RESUMEN

The GluN3A subunit of N-methyl-D-aspartate receptors (NMDARs) plays an established role in synapse development, but its contribution to neural circuits in the adult brain is less clear. Recent work has demonstrated that in select cell populations, GluN3A assembles with GluN1 to form GluN1/GluN3A receptors that are insensitive to glutamate and instead serve as functional excitatory glycine receptors (eGlyRs). Our understanding of these eGlyRs, and how they contribute to intrinsic excitability and synaptic communication within relevant networks of the developing and the mature brain, is only beginning to be uncovered. Here, using male and female mice, we demonstrate that GluN3A subunits are enriched in the adult ventral hippocampus (VH), where they localize to synaptic and extrasynaptic sites and can assemble as functional eGlyRs on CA1 pyramidal cells. GluN3A expression was barely detectable in the adult dorsal hippocampus (DH). We also observed a high GluN2B content in the adult VH, characterized by slow NMDAR current decay kinetics and a high sensitivity to the GluN2B-containing NMDAR antagonist ifenprodil. Interestingly, the GluN2B enrichment in the adult VH was dependent on GluN3A as GluN3A deletion accelerated NMDAR decay and reduced ifenprodil sensitivity in the VH, suggesting that GluN3A expression can regulate the balance of conventional NMDAR subunit composition at synaptic sites. Lastly, we found that GluN3A knockout also enhanced both NMDAR-dependent calcium influx and NMDAR-dependent long-term potentiation in the VH. Together, these data reveal a novel role for GluN3A and eGlyRs in the control of ventral hippocampal circuits in the mature brain.Significance statement Glutamate is the brain's most abundant excitatory neurotransmitter. One of its most common receptors is the NMDA receptor (NMDAR), composed of a variety of subunits. Here, we show that an atypical NMDAR subunit, GluN3A, is enriched in the ventral hippocampus, where it forms a receptor that is no longer sensitive to glutamate. Instead, it acts as an excitatory receptor for glycine, normally an inhibitory transmitter. We show that GluN3A plays essential roles in the adult ventral hippocampus, modulating transmitter release probability, conventional NMDAR subunit composition, calcium entry, and synaptic plasticity. These findings are important as they shed light on the role of GluN3A and these newly discovered excitatory glycine receptors in the mature brain.

RESUMEN

Hypothalamic inflammation reduces appetite and body weight during inflammatory diseases, while promoting weight gain when induced by high-fat diet (HFD). How hypothalamic inflammation can induce opposite energy balance outcomes remains unclear. We found that prostaglandin E2 (PGE2), a key hypothalamic inflammatory mediator of sickness, also mediates diet-induced obesity (DIO) by activating appetite-promoting melanin-concentrating hormone (MCH) neurons in the hypothalamus in rats and mice. The effect of PGE2 on MCH neurons is excitatory at low concentrations while inhibitory at high concentrations, indicating that these neurons can bidirectionally respond to varying levels of inflammation. During prolonged HFD, endogenous PGE2 depolarizes MCH neurons through an EP2 receptor-mediated inhibition of the electrogenic Na+/K+-ATPase. Disrupting this mechanism by genetic deletion of EP2 receptors on MCH neurons is protective against DIO and liver steatosis in male and female mice. Thus, an inflammatory mediator can directly stimulate appetite-promoting neurons to exacerbate DIO and fatty liver.

Asunto(s)

Hígado Graso , Obesidad , Ratones , Ratas , Masculino , Femenino , Animales , Obesidad/genética , Melaninas/genética , Hipotálamo , Inflamación , Dieta Alta en Grasa/efectos adversos , Neuronas , Mediadores de Inflamación , Prostaglandinas

RESUMEN

Most research on glutamate spillover focuses on the deleterious consequences of postsynaptic glutamate receptor overactivation. However, two decades ago, it was noted that the glial coverage of hippocampal synapses is asymmetric: astrocytic coverage of postsynaptic sites exceeds coverage of presynaptic sites by a factor of four. The fundamental relevance of this glial asymmetry remains poorly understood. Here, we used the glutamate biosensor iGluSnFR, and restricted its expression to either CA3 or CA1 neurons to visualize glutamate dynamics at pre- and postsynaptic microenvironments, respectively. We demonstrate that inhibition of the primarily astrocytic glutamate transporter-1 (GLT-1) slows glutamate clearance to a greater extent at presynaptic compared to postsynaptic membranes. GLT-1 expression was reduced early in a mouse model of AD, resulting in slower glutamate clearance rates at presynaptic but not postsynaptic membranes that opposed presynaptic short-term plasticity. Overall, our data demonstrate that the presynapse is particularly vulnerable to GLT-1 dysfunction and may have implications for presynaptic impairments in a variety of brain diseases.

Asunto(s)

Enfermedad de Alzheimer , Ácido Glutámico , Ratones , Animales , Ácido Glutámico/metabolismo , Enfermedad de Alzheimer/metabolismo , Sinapsis/metabolismo , Neuronas/metabolismo , Hipocampo/metabolismo

RESUMEN

Huntington disease (HD) is a monogenic disease that results in a combination of motor, psychiatric and cognitive symptoms. HD is caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene, which results in the production of a pathogenic mutant HTT protein (mHTT). Although there is no cure at present for HD, a number of RNA-targeting therapies have recently entered clinical trials which aim to lower mHTT production through the use of antisense oligonucleotides (ASOs) and RNAi. However, many of these treatment strategies are non-selective in that they cannot differentiate between non-pathogenic wild type HTT (wtHTT) and the mHTT variant. As HD patients are already born with decreased levels of wtHTT, these genetic therapies may result in critically low levels of wtHTT. The consequence of wtHTT reduction in the adult brain is currently under debate, and here we argue that wtHTT loss is not well-tolerated at the synaptic level. Synaptic dysfunction is an extremely sensitive measure of subsequent cell death, and is known to precede neurodegeneration in numerous brain diseases including HD. The present review focuses on the prominent role of wtHTT at the synapse and considers the consequences of wtHTT loss on both pre- and postsynaptic function. We discuss how wtHTT is implicated in virtually all major facets of synaptic neurotransmission including anterograde and retrograde transport of proteins to/from terminal buttons and dendrites, neurotransmitter release, endocytic vesicle recycling, and postsynaptic receptor localization and recycling. We conclude that wtHTT presence is essential for proper synaptic function.

RESUMEN

Pharmacological upregulation of glutamate transporter-1 (GLT-1), commonly achieved using the beta-lactam antibiotic ceftriaxone, represents a promising therapeutic strategy to accelerate glutamate uptake and prevent excitotoxic damage in neurological conditions. While excitotoxicity is indeed implicated in numerous brain diseases, it is typically restricted to select vulnerable brain regions, particularly in early disease stages. In healthy brain tissue, the speed of glutamate uptake is not constant and rather varies in both an activity- and region-dependent manner. Despite the widespread use of ceftriaxone in disease models, very little is known about how such treatments impact functional measures of glutamate uptake in healthy tissue, and whether GLT-1 upregulation can mask the naturally occurring activity-dependent and regional heterogeneities in uptake. Here, we used two different compounds, ceftriaxone and LDN/OSU-0212320 (LDN), to upregulate GLT-1 in healthy wild-type mice. We then used real-time imaging of the glutamate biosensor iGluSnFR to investigate functional consequences of GLT-1 upregulation on activity- and regional-dependent variations in glutamate uptake capacity. We found that while both ceftriaxone and LDN increased GLT-1 expression in multiple brain regions, they did not prevent activity-dependent slowing of glutamate clearance nor did they speed basal clearance rates, even in areas characterized by slow uptake (e.g., striatum). Unexpectedly, ceftriaxone but not LDN decreased glutamate release in the cortex, suggesting that ceftriaxone may alter release properties independent of its effects on GLT-1 expression. In sum, our data demonstrate the complexities of glutamate uptake by showing that GLT-1 expression does not necessarily translate to accelerated uptake. Furthermore, these data suggest that the mechanisms underlying activity- and regional-dependent differences in glutamate dynamics are independent of GLT-1 expression levels.

RESUMEN

Synaptic structure and function are compromised prior to cell death and symptom onset in a variety of neurodegenerative diseases. In Huntington disease (HD), a CAG repeat expansion in the gene encoding the huntingtin protein results in a presymptomatic stage that typically spans multiple decades and is followed by striking degeneration of striatal tissue and the progression of debilitating motor symptoms. Many lines of evidence demonstrate that the HD presymptomatic window is associated with injurious effects to striatal synapses, many of which appear to be prerequisites to subsequent cell death. While the striatum is the most vulnerable region in the HD brain, it is widely recognized that HD is a brain-wide disease, affecting numerous extrastriatal regions that contribute to debilitating non-motor symptoms including cognitive dysfunction. Currently, we have a poor understanding of the synaptic integrity, or lack thereof, in extrastriatal regions in the presymptomatic HD brain. If early therapeutic intervention seeks to maintain healthy synaptic function, it is important to understand early HD-associated synaptopathy at a brain-wide, rather than striatal-exclusive, level. Here, we focused on the hippocampus as this structure is generally thought to be affected only in manifest HD despite the subtle cognitive deficits known to emerge in prodromal HD. We used super-resolution microscopy and multi-electrode array electrophysiology as sensitive measures of excitatory synapse structure and function, respectively, in the hippocampus of presymptomatic heterozygous HD mice (Q175FDN model). We found clear evidence for enhanced AMPA receptor subunit clustering and hyperexcitability well before the onset of a detectable HD-like behavioral phenotype. In addition, activity-dependent re-organization of synaptic protein nanostructure, and functional measures of synaptic plasticity were impaired in presymptomatic HD mice. These data demonstrate that synaptic abnormalities in the presymptomatic HD brain are not exclusive to the striatum, and highlight the need to better understand the region-dependent complexities of early synaptopathy in the HD brain.

Asunto(s)

Hipocampo/fisiopatología , Enfermedad de Huntington/fisiopatología , Receptores AMPA/ultraestructura , Sinapsis/patología , Sinapsis/ultraestructura , Animales , Femenino , Hipocampo/patología , Hipocampo/ultraestructura , Enfermedad de Huntington/patología , Masculino , Ratones , Plasticidad Neuronal/fisiología , Síntomas Prodrómicos , Receptores AMPA/metabolismo

RESUMEN

Glutamate transporter proteins, expressed on both neurons and glia, serve as the main gatekeepers that dictate the spatial and temporal actions of extracellular glutamate. Glutamate is essential to the function of the healthy brain yet paradoxically contributes to the toxicity associated with many neurodegenerative diseases. Rapid transporter-mediated glutamate uptake, primarily occurring at astrocytic processes, tightens the efficiency of excitatory network activity and prevents toxic glutamate build-up in the extracellular space. Glutamate transporter dysfunction is thought to underlie myriad central nervous system (CNS) diseases including Alzheimer and Huntington disease. Over the past few decades, techniques such as biochemical uptake assays and electrophysiological recordings of transporter currents from individual astrocytes have revealed the remarkable ability of the CNS to efficiently clear extracellular glutamate. In more recent years, the rapidly evolving glutamate-sensing "sniffers" now allow researchers to visualize real-time glutamate transients on a millisecond time scale with single synapse spatial resolution in defined cell populations. As we transition to an increased reliance on optical-based methods of glutamate visualization and quantification, it is of utmost importance to understand not only the advantages that glutamate biosensors bring to the table but also the associated caveats and their implications for data interpretation. In this review, we summarize the strengths and limitations of the commonly used methods to quantify glutamate uptake. We then discuss what these techniques, when viewed as a complementary whole, have told us about the brain's ability to regulate glutamate levels, in both health and in the context of neurodegenerative disease.

Asunto(s)

Química Encefálica , Ácido Glutámico/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Animales , Humanos

RESUMEN

The pool of ß-Amyloid (Aß) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for Aß peptides. We examined how a naturally occurring variant, e.g. Aß(1-38), interacts with the AD-related variant, Aß(1-42), and the predominant physiological variant, Aß(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that Aß(1-38) interacts differently with Aß(1-40) and Aß(1-42) and, in general, Aß(1-38) interferes with the conversion of Aß(1-42) to a ß-sheet-rich aggregate. Functionally, Aß(1-38) reverses the negative impact of Aß(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an Aß(1-42) phenotype in Caenorhabditis elegans. Aß(1-38) also reverses any loss of MTT conversion induced by Aß(1-40) and Aß(1-42) in HT-22 hippocampal neurons and APOE ε4-positive human fibroblasts, although the combination of Aß(1-38) and Aß(1-42) inhibits MTT conversion in APOE ε4-negative fibroblasts. A greater ratio of soluble Aß(1-42)/Aß(1-38) [and Aß(1-42)/Aß(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that Aß(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant Aß(1-42).

Asunto(s)

Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/efectos adversos , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/farmacología , Fragmentos de Péptidos/efectos adversos , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Edad de Inicio , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Encéfalo/patología , Caenorhabditis elegans , Células Cultivadas , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo

RESUMEN

Glutamate transporters, particularly glutamate transporter 1 (GLT-1), help to prevent the adverse effects associated with glutamate toxicity by rapidly clearing glutamate from the extracellular space. Since GLT-1 expression and/or function are reduced in many neurodegenerative diseases, upregulation of GLT-1 is a favorable approach to treat the symptoms of these diseases. Ceftriaxone, a ß-lactam antibiotic reported to increase GLT-1 expression, can exert neuroprotective effects in a variety of neurodegenerative diseases; however, many of these diseases do not exhibit uniform brain pathology. In contrast, as a drug that readily crosses the blood-brain barrier, ceftriaxone administration is likely to increase GLT-1 levels globally throughout the neuroaxis. In Huntington disease (HD), low GLT-1 expression is observed in the striatum in postmortem tissue and animal models. While ceftriaxone was reported to increase striatal GLT-1 and ameliorate the motor symptoms in a mouse model of HD, the extrastriatal effects of ceftriaxone in HD are unknown. Using electrophysiology and high-speed imaging of the glutamate biosensor iGluSnFR, we quantified real-time glutamate dynamics and synaptic plasticity in the hippocampus of the Q175FDN mouse model of HD, following intraperitoneal injections of either saline or ceftriaxone. We observed an activity-dependent increase in extracellular glutamate accumulation within the HD hippocampus, which was not the result of reduced GLT-1 expression. Surprisingly, ceftriaxone had little effect on glutamate clearance rates and negatively impacted synaptic plasticity. These data provide evidence for glutamate dysregulation in the HD hippocampus but also caution the use of ceftriaxone as a treatment for HD.

Asunto(s)

Ceftriaxona , Enfermedad de Huntington , Animales , Ceftriaxona/farmacología , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico , Hipocampo/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Ratones

RESUMEN

OBJECTIVE: Orexin (ORX) and melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus are critical regulators of energy homeostasis and are thought to differentially contribute to diet-induced obesity. However, it is unclear whether the synaptic properties of these cells are altered by obesogenic diets over time. METHODS: Rats and mice were fed a control chow or palatable high-fat diet (HFD) for various durations and then synaptic properties of ORX and MCH neurons were examined using exvivo whole-cell patch clamp recording. Confocal imaging was performed to assess the number of excitatory synaptic contacts to these neurons. RESULTS: ORX neurons exhibited a transient increase in spontaneous excitatory transmission as early as 1 day up to 1 week of HFD, which returned to control levels with prolonged feeding. Conversely, HFD induced a delayed increase in excitatory synaptic transmission to MCH neurons, which progressively increased as HFD became chronic. This increase occurred before the onset of significant weight gain. These synaptic changes appeared to be due to altered postsynaptic sensitivity or the number of active synaptic contacts depending on cell type and feeding duration. However, HFD induced no change in inhibitory transmission in either cell type at any time point. CONCLUSIONS: These results suggest that the effects of HFD on feeding-related neurons are cell type-specific and dynamic. This highlights the importance of considering the feeding duration for research and weight loss interventions. ORX neurons may contribute to early hyperphagia, whereas MCH neurons may play a role in the onset and long-term maintenance of diet-induced obesity.

Asunto(s)

Dieta Alta en Grasa/efectos adversos , Área Hipotalámica Lateral/metabolismo , Plasticidad Neuronal/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Metabolismo Energético , Homeostasis/fisiología , Área Hipotalámica Lateral/efectos de los fármacos , Hormonas Hipotalámicas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Melaninas/metabolismo , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal/efectos de los fármacos , Neuronas/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Orexinas/metabolismo , Técnicas de Placa-Clamp/métodos , Hormonas Hipofisarias/metabolismo , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología

RESUMEN

The spatiotemporal dynamics of excitatory neurotransmission must be tightly regulated to achieve efficient synaptic communication. By limiting spillover, glutamate transporters are believed to prevent excessive activation of extrasynaptically located receptors that can impair synaptic plasticity. While glutamate transporter expression is reduced in numerous neurodegenerative diseases, the contributions of transporter dysfunction to disease pathophysiology remain ambiguous as the fundamental relationship between glutamate dynamics and plasticity, and the mechanisms linking these two phenomena, remain poorly understood. Here, we combined electrophysiology and real-time high-speed imaging of extracellular glutamate transients during LTP induction and characterized the sensitivity of the relationship between glutamate dynamics during theta burst stimulation (TBS) and the resulting magnitude of LTP consolidation, both in control conditions and following selective and nonselective glutamate transporter blockade. Glutamate clearance times were negatively correlated with LTP magnitude following nonselective glutamate transporter inhibition but not following selective blockade of a majority of GLT-1, the brain's most abundant glutamate transporter. Although glutamate transporter inhibition reduced the postsynaptic population response to TBS, calcium responses to TBS were greatly exaggerated. The source of excess calcium was dependent on NMDARs, L-type VGCCs, GluA2-lacking AMPARs, and internal calcium stores. Surprisingly, inhibition of L-type VGCCs, but not GluA2-lacking AMPARs or ryanodine receptors, was required to restore robust LTP. In all, these data provide a detailed understanding of the relationship between glutamate dynamics and plasticity and uncover important mechanisms by which poor glutamate uptake can negatively impact LTP consolidation.SIGNIFICANCE STATEMENT Specific patterns of neural activity can promote long-term changes in the strength of synaptic connections through a phenomenon known as synaptic plasticity. Synaptic plasticity is well accepted to represent the cellular mechanisms underlying learning and memory, and many forms of plasticity are initiated by the excitatory neurotransmitter glutamate. While essential for rapid cellular communication in the brain, excessive levels of extracellular glutamate can negatively impact brain function. In this study, we demonstrate that pharmacological manipulations that increase the availability of extracellular glutamate during neural activity can have profoundly negative consequences on synaptic plasticity. We identify mechanisms through which excess glutamate can negatively influence synaptic plasticity, and we discuss the relevance of these findings to neurodegenerative diseases and in the aging brain.

Asunto(s)

Ácido Glutámico/fisiología , Potenciación a Largo Plazo/fisiología , Neuronas/fisiología , Transmisión Sináptica/fisiología , Animales , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL

RESUMEN

Huntington disease (HD) is an inherited neurodegenerative disease characterized by a clinical triad of motor, psychiatric and cognitive symptoms. HD is caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Homozygosity for the HD-causing mutation is extremely rare; thus, the majority of HD patients express the mutant huntingtin protein in addition to reduced levels of the non-pathogenic huntingtin protein. Deficits in synaptic plasticity, including hippocampal long-term potentiation (LTP), have been identified in various mouse models of HD and are thought to contribute to the debilitating cognitive symptoms associated with the disease. However, the bulk of these studies used N-terminal fragment or homozygous knock-in mouse models of HD at symptomatic ages, and our understanding of the onset and progression of synaptic plasticity deficits in the HD brain is lacking. To better understand the time-course of synaptic plasticity deficits in HD, as well as the impact of heterozygous and homozygous huntingtin mutations, we quantified basal synaptic connectivity, presynaptic release probability, presynaptically mediated post-tetanic potentiation (PTP) and postsynaptically mediated LTP at presymptomatic, early symptomatic and late symptomatic ages in heterozygous and homozygous Q175FDN knock-in HD mice. Our results demonstrate clear age-dependent effects of the HD-causing mutation on both short and long-term plasticity that generally emerge earlier in homozygous mice. Interestingly, deficits in presynaptic short-term plasticity were more closely linked to disease progression than deficits in postsynaptic LTP, and heterozygous mice were more susceptible to an LTP deficit when induced by high frequency stimulation compared to theta burst stimulation. To the best of our knowledge, the present study represents the most thorough characterization to date of the onset and progression of hippocampal synaptic plasticity deficits in a mouse model of HD, and should prove valuable to future studies exploring cellular mechanisms underlying the debilitating cognitive decline in HD.

RESUMEN

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin (HTT) protein, resulting in acquisition of toxic functions. Previous studies have shown that lowering mutant HTT has the potential to be broadly beneficial. We previously identified HTT single-nucleotide polymorphisms (SNPs) tightly linked to the HD mutation and developed antisense oligonucleotides (ASOs) targeting HD-SNPs that selectively suppress mutant HTT. We tested allele-specific ASOs in a mouse model of HD. Both early and late treatment reduced cognitive and behavioral impairments in mice. To determine the translational potential of the treatment, we examined the effect of ASO administration on HTT brain expression in nonhuman primates. The treatment induced robust HTT suppression throughout the cortex and limbic system, areas implicated in cognition and psychiatric function. The results suggest that ASOs specifically targeting mutated HTT might have therapeutic effects on HD-mediated cognitive impairments.

Asunto(s)

Cognición , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/fisiopatología , Animales , Ansiedad/complicaciones , Ansiedad/patología , Ansiedad/fisiopatología , Atrofia/patología , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Femenino , Humanos , Enfermedad de Huntington/complicaciones , Enfermedad de Huntington/patología , Sistema Límbico/patología , Masculino , Proteínas Mutantes/metabolismo , Oligonucleótidos Antisentido/farmacología , Primates

RESUMEN

Transporter-mediated glutamate uptake plays an essential role in shaping synaptic neurotransmission. The rapid removal of synaptically released glutamate ensures the high temporal dynamics characteristic of fast excitatory chemical neurotransmission and prevents the overexcitation of extrasynaptic NMDA receptors that have been implicated in synaptic plasticity impairments and cell death. Despite clear regional differences in plasticity and excitotoxic thresholds, few studies have compared extracellular glutamate dynamics across different brain regions and in response to a range of neural activity including plasticity-inducing stimuli. Here, we used the rapid extracellular fluorescent glutamate sensor iGluSnFR (intensity-based glutamate-sensing fluorescent reporter) and high-speed imaging (205 frames per second) to quantify relative differences in glutamate clearance rates over a wide range of presynaptic activity in situ in the hippocampus, cortex, and striatum of male C57/BL6NCrl mice. We found that the hippocampus was significantly more efficient than the cortex and striatum at clearing synaptically released glutamate and that this efficiency could be attributed, at least in part, to faster glutamate diffusion away from the release site. In addition, we found that pharmacological inhibition of GLT-1, the brain's most abundant glutamate transporter, slowed clearance rates to only a fraction (∼20-25%) of the effect induced by nonselective transporter blockade, regardless of the brain region and the duration of presynaptic activity. In all, our data reveal clear regional differences in glutamate dynamics after neural activity and suggest that non-GLT-1 transporters can make a large contribution to the rate of glutamate clearance in the hippocampus, cortex, and striatum.SIGNIFICANCE STATEMENT Glutamate is the brain's most abundant neurotransmitter, and although essential for rapid cell-cell communication, too much glutamate can negatively impact cellular health. Extracellular glutamate levels are tightly regulated by membrane-bound transporters that rapidly remove the glutamate that is released during neural activity, thereby shaping both the spatial and temporal dynamics of excitatory neurotransmission. Using high-speed imaging of an optical sensor of extracellular glutamate, we show that glutamate dynamics vary widely from one brain region to the next and are highly dependent on the duration of synaptic activity. Our data demonstrate the heterogeneous nature of glutamate regulation in the brain and suggest that such regional differences can dramatically affect both the localization and duration of postsynaptic receptor activation during synaptic neurotransmission.

Asunto(s)

Sistema de Transporte de Aminoácidos X-AG/metabolismo , Encéfalo/metabolismo , Ácido Glutámico/metabolismo , Transmisión Sináptica/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL

RESUMEN

BACKGROUND: Palmitoylation, the addition of palmitate to proteins by palmitoyl acyltransferases (PATs), is an important regulator of synaptic protein localization and function. Many palmitoylated proteins and PATs have been implicated in neuropsychiatric diseases, including Huntington disease, schizophrenia, amyotrophic lateral sclerosis, Alzheimer disease, and X-linked intellectual disability. HIP14/DHHC17 is the most conserved PAT that palmitoylates many synaptic proteins. Hip14 hypomorphic mice have behavioral and synaptic deficits. However, the phenotype is developmental; thus, a model of post-developmental loss of Hip14 was generated to examine the role of HIP14 in synaptic function in the adult. RESULTS: Ten weeks after Hip14 deletion (iHip14 Δ/Δ ), mice die suddenly from rapidly progressive paralysis. Prior to death the mice exhibit motor deficits, increased escape response during tests of anxiety, anhedonia, a symptom indicative of depressive-like behavior, and striatal synaptic deficits, including reduced probability of transmitter release and increased amplitude but decreased frequency of spontaneous post-synaptic currents. The mice also have increased brain weight due to microgliosis and astrogliosis in the cortex. CONCLUSIONS: Behavioral changes and electrophysiological measures suggest striatal dysfunction in iHip14 Δ/Δ mice, and increased cortical volume due to astrogliosis and microgliosis suggests a novel role for HIP14 in glia. These data suggest that HIP14 is essential for maintenance of life and neuronal integrity in the adult mouse.

Asunto(s)

Aciltransferasas/genética , Muerte Súbita , Eliminación de Gen , Aciltransferasas/metabolismo , Animales , Peso Corporal , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Lipoilación , Masculino , Ratones , Ratones Noqueados , Neuroglía/patología , Tamaño de los Órganos

RESUMEN

Huntington disease (HD) model mice with heterozygous knock-in (KI) of an expanded CAG tract in exon 1 of the mouse huntingtin (Htt) gene homolog genetically recapitulate the mutation that causes HD, and might be favoured for preclinical studies. However, historically these mice have failed to phenotypically recapitulate the human disease. Thus, homozygous KI mice, which lack wildtype Htt, and are much less relevant to human HD, have been used. The zQ175 model was the first KI mouse to exhibit significant HD-like phenotypes when heterozygous. In an effort to exacerbate HD-like phenotypes and enhance preclinical utility, we have backcrossed zQ175 mice to FVB/N, a strain highly susceptible to neurodegeneration. These Q175F mice display significant HD-like phenotypes along with sudden early death from fatal seizures. The zQ175 KI allele retains a floxed neomycin resistance cassette upstream of the Htt gene locus and produces dramatically reduced mutant Htt as compared to the endogenous wildtype Htt allele. By intercrossing with mice expressing cre in germ line cells, we have excised the neo cassette from Q175F mice generating a new line, Q175FΔneo (Q175FDN). Removal of the neo cassette resulted in a ∼2 fold increase in mutant Htt and rescue of fatal seizures, indicating that the early death phenotype of Q175F mice is caused by Htt deficiency rather than by mutant Htt. Additionally, Q175FDN mice exhibit earlier onset and a greater variety and severity of HD-like phenotypes than Q175F mice or any previously reported KI HD mouse model, making them valuable for preclinical studies.

Asunto(s)

Técnicas de Sustitución del Gen/métodos , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Mutación , Animales , Conducta Animal , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Heterocigoto , Humanos , Enfermedad de Huntington/patología , Ratones , Fenotipo

RESUMEN

Corticostriatal cocultures are utilized to recapitulate the cortex-striatum connection in vitro as a convenient model to investigate the development, function, and regulation of synapses formed between cortical and striatal neurons. However, optimization of this dissociated neuronal system to more closely reproduce in vivo circuits has not yet been explored. We studied the effect of varying the plating ratio of cortical to striatal neurons on striatal spiny projection neuron (SPN) characteristics in primary neuronal cocultures. Despite the large difference in cortical-striatal neuron ratio (1:1 vs. 1:3) at day of plating, by 18 days in vitro the difference became modest (∼25% lower cortical-striatal neuron ratio in 1:3 cocultures) and the neuronal density was lower in the 1:3 cocultures, indicating enhanced loss of striatal SPNs. Comparing SPNs in cocultures plated at a 1:1 vs. 1:3 ratio, we found that resting membrane potential, input resistance, current injection-induced action potential firing rates, and input-output curves were similar in the two conditions. However, SPNs in the cocultures plated at the lower cortical ratio exhibited reduced membrane capacitance along with significantly shorter total dendritic length, decreased dendritic complexity, and fewer excitatory synapses, consistent with their trend toward reduced miniature excitatory postsynaptic current frequency. Strikingly, the proportion of NMDA receptors found extrasynaptically in recordings from SPNs was significantly higher in the less cortical coculture. Consistently, SPNs in cocultures with reduced cortical input showed decreased basal pro-survival signaling through cAMP response element binding protein and enhanced sensitivity to NMDA-induced apoptosis. Altogether, our study indicates that abundance of cortical input regulates SPN dendritic arborization and survival/death signaling.

Asunto(s)

Dendritas/efectos de los fármacos , Dendritas/fisiología , Agonistas de Aminoácidos Excitadores/farmacología , N-Metilaspartato/farmacología , Neuronas/citología , Sinapsis/fisiología , Animales , Apoptosis/efectos de los fármacos , Proteína de Unión a CREB/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Técnicas de Cocultivo , Cuerpo Estriado/citología , Homólogo 4 de la Proteína Discs Large , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Embrión de Mamíferos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Guanilato-Quinasas/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/efectos de los fármacos

RESUMEN

It has become well accepted that Huntington disease (HD) is associated with impaired glutamate uptake, resulting in a prolonged time-course of extracellular glutamate that contributes to excitotoxicity. However, the data supporting this view come largely from work in synaptosomes, which may overrepresent nerve-terminal uptake over astrocytic uptake. Here, we quantify real-time glutamate dynamics in HD mouse models by high-speed imaging of an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) and electrophysiological recordings of synaptically activated transporter currents in astrocytes. These techniques reveal a disconnect between the results obtained in synaptosomes and those in situ. Exogenous glutamate uptake is impaired in synaptosomes, whereas real-time measures of glutamate clearance in the HD striatum are normal or even accelerated, particularly in the aggressive R6/2 model. Our results highlight the importance of quantifying glutamate dynamics under endogenous release conditions, and suggest that the widely cited uptake impairment in HD does not contribute to pathogenesis.

Asunto(s)

Astrocitos/metabolismo , Cuerpo Estriado/metabolismo , Ácido Glutámico/metabolismo , Enfermedad de Huntington/metabolismo , Imagen Óptica/métodos , Sinapsis/metabolismo , Animales , Astrocitos/patología , Transporte Biológico , Cuerpo Estriado/patología , Dependovirus/genética , Modelos Animales de Enfermedad , Genes Reporteros , Vectores Genéticos , Humanos , Enfermedad de Huntington/patología , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Transgénicos , Sinaptosomas/metabolismo

RESUMEN

Huntington's disease (HD) is a genetically inherited neurodegenerative disease caused by a mutation in the gene encoding the huntingtin protein. This mutation results in progressive cell death that is particularly striking in the striatum. Recent evidence indicates that early HD is initially a disease of the synapse, in which subtle alterations in synaptic neurotransmission, particularly at the cortico-striatal (C-S) synapse, can be detected well in advance of cell death. Here, we used a cell culture model in which striatal neurons are co-cultured with cortical neurons, and monitored the development of C-S connectivity up to 21days in vitro (DIV) in cells cultured from either the YAC128 mouse model of HD or the background strain, FVB/N (wild-type; WT) mice. Our data demonstrate that while C-S connectivity in WT co-cultures develops rapidly and continuously from DIV 7 to 21, YAC128 C-S connectivity shows no significant growth from DIV 14 onward. Morphological and electrophysiological data suggest that a combination of pre- and postsynaptic mechanisms contribute to this effect, including a reduction in both the postsynaptic dendritic arborization and the size and replenishment rate of the presynaptic readily releasable pool of excitatory vesicles. Moreover, a chimeric culture strategy confirmed that the most robust impairment in C-S connectivity was only observed when mutant huntingtin was expressed both pre- and postsynaptically. In all, our data demonstrate a progressive HD synaptic phenotype in this co-culture system that may be exploited as a platform for identifying promising therapeutic strategies to prevent early HD-associated synaptopathy.

Asunto(s)

Corteza Cerebral/fisiopatología , Cuerpo Estriado/fisiopatología , Enfermedad de Huntington/fisiopatología , Sinapsis/fisiología , Animales , Células Cultivadas , Corteza Cerebral/patología , Técnicas de Cocultivo , Cuerpo Estriado/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dendritas/patología , Dendritas/fisiología , Modelos Animales de Enfermedad , Potenciales Postsinápticos Excitadores/fisiología , Ácido Glutámico/metabolismo , Humanos , Proteína Huntingtina , Enfermedad de Huntington/patología , Ratones Transgénicos , Potenciales Postsinápticos Miniatura/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Inhibición Neural/fisiología , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Técnicas de Placa-Clamp , Sinapsis/patología , Vesículas Sinápticas/patología , Vesículas Sinápticas/fisiología

RESUMEN

Existing models of Huntington's disease posit that deficits in BDNF delivery to the striatum contribute to atrophy and motor impairment. In this issue of Neuron, Plotkin et al. (2014) show that BDNF delivery is normal but downstream signaling via TrkB and p75 is impaired, leading to corticostriatal synaptic dysfunction.

Asunto(s)

Corteza Cerebral/fisiopatología , Cuerpo Estriado/fisiopatología , Modelos Animales de Enfermedad , Enfermedad de Huntington/fisiopatología , Receptor trkB/deficiencia , Transducción de Señal/genética , Animales