020 Sep 3.

Synaptic Structure Laboratory, Instituto de Investigación en Discapacidades Neurológicas (IDINE), Dept. Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Campus Biosanitario, C/Almansa 14, 02008 Albacete, Spain.

The K channel interacting proteins (KChIPs) are a family of cytosolic proteins that interact with Kv4 channels, leading to higher current density, modulation of channel inactivation and faster recovery from inactivation. Using immunohistochemical techniques at the light and electron microscopic level combined with quantitative analysis, we investigated the cellular and subcellular localisation of KChIP3 and KChIP4 to compare their distribution patterns with those for Kv4.2 and Kv4.3 in the cerebellar cortex. Immunohistochemistry at the light microscopic level demonstrated that KChIP3, KChIP4, Kv4.2 and Kv4.3 proteins were widely expressed in the cerebellum, with mostly overlapping patterns. Immunoelectron microscopic techniques showed that KChIP3, KChIP4, Kv4.2 and Kv4.3 shared virtually the same somato-dendritic domains of Purkinje cells and granule cells. Application of quantitative approaches showed that KChIP3 and KChIP4 were mainly membrane-associated, but also present at cytoplasmic sites close to the plasma membrane, in dendritic spines and shafts of Purkinje cells (PCs) and dendrites of granule cells (GCs). Similarly, immunoparticles for Kv4.2 and Kv4.3 were observed along the plasma membrane and at intracellular sites in the same neuron populations. In addition to the preferential postsynaptic distribution, KChIPs and Kv4 were also distributed presynaptically in parallel fibres and mossy fibres. Immunoparticles for KChIP3, KChIP4 and Kv4.3 were detected in parallel fibres, and KChIP3, KChIP4, Kv4.2 and Kv4.3 were found in parallel fibres, indicating that composition of KChIP and Kv4 seems to be input-dependent. Together, our findings unravelled previously uncharacterised KChIP and Kv4 subcellular localisation patterns in neurons, revealed that KChIP have additional Kv4-unrelated functions in the cerebellum and support the formation of macromolecular complexes between KChIP3 and KChIP4 with heterotetrameric Kv4.2/Kv4.3 channels.
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http://dx.doi.org/10.3390/ijms21176403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503578PMC
September 2020

PLA2G4E, a candidate gene for resilience in Alzheimer´s disease and a new target for dementia treatment.

Prog Neurobiol 2020 08 5;191:101818. Epub 2020 May 5.

Alzheimer´s Disease, Neurosciences Program, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain; IdiSNA (Navarra Institute for Health Research) Pamplona, Spain. Electronic address:

Clinical studies revealed that some aged-individuals accumulate a significant number of histopathological Alzheimer´s disease (AD) lesions in their brain, yet without developing any signs of dementia. Animal models of AD represent suitable tools to identify genes that might promote cognitive resilience and hence, this study first set out to identify cognitively resilient individuals in the aged-Tg2576 mouse model. A transcriptomic analysis of these mice identified PLA2G4E as a gene that might confer resistance to dementia. Indeed, a significant decrease in PLA2G4E is evident in the brain of late-stage AD patients, whereas no such changes are observed in early stage patients with AD neuropathological lesions but no signs of dementia. We demonstrated that adeno-associated viral vector-mediated overexpression of PLA2G4E in hippocampal neurons completely restored cognitive deficits in elderly APP/PS1 mice, without affecting the amyloid or tau pathology. These PLA2G4E overexpressing APP/PS1 mice developed significantly more dendritic spines than sham-injected mice, coinciding with the cognitive improvement observed. Hence, these results support the idea that a loss of PLA2G4E might play a key role in the onset of dementia in AD, highlighting the potential of PLA2G4E overexpression as a novel therapeutic strategy to manage AD and other disorders that course with memory deficits.
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http://dx.doi.org/10.1016/j.pneurobio.2020.101818DOI Listing
August 2020

Density of GABA Receptors Is Reduced in Granule Cells of the Hippocampus in a Mouse Model of Alzheimer's Disease.

Int J Mol Sci 2020 Apr 2;21(7). Epub 2020 Apr 2.

Synaptic Structure Laboratory, Instituto de Investigación en Discapacidades Neurológicas (IDINE), Dept. Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Campus Biosanitario, C/Almansa 14, 02008 Albacete, Spain.

Metabotropic γ-aminobutyric acid (GABA) receptors contribute to the control of network activity and information processing in hippocampal circuits by regulating neuronal excitability and synaptic transmission. The dysfunction in the dentate gyrus (DG) has been implicated in Alzheimer´s disease (AD). Given the involvement of GABA receptors in AD, to determine their subcellular localisation and possible alteration in granule cells of the DG in a mouse model of AD at 12 months of age, we used high-resolution immunoelectron microscopic analysis. Immunohistochemistry at the light microscopic level showed that the regional and cellular expression pattern of GABA was similar in an AD model mouse expressing mutated human amyloid precursor protein and presenilin1 (APP/PS1) and in age-matched wild type mice. High-resolution immunoelectron microscopy revealed a distance-dependent gradient of immunolabelling for GABA receptors, increasing from proximal to distal dendrites in both wild type and APP/PS1 mice. However, the overall density of GABA receptors at the neuronal surface of these postsynaptic compartments of granule cells was significantly reduced in APP/PS1 mice. Parallel to this reduction in surface receptors, we found a significant increase in GABA at cytoplasmic sites. GABA receptors were also detected at presynaptic sites in the molecular layer of the DG. We also found a decrease in plasma membrane GABA receptors in axon terminals contacting dendritic spines of granule cells, which was more pronounced in the outer than in the inner molecular layer. Altogether, our data showing post- and presynaptic reduction in surface GABA receptors in the DG suggest the alteration of the GABA-mediated modulation of excitability and synaptic transmission in granule cells, which may contribute to the cognitive dysfunctions in the APP/PS1 model of AD.
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http://dx.doi.org/10.3390/ijms21072459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177735PMC
April 2020

Sensory neuron-derived Na1.7 contributes to dorsal horn neuron excitability.

Sci Adv 2020 02 19;6(8):eaax4568. Epub 2020 Feb 19.

Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK.

Expression of the voltage-gated sodium channel Na1.7 in sensory neurons is required for pain sensation. We examined the role of Na1.7 in the dorsal horn of the spinal cord using an epitope-tagged Na1.7 knock-in mouse. Immuno-electron microscopy showed the presence of Na1.7 in dendrites of superficial dorsal horn neurons, despite the absence of mRNA. Rhizotomy of L5 afferent nerves lowered the levels of Na1.7 in the dorsal horn. Peripheral nervous system-specific Na1.7 null mutant mice showed central deficits, with lamina II dorsal horn tonic firing neurons more than halved and single spiking neurons more than doubled. Na1.7 blocker PF05089771 diminished excitability in dorsal horn neurons but had no effect on Na1.7 null mutant mice. These data demonstrate an unsuspected functional role of primary afferent neuron-generated Na1.7 in dorsal horn neurons and an expression pattern that would not be predicted by transcriptomic analysis.
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http://dx.doi.org/10.1126/sciadv.aax4568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7030926PMC
February 2020

Dopamine-Evoked Synaptic Regulation in the Nucleus Accumbens Requires Astrocyte Activity.

Neuron 2020 03 15;105(6):1036-1047.e5. Epub 2020 Jan 15.

Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

Dopamine is involved in physiological processes like learning and memory, motor control and reward, and pathological conditions such as Parkinson's disease and addiction. In contrast to the extensive studies on neurons, astrocyte involvement in dopaminergic signaling remains largely unknown. Using transgenic mice, optogenetics, and pharmacogenetics, we studied the role of astrocytes on the dopaminergic system. We show that in freely behaving mice, astrocytes in the nucleus accumbens (NAc), a key reward center in the brain, respond with Ca elevations to synaptically released dopamine, a phenomenon enhanced by amphetamine. In brain slices, synaptically released dopamine increases astrocyte Ca, stimulates ATP/adenosine release, and depresses excitatory synaptic transmission through activation of presynaptic A receptors. Amphetamine depresses neurotransmission through stimulation of astrocytes and the consequent A receptor activation. Furthermore, astrocytes modulate the acute behavioral psychomotor effects of amphetamine. Therefore, astrocytes mediate the dopamine- and amphetamine-induced synaptic regulation, revealing a novel cellular pathway in the brain reward system.
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http://dx.doi.org/10.1016/j.neuron.2019.12.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322729PMC
March 2020

Reduction in the neuronal surface of post and presynaptic GABA receptors in the hippocampus in a mouse model of Alzheimer's disease.

Brain Pathol 2020 05 12;30(3):554-575. Epub 2019 Dec 12.

Synaptic Structure Laboratory, Instituto de Investigación en Discapacidades Neurológicas (IDINE), Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Campus Biosanitario, C/ Almansa 14, 02008, Albacete, Spain.

The hippocampus plays key roles in learning and memory and is a main target of Alzheimer's disease (AD), which causes progressive memory impairments. Despite numerous investigations about the processes required for the normal hippocampal functions, the neurotransmitter receptors involved in the synaptic deficits by which AD disables the hippocampus are not yet characterized. By combining histoblots, western blots, immunohistochemistry and high-resolution immunoelectron microscopic methods for GABA receptors, this study provides a quantitative description of the expression and the subcellular localization of GABA in the hippocampus in a mouse model of AD at 1, 6 and 12 months of age. Western blots and histoblots showed that the total amount of protein and the laminar expression pattern of GABA were similar in APP/PS1 mice and in age-matched wild-type mice. In contrast, immunoelectron microscopic techniques showed that the subcellular localization of GABA subunit did not change significantly in APP/PS1 mice at 1 month of age, was significantly reduced in the stratum lacunosum-moleculare of CA1 pyramidal cells at 6 months of age and significantly reduced at the membrane surface of CA1 pyramidal cells at 12 months of age. This reduction of plasma membrane GABA was paralleled by a significant increase of the subunit at the intracellular sites. We further observed a decrease of membrane-targeted GABA receptors in axon terminals contacting CA1 pyramidal cells. Our data demonstrate compartment- and age-dependent reduction of plasma membrane-targeted GABA receptors in the CA1 region of the hippocampus, suggesting that this decrease might be enough to alter the GABA -mediated synaptic transmission taking place in AD.
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http://dx.doi.org/10.1111/bpa.12802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317930PMC
May 2020

Corrigendum to: -PTEN activity defines an axis for plasticity at cortico-amygdala synapses and influences social behavior.

Cereb Cortex 2020 03;30(2):849

Molecular Cognition Laboratory, Biophysics Institute, Consejo Superior de Investigaciones Cientificas (CSIC)-University of the Basque Country (UPV)/Euskal Herriko University (EHU), Campus Universidad del País Vasco, 48940 Leioa, Spain.

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http://dx.doi.org/10.1093/cercor/bhz232DOI Listing
March 2020

Excitotoxic targeting of Kidins220 to the Golgi apparatus precedes calpain cleavage of Rap1-activation complexes.

Cell Death Dis 2019 07 11;10(7):535. Epub 2019 Jul 11.

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), C/ Arturo Duperier, 4, 28029, Madrid, Spain.

Excitotoxic neuronal death induced by high concentrations of glutamate is a pathological event common to multiple acute or chronic neurodegenerative diseases. Excitotoxicity is mediated through overactivation of the N-Methyl-D-aspartate type of ionotropic glutamate receptors (NMDARs). Physiological stimulation of NMDARs triggers their endocytosis from the neuronal surface, inducing synaptic activity and survival. However almost nothing is known about the internalization of overactivated NMDARs and their interacting proteins, and how this endocytic process is connected with neuronal death has been poorly explored. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a component of NMDAR complexes essential for neuronal viability by the control of ERK activation. Here we have investigated Kidins220 endocytosis induced by NMDAR overstimulation and the participation of this internalization step in the molecular mechanisms of excitotoxicity. We show that excitotoxicity induces Kidins220 and GluN1 traffic to the Golgi apparatus (GA) before Kidins220 is degraded by the protease calpain. We also find that excitotoxicity triggers an early activation of Rap1-GTPase followed by its inactivation. Kidins220 excitotoxic endocytosis and subsequent calpain-mediated downregulation governs this late inactivation of Rap1 that is associated to decreases in ERK activity preceding neuronal death. Furthermore, we identify the molecular mechanisms involved in the excitotoxic shutoff of Kidins220/Rap1/ERK prosurvival cascade that depends on calpain processing of Rap1-activation complexes. Our data fit in a model where Kidins220 targeting to the GA during early excitotoxicity would facilitate Rap1 activation and subsequent stimulation of ERK. At later times, activation of Golgi-associated calpain, would promote the degradation of GA-targeted Kidins220 and two additional components of the specific Rap1 activation complex, PDZ-GEF1, and S-SCAM. In this way, late excitotoxicity would turn off Rap1/ERK cascade and compromise neuronal survival.
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http://dx.doi.org/10.1038/s41419-019-1766-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624258PMC
July 2019

PTEN Activity Defines an Axis for Plasticity at Cortico-Amygdala Synapses and Influences Social Behavior.

Cereb Cortex 2020 03;30(2):505-524

Molecular Cognition Laboratory, Biophysics Institute, Consejo Superior de Investigaciones Cientificas (CSIC)-University of the Basque Country (UPV)/Euskal Herriko University (EHU), Campus Universidad del País Vasco, 48940 Leioa, Spain.

Phosphatase and tensin homolog on chromosome 10 (PTEN) is a tumor suppressor and autism-associated gene that exerts an important influence over neuronal structure and function during development. In addition, it participates in synaptic plasticity processes in adulthood. As an attempt to assess synaptic and developmental mechanisms by which PTEN can modulate cognitive function, we studied the consequences of 2 different genetic manipulations in mice: presence of additional genomic copies of the Pten gene (Ptentg) and knock-in of a truncated Pten gene lacking its PDZ motif (Pten-ΔPDZ), which is required for interaction with synaptic proteins. Ptentg mice exhibit substantial microcephaly, structural hypoconnectivity, enhanced synaptic depression at cortico-amygdala synapses, reduced anxiety, and intensified social interactions. In contrast, Pten-ΔPDZ mice have a much more restricted phenotype, with normal synaptic connectivity, but impaired synaptic depression at cortico-amygdala synapses and virtually abolished social interactions. These results suggest that synaptic actions of PTEN in the amygdala contribute to specific behavioral traits, such as sociability. Also, PTEN appears to function as a bidirectional rheostat in the amygdala: reduction in PTEN activity at synapses is associated with less sociability, whereas enhanced PTEN activity accompanies hypersocial behavior.
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http://dx.doi.org/10.1093/cercor/bhz103DOI Listing
March 2020

Circuit-specific control of the medial entorhinal inputs to the dentate gyrus by atypical presynaptic NMDARs activated by astrocytes.

Proc Natl Acad Sci U S A 2019 07 31;116(27):13602-13610. Epub 2019 May 31.

Département de Neurosciences Fondamentales, Université de Lausanne, 1005 Lausanne, Switzerland;

Here, we investigated the properties of presynaptic -methyl-d-aspartate receptors (pre-NMDARs) at corticohippocampal excitatory connections between perforant path (PP) afferents and dentate granule cells (GCs), a circuit involved in memory encoding and centrally affected in Alzheimer's disease and temporal lobe epilepsy. These receptors were previously reported to increase PP release probability in response to gliotransmitters released from astrocytes. Their activation occurred even under conditions of elevated Mg and lack of action potential firing in the axons, although how this could be accomplished was unclear. We now report that these pre-NMDARs contain the GluN3a subunit conferring them low Mg sensitivity. GluN3a-containing NMDARs at PP-GC synapses are preponderantly presynaptic vs. postsynaptic and persist beyond the developmental period. Moreover, they are expressed selectively at medial-not lateral-PP axons and act to functionally enhance release probability specifically of the medial perforant path (MPP) input to GC dendrites. By controlling release probability, GluN3a-containing pre-NMDARs also control the dynamic range for long-term potentiation (LTP) at MPP-GC synapses, an effect requiring Ca signaling in astrocytes. Consistent with the functional observations, GluN3a subunits in MPP terminals are localized at sites away from the presynaptic release sites, often facing astrocytes, in line with a primary role for astrocytic inputs in their activation. Overall, GluN3A-containing pre-NMDARs emerge as atypical modulators of dendritic computations in the MPP-GC memory circuit.
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http://dx.doi.org/10.1073/pnas.1816013116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612919PMC
July 2019

Expression, Cellular and Subcellular Localisation of Kv4.2 and Kv4.3 Channels in the Rodent Hippocampus.

Int J Mol Sci 2019 Jan 9;20(2). Epub 2019 Jan 9.

Synaptic Structure Laboratory, Instituto de Investigación en Discapacidades Neurológicas (IDINE), Dept. Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Campus Biosanitario, C/Almansa 14, 02008 Albacete, Spain.

The Kv4 family of voltage-gated K⁺ channels underlie the fast transient (A-type) outward K⁺ current. Although A-type currents are critical to determine somato-dendritic integration in central neurons, relatively little is known about the precise subcellular localisation of the underlying channels in hippocampal circuits. Using histoblot and immunoelectron microscopic techniques, we investigated the expression, regional distribution and subcellular localisation of Kv4.2 and Kv4.3 in the adult brain, as well as the ontogeny of their expression during postnatal development. Histoblot demonstrated that Kv4.2 and Kv4.3 proteins were widely expressed in the brain, with mostly non-overlapping patterns. During development, levels of Kv4.2 and Kv4.3 increased with age but showed marked region- and developmental stage-specific differences. Immunoelectron microscopy showed that labelling for Kv4.2 and Kv4.3 was differentially present in somato-dendritic domains of hippocampal principal cells and interneurons, including the synaptic specialisation. Quantitative analyses indicated that most immunoparticles for Kv4.2 and Kv4.3 were associated with the plasma membrane in dendritic spines and shafts, and that the two channels showed very similar distribution patterns in spines of principal cells and along the surface of granule cells. Our data shed new light on the subcellular localisation of Kv4 channels and provide evidence for their non-uniform distribution over the plasma membrane of hippocampal neurons.
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http://dx.doi.org/10.3390/ijms20020246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359635PMC
January 2019

Adenosine A-Cannabinoid CB Receptor Heteromers in the Hippocampus: Cannabidiol Blunts Δ-Tetrahydrocannabinol-Induced Cognitive Impairment.

Mol Neurobiol 2019 Aug 4;56(8):5382-5391. Epub 2019 Jan 4.

Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL-Universitat de Barcelona, C/Feixa Llarga s/n, 08907, L'Hospitalet de Llobregat, Spain.

At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ-tetrahydrocannabinol (Δ-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ-THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ-THC-induced cognitive impairment in an adenosine A receptor (AR)-dependent manner. Furthermore, we reveal the existence of AR and cannabinoid CB receptor (CBR) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent AR-CBR functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal AR/CBR complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of AR-CBR interactions in the brain.
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http://dx.doi.org/10.1007/s12035-018-1456-3DOI Listing
August 2019

Inhibitory Signaling to Ion Channels in Hippocampal Neurons Is Differentially Regulated by Alternative Macromolecular Complexes of RGS7.

J Neurosci 2018 11 12;38(46):10002-10015. Epub 2018 Oct 12.

Department of Neuroscience, The Scripps Research Institute, Jupiter, Florida 33458,

The neuromodulatory effects of GABA on pyramidal neurons are mediated by GABA receptors (GABARs) that signal via a conserved G-protein-coupled pathway. Two prominent effectors regulated by GABARs include G-protein inwardly rectifying K (GIRK) and P/Q/N type voltage-gated Ca (Ca2) ion channels that control excitability and synaptic output of these neurons, respectively. Regulator of G-protein signaling 7 (RGS7) has been shown to control GABA effects, yet the specificity of its impacts on effector channels and underlying molecular mechanisms is poorly understood. In this study, we show that hippocampal RGS7 forms two distinct complexes with alternative subunit configuration bound to either membrane protein R7BP (RGS7 binding protein) or orphan receptor GPR158. Quantitative biochemical experiments show that both complexes account for targeting nearly the entire pool of RGS7 to the plasma membrane. We analyzed the effect of genetic elimination in mice of both sexes and overexpression of various components of RGS7 complex by patch-clamp electrophysiology in cultured neurons and brain slices. We report that RGS7 prominently regulates GABAR signaling to Ca2, in addition to its known involvement in modulating GIRK. Strikingly, only complexes containing R7BP, but not GPR158, accelerated the kinetics of both GIRK and Ca2 modulation by GABARs. In contrast, GPR158 overexpression exerted the opposite effect and inhibited RGS7-assisted temporal modulation of GIRK and Ca2 by GABA. Collectively, our data reveal mechanisms by which distinctly composed macromolecular complexes modulate the activity of key ion channels that mediate the inhibitory effects of GABA on hippocampal CA1 pyramidal neurons. This study identifies the contributions of distinct macromolecular complexes containing a major G-protein regulator to controlling key ion channel function in hippocampal neurons with implications for understanding molecular mechanisms underlying synaptic plasticity, learning, and memory.
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http://dx.doi.org/10.1523/JNEUROSCI.1378-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234300PMC
November 2018

SK2 Channels Associate With mGlu Receptors and Ca2.1 Channels in Purkinje Cells.

Front Cell Neurosci 2018 19;12:311. Epub 2018 Sep 19.

Division of Brain Structure and Function, Research Center for Child Mental Development, Life Science Advancement Program, Faculty of Medical Science, University of Fukui, Fukui, Japan.

The small-conductance, Ca-activated K (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca: Ca2.1 channels and mGlu receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for Ca2.1 channels and mGlu receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to Ca2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca, and suggest that the ultrastructural association of SK2 with Ca2.1 and mGlu provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca transients in PCs.
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http://dx.doi.org/10.3389/fncel.2018.00311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156379PMC
September 2018

Metabotropic glutamate type 5 receptor requires contactin-associated protein 1 to control memory formation.

Hum Mol Genet 2018 10;27(20):3528-3541

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Spain.

The hippocampus is a key brain region for memory formation. Metabotropic glutamate type 5 receptors (mGlu5R) are strongly expressed in CA1 pyramidal neurons and fine-tune synaptic plasticity. Accordingly, mGlu5R pharmacological manipulation may represent an attractive therapeutic strategy to manage hippocampal-related neurological disorders. Here, by means of a membrane yeast two-hybrid screening, we identified contactin-associated protein 1 (Caspr1), a type I transmembrane protein member of the neurexin family, as a new mGlu5R partner. We report that mGlu5R and Caspr1 co-distribute and co-assemble both in heterologous expression systems and in rat brain. Furthermore, downregulation of Caspr1 in rat hippocampal primary cultures decreased mGlu5R-mediated signaling. Finally, silencing Caspr1 expression in the hippocampus impaired the impact of mGlu5R on spatial memory. Our results indicate that Caspr1 plays a pivotal role controlling mGlu5R function in hippocampus-dependent memory formation. Hence, this new protein-protein interaction may represent novel target for neurological disorders affecting hippocampal glutamatergic neurotransmission.
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http://dx.doi.org/10.1093/hmg/ddy264DOI Listing
October 2018

Transcriptome analysis in tissue sectors with contrasting crocins accumulation provides novel insights into apocarotenoid biosynthesis and regulation during chromoplast biogenesis.

Sci Rep 2018 02 12;8(1):2843. Epub 2018 Feb 12.

Instituto Botánico, Departamento de Ciencia y Tecnología Agroforestal y Genética, Facultad de Farmacia, Universidad de Castilla-La Mancha, Campus Universitario s/n, 02071, Albacete, Spain.

Crocins, the red soluble apocarotenoids of saffron, accumulate in the flowers of Crocus species in a developmental and tissue-specific manner. In Crocus sieberi, crocins accumulate in stigmas but also in a distinct yellow tepal sector, which we demonstrate contains chromoplast converted from amyloplasts. Secondary metabolites were analysed by LC-DAD-HRMS, revealing the progressive accumulation of crocetin and crocins in the yellow sector, which were also localized in situ by Raman microspectroscopy. To understand the underlying mechanisms of crocin biosynthesis, we sequenced the C. sieberi tepal transcriptome of two differentially pigmented sectors (yellow and white) at two developmental stages (6 and 8) by Illumina sequencing. A total of 154 million high-quality reads were generated and assembled into 248,099 transcripts. Differentially expressed gene analysis resulted in the identification of several potential candidate genes involved in crocin metabolism and regulation. The results provide a first profile of the molecular events related to the dynamics of crocetin and crocin accumulation during tepal development, and present new information concerning apocarotenoid biosynthesis regulators and their accumulation in Crocus. Further, reveals genes that were previously unknown to affect crocin formation, which could be used to improve crocin accumulation in Crocus plants and the commercial quality of saffron spice.
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http://dx.doi.org/10.1038/s41598-018-21225-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809551PMC
February 2018

Bidirectional modulation of glutamatergic synaptic transmission by metabotropic glutamate type 7 receptors at Schaffer collateral-CA1 hippocampal synapses.

J Physiol 2018 03 25;596(5):921-940. Epub 2018 Jan 25.

Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, 28040, Madrid, Spain.

Key Points: Neurotransmitter release is inhibited by metabotropic glutamate type 7 (mGlu ) receptors that reduce Ca influx, yet synapses lacking this receptor also produce weaker release, suggesting that mGlu receptors may also prime synaptic vesicles for release. Prolonged activation of mGlu receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs through a presynaptic effect. The inhibitory response is blocked by pertussis toxin, while the potentiating response is prevented by a phospholipase C inhibitor (U73122) and an inhibitor of diacylglycerol (DAG) binding (calphostin C), suggesting that this receptor also couples to pathways that generate DAG. Release potentiation is associated with an increase in the number of synaptic vesicles close to the plasma membrane, which was dependent on the Munc13-2 and RIM1α proteins. The Glu7 receptors activated by the glutamate released following high frequency stimulation provoke a bidirectional modulation of synaptic transmission.

Abstract: Neurotransmitter release is driven by Ca influx at synaptic boutons that acts on synaptic vesicles ready to undergo exocytosis. Neurotransmitter release is inhibited when metabotropic glutamate type 7 (mGlu ) receptors provoke a reduction in Ca influx, although the reduced release from synapses lacking this receptor suggests that they may also prime synaptic vesicles for release. These mGlu receptors activate phospholipase C (PLC) and generate inositol trisphosphate, which in turn releases Ca from intracellular stores and produces diacylglycerol (DAG), an activator of proteins containing DAG-binding domains such as Munc13 and protein kinase C (PKC). However, the full effects of mGlu receptor signalling on synaptic transmission are unclear. We found that prolonged activation of mGlu receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs, a presynaptic effect that changes the frequency but not the amplitude of the mEPSCs and the paired pulse ratio. Pertussis toxin blocks the inhibitory response, while the PLC inhibitor U73122, and the inhibitor of DAG binding calphostin C, prevent receptor mediated potentiation. Moreover, this DAG-dependent potentiation of the release machinery brings more synaptic vesicles closer to the active zone plasma membrane in a Munc13-2- and RIM1α-dependent manner. Electrically evoked release of glutamate that activates mGlu receptors also bidirectionally modulates synaptic transmission. In these conditions, potentiation now occurs rapidly and it overcomes any inhibition, such that potentiation prevails unless it is suppressed with the PLC inhibitor U73122.
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http://dx.doi.org/10.1113/JP275371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830420PMC
March 2018

Differential association of GABA receptors with their effector ion channels in Purkinje cells.

Brain Struct Funct 2018 Apr 25;223(3):1565-1587. Epub 2017 Nov 25.

Department of Histological and Physiological Sciences, Faculty of Medical Science, University of Fukui, Yoshida, Fukui, 910-1193, Japan.

Metabotropic GABA receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABA receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABA was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABA receptors with two key effector ion channels, the G protein-gated inwardly rectifying K (GIRK/Kir3) channel and the voltage-dependent Ca channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABA receptors co-assembled with GIRK and Ca2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABA and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABA and Ca2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABA and GIRK2 or Ca2.1 channels was detected, inter-cluster distance for GABA and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABA and Ca2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABA receptors are associated with GIRK and Ca2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABA receptors and their effector ion channels in the cerebellar network.
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http://dx.doi.org/10.1007/s00429-017-1568-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869904PMC
April 2018

Monitoring the colonization and infection of legume nodules by Micromonospora in co-inoculation experiments with rhizobia.

Sci Rep 2017 09 8;7(1):11051. Epub 2017 Sep 8.

Departamento de Microbiología y Genética, Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, Salamanca, Spain.

The discovery that the actinobacterium Micromonospora inhabits nitrogen-fixing nodules raised questions as to its potential ecological role. The capacity of two Micromonospora strains to infect legumes other than their original host, Lupinus angustifolius, was investigated using Medicago and Trifolium as test plants. Compatible rhizobial strains were used for coinoculation of the plants because Micromonospora itself does not induce nodulation. Over 50% of nodules from each legume housed Micromonospora, and using 16S rRNA gene sequence identification, we verified that the reisolated strains corresponded to the microorganisms inoculated. Entry of the bacteria and colonization of the plant hosts were monitored using a GFP-tagged Lupac 08 mutant together with rhizobia, and by using immunogold labeling. Strain Lupac 08 was localized in plant tissues, confirming its capacity to enter and colonize all hosts. Based on studying three different plants, our results support a non-specific relationship between Micromonospora and legumes. Micromonospora Lupac 08, originally isolated from Lupinus re-enters root tissue, but only when coinoculated with the corresponding rhizobia. The ability of Micromonospora to infect and colonize different legume species and function as a potential plant-growth promoting bacterium is relevant because this microbe enhances the symbiosis without interfering with the host and its nodulating and nitrogen-fixing microbes.
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http://dx.doi.org/10.1038/s41598-017-11428-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591303PMC
September 2017

The Parkinson's disease-associated GPR37 receptor interacts with striatal adenosine A receptor controlling its cell surface expression and function in vivo.

Sci Rep 2017 08 25;7(1):9452. Epub 2017 Aug 25.

Unitat de Farmacologia, Departament Patologia i Terapéutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

G protein-coupled receptor 37 (GPR37) is an orphan receptor associated to Parkinson's disease (PD) neuropathology. Here, we identified GPR37 as an inhibitor of adenosine A receptor (AR) cell surface expression and function in vivo. In addition, we showed that GPR37 and AR do oligomerize in the striatum. Thus, a close proximity of GPR37 and AR at the postsynaptic level of striatal synapses was observed by double-labelling post-embedding immunogold detection. Indeed, the direct receptor-receptor interaction was further substantiated by proximity ligation in situ assay. Interestingly, GPR37 deletion promoted striatal AR cell surface expression that correlated well with an increased AR agonist-mediated cAMP accumulation, both in primary striatal neurons and nerve terminals. Furthermore, GPR37-/- mice showed enhanced AR agonist-induced catalepsy and an increased response to AR antagonist-mediated locomotor activity. Overall, these results revealed a key role for GPR37 controlling AR biology in the striatum, which may be relevant for PD management.
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http://dx.doi.org/10.1038/s41598-017-10147-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573386PMC
August 2017

HCN1 channels reduce the rate of exocytosis from a subset of cortical synaptic terminals.

Sci Rep 2017 01 10;7:40257. Epub 2017 Jan 10.

UCL School of Pharmacy, University College London, London, WC1N 1AX, UK.

The hyperpolarization-activated cyclic nucleotide-gated (HCN1) channels are predominantly located in pyramidal cell dendrites within the cortex. Recent evidence suggests these channels also exist pre-synaptically in a subset of synaptic terminals within the mature entorhinal cortex (EC). Inhibition of pre-synaptic HCN channels enhances miniature excitatory post-synaptic currents (mEPSCs) onto EC layer III pyramidal neurons, suggesting that these channels decrease the release of the neurotransmitter, glutamate. Thus, do pre-synaptic HCN channels alter the rate of synaptic vesicle exocytosis and thereby enhance neurotransmitter release? To address this, we imaged the release of FM1-43, a dye that is incorporated into synaptic vesicles, from EC synaptic terminals using two photon microscopy in slices obtained from forebrain specific HCN1 deficient mice, global HCN1 knockouts and their wildtype littermates. This coupled with electrophysiology and pharmacology showed that HCN1 channels restrict the rate of exocytosis from a subset of cortical synaptic terminals within the EC and in this way, constrain non-action potential-dependent and action potential-dependent spontaneous release as well as synchronous, evoked release. Since HCN1 channels also affect post-synaptic potential kinetics and integration, our results indicate that there are diverse ways by which HCN1 channels influence synaptic strength and plasticity.
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http://dx.doi.org/10.1038/srep40257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223132PMC
January 2017

Cellular and Subcellular Localization of the RGS7/Gβ5/R7BP Complex in the Cerebellar Cortex.

Front Neuroanat 2016 30;10:114. Epub 2016 Nov 30.

Synaptic Structure Laboratory, Instituto de Investigación en Discapacidades Neurológicas (IDINE), Departamento Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha Albacete, Spain.

A member of regulator of G-protein signaling family, RGS7, is an essential modulator of signaling through GABA receptors. RGS7 functions as a macromolecular complex with type 5 G protein β (Gβ5) and R7 binding protein (R7BP) to control the localization and function of the resultant heterotrimeric complexes. Here, we used co-immunoprecipitation, hybridization, histoblot and immunohistochemical techniques at the light and electron microscopic level to advance understanding of RGS7-Gβ5-R7BP complexes in the central nervous system, focusing on distinct neuronal populations in the cerebellar cortex. Histoblot analysis showed that RGS7, Gβ5 and R7BP proteins were widely expressed in the brain, with mostly an overlapping pattern and showing a high expression level in the molecular layer of the cerebellar cortex. Co-immunoprecipitation experiments established that the RGS7/Gβ5 forms complexes with R7BP in the cerebellum. At the cellular level, RGS7 and R7BP mRNAs were expressed at the highest level in Purkinje cells (PCs) and Golgi cells, and at low levels in granule cells. Immunohistochemistry confirmed that labeling for RGS7, Gβ5 and R7BP were present in the three neuronal populations and concentrated in dendrites and spines. At the electron microscopic level, immunolabeling for RGS7, Gβ5 and R7BP proteins was found both at postsynaptic and presynaptic sites and showed similar distribution patterns. Immunoreactivity for the three proteins was mostly localized along the extrasynaptic plasma membrane of dendritic shafts and spines of PCs and to a lesser extent, in axon terminals (AT) establishing excitatory synapses. Quantitative analysis of immunogold particles for RGS7, Gβ5 and R7BP revealed that they are non-uniformly distributed along the surface of PCs, and show enrichment around excitatory synapses on dendritic spines. We further report that deletion of R7BP in mice reduced the targeting of both RGS7 and Gβ5 to the plasma membrane. Altogether, these data support the existence of macromolecular complexes composed of RGS7-Gβ5-R7BP in PCs. The location at post- and pre-synaptic sites in PCs spines-parallel fiber synapses suggests their involvement in the modulation of glutamatergic neurotransmission in the cerebellar cortex.
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http://dx.doi.org/10.3389/fnana.2016.00114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127842PMC
November 2016

Excitatory and Inhibitory Neurons in the Hippocampus Exhibit Molecularly Distinct Large Dense Core Vesicles.

Front Cell Neurosci 2016 31;10:202. Epub 2016 Aug 31.

Department of Pharmacology, University of Maryland School of Medicine Baltimore, MD, USA.

Hippocampal interneurons comprise a diverse family of inhibitory neurons that are critical for detailed information processing. Along with gamma-aminobutyric acid (GABA), interneurons secrete a myriad of neuroactive substances via secretory vesicles but the molecular composition and regulatory mechanisms remain largely unknown. In this study, we have carried out an immunohistofluorescence analysis to describe the molecular content of vesicles in distinct populations of hippocampal neurons. Our results indicate that phogrin, an integral protein of secretory vesicles in neuroendocrine cells, is highly enriched in parvalbumin-positive interneurons. Consistently, immunoelectron microscopy revealed phogrin staining in axon terminals of symmetrical synapses establishing inhibitory contacts with cell bodies of CA1 pyramidal neurons. Furthermore, phogrin is highly expressed in CA3 and dentate gyrus (DG) interneurons which are both positive for PV and neuropeptide Y. Surprisingly, chromogranin B a canonical large dense core vesicle marker, is excluded from inhibitory cells in the hippocampus but highly expressed in excitatory CA3 pyramidal neurons and DG granule cells. Our results provide the first evidence of phogrin expression in hippocampal interneurons and suggest the existence of molecularly distinct populations of secretory vesicles in different types of inhibitory neurons.
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http://dx.doi.org/10.3389/fncel.2016.00202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5005380PMC
September 2016

Ontogenic Changes and Differential Localization of T-type Ca(2+) Channel Subunits Cav3.1 and Cav3.2 in Mouse Hippocampus and Cerebellum.

Front Neuroanat 2016 26;10:83. Epub 2016 Aug 26.

Synaptic Structure Laboratory, Department Ciencias Médicas, Instituto de Investigación en Discapacidades Neurológicas (IDINE), Facultad de Medicina, Universidad Castilla-La Mancha Albacete, Spain.

T-type calcium (Ca(2+)) channels play a central role in regulating membrane excitability in the brain. Although the contributions of T-type current to neuron output is often proposed to reflect a differential distribution of T-type channel subtypes to somato-dendritic compartments, their precise subcellular distributions in central neurons are not fully determined. Using histoblot and high-resolution immunoelectron microscopic techniques, we have investigated the expression, regional distribution and subcellular localization of T-type Cav3.1 and Cav3.2 channel subunits in the adult brain, as well as the ontogeny of expression during postnatal development. Histoblot analysis showed that Cav3.1 and Cav3.2 proteins were widely expressed in the brain, with mostly non-overlapping patterns. Cav3.1 showed the highest expression level in the molecular layer (ml) of the cerebellum (Cb), and Cav3.2 in the hippocampus (Hp) and the ml of Cb. During development, levels of Cav3.1 and Cav3.2 increased with age, although there were marked region- and developmental stage-specific differences in their expression. At the cellular and subcellular level, immunoelectron microscopy showed that labeling for Cav3.1 was present in somato-dendritic domains of hippocampal interneurons and Purkinje cells (PCs), while Cav3.2 was present in somato-dendritic domains of CA1 pyramidal cells, hippocampal interneurons and PCs. Most of the immunoparticles for Cav3.1 and Cav3.2 were either associated with the plasma membrane or the intracellular membranes, with notable differences depending on the compartment. Thus, Cav3.1 was mainly located in the plasma membrane of interneurons, whereas Cav3.2 was mainly located in the plasma membrane of dendritic spines and had a major intracellular distribution in dendritic shafts. In PCs, Cav3.1 and Cav3.2 showed similar distribution patterns. In addition to its main postsynaptic distribution, Cav3.2 but not Cav3.1 was also detected in axon terminals establishing excitatory synapses. These results shed new light on the subcellular localization of T-type channel subunits and provide evidence for the non-uniform distribution of Cav3.1 and Cav3.2 subunits over the plasma membrane of central neurons, which may account for the functional heterogeneity of T-type mediated current.
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http://dx.doi.org/10.3389/fnana.2016.00083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999439PMC
September 2016

L-DOPA Oppositely Regulates Synaptic Strength and Spine Morphology in D1 and D2 Striatal Projection Neurons in Dyskinesia.

Cereb Cortex 2016 10;26(11):4253-4264

Instituto Cajal, Consejo Superior de Investigaciones Científicas, CSIC, 28002 Madrid, Spain.

Dopamine depletion in Parkinson's disease (PD) produces dendritic spine loss in striatal medium spiny neurons (MSNs) and increases their excitability. However, the synaptic changes that occur in MSNs in PD, in particular those induced by chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, are still poorly understood. We exposed BAC-transgenic D1-tomato and D2-eGFP mice to PD and dyskinesia model paradigms, enabling cell type-specific assessment of changes in synaptic physiology and morphology. The distinct fluorescence markers allowed us to identify D1 and D2 MSNs for analysis using intracellular sharp electrode recordings, electron microscopy, and 3D reconstructions with single-cell Lucifer Yellow injections. Dopamine depletion induced spine pruning in both types of MSNs, affecting mushroom and thin spines equally. Dopamine depletion also increased firing rate in both D1- and D2-MSNs, but reduced evoked-EPSP amplitude selectively in D2-MSNs. L-DOPA treatment that produced dyskinesia differentially affected synaptic properties in D1- and D2-MSNs. In D1-MSNs, spine density remained reduced but the remaining spines were enlarged, with bigger heads and larger postsynaptic densities. These morphological changes were accompanied by facilitation of action potential firing triggered by synaptic inputs. In contrast, although L-DOPA restored the number of spines in D2-MSNs, it resulted in shortened postsynaptic densities. These changes in D2-MSNs correlated with a decrease in synaptic transmission. Our findings indicate that L-DOPA-induced dyskinesia is associated with abnormal spine morphology, modified synaptic transmission, and altered EPSP-spike coupling, with distinct effects in D1- and D2-MSNs.
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http://dx.doi.org/10.1093/cercor/bhw263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066835PMC
October 2016

Examination of the hippocampal contribution to serotonin 5-HT2A receptor-mediated facilitation of object memory in C57BL/6J mice.

Neuropharmacology 2016 10 22;109:332-340. Epub 2016 Apr 22.

Department of Psychology and Life Science Initiative, Charles E. Schmidt College of Science, Florida Atlantic University, 5353 Parkside Drive, Jupiter, FL 33458, USA. Electronic address:

The rodent hippocampus supports non-spatial object memory. Serotonin 5-HT2A receptors (5-HT2AR) are widely expressed throughout the hippocampus. We previously demonstrated that the activation of 5-HT2ARs enhanced the strength of object memory assessed 24 h after a limited (i.e., weak memory) training procedure. Here, we examined the subcellular distribution of 5-HT2ARs in the hippocampal CA1 region and underlying mechanisms of 5-HT2AR-mediated object memory consolidation. Analyses with immuno-electron microscopy revealed the presence of 5-HT2ARs on the dendritic spines and shafts of hippocampal CA1 neurons, and presynaptic terminals in the CA1 region. In an object recognition memory procedure that places higher demand on the hippocampus, only post-training systemic or intrahippocampal administration of the 5-HT2AR agonist TCB-2 enhanced object memory. Object memory enhancement by TCB-2 was blocked by the 5-HT2AR antagonist, MDL 11,937. The memory-enhancing dose of systemic TCB-2 increased extracellular glutamate levels in hippocampal dialysate samples, and increased the mean in vivo firing rate of hippocampal CA1 neurons. In summary, these data indicate a pre- and post-synaptic distribution of 5-HT2ARs, and activation of 5-HT2ARs selectively enhanced the consolidation of object memory, without affecting encoding or retrieval. The 5-HT2AR-mediated facilitation of hippocampal memory may be associated with an increase in hippocampal neuronal firing and glutamate efflux during a post-training time window in which recently encoded memories undergo consolidation.
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http://dx.doi.org/10.1016/j.neuropharm.2016.04.033DOI Listing
October 2016

Distinct expression patterns of inwardly rectifying potassium currents in developing cerebellar granule cells of the hemispheres and the vermis.

Eur J Neurosci 2016 06 29;43(11):1460-73. Epub 2016 Mar 29.

Department of Biology and Biotechnology, University of Pavia, via Ferrata 9, 27100 Pavia, Italy.

G-protein-coupled inwardly rectifying potassium (GIRK) channels play a crucial role during the migration and maturation of cerebellar granule cells (GCs) in the vermis. In the cerebellar hemispheres, however, only minor effects on the development of GCs are observed in mice with GIRK channel impairment. This regional difference may reflect distinct ontogenetic expression patterns of GIRK channels. Therefore, inwardly rectifying responses in mice were characterized at different stages of development in the vermis and the hemispheres. In the vermis, GCs in the premigratory zone (PMZ) at P7-P15 exhibit GIRK current but not constitutive inwardly rectifying potassium (CIRK) current, and are relatively depolarized at rest. In contrast, premigratory GCs in the hemispheres express only CIRK channels, which accounts for their more hyperpolarized resting membrane potential. Furthermore, the pattern of voltage-dependent inward currents in the PMZ GCs of cerebellar hemispheres is consistent with a more mature stage of development than the corresponding GCs in the vermis, resulting in robust firing properties mediated by sodium channels. Later in development (P21-P22), CIRK current is then observed in the majority of vermis GCs. This developmental pattern, revealed by electrophysiological recordings, was confirmed by immunohistological experiments that showed greater reactivity for GIRK2 in the PMZ of the vermis than in the hemispheres during development (P7-P15). These findings suggest that regional differences in development are responsible for the differential expression of inwardly rectifying potassium channels in the vermis and in the hemispheres.
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http://dx.doi.org/10.1111/ejn.13219DOI Listing
June 2016

Membrane palmitoylated protein 2 is a synaptic scaffold protein required for synaptic SK2-containing channel function.

Elife 2016 Feb 12;5. Epub 2016 Feb 12.

Vollum Institute, Oregon Health and Science University, Portland, United States.

Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca(2+) influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.
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http://dx.doi.org/10.7554/eLife.12637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764564PMC
February 2016