Tabla espec. clave
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, R, M, Fe||IHC, WB||Rb||Purified||Polyclonal Antibody|
|Presentation||Purified rabbit polyclonal in buffer containing PBS-BSA containing 0.1% sodium azide.|
|Safety Information according to GHS|
|Material Size||50 µL|
Ficha datos de seguridad (MSDS)
Referencias bibliográficas | 71 Disponible | Ver todas las referencias
|Visión general referencias||Aplicación||Especie||Pub Med ID|
|Neurovascular crosstalk between interneurons and capillaries is required for vision. |
Usui, Y; Westenskow, PD; Kurihara, T; Aguilar, E; Sakimoto, S; Paris, LP; Wittgrove, C; Feitelberg, D; Friedlander, MS; Moreno, SK; Dorrell, MI; Friedlander, M
The Journal of clinical investigation 125 2335-46 2015
Functional interactions between neurons, vasculature, and glia within neurovascular units are critical for maintenance of the retina and other CNS tissues. For example, the architecture of the neurosensory retina is a highly organized structure with alternating layers of neurons and blood vessels that match the metabolic demand of neuronal activity with an appropriate supply of oxygen within perfused blood. Here, using murine genetic models and cell ablation strategies, we have demonstrated that a subset of retinal interneurons, the amacrine and horizontal cells, form neurovascular units with capillaries in 2 of the 3 retinal vascular plexuses. Moreover, we determined that these cells are required for generating and maintaining the intraretinal vasculature through precise regulation of hypoxia-inducible and proangiogenic factors, and that amacrine and horizontal cell dysfunction induces alterations to the intraretinal vasculature and substantial visual deficits. These findings demonstrate that specific retinal interneurons and the intraretinal vasculature are highly interdependent, and loss of either or both elicits profound effects on photoreceptor survival and function.
|Genome-Wide Definition of Promoter and Enhancer Usage during Neural Induction of Human Embryonic Stem Cells. |
Poletti, V; Delli Carri, A; Malagoli Tagliazucchi, G; Faedo, A; Petiti, L; Mazza, EM; Peano, C; De Bellis, G; Bicciato, S; Miccio, A; Cattaneo, E; Mavilio, F
PloS one 10 e0126590 2015
Genome-wide mapping of transcriptional regulatory elements is an essential tool for understanding the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of transcription start sites with genome-wide profiling of histones modifications to map active promoters and enhancers in embryonic stem cells (ESCs) induced to neuroepithelial-like stem cells (NESCs). Our analysis showed that most promoters are active in both cell types while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a "bivalent" histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provides a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and of gene expression programs characterizing the transition from a pluripotent to a neural-restricted cell fate.
|Repeated neonatal propofol administration induces sex-dependent long-term impairments on spatial and recognition memory in rats. |
Gonzales, EL; Yang, SM; Choi, CS; Mabunga, DF; Kim, HJ; Cheong, JH; Ryu, JH; Koo, BN; Shin, CY
Biomolecules & therapeutics 23 251-60 2015
Propofol is an anesthetic agent that gained wide use because of its fast induction of anesthesia and rapid recovery post-anesthesia. However, previous studies have reported immediate neurodegeneration and long-term impairment in spatial learning and memory from repeated neonatal propofol administration in animals. Yet, none of those studies has explored the sex-specific long-term physical changes and behavioral alterations such as social (sociability and social preference), emotional (anxiety), and other cognitive functions (spatial working, recognition, and avoidance memory) after neonatal propofol treatment. Seven-day-old Wistar-Kyoto (WKY) rats underwent repeated daily intraperitoneal injections of propofol or normal saline for 7 days. Starting fourth week of age and onwards, rats were subjected to behavior tests including open-field, elevated-plus-maze, Y-maze, 3-chamber social interaction, novel-object-recognition, passive-avoidance, and rotarod. Rats were sacrificed at 9 weeks and hippocampal protein expressions were analyzed by Western blot. Results revealed long-term body weight gain alterations in the growing rats and sex-specific impairments in spatial (female) and recognition (male) learning and memory paradigms. A markedly decreased expression of hippocampal NMDA receptor GluN1 subunit in female- and increased expression of AMPA GluR1 subunit protein expression in male rats were also found. Other aspects of behaviors such as locomotor activity and coordination, anxiety, sociability, social preference and avoidance learning and memory were not generally affected. These results suggest that neonatal repeated propofol administration disrupts normal growth and some aspects of neurodevelopment in rats in a sex-specific manner.
|Transmitter inputs to different motoneuron subgroups in the oculomotor and trochlear nucleus in monkey. |
Zeeh, C; Mustari, MJ; Hess, BJ; Horn, AK
Frontiers in neuroanatomy 9 95 2015
In all vertebrates the eyes are moved by six pairs of extraocular muscles enabling horizontal, vertical and rotatory movements. Recent work showed that each extraocular muscle is controlled by two motoneuronal groups: (1) Motoneurons of singly-innervated muscle fibers (SIF) that lie within the boundaries of motonuclei mediating a fast muscle contraction; and (2) motoneurons of multiply-innervated muscle fibers (MIF) in the periphery of motonuclei mediating a tonic muscle contraction. Currently only limited data about the transmitter inputs to the SIF and MIF motoneurons are available. Here we performed a quantitative study on the transmitter inputs to SIF and MIF motoneurons of individual muscles in the oculomotor and trochlear nucleus in monkey. Pre-labeled motoneurons were immunostained for GABA, glutamate decarboxylase, GABA-A receptor, glycine transporter 2, glycine receptor 1, and vesicular glutamate transporters 1 and 2. The main findings were: (1) the inhibitory control of SIF motoneurons for horizontal and vertical eye movements differs. Unlike in previous primate studies a considerable GABAergic input was found to all SIF motoneuronal groups, whereas a glycinergic input was confined to motoneurons of the medial rectus (MR) muscle mediating horizontal eye movements and to those of the levator palpebrae (LP) muscle elevating the upper eyelid. Whereas SIF and MIF motoneurons of individual eye muscles do not differ numerically in their GABAergic, glycinergic and vGlut2 input, vGlut1 containing terminals densely covered the supraoculomotor area (SOA) targeting MR MIF motoneurons. It is reasonable to assume that the vGlut1 input affects the near response system in the SOA, which houses the preganglionic neurons mediating pupillary constriction and accommodation and the MR MIF motoneurones involved in vergence.
|Human Induced Pluripotent Stem Cell Derived Neuronal Cells Cultured on Chemically-Defined Hydrogels for Sensitive In Vitro Detection of Botulinum Neurotoxin. |
Pellett, S; Schwartz, MP; Tepp, WH; Josephson, R; Scherf, JM; Pier, CL; Thomson, JA; Murphy, WL; Johnson, EA
Scientific reports 5 14566 2015
Botulinum neurotoxin (BoNT) detection provides a useful model for validating cell-based neurotoxicity screening approaches, as sensitivity is dependent on functionally competent neurons and clear quantitative endpoints are available for correlating results to approved animal testing protocols. Here, human induced pluripotent stem cell (iPSC)-derived neuronal cells were cultured on chemically-defined poly(ethylene glycol) (PEG) hydrogels formed by "thiol-ene" photopolymerization and tested as a cell-based neurotoxicity assay by determining sensitivity to active BoNT/A1. BoNT/A1 sensitivity was comparable to the approved in vivo mouse bioassay for human iPSC-derived neurons and neural stem cells (iPSC-NSCs) cultured on PEG hydrogels or treated tissue culture polystyrene (TCP) surfaces. However, maximum sensitivity for BoNT detection was achieved two weeks earlier for iPSC-NSCs that were differentiated and matured on PEG hydrogels compared to TCP. Therefore, chemically-defined synthetic hydrogels offer benefits over standard platforms when optimizing culture conditions for cell-based screening and achieve sensitivities comparable to an approved animal testing protocol.
|microRNA-125 distinguishes developmentally generated and adult-born olfactory bulb interneurons. |
Akerblom, M; Petri, R; Sachdeva, R; Klussendorf, T; Mattsson, B; Gentner, B; Jakobsson, J
Development (Cambridge, England) 141 1580-8 2014
New neurons, originating from the subventricular zone, are continuously integrating into neuronal circuitry in the olfactory bulb (OB). Using a transgenic sensor mouse, we found that adult-born OB interneurons express microRNA-125 (miR-125), whereas the pre-existing developmentally generated OB interneurons represent a unique population of cells in the adult brain, without miR-125 activity. Stable inhibition of miR-125 in newborn OB neurons resulted in enhanced dendritic morphogenesis, as well as in increased synaptic activation in response to odour sensory stimuli. These data demonstrate that miR-125 controls functional synaptic integration of adult-born OB interneurons. Our results also suggest that absence of an otherwise broadly expressed miRNA is a novel mechanism with which to achieve neuronal subtype specification.
|CCL2-ethanol interactions and hippocampal synaptic protein expression in a transgenic mouse model. |
Gruol, DL; Vo, K; Bray, JG; Roberts, AJ
Frontiers in integrative neuroscience 8 29 2014
Chronic exposure to ethanol produces a number of detrimental effects on behavior. Neuroadaptive changes in brain structure or function underlie these behavioral effects and may be transient or persistent in nature. Central to the functional changes are alterations in the biology of neuronal and glial cells of the brain. Recent data show that ethanol induces glial cells of the brain to produce elevated levels of neuroimmune factors including CCL2, a key innate immune chemokine. Depending on the conditions of ethanol exposure, the upregulated levels of CCL2 can be transient or persistent and outlast the period of ethanol exposure. Importantly, results indicate that the upregulated levels of CCL2 may lead to CCL2-ethanol interactions that mediate or regulate the effects of ethanol on the brain. Glial cells are in close association with neurons and regulate many neuronal functions. Therefore, effects of ethanol on glial cells may underlie some of the effects of ethanol on neurons. To investigate this possibility, we are studying effects of chronic ethanol on hippocampal synaptic function in a transgenic mouse model that expresses elevated levels of CCL2 in the brain through enhanced glial expression, a situation know to occur in alcoholics. Both CCL2 and ethanol have been reported to alter synaptic function in the hippocampus. In the current study, we determined if interactions are evident between CCL2 and ethanol at the level of hippocampal synaptic proteins. Two ethanol exposure paradigms were used; the first involved ethanol exposure by drinking and the second involved ethanol exposure in a paradigm that combines drinking plus ethanol vapor. The first paradigm does not produce dependence on ethanol, whereas the second paradigm is commonly used to produce ethanol dependence. Results show modest effects of both ethanol exposure paradigms on the level of synaptic proteins in the hippocampus of CCL2 transgenic mice compared with their non-transgenic littermate controls, consistent with ethanol-CCL2 interactions. No evidence of toxic effects of CCL2 or CCL2-ethanol interactions was observed. Taken together, these results support the idea that ethanol induced astrocyte production of CCL2 can result in neuroadaptive changes that interact with the actions of ethanol.
|In utero exposure to valproic acid changes sleep in juvenile rats: a model for sleep disturbances in autism. |
Cusmano, DM; Mong, JA
Sleep 37 1489-99 2014
To determine whether sleep disturbances are found in the valproic acid model of autism spectrum disorders (ASD).Comparative study for sleep behavior, sleep architecture, electroencephalogram (EEG) spectral analysis, and glutamic acid decarboxylase (GAD) 65/67 protein expression in juvenile rats exposed to valproic acid (VPA), sodium salt, or saline in utero.N/A.Juvenile (postnatal day 32) male and female Sprague-Dawley rats.In utero exposure to either saline or 400 mg/kg VPA administered intraperitoneally to the dams on gestational day 12.5. On postnatal days 22-24, all rats were implanted with transmitters to record EEG and electromyogram (EMG) activity.During the light phase, when nocturnal animals are typically quiescent, the VPA-exposed animals spent significantly more time in wake (∼35 min) and significantly less time in non-rapid eye movement (NREM) sleep (∼26 min) compared to the saline controls. Furthermore, spectral analysis of the EEG revelled that VPA-exposed animals exhibited increased high-frequency activity during wake and rapid eye movement (REM) sleep and reduced theta power across all vigilance states. Interestingly, the gamma-aminobutyric acid (GABA)-ergic system, which modulates the induction and maintenance of sleep states, was also disrupted, with reduced levels of both GAD 65 and GAD67 in the cortical tissue of VPA-exposed animals compared to saline controls.To date, the current animal models of ASD have been underutilized in the investigation of associated sleep disturbances. The VPA animal model recapitulates aspects of sleep disruptions reported clinically, providing a tool to investigate cellular and molecular dysregulation contributing to sleep disruptions in ASD.
|Increased astrocyte expression of IL-6 or CCL2 in transgenic mice alters levels of hippocampal and cerebellar proteins. |
Gruol, DL; Vo, K; Bray, JG
Frontiers in cellular neuroscience 8 234 2014
Emerging research has identified that neuroimmune factors are produced by cells of the central nervous system (CNS) and play critical roles as regulators of CNS function, directors of neurodevelopment and responders to pathological processes. A wide range of neuroimmune factors are produced by CNS cells, primarily the glial cells, but the role of specific neuroimmune factors and their glial cell sources in CNS biology and pathology have yet to be fully elucidated. We have used transgenic mice that express elevated levels of a specific neuroimmune factor, the cytokine IL-6 or the chemokine CCL2, through genetic modification of astrocyte expression to identify targets of astrocyte produced IL-6 or CCL2 at the protein level. We found that in non-transgenic mice constitutive expression of IL-6 and CCL2 occurs in the two CNS regions studied, the hippocampus and cerebellum, as measured by ELISA. In the CCL2 transgenic mice elevated levels of CCL2 were evident in the hippocampus and cerebellum, whereas in the IL-6 transgenic mice, elevated levels of IL-6 were only evident in the cerebellum. Western blot analysis of the cellular and synaptic proteins in the hippocampus and cerebellum of the transgenic mice showed that the elevated levels of CCL2 or IL-6 resulted in alterations in the levels of specific proteins and that these actions differed for the two neuroimmune factors and for the two brain regions. These results are consistent with cell specific profiles of action for IL-6 and CCL2, actions that may be an important aspect of their respective roles in CNS physiology and pathophysiology.
|Leptin potentiates GABAergic synaptic transmission in the developing rodent hippocampus. |
Guimond, D; Diabira, D; Porcher, C; Bader, F; Ferrand, N; Zhu, M; Appleyard, SM; Wayman, GA; Gaiarsa, JL
Frontiers in cellular neuroscience 8 235 2014
It is becoming increasingly clear that leptin is not only a hormone regulating energy homeostasis but also a neurotrophic factor impacting a number of brain regions, including the hippocampus. Although leptin promotes the development of GABAergic transmission in the hypothalamus, little is known about its action on the GABAergic system in the hippocampus. Here we show that leptin modulates GABAergic transmission onto developing CA3 pyramidal cells of newborn rats. Specifically, leptin induces a long-lasting potentiation (LLP-GABAA) of miniature GABAA receptor-mediated postsynaptic current (GABAA-PSC) frequency. Leptin also increases the amplitude of evoked GABAA-PSCs in a subset of neurons along with a decrease in the coefficient of variation and no change in the paired-pulse ratio, pointing to an increased recruitment of functional synapses. Adding pharmacological blockers to the recording pipette showed that the leptin-induced LLP-GABAA requires postsynaptic calcium released from internal stores, as well as postsynaptic MAPK/ERK kinases 1 and/or 2 (MEK1/2), phosphoinositide 3 kinase (PI3K) and calcium-calmodulin kinase kinase (CaMKK). Finally, study of CA3 pyramidal cells in leptin-deficient ob/ob mice revealed a reduction in the basal frequency of miniature GABAA-PSCs compared to wild type littermates. In addition, presynaptic GAD65 immunostaining was reduced in the CA3 stratum pyramidale of mutant animals, both results converging to suggest a decreased number of functional GABAergic synapses in ob/ob mice. Overall, these results show that leptin potentiates and promotes the development of GABAergic synaptic transmission in the developing hippocampus likely via an increase in the number of functional synapses, and provide insights into the intracellular pathways mediating this effect. This study further extends the scope of leptin's neurotrophic action to a key regulator of hippocampal development and function, namely GABAergic transmission.
|Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research. |
Ray, B; Chopra, N; Long, JM; Lahiri, DK
Molecular brain 7 63 2014
Culturing primary cortical neurons is an essential neuroscience technique. However, most cultures are derived from rodent brains and standard protocols for human brain cultures are sparse. Herein, we describe preparation, maintenance and major characteristics of a primary human mixed brain culture, including neurons, obtained from legally aborted fetal brain tissue. This approach employs standard materials and techniques used in the preparation of rodent neuron cultures, with critical modifications.This culture has distinct differences from rodent cultures. Specifically, a significant numbers of cells in the human culture are derived from progenitor cells, and the yield and survival of the cells grossly depend on the presence of bFGF. In the presence of bFGF, this culture can be maintained for an extended period. Abundant productions of amyloid-β, tau and proteins make this a powerful model for Alzheimer's research. The culture also produces glia and different sub-types of neurons.We provide a well-characterized methodology for human mixed brain cultures useful to test therapeutic agents under various conditions, and to carry forward mechanistic and translational studies for several brain disorders.
|Vascular endothelial growth factor increases during blood-brain barrier-enhanced permeability caused by Phoneutria nigriventer spider venom. |
Mendonça, MC; Soares, ES; Stávale, LM; Kalapothakis, E; Cruz-Höfling, MA
BioMed research international 2014 721968 2014
Phoneutria nigriventer spider accidental envenomation provokes neurotoxic manifestations, which when critical, results in epileptic-like episodes. In rats, P. nigriventer venom (PNV) causes blood-brain barrier breakdown (BBBb). The PNV-induced excitotoxicity results from disturbances on Na(+), K(+) and Ca(2+) channels and glutamate handling. The vascular endothelial growth factor (VEGF), beyond its angiogenic effect, also, interferes on synaptic physiology by affecting the same ion channels and protects neurons from excitotoxicity. However, it is unknown whether VEGF expression is altered following PNV envenomation. We found that adult and neonates rats injected with PNV showed immediate neurotoxic manifestations which paralleled with endothelial occludin, β-catenin, and laminin downregulation indicative of BBBb. In neonate rats, VEGF, VEGF mRNA, and Flt-1 receptors, glutamate decarboxylase, and calbindin-D28k increased in Purkinje neurons, while, in adult rats, the BBBb paralleled with VEGF mRNA, Flk-1, and calbindin-D28k increases and Flt-1 decreases. Statistically, the variable age had a role in such differences, which might be due to age-related unequal maturation of blood-brain barrier (BBB) and thus differential cross-signaling among components of the glial neurovascular unit. The concurrent increases in the VEGF/Flt-1/Flk-1 system in the cerebellar neuron cells and the BBBb following PNV exposure might imply a cytokine modulation of neuronal excitability consequent to homeostatic perturbations induced by ion channels-acting PNV neuropeptides. Whether such modulation represents neuroprotection needs further investigation.
|Ischemia induces different levels of hypoxia inducible factor-1α protein expression in interneurons and pyramidal neurons. |
Ramamoorthy, P; Shi, H
Acta neuropathologica communications 2 51 2014
Pyramidal (glutamatergic) neurons and interneurons are morphologically and functionally well defined in the central nervous system. Although it is known that glutamatergic neurons undergo immediate cell death whereas interneurons are insensitive or survive longer during cerebral ischemia, the protection mechanisms responsible for this interneuronal survival are not well understood. Hypoxia inducible factor-1 (HIF-1) plays an important role in protecting neurons from hypoxic/ischemic insults. Here, we studied the expression of HIF-1α, the regulatable subunit of HIF-1, in the different neuronal phenotypes under in vitro and in vivo ischemia.In a primary cortical culture, HIF-1α expression was observed in neuronal somata after hypoxia (1% oxygen) in the presence of 5 or 25 mM glucose but not under normoxia (21% oxygen). Interestingly, only certain MAP2-positive neurons containing round somata (interneuron-like morphology) co-localized with HIF-1α staining. Other neurons such as pyramidal-like neurons showed no expression of HIF-1α under either normoxia or hypoxia. The HIF-1α positive neurons were GAD65/67 positive, confirming that they were interneuron-type cells. The HIF-1α expressing GAD65/67-positive neurons also possessed high levels of glutathione. We further demonstrated that ischemia induced significant HIF-1α expression in interneurons but not in pyramidal neurons in a rat model of middle cerebral artery occlusion.These results suggest that HIF-1α protein expression induced by ischemia is neuron-type specific and that this specificity may be related to the intracellular level of glutathione (GSH).
|Cross-modal plasticity results in increased inhibition in primary auditory cortical areas. |
Mao, YT; Pallas, SL
Neural plasticity 2013 530651 2013
Loss of sensory input from peripheral organ damage, sensory deprivation, or brain damage can result in adaptive or maladaptive changes in sensory cortex. In previous research, we found that auditory cortical tuning and tonotopy were impaired by cross-modal invasion of visual inputs. Sensory deprivation is typically associated with a loss of inhibition. To determine whether inhibitory plasticity is responsible for this process, we measured pre- and postsynaptic changes in inhibitory connectivity in ferret auditory cortex (AC) after cross-modal plasticity. We found that blocking GABAA receptors increased responsiveness and broadened sound frequency tuning in the cross-modal group more than in the normal group. Furthermore, expression levels of glutamic acid decarboxylase (GAD) protein were increased in the cross-modal group. We also found that blocking inhibition unmasked visual responses of some auditory neurons in cross-modal AC. Overall, our data suggest a role for increased inhibition in reducing the effectiveness of the abnormal visual inputs and argue that decreased inhibition is not responsible for compromised auditory cortical function after cross-modal invasion. Our findings imply that inhibitory plasticity may play a role in reorganizing sensory cortex after cross-modal invasion, suggesting clinical strategies for recovery after brain injury or sensory deprivation.
|Endothelial VEGF sculpts cortical cytoarchitecture. |
Li, S; Haigh, K; Haigh, JJ; Vasudevan, A
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 14809-15 2013
Current models of brain development support the view that VEGF, a signaling protein secreted by neuronal cells, regulates angiogenesis and neuronal development. Here we demonstrate an autonomous and pivotal role for endothelial cell-derived VEGF that has far-reaching consequences for mouse brain development. Selective deletion of Vegf from endothelial cells resulted in impaired angiogenesis and marked perturbation of cortical cytoarchitecture. Abnormal cell clusters or heterotopias were detected in the marginal zone, and disorganization of cortical cells induced several malformations, including aberrant cortical lamination. Critical events during brain development-neuronal proliferation, differentiation, and migration were significantly affected. In addition, axonal tracts in the telencephalon were severely defective in the absence of endothelial VEGF. The unique roles of endothelial VEGF cannot be compensated by neuronal VEGF and underscores the high functional significance of endothelial VEGF for cerebral cortex development and from disease perspectives.
|Adolescent female C57BL/6 mice with vulnerability to activity-based anorexia exhibit weak inhibitory input onto hippocampal CA1 pyramidal cells. |
Chowdhury, TG; Wable, GS; Sabaliauskas, NA; Aoki, C
Neuroscience 241 250-67 2013
Anorexia nervosa (AN) is an eating disorder characterized by self-imposed severe starvation and often linked with excessive exercise. Activity-based anorexia (ABA) is an animal model that reproduces some of the behavioral phenotypes of AN, including the paradoxical increase in voluntary exercise following food restriction (FR). Although certain rodents have been used successfully in this animal model, C57BL/6 mice are reported to be less susceptible to ABA. We re-examined the possibility that female C57BL/6 mice might exhibit ABA vulnerability during adolescence, the developmental stage/sex among the human population with particularly high AN vulnerability. After introducing the running wheel to the cage for 3 days, ABA was induced by restricting food access to 1h per day (ABA1, N=13) or 2 h per day (ABA2, N=10). All 23 exhibited increased voluntary wheel running (pless than 0.005) and perturbed circadian rhythm within 2 days. Only one out of five survived ABA1 for 3 days, while 10 out of 10 survived ABA2 for 3 days and could subsequently restore their body weight and circadian rhythm. Exposure of recovered animals to a second ABA2 induction revealed a large range of vulnerability, even within littermates. To look for the cellular substrate of differences in vulnerability, we began by examining synaptic patterns in the hippocampus, a brain region that regulates anxiety as well as plasticity throughout life. Quantitative EM analysis revealed that CA1 pyramidal cells of animals vulnerable to the second ABA2 exhibit less GABAergic innervation on cell bodies and dendrites, relative to the animals resilient to the second ABA (pless than 0.001) or controls (pless than 0.05). These findings reveal that C57BL/6J adolescent females can be used to capture brain changes underlying ABA vulnerability, and that GABAergic innervation of hippocampal pyramidal neurons is one important cellular substrate to consider for understanding the progression of and resilience to AN.
|PMCA2 via PSD-95 controls calcium signaling by α7-containing nicotinic acetylcholine receptors on aspiny interneurons. |
Gómez-Varela, D; Schmidt, M; Schoellerman, J; Peters, EC; Berg, DK
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 6894-905 2012
Local control of calcium concentration within neurons is critical for signaling and regulation of synaptic communication in neural circuits. How local control can be achieved in the absence of physical compartmentalization is poorly understood. Challenging examples are provided by nicotinic acetylcholine receptors that contain α7 nicotinic receptor subunits (α7-nAChRs). These receptors are highly permeable to calcium and are concentrated on aspiny dendrites of interneurons, which lack obvious physical compartments for constraining calcium diffusion. Using functional proteomics on rat brain, we show that α7-nAChRs are associated with plasma membrane calcium-ATPase pump isoform 2 (PMCA2). Analysis of α7-nAChR function in hippocampal interneurons in culture shows that PMCA2 activity limits the duration of calcium elevations produced by the receptors. Unexpectedly, PMCA2 inhibition triggers rapid calcium-dependent loss of α7-nAChR clusters. This extreme regulatory response is mediated by CaMKII, involves proteasome activity, depends on the second intracellular loop of α7-nAChR subunits, and is specific in that it does not alter two other classes of calcium-permeable ionotropic receptors on the same neurons. A critical link is provided by the scaffold protein PSD-95 (postsynaptic density-95), which is associated with α7-nAChRs and constrains their mobility as revealed by single-particle tracking on neurons. The PSD-95 link is required for PMCA2-mediated removal of α7-nAChR clusters. This three-component combination of PMCA2, PSD-95, and α7-nAChR offers a novel mechanism for tight control of calcium dynamics in neurons.
|Altered synaptic transmission in the hippocampus of transgenic mice with enhanced central nervous systems expression of interleukin-6. |
Nelson, TE; Olde Engberink, A; Hernandez, R; Puro, A; Huitron-Resendiz, S; Hao, C; De Graan, PN; Gruol, DL
Brain, behavior, and immunity 26 959-71 2012
Elevated levels of the inflammatory cytokine interleukin-6 (IL-6) occur in a number of CNS disorders. However, little is known about how this condition affects CNS neuronal function. Transgenic mice that express elevated levels of IL-6 in the CNS show cognitive changes, increased propensity for hippocampal seizures and reduced number of inhibitory interneurons, suggesting that elevated levels of IL-6 can cause neuroadaptive changes that alter hippocampal function. To identify these neuroadaptive changes, we measured the levels of protein expression using Western blot analysis and synaptic function using field potential recordings in hippocampus from IL-6 transgenic mice (IL-6 tg) and their non-transgenic (non-tg) littermates. Western blot analysis showed enhanced levels of the GFAP and STAT3 in the IL-6 tg hippocampus compared with the non-tg hippocampus, but no difference for several other proteins. Field potential recordings of synaptic transmission at the Schaffer collateral to CA1 synapse showed enhanced dendritic excitatory postsynaptic potentials and somatic population spikes in the CA1 region of hippocampal slices from IL-6 tg mice compared with slices from non-tg littermate controls. No differences were observed for several forms of short-term and long-term synaptic plasticity between hippocampal slices from IL-6 tg and non-tg mice. These results demonstrate that elevated levels of IL-6 can alter mechanisms involved in the excitability of hippocampal neurons and synapses, an effect consistent with recent evidence indicating that elevated production of IL-6 plays an important role in conditions associated with seizure activity and in other impairments observed in CNS disorders with a neuroinflammatory component.
|Direction-selective retinal ganglion cells arise from molecularly specified multipotential progenitors. |
De la Huerta, I; Kim, IJ; Voinescu, PE; Sanes, JR
Proceedings of the National Academy of Sciences of the United States of America 109 17663-8 2012
Single progenitors can give rise to any and all of the main retinal cell types: photoreceptors, interneurons (horizontal, bipolar, and amacrine cells), retinal ganglion cells (RGCs), and glia. Many of these types are divisible into multiple functionally, structurally, and molecularly distinct subtypes (e.g., ~25 for RGCs). It remains unknown when and how progenitors become committed to generate such subtypes. Here, we determine the origin of RGCs that respond selectively to vertical motion and express cadherin 6 (cdh6). Using Cre recombinase-based lineage tracing, we show that these RGCs arise from progenitors that themselves express cdh6. These progenitors are capable of generating all major retinal cell types, but the RGCs they generate are predominantly of the single direction-selective subtype. In contrast, cdh6-positive progenitors retain the ability to generate multiple subtypes of amacrine and bipolar cells. Our results demonstrate that type and subtype specification are regulated in different ways and suggest that multipotential but fate-restricted progenitors contribute to subtype specification in retina.
|Diverse populations of intrinsic cholinergic interneurons in the mouse olfactory bulb. |
Krosnowski, K; Ashby, S; Sathyanesan, A; Luo, W; Ogura, T; Lin, W
Neuroscience 213 161-78 2012
Cholinergic activities affect olfactory bulb (OB) information processing and associated learning and memory. However, the presence of intrinsic cholinergic interneurons in the OB remains controversial. As a result, morphological and functional properties of these cells are largely undetermined. We characterized cholinergic interneurons using transgenic mice that selectively mark choline acetyltransferase (ChAT)-expressing cells and immunolabeling. We found a significant number of intrinsic cholinergic interneurons in the OB. These interneurons reside primarily in the glomerular layer (GL) and external plexiform layer (EPL) and exhibit diverse distribution patterns of nerve processes, indicating functional heterogeneity. Further, we found these neurons express ChAT and vesicular acetylcholine transporter (VAChT), but do not immunoreact to glutamatergic, GABAergic or dopaminergic markers and are distinct from calretinin-expressing interneurons. Interestingly, the cholinergic population partially overlaps with the calbindin D28K-expressing interneuron population, revealing the neurotransmitter identity of this sub-population. Additionally, we quantitatively determined the density of VAChT labeled cholinergic nerve fibers in various layers of the OB, as well as the intensity of VAChT immunoreactivity within the GL, suggesting primary sites of cholinergic actions. Taken together, our results provide clear evidence showing the presence of a significant number of cholinergic interneurons and that these morphologically and distributionally diverse interneurons make up complex local cholinergic networks in the OB. Thus, our results suggest that olfactory information processing is modulated by dual cholinergic systems of local interneuron networks and centrifugal projections.
|BACE1 elevation is involved in amyloid plaque development in the triple transgenic model of Alzheimer's disease: differential Aβ antibody labeling of early-onset axon terminal pathology. |
Cai, Y; Zhang, XM; Macklin, LN; Cai, H; Luo, XG; Oddo, S; Laferla, FM; Struble, RG; Rose, GM; Patrylo, PR; Yan, XX
Neurotoxicity research 21 160-74 2012
β-amyloid precursor protein (APP) and presenilins mutations cause early-onset familial Alzheimer's disease (FAD). Some FAD-based mouse models produce amyloid plaques, others do not. β-Amyloid (Aβ) deposition can manifest as compact and diffuse plaques; it is unclear why the same Aβ molecules aggregate in different patterns. Is there a basic cellular process governing Aβ plaque pathogenesis? We showed in some FAD mouse models that compact plaque formation is associated with a progressive axonal pathology inherent with increased expression of β-secretase (BACE1), the enzyme initiating the amyloidogenic processing of APP. A monoclonal Aβ antibody, 3D6, visualized distinct axon terminal labeling before plaque onset. The present study was set to understand BACE1 and axonal changes relative to diffuse plaque development and to further characterize the novel axonal Aβ antibody immunoreactivity (IR), using triple transgenic AD (3xTg-AD) mice as experimental model. Diffuse-like plaques existed in the forebrain in aged transgenics and were regionally associated with increased BACE1 labeled swollen/sprouting axon terminals. Increased BACE1/3D6 IR at axon terminals occurred in young animals before plaque onset. These axonal elements were also co-labeled by other antibodies targeting the N-terminal and mid-region of Aβ domain and the C-terminal of APP, but not co-labeled by antibodies against the Aβ C-terminal and APP N-terminal. The results suggest that amyloidogenic axonal pathology precedes diffuse plaque formation in the 3xTg-AD mice, and that the early-onset axonal Aβ antibody IR in transgenic models of AD might relate to a cross-reactivity of putative APP β-carboxyl terminal fragments.
|Growth arrest and DNA-damage-inducible, beta (GADD45b)-mediated DNA demethylation in major psychosis. |
Gavin, DP; Sharma, RP; Chase, KA; Matrisciano, F; Dong, E; Guidotti, A
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 37 531-42 2012
Aberrant neocortical DNA methylation has been suggested to be a pathophysiological contributor to psychotic disorders. Recently, a growth arrest and DNA-damage-inducible, beta (GADD45b) protein-coordinated DNA demethylation pathway, utilizing cytidine deaminases and thymidine glycosylases, has been identified in the brain. We measured expression of several members of this pathway in parietal cortical samples from the Stanley Foundation Neuropathology Consortium (SFNC) cohort. We find an increase in GADD45b mRNA and protein in patients with psychosis. In immunohistochemistry experiments using samples from the Harvard Brain Tissue Resource Center, we report an increased number of GADD45b-stained cells in prefrontal cortical layers II, III, and V in psychotic patients. Brain-derived neurotrophic factor IX (BDNF IXabcd) was selected as a readout gene to determine the effects of GADD45b expression and promoter binding. We find that there is less GADD45b binding to the BDNF IXabcd promoter in psychotic subjects. Further, there is reduced BDNF IXabcd mRNA expression, and an increase in 5-methylcytosine and 5-hydroxymethylcytosine at its promoter. On the basis of these results, we conclude that GADD45b may be increased in psychosis compensatory to its inability to access gene promoter regions.
|High LRRK2 levels fail to induce or exacerbate neuronal alpha-synucleinopathy in mouse brain. |
Herzig, MC; Bidinosti, M; Schweizer, T; Hafner, T; Stemmelen, C; Weiss, A; Danner, S; Vidotto, N; Stauffer, D; Barske, C; Mayer, F; Schmid, P; Rovelli, G; van der Putten, PH; Shimshek, DR
PloS one 7 e36581 2012
The G2019S mutation in the multidomain protein leucine-rich repeat kinase 2 (LRRK2) is one of the most frequently identified genetic causes of Parkinson's disease (PD). Clinically, LRRK2(G2019S) carriers with PD and idiopathic PD patients have a very similar disease with brainstem and cortical Lewy pathology (α-synucleinopathy) as histopathological hallmarks. Some patients have Tau pathology. Enhanced kinase function of the LRRK2(G2019S) mutant protein is a prime suspect mechanism for carriers to develop PD but observations in LRRK2 knock-out, G2019S knock-in and kinase-dead mutant mice suggest that LRRK2 steady-state abundance of the protein also plays a determining role. One critical question concerning the molecular pathogenesis in LRRK2(G2019S) PD patients is whether α-synuclein (aSN) has a contributory role. To this end we generated mice with high expression of either wildtype or G2019S mutant LRRK2 in brainstem and cortical neurons. High levels of these LRRK2 variants left endogenous aSN and Tau levels unaltered and did not exacerbate or otherwise modify α-synucleinopathy in mice that co-expressed high levels of LRRK2 and aSN in brain neurons. On the contrary, in some lines high LRRK2 levels improved motor skills in the presence and absence of aSN-transgene-induced disease. Therefore, in many neurons high LRRK2 levels are well tolerated and not sufficient to drive or exacerbate neuronal α-synucleinopathy.
|Immunocytochemical evidence of the localization of the Crumbs homologue 3 protein (CRB3) in the developing and mature mouse retina. |
Herranz-Martín, S; Jimeno, D; Paniagua, AE; Velasco, A; Lara, JM; Aijón, J; Lillo, C
PloS one 7 e50511 2012
CRB3 (Crumbs homologue 3), a member of the CRB protein family (homologous to the Drosophila Crumbs), is expressed in different epithelium-derived cell types in mammals, where it seems to be involved in regulating the establishment and stability of tight junctions and in ciliogenesis. This protein has been also detected in the retina, but little is known about its localization and function in this tissue. Our goal here was to perform an in-depth study of the presence of CRB3 protein in the mouse retina and to analyze its expression during photoreceptor ciliogenesis and the establishment of the plexiform retinal layers. Double immunofluorescence experiments for CRB3 and well-known markers for the different retinal cell types were performed to study the localization of the CRB3 protein. According to our results, CRB3 is present from postnatal day 0 (P0) until adulthood in the mouse retina. It is localized in the inner segments (IS) of photoreceptor cells, especially concentrated in the area where the connecting cilium is located, in their synaptic terminals in the outer plexiform layer (OPL), and in sub-populations of amacrine and bipolar cells in the inner plexiform layer (IPL).
|Methamphetamine-evoked depression of GABA(B) receptor signaling in GABA neurons of the VTA. |
Padgett, CL; Lalive, AL; Tan, KR; Terunuma, M; Munoz, MB; Pangalos, MN; Martínez-Hernández, J; Watanabe, M; Moss, SJ; Luján, R; Lüscher, C; Slesinger, PA
Neuron 73 978-89 2012
Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.
|Functional nicotinic acetylcholine receptors containing α6 subunits are on GABAergic neuronal boutons adherent to ventral tegmental area dopamine neurons. |
Yang, K; Buhlman, L; Khan, GM; Nichols, RA; Jin, G; McIntosh, JM; Whiteaker, P; Lukas, RJ; Wu, J
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 2537-48 2011
Diverse nicotinic acetylcholine receptor (nAChR) subtypes containing different subunit combinations can be placed on nerve terminals or soma/dendrites in the ventral tegmental area (VTA). nAChR α6 subunit message is abundant in the VTA, but α6*-nAChR cellular localization, function, pharmacology, and roles in cholinergic modulation of dopaminergic (DA) neurons within the VTA are not well understood. Here, we report evidence for α6β2*-nAChR expression on GABA neuronal boutons terminating on VTA DA neurons. α-Conotoxin (α-Ctx) MII labeling coupled with immunocytochemical staining localizes putative α6*-nAChRs to presynaptic GABAergic boutons on acutely dissociated, rat VTA DA neurons. Functionally, acetylcholine (ACh) induces increases in the frequency of bicuculline-, picrotoxin-, and 4-aminopyridine-sensitive miniature IPSCs (mIPSCs) mediated by GABA(A) receptors. These increases are abolished by α6*-nAChR-selective α-Ctx MII or α-Ctx PIA (1 nm) but not by α7 (10 nm methyllycaconitine) or α4* (1 μm dihydro-β-erythroidine)-nAChR-selective antagonists. ACh also fails to increase mIPSC frequency in VTA DA neurons prepared from nAChR β2 knock-out mice. Moreover, ACh induces an α-Ctx PIA-sensitive elevation in intraterminal Ca(2+) in synaptosomes prepared from the rat VTA. Subchronic exposure to 500 nm nicotine reduces ACh-induced GABA release onto the VTA DA neurons, as does 10 d of systemic nicotine exposure. Collectively, these results indicate that α6β2*-nAChRs are located on presynaptic GABAergic boutons within the VTA and modulate GABA release onto DA neurons. These presynaptic α6β2*-nAChRs likely play important roles in nicotinic modulation of DA neuronal activity.
|Neuroadaptive changes in cerebellar neurons induced by chronic exposure to IL-6. |
Gruol, DL; Puro, A; Hao, C; Blakely, P; Janneke, E; Vo, K
Journal of neuroimmunology 239 28-36 2011
IL-6 is an important signaling molecule in the CNS. CNS neurons express IL-6 receptors and their signal transduction molecules, consistent with a role for IL-6 in neuronal physiology. Research indicates that IL-6 levels are low in the normal brain but can be significantly elevated in CNS injury and disease. Relatively little is known about how the elevated levels of IL-6 affect neurons. In the current study we show that under conditions of chronic exposure, IL-6 induces alterations in the level of protein expression in developing CNS cells. Such changes may play a role in the altered CNS function observed in CNS conditions associated with elevated levels of IL-6 in the CNS.
|Microglial cells contribute to endogenous brain defenses after acute neonatal focal stroke. |
Faustino JV, Wang X, Johnson CE, Klibanov A, Derugin N, Wendland MF, Vexler ZS
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 12992-3001. 2011
Macrophages are viewed as amplifiers of ischemic brain injury, but the origin of injury-producing macrophages is poorly defined. The role of resident brain macrophages-microglial cells-in stroke remains controversial. To determine whether microglial cells exert injurious effects after neonatal focal stroke, we selectively depleted these cells with intracerebral injection of liposome-encapsulated clodronate before transient middle cerebral artery occlusion in postnatal day 7 rats. Phagocytosis of apoptotic neurons by activated microglia was poor in animals with unmanipulated microglia, and depletion of these cells did not increase the number of apoptotic neurons. Lack of microglia increased the brain levels of several cytokines and chemokines already elevated by ischemia-reperfusion, and also increased the severity and volume of injury, suggesting that microglial cells contribute to endogenous protection during the subacute injury phase. Then, to determine whether accumulation of reactive oxygen species in microglia adversely affects phagocytosis of dying neurons and contributes to injury, we delivered reduced glutathione (GSH) into microglia, again using liposomes. Remarkably, pharmacologically increased intracellular GSH concentrations in microglia induced superoxide accumulation in lipid rafts in these cells, further increased the brain levels of macrophage chemoattractants, and exacerbated injury. Together, these data show that microglia are part of the endogenous defense mechanisms and that, while antioxidants can protect the injured neonatal brain, high levels of reducing equivalents in activated microglia, GSH, trigger superoxide production, favor the reorganization of lipids, amplify local inflammation and exacerbate injury.
|Cigarette Smoke Induces DNA Damage and Alters Base-Excision Repair and Tau Levels in the Brain of Neonatal Mice. |
La Maestra S, Kisby GE, Micale RT, Johnson J, Kow YW, Bao G, Sheppard C, Stanfield S, Tran H, Woltjer RL, D\'Agostini F, Steele VE, De Flora S
Toxicological sciences : an official journal of the Society of Toxicology 2011
The prenatal and perinatal periods of brain development are especially vulnerable to insults by environmental agents. Early life exposure to cigarette smoke (CS), which contains both genotoxicants and oxidants, is considered an important risk factor for both neurodevelopmental and neurodegenerative disorders. Yet, little is known regarding the underlying pathogenetic mechanisms. In the present study, neonatal Swiss ICR (CD-1(®)) albino mice were exposed to various concentrations of CS for 4 weeks and the brain examined for lipid peroxides, DNA damage, base-excision repair (BER) enzymes, apoptosis, and levels of the microtubule protein tau. CS induced a dose-dependent increase in both malondialdehyde and various types of DNA damage, including single-strand breaks, double-strand breaks, and DNA-protein crosslinks. However, the CS-induced DNA damage in the brain returned to basal levels one week after smoking cessation. CS also modulated the activity and distribution of the BER enzymes 8-oxoguanine-DNA-glycosylase (OGG1) and apyrimidinic/apurinic endonuclease (APE1) in several brain regions. Normal tau (i.e., three-repeat tau, 3R tau) and various pathological forms of tau were also measured in the brain of CS exposed neonatal mice, but only 3R tau and tau phosphorylated at serine199 were significantly elevated. The oxidative stress, genomic dysregulation and alterations in tau metabolism caused by CS during a critical period of brain development could explain why CS is an important risk factor for both neurodevelopmental and neurodegenerative disorders appearing in later life.
|Neurochemical characterization of the tree shrew dorsal striatum. |
Rice, MW; Roberts, RC; Melendez-Ferro, M; Perez-Costas, E
Frontiers in neuroanatomy 5 53 2011
The striatum is a major component of the basal ganglia and is associated with motor and cognitive functions. Striatal pathologies have been linked to several disorders, including Huntington's, Tourette's syndrome, obsessive-compulsive disorders, and schizophrenia. For the study of these striatal pathologies different animal models have been used, including rodents and non-human primates. Rodents lack on morphological complexity (for example, the lack of well defined caudate and putamen nuclei), which makes it difficult to translate data to the human paradigm. Primates, and especially higher primates, are the closest model to humans, but there are ever-increasing restrictions to the use of these animals for research. In our search for a non-primate animal model with a striatum that anatomically (and perhaps functionally) can resemble that of humans, we turned our attention to the tree shrew. Evolutionary genetic studies have provided strong data supporting that the tree shrews (Scadentia) are one of the closest groups to primates, although their brain anatomy has only been studied in detail for specific brain areas. Morphologically, the tree shrew striatum resembles the primate striatum with the presence of an internal capsule separating the caudate and putamen, but little is known about its neurochemical composition. Here we analyzed the expression of calcium-binding proteins, the presence and distribution of the striosome and matrix compartments (by the use of calbindin, tyrosine hydroxylase, and acetylcholinesterase immunohistochemistry), and the GABAergic system by immunohistochemistry against glutamic acid decarboxylase and Golgi impregnation. In summary, our results show that when compared to primates, the tree shrew dorsal striatum presents striking similarities in the distribution of most of the markers studied, while presenting some marked divergences when compared to the rodent striatum.
|A Role for GAT-1 in Presynaptic GABA Homeostasis? |
Conti, F; Melone, M; Fattorini, G; Bragina, L; Ciappelloni, S
Frontiers in cellular neuroscience 5 2 2011
In monoamine-releasing terminals, neurotransmitter transporters - in addition to terminating synaptic transmission by clearing released transmitters from the extracellular space - are the primary mechanism for replenishing transmitter stores and thus regulate presynaptic homeostasis. Here, we analyze whether GAT-1, the main plasma membrane GABA transporter, plays a similar role in GABAergic terminals. Re-examination of existing literature and recent data gathered in our laboratory show that GABA homeostasis in GABAergic terminals is dominated by the activity of the GABA synthesizing enzyme and that GAT-1-mediated GABA transport contributes to cytosolic GABA levels. However, analysis of GAT-1 KO, besides demonstrating the effects of reduced clearance, reveals the existence of changes compatible with an impaired presynaptic function, as miniature IPSCs frequency is reduced by one-third and glutamic acid decarboxylases and phosphate-activated glutaminase levels are significantly up-regulated. Although the changes observed are less robust than those reported in mice with impaired dopamine, noradrenaline, and serotonin plasma membrane transporters, they suggest that in GABAergic terminals GAT-1 impacts on presynaptic GABA homeostasis, and may contribute to the activity-dependent regulation of inhibitory efficacy.
|Ultrastructural analysis of the synaptic connectivity of TRPV1-expressing primary afferent terminals in the rat trigeminal caudal nucleus. |
Eun Jin Yeo,Yi Sul Cho,Sang Kyoo Paik,Atsushi Yoshida,Mae Ja Park,Dong Kuk Ahn,Cheil Moon,Yun Sook Kim,Yong Chul Bae
The Journal of comparative neurology 518 2010
Trigeminal primary afferents that express the transient receptor potential vanilloid 1 (TRPV1) are important for the transmission of orofacial nociception. However, little is known about how the TRPV1-mediated nociceptive information is processed at the first relay nucleus in the central nervous system (CNS). To address this issue, we studied the synaptic connectivity of TRPV1-positive (+) terminals in the rat trigeminal caudal nucleus (Vc) by using electron microscopic immunohistochemistry and analysis of serial thin sections. Whereas the large majority of TRPV1+ terminals made synaptic contacts of an asymmetric type with one or two postsynaptic dendrites, a considerable fraction also participated in complex glomerular synaptic arrangements. A few TRPV1+ terminals received axoaxonic contacts from synaptic endings that contained pleomorphic synaptic vesicles and were immunolabeled for glutamic acid decarboxylase, the synthesizing enzyme for the inhibitory neurotransmitter γ-aminobutyric acid (GABA). We classified the TRPV1+ terminals into an S-type, containing less than five dense-core vesicles (DCVs), and a DCV-type, containing five or more DCVs. The number of postsynaptic dendrites was similar between the two types of terminals; however, whereas axoaxonic contacts were frequent on the S-type, the DCV-type did not receive axoaxonic contacts. In the sensory root of the trigeminal ganglion, TRPV1+ axons were mostly unmyelinated, and a small fraction was small myelinated. These results suggest that the TRPV1-mediated nociceptive information from the orofacial region is processed in a specific manner by two distinct types of synaptic arrangements in the Vc, and that the central input of a few TRPV1+ afferents is presynaptically modulated via a GABA-mediated mechanism.
|Collagen XIX is expressed by interneurons and contributes to the formation of hippocampal synapses. |
Su, J; Gorse, K; Ramirez, F; Fox, MA
The Journal of comparative neurology 518 229-53 2010
Extracellular matrix (ECM) molecules contribute to the formation and maintenance of synapses in the mammalian nervous system. We previously discovered a family of nonfibrillar collagens that organize synaptic differentiation at the neuromuscular junction (NMJ). Although many NMJ-organizing cues contribute to central nervous system (CNS) synaptogenesis, whether similar roles for collagens exist at central synapses remained unclear. In the present study we discovered that col19a1, the gene encoding nonfibrillar collagen XIX, is expressed by subsets of hippocampal neurons. Colocalization with the interneuron-specific enzyme glutamate decarboxylase 67 (Gad67), but not other cell-type-specific markers, suggests that hippocampal expression of col19a1 is restricted to interneurons. However, not all hippocampal interneurons express col19a1 mRNA; subsets of neuropeptide Y (NPY)-, somatostatin (Som)-, and calbindin (Calb)-immunoreactive interneurons express col19a1, but those containing parvalbumin (Parv) or calretinin (Calr) do not. To assess whether collagen XIX is required for the normal formation of hippocampal synapses, we examined synaptic morphology and composition in targeted mouse mutants lacking collagen XIX. We show here that subsets of synaptotagmin 2 (Syt2)-containing hippocampal nerve terminals appear malformed in the absence of collagen XIX. The presence of Syt2 in inhibitory hippocampal synapses, the altered distribution of Gad67 in collagen XIX-deficient subiculum, and abnormal levels of gephyrin in collagen XIX-deficient hippocampal extracts all suggest inhibitory synapses are affected by the loss of collagen XIX. Together, these data not only reveal that collagen XIX is expressed by central neurons, but show for the first time that a nonfibrillar collagen is necessary for the formation of hippocampal synapses.Artículo Texto completo
|β-Secretase-1 elevation in aged monkey and Alzheimer's disease human cerebral cortex occurs around the vasculature in partnership with multisystem axon terminal pathogenesis and β-amyloid accumulation. |
Cai, Y; Xiong, K; Zhang, XM; Cai, H; Luo, XG; Feng, JC; Clough, RW; Struble, RG; Patrylo, PR; Chu, Y; Kordower, JH; Yan, XX
The European journal of neuroscience 32 1223-38 2010
Alzheimer's disease (AD) is the most common dementia-causing disorder in the elderly; it may be related to multiple risk factors, and is characterized pathologically by cerebral hypometabolism, paravascular β-amyloid peptide (Aβ) plaques, neuritic dystrophy, and intra-neuronal aggregation of phosphorylated tau. To explore potential pathogenic links among some of these lesions, we examined β-secretase-1 (BACE1) alterations relative to Aβ deposition, neuritic pathology and vascular organization in aged monkey and AD human cerebral cortex. Western blot analyses detected increased levels of BACE1 protein and β-site-cleavage amyloid precursor protein C-terminal fragments in plaque-bearing human and monkey cortex relative to controls. In immunohistochemistry, locally elevated BACE1 immunoreactivity (IR) occurred in AD but not in control human cortex, with a trend for increased overall density among cases with greater plaque pathology. In double-labeling preparations, BACE1 IR colocalized with immunolabeling for Aβ but not for phosphorylated tau. In perfusion-fixed monkey cortex, locally increased BACE1 IR co-existed with intra-axonal and extracellular Aβ IR among virtually all neuritic plaques, ranging from primitive to typical cored forms. This BACE1 labeling localized to swollen/sprouting axon terminals that might co-express one or another neuronal phenotype markers (GABAergic, glutamatergic, cholinergic, or catecholaminergic). Importantly, these BACE1-labeled dystrophic axons resided near to or in direct contact with blood vessels. These findings suggest that plaque formation in AD or normal aged primates relates to a multisystem axonal pathogenesis that occurs in partnership with a potential vascular or metabolic deficit. The data provide a mechanistic explanation for why senile plaques are present preferentially near the cerebral vasculature.Artículo Texto completo
|Functional deprivation promotes amyloid plaque pathogenesis in Tg2576 mouse olfactory bulb and piriform cortex. |
Zhang, XM; Xiong, K; Cai, Y; Cai, H; Luo, XG; Feng, JC; Clough, RW; Patrylo, PR; Struble, RG; Yan, XX
The European journal of neuroscience 31 710-21 2010
Cerebral hypometabolism and amyloid accumulation are principal neuropathological manifestations of Alzheimer's disease (AD). Whether and how brain/neuronal activity might modulate certain pathological processes of AD are interesting topics of recent clinical and basic research in the field, and may be of potential medical relevance in regard to both the disease etiology and intervention. Using the Tg2576 transgenic mouse model of AD, this study characterized a promotive effect of neuronal hypoactivity associated with functional deprivation on amyloid plaque pathogenesis in the olfactory pathway. Unilateral naris-occlusion caused beta-secretase-1 (BACE1) elevation in neuronal terminals in the deprived relative to the non-deprived bulb and piriform cortex in young adult mice. In parallel with the overall age-related plaque development in the forebrain, locally increased BACE1 immunoreactivity co-occurred with amyloid deposition first in the piriform cortex then within the bulb, more prominent on the deprived relative to the non-deprived side. Biochemical analyses confirmed elevated BACE1 protein levels, enzymatic activity and products in the deprived relative to non-deprived bulbs. Plaque-associated BACE1 immunoreactivity in the bulb and piriform cortex was localized preferentially to swollen/sprouting glutamatergic axonal terminals, with Abeta immunoreactivity occurring inside as well as around these terminals. Together, these findings suggest that functional deprivation or neuronal hypoactivity facilitates amyloid plaque formation in the forebrain in a transgenic model of AD, which operates synergistically with age effect. The data also implicate an intrinsic association of amyloid accumulation and plaque formation with progressive axonal pathology.Artículo Texto completo
|Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers. |
Micheva, KD; Busse, B; Weiler, NC; O'Rourke, N; Smith, SJ
Neuron 68 639-53 2010
A lack of methods for measuring the protein compositions of individual synapses in situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here, we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate among 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.Artículo Texto completo
|SDF-1alpha/CXCL12 enhances GABA and glutamate synaptic activity at serotonin neurons in the rat dorsal raphe nucleus. |
Heinisch, S; Kirby, LG
Neuropharmacology 58 501-14 2010
The serotonin (5-hydroxytryptamine; 5-HT) system has a well-characterized role in depression. Recent reports describe comorbidities of mood-immune disorders, suggesting an immunological component may contribute to the pathogenesis of depression as well. Chemokines, immune proteins which mediate leukocyte trafficking, and their receptors are widely distributed in the brain, mediate neuronal patterning, and modulate various neuropathologies. The purpose of this study was to investigate the neuroanatomical relationship and functional impact of the chemokine stromal cell-derived factor-1alpha/CXCL12 and its receptor, CXCR4, on the serotonin dorsal raphe nucleus (DRN) system in the rat using anatomical and electrophysiological techniques. Immunohistochemical analysis indicates that over 70% of 5-HT neurons colocalize with CXCL12 and CXCR4. At a subcellular level, CXCL12 localizes throughout the cytoplasm whereas CXCR4 concentrates to the outer membrane and processes of 5-HT neurons. CXCL12 and CXCR4 also colocalize on individual DRN cells. Furthermore, electrophysiological studies demonstrate CXCL12 depolarization of 5-HT neurons indirectly via glutamate synaptic inputs. CXCL12 also enhances the frequency of spontaneous inhibitory and excitatory postsynaptic currents (sIPSC and sEPSC). CXCL12 concentration-dependently increases evoked IPSC amplitude and decreases evoked IPSC paired-pulse ratio selectively in 5-HT neurons, effects blocked by the CXCR4 antagonist AMD3100. These data indicate presynaptic enhancement of GABA and glutamate release at 5-HT DRN neurons by CXCL12. Immunohistochemical analysis further shows CXCR4 localization to DRN GABA neurons, providing an anatomical basis for CXCL12 effects on GABA release. Thus, CXCL12 indirectly modulates 5-HT neurotransmission via GABA and glutamate synaptic afferents. Future therapies targeting CXCL12 and other chemokines may treat serotonin related mood disorders, particularly depression experienced by immune-compromised individuals.
|Classical MHCI molecules regulate retinogeniculate refinement and limit ocular dominance plasticity. |
Datwani, A; McConnell, MJ; Kanold, PO; Micheva, KD; Busse, B; Shamloo, M; Smith, SJ; Shatz, CJ
Neuron 64 463-70 2009
Major histocompatibility complex class I (MHCI) genes were discovered unexpectedly in healthy CNS neurons in a screen for genes regulated by neural activity. In mice lacking just 2 of the 50+ MHCI genes H2-K(b) and H2-D(b), ocular dominance (OD) plasticity is enhanced. Mice lacking PirB, an MHCI receptor, have a similar phenotype. H2-K(b) and H2-D(b) are expressed not only in visual cortex, but also in lateral geniculate nucleus (LGN), where protein localization correlates strongly with synaptic markers and complement protein C1q. In K(b)D(b-/-) mice, developmental refinement of retinogeniculate projections is impaired, similar to C1q(-/-) mice. These phenotypes in K(b)D(b-/-) mice are strikingly similar to those in beta2 m(-/-)TAP1(-/-) mice, which lack cell surface expression of all MHCIs, implying that H2-K(b) and H2-D(b) can account for observed changes in synapse plasticity. H2-K(b) and H2-D(b) ligands, signaling via neuronal MHCI receptors, may enable activity-dependent remodeling of brain circuits during developmental critical periods.Artículo Texto completo
|Associative pairing involving monocular stimulation selectively mobilizes a subclass of GABAergic interneurons in the mouse visual cortex. |
Monika Liguz-Lecznar,Wioletta J Waleszczyk,Renata Zakrzewska,Jolanta Skangiel-Kramska,Malgorzata Kossut
The Journal of comparative neurology 516 2009
Levels of gamma-aminobutyric acid (GABA) and its synthesizing enzyme in cerebral cortex are regulated by sensory experience. Previously we found that associative pairing of vibrissae stimulation and tail shock results in upregulation of GABAergic markers in the mouse barrel cortex. In order to ascertain whether GABAergic upregulation also accompanies associative pairing in other sensory modalities, we examined the mouse visual cortex after analogous training with visual stimulus. During pairing, visual stimulus (CS) was coupled with a tail shock (UCS). We examined the density of cells expressing glutamic acid decarboxylase (GAD) and parvalbumin (PV) in monocular and binocular segments of the primary visual cortex (V1). The auditory cortex was used as a control. After monocular training, the density of cells expressing GAD rose significantly in the monocular segment of V1 contralateral to the stimulated eye, compared with the opposite hemisphere. This effect was due to the association of CS and UCS, as no changes were found after visual stimulation alone or in the auditory cortex. No changes were noted in the density of PV(+) neurons, so the effect was attributed to GAD(+)/PV(-) neurons. Mobilization of a specific subclass of GABAergic cells, observed after associative pairing in the somatosensory and visual cortices, may reflect the necessity to restrict the activity of circuits involved in sensory association.
|Chronic CXCL10 alters neuronal properties in rat hippocampal culture. |
Cho, J; Nelson, TE; Bajova, H; Gruol, DL
Journal of neuroimmunology 207 92-100 2009
The chemokine CXCL10 is expressed in the central nervous system (CNS) during neuroinflammatory conditions. Neurons express CXCR3, the receptor for CXCL10, and neuronal function has been shown to be altered by acute exposure to CXCL10. Little is known about the effects of chronic exposure to CXCL10 on neuronal function. Results from our studies show that chronic exposure of cultured rat hippocampal neurons to CXCL10 results in altered levels of protein for GABA and glutamate receptors and altered synaptic network activity. These effects of CXCL10 may contribute to altered CNS function that occurs in some chronic neuroinflammatory conditions.
|Co-transmission of dopamine and GABA in periglomerular cells. |
Maher, BJ; Westbrook, GL
Journal of neurophysiology 99 1559-64 2008
Most central neurons package and release a single transmitter. However co-transmission of fast-acting and modulatory transmitters has been observed in vertebrate and invertebrate systems. Here we describe a population of periglomerular cells in mouse brain slices (PND14-21) that co-release dopamine and GABA. We made whole cell recordings from periglomerular cells that expressed enhanced green fluorescent protein (EGFP) under the control of the tyrosine hyrdoxylase (TH) promoter. Immunolabeling confirmed that EGFP+ periglomerular cells synthesized TH as well as glutamic acid decarboxylase (GAD). Stimulation of olfactory receptor neuron (ORN) afferent input evoked excitatory postsynaptic currents (EPSCs) in EGFP+ cells that were inhibited by cocaine, which blocks dopamine transport. These effects were reversed by the D2 receptor antagonist sulpiride. Cocaine also increased the paired-pulse ratio of ORN-evoked EPSCs. These results demonstrate that TH+ periglomerular cells spontaneously release dopamine. In addition to dopamine, TH-EGFP+ cells also released GABA. Brief depolarizing voltage steps in labeled cells evoked a tail current that was completely blocked by the GABA(A) receptor antagonist gabazine and by cadmium, indicative of calcium-dependent self-inhibition in periglomerular cells. However, similar voltage steps were insufficient to cause D2-receptor mediated inhibition of ORN terminals. Our results indicate that TH+ periglomerular cells are directly activated by ORN input and release both dopamine and GABA. We suggest that concerted activation of multiple periglomerular cells may be required to detect dopamine release under normal physiological conditions.
|Hearing loss alters the subcellular distribution of presynaptic GAD and postsynaptic GABAA receptors in the auditory cortex. |
Sarro, EC; Kotak, VC; Sanes, DH; Aoki, C
Cerebral cortex (New York, N.Y. : 1991) 18 2855-67 2008
We have shown previously that auditory experience regulates the maturation of excitatory synapses in the auditory cortex (ACx). In this study, we used electron microscopic immunocytochemistry to determine whether the heightened excitability of the ACx following neonatal sensorineural hearing loss (SNHL) also involves pre- or postsynaptic alterations of GABAergic synapses. SNHL was induced in gerbils just prior to the onset of hearing (postnatal day 10). At P17, the gamma-aminobutyri acid type A (GABA(A)) receptor's beta2/3-subunit (GABA(A)beta2/3) clusters residing at plasma membranes in layers 2/3 of ACx was reduced significantly in size (P less than 0.05) and number (P less than 0.005), whereas the overall number of immunoreactive puncta (intracellular + plasmalemmal) remained unchanged. The reduction of GABA(A)beta2/3 was observed along perikaryal plasma membranes of excitatory neurons but not of GABAergic interneurons. This cell-specific change can contribute to the enhanced excitability of SNHL ACx. Presynaptically, GABAergic axon terminals were significantly larger but less numerous and contained 47% greater density of glutamic acid decarboxylase immunoreactivity (P less than 0.05). This suggests that GABA synthesis may be upregulated by a retrograde signal arising from lowered levels of postsynaptic GABA(A)R. Thus, both, the pre- and postsynaptic sides of inhibitory synapses that form upon pyramidal neurons of the ACx are regulated by neonatal auditory experience.Artículo Texto completo
|Functional expression of GABAB receptors in airway epithelium. |
Kentaro Mizuta, Yoko Osawa, Fumiko Mizuta, Dingbang Xu, Charles W Emala, Kentaro Mizuta, Yoko Osawa, Fumiko Mizuta, Dingbang Xu, Charles W Emala
American journal of respiratory cell and molecular biology 39 296-304 2008
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABA(A)) and metabotropic (GABA(B)) receptors. The GABA(B) receptor is a dimer composed of R1 and R2 components and classically couples to the heterotrimeric G(i) protein. In addition to their location on neurons, GABA and functional GABA(B) receptors have been detected in peripheral tissue such as airway smooth muscle. We questioned whether airway epithelium expresses receptors that could respond to GABA. We detected the mRNA encoding multiple-splice variants of the GABA(B)R1 and GABA(B)R2 in total RNA isolated from native human and guinea pig airway epithelium and human airway epithelial cell lines (BEAS-2B and H441). Immunoblots identified the GABA(B)R1 and GABA(B)R2 proteins in both guinea pig airway epithelium and BEAS-2B cells. The expression of GABA(B)R1 protein was immunohistochemically localized to basal mucin-secreting and ciliated columnar epithelial cells in guinea pig trachea. Baclofen inhibited adenylyl cyclase activity, induced ERK phosphorylation and cross-regulated phospholipase C, leading to increased inositol phosphates in BEAS-2B cells in a pertussis toxin-sensitive manner, implicating G(i) protein coupling. Thus, these receptors couple to G(i) and cross-regulate the phospholipase C/inositol phosphate pathway. The second messengers of these pathways, cyclic AMP and calcium, play pivotal roles in airway epithelial cell primary functions of mucus clearance. Furthermore, the enzyme that synthesizes GABA, glutamic acid decarboxylase (GAD65/67), was also localized to airway epithelium. GABA may modulate an uncharacterized signaling cascade via GABA(B) receptors coupled to G(i) protein in airway epithelium.Artículo Texto completo
|Selective resistance of taurine-fed mice to isoniazide-potentiated seizures: in vivo functional test for the activity of glutamic acid decarboxylase. |
A El Idrissi,
Neuroscience 156 693-9 2008
Taurine, 2-aminoethanesulfonic acid, is one of the most abundant free amino acids especially in excitable tissues, with wide physiological actions. We have previously reported that in mice, supplementation of the drinking water with taurine induces alterations in the inhibitory GABAergic system. In taurine-fed mice we found that the expression level of glutamic acid decarboxylase (GAD), the enzyme responsible for GABA synthesis, is elevated. Increased expression of GAD was accompanied by increased levels of GABA. Here, we investigated pharmacologically the functional significance of taurine-induced increase in GAD expression by determining the threshold for kainic acid-induced seizures after partial inhibition of GAD activity with isoniazide. We found that taurine-fed mice have elevated GAD expression and showed a higher threshold for seizure onset when compared with age-matched controls. Thus, taurine-fed mice have a functional increase in GAD activity which offers some protection in this seizure model. Furthermore, this pharmacological manipulation can be used to determine the level of GAD activity in other model systems that show alterations in GAD expression.
|Mice lacking the transcription factor Ikaros display behavioral alterations of an anti-depressive phenotype. |
Tim-Rasmus Kiehl, Sandra E Fischer, Shereen Ezzat, Sylvia L Asa
Experimental neurology 211 107-14 2008
The Ikaros (Ik) family of transcription factors has critical functions in immune regulation, lymphohematopoiesis and the hypothalamic-pituitary axis. Ik influences cell fate decisions through transcriptional activation of target genes and its interaction with chromatin remodeling complexes. While Ik is well-described in the lymphoid system and pituitary, its presence and function in the brain has received limited attention to date. This study describes the transient spatio-temporal expression of Ik in striatal medium spiny neurons of the developing murine CNS. To determine the impact of Ik deficiency, standardized behavioral tests were performed. In the elevated plus-maze and contextual fear conditioning tests, homozygous Ik-deficient mice performed similarly to wild-type or heterozygote mice. However, significant differences were observed in Ik-null mice in several behavioral tests. Pinch-induced catalepsy was markedly extended. In the Porsolt forced swim test, Ik-null mice showed reduced immobility, consistent with an anti-depressive effect. The acoustic startle response of Ik-null mice was also markedly diminished. Our findings extend the role of the Ikaros zinc-finger protein to the maturation and differentiation of striatal medium spiny neurons and indicate important actions for Ik in the development of neurocognitive functions and affecting depressive behaviors.
|Molecular mechanisms supporting a paracrine role of GABA in rat adrenal medullary cells. |
Matsuoka, H; Harada, K; Endo, Y; Warashina, A; Doi, Y; Nakamura, J; Inoue, M
The Journal of physiology 586 4825-42 2008
GABA is known to produce membrane depolarization and secretion in adrenal medullary (AM) cells in various species. However, whether the GABAergic system is intrinsic or extrinsic or both in the adrenal medulla and the role that GABA plays are controversial. Therefore, these issues were addressed by combining a biochemical and functional analysis. Glutamic acid decarboxylase (GAD), a GABA synthesizing enzyme, and vesicular GABA transporter (VGAT) were expressed in rat AM cells at the mRNA and protein levels, and the adrenal medulla had no nerve fibre-like structures immunoreactive to an anti-GAD Ab. The double staining for VGAT and chromogranin A indicates that GABA was stored in chromaffin granules. The alpha1, alpha3, beta2/3, gamma2 and delta subunits of GABA(A) receptors were identified in AM cells at the mRNA and protein levels. Pharmacological properties of GABA-induced Cl(-) currents, immunoprecipitation experiments and immunocytochemistry indicated the expression of not only gamma2-, but also delta-containing GABA(A) receptors, which have higher affinities for GABA and neurosteroids. Expression of GATs, which are involved in the clearance of GABA at GABAergic synapses, were conspicuously suppressed in the adrenal medulla, compared with expression levels of GABA(A) receptors. Increases in Ca(2+) signal in AM cells evoked trans-synaptically by nerve stimulation were suppressed during the response to GABA, and this suppression was attributed to the shunt effect of the GABA-induced increase in conductance. Overall Ca(2+) responses to electrical stimulation and GABA in AM cells were larger or smaller than those to electrical stimulation alone, depending on the frequency of stimulation. The results indicate that GABA functions as a paracrine in rat AM cells and this function may be supported by the suppression of GAT expression and the expression of not only gamma2-, but also delta-GABA(A) receptors.
|Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. |
Michael G Kaplitt, Andrew Feigin, Chengke Tang, Helen L Fitzsimons, Paul Mattis, Patricia A Lawlor, Ross J Bland, Deborah Young, Kristin Strybing, David Eidelberg, Matthew J During
Lancet 369 2097-105 2007
BACKGROUND: Dopaminergic neuronal loss in Parkinson's disease leads to changes in the circuitry of the basal ganglia, such as decreased inhibitory GABAergic input to the subthalamic nucleus. We aimed to measure the safety, tolerability, and potential efficacy of transfer of glutamic acid decarboxylase (GAD) gene with adeno-associated virus (AAV) into the subthalamic nucleus of patients with Parkinson's disease. METHODS: We did an open label, safety and tolerability trial of unilateral subthalamic viral vector (AAV-GAD) injection in 11 men and 1 woman with Parkinson's disease (mean age 58.2, SD=5.7 years). Four patients received low-dose, four medium-dose, and four high-dose AAV-GAD at New York Presbyterian Hospital. Inclusion criteria consisted of Hoehn and Yahr stage 3 or greater, motor fluctuations with substantial off time, and age 70 years or less. Patients were assessed clinically both off and on medication at baseline and after 1, 3, 6, and 12 months at North Shore Hospital. Efficacy measures included the Unified Parkinson's Disease Rating Scale (UPDRS), scales of activities of daily living (ADL), neuropsychological testing, and PET imaging with 18F-fluorodeoxyglucose. The trial is registered with the ClinicalTrials.gov registry, number NCT00195143. FINDINGS: All patients who enrolled had surgery, and there were no dropouts or patients lost to follow-up. There were no adverse events related to gene therapy. Significant improvements in motor UPDRS scores (p=0.0015), predominantly on the side of the body that was contralateral to surgery, were seen 3 months after gene therapy and persisted up to 12 months. PET scans revealed a substantial reduction in thalamic metabolism that was restricted to the treated hemisphere, and a correlation between clinical motor scores and brain metabolism in the supplementary motor area. INTERPRETATION: AAV-GAD gene therapy of the subthalamic nucleus is safe and well tolerated by patients with advanced Parkinson's disease, suggesting that in-vivo gene therapy in the adult brain might be safe for various neurodegenerative diseases.
|HVC interneurons are not renewed in adult male zebra finches. |
Sophie Scotto-Lomassese, Christelle Rochefort, Arpenik Nshdejan, Constance Scharff, Sophie Scotto-Lomassese, Christelle Rochefort, Arpenik Nshdejan, Constance Scharff
The European journal of neuroscience 25 1663-8 2007
Adult neurogenesis is a widespread phenomenon in many species, from invertebrates to humans. In songbirds, the telencephalic region, high vocal center (HVC), continuously integrates new neurons in adulthood. This nucleus consists of a heterogenous population of inhibitory interneurons (HVC(IN)) and two populations of projection neurons that send axons towards either the robust nucleus of the arcopallium (HVC(RA)) or the striatal nucleus area X (HVC(X)). New HVC neurons were initially inferred to be interneurons, because they lacked retrograde labelling from the HVC's targets. Later studies using different tracers demonstrated that HVC(RA) are replaced but HVC(X) are not. Whether interneurons are also renewed became an open question. As the HVC's neuronal populations display different physiological properties and functions, we asked whether adult HVC indeed recruits two neuronal populations or whether only the HVC(RA) undergo renewal in adult male zebra finches. We show that one month after being born in the lateral ventricle, 42% of the newborn HVC neurons were retrogradely labelled by tracer injections into the RA. However, the remaining 58% were not immunoreactive for the neurotransmitter GABA, nor for the calcium-binding proteins, parvalbumin (PA), calbindin (CB) and calretinin (CR) that characterize different classes of HVC(IN). We further established that simultaneous application of parvalbumin, calbindin and calretinin antibodies to HVC revealed approximately the same fraction of HVC neurons, i.e. 10%, as could be detected by GABA immunoreactivity. This implies that the sum of HVC(IN) expressing the different calcium-binding proteins constitute all inhibitory HVC(IN). Together these results strongly suggest that only HVC(RA) are recruited into the adult HVC.
|GABAergic pump cells of solitary tract nucleus innervate retrotrapezoid nucleus chemoreceptors. |
Takakura, AC; Moreira, TS; West, GH; Gwilt, JM; Colombari, E; Stornetta, RL; Guyenet, PG
Journal of neurophysiology 98 374-81 2007
The retrotrapezoid nucleus (RTN) contains central respiratory chemoreceptors that are inhibited by activation of slowly adapting pulmonary stretch receptors (SARs). Here we examine whether RTN inhibition by lung inflation could be mediated by a direct projection from SAR second-order neurons (pump cells). Pump cells (n = 56 neurons, 13 rats) were recorded in the nucleus of solitary tract (NTS) of halothane-anesthetized rats with intact vagus nerves. Pump cells had discharges that coincided with lung inflation as monitored by the tracheal pressure. Their activity increased when end-expiratory pressure was raised and stopped instantly when ventilation was interrupted in expiration. Many pump cells could be antidromically activated from RTN (12/36). Nine of those were labeled with biotinamide. Of these nine cells, eight contained glutamic acid decarboxylase 67 (GAD67) mRNA and seven were found to reside in the lower half of the interstitial subnucleus of NTS (iNTS). Using the retrograde tracer cholera toxin-B, we confirmed that neurons located in or close to iNTS innervate RTN (two rats). Many such neurons contained GAD67 mRNA and a few contained glycine transporter2 (GLYT2) mRNA. Anterograde tract tracing with biotinylated dextranamide (four rats) applied to iNTS also confirmed that this region innervates RTN by a predominantly GABAergic projection. This work confirms that many rat NTS pump cells are located in and around the interstitial subnucleus at area postrema level. We demonstrate that a GABAergic subset of these pump cells innervates the RTN region. We conclude that these inhibitory neurons probably contact RTN chemoreceptors and mediate their inhibition by lung inflation.
|Vesicular glutamate transporter 2 protein and mRNA containing neurons in the hypothalamic suprachiasmatic nucleus of the rat. |
József Kiss, Béla Halász, Agnes Csáki, Zsolt Liposits, Erik Hrabovszky
Brain research bulletin 74 397-405 2007
The hypothalamic suprachiasmatic nucleus is the key structure of the control of circadian rhythms and has a rich glutamatergic innervation. Besides the presence of glutamatergic afferents, several findings also suggest the existence of glutamatergic efferents from the suprachiasmatic nucleus to its target neurons in various prominent hypothalamic cell groups. However, there is no direct neuromorphological evidence for the presence of glutamatergic neurons in the suprachiasmatic nucleus. Therefore, the purpose of the present investigations was to try to clarify this question. Immunocytochemistry was used at the light and electron microscopy level to identify vesicular glutamate transporter type 2 (VGluT2) immunopositive (presumed glutamatergic) neurons in the rat suprachiasmatic nucleus. In addition VGluT2 mRNA expression in neurons of the nucleus was also addressed with radioisotopic in situ hybridization. Both at the light and electron microscopy level we detected VGluT2 positive neurons, which did not contain GABA, vasoactive intestinal polypeptide or vasopressin. Further, we demonstrated the expression of VGluT2 mRNA in a few cells within the suprachiasmatic nucleus; these glutamatergic cells were distinct from somatostatin mRNA expressing neurons. As VGluT2 is a selective marker of glutamatergic neuronal elements, the present observations provide direct neuromorphological evidence for the presence of glutamatergic neurons in the cell group.
|Brain-derived neurotrophic factor rescues synaptic plasticity in a mouse model of fragile X syndrome. |
Lauterborn, JC; Rex, CS; Kramár, E; Chen, LY; Pandyarajan, V; Lynch, G; Gall, CM
The Journal of neuroscience : the official journal of the Society for Neuroscience 27 10685-94 2007
Mice lacking expression of the fragile X mental retardation 1 (Fmr1) gene have deficits in types of learning that are dependent on the hippocampus. Here, we report that long-term potentiation (LTP) elicited by threshold levels of theta burst afferent stimulation (TBS) is severely impaired in hippocampal field CA1 of young adult Fmr1 knock-out mice. The deficit was not associated with changes in postsynaptic responses to TBS, NMDA receptor activation, or levels of punctate glutamic acid decarboxylase-65/67 immunoreactivity. TBS-induced actin polymerization within dendritic spines was also normal. The LTP impairment was evident within 5 min of induction and, thus, may not be secondary to defects in activity-initiated protein synthesis. Protein levels for both brain-derived neurotrophic factor (BDNF), a neurotrophin that activates pathways involved in spine cytoskeletal reorganization, and its TrkB receptor were comparable between genotypes. BDNF infusion had no effect on baseline transmission or on postsynaptic responses to theta burst stimulation, but nonetheless fully restored LTP in slices from fragile X mice. These results indicate that the fragile X mutation produces a highly selective impairment to LTP, possibly at a step downstream of actin filament assembly, and suggest a means for overcoming this deficit. The possibility of a pharmacological therapy based on these results is discussed.
|Nitric oxide-producing microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain slice culture. |
Hiroshi Katsuki, Mitsugi Okawara, Haruki Shibata, Toshiaki Kume, Akinori Akaike
Journal of neurochemistry 97 1232-42 2006
Activated microglia are considered to play important roles in degenerative processes of midbrain dopaminergic neurons. Here we examined mechanisms of neurotoxicity of thrombin, a protease known to trigger microglial activation, in organotypic midbrain slice cultures. Thrombin induced a progressive decline in the number of dopaminergic neurons, an increase in nitric oxide (NO) production, and whole tissue injury indicated by lactate dehydrogenase release and propidium iodide uptake. Microglia expressed inducible NO synthase (iNOS) in response to thrombin, and inhibition of iNOS rescued dopaminergic neurons without affecting whole tissue injury. Inhibitors of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) attenuated thrombin-induced iNOS induction and dopaminergic cell death. Whole tissue injury was also attenuated by inhibition of ERK and p38 MAPK. Moreover, depletion of resident microglia from midbrain slices abrogated thrombin-induced NO production and dopaminergic cell death, but did not inhibit tissue injury. Finally, antioxidative drugs prevented thrombin-induced dopaminergic cell death without affecting whole tissue injury. Hence, NO production resulting from MAPK-dependent microglial iNOS induction is a crucial event in thrombin-induced dopaminergic neurodegeneration, whereas damage of other midbrain cells is MAPK-dependent but is NO-independent.
|Projections of the nucleus lentiformis mesencephali in pigeons (Columba livia): a comparison of the morphology and distribution of neurons with different efferent projections. |
Janelle M P Pakan, Kimberly Krueger, Erin Kelcher, Sarah Cooper, Kathryn G Todd, Douglas R W Wylie
The Journal of comparative neurology 495 84-99 2006
The avian nucleus lentiformis mesencephali (LM) is a visual structure involved in the optokinetic response. The LM consists of several morphologically distinct cell types. In the present study we sought to determine if different cell types had differential projections. Using retrograde tracers, we examined the morphology and distribution of LM neurons projecting to the vestibulocerebellum (VbC), inferior olive (IO), dorsal thalamus, nucleus of the basal optic root (nBOR), and midline mesencephalon. From injections into the latter two structures, small LM cells were labeled. More were localized to the lateral LM as opposed to medial LM. From injections into the dorsal thalamus, small neurons were found throughout LM. From injections into the VbC, large multipolar cells were found throughout LM. From injections into IO, a strip of medium-sized fusiform neurons along the border of the medial and lateral subnuclei was labeled. To investigate if neurons project to multiple targets we used fluorescent retrograde tracers. After injections into IO and VbC, double-labeled neurons were not observed in LM. Likewise, after injections into nBOR and IO, double-labeled neurons were not observed. Finally, we processed sections through LM for glutamic acid decarboxylase (GAD). Small neurons, mostly in the lateral LM, were labeled, suggesting that projections from LM to nBOR and midline mesencephalon are GABAergic. We conclude that two efferents of LM, VbC and IO, receive input from morphologically distinct neurons: large multipolar and medium-sized fusiform neurons, respectively. The dorsal thalamus, nBOR, and midline mesencephalon receive input from small neurons, some of which are likely GABAergic.
|Slow progressive degeneration of nigral dopaminergic neurons in postnatal Engrailed mutant mice. |
Sgadò, P; Albéri, L; Gherbassi, D; Galasso, SL; Ramakers, GM; Alavian, KN; Smidt, MP; Dyck, RH; Simon, HH
Proceedings of the National Academy of Sciences of the United States of America 103 15242-7 2006
The homeobox transcription factors Engrailed-1 and Engrailed-2 are required for the survival of mesencephalic dopaminergic neurons in a cell-autonomous and gene-dose-dependent manner. Because of this requirement, the cells die by apoptosis when all four alleles of the Engrailed genes are genetically ablated (En1-/-;En2-/-). In the present study, we show that viable and fertile mice, heterozygous null for Engrailed-1 and homozygous null for Engrailed-2 (En1+/-;En2-/-), have an adult phenotype that resembles key pathological features of Parkinson's disease. Specifically, postnatal mutant mice exhibit a progressive degeneration of dopaminergic neurons in the substantia nigra during the first 3 mo of their lives, leading to diminished storage and release of dopamine in the caudate putamen, motor deficits similar to akinesia and bradykinesia, and a lower body weight. This genetic model may provide access to the molecular etiology for Parkinson's disease and could assist in the development of novel treatments for this neurodegenerative disorder.
|Quinolinic acid toxicity on orexin neurons blocked by gamma aminobutyric acid type A receptor stimulation. |
Hiroshi Katsuki, Akinori Akaike
Neuroreport 16 1157-61 2005
Selective degeneration of hypothalamic orexin neurons, a hallmark of pathology in narcolepsy patients, is in part reproduced in hypothalamic slice cultures by application of an endogenous excitotoxin quinolinic acid. Depolarized membrane potential may be responsible for the vulnerability of orexin neurons to excitotoxicity. We show that stimulation of gamma-aminobutyric acid type A receptors, which is known to hyperpolarize orexin neurons, by muscimol or isoguvacine potently inhibits quinolinic acid cytotoxicity on orexin neurons. In addition, the protective effect of gamma-aminobutyric acid and a gamma-aminobutyric acid uptake blocker nipecotic acid is abolished by a gamma-aminobutyric acid type A antagonist picrotoxin. Norepinephrine and serotonin do not provide a neuroprotective effect. Thus, GABAergic inhibitory control may be a decisive factor regulating survival of orexin neurons under excitotoxic insults.
|Incongruent restoration of inhibitory transmission and general metabolic activity during reorganization of somatosensory cortex. |
Liisa A Tremere, Raphael Pinaud
The International journal of neuroscience 115 1003-15 2005
Activity markers cytochrome oxidase (CO) and glutamic acid decarboxylase (GAD) were analyzed in the primary somatosensory cortex of raccoons that underwent digit amputation. Subjects recovered for 2, 15, and 23 weeks following amputation of the fourth forepaw digit. Histochemistry was used to assess relative activity levels of both enzymes. We found a pronounced decrease in the numbers of CO intense patches in the cortical gyrus that had lost its original sensory input from the fourth digit. This decrease in CO activity was still apparent 15 weeks post-amputation. Conversely, no clear decrease in GAD levels could be identified in connection with the amputation procedure. Our findings present evidence that a significant decrease in metabolic activity results from the loss of the primary afferent sensory drive. The remaining GAD activity suggests that the absence of electrical activity, characteristic of reorganizing cortex, is likely to depend in part on lateral inhibitory cortical connections.
|Dendritic cells in human thymus and periphery display a proinsulin epitope in a transcription-dependent, capture-independent fashion. |
Carlos A Garcia, Kamalaveni R Prabakar, Juan Diez, Zhu Alexander Cao, Gloria Allende, Markus Zeller, Rajpreet Dogra, Armando Mendez, Eliot Rosenkranz, Ulf Dahl, Camillo Ricordi, Douglas Hanahan, Alberto Pugliese
Journal of immunology (Baltimore, Md. : 1950) 175 2111-22 2005
The natural expression of tissue-specific genes in the thymus, e.g., insulin, is critical for self-tolerance. The transcription of tissue-specific genes is ascribed to peripheral Ag-expressing (PAE) cells, which discordant studies identified as thymic epithelial cells (TEC) or CD11c+ dendritic cells (DC). We hypothesized that, consistent with APC function, PAE-DC should constitutively display multiple self-epitopes on their surface. If recognized by Abs, such epitopes could help identify PAE cells to further define their distribution, nature, and function. We report that selected Abs reacted with self-epitopes, including a proinsulin epitope, on the surface of CD11c+ cells. We find that Proins+ CD11c+ PAE cells exist in human thymus, spleen, and also circulate in blood. Human thymic Proins+ cells appear as mature DC but express CD8alpha, CD20, CD123, and CD14; peripheral Proins+ cells appear as immature DC. However, DC derived in vitro from human peripheral blood monocytes include Proins+ cells that uniquely differentiate and mature into thymic-like PAE-DC. Critically, we demonstrate that human Proins+ CD11c+ cells transcribe the insulin gene in thymus, spleen, and blood. Likewise, we show that mouse thymic and peripheral CD11c+ cells transcribe the insulin gene and display the proinsulin epitope; moreover, by using knockout mice, we show that the display of this epitope depends upon insulin gene transcription and is independent of Ag capturing. Thus, we propose that PAE cells include functionally distinct DC displaying self-epitopes through a novel, transcription-dependent mechanism. These cells might play a role in promoting self-tolerance, not only in the thymus but also in the periphery.
|Axodendritic contacts onto calcium/calmodulin-dependent protein kinase type II-expressing neurons in the barn owl auditory space map. |
Rodriguez-Contreras, A; Liu, XB; DeBello, WM
The Journal of neuroscience : the official journal of the Society for Neuroscience 25 5611-22 2005
In the owl midbrain, a map of auditory space is synthesized in the inferior colliculus (IC) and conveyed to the optic tectum (OT). Ascending auditory information courses through these structures via topographic axonal projections. Little is known about the molecular composition of projection neurons or their postsynaptic targets. To visualize axodendritic contacts between identified cell types, we used double-label immunohistochemistry, in vivo retrograde tracing, in vitro anterograde tracing, high-resolution confocal microscopy, three-dimensional reconstruction and fly-through visualization. We discovered a major class of IC neurons that strongly expressed calcium/calmodulin-dependent protein kinase type II, alpha subunit (CaMKII). The distribution of these cells within the IC was mostly restricted to the external nucleus of the IC (ICX), in which the auditory space map is assembled. A large proportion of ICX-OT projection neurons were CaMKII positive. In addition to being the principal outputs, CaMKII cells were in direct contact with axonal boutons emanating from the main source of input to ICX, the lateral shell of the central nucleus of the inferior colliculus (ICCls). Numerous sites of putative synaptic contact were found on the somata, proximal dendrites, and distal dendrites. Double-label immunoelectron microscopy confirmed the existence of synapses between ICCls axons and the dendrites of CaMKII cells. Collectively, our data indicate that CaMKII ICX neurons are a cellular locus for the computation of auditory space-specific responses. Because the ICCls-ICX projection is physically altered during experience-dependent plasticity, these results lay the groundwork for probing microanatomical rearrangements that may underlie plasticity and learning.
|Functional integration of embryonic stem cell-derived neurons in vivo. |
Wernig, Marius, et al.
J. Neurosci., 24: 5258-68 (2004) 2004
Pluripotency and the potential for continuous self-renewal make embryonic stem (ES) cells an attractive donor source for neuronal cell replacement. Despite recent encouraging results in this field, little is known about the functional integration of transplanted ES cell-derived neurons on the single-cell level. To address this issue, ES cell-derived neural precursors exhibiting neuron-specific enhanced green fluorescent protein (EGFP) expression were introduced into the developing brain. Donor cells implanted into the cerebral ventricles of embryonic rats migrated as single cells into a variety of brain regions, where they acquired complex morphologies and adopted excitatory and inhibitory neurotransmitter phenotypes. Synaptic integration was suggested by the expression of PSD-95 (postsynaptic density-95) on donor cell dendrites, which in turn were approached by multiple synaptophysin-positive host axon terminals. Ultrastructural and electrophysiological data confirmed the formation of synapses between host and donor cells. Ten to 21 d after birth, all EGFP-positive donor cells examined displayed active membrane properties and received glutamatergic and GABAergic synaptic input from host neurons. These data demonstrate that, at the single-cell level, grafted ES cell-derived neurons undergo morphological and functional integration into the host brain circuitry. Antibodies to the region-specific transcription factors Bf1, Dlx, En1, and Pax6 were used to explore whether functional donor cell integration depends on the acquisition of a regional phenotype. Our data show that incorporated neurons frequently exhibit a lacking or ectopic expression of these transcription factors. Thus, the lack of an appropriate regional "code" does not preclude morphological and synaptic integration of ES cell-derived neurons.
|Taurine as a modulator of excitatory and inhibitory neurotransmission. |
Abdeslem El Idrissi, Ekkehart Trenkner
Neurochemical research 29 189-97 2004
We present data that summarize our findings on the role of taurine in the central nervous system and in particular taurine's interaction with the inhibitory and excitatory systems. In taurine-fed mice, the expression level of glutamic acid decarboxylase (GAD), the enzyme responsible for GABA synthesis, is elevated. Increased expression of GAD was accompanied by increased levels of GABA. We also found in vitro, that taurine regulates neuronal calcium homeostasis and calcium-dependent processes, such as protein kinase C (PKC) activity. This calcium-dependent kinase was regulated by taurine, whereas the activity of protein kinase A (PKA), a cAMP-dependent, calcium-independent kinase, was not affected. Furthermore, as a consequence of calcium regulation, taurine counteracted glutamate-induced mitochondrial damage and cell death.
|GAD67 and GAD65 mRNA and protein expression in cerebrocortical regions of elderly patients with schizophrenia. |
Stella Dracheva, Sharif L Elhakem, Susan R McGurk, Kenneth L Davis, Vahram Haroutunian
Journal of neuroscience research 76 581-92 2004
Gamma-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter of CNS, has been consistently implicated in the pathophysiology of schizophrenia. GABA is synthesized from glutamate by the enzyme glutamic acid decarboxylase (GAD). Two isoforms of GAD have been identified and have been named GAD65 and GAD67 based on their apparent molecular weights. In this study, GAD65 and GAD67 mRNA and protein levels were measured by using real-time RT-PCR and immunoblotting, respectively, in post-mortem brain tissue from the dorsolateral prefrontal cortex (DLPFC) and the occipital cortex of the elderly persons with schizophrenia and matched normal controls. In addition, the mRNA expression of GAT-1, one of the principal transporters of GABA, was also studied in the same subjects. Expression of GAD65 and GAD67 mRNA in the DLPFC and in the occipital cortex was significantly elevated in patients with schizophrenia, whereas the expression of the corresponding proteins and GAT-1 mRNA was unchanged. Although the levels of GAD65 and GAD67 messages were increased in schizophrenia subjects, the proportion of the two GAD isoforms remained constant in controls and schizophrenics. In the human DLPFC, GAD65 mRNA was found to be expressed significantly less than the message for GAD67, approximately 16% of that observed for GAD67. On the contrary, the abundance of GAD65 protein in the DLPFC was about 350% of that observed for GAD67. The results suggest a substantial dysregulation of GAD mRNA expression in schizophrenia and, taken together with the results of protein expression studies, raise the possibility that both cortical and subcortical GABA function may be compromised in the disease.
|Opioid modulation of GABA release in the rat inferior colliculus. |
Tongjaroenbungam, W; Jongkamonwiwat, N; Cunningham, J; Phansuwan-Pujito, P; Dodson, HC; Forge, A; Govitrapong, P; Casalotti, SO
BMC neuroscience 5 31 2004
The inferior colliculus, which receives almost all ascending and descending auditory signals, plays a crucial role in the processing of auditory information. While the majority of the recorded activities in the inferior colliculus are attributed to GABAergic and glutamatergic signalling, other neurotransmitter systems are expressed in this brain area including opiate peptides and their receptors which may play a modulatory role in neuronal communication.Using a perfusion protocol we demonstrate that morphine can inhibit KCl-induced release of [3H]GABA from rat inferior colliculus slices. DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin) but not DADLE ([D-Ala2, D-Leu5]-enkephalin or U69593 has the same effect as morphine indicating that micro rather than delta or kappa opioid receptors mediate this action. [3H]GABA release was diminished by 16%, and this was not altered by the protein kinase C inhibitor bisindolylmaleimide I. Immunostaining of inferior colliculus cryosections shows extensive staining for glutamic acid decarboxylase, more limited staining for micro opiate receptors and relatively few neurons co-stained for both proteins.The results suggest that micro-opioid receptor ligands can modify neurotransmitter release in a sub population of GABAergic neurons of the inferior colliculus. This could have important physiological implications in the processing of hearing information and/or other functions attributed to the inferior colliculus such as audiogenic seizures and aversive behaviour.
|Manganese ethylene-bis-dithiocarbamate and selective dopaminergic neurodegeneration in rat: a link through mitochondrial dysfunction. |
Zhang, Jing, et al.
J. Neurochem., 84: 336-46 (2003) 2003
Manganese ethylene-bis-dithiocarbamate (Mn-EBDC) is the major active element of maneb, a pesticide linked to parkinsonism in certain individuals upon chronic exposure. Additionally, it has been shown to produce dopaminergic neurodegeneration in mice systemically coexposed to another pesticide, 1,1'-dimethyl-4,4'-bipyridinium (paraquat). Here, we described a rat model in which selective dopaminergic neurodegeneration was produced by delivering Mn-EBDC directly to the lateral ventricles. After establishing this model, we tested whether Mn-EBDC provoked dopamine efflux in the striatum, a well-known phenomenon produced by the mitochondrial inhibitor 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that causes parkinsonism in humans, as well as in some animals. Finally, we investigated whether Mn-EBDC directly inhibited mitochondrial function in vitro using isolated brain mitochondria. Our data demonstrated that Mn-EBDC induced extensive striatal dopamine efflux that was comparable with that induced by MPP+, and that Mn-EBDC preferentially inhibited mitochondrial complex III. As mitochondrial dysfunction is pivotal in the pathogenesis of Parkinson's disease (PD), our results support the proposal that exposure to pesticides such as maneb, or other naturally occurring compounds that inhibit mitochondrial function, may contribute to PD development.
|Nicotinic alpha 7 receptor clusters on hippocampal GABAergic neurons: regulation by synaptic activity and neurotrophins. |
Kawai, Hideki, et al.
J. Neurosci., 22: 7903-12 (2002) 2002
Nicotinic acetylcholine receptors containing the alpha7 gene product are expressed at substantial levels in the hippocampus. Because of their specific locations and their high relative calcium permeability, the receptors not only mediate cholinergic transmission in the hippocampus but also influence signaling at noncholinergic synapses. We have used fluorescently labeled alpha-bungarotoxin to image alpha7-containing receptors on hippocampal neurons and to examine their regulation in culture. The highest levels of staining for such receptors were most commonly found on GABAergic interneurons identified immunohistochemically. The receptors were distributed in clusters on the soma and dendrites and were localized in part at GABAergic synapses. A 3 d blockade of electrical activity with tetrodotoxin or NMDA receptors with APV dramatically reduced the proportion of GABAergic neurons expressing high levels of receptor staining and reduced the mean number of distinguishable receptor clusters on individual neurons. Blockade of either GABA(A) receptors with bicuculline or nicotinic receptors with d-tubocurarine had no effect, although exposure to nicotine could increase the level of receptor staining. Anti-BDNF and anti-NGF antibodies produced decrements equivalent to those of tetrodotoxin and APV, whereas addition of BDNF and NGF each increased staining levels and increased the number of distinguishable receptor clusters on GABAergic neurons. The exogenous neurotrophins could not, however, overcome the effects of either tetrodotoxin or APV. The results indicate that both NMDA receptor activation and the neurotrophins BDNF and NGF are necessary to sustain the distribution patterns of alpha7-containing nicotinic receptors on GABAergic hippocampal neurons.
|Brain-derived neurotrophic factor promotes the maturation of GABAergic mechanisms in cultured hippocampal neurons. |
Maki K Yamada, Kohsuke Nakanishi, Shizu Ohba, Takeshi Nakamura, Yuji Ikegaya, Nobuyoshi Nishiyama, Norio Matsuki
The Journal of neuroscience : the official journal of the Society for Neuroscience 22 7580-5 2002
Brain-derived neurotrophic factor (BDNF) has been implicated in activity-dependent plasticity of neuronal function and network arrangement. To clarify how BDNF exerts its action, we evaluated the physiological, histological, and biochemical characteristics of cultured hippocampal neurons after long-term treatment with BDNF. Here we show that BDNF facilitates high K(+)-elicited release of GABA but not of glutamate and induces an increase in immunoreactive signals of glutamic acid decarboxylase, a GABA-synthesizing enzyme. The soma size of GABAergic neurons was enlarged in BDNF-treated cultures, whereas the average soma size of all neurons was virtually unchanged. BDNF also upregulated protein levels of GABA(A) receptors but not of glutamate receptors. These data imply that BDNF selectively advances the maturation of GABAergic synapses. However, immunocytochemical analyses revealed that a significant expression of TrkB, a high-affinity receptor for BDNF, was detected in non-GABAergic as well as GABAergic neurons. BDNF also increased to total amount of synaptic vesicle-associated proteins without affecting the number of presynaptic vesicles that can be labeled with FM1-43 after K(+) depolarization. Together, our findings indicate that BDNF principally promotes GABAergic maturation but may also potentially contribute to excitatory synapse development via increasing resting synaptic vesicles.
|Metabolic distinction between vesicular and cytosolic GABA in cultured GABAergic neurons using 13C magnetic resonance spectroscopy. |
H S Waagepetersen, U Sonnewald, G Gegelashvili, O M Larsson, A Schousboe, H S Waagepetersen, U Sonnewald, G Gegelashvili, O M Larsson, A Schousboe
Journal of neuroscience research 63 347-55 2001
GABA exists in at least two different intracellular pools, i.e., a cytoplasmic or metabolic pool and a vesicular pool. This study was performed to gain information about the quantitative role of the tricarboxylic acid (TCA) cycle in biosynthesis of GABA from glutamine when GABA was selectively released from either one of these two pools. Cultured cerebral cortical neurons (GABAergic) were incubated in a medium containing 0.5 mM [U-13C]glutamine and subsequently depolarized for release of GABA from either the vesicular or the cytoplasmic pool. The vesicular release was induced by 55 mM K+ in the presence of tiagabine, a nontransportable inhibitor of the plasma membrane GABA carriers, whereas the cytoplasmic release via a reversal of the GABA carrier was induced by exposure to N-methyl-D-aspartate (NMDA; 50 microM) in the presence of (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA; 50 microM). Cell extracts were analyzed by 13C magnetic resonance spectroscopy subsequent to the incubation or depolarization. The percentage of GABA generated from glutamine via the TCA cycle decreased from 60% to 46% during depolarization, inducing GABA release from the cytoplasmic pool, whereas a significant change in this parameter was not observed after release from the vesicular pool. These observations indicate that, during release from the cytoplasmic pool, the fraction of GABA synthesized directly from glutamine without involvement of the TCA cycle is more pronounced than that occurring during resting conditions and when release occurs from the vesicular pool. This might be explained by differences in the regulation of the two isoforms of glutamate decarboxylase (GAD(65) and GAD(67)), which presumably play different roles in the maintenance of GABA in the two pools. Both isoforms were found in the cultured cerebral cortical neurons, as shown by Western blotting employing an antibody recognizing GAD(65) as well as GAD(67).
|Ventrally located commissural neurons express the GABAergic phenotype in developing rat spinal cord. |
Phelps, P E, et al.
J. Comp. Neurol., 409: 285-98 (1999) 1999
Early-forming commissural neurons are studied intensively as a model of axonal outgrowth and pathfinding, yet the neurotransmitter phenotype of the majority of these neurons is not known. The present study has determined that a substantial number of commissural neurons express the 65-kDa isoform of glutamic acid decarboxylase (GAD65) as early as embryonic day 12 (E 12). Patterns of GAD65 localization were compared with those of TAG-1, the Transiently expressed Axonal Glycoprotein that is the best known marker of commissural axons. On E13, both GAD65- and TAG-1-labeled commissural axons emanate from similar lateral and ventromedial regions. However, dorsally located TAG-1-positive commissural axons were GAD65-negative. These results suggest that commissural neurons have both gamma-aminobutyric acid (GABA)ergic and non-GABAergic phenotypes. The intensity of GAD65 staining within commissural somata and axons decreased between E14-15 and continued to decline during embryonic development, whereas terminal-like structures in surrounding neuropil increased dramatically. This sudden loss of somatic and axonal GAD65 staining was unexpected and could be interpreted as commissural neurons only transiently expressing the GABAergic phenotype. Further experiments were undertaken to identify commissural neurons with other established GABAergic markers, GAD67 and GABA. When antibody labeling of the two GAD isoforms was compared, GAD67 was detected 1 day later than GAD65, and in a different subcellular distribution. In contrast to GAD65, GAD67 intensely stained somata but labeled few commissural axons. GABA immunoreactivity also was detected in commissural axons 1 day after GAD65, and the labeling pattern between E13 and E16 resembled that of GAD67 rather than GAD65. When GAD and GABA results were compared, it was clear that a number of ventrally located commissural neurons expressed and maintained the GABAergic phenotype during embryonic development. However, the early expression and subcellular redistribution of GAD65 suggests that the GAD isoforms are differentially regulated. The function of the transient GAD65 expression in commissural somata and axons is unknown, but its temporal expression pattern parallels the transient expression of TAG-1, as both are expressed during the early stages of commissural axon outgrowth and pathfinding.
|Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10. |
Karlsen, A E, et al.
Proc. Natl. Acad. Sci. U.S.A., 88: 8337-41 (1991) 1991
Glutamic acid decarboxylase (GAD; glutamate decarboxylase, L-glutamate 1-carboxy-lyase, EC 18.104.22.168), which catalyzes formation of gamma-aminobutyric acid from L-glutamic acid, is detectable in different isoforms with distinct electrophoretic and kinetic characteristics. GAD has also been implicated as an autoantigen in the vastly differing autoimmune disease stiff-man syndrome and insulin-dependent diabetes mellitus. Despite the differing GAD isoforms, only one type of GAD cDNA (GAD-1), localized to a syntenic region of chromosome 2, has been isolated from rat, mouse, and cat. Using sequence information from GAD-1 to screen a human pancreatic islet cDNA library, we describe the isolation of an additional GAD cDNA (GAD-2), which was mapped to the short arm of human chromosome 10. Genomic Southern blotting with GAD-2 demonstrated a hybridization pattern different from that detected by GAD-1. GAD-2 recognizes a 5.6-kilobase transcript in both islets and brain, in contrast to GAD-1, which detects a 3.7-kilobase transcript in brain only. The deduced 585-amino acid sequence coded for by GAD-2 shows less than 65% identity to previously published, highly conserved GAD-1 brain sequences, which show greater than 96% deduced amino acid sequence homology among the three species. The function of this additional islet GAD isoform and its importance as an autoantigen in insulin-dependent diabetes remain to be determined.
|Two genes encode distinct glutamate decarboxylases. |
Erlander, M G, et al.
Neuron, 7: 91-100 (1991) 1991
gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.
|Characterization of a cDNA coding for rat glutamic acid decarboxylase. |
Wyborski, R J, et al.
Brain Res. Mol. Brain Res., 8: 193-8 (1990) 1990
cDNA clones have been isolated for rat glutamic acid decarboxylase (glutamate decarboxylase; EC 22.214.171.124) (GAD) and 3216 bp of the sequence have been determined. This sequence extends the previously reported feline GAD cDNA sequence both in the 5' (67 bp) and 3' (887 bp) directions and contains the polyadenylation signal and tail. The cDNA codes for a 67 kDa mol. wt. protein beginning from the putative initiator methionine found in the feline sequence. Extensive homology to feline GAD was identified at the amino acid level (97% identity) within the coding region. This interspecies homology is high compared to other neurotransmitter synthesizing enzymes and suggests selective pressure to maintain the primary sequence throughout the full length of the protein. Homology is found 5' to the putative initiator methionine. Extensive stretches of homology are also found in the 3' non-coding region. These conserved non-coding regions may play a role in GAD mRNA regulation. The rat cDNA sequence will facilitate investigations into the structure and regulation of the GAD gene.
|Rat brain glutamic acid decarboxylase sequence deduced from a cloned cDNA. |
Julien, J F, et al.
J. Neurochem., 54: 703-5 (1990) 1990
A cDNA clone complementary to the rat brain glutamic acid decarboxylase mRNA was isolated from a rat brain cDNA expression library using an antibody specific to the enzyme. The cDNA insert has been shown to direct the synthesis of an active protein in Escherichia coli. In this study, the nucleotide sequence of this clone, which includes the complete coding region, is presented. The predicted protein is 593 amino acids in length. The first 557 residues display a 95% identity when compared with the corresponding cat sequence. However, the deduced amino acid sequence of the carboxy-terminal end of the rat protein, downstream of residue 557, is totally different from the cat, whereas it agrees with a published partial peptidic sequence of the rat protein.
|Anti-Glutamate Decarboxylase 65 & 67 - Data Sheet|