Glutamate transporter contribution to retinal ganglion cell vulnerability in a rat model of multiple sclerosis.

Glial glutamate transporters actively participate in neurotransmission and have a fundamental role in determining the ambient glutamate concentration in the extracellular space. Their expression is dynamically regulated in many diseases, including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In EAE, a downregulation has been reported which may render neurons more susceptible to glutamate excitotoxicity. In this study, we have investigated the expression of GLAST (EAAT1) and GLT-1 (EAAT2) in the retina of Brown Norway rats following induction of myelin oligodendrocyte glycoprotein (MOG)-EAE, which results in retinal ganglion cell (RGC) degeneration and dysfunction. In addition, we tested whether AAV-mediated overexpression of GLAST in the retina can protect RGCs from degeneration. To address the impact of glutamate transporter modulation on RGCs, we performed whole-cell recordings and measured tonic NMDA receptor-mediated currents in the absence and presence of a glutamate-uptake blocker. We report that αOFF-RGCs show larger tonic glutamate-induced currents than αON-RGCs, in line with their greater vulnerability under neuroinflammatory conditions. We further show that increased AAV-mediated expression of GLAST in the retina does indeed protect RGCs from degeneration during the inflammatory disease. Collectively, our study highlights the neuroprotective role of glutamate transporters in the EAE retina and provides a characterization of tonic glutamate-currents of αRGCs. The larger effects of increased extracellular glutamate concentration on the αOFF-subtype may underlie its enhanced vulnerability to degeneration.

Activated glia cells cause bioenergetic impairment of neurons that can be rescued by knock-down of the mitochondrial calcium uniporter.

Neuroinflammation is a hallmark of various neurological disorders including autoimmune-, neurodegenerative and neuropsychiatric diseases. In neuroinflammation, activated microglia and astrocytes release soluble mediators such as cytokines, glutamate, and reactive oxygen species that negatively affect neuronal function and viability, and thus contribute to neurodegeneration during disease progression. Therefore, the development of neuroprotective strategies might be important in addition to treating inflammation in these diseases. Mitochondria are promising cellular targets for neuroprotective interventions: They are among the first structures affected in many neuroinflammatory diseases, with mitochondrial impairment ranging from impaired respiratory activity and reduced mitochondrial membrane potential to mitochondrial oxidation and fragmentation. Therefore, we developed a cell culture model that resembles an early state of inflammation-induced neuronal mitochondrial dysfunction preceding neuronal cell death, and can be used to test mito- and neuroprotective strategies. Rat primary cortical neurons were challenged with conditioned medium from mixed primary cultures of rat microglia and astrocytes that had been activated with lipopolysaccharide and ATP. When sublethal amounts of glia-conditioned medium were added to neurons for 24 h, mitochondrial membrane potential and ATP levels were decreased, whereas mitochondrial redox state remained unaffected. Effects on mitochondrial membrane potential and ATP levels were ameliorated by knock-down of the mitochondrial calcium uniporter in neurons. This study suggests that neuronal bioenergetic failure is an early event during neuroinflammation and it identifies the mitochondrial calcium uniporter as a candidate target for neuroprotection in this context.

Disrupted expression of mitochondrial NCLX sensitizes neuroglial networks to excitotoxic stimuli and renders synaptic activity toxic.

The mitochondrial solute carrier family 8 sodium/calcium/lithium exchanger, member B1 (NCLX) is an important mediator of calcium extrusion from mitochondria. In this study, we tested the hypothesis that physiological expression levels of NCLX are essential for maintaining neuronal resilience in the face of excitotoxic challenge. Using an shRNA-mediated approach, we showed that reduced NCLX expression exacerbates neuronal mitochondrial calcium dysregulation, mitochondrial membrane potential (ΔΨm) breakdown, and reactive oxygen species generation during excitotoxic stimulation of primary hippocampal cultures. Moreover, NCLX knockdown-which affected both neurons and glia-resulted not only in enhanced neurodegeneration following an excitotoxic insult but also in neuronal and astrocytic cell death under basal conditions. Our data also revealed that synaptic activity, which promotes neuroprotective signaling, can become lethal upon NCLX depletion; expression of NCLX-targeted shRNA impaired the clearance of mitochondrial calcium following action potential bursts, and was associated both with ΔΨm breakdown and substantial neurodegeneration in hippocampal cultures undergoing synaptic activity. Finally, we showed that NCLX knockdown within the hippocampal cornu ammonis 1 region in vivo causes substantial neurodegeneration and astrodegeneration. In summary, we demonstrated that dysregulated NCLX expression not only sensitizes neuroglial networks to excitotoxic stimuli but also notably renders otherwise neuroprotective synaptic activity toxic. These findings may explain the emergence of neurodegeneration and astrodegeneration in patients with disorders characterized by disrupted NCLX expression or function, and suggest that treatments aimed at enhancing or restoring NCLX function may prevent central nervous system damage in these disease states.

Neuronal Mitochondrial Dysfunction and Bioenergetic Failure in Inflammation-Associated Depression.

Depression is a leading cause of disability and affects more than 4% of the population worldwide. Even though its pathophysiology remains elusive, it is now well accepted that peripheral inflammation might increase the risk of depressive episodes in a subgroup of patients. However, there is still insufficient knowledge about the mechanisms by which inflammation induces alterations in brain function. In neurodegenerative and neuroinflammatory diseases, extensive studies have reported that inflammation negatively impacts mitochondrial health, contributing to excitotoxicity, oxidative stress, energy deficits, and eventually neuronal death. In addition, damaged mitochondria can release a wide range of damage-associated molecular patterns that are potent activators of the inflammatory response, creating a feed-forward cycle between oxidative stress, mitochondrial impairment, inflammation, and neuronal dysfunction. Surprisingly, the possible involvement of this vicious cycle in the pathophysiology of inflammation-associated depression remains understudied. In this mini-review we summarize the research supporting the association between neuroinflammation, mitochondrial dysfunction, and bioenergetic failure in inflammation-associated depression to highlight the relevance of further studies addressing this crosstalk.

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator.

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

A microscopy-based small molecule screen in primary neurons reveals neuroprotective properties of the FDA-approved anti-viral drug Elvitegravir.

Glutamate toxicity is a pathomechanism that contributes to neuronal cell death in a wide range of acute and chronic neurodegenerative and neuroinflammatory diseases. Activation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor and breakdown of the mitochondrial membrane potential are key events during glutamate toxicity. Due to its manifold functions in nervous system physiology, however, the NMDA receptor is not well suited as a drug target. To identify novel compounds that act downstream of toxic NMDA receptor signaling and can protect mitochondria from glutamate toxicity, we developed a cell viability screening assay in primary mouse cortical neurons. In a proof-of-principle screen we tested 146 natural products and 424 FDA-approved drugs for their ability to protect neurons against NMDA-induced cell death. We confirmed several known neuroprotective drugs that include Dutasteride, Enalapril, Finasteride, Haloperidol, and Oxybutynin, and we identified neuroprotective properties of Elvitegravir. Using live imaging of tetramethylrhodamine ethyl ester-labelled primary cortical neurons, we found that Elvitegravir, Dutasteride, and Oxybutynin attenuated the NMDA-induced breakdown of the mitochondrial membrane potential. Patch clamp electrophysiological recordings in NMDA receptor-expressing HEK293 cell lines and primary mouse hippocampal neurons revealed that Elvitegravir does not act at the NMDA receptor and does not affect the function of glutamatergic synapses. In summary, we have developed a cost-effective and easy-to-implement screening assay in primary neurons and identified Elvitegravir as a neuro- and mitoprotective drug that acts downstream of the NMDA receptor.

A microRNA signature of toxic extrasynaptic N-methyl-D-aspartate (NMDA) receptor signaling.

The cellular consequences of N-Methyl-D-Aspartate receptor (NMDAR) stimulation depend on the receptors' subcellular localization. Synaptic NMDARs promote plasticity and survival whereas extrasynaptic NMDARs mediate excitotoxicity and contribute to cell death in neurodegenerative diseases. The mechanisms that couple activation of extrasynaptic NMDARs to cell death remain incompletely understood. We here show that activation of extrasynaptic NMDARs by bath application of NMDA or L-glutamate leads to the upregulation of a group of 19 microRNAs in cultured mouse hippocampal neurons. In contrast, none of these microRNAs is induced upon stimulation of synaptic activity. Increased microRNA expression depends on the pri-miRNA processing enzyme Drosha, but not on de novo gene transcription. These findings suggest that toxic NMDAR signaling involves changes in the expression levels of particular microRNAs.

Functional Consequences of Calcium-Dependent Synapse-to-Nucleus Communication: Focus on Transcription-Dependent Metabolic Plasticity.

In the nervous system, calcium signals play a major role in the conversion of synaptic stimuli into transcriptional responses. Signal-regulated gene transcription is fundamental for a range of long-lasting adaptive brain functions that include learning and memory, structural plasticity of neurites and synapses, acquired neuroprotection, chronic pain, and addiction. In this review, we summarize the diverse mechanisms governing calcium-dependent transcriptional regulation associated with central nervous system plasticity. We focus on recent advances in the field of synapse-to-nucleus communication that include studies of the signal-regulated transcriptome in human neurons, identification of novel regulatory mechanisms such as activity-induced DNA double-strand breaks, and the identification of novel forms of activity- and transcription-dependent adaptations, in particular, metabolic plasticity. We summarize the reciprocal interactions between different kinds of neuroadaptations and highlight the emerging role of activity-regulated epigenetic modifiers in gating the inducibility of signal-regulated genes.

The mitochondrial calcium uniporter is crucial for the generation of fast cortical network rhythms.

The role of the mitochondrial calcium uniporter (MCU) gene (Mcu) in cellular energy homeostasis and generation of electrical brain rhythms is widely unknown. We investigated this issue in mice and rats using Mcu-knockout and -knockdown strategies in vivo and in situ and determined the effects of these genetic manipulations on hippocampal gamma oscillations (30-70 Hz) and sharp wave-ripples. These physiological network states require precise neurotransmission between pyramidal cells and inhibitory interneurons, support spike-timing and synaptic plasticity and are associated with perception, attention and memory. Absence of the MCU resulted in (i) gamma oscillations with decreased power (by >40%) and lower synchrony, including less precise neural action potential generation ('spiking'), (ii) sharp waves with decreased incidence (by about 22%) and decreased fast ripple frequency (by about 3%) and (iii) lack of activity-dependent pyruvate dehydrogenase dephosphorylation. However, compensatory adaptation in gene expression related to mitochondrial function and glucose metabolism was not detected. These data suggest that the neuronal MCU is crucial for the generation of network rhythms, most likely by influences on oxidative phosphorylation and perhaps by controlling cytoplasmic Ca2+ homeostasis. This work contributes to an increased understanding of mitochondrial Ca2+ uptake in cortical information processing underlying cognition and behaviour.

Preclinical stress originates in the rat optic nerve head during development of autoimmune optic neuritis.

Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Although it is the presenting symptom in many cases, the initial events are currently unknown. However, in the earliest stages of autoimmune optic neuritis in rats, pathological changes are already apparent such as microglial activation and disturbances in myelin ultrastructure of the optic nerves. αB-crystallin is a heat-shock protein induced in cells undergoing cellular stress and has been reported to be up-regulated in both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Therefore, we wished to investigate the timing and localization of its expression in autoimmune optic neuritis. Although loss of oligodendrocytes was not observed until the later disease stages accompanying immune cell infiltration and demyelination, an increase in oligodendrocyte αB-crystallin was observed during the preclinical stages. This was most pronounced within the optic nerve head and was associated with areas of IgG deposition. Since treatment of isolated oligodendrocytes with sera from myelin oligodendrocyte glycoprotein (MOG)-immunized animals induced an increase in αB-crystallin expression, as did passive transfer of sera from MOG-immunized animals to unimmunized recipients, we propose that the partially permeable blood-brain barrier of the optic nerve head may present an opportunity for blood-borne components such as anti-MOG antibodies to come into contact with oligodendrocytes as one of the earliest events in disease development.

Synaptic Activity Protects Neurons Against Calcium-Mediated Oxidation and Contraction of Mitochondria During Excitotoxicity.

AIMS: Excitotoxicity triggered by extrasynaptic N-methyl-d-aspartate-type glutamate receptors has been implicated in many neurodegenerative conditions, including Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and stroke. Mitochondrial calcium overload leading to mitochondrial dysfunction represents an early event in excitotoxicity. Neurons are rendered resistant to excitotoxicity by previous periods of synaptic activity that activates a nuclear calcium-driven neuroprotective gene program. This process, termed acquired neuroprotection, involves transcriptional repression of the mitochondrial calcium uniporter leading to a reduction in excitotoxcity-associated mitochondrial calcium load. As mitochondrial calcium and the production of reactive oxygen species may be linked, we monitored excitotoxicity-associated changes in the mitochondrial redox status using the ratiometric glutathione redox potential indicator, glutaredoxin 1 (GRX1)-redox-sensitive green fluorescent protein (roGFP)2, targeted to the mitochondrial matrix. Aim of this study was to investigate if suppression of oxidative stress underlies mitoprotection afforded by synaptic activity. RESULTS: We found that synaptic activity protects primary rat hippocampal neurons against acute excitotoxicity-induced mitochondrial oxidative stress and mitochondrial contraction associated with it. Downregulation of the mitochondrial uniporter by genetic means mimics the protective effect of synaptic activity on mitochondrial redox status. These findings indicate that oxidative stress acts downstream of mitochondrial calcium overload in excitotoxicity. Innovation and Conclusion: We established mito-GRX1-roGFP2 as a reliable and sensitive tool to monitor rapid redox changes in mitochondria during excitotoxicity. Our results highlight the importance of developing means of blocking mitochondrial calcium overload for therapeutic targeting of oxidative stress and mitochondrial dysfunction in neurodegenerative diseases. Antioxid. Redox. Signal. 29, 1109-1124.

Synaptic Activity Drives a Genomic Program That Promotes a Neuronal Warburg Effect.

Synaptic activity drives changes in gene expression to promote long lasting adaptations of neuronal structure and function. One example of such an adaptive response is the buildup of acquired neuroprotection, a synaptic activity- and gene transcription-mediated increase in the resistance of neurons against harmful conditions. A hallmark of acquired neuroprotection is the stabilization of mitochondrial structure and function. We therefore re-examined previously identified sets of synaptic activity-regulated genes to identify genes that are directly linked to mitochondrial function. In mouse and rat primary hippocampal cultures, synaptic activity caused an up-regulation of glycolytic genes and a concomitant down-regulation of genes required for oxidative phosphorylation, mitochondrial biogenesis, and maintenance. Changes in metabolic gene expression were induced by action potential bursting, but not by glutamate bath application activating extrasynaptic NMDA receptors. The specific and coordinate pattern of gene expression changes suggested that synaptic activity promotes a shift of neuronal energy metabolism from oxidative phosphorylation toward aerobic glycolysis, also known as the Warburg effect. The ability of neurons to up-regulate glycolysis has, however, been debated. We therefore used FACS sorting to show that, in mixed neuron glia co-cultures, activity-dependent regulation of metabolic gene expression occurred in neurons. Changes in gene expression were accompanied by changes in the phosphorylation-dependent regulation of the key metabolic enzyme, pyruvate dehydrogenase. Finally, increased synaptic activity caused an increase in the ratio of l-lactate production to oxygen consumption in primary hippocampal cultures. Based on these data we suggest the existence of a synaptic activity-mediated neuronal Warburg effect that may promote mitochondrial homeostasis and neuroprotection.

The calmodulin-binding transcription activator CAMTA1 is required for long-term memory formation in mice.

The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by calcium/calmodulin signaling in the nucleus. Recent studies have drawn attention to a new family of transcriptional regulators, the so-called calmodulin-binding transcription activator (CAMTA) proteins. CAMTAs are expressed at particularly high levels in the mouse and human brain, and we reasoned that, as calmodulin-binding transcription factors, CAMTAs may regulate the formation of long-term memory by coupling synaptic activity and calcium/calmodulin signaling to memory-related transcriptional responses. This hypothesis is supported by genetic studies that reported a correlation between Camta gene polymorphisms or mutations and cognitive capability in humans. Here, we show that acute knockdown of CAMTA1, but not CAMTA2, in the hippocampus of adult mice results in impaired performance in two memory tests, contextual fear conditioning and object-place recognition test. Short-term memory and neuronal morphology were not affected by CAMTA knockdown. Gene expression profiling in the hippocampus of control and CAMTA knockdown mice revealed a number of putative CAMTA1 target genes related to synaptic transmission and neuronal excitability. Patch clamp recordings in organotypic hippocampal slice cultures provided further evidence for CAMTA1-dependent changes in electrophysiological properties. In summary, our study provides experimental evidence that confirms previous human genetic studies and establishes CAMTA1 as a regulator of long-term memory formation.

The nuclear calcium signaling target, activating transcription factor 3 (ATF3), protects against dendrotoxicity and facilitates the recovery of synaptic transmission after an excitotoxic insult.

The focal swellings of dendrites ("dendritic beading") are an early morphological hallmark of neuronal injury and dendrotoxicity. They are associated with a variety of pathological conditions, including brain ischemia, and cause an acute disruption of synaptic transmission and neuronal network function, which contribute to subsequent neuronal death. Here, we show that increased synaptic activity prior to excitotoxic injury protects, in a transcription-dependent manner, against dendritic beading. Expression of activating transcription factor 3 (ATF3), a nuclear calcium-regulated gene and member of the core gene program for acquired neuroprotection, can protect against dendritic beading. Conversely, knockdown of ATF3 exacerbates dendritic beading. Assessment of neuronal network functions using microelectrode array recordings revealed that hippocampal neurons expressing ATF3 were able to regain their ability for functional synaptic transmission and to participate in coherent neuronal network activity within 48 h after exposure to toxic concentrations of NMDA. Thus, in addition to attenuating cell death, synaptic activity and expression of ATF3 render hippocampal neurons more resistant to acute dendrotoxicity and loss of synapses. Dendroprotection can enhance recovery of neuronal network functions after excitotoxic insults.

Synaptopodin regulates denervation-induced homeostatic synaptic plasticity.

Synaptopodin (SP) is a marker and essential component of the spine apparatus (SA), an enigmatic cellular organelle composed of stacked smooth endoplasmic reticulum that has been linked to synaptic plasticity. However, SP/SA-mediated synaptic plasticity remains incompletely understood. To study the role of SP/SA in homeostatic synaptic plasticity we here used denervation-induced synaptic scaling of mouse dentate granule cells as a model system. This form of plasticity is of considerable interest in the context of neurological diseases that are associated with the loss of neurons and subsequent denervation of connected brain regions. In entorhino-hippocampal slice cultures prepared from SP-deficient mice, which lack the SA, a compensatory increase in excitatory synaptic strength was not observed following partial deafferentation. In line with this finding, prolonged blockade of sodium channels with tetrodotoxin induced homeostatic synaptic scaling in wild-type, but not SP-deficient, slice cultures. By crossing SP-deficient mice with a newly generated transgenic mouse strain that expresses GFP-tagged SP under the control of the Thy1.2 promoter, the ability of dentate granule cells to form the SA and to homeostatically strengthen excitatory synapses was rescued. Interestingly, homeostatic synaptic strengthening was accompanied by a compensatory increase in SP cluster size/stability and SA stack number, suggesting that activity-dependent SP/SA remodeling could be part of a negative feedback mechanism that aims at adjusting the strength of excitatory synapses to persisting changes in network activity. Thus, our results disclose an important role for SP/SA in homeostatic synaptic plasticity.

The divergence-convergence model of acquired neuroprotection.

It is commonly known that mental activity helps to maintain a healthy brain. Recent research has unraveled the underlying molecular mechanisms that explain why an active brain lives longer. These mechanisms involve the activation of a comprehensive transcriptional program that is triggered by enhanced synaptic activity and renders neurons resistant to harmful conditions. Functionally, this state of acquired neuroprotection may be achieved mainly via one mechanism, which is the stabilization of mitochondria. In this review we propose a model that describes the signaling network that links synaptic activity to neuroprotection. We suggest that the divergent-convergent architecture of this signaling network ensures both robust and reliable as well as persistent activation of the neuroprotective machinery.

Time-lapse imaging of granule cells in mouse entorhino-hippocampal slice cultures reveals changes in spine stability after entorhinal denervation.

Principal neurons that are partially denervated after brain injury remodel their synaptic connections and show biphasic changes in their dendritic spine density: during an early phase after denervation spine density decreases and during a late phase spine density recovers again. It has been hypothesized that these changes in spine density are caused by a period of increased spine loss followed by a period of increased spine formation. We have tested this hypothesis, which is based on data from fixed tissues, by using time-lapse imaging of denervated dentate granule cells in organotypic entorhino-hippocampal slice cultures of Thy1-GFP mice. Our data show that nondenervated granule cells turn over spines spontaneously while keeping their spine density constant. Denervation influenced this equilibrium and induced biphasic changes in the spine loss rate but not in the rate of spine formation: during the early phase after denervation the spine loss rate was increased and during the late phase after denervation the spine loss rate was decreased compared with nondenervated control cultures. In line with these observations, time-lapse imaging of identified spines formed after the lesion revealed that the stability of these spines was decreased during the early phase and increased during the late phase after the lesion. We conclude that biphasic changes in spine loss rate and spine stability but not in the rate of spine formation play a central role in the reorganization of dentate granule cells after entorhinal denervation in vitro.

Neurotrophin-mediated dendrite-to-nucleus signaling revealed by microfluidic compartmentalization of dendrites.

Signaling from dendritic synapses to the nucleus regulates important aspects of neuronal function, including synaptic plasticity. The neurotrophin brain-derived neurotrophic factor (BDNF) can induce long-lasting strengthening of synapses in vivo and this effect is dependent on transcription. However, the mechanism of signaling to the nucleus is not well understood. Here we describe a microfluidic culture device to investigate dendrite-to-nucleus signaling. Using these microfluidic devices, we demonstrate that BDNF can act directly on dendrites to elicit an anterograde signal that induces transcription of the immediate early genes, Arc and c-Fos. Induction of Arc is dependent on dendrite- and cell body-derived calcium, whereas induction of c-Fos is calcium-independent. In contrast to retrograde neurotrophin-mediated axon-to-nucleus signaling, which is MEK5-dependent, BDNF-mediated anterograde dendrite-to-nucleus signaling is dependent on MEK1/2. Intriguingly, the activity of TrkB, the BDNF receptor, is required in the cell body for the induction of Arc and c-Fos mediated by dendritically applied BDNF. These results are consistent with the involvement of a signaling endosome-like pathway that conveys BDNF signals from the dendrite to the nucleus.

Impairment of in vivo theta-burst long-term potentiation and network excitability in the dentate gyrus of synaptopodin-deficient mice lacking the spine apparatus and the cisternal organelle.

The function of the spine apparatus in dendritic spines and the cisternal organelles in axon initial segments is little understood. The actin-associated protein, synaptopodin, is essential for the formation of these organelles which are absent in synaptopodin -/- mice. Here, we used synaptopodin -/- mice to explore the role of the spine apparatus and the cisternal organelle in synaptic plasticity and local circuit excitability in response to activation of the perforant path input to the dentate gyrus in vivo. We found impaired long-term potentiation following theta-burst stimulation, whereas tetanus-evoked LTP was unaffected. Furthermore, paired-pulse inhibition of the population spike was reduced and granule cell excitability was enhanced in mutants, hence revealing an impairment of local network inhibition. In summary, our data represent the first electrophysiological evidence that the lack of the spine apparatus and the cisternal organelle leads to a defect in long-term synaptic plasticity and alterations in local circuit control of granule cell excitability under adult in vivo conditions.

3D-reconstruction and functional properties of GFP-positive and GFP-negative granule cells in the fascia dentata of the Thy1-GFP mouse.

Granule cells of the mouse fascia dentata are widely used in studies on neuronal development and plasticity. In contrast to the rat, however, high-resolution morphometric data on these cells are scarce. Thus, we have analyzed granule cells in the fascia dentata of the adult Thy1-GFP mouse (C57BL/6 background). In this mouse line, single neurons in the granule cell layer are GFP-labeled, making them amenable to high-resolution 3D-reconstruction. First, calbindin or parvalbumin-immunofluorescence was used to identify GFP-positive cells as granule cells. Second, the dorsal-ventral distribution of GFP-positive granule cells was studied: In the dorsal part of the fascia dentata 11% and in the ventral part 15% of all granule cells were GFP-positive. Third, GFP-positive and GFP-negative granule cells were compared using intracellular dye-filling (fixed slice technique) and patch-clamp recordings; no differences were observed between the cells. Finally, GFP-positive granule cells (dorsal and ventral fascia dentata) were imaged at high resolution with a confocal microscope, 3D-reconstructed in their entirety and analyzed for soma size, total dendritic length, number of segments, total number of spines and spine density. Sholl analysis revealed that dendritic complexity of granule cells is maximal 150-200 mum from the soma. Granule cells located in the ventral part of the hippocampus revealed a greater degree of dendritic complexity compared to cells in the dorsal part. Taken together, this study provides morphometric data on granule cells of mice bred on a C57BL/6 background and establishes the Thy1-GFP mouse as a tool to study granule cell neurobiology.