Density Of Glutamatergic Synapses Research Articles

Elevated insulin growth factor-1 in dentate gyrus induces cognitive deficits in pre-term newborns.

Prematurely born infants are deprived of maternal hormones and cared for in the stressful environment of Neonatal Intensive Care Units (NICUs). They suffer from long-lasting deficits in learning and memory. Here, we show that prematurity and associated neonatal stress disrupt dentate gyrus (DG) development and induce long-term cognitive deficits and that these effects are mediated by insulin growth factor-1 (IGF1). Nonmaternal care of premature rabbits increased the number of granule cells and interneurons and reduced neurogenesis, suggesting accelerated premature maturation of DG. However, the density of glutamatergic synapses, mature dendritic spines, and synaptic transmission were reduced in preterm kits compared with full-term controls, indicating that premature synaptic maturation was abnormal. These findings were consistent with cognitive deficits observed in premature rabbits and appeared to be driven by transcriptomic changes in the granule cells. Preterm kits displayed reduced weight, elevated serum cortisol and growth hormone, and higher IGF1 expression in the liver and DG relative to full-term controls. Importantly, blocking IGF-1 receptor in premature kits restored cognitive deficits, increased the density of glutamatergic puncta, and rescued NR2B and PSD95 levels in the DG. Hence, IGF1 inhibition alleviates prematurity-induced cognitive dysfunction and synaptic changes in the DG through modulation of NR2B and PSD95. The study identifies a novel strategy to potentially rescue DG maldevelopment and cognitive dysfunction in premature infants under stress in NICUs.

P140Cap Controls Female Fertility in Mice Acting via Glutamatergic Afference on Hypothalamic Gonadotropin-Releasing Hormone Neurons.

p140Cap, encoded by the gene SRCIN1 (SRC kinase signaling inhibitor 1), is an adaptor/scaffold protein highly expressed in the mouse brain, participating in several pre- and post-synaptic mechanisms. p140Cap knock-out (KO) female mice show severe hypofertility, delayed puberty onset, altered estrus cycle, reduced ovulation, and defective production of luteinizing hormone and estradiol during proestrus. We investigated the role of p140Cap in the development and maturation of the hypothalamic gonadotropic system. During embryonic development, migration of Gonadotropin-Releasing Hormone (GnRH) neurons from the nasal placode to the forebrain in p140Cap KO mice appeared normal, and young p140Cap KO animals showed a normal number of GnRH-immunoreactive (-ir) neurons. In contrast, adult p140Cap KO mice showed a significant loss of GnRH-ir neurons and a decreased density of GnRH-ir projections in the median eminence, accompanied by reduced levels of GnRH and LH mRNAs in the hypothalamus and pituitary gland, respectively. We examined the number of kisspeptin (KP) neurons in the rostral periventricular region of the third ventricle, the number of KP-ir fibers in the arcuate nucleus, and the number of KP-ir punctae on GnRH neurons but we found no significant changes. Consistently, the responsiveness to exogenous KP in vivo was unchanged, excluding a cell-autonomous defect on the GnRH neurons at the level of KP receptor or its signal transduction. Since glutamatergic signaling in the hypothalamus is critical for both puberty onset and modulation of GnRH secretion, we examined the density of glutamatergic synapses in p140Cap KO mice and observed a significant reduction in the density of VGLUT-ir punctae both in the preoptic area and on GnRH neurons. Our data suggest that the glutamatergic circuitry in the hypothalamus is altered in the absence of p140Cap and is required for female fertility.

Prenatal interleukin 6 elevation increases glutamatergic synapse density and disrupts hippocampal connectivity in offspring

SummaryEarly prenatal inflammatory conditions are thought to be a risk factor for different neurodevelopmental disorders. Maternal interleukin-6 (IL-6) elevation during pregnancy causes abnormal behavior in offspring, but whether these defects result from altered synaptic developmental trajectories remains unclear. Here we showed that transient IL-6 elevation via injection into pregnant mice or developing embryos enhanced glutamatergic synapses and led to overall brain hyperconnectivity in offspring into adulthood. IL-6 activated synaptogenesis gene programs in glutamatergic neurons and required the transcription factor STAT3 and expression of the RGS4 gene. The STAT3-RGS4 pathway was also activated in neonatal brains during poly(I:C)-induced maternal immune activation, which mimics viral infection during pregnancy. These findings indicate that IL-6 elevation at early developmental stages is sufficient to exert a long-lasting effect on glutamatergic synaptogenesis and brain connectivity, providing a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders.

Glutamate transporter 1-expressing glia in the rat substantia nigra-Morphometric analysis and relationships to synapses.

Glial cells have a major role in protecting neurons against various forms of stress. Especially, astrocytes mediate the bulk of glutamate clearance in the brain via specific membrane transporters (GLAST and GLT1), thereby preventing the occurrence of excitotoxic events. Although glutamate-mediated mechanisms are thought to contribute to nigral dopaminergic neuron degeneration in Parkinson's disease, detailed information on the organization of glia in the substantia nigra is still lacking. The present study was performed to provide quantitative information on the organization of astroglia and on the relationships between astrocytes and excitatory synapses in the rat substantia nigra. Using immunolabeling of GLT1 and confocal imaging, we found that the substantia nigra was filled with a dense meshwork of immunoreactive astrocyte processes. Stereological analysis performed on electron microscope images revealed that the density of immunoreactive astrocyte plasma membranes was substantial, close to 1 μm2 /μm3 , in the substantia nigra neuropil, both in the pars compacta and the pars reticulata. Excitatory synapses had on average two thirds of their perimeters free from glia, a disposition that may favor transmitter spillover. The density of glutamatergic synapses, as quantified on confocal images by the simultaneous detection of bassoon and of vesicular glutamate transporter 1 or 2, was very low (0.01 and 0.025 per μm3 in the reticulata and compacta subdivisions, respectively). Thus the ratio of GLT1-expressing glial membrane surface to glutamatergic synapses was very high (40-100 μm2 ), suggesting an efficient regulation of extracellular glutamate concentrations.

The Role of AMPARs in the Maturation and Integration of Caudal Ganglionic Eminence-Derived Interneurons into Developing Hippocampal Microcircuits

In the hippocampal CA1, caudal ganglionic eminence (CGE)-derived interneurons are recruited by activation of glutamatergic synapses comprising GluA2-containing calcium-impermeable AMPARs and exert inhibitory regulation of the local microcircuit. However, the role played by AMPARs in maturation of the developing circuit is unknown. We demonstrate that elimination of the GluA2 subunit (GluA2 KO) of AMPARs in CGE-derived interneurons, reduces spontaneous EPSC frequency coupled to a reduction in dendritic glutamatergic synapse density. Removal of GluA1&2&3 subunits (GluA1-3 KO) in CGE-derived interneurons, almost completely eliminated sEPSCs without further reducing synapse density, but increased dendritic branching. Moreover, in GluA1-3 KOs, the number of interneurons invading the hippocampus increased in the early postnatal period but converged with WT numbers later due to increased apoptosis. However, the CCK-containing subgroup increased in number, whereas the VIP-containing subgroup decreased. Both feedforward and feedback inhibitory input onto pyramidal neurons was decreased in GluA1-3 KO. These combined anatomical, synaptic and circuit alterations, were accompanied with a wide range of behavioural abnormalities in GluA1-3 KO mice compared to GluA2 KO and WT. Thus, AMPAR subunits differentially contribute to numerous aspects of the development and maturation of CGE-derived interneurons and hippocampal circuitry that are essential for normal behaviour.

706. Wnt/β-Catenin Pathway Contributions to Dendritic Spine and Glutamatergic Synapse Formation Responsive to Lithium-Mediated GSK3 Inhibition

Background A major direct target of lithium is the metalloenzyme GSK3, the central kinase in the Wnt/β-catenin pathway broadly involved in neural development. Methods We have investigated neurodevelopmental and behavioral functions of DIXDC1, which regulates Wnt/β-catenin signaling including late in neural development and postnatally. We have studied a Dixdc1 knock-out (KO) mouse line, applying in vitro and in vivo experimental approaches to assess neurodevelopment, neural activity, dendritic spine dynamics, animal behavior, and responsiveness to lithium and a selective GSK3 inhibitor. This has been accompanied by sequencing of the human DIXDC1 locus in several psychiatric disorders including bipolar disorder, and rescue and gain-of-function experiments involving rare human missense single nucleotide variants (SNVs) that alter the protein׳s signaling activity. Results In mice, loss of Dixdc1 leads to dose-sensitive deficits in behavioral assays including models of depression; this is responsive to lithium as well as to a selective GSK3-inhibitor. Forebrain cortical pyramidal neurons from Dixdc1KO mice have reduced dendritic spine and glutamatergic synapse density correctable by lithium or a selective GSK3 inhibitor. Many of the rare SNVs found more frequently in psychiatric patients affect Wnt/β-catenin activity of the encoded protein. They also alter the above neurodevelopmental parameters when expressed in differentiating pyramidal neurons. Conclusions Our data support a role for DIXDC1 in formation and/or stability of dendritic spines and synapses in forebrain pyramidal neurons upstream of complex behaviors including depression. Our data further suggest that this occurs through the protein’s activity in the Wnt/β-catenin signaling pathway within neurons, and that this responds to GSK3 inhibition by lithium.

DIXDC1 contributes to psychiatric susceptibility by regulating dendritic spine and glutamatergic synapse density via GSK3 and Wnt/β-catenin signaling.

Mice lacking DIX domain containing-1 (DIXDC1), an intracellular Wnt/β-catenin signal pathway protein, have abnormal measures of anxiety, depression and social behavior. Pyramidal neurons in these animals’ brains have reduced dendritic spines and glutamatergic synapses. Treatment with lithium or a Glycogen Synthase Kinase-3 (GSK3) inhibitor corrects behavioral and neurodevelopmental phenotypes in these animals. Analysis of DIXDC1 in over 9,000 cases of autism, bipolar disorder and schizophrenia reveals higher rates of rare inherited sequence-disrupting single nucleotide variants (SNVs) in these individuals compared to psychiatrically-unaffected controls. Many of these SNVs alter Wnt/β-catenin signaling activity of the neurally-predominant DIXDC1 isoform; a subset that hyperactivate this pathway cause dominant neurodevelopmental effects. We propose that rare missense SNVs in DIXDC1 contribute to psychiatric pathogenesis by reducing spine and glutamatergic synapse density downstream of GSK3 in the Wnt/β-catenin pathway.

MHCI negatively regulates synapse density during the establishment of cortical connections

Major histocompatibility complex class I (MHCI) molecules modulate activity-dependent refinement and plasticity. We found that MHCI also negatively regulates the density and function of cortical synapses during their initial establishment both in vitro and in vivo. MHCI molecules are expressed on cortical neurons before and during synaptogenesis. In vitro, decreasing surface MHCI (sMHCI) on neurons increased glutamatergic and GABAergic synapse density, whereas overexpression decreased it. In vivo, synapse density was higher throughout development in β2m(-/-) mice. MHCI also negatively regulated the strength of excitatory, but not inhibitory, synapses and controlled the balance of excitation and inhibition onto cortical neurons. sMHCI levels were modulated by activity and were necessary for activity to negatively regulate glutamatergic synapse density. Finally, acute changes in sMHCI and activity altered synapse density exclusively during early postnatal development. These results identify a previously unknown function for immune proteins in the negative regulation of the initial establishment and function of cortical connections.

P1-049: Chronic Memantine administration reduces APP expression, normalizes glutamatergic synapse density, and improves spatial learning in the Ts65Dn mouse model for down syndrome

Trabajo presentado a la International Conference on Alzheimer´s Disease celebrada en Chicago del 26 al 31 de julio de 2008.

The Dual Roles of GABA in Seizures and Epilepsy Generate More Excitement

Anomalous Levels of Cl– Transporters in the Hippocampal Subiculum from Temporal Lobe Epilepsy Patients Make GABA Excitatory. Palma E, Amici M, Sobrero F, Spinelli G, Di Angelantonio S, Ragozzino D, Mascia A, Scoppetta C, Esposito V, Miledi R, Eusebi F. Proc Natl Acad Sci USA 2006;103:8465–8468. Erratum in Proc Natl Acad Sci USA 2006;103:11814. The mRNA levels of NKCC1, an inwardly directed Na+, K+-2Cl– cotransporter that facilitates the accumulation of intracellular Cl–, and of KCC2, an outwardly directed K+-Cl– cotransporter that extrudes Cl–, were studied in surgically resected brain specimens from drug-resistant temporal lobe (TL) epilepsy (TLE) patients. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analyses of the mRNAs extracted from the human TLE-associated brain regions revealed an upregulation of NKCC1 mRNA and a downregulation of KCC2 mRNA in the hippocampal subiculum, compared with the hippocampus proper or the TL neocortex, suggesting an abnormal transcription of Cl– transporters in the TLE subiculum. In parallel experiments, cell membranes isolated from the same TLE-associated brain regions were injected into Xenopus oocytes that rapidly incorporated human GABAA receptors into their surface membrane. The GABA currents elicited in oocytes injected with membranes from the subiculum had a more depolarized reversal potential ( EGABA) compared with the hippocampus proper or the neocortex. The NKCC1 blocker bumetanide or a temperature decrease of 10°C shifted the GABA-current EGABA more negative in oocytes injected with membranes from TLE hippocampal subiculum, matching the EGABA of TL neocortex-injected oocytes. We conclude that the anomalous expression of both Cl– transporters, KCC1 and NKCC2, in TLE hippocampal subiculum probably causes altered Cl– transport in the “epileptic” neurons, as revealed in the microtransplanted Xenopus oocytes, and renders GABA aberrantly “exciting,” a feature that may contribute to the precipitation of epileptic seizures. The authors note that the last sentence of the abstract should read: “We conclude that the anomalous expression of both Cl– transporters, NKCC1 and KCC2, in TLE hippocampal subiculum probably causes altered Cl– transport in the ‘epileptic’ neurons, as revealed in the microtransplanted Xenopus oocytes, and renders GABA aberrantly “exciting,” a feature that may contribute to the precipitation of epileptic seizures. This error does not affect the conclusions of the article. Epileptogenic Actions of GABA and Fast Oscillations in the Developing Hippocampus. Khalilov I, Le Van Quyen M, Gozlan H, Ben-Ari Y. Neuron 2005;48:787–796. GABA excites immature neurons and inhibits adult ones, but whether this contributes to seizures in the developing brain is not known. We now report that in the developing, but not the adult, hippocampus, seizures beget seizures only if GABAergic synapses are functional. In the immature hippocampus, seizures generated with functional GABAergic synapses include fast oscillations that are required to transform a naive network to an epileptic one: blocking GABA receptors prevents the long-lasting sequels of seizures. In contrast, in adult neurons, full blockade of GABA(A) receptors generates epileptogenic high-frequency seizures. Therefore, purely glutamatergic seizures are not epileptogenic in the developing hippocampus. We suggest that the density of glutamatergic synapses is not sufficient for epileptogenesis in immature neurons; excitatory GABAergic synapses are required for that purpose. We suggest that the synergistic actions of GABA and NMDA receptors trigger the cascades involved in epileptogenesis in the developing hippocampus.

Basic developmental rules and their implications for epilepsy in the immature brain

ABSTRACT The construction of the human brain with its 10 15 synapses follows a set of complex developmentally and environmentally regulated steps. A series of sequences have been described that are instrumental, in the sense that a failure of any one of them leads to dramatic, life‐long consequences. Hence the importance of determining the sequential maturation of neurons, synapses and cortical maps. It is also important to determine how network‐driven events become installed, as neuronal activity intervenes in all of these steps and modulates, for better or worse, the outcome. A fundamental consequence of these sequential events is that any disruption will have very different consequences depending on when it occurs, indeed, “when is as important as what”. An obvious aspect of these general features is related to seizures. In fact, the developing brain has both a higher incidence of seizures in human and animal models, and experiences seizures that can produce long‐lasting consequences that are also stage‐dependent. This seminar and the series of slides presented are an introduction to these issues, summing up several studies made notably by INMED researchers during the last two decades ( http://www.inmednet.com ). It concentrates on four basic developmental rules: i) the generation by very immature neurons, of very large currents mediated by the activation of receptors in neurons that bear no synapses. This is due to the release of GABA that diffuses to distal sites and acts as a paracrine factor; ii) the excitatory/inhibitory shift of the actions of GABA during development because of a progressive reduction in the intracellular chloride concentration; iii) the sequential formation of GABAergic synapses and networks before glutamatergic ones, implying that, at an early stage, all the excitatory drive will be GABAergic; iv) the presence, at an early stage, of a unique, primitive pattern in all developing structures, this pattern disappears when most GABAergic synapses have shifted to their adult configuration‐ .Several consequences of these sequences are described including: i) a control of neuronal migration by GABA‐acting drugs, and the possibility that migration disorders are also generated by environmental factors that include the effects of GABA‐acting agents; ii) If GABA excites immature neurons and inhibits adult one, then GABA‐acting agents will also produce different effects on the mother and the embryo; iii) early brain oscillations are generated by the periphery and propagate centrally – notably to the sensory‐motor cortex, suggesting that peripherally‐generated movements may provide an important signal for the formation of cortical maps, in keeping with the importance of embryonic movements; iv) “seizures beget seizures” in the developing brain. This has now been shown in a triple chamber with the two intact hippocampi that we developed, and with which it has been possible to show that only recurrent seizures that include high frequency oscillations can transform the naïve, contralateral hippocampus to an epileptic one that seizes spontaneously. Most interestingly, at an early developmental stage, when GABA excites many neurons and the density of glutamatergic synapses is not sufficiently high, purely glutamatergic seizures cannot lead to long‐term consequences, the additional excitatory drive provided by GABAergic synapses is needed. In other words, at that stage, blocking GABA synapses generates seizures, as in adults, but these do not lead to long‐term consequences. The mechanisms that underlie these differences is due to the need for high frequency oscillations (> 80 Hz or so), and these can only be generated when GABA synapses are operative in the developing brain: GABA receptor antagonists are ictogenic, but not epileptogenic. To facilitate teaching purposes the paper is published together with supplemental data (as a PowerPoint presentation included in the accompanying DVD), thus allowing an overview of important developmental steps and their implications. [ Published with supplemental data on CD ]

Epileptogenic Actions of GABA and Fast Oscillations in the Developing Hippocampus

GABA excites immature neurons and inhibits adult ones, but whether this contributes to seizures in the developing brain is not known. We now report that in the developing, but not the adult, hippocampus, seizures beget seizures only if GABAergic synapses are functional. In the immature hippocampus, seizures generated with functional GABAergic synapses include fast oscillations that are required to transform a naive network to an epileptic one: blocking GABA receptors prevents the long-lasting sequels of seizures. In contrast, in adult neurons, full blockade of GABA(A) receptors generates epileptogenic high-frequency seizures. Therefore, purely glutamatergic seizures are not epileptogenic in the developing hippocampus. We suggest that the density of glutamatergic synapses is not sufficient for epileptogenesis in immature neurons; excitatory GABAergic synapses are required for that purpose. We suggest that the synergistic actions of GABA and NMDA receptors trigger the cascades involved in epileptogenesis in the developing hippocampus.