NMDA Receptors in GABAergic Synapses during Postnatal Development

GABA (gamma-aminobutyric-acid), the main inhibitory neurotransmitter in the adult brain, exerts depolarizing (excitatory) actions during development and this GABAergic depolarization cooperates with NMDARs (N-methyl-D-aspartate receptors) to drive spontaneous synchronous activity (SSA) that is fundamentally important for developing neuronal networks. Although GABAergic depolarization is known to assist in the activation of NMDARs during development, the subcellular localization of NMDARs relative to GABAergic synapses is still unknown. Here, we investigated the subcellular distribution of NMDARs in association with GABAergic synapses at the developmental stage when SSA is most prominent in mice. Using multiple immunofluorescent labeling and confocal laser-scanning microscopy in the developing mouse hippocampus, we found that NMDARs were associated with both glutamatergic and GABAergic synapses at postnatal day 6–7 and we observed a direct colocalization of GABAA- and NMDA-receptor labeling in GABAergic synapses. Electron microscopy of pre-embedding immunogold-immunoperoxidase reactions confirmed that GluN1, GluN2A and GluN2B NMDAR subunits were all expressed in glutamatergic and GABAergic synapses postsynaptically. Finally, quantitative post-embedding immunogold labeling revealed that the density of NMDARs was 3 times higher in glutamatergic than in GABAergic synapses. Since GABAergic synapses were larger, there was little difference in the total number of NMDA receptors in the two types of synapses. In addition, receptor density in synapses was substantially higher than extrasynaptically. These data can provide the neuroanatomical basis of a new interpretation of previous physiological data regarding the GABAAR-NMDAR cooperation during early development. We suggest that during SSA, synaptic GABAAR-mediated depolarization assists NMDAR activation right inside GABAergic synapses and this effective spatial cooperation of receptors and local change of membrane potential will reach developing glutamatergic synapses with a higher probability and efficiency even further away on the dendrites. This additional level of cooperation that operates within the depolarizing GABAergic synapse, may also allow its own modification triggered by Ca2+-influx through the NMDA receptors.

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title: Mechanisms underlying spontaneous patterned activity in developing neural circuits
abstract: Patterned, spontaneous activity occurs in many developing neural circuits, including the retina, the cochlea, the spinal cord, the cerebellum and the hippocampus, where it provides signals that are important for the development of neurons and their connections. Despite there being differences in adult architecture and output across these various circuits, the patterns of spontaneous network activity and the mechanisms that generate it are remarkably similar. The mechanisms can include a depolarizing action of GABA (γ-aminobutyric acid), transient synaptic connections, extrasynaptic transmission, gap junction coupling and the presence of pacemaker-like neurons. Interestingly, spontaneous activity is robust; if one element of a circuit is disrupted another will generate similar activity. This research suggests that developing neural circuits exhibit transient and tunable features that maintain a source of correlated activity during crucial stages of development.

title: Developing networks play a similar melody
abstract: During development, when synapses start to be established, a primitive form of network-driven activity provides most of the synaptic activity. This pattern enables a high degree of synchrony in immature neurons in spite of the small number of functional synapses and could participate in activity-dependent growth and synapse formation. Relying on the giant depolarizing potentials that provide most of the synaptic activity in the developing hippocampus, this article reviews the common properties and generating mechanisms of these patterns, and particularly the role of the early depolarizing action of GABA(A) and glycine receptors and the sequential expression of GABA and glutamate synapses. Patterns similar to giant depolarizing potentials have been observed in a wide range of structures and species suggesting that there is a temporal template throughout evolution that constitutes an essential step in the formation of functional networks.

title: Ca2+ Oscillations Mediated by the Synergistic Excitatory Actions of GABAA and NMDA Receptors in the Neonatal Hippocampus
abstract: We asked whether GABA(A) and NMDA receptors may act in synergy in neonatal hippocampal slices, at a time when GABA exerts a depolarizing action. The GABA(A) receptor agonist isoguvacine reduced the voltage-dependent Mg2+ block of single NMDA channels recorded in cell-attached configuration from P(2-5) CA3 pyramidal neurons and potentiated the Ca2+ influx through NMDA channels. The synaptic response evoked by electrical stimulation of stratum radiatum was mediated by a synergistic interaction between GABA(A) and NMDA receptors. Network-driven Giant Depolarizing Potentials, which are a typical feature of the neonatal hippocampal network, provided coactivation of GABA(A) and NMDA receptors and were associated with spontaneous and synchronous Ca2+ increases in CA3 pyramidal neurons. Thus, at the early stages of development, GABA is a major excitatory transmitter that acts in synergy with NMDA receptors. This provides in neonatal neurons a hebbian stimulation that may be involved in neuronal plasticity and network formation in the developing hippocampus.

title: GABA Regulates Excitatory Synapse Formation in the Neocortex via NMDA Receptor Activation
abstract: The development of a balance between excitatory and inhibitory synapses is a critical process in the generation and maturation of functional circuits. Accumulating evidence suggests that neuronal activity plays an important role in achieving such a balance in the developing cortex, but the mechanism that regulates this process is unknown. During development, GABA, the primary inhibitory neurotransmitter in adults, excites neurons as a result of high expression of the Na+-K+-2Cl− cotransporter (NKCC1). Using NKCC1 RNA interference knockdown in vivo, we show that GABA-induced depolarization is necessary for proper excitatory synapse formation and dendritic development of newborn cortical neurons. Blocking NKCC1 with the diuretic bumetanide during development leads to similar persistent changes in cortical circuitry in the adult. Interestingly, expression of a voltage-independent NMDA receptor rescues the failure of NKCC1 knockdown neurons to develop excitatory AMPA transmission, indicating that GABA depolarization cooperates with NMDA receptor activation to regulate excitatory synapse formation. Our study identifies an essential role for GABA in the synaptic integration of newborn cortical neurons and suggests an activity-dependent mechanism for achieving the balance between excitation and inhibition in the developing cortex.

title: A Parturition-Associated Nonsynaptic Coherent Activity Pattern in the Developing Hippocampus
abstract: Correlated neuronal activity is instrumental in the formation of networks, but its emergence during maturation is poorly understood. We have used multibeam two-photon calcium microscopy combined with targeted electrophysiological recordings in order to determine the development of population coherence from embryonic to postnatal stages in the hippocampus. At embryonic stages (E16-E19), synchronized activity is absent, and neurons are intrinsically active and generate L-type channel-mediated calcium spikes. At birth, small cell assemblies coupled by gap junctions spontaneously generate synchronous nonsynaptic calcium plateaus associated to recurrent burst discharges. The emergence of coherent calcium plateaus at birth is controlled by oxytocin, a maternal hormone initiating labour, and progressively shut down a few days later by the synapse-driven giant depolarizing potentials (GDPs) that synchronize the entire network. Therefore, in the developing hippocampus, delivery is an important signal that triggers the first coherent activity pattern, which is silenced by the emergence of synaptic transmission.

title: Three Patterns of Oscillatory Activity Differentially Synchronize Developing Neocortical Networks In Vivo
abstract: Coordinated patterns of electrical activity are important for the early development of sensory systems. The spatiotemporal dynamics of these early activity patterns and the role of the peripheral sensory input for their generation are essentially unknown. We performed extracellular multielectrode recordings in the somatosensory cortex of postnatal day 0 to 7 rats in vivo and observed three distinct patterns of synchronized oscillatory activity. (1) Spontaneous and periphery-driven spindle bursts of 1–2 s in duration and ∼10 Hz in frequency occurred approximately every 10 s. (2) Spontaneous and sensory-driven gamma oscillations of 150–300 ms duration and 30–40 Hz in frequency occurred every 10–30 s. (3) Long oscillations appeared only every ∼20 min and revealed the largest amplitude (250–750 μV) and longest duration (>40 s). These three distinct patterns of early oscillatory activity differently synchronized the neonatal cortical network. Whereas spindle bursts and gamma oscillations did not propagate and synchronized a local neuronal network of 200–400 μm in diameter, long oscillations propagated with 25–30 μm/s and synchronized 600–800 μm large ensembles. All three activity patterns were triggered by sensory activation. Single electrical stimulation of the whisker pad or tactile whisker activation elicited neocortical spindle bursts and gamma activity. Long oscillations could be only evoked by repetitive sensory stimulation. The neonatal oscillatory patterns in vivo depended on NMDA receptor-mediated synaptic transmission and gap junctional coupling. Whereas spindle bursts and gamma oscillations may represent an early functional columnar-like pattern, long oscillations may serve as a propagating activation signal consolidating these immature neuronal networks.

title: Brief Alteration of NMDA or GABAA Receptor-mediated Neurotransmission Has Long Term Effects on the Developing Cerebral Cortex *S
abstract: Neurotransmitter signaling is essential for physiologic brain development. Sedative and anticonvulsant agents that reduce neuronal excitability via antagonism at N-methyl-d-aspartate receptors (NMDARs) and/or agonism at γ-aminobutyric acid subtype A receptors (GABAARs) are applied frequently in obstetric and pediatric medicine. We demonstrated that a 1-day treatment of infant mice at postnatal day 6 (P6) with the NMDAR antagonist dizocilpine or the GABAAR agonist phenobarbital not only has acute but also long term effects on the cerebral cortex. Changes of the cerebral cortex proteome 1 day (P7), 1 week (P14), and 4 weeks (P35) following treatment at P6 suggest that a suppression of synaptic neurotransmission during brain development dysregulates proteins associated with apoptosis, oxidative stress, inflammation, cell proliferation, and neuronal circuit formation. These effects appear to be age-dependent as most protein changes did not occur in mice subjected to such pharmacological treatment in adulthood. Previously performed histological evaluations of the brains revealed widespread apoptosis and decreased cell proliferation following such a drug treatment in infancy and are thus consistent with brain protein changes reported in this study. Our results point toward several pathways modulated by a reduction of neuronal excitability that might interfere with critical developmental events and thus affirm concerns about the impact of NMDAR- and/or GABAAR-modulating drugs on human brain development.

title: NMDA Receptor-Dependent Pattern Transfer from Afferents to Postsynaptic Cells and Dendritic Differentiation in the Barrel Cortex
abstract: N-Methyl-D-aspartate receptors (NMDARs) are important for synaptic refinement during development. In CxNR1KO mice, cortical excitatory neurons lack NR1, the essential subunit of the NMDAR, and in their primary somatosensory (S1) cortex whisker-specific cellular patterns, "barrels," are absent. Despite this cytoarchitectural defect, thalamocortical axons (TCAs) representing the mystacial vibrissae form topographically organized patterns and undergo critical period plasticity. This region-specific knockout mouse model allows for dissection of the mechanisms underlying patterning of the pre- and postsynaptic neural elements in the S1 cortex. In the absence of functional NMDARs, layer IV cell numbers are unaltered, but these cells fail to segregate into barrels. Furthermore, the dendritic fields of spiny stellate cells do not orient toward TCA terminal patches as in normal mice. Instead, they radiate in all directions covering larger territories, exhibiting profuse branching with increased spine density. Comparison of TCA patches with serotonin transporter (5-HTT) immunohistochemistry or Dil labeling also indicates that in the CxNR1KO cortex TCAs form smaller patches and individual axon terminal branching is not as well developed as in control cortex. Our results suggest that postsynaptic NMDAR activation is critical in communicating periphery-related sensory patterns from TCAs to barrel cells. When postsynaptic NMDAR function is disrupted, layer IV spiny stellate cells remain imperceptive to patterning of their presynaptic inputs and elaborate exuberant dendritic specializations.

title: Neurochemical, behavioral and architectural changes after chronic inactivation of NMDA receptors in mice
abstract: Schizophrenia is a psychotic illness characterized by problems in perception, learning, and memory. Post-mortem clinical data revealed abnormalities in neuronal organization, reduced soma and dendritic tree size. In rodents, reduction of glutamatergic neurotransmission by NMDA receptor antagonists mimics symptoms of schizophrenia. However, the dosage, treatment and species used in previous studies have not been consistent, leading to a lack of correlation between the findings reported in low-dose, long-term treatment models and the results in acute or chronic high dose administration. Thus, the present study investigates whether long-term, low-dose blockade of NMDA receptors with MK-801 in the early postnatal period results in molecular, cellular, morphological and behavioral changes in the mouse, alterations that have been singly described by using different drugs and dosages in either mice or rats. We found that early postnatal administration of 0.1mg/kg MK-801 for 15 days altered protein translation, synapse formation, hippocampus-dependent learning and neuronal development, resembling findings reported in schizophrenia. These results suggest that there are strong parallels between this animal model and schizophrenia, which validates it as an animal model for this condition and lends further strength of the NMDA receptor hypofunction as a useful model for the study of psychosis.

title: Long‐term behavioural, molecular and morphological effects of neonatal NMDA receptor antagonism
abstract: Brief N‐methyl‐d‐aspartate (NMDA) receptor blockade in neonatal rats has been reported to increase neuronal apoptosis. We replicated this finding using MK‐801 (0.5 mg/kg) administered twice on postnatal day 7, and then studied the long‐term consequences. In adulthood, treated rats showed reduced volume and neuronal number within the hippocampus, and altered hippocampal NMDA receptor (NR1 subunit) expression. Synaptophysin mRNA was decreased in the thalamus (laterodorsal nucleus). Adult MK‐801‐treated females had prepulse inhibition deficits and increased locomotor activity. The data show that a transient and limited glutamatergic intervention during development can have chronic behavioural, structural and molecular effects. The effects are reminiscent of alterations reported in schizophrenia and, as such, are consistent with hypotheses advocating a role for NMDA receptor hypofunction, and aberrant apoptosis, in the neurodevelopmental pathogenesis of the disorder.

title: Activation of Early Silent Synapses by Spontaneous Synchronous Network Activity Limits the Range of Neocortical Connections
abstract: During the early development of neocortical networks, many glutamatergic synapses lack AMPA receptors and are physiologically silent. We show in neocortical cultures that spontaneous synchronous network activity is able to convert silent synapses to active synapses by the incorporation of AMPA receptors into synaptic complexes throughout the network within a few minutes. To test the effect of synaptic activation on the connectivity of neuronal populations, we created separated neuronal networks that could innervate each other. We allowed outgrowing axons to invade the neighboring network either before or after the onset of synchronous network activity. In the first case, both subnetworks connected to each other and synchronized their activity, whereas in the second case, axonal connections failed to form and network activity did not synchronize between compartments. We conclude that early spontaneous synchronous network activity triggers a global AMPAfication of immature synapses, which in turn prevents later-arriving axons from forming afferent connections. This activity-dependent process may set the range of corticocortical connections during early network development before experience-dependent mechanisms begin elaborating the mature layout of the neocortical connections and modules.

title: AP5 blocks LTP in developing rat dentate gyrus and unmasks LTD
abstract: The hippocampal dentate gyrus undergoes active neuronogenesis as well as growth and regression of neuronal elements and connections during the early postnatal period. In some brain regions, most notably in the visual system, both activity-dependent synaptic plasticity and NMDA receptor activation are candidate mechanisms by which neuronal architecture may be refined during brain maturation. To investigate whether similar mechanisms might obtain in developing dentate, we studied the effects of tetanic stimulation before and after NMDA receptor blockade in hippocampal slices from rats at 7-33 days. Field potentials were recorded in the suprapyramidal granule cell layer in response to stimulation of the medial perforant path. Robust long-term potentiation (LTP) of population spike amplitude (approximately 200% of baseline) was produced by a single tetanus (100 Hz, 2 s, 200 microseconds) at all ages studied. Application of 10 microM AP5 depressed population spike amplitude only in the younger slices (approximately 81% of baseline at 8-15 days; approximately 86% of baseline at 16-24 days), suggesting that the NMDA receptor-mediated component of normal synaptic transmission is higher in early development and decreases with maturation. AP5 prevented or significantly diminished LTP at all ages, establishing the NMDA dependence of LTP induction in the medial perforant path throughout development. AP5 also unmasked tetanus-induced homosynaptic long-term depression (62-75% of baseline) in the younger slices (8-24 days). Thus, prominent NMDA receptor-mediated activity and the capacity for bidirectional synaptic plasticity are characteristic of immature dentate. These processes may influence dentate morphogenesis by contributing to the growth, regression, and stabilization of neuronal elements.

title: Silent Synapses Speak Up
abstract: *Department of Psychiatry The existence of silent or ineffective synapses in the †Department of Cellular and Molecular spinal cordwas proposed over 20 years ago (Wall, 1977), Pharmacology based on experimentally induced rapid changes in the Department of Physiology ability of afferents to evoke single unit activity. However, University of California a limitation of this approach was that it was impossible San Francisco, California 94143 to distinguish silent synapses from functional ones that simply were not adequate to cause action potential dis-

title: Synergistic action of GABA‐A and NMDA receptors in the induction of long‐term depression in glutamatergic synapses in the newborn rat hippocampus
abstract: We show that activation of GABAA receptors (GABAARs) promotes induction of N‐methyl‐d‐aspartate (NMDA) receptor (NMDAR)‐dependent long‐term depression (LTD) of glutamatergic synapses in the newborn rat hippocampal area CA1 in a developmentally restricted manner. In the newborn rat hippocampus two mechanistically different types of LTD of glutamatergic synapses could be induced under similar experimental conditions. The form of the LTD induced depended on the stimulation protocol and on the age of the animal. Low‐frequency stimulation (1 Hz) with 100 stimuli induced a robust homosynaptic, reversible LTD at postnatal days 2–8 (P2–P8) but not at P14. This LTD was blocked by the NMDAR antagonist AP5 or by the GABAAR antagonist picrotoxin. Use of a low‐chloride solution in the patch pipette resulting in EGABA‐A < –70 mV blocked the NMDAR‐dependent LTD, whereas clamping the cell to –40 mV during induction rescued it. In addition, it was possible to induce LTD at P14 with 100 stimuli if the cells were clamped to –40 mV during induction. Low‐frequency stimulation with 900 stimuli induced a robust homosynaptic, reversible LTD both at P2–P8 and at P14. However, neither AP5 nor picrotoxin affected the LTD induced by 900 pulses at P2–P8. Instead, the 900 stimuli‐induced LTD was blocked by the metabotropic glutamate receptor antagonists when co‐applied with AP5. We suggest that during the first postnatal week postsynaptic depolarization provided by the activation of GABAARs shifts the threshold for the LTD induction, making the synapses more prone to activity‐induced plasticity. From the second postnatal week onwards, when the GABAA responses are already hyperpolarizing, different mechanisms for LTD induction prevail.

title: Roles of glutamate and GABA receptors in setting the developmental timing of spontaneous synchronized activity in the developing mouse cortex
abstract: Spontaneous, synchronized electrical activity (SSA) plays important roles in nervous system development, but it is not clear what causes it to start and stop at the appropriate times. In previous work, we showed that when SSA in neonatal mouse cortex is blocked by TTX in cultured slices during its normal time of occurrence (E17–P3), it fails to stop at P3 as it does in control cultured slices, but instead persists through at least P10. This indicates that SSA is self‐extinguishing. Here we use whole‐cell recordings and [Ca2+]i imaging to compare control and TTX‐treated slices to isolate the factors that normally extinguish SSA on schedule. In TTX‐treated slices, SSA bursts average 4 s in duration, and have two components. The first, lasting about 1 s, is mediated by AMPA receptors; the second, which extends the burst to 4 s and is responsible for most of the action potential generation during the burst, is mediated by NMDA receptors. In later stage (P5–P9) control slices, after SSA has declined to about 4% of its peak frequency, bursts lack this long NMDA component. Blocking this NMDA component in P5–P9 TTX‐treated slices reduces SSA frequency, but not to the low values found in control slices, implying that additional factors help extinguish SSA. GABAA inhibitors restore SSA in control slices, indicating that the emergence of GABAA‐mediated inhibition is another major factor that helps terminate SSA.

title: Selective acquisition of AMPA receptors over postnatal development suggests a molecular basis for silent synapses
abstract: Early in postnatal development, glutamatergic synapses transmit primarily through NMDA receptors. As development progresses, synapses acquire AMPA receptor function. The molecular basis of these physiological observations is not known. Here we examined single excitatory synapses with immunogold electron–microscopic analysis of AMPA and NMDA receptors along with electrophysiological measurements. Early in postnatal development, a significant fraction of excitatory synapses had NMDA receptors and lacked AMPA receptors. As development progressed, synapses acquired AMPA receptors with little change in NMDA receptor number. Thus, synapses with NMDA receptors but no AMPA receptors can account for the electrophysiologically observed 'silent synapse'.

title: GABA regulates synaptic integration of newly generated neurons in the adult brain
abstract: Adult neurogenesis, the birth and integration of new neurons from adult neural stem cells, is a striking form of structural plasticity and highlights the regenerative capacity of the adult mammalian brain. Accumulating evidence suggests that neuronal activity regulates adult neurogenesis and that new neurons contribute to specific brain functions. The mechanism that regulates the integration of newly generated neurons into the pre-existing functional circuitry in the adult brain is unknown. Here we show that newborn granule cells in the dentate gyrus of the adult hippocampus are tonically activated by ambient GABA (γ-aminobutyric acid) before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA, the major inhibitory neurotransmitter in the adult brain, initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic chloride ion content. Conversion of GABA-induced depolarization (excitation) into hyperpolarization (inhibition) in newborn neurons leads to marked defects in their synapse formation and dendritic development in vivo. Our study identifies an essential role for GABA in the synaptic integration of newly generated neurons in the adult brain, and suggests an unexpected mechanism for activity-dependent regulation of adult neurogenesis, in which newborn neurons may sense neuronal network activity through tonic and phasic GABA activation.

title: Early Development of Neuronal Activity in the Primate HippocampusIn Utero
abstract: Morphological studies suggest that the primate hippocampus develops extensively before birth, but little is known about its functional development. Patch-clamp recordings of hippocampal neurons and reconstruction of biocytin-filled pyramidal cells were performed in slices of macaque cynomolgus fetuses delivered by cesarean section. We found that during the second half of gestation, axons and dendrites of pyramidal cells grow intensively by hundreds of micrometers per day to attain a high level of maturity near term. Synaptic currents appear around midgestation and are correlated with the level of morphological differentiation of pyramidal cells: the first synapses are GABAergic, and their emergence correlates with the growth of apical dendrite into stratum radiatum. A later occurrence of glutamatergic synaptic currents correlates with a further differentiation of the axodendritic tree and the appearance of spines. Relying on the number of dendritic spines, we estimated that hundreds of new glutamatergic synapses are established every day on a pyramidal neuron during the last third of gestation. Most of the synaptic activity is synchronized in spontaneous slow (≈0.1 Hz) network oscillations reminiscent of the giant depolarizing potentials in neonatal rodents. Epileptiform discharges can be evoked by the GABA(A) receptor antagonist bicuculline by the last third of gestation, and postsynaptic GABA(B) receptors contribute to the termination of epileptiform discharges. Comparing the results obtained in primates and rodents, we conclude that the template of early hippocampal network development is conserved across the mammalian evolution but that it is shifted toward fetal life in primate.

title: The Establishment of GABAergic and Glutamatergic Synapses on CA1 Pyramidal Neurons is Sequential and Correlates with the Development of the Apical Dendrite
abstract: We have performed a morphofunctional analysis of CA1 pyramidal neurons at birth to examine the sequence of formation of GABAergic and glutamatergic postsynaptic currents (PSCs) and to determine their relation to the dendritic arborization of pyramidal neurons. We report that at birth pyramidal neurons are heterogeneous. Three stages of development can be identified: (1) the majority of the neurons (80%) have small somata, an anlage of apical dendrite, and neither spontaneous nor evoked PSCs; (2) 10% of the neurons have a small apical dendrite restricted to the stratum radiatum and PSCs mediated only by GABAA receptors; and (3) 10% of the neurons have an apical dendrite that reaches the stratum lacunosum moleculare and PSCs mediated both by GABAA and glutamate receptors. These three groups of pyramidal neurons can be differentiated by their capacitance (Cm = 17.9 ± 0.8; 30.2 ± 1.6; 43.2 ± 3.0 pF, respectively). At birth, the synaptic markers synapsin-1 and synaptophysin labeling are present in dendritic layers but not in the stratum pyramidale, suggesting that GABAergic peridendritic synapses are established before perisomatic ones. The present observations demonstrate that GABAergic and glutamatergic synapses are established sequentially with GABAergic synapses being established first most likely on the apical dendrites of the principal neurons. We propose that different sets of conditions are required for the establishment of functional GABA and glutamate synapses, the latter necessitating more developed neurons that have apical dendrites that reach the lacunosum moleculare region.

title: NMDA Receptors in Hippocampal GABAergic Synapses and Their Role in Nitric Oxide Signaling
abstract: GABAergic inhibition plays a central role in the control of pyramidal cell ensemble activities; thus, any signaling mechanism that regulates inhibition is able to fine-tune network patterns. Here, we provide evidence that the retrograde nitric oxide (NO)–cGMP cascade triggered by NMDA receptor (NMDAR) activation plays a role in the control of hippocampal GABAergic transmission in mice. GABAergic synapses express neuronal nitric oxide synthase (nNOS) postsynaptically and NO receptors (NO-sensitive guanylyl cyclase) in the presynaptic terminals. We hypothesized that—similar to glutamatergic synapses—the Ca2+ transients required to activate nNOS were provided by NMDA receptor activation. Indeed, administration of 5 μm NMDA induced a robust nNOS-dependent cGMP production in GABAergic terminals, selectively in the CA1 and CA3c areas. Furthermore, using preembedding, postembedding, and SDS-digested freeze–fracture replica immunogold labeling, we provided quantitative immunocytochemical evidence that NMDAR subunits GluN1, GluN2A, and GluN2B were present in most somatic GABAergic synapses postsynaptically. These data indicate that NMDARs can modulate hippocampal GABAergic inhibition via NO–cGMP signaling in an activity-dependent manner and that this effect is subregion specific in the mouse hippocampus.

title: Developmental changes in distribution of NMDA receptor channel subunit mRNAs.
abstract: In situ hybridization analyses have revealed drastic changes in expression and distribution of five subunit mRNAs of the mouse NMDA receptor channel during brain development. The epsilon 1 subunit mRNA is expressed postnatally and widely in the brain. On the other hand, the epsilon 2 subunit mRNA is found throughout the entire embryonic brain, but its expression becomes restricted to the forebrain at postnatal stages. The epsilon 3 subunit mRNA appears postnatally and predominantly in the cerebellum, whereas the epsilon 4 subunit mRNA is abundantly expressed in the diencephalon and the brainstem at embryonic and neonatal stages. In contrast, the zeta 1 subunit mRNA distributes ubiquitously in the brain throughout development. These findings suggest that changes in the subunit composition of the NMDA receptor channel take place during brain development.

title: NMDA Receptor Heterogeneity During Postnatal Development of the Rat Brain: Differential Expression of the NR2A, NR2B, and NR2C Subunit Proteins
abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit‐specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area‐specific rearrangement of NR2B subunit expression following birth.

title: Distribution of N-methyl-D-aspartate receptors (NMDARs) in the developing rat brain.
abstract: The N-methyl-D-aspartate receptor (NMDAR), which is one of the glutamate receptors, is considered to have a close relationship to synaptic plasticity in the developing brain. In addition, it is known that the excessive stimulation of NMDARs can trigger neuronal apoptosis. In this study, we examined the distribution of NMDAR subunits [anti-NR1, NR2(A-C)] in the developing rat brain immunohistochemically. As a result, NR1, an essential subunit for the formation of a functional NMDAR complex, was mildly expressed in the restricted areas such as the temporal region of the cerebral cortex and the hippocampus in the fetal brain at Embryonal Days 18 and 20. On the other hand, in neonates, NR1 was expressed widely throughout the whole brain. The distributions of NR2A and NR2C showed temporal and spatial similarities to that of NR1, while the expression of NR2B showed differences in the intensity and distribution. A progressive change in subunit expression seen prenatally and postnatally could contribute to variation of NMDARs and synaptic plasticity during the developing period.

title: Organization of NMDA receptors at extrasynaptic locations
abstract: NMDA receptors are found in neurons both at synapses and in extrasynaptic locations. Extrasynaptic locations are poorly characterized. Here we used preembedding immunoperoxidase and postembedding immunogold electron microscopy and fluorescence light microscopy to characterize extrasynaptic NMDA receptor locations in dissociated hippocampal neurons in vitro and in the adult and postnatal hippocampus in vivo. We found that extrasynaptic NMDA receptors on neurons in vivo and in vitro were usually concentrated at points of contact with adjacent processes, which were mainly axons, axon terminals, or glia. Many of these contacts were shown to contain adhesion factors such as cadherin and catenin. We also found associations of extrasynaptic NMDA receptors with the membrane associated guanylate kinase (MAGUKs), postsynaptic density (PSD)-95 and SAP102. Developmental differences were also observed. At postnatal day 2 in vivo, extrasynaptic NMDA receptors could often be found at sites with distinct densities whereas dense material was seen only rarely at sites of extrasynaptic NMDA receptors in the adult hippocampus in vivo. This difference probably indicates that many sites of extrasynaptic NMDA receptors in early postnatal ages represent synapse formation or possibly sites for synapse elimination. At all ages, as suggested in both in vivo and in vitro studies, extrasynaptic NMDA receptors on dendrites or the sides of spines may form complexes with other proteins, in many cases, at stable associations with adjacent cell processes. These associations may facilitate unique functions for extrasynaptic NMDA receptors.

title: Heteromeric NMDA Receptors: Molecular and Functional Distinction of Subtypes
abstract: The N-methyl d-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% ientical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate-and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.

title: Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice
abstract: Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system, and the N-methyl-d-aspartate-selective glutamate receptor (NR) consisting of the NR1 subunit and an NR2 or NR3 subunit plays crucial roles in synaptic transmission, plasticity, and learning and memory. By using a knockout mouse strain, in which the NR1 gene deletion is primarily targeted to the CA1 pyramidal cells of the hippocampus, we investigated the in vivo effect of the loss of the NR1 subunit on the cellular expression and intracellular distribution of the NR2 subunits. The NR1 gene deletion had no apparent effect on the levels of NR2A or NR2B mRNA but led to severe reductions of NR2A and NR2B protein in dendrites of CA1 pyramidal cells. This reduced dendritic distribution of the NR2 subunits accompanied their robust accumulation in perikarya, where they were condensed in the lumen of the endoplasmic reticulum as electron-dense granules. These granules were also observed in CA1 pyramidal cells of the control mice but they were much fewer and contained no detectable levels of the NR2 subunit. The effect of the NR1 knockout on intracellular localization of the NR2 subunits was specific in that no such effect was observed for the GluR1 and PSD-95, two other major postsynaptic proteins. These results suggest that the NR1 subunit plays a crucial role in the release of the NR2 subunit from the endoplasmic reticulum in hippocampal pyramidal cells in vivo, and when the NR1 subunit is unavailable, the NR2 subunits are retained and aggregate into intracisternal granules.

title: Role of giant depolarizing potentials in shaping synaptic currents in the developing hippocampus.
abstract: Early in development, network activity in the hippocampus is characterized by giant depolarizing potentials (GDPs). These potentials consist of recurrent membrane depolarizations with superimposed fast action potentials separated by quiescent intervals. They are generated by the interplay of glutamate and gamma-aminobutyric acid (GABA) that, in the immediate postnatal period, is depolarizing and excitatory. Here, we review some recent data concerning the functional role of GDPs in shaping synaptic currents at low-probability mossy-fiber (MF)-CA3 synapses. A pairing procedure was used to correlate GDPs-associated calcium increase in the postsynaptic cell with stimulation of afferent inputs. The pairing protocol caused the appearance of synaptic responses or persistently enhanced the number of successes in "presynaptically" silent or low-probability synapses, respectively. In double-pulses experiments, this effect was associated with a significant reduction in the paired-pulse ratio and a significant increase in the inverse squared value of the coefficient of variation of response amplitude, suggesting that long-term potentiation (LTP) expression was due to the increased probability of transmitter released. In the absence of pairing, no significant changes in synaptic efficacy could be detected. When the interval between GDPs and MF stimulation was increased, the potentiating effect progressively declined and reached the control level in less than 4 s. Mossy-fiber responses were identified on the basis of their paired-pulse facilitation, short-term frequency facilitation, and sensitivity to the group III metabotropic glutamate receptor (mGluR) agonist, 2-amino-4-phosphonobutyric acid (L-AP4). Using these criteria, we found that MFs release mainly GAB A onto CA3 pyramidal cells or GABAergic interneurons. In line with their GABAergic nature, MF responses were blocked by the GABAA receptor antagonists bicuculline or gabazine and were potentiated by NO-711, a blocker of the GABA transporter GAT-1, and by flurazepam, an allosteric modulator of GABAA receptors. In addition, chemical stimulation of granule cell dendrites with glutamate in the presence of 6,7-dinitroquinoxaline-2,3-dione (DNQX) induced into target neurons barrages of L-AP4-sensitive GABAA-mediated postsynaptic currents, further supporting the GABAergic phenotype of granule cells. As in MF, pairing GDPs with Schaffer collateral stimulation induced a persistent potentiation of spontaneous and evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-mediated responses at poorly developed CA3-CA1 synapses. This effect was mediated by an increase in calcium in the postsynaptic cell via voltage-dependent calcium channels activated by the depolarizing action of GABA during GDPs. We provide evidence also that, at these connections, cyclic AMP-dependent protein kinase A (PKA) is the signaling molecule necessary for enhancing synaptic efficacy, since GDPs-induced potentiation was prevented by the membrane permeable PKA inhibitor (PKI 14-22) applied in the bath or by the membrane impermeable form of PKI (PKI 6-22) applied via the patch pipette. In conclusion, it is suggested that GDPs translate specific patterns of pre- and postsynaptic activity into long-lasting changes in synaptic strength and stabilize synaptic connections, thus contributing to the structural refinement of the hippocampal circuit.

title: Early patterns of electrical activity in the developing cerebral cortex of humans and rodents
abstract: During prenatal and early postnatal development, the cerebral cortex exhibits synchronized oscillatory network activity that is believed to be essential for the generation of neuronal cortical circuits. The nature and functional role of these early activity patterns are of central interest in neuroscience. Much of the research is performed in rodents and in vitro, but how closely do these model systems relate to the human fetal brain? In this review, we compare observations in humans with in vivo and in vitro rodent data, focusing on particular oscillatory activity patterns that share many common features: delta brushes, spindle bursts and spindle-like oscillations. There is considerable evidence that the basic functional properties of immature cortical networks are conserved through mammalian evolution, making the neonatal rodent an excellent model for studying early cortical activity and associated plasticity during the developmental period corresponding to the human fetal stage. This review is part of the INMED/TINS special issue "Nature and nurture in brain development and neurological disorders", based on presentations at the annual INMED/TINS symposium (http://inmednet.com/).

title: The Depolarizing Action of GABA Controls Early Network Activity in the Developing Hippocampus
abstract: Early in postnatal life γ-aminobutyric acid (GABA), the primary inhibitory transmitter in adults, excites targeted neurons by an outwardly directed flux of chloride which results from the unbalance between the cation–chloride cotransporters NKCC1 and KCC2, involved in chloride uptake and extrusion, respectively. This effect contributes to generate synchronized network activity or giant depolarizing potentials (GDPs) in the developing hippocampus. Here, we review some recent data concerning the mechanisms by which GDPs are generated and their functional role in enhancing synaptic efficacy at poorly developed GABAergic and glutamatergic synapses. In adulthood, reshaping neuronal circuits due to changes in chloride homeostasis and to the shift of GABA from hyperpolarizing to depolarizing, has been implicated in several neurological disorders, including epilepsy. Evidence has been recently provided that in chronically nerve growth factor-deprived mice expressing a progressive age-dependent neurodegenerative pathology resembling that observed in patients with Alzheimer’s disease, the reduced expression of mRNA encoding for the Kcc2 gene and the depolarizing action of GABA lead to the reorganization of the neuronal hippocampal network. This may represent a novel mechanism by which GABAergic signaling counterbalances the loss of synaptic activity in neurodegenerative diseases.

title: The cation‐chloride cotransporter NKCC1 promotes sharp waves in the neonatal rat hippocampus
abstract: Earlier studies indicate a crucial role for the interconnected network of intrinsically bursting CA3 pyramidal neurons in the generation of in vivo hippocampal sharp waves (SPWs) and their proposed neonatal in vitro counterparts, the giant depolarizing potentials (GDPs). While mechanisms involving ligand‐ and voltage‐gated channels have received lots of attention in the generation of CA3 network events in the immature hippocampus, the contribution of ion‐transport mechanisms has not been extensively studied. Here, we show that bumetanide, a selective inhibitor of neuronal Cl− uptake mediated by the Na+–K+–2Cl− cotransporter isoform 1 (NKCC1), completely and reversibly blocks SPWs in the neonate (postnatal days 7–9) rat hippocampus in vivo, an action also seen on GDPs in slices (postnatal days 1–8). These findings strengthen the view that GDPs and early SPWs are homologous events. Gramicidin‐perforated patch recordings indicated that NKCC1 accounts for a large (∼10 mV) depolarizing driving force for the GABAA current in the immature CA3 pyramids. Consistent with a reduction in the depolarization mediated by endogenous GABAA‐receptor activation, bumetanide inhibited the spontaneous bursts of individual neonatal CA3 pyramids, but it slightly increased the interneuronal activity as seen in the frequency of spontaneous GABAergic currents. An inhibitory effect of bumetanide was seen on the in vitro population events in the absence of synaptic GABAA receptor‐mediated transmission, provided that a tonic GABAA receptor‐mediated current was present. Our work indicates that NKCC1 expressed in CA3 pyramidal neurons promotes network activity in the developing hippocampus.

title: Targeted disruption of NMDA receptor 1 gene abolishes NMDA response and results in neonatal death
abstract: In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR1 allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NR1-/-). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1-/- mice, demonstrating that there is an interdependence of subunit expression. NR1-/- mice died 8-15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1-/- mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death.

title: Whisker-related neuronal patterns fail to develop in the trigeminal brainstem nuclei of NMDAR1 knockout mice
abstract: Sensory pathways of the brain generally develop from crudely wired networks to precisely organized systems. Several studies have implicated neural activity-dependent mechanisms, including N-methyl-D-aspartate (NMDA) receptors, in this refinement process. We applied the gene targeting to the NMDAR1 gene and created a mutant mouse that lacks functional NMDA receptors. The development of whisker-related patterns in the trigeminal nuclei of the mutant mice and their normal littermates was compared. We show that in the mutant mice pathfinding, initial targeting, and crude topographic projection of trigeminal axons in the brainstem are unaffected, but that whisker-specific patches fail to form. Our results provide a direct demonstration of the involvement of the NMDA receptor in the formation of periphery-related neural patterns in the mammalian brain.

title: Impairment of Suckling Response, Trigeminal Neuronal Pattern Formation, and Hippocampal LTD in NMDA Receptor ε2 Subunit Mutant Mice
abstract: Multiple epsilon subunits are major determinants of the NMDA receptor channel diversity. Based on their functional properties in vitro and distributions, we have proposed that the epsilon 1 and epsilon 2 subunits play a role in synaptic plasticity. To investigate the physiological significance of the NMDA receptor channel diversity, we generated mutant mice defective in the epsilon 2 subunit. These mice showed no suckling response and died shortly after birth but could survive by hand feeding. The mutation hindered the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brainstem trigeminal nucleus. In the hippocampus of the mutant mice, synaptic NMDA responses and longterm depression were abolished. These results suggest that the epsilon 2 subunit plays an essential role in both neuronal pattern formation and synaptic plasticity.

title: Early brain development disruption from NMDA receptor hypofunction: Relevance to schizophrenia
abstract: Disruption to brain development at an early stage can potentially alter chemically coded neural networks and can affect behavior in later life. During early brain development antagonism of glutamate NMDA receptors, which play an important role in neuronal outgrowth and survival, leads to neuronal damage in several brain regions and causes behavioral alterations in rodents that mimic schizophrenia symptoms and endophenotypes. There are several lines of evidence implicating involvement of a dysfunctional glutamate system in schizophrenia. In normal subjects, NMDA receptor antagonists produce behavioral and neurochemical changes that mimic schizophrenia symptoms better than any other psychotomimetic drug. Moreover, these drugs worsen symptoms in schizophrenia patients and can trigger a recrudescence of the acute psychotic state in patients with stable chronic schizophrenia. In addition, genes consistently reported as being altered in schizophrenia play roles in development, neuroplasticity and glutamate/GABAergic neurotransmission. Perinatal NMDA receptor antagonist treatment is a useful model for studying the neurodevelopmental and NMDA receptor hypofunction hypotheses of schizophrenia because neurochemical and behavioral changes, reminiscent of those seen in schizophrenia, are present long after cessation of drug administration, which suggests that a permanent change in brain structure and organization has occurred during brain development.

title: Maturation of a Recurrent Excitatory Neocortical Circuit by Experience-Dependent Unsilencing of Newly Formed Dendritic Spines
abstract: Local recurrent excitatory circuits are ubiquitous in neocortex, yet little is known about their development or architecture. Here we introduce a quantitative technique for efficient single-cell resolution circuit mapping using 2-photon (2P) glutamate uncaging and analyze experience-dependent neonatal development of the layer 4 barrel cortex local excitatory circuit. We show that sensory experience specifically drives a 3-fold increase in connectivity at postnatal day (P) 9, producing a highly recurrent network. A profound dendritic spinogenesis occurs concurrent with the connectivity increase, but this is not experience dependent. However, in experience-deprived cortex, a much greater proportion of spines lack postsynaptic AMPA receptors (AMPARs) and synaptic connectivity via NMDA receptors (NMDARs) is the same as in normally developing cortex. Thus we describe a approach for quantitative circuit mapping and show that sensory experience sculpts an intrinsically developing template network, which is based on NMDAR-only synapses, by driving AMPARs into newly formed silent spines.

title: Silent synapses and the emergence of a postsynaptic mechanism for LTP
abstract: Silent synapses abound in the young brain, representing an early step in the pathway of experience-dependent synaptic development. Discovered amidst the debate over whether long-term potentiation reflects a presynaptic or a postsynaptic modification, silent synapses — which in the hippocampal CA1 subfield are characterized by the presence of NMDA receptors but not AMPA receptors — have stirred some mechanistic controversy of their own. Out of this literature has emerged a model for synapse unsilencing that highlights the central role for postsynaptic AMPA-receptor trafficking in the expression of excitatory synaptic plasticity.

title: NKCC1 transporter facilitates seizures in the developing brain
abstract: During development, activation of Cl−-permeable GABAA receptors (GABAA-R) excites neurons as a result of elevated intracellular Cl− levels and a depolarized Cl− equilibrium potential (ECl). GABA becomes inhibitory as net outward neuronal transport of Cl− develops in a caudal-rostral progression. In line with this caudal-rostral developmental pattern, GABAergic anticonvulsant compounds inhibit motor manifestations of neonatal seizures but not cortical seizure activity. The Na+-K+-2Cl− cotransporter (NKCC1) facilitates the accumulation of Cl− in neurons. The NKCC1 blocker bumetanide shifted ECl negative in immature neurons, suppressed epileptiform activity in hippocampal slices in vitro and attenuated electrographic seizures in neonatal rats in vivo. Bumetanide had no effect in the presence of the GABAA-R antagonist bicuculline, nor in brain slices from NKCC1-knockout mice. NKCC1 expression level versus expression of the Cl−-extruding transporter (KCC2) in human and rat cortex showed that Cl− transport in perinatal human cortex is as immature as in the rat. Our results provide evidence that NKCC1 facilitates seizures in the developing brain and indicate that bumetanide should be useful in the treatment of neonatal seizures.

title: Slow endogenous activity transients and developmental expression of K+–Cl− cotransporter 2 in the immature human cortex
abstract: Spontaneous transients of correlated activity are a characteristic feature of immature brain structures, where they are thought to be crucial for the establishment of precise neuronal connectivity. Studies on experimental animals have shown that this kind of early activity in cortical structures is composed of long‐lasting, intermittent network events, which undergo a developmental decline that is closely paralleled by the maturation of GABAergic inhibition. In order to examine whether similar events occur in the immature human cortex, we performed direct current‐coupled electroencephalography (EEG) recordings from sleeping preterm babies. We show now that much of the preterm EEG activity is confined to spontaneous, slow activity transients. These transients are characterized by a large voltage deflection that nests prominent oscillatory activity in several frequency bands covering the whole frequency spectrum of the preterm EEG (<0.1–30 Hz). The slow voltage deflections had an amplitude of up to 800 µV. Most of these ‘giant’ events originated in the temporo‐occipital areas, with a maximum rate of about 8/min, and their occurrence as well as amplitude showed a decline by the time of normal birth. In age‐matched fetal brain tissue, this decrease in the spontaneous activity transients was associated with a developmental up‐regulation of the neuronal chloride extruder K+–Cl− cotransporter 2, a crucial molecule for the generation of inhibitory GABAergic Cl– currents. Our work indicates that slow endogenous activity transients in the immature human neocortex are mostly confined to the prenatal stage and appear to be terminated in parallel with the maturation of functional GABAergic inhibition.

title: Excitatory GABA Action Is Essential for Morphological Maturation of Cortical Neurons In Vivo
abstract: GABA exerts excitatory actions on embryonic and neonatal cortical neurons, but the in vivo function of this GABA excitation is essentially unknown. Using in utero electroporation, we eliminated the excitatory action of GABA in a subpopulation of rat ventricular progenitors and cortical neurons derived from these progenitors by premature expression of the Cl− transporter KCC2, as confirmed by the changes in the reversal potential of GABA-induced currents and the resting membrane potential after GABAA receptor blockade. We found that radial migration to layer II/III of the somatosensory cortex of neurons derived from the transfected progenitors was not significantly affected, but their morphological maturation was markedly impaired. Furthermore, reducing neuronal excitability of cortical neurons in vivo by overexpressing an inward-rectifying K+ channel, which lowered the resting membrane potential, mimicked the effect of premature KCC2 expression. Thus, membrane depolarization caused by early GABA excitation is critical for morphological maturation of neonatal cortical neurons in vivo.

title: GABAA, NMDA and AMPA receptors: a developmentally regulated `ménage à trois'
abstract: The main ionotropic receptors (GABAA, NMDA and AMPA) display a sequential participation in neuronal excitation in the neonatal hippocampus. GABA, the principal inhibitory transmitter in the adult CNS, acts as an excitatory transmitter in early postnatal stage. Glutamatergic synaptic transmission is first purely NMDA-receptor based and lacks functional AMPA receptors. Therefore, initially glutamatergic synapses are 'silent' at resting membrane potential, NMDA channels being blocked by Mg2+. However, when GABA and glutamatergic synapses are coactivated during the physiological patterns of activity, GABAA receptors can facilitate the activation of NMDA receptors, playing the role conferred to AMPA receptors later on in development. Determining the mechanisms underlying the development of this 'ménage à trois' will shed light not only on the wide range of trophic roles of glutamate and GABA in the developing brain, but also on the significance of the transition from neonatal to adult forms of plasticity.

title: Expression of NMDA receptor NR1, NR2A and NR2B subunit mRNAs during development of the human hippocampal formation
abstract: The N‐methyl‐d‐aspartate receptor plays a critical role in the formation and maintenance of synapses during brain development. In the rodent, changes in subunit expression and assembly of the heteromeric receptor complex accompany these maturational processes. However, little is known about N‐methyl‐d‐aspartate receptor subunit expression during human brain development. We used in situ hybridization to examine the distribution and relative abundance of NR1, NR2A and NR2B subunit messenger ribonucleic acids in the hippocampal formation and adjacent cortex of 34 human subjects at five stages of life (neonate, infant, adolescent, young adult and adult). At all ages, the three messenger ribonucleic acids were expressed in all subfields, predominantly by pyramidal neurons, granule cells and polymorphic hilar cells. However, their abundance varied across ontogeny. Levels of NR1 messenger ribonucleic acid in CA4, CA3 and CA2 subfields were significantly lower in the neonate than all other age groups. In the dentate gyrus, subiculum and parahippocampal gyrus, NR2B messenger ribonucleic acid levels were higher in the neonate than in older age groups. NR2A messenger ribonucleic acid levels remained constant, leading to an age‐related increase in NR2A/2B transcript ratio. We conclude that N‐methyl‐d‐aspartate receptor subunit messenger ribonucleic acids are differentially expressed during postnatal development of the human hippocampus, with a pattern similar but not identical to that seen in the rodent. Changes in subunit composition may thus contribute to maturational differences in human hippocampal N‐methyl‐d‐aspartate receptor function, and to their role in the pathophysiology of schizophrenia and other neurodevelopmental disorders.

title: Synaptic scaffolding molecule (S‐SCAM) membrane‐associated guanylate kinase with inverted organization (MAGI)‐2 is associated with cell adhesion molecules at inhibitory synapses in rat hippocampal neurons
abstract: Synaptic scaffolding molecule (S‐SCAM) is a synaptic protein, which harbors five or six PSD‐95/Discs large/ZO‐1 (PDZ), a guanylate kinase and two WW domains. It interacts with NMDA receptor subunits, neuroligin and β‐catenin, and is involved in the accumulation of neuroligin at excitatory synapses. In this study, we have demonstrated S‐SCAM is localized at inhibitory synapses in rat primary cultured hippocampal neurons. We have identified β‐dystroglycan (β‐DG) as a binding partner for S‐SCAM at inhibitory synapses. WW domains of S‐SCAM bind to three sequences of β‐DG. We have also revealed that S‐SCAM can interact with neuroligin 2, which is known to be exclusively localized at inhibitory synapses. The WW domains and the second PDZ domain of S‐SCAM are involved in the interaction with neuroligin 2. β‐DG, neuroligin 2 and S‐SCAM form a tripartite complex in vitro. Neuroligin 2 is detected in the immunoprecipitates by anti‐β‐DG antibody from rat brain. S‐SCAM, β‐DG and neuroligin 2 are partially co‐localized in rat hippocampal neurons. These data suggest that S‐SCAM is associated with β‐DG and neuroligin 2 at inhibitory synapses, and functions as a linker between the dystrophin glycoprotein complex and the neurexin–neuroligin complex.

title: A Novel Multiple PDZ Domain-containing Molecule Interacting withN-Methyl-d-aspartateReceptors and Neuronal Cell Adhesion Proteins*
abstract: At synaptic junctions, pre- and postsynaptic membranes are connected by cell adhesion and have distinct structures for specialized functions. The presynaptic membranes have a machinery for fast neurotransmitter release, and the postsynaptic membranes have clusters of neurotransmitter receptors. The molecular mechanism of the assembly of synaptic junctions is not yet clear. Pioneering studies identified postsynaptic density (PSD)-95/SAP90 as a prototypic synaptic scaffolding protein to maintain the structure of synaptic junctions. PSD-95/SAP90 belongs to a family of membrane-associated guanylate kinases and binds N-methyl-d-aspartate receptors, potassium channels, and neuroligins through the PDZ domains and GKAP/SAPAP/DAP through the guanylate kinase (GK) domain. We performed here a yeast two-hybrid screening for SAPAP-interacting molecules and identified a novel protein that has an inverse structure of membrane-associated guanylate kinases with an NH2-terminal GK-like domain followed by two WW and five PDZ domains. It binds SAPAP through the GK-like domain and NMDA receptors and neuroligins through the PDZ domains. We named this protein S-SCAM (synaptic scaffolding molecule) because S-SCAM may assemble receptors and cell adhesion proteins at synaptic junctions.

title: A Four PDZ Domain-containing Splice Variant Form of GRIP1 Is Localized in GABAergic and Glutamatergic Synapses in the Brain*
abstract: We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is γ-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with γ-aminobutyric acid, type A, receptor (GABAAR) clusters than with α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABAAR clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses.

title: Gephyrin interacts with the glutamate receptor interacting protein 1 isoforms at GABAergic synapses
abstract: We have previously shown that the glutamate receptor interacting protein 1 (GRIP1) splice forms GRIP1a/b and GRIP1c4–7 are present at the GABAergic post‐synaptic complex. Nevertheless, the role that these GRIP1 protein isoforms play at the GABAergic post‐synaptic complex is not known. We are now showing that GRIP1c4–7 and GRIP1a/b interact with gephyrin, the main post‐synaptic scaffold protein of GABAergic and glycinergic synapses. Gephyrin coprecipitates with GRIP1c4–7 or GRIP1a/b from rat brain extracts and from extracts of human embryonic kidney 293 cells that have been cotransfected with gephyrin and one of the GRIP1 protein isoforms. Moreover, purified gephyrin binds to purified GRIP1c4–7 or GRIP1a/b, indicating that gephyrin directly interacts with the common region of these GRIP1 proteins, which includes PDZ domains 4–7. An engineered deletion construct of GRIP1a/b (GRIP1a4–7), which both contains the aforementioned common region and binds to gephyrin, targets to the post‐synaptic GABAergic complex of transfected cultured hippocampal neurons. In these hippocampal cultures, endogenous gephyrin colocalizes with endogenous GRIP1c4–7 and GRIP1a/b in over 90% of the GABAergic synapses. Double‐labeling electron microscopy immunogold reveals that in the rat brain GRIP1c4–7 and GRIP1a/b colocalize with gephyrin at the post‐synaptic complex of individual synapses. These results indicate that GRIP1c4–7 and GRIP1a/b colocalize and interact with gephyrin at the GABAergic post‐synaptic complex and suggest that this interaction plays a role in GABAergic synaptic function.

title: Association of GRIP1 with a GABA(A) receptor associated protein suggests a role for GRIP1 at inhibitory synapses.
abstract: GABA(A) receptors mediate the majority of fast synaptic inhibition in the mammalian central nervous system. GABA(A) receptors associate with a number of cytosolic proteins important for regulating their function including the GABA(A) receptor gamma2 subunit associated protein GABARAP. Here we show GABARAP associates with the synaptic PDZ domain containing protein GRIP1. GRIP1 has been localized to inhibitory synapses however the role of this protein with respect to neuronal inhibition remains unclear. Using in vitro protein interaction assays we show that GABARAP interacts directly with PDZ domains 4-6 of GRIP1. Furthermore, using coimmunoprecipitation assays we show that GABARAP interacts with GRIP1 in vivo. Finally, we show that GRIP1 colocalizes with gamma2 subunit containing GABA(A) receptors in cultured hippocampal neurons. Our findings provide evidence that GRIP1 can associate with proteins important for regulating GABA(A) receptor function and suggest that GRIP1 may play a role at inhibitory synapses.

title: Evidence that GRIP, a PDZ-domain protein which is expressed in the embryonic forebrain, co-activates transcription with DLX homeodomain proteins.
abstract: The DLX homeodomain proteins control development of the basal ganglia and branchial arches. To identify co-factors that regulate DLX function we utilized the yeast two-hybrid assay, and found a DLX interacting protein (DIP2) which binds to the N-terminal region of DLX2 via a PDZ domain. DIP2 appears to be an alternatively spliced form of GRIP1, a protein known to bind AMPA glutamate receptors via PDZ domains. Thus, we named it GRIP1b. We provide evidence that GRIP1b can function as a transcriptional co-activator of DLX2 and DLX5. Glutamate receptors inhibit this co-activation. These results suggest that some PDZ proteins may regulate transcription via their interactions with homeodomain proteins. Furthermore, these results suggest a link between glutamate receptors, PDZ proteins and the DLX transcription factors, all of which are co-expressed in the developing basal ganglia.

title: Epidermolysis bullosa and embryonic lethality in mice lacking the multi-PDZ domain protein GRIP1
abstract: Glutamate receptor-interacting protein 1 (GRIP1) is an adaptor protein composed of seven PDZ (postsynaptic density-95/Discs large/zona occludens-1) domains, capable of mediating diverse protein–protein interactions. GRIP1 has been implicated in the regulation of neuronal synaptic function, but its physiologic roles have not been defined in vivo. We find that elimination of murine GRIP1 results in embryonic lethality. GRIP1−/− embryos develop abnormalities of the dermo-epidermal junction, resulting in extensive skin blistering around day 12 of embryonic life. Ultra-structural characterization of the blisters (or bullae) revealed cleavage of the dermo-epidermal junction below the lamina densa, an alteration reminiscent of the dystrophic form of human epidermolysis bullosa. Blisters were also observed in the lateral ventricle of the brain and in the meninges covering the cerebral cortex. These genetic data suggest that the GRIP1 scaffolding protein is required for the formation and integrity of the dermo-epidermal junction and reveal the importance of PDZ domains in the organization of supramolecular structures essential for mammalian embryonic development.

title: Glutamate co-release at GABA/glycinergic synapses is crucial for the refinement of an inhibitory map
abstract: Many nonglutamatergic synaptic terminals in the mammalian brain contain the vesicular glutamate transporter 3 (VGLUT3), indicating that they co-release the excitatory neurotransmitter glutamate. However, the functional role of glutamate co-transmission at these synapses is poorly understood. In the auditory system, VGLUT3 expression and glutamate co-transmission are prominent in a developing GABA/glycinergic sound-localization pathway. We found that mice with a genetic deletion of Vglut3 (also known as Slc17a8) had disrupted glutamate co-transmission and severe impairment in the refinement of this inhibitory pathway. Specifically, loss of glutamate co-transmission disrupted synaptic silencing and the strengthening of GABA/glycinergic connections that normally occur with maturation. Functional mapping studies further revealed that these deficits markedly degraded the precision of tonotopy in this inhibitory auditory pathway. These results indicate that glutamate co-transmission is crucial for the synaptic reorganization and topographic specification of a developing inhibitory circuit.

title: Developmentally regulated expression of VGLUT3 during early post-natal life
abstract: Three subtypes of vesicular glutamate transporters, named VGLUT1-3, accumulate glutamate into synaptic vesicles. In this study, the post-natal expression of VGLUT3 was determined with specific probes and antiserums in the rat brain and compared with that of VGLUT1 and VGLUT2. The expression of VGLUT1 and VGLUT2 increases linearly during post-natal development. In contrast, VGLUT3 developmental pattern appears to have a more or less biphasic profile. A first peak of expression is centered around post-natal day 10 (P10) while the second one is reached in the adult brain. Between P1 and P15, VGLUT3 is observed in the frontal brain (striatum, accumbens, and hippocampus) and in the caudal brain (colliculi, pons and cerebellum). During a second phase extending from P15 to adulthood, the labeling of the caudal brain fades away. The adult pattern is reached at P21. We further analyzed the transient expression of VGLUT3 in the cerebellum and found it to correspond to a temporary expression in Purkinje cells. At P10 VGLUT3 immunoreactivity was present both in the soma and terminals of Purkinje cells (PC), where it colocalized with the vesicular inhibitory amino acid transporter (VIAAT). In agreement with data from the literature [Gillespie, D.C., Kim, G., Kandler, K., 2005. Inhibitory synapses in the developing auditory system are glutamatergic. ], our results suggest that during the first 2 weeks of post-natal life PC may have the potential to transiently release simultaneously GABA and glutamate.

title: GABAergic synapses in hippocampus exocytose aspartate on to NMDA receptors: quantitative immunogold evidence for co-transmission
abstract: We previously found evidence for the exocytosis of aspartate from excitatory nerve terminals in hippocampus [ (1998) 6059]. Here we show, by immunogold electron microscopy in hippocampal slices that aspartate is co-localized and co-exocytosed with GABA from synaptic vesicles in nerve endings assumed to be inhibitory on dentate granule cells and CA1 pyramidal cells. By immunogold double labeling cytochemistry in perfusion fixed hippocampus, we further find that GABA-positive terminals forming symmetric synaptic specializations on perikarya of granule and pyramidal cells express NMDA receptors. In addition, NMDA receptors are present at synapses on granule cell bodies that have asymmetric synaptic specializations and contain high levels of GABA. Glutamate levels are low in the described types of GABA-positive nerve terminals, but high in terminals making asymmetric synapses on dendritic spines, whereas aspartate is localized with high levels in all of these types of terminal. We propose that aspartate is exocytotically released not only from glutamatergic terminals, but also from GABAergic terminals to act on NMDA receptors and may have a role in the regulation of synaptic transmission. Under pathological conditions, release of an excitatory transmitter at an inhibitory synapse could contribute to the development of, for example, epilepsy.

title: GABA release from mouse axonal growth cones
abstract: 1 Using developing hypothalamic neurons from transgenic mice that express high levels of green fluorescent protein in growing axons, and an outside‐out patch from mature neuronal membranes that contain neurotransmitter receptors as a sensitive detector, we found that GABA is released by a vesicular mechanism from the growth cones of developing axons prior to synapse formation. 2 A low level of GABA release occurs spontaneously from the growth cone, and this is substantially increased by evoked action potentials. 3 Neurotransmitters such as acetylcholine can enhance protein kinase C (PKC) activity even prior to synapse formation; PKC activation caused a substantial increase in spontaneous GABA release from the growth cone, probably acting at the axon terminal. 4 These data indicate that GABA is secreted from axons during a stage of neuronal development when GABA is excitatory, and that neuromodulators could alter GABA release from the growing axon, potentially enabling other developing neurons of different transmitter phenotype to modulate the early actions of GABA.

title: Spontaneous Acetylcholine Secretion from Developing Growth Cones of Drosophila Central Neurons in Culture: Effects of cAMP-Pathway Mutations
abstract: We describe a novel bioassay system that usesXenopus embryonic myocytes (myoballs) to detect the release of acetylcholine from Drosophila CNS neurons. When a voltage-clamped Xenopus myoball was manipulated into contact with cultured Drosophila “giant” neurons, spontaneous synaptic current-like events were registered. These events were observed within seconds after contact and were blocked by curare and α-bungarotoxin, but not by TTX and Cd2+, suggesting that they are caused by the spontaneous quantal release of acetylcholine (ACh). The secretion occurred not only at the growth cone, but also along the neurite and at the soma, with significantly different release parameters among various regions. The amplitude of these currents displayed a skewed distribution. These features are distinct from synaptic transmission at more mature synapses or autapses formed in this culture system and are reminiscent of the transmitter release process during early development in other preparations. The usefulness of this coculture system in studying presynaptic secretion mechanisms is illustrated by a series of studies on the cAMP pathway mutations, dunce(dnc) and PKA-RI, which disrupt a cAMP-specific phosphodiesterase and the regulatory subunit of cAMP-dependent protein kinase A, respectively. We found that these mutations affected the ACh current kinetics, but not the quantal ACh packet, and that the release frequency was greatly enhanced by repetitive neuronal activity in dnc, but not wild-type, growth cones. These results suggest that the cAMP pathway plays an important role in the activity-dependent regulation of transmitter release not only in mature synapses as previously shown, but also in developing nerve terminals before synaptogenesis.

title: Neurotransmitter release from growth cones of rat dorsal root ganglion neurons in culture
abstract: Growing neurites of rat dorsal root ganglion neurons in culture formed growth cones at the tips. Possible release of glutamate from these growth cones was investigated by using a whole-cell patch-clamp recording from an acutely dissociated hippocampal neuron containing glutamate receptors. The hippocampal neuron was placed in contact to various regions of the dorsal root ganglion neurons. Inward currents were recorded from the hippocampal neuron positioned on the growth cones of the dorsal root ganglion neurons (diameter, 12-16 microm) in response to the dorsal root ganglion cell body stimulation. The inward currents were associated with an increase in membrane conductance, and the reversal potential was estimated at -6.5 mV (n=8). The inward currents were blocked by 6-cyano-7-nitroquinoxaline (10 microM), but not blocked by 2-amino-5-phosphonovaleric acid (50 microM) and bicuculline (10 microM). The inward currents were abolished by tetrodotoxin (1 microM), EGTA-buffered Ca2+-free external solution or omega-agatoxin IVA (300 nM), and were inhibited by omega-conotoxin GVIA (3 microM), but were not affected by nicardipine (10 microM). Intracellular calcium ion concentration ([Ca2+]i) in growth cones of the dorsal root ganglion neurons increased in response to dorsal root ganglion cell body stimulation, whereas the elevation of [Ca2+]i was not observed either in the presence of tetrodotoxin (1 microM) or in a Ca2+-free external solution. These results indicate that the inward currents were evoked by glutamate released from the growth cones via a Ca2+-dependent process, and suggest that the growth cones are already endowed with much of the machinery for neurotransmitter release, even before making a structure for synaptic transmission.

title: Functional characterization of calcium channels localized on the growth cones of cultured rat dorsal root ganglion cells
abstract: Ca2+ channels on growth cones of cultured rat dorsal root ganglion (DRG) neurons were functionally characterized with an optical method using Fura-2. An increase in intracellular calcium concentration ([Ca2+](i)) of the growth cone was induced in response to electrical stimulation to the DRG cell body. The ([Ca2+](i))-increase was partly inhibited by either of omega - conotoxin GVIA (omega - CgTx, 3 microM) or omega - agatoxin IVA (omega - aga IVA, 300 nM) and completely blocked by both present at the same time, but was not affected by nicardipine (30 microM). The omega - CgTx - as well as omega - aga IVA - sensitive Ca2+ channels were immunologically localized on the growth cones using field emission scanning electron microscopy. It is concluded that the omega - CgTx - as well as omea - aga IVA - sensitive Ca2+ channels are involved in the ([Ca2+](i))-increase in the growth cones of the cultured DRG neurons, leading to glutamate release before synapse formation.

title: Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate
abstract: Astrocytes establish rapid cell-to-cell communication through the release of chemical transmitters. The underlying mechanisms and functional significance of this release are, however, not well understood. Here we identify an astrocytic vesicular compartment that is competent for glutamate exocytosis. Using postembedding immunogold labeling of the rat hippocampus, we show that vesicular glutamate transporters (VGLUT1/2) and the vesicular SNARE protein, cellubrevin, are both expressed in small vesicular organelles that resemble synaptic vesicles of glutamatergic terminals. Astrocytic vesicles, which are not as densely packed as their neuronal counterparts, can be observed in small groups at sites adjacent to neuronal structures bearing glutamate receptors. Fluorescently tagged VGLUT-containing vesicles were studied dynamically in living astrocytes by total internal reflection fluorescence (TIRF) microscopy. After activation of metabotropic glutamate receptors, astrocytic vesicles underwent rapid (milliseconds) Ca2+- and SNARE-dependent exocytic fusion that was accompanied by glutamate release. These data document the existence of a Ca2+-dependent quantal glutamate release activity in glia that was previously considered to be specific to synapses.

title: Morphological evidence for vesicular glutamate release from astrocytes
abstract: There is now growing evidence that astrocytes, like neurons, can release transmitters. One transmitter that in a vast number of studies has been shown to be released from astrocytes is glutamate. Although asytrocytic glutamate may be released by several mechanisms, the evidence in favor of exocytosis is most compelling. Astrocytes may respond to neuronal activity by such exocytotic release of glutamate. The astrocyte derived glutamate can in turn activate neuronal glutamate receptors, in particular N-methyl-D-aspartate (NMDA) receptors. Here we review the morphological data supporting that astrocytes possess the machinery for exocytosis of glutamate. We describe the presence of small synaptic-like microvesicles, SNARE proteins and vesicular glutamate transporters in astrocytes, as well as NMDA receptors situated in vicinity of the astrocytic vesicles.

title: Immunogold detection of L-glutamate and D-serine in small synaptic-like microvesicles in adult hippocampal astrocytes.
abstract: Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

title: Glutamate exocytosis from astrocytes controls synaptic strength
abstract: The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity–dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.

title: Astrocyte-mediated potentiation of inhibitory synaptic transmission
abstract: We investigated the role of astrocytes in activity-dependent modulation of inhibitory synaptic transmission in hippocampal slices. Repetitive firing of an interneuron decreased the probability of synaptic failures in spike-evoked inhibitory postsynaptic currents (unitary IPSCs) in CA1 pyramidal neurons. The GABAB-receptor antagonist CGP55845A abolished this effect. Direct stimulation of astrocytes, or application of the GABAB-receptor agonist baclofen, potentiated miniature inhibitory postsynaptic currents (mIPSCs) in pyramidal neurons. These effects were blocked by inhibition of astrocytic calcium signaling with the calcium chelator BAPTA or by antagonists of the ionotropic glutamate receptors. These observations suggest that interneuronal firing elicits a GABAB-receptor-mediated elevation of calcium in surrounding astrocytes, which in turn potentiates inhibitory transmission. Astrocytes may therefore be a necessary intermediary in activity-dependent modulation of inhibitory synapses in the hippocampus.

title: Neuronal Activity Regulates Correlated Network Properties of Spontaneous Calcium Transients in Astrocytes In Situ
abstract: Spontaneous neuronal activity is essential to neural development. Until recently, neurons were believed to be the only excitable cells to display spontaneous activity. However, cultured astrocytes and, more recently, astrocytes in situ are now known to exhibit spontaneous Ca2+ transients. Here we used Ca2+ imaging of astrocytes from transgenic mice for the simultaneous monitoring of [Ca2+]ichanges in large numbers of astrocytes. We found that spontaneous activity is a common property of most brain astrocytes that is lost in response to a lesion. These spontaneous [Ca2+]i oscillations require extracellular and intracellular Ca2+. Moreover, network analysis revealed that most astrocytes formed correlated networks of dozens of these cells, which were synchronous with both astrocytes and neurons. We found that decreasing spontaneous [Ca2+]i transients in neurons by TTX does not alter the number of active astrocytes, although it impairs their synchronous network activity. Conversely, bicuculline-induced epileptic patterns of [Ca2+]itransients in neurons cause an increase in the number of active astrocytes and in their network synchrony. Furthermore, activation of non-NMDA and NMDA ionotropic glutamate receptors is required to correlate astrocytic networks. These results show that spontaneous activity in astrocytes and neurons is patterned into correlated neuronal/astrocytic networks in which neuronal activity regulates the network properties of astrocytes. This network activity may be essential for neural development and synaptic plasticity.

title: The Relative Roles of Diffusion and Uptake in Clearing Synaptically Released Glutamate Change during Early Postnatal Development
abstract: Glutamate uptake by transporters expressed in astrocytes combines with synaptic structure to regulate the time that synaptically released glutamate remains in the extracellular space and, consequently, the duration and location of postsynaptic receptor activation. Both factors change greatly in the rodent hippocampus during the second postnatal week when most synapses become established and begin to mature, processes that are influenced by synaptically released glutamate. Transporter expression increases, potentially speeding removal of synaptically released glutamate, whereas extracellular space decreases, thereby slowing dilution. We investigated whether these competing changes influence the glutamate concentration time course and postsynaptic responses in the CA1 region of the mouse hippocampus during this critical period of synaptic development. Our results suggest that the glutamate concentration time course remains relatively consistent over this period, although the primary mechanisms regulating glutamate clearance change. Before the second postnatal week, clearance of synaptically released glutamate depends primarily on diffusion into large extracellular spaces, whereas later in development it relies more on increased uptake capacity. Thus, increased transporter expression during this period accompanies structural changes in the neuropil, preserving a relatively consistent glutamate concentration time course and ensuring that postsynaptic receptor activation remains brief and primarily localized to receptors close to release sites.

title: Long-term potentiation in the dentate gyrus is not linked to increased extracellular glutamate concentration.
abstract: Long-term potentiation (LTP) of excitatory transmission is a likely candidate for the encoding and storage of information in the mammalian brain. There is a general agreement that LTP involves an increase in synaptic strength, but the mechanisms underlying this persistent change are unclear and controversial. Synaptic efficacy may be enhanced because more transmitter glutamate is released or because postsynaptic responsiveness increases or both. The purpose of this study was to examine whether increased extracellular glutamate concentration was associated with the robust and well-characterized LTP that can be induced in the rat dentate gyrus. To favor the detection of any putative change in extracellular glutamate associated with LTP, our experimental strategy included the following features. 1) Two separate series of experiments were carried out with animals under pentobarbital or urethan anesthesia; 2) changes in extracellular concentration of glutamate were monitored continuously by microdialysis coupled to enzyme amperometry; and 3) dialysate glutamate levels and changes in the slope of excitatory postsynaptic potential evoked by activation of the perforant path were recorded precisely at the same site. Tetanic stimulation of the perforant path increased persistently test-evoked responses in the dentate gyrus (by 19 and 14% in barbiturate and urethan group, respectively), but there was no glutamate change either during or after LTP induction and no indication of increased glutamate efflux when low-frequency stimulation was applied. The results do not rule out a possible contribution of enhanced glutamate exocytosis to LTP induction and/or maintenance because such a presynaptic change may not be detectable extracellularly. However, our findings and other data supporting the notion that neurotransmitter glutamate may hardly leak out of the synaptic cleft conflict with the hypothesis that LTP could also involve a broad synaptic spillover of glutamate.

title: Effects of Cocaine-Kindling on the Expression of NMDA Receptors and Glutamate Levels in Mouse Brain
abstract: In the present study we examined the effects of cocaine seizure kindling on the expression of NMDA receptors and levels of extracellular glutamate in mouse brain. Quantitative autoradiography did not reveal any changes in binding of [3H] MK-801 to NMDA receptors in several brain regions. Likewise, in situ hybridization and Western blotting revealed no alteration in expression of the NMDA receptor subunits, NR1 and NR2B. Basal overflow of glutamate in the ventral hippocampus determined by microdialysis in freely moving animals also did not differ between cocaine-kindled and control groups. Perfusion with the selective excitatory amino acid transporter inhibitor, pyrrolidine-2,4-dicarboxylic acid (tPDC, 0.6 mM), increased glutamate overflow confirming transport inhibition. Importantly, KCl-evoked glutamate overflow under tPDC perfusion was significantly higher in cocaine-kindled mice than in control mice. These data suggest that enhancement of depolarization stimulated glutamate release may be one of the mechanisms underlying the development of increased seizure susceptibility after cocaine kindling.

title: Reduction of Endogenous Kynurenic Acid Formation Enhances Extracellular Glutamate, Hippocampal Plasticity, and Cognitive Behavior
abstract: At endogenous brain concentrations, the astrocyte-derived metabolite kynurenic acid (KYNA) antagonizes the α7 nicotinic acetylcholine receptor and, possibly, the glycine co-agonist site of the NMDA receptor. The functions of these two receptors, which are intimately involved in synaptic plasticity and cognitive processes, may, therefore, be enhanced by reductions in brain KYNA levels. This concept was tested in mice with a targeted deletion of kynurenine aminotransferase II (KAT II), a major biosynthetic enzyme of brain KYNA. At 21 days of age, KAT II knock-out mice had reduced hippocampal KYNA levels (−71%) and showed significantly increased performance in three cognitive paradigms that rely in part on the integrity of hippocampal function, namely object exploration and recognition, passive avoidance, and spatial discrimination. Moreover, compared with wild-type controls, hippocampal slices from KAT II-deficient mice showed a significant increase in the amplitude of long-term potentiation in vitro. These functional changes were accompanied by reduced extracellular KYNA (−66%) and increased extracellular glutamate (+51%) concentrations, measured by hippocampal microdialysis in vivo. Taken together, a picture emerges in which a reduction in the astrocytic formation of KYNA increases glutamatergic tone in the hippocampus and enhances cognitive abilities and synaptic plasticity. Our studies raise the prospect that interventions aimed specifically at reducing KYNA formation in the brain may constitute a promising molecular strategy for cognitive improvement in health and disease.

title: The glutamate receptor ion channels.
abstract: The ionotropic glutamate receptors are ligand-gated ion channels that mediate the vast majority of excitatory neurotransmission in the brain. The cloning of cDNAs encoding glutamate receptor subunits, which occurred mainly between 1989 and 1992 ([Hollmann and Heinemann, 1994][1]), stimulated this

title: Hippocampal GABAergic Synapses Possess the Molecular Machinery for Retrograde Nitric Oxide Signaling
abstract: Nitric oxide (NO) plays an important role in synaptic plasticity as a retrograde messenger at glutamatergic synapses. Here we describe that, in hippocampal pyramidal cells, neuronal nitric oxide synthase (nNOS) is also associated with the postsynaptic active zones of GABAergic symmetrical synapses terminating on their somata, dendrites, and axon initial segments in both mice and rats. The NO receptor nitric oxide-sensitive guanylyl cyclase (NOsGC) is present in the brain in two functional subunit compositions: α1β1 and α2β1. The β1 subunit is expressed in both pyramidal cells and interneurons in the hippocampus. Using immunohistochemistry and in situ hybridization methods, we describe that the α1 subunit is detectable only in interneurons, which are always positive for β1 subunit as well; however, pyramidal cells are labeled only for β1 and α2 subunits. With double-immunofluorescent staining, we also found that most cholecystokinin- and parvalbumin-positive and smaller proportion of the somatostatin- and nNOS-positive interneurons are α1 subunit positive. We also found that the α1 subunit is present in parvalbumin- and cholecystokinin-positive interneuron terminals that establish synapses on somata, dendrites, or axon initial segments. Our results demonstrate that NOsGC, composed of α1β1 subunits, is selectively expressed in different types of interneurons and is present in their presynaptic GABAergic terminals, in which it may serve as a receptor for NO produced postsynaptically by nNOS in the very same synapse.

title: Nitric Oxide Signaling Modulates Synaptic Transmission during Early Postnatal Development
abstract: Early γ-aminobutyric acid mediated (GABAergic) synaptic transmission and correlated neuronal activity are fundamental to network formation; however, their regulation during early postnatal development is poorly understood. Nitric oxide (NO) is an important retrograde messenger at glutamatergic synapses, and it was recently shown to play an important role also at GABAergic synapses in the adult brain. The subcellular localization and network effect of this signaling pathway during early development are so far unexplored, but its disruption at this early age is known to lead to profound morphological and functional alterations. Here, we provide functional evidence—using whole-cell recording—that NO signaling modulates not only glutamatergic but also GABAergic synaptic transmission in the mouse hippocampus during the early postnatal period. We identified the precise subcellular localization of key elements of the underlying molecular cascade using immunohistochemistry at the light—and electron microscopic levels. As predicted by these morpho-functional data, multineuron calcium imaging in acute slices revealed that this NO-signaling machinery is involved also in the control of synchronous network activity patterns. We suggest that the retrograde NO-signaling system is ideally suited to fulfill a general presynaptic regulatory role and may effectively fine-tune network activity during early postnatal development, while GABAergic transmission is still depolarizing.

title: Immunohistochemical localization of GAD67‐expressing neurons and processes in the rat brainstem: Subregional distribution in the nucleus tractus solitarius
abstract: The role of γ‐aminobutyric acid (GABA) in homeostatic control in the brainstem, in particular, in the nucleus tractus solitarius (NTS), is well established. However, to date, there is no detailed description of the distribution of GABAergic neurons within the NTS. The goal of the current study was to reexamine the efficacy of immunohistochemical localization of glutamic acid decarboxylase (GAD) protein, specifically the 67‐kDa isoform (GAD67), as a marker for GABAergic neurons in the medulla and to provide a detailed map of GAD67‐immunoreactive (‐ir) cells within rat NTS by using a recently developed mouse monoclonal antibody. We describe a distribution of GAD67‐ir cells in the medulla similar to that reported previously from in situ hybridization study. GAD67‐ir cells were localized in regions known to contain high GABA content, including the ventrolateral medulla, raphe nuclei, and area postrema, but were absent from all motor nuclei, although dense terminal labeling was discerned in these regions. In the NTS, GAD67‐ir was localized in all subregions. Semiquantitative analysis of the GAD67‐ir distribution in the NTS revealed greater numbers of GAD67‐ir cells medial to the solitary tract. Finally, dense GAD67 terminal labeling was found in the medial, central, intermediate, commissural, and subpostremal subregions, whereas sparse labeling was observed in the ventral subregion. Our findings support the use of immunohistochemistry for GAD67 as a marker for the localization of GABAergic cells and terminal processes in the rat brainstem. Furthermore, the reported heterogeneous distribution of GAD67‐ir in the NTS suggests differential inhibitory modulation of sensory processing.  2005.

title: Decrease of glutamate decarboxylase (GAD)-immunoreactive nerve terminals in the substantia nigra after kainic acid lesion of the striatum.
abstract: Antiserum sheep 3 against rat brain glutamate decarboxylase (GAD) was employed for the immunohistochemical localization of GAD-immunoreactive nerve terminals in the substantia nigra (SN). To test whether the antiserum specifically localized GAD-containing axon terminals, the effect of kainic acid-induced striatal lesions on the reactive nerve endings in the SN was investigated. Seven days after the injection of 1 microgram kainic acid into the striatum, a 65% decrease in GAD-enzyme activity occurred in the ipsilateral SN. On immunohistochemical examination there was correspondingly a marked reduction of GAD-positive terminals. The parallel decrease in biochemical and immunohistochemical GAD activity indicates that antiserum sheep 3 can be used as a specific immunohistochemical probed for GAD-containing elements.

title: Immunocytochemical localization of glutamate decar☐ylase in rat cerebellum with a new antiserum
abstract: An antiserum prepared in sheep against rat brain glutamic acid decar☐ylase, the biosynthetic enzyme for γ-aminobutyric acid, has been employed to localize, at the light and electronmicroscopic levels, neurons containing glutamate decar☐ylase in the cerebellum of adult rats using the unlabeled antibody enzyme method of S ternberger (1979). With high dilutions of the primary antiserum (1:4000 and higher) glutamate decar☐ylase-immunoreactivity was localized in synaptic terminals of basket, stellate and Golgi cells and Purkinje cell axon recurrent collaterals in the cerebellar cortex, as well as in axosomatic and axodendritic synaptic terminals in the deep cerebellar nuclei, all of which contain pleomorphic synaptic vesicles. The reaction product was present in the axoplasm and at the cytoplasmic aspect of the plasmamembrane, the membrane of synaptic vesicles and axoplasmic reticulum, the outer mitochondrial membrane and also the microtubules. Lower dilutions of antiserum (1:1500) revealed light, specific immunoreactivity in somata of Purkinje, basket, stellate and Golgi-cells of the cerebellar cortex. We obtained specimens in which the vast majority of the members of these cells were stained. Staining of stellate cells was the least intense of all positive neurons in the cerebellar cortex. Very light labeling of some small cell bodies of the deep cerebellar nuclei was also observed. Granule cells, parallel fibers, mossy fibers and climbing fibers in the cerebellar cortex, the large neurons of the cerebellar nuclei, and glial cells were negative. When the antiserum was absorbed against acetone-dried rat liver powder, the same staining pattern was obtained, however, dilutions of 1:500 were necessary to obtain equally strong staining of neuronal cell bodies. These data indicate the specificity of the new anti-serum to rat brain glutamate decar☐ylase. They confirm and complement earlier immunocytochemical work with an antiserum to mouse brain glutamate decar☐ylase. The new antiserum not only shows that nearly all inhibitory axons in the cerebellum are glutamate decar☐ylase positive but also detects immunoreactivity in neuronal cell bodies in the cerebellar cortex without administration of blockers of axoplasmic transport.

title: Regulatory properties of brain glutamate decarboxylase (GAD): the apoenzyme of GAD is present principally as the smaller of two molecular forms of GAD in brain
abstract: The apoenzyme of glutamate decarboxylase [enzyme without bound cofactor, pyridoxal 5′-phosphate (pyridoxal-P)] serves as a reservoir of inactive glutamate decarboxylase (GAD) that can be activated when additional GABA synthesis is required. We have investigated which of two molecular forms of GAD is present as apoenzyme in synaptosomes and in cortex, caudate nucleus, hippocampus, and cerebellum of rat brain. Endogenous glutamate apodecarboxylase (apoGAD) was labeled by incubating extracts of synaptosomes or punches of each region with 32P- pyridoxal-P, followed by reduction with NaBH4, to link covalently the 32P-pyridoxal-P to GAD. Proteins were separated by SDS-PAGE. Punches from all four brain regions and forebrain synaptosomes contained two forms of GAD with apparent Mrs of 63 and 65 kDa as identified by immunoblotting with four antiGAD sera. Punches and synaptosomes contained a major 32P-pyridoxal-P-labeled band with an apparent Mr of 63 kDa that was stained on immunoblots by the antiGAD serum 1440 and the monoclonal antibody GAD-6, and a minor labeled band at 65 kDa that was stained by the 1440, 6799, and K2 antisera. Synaptosomes contained remarkably few other strongly labeled proteins, but punches contained several other labeled bands. Three additional lines of evidence indicate that the labeled 63-kDa protein is apoGAD: (1) it was purified by immunoaffinity chromatography with the GAD-1 monoclonal antibody; (2) it yielded one major labeled peptide when digested with chymotrypsin, and that peptide appeared identical in peptide-mapping experiments to the labeled active-site peptide isolated from chromatographically prepared rat brain GAD; and (3) its labeling was selectively blocked by 4-deoxypyridoxine 5′-phosphate, a competitive inhibitor of the binding of pyridoxal-P to GAD.

title: Light microscopic identification and immunocytochemical characterization of glutamatergic synapses in brain sections
abstract: Presynaptic proteins are readily identified by light microscopic immunocytochemistry, but immunodetection of postsynaptic proteins in brain sections proves difficult. We performed immunofluorescent double labeling for the NR1 subunit of the N‐methyl‐D‐aspartate receptor (NMDAR) and the vesicular glutamate transporter 1 (VGLUT1). In material fixed with 4% paraformaldehyde, NMDAR staining in somatosensory cortex was restricted to the section surface, whereas presynaptic staining extended deeper into the tissue. Staining for postsynaptic proteins was enhanced in weakly fixed material and in tissue treated with pepsin, as previously reported, but tissue quality was impaired. Staining was also markedly enhanced, and without impairment of tissue quality, by treatment during perfusion with a mixture of inhibitors of proteases and the ubiquitin/proteosome system. We performed quantitative analysis of confocal images to study how immunostaining varies with depth into the tissue. Virtually all puncta immunopositive for VGLUT1 colocalized with synaptophysin puncta; these presynaptic puncta were most numerous 1–2 μm beneath the section surface. In contrast, puncta immunopositive for the NR1 subunit were most numerous at the surface, as were puncta immunopositive for the NR2 subunit, SynGAP, and CaMKII. Punctate staining for all postsynaptic proteins, but not presynaptic markers, was substantially enhanced in material pretreated with antiproteolytic agents. The large majority of NR1‐positive puncta at the surface associated with VGLUT1 in this material are likely to represent synaptic contacts. Approximately eighty‐five percent of VGLUT1‐positive puncta in layers II–III of SI are associated with NR1‐positive puncta, and ∼80% are associated with NR2, SynGAP, and CaMKII. This approach may permit systematic analysis of the chemistry of glutamatergic synapses with light microscopic immunocytochemistry.  2005.

title: The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn
abstract: Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid‐lumbar spinal cord of the rat. The great majority (94–100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2‐immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin‐ and enkephalin‐containing boutons (including somatostatin‐immunoreactive boutons that lacked calcitonin gene‐related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present.

title: Bassoon, a Novel Zinc-finger CAG/Glutamine-repeat Protein Selectively Localized at the Active Zone of Presynaptic Nerve Terminals
abstract: The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 M r, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.

title: Presynaptic cytomatrix protein Bassoon is localized at both excitatory and inhibitory synapses of rat brain
abstract: Bassoon is a 420‐kDa protein specifically localized at the active zone of presynaptic nerve terminals. It is thought to be involved in the structural organization of the neurotransmitter release site. We studied the distribution of Bassoon transcripts and protein in rat brain and assessed which types of presynaptic terminals contain the protein. As shown by in situ hybridization, Bassoon transcripts are widely distributed in the brain and occur primarily in excitatory neurons. In addition, examples of γ‐aminobutyric acid (GABA)‐ergic neurons expressing Bassoon are detected. At the light microscopic level, Bassoon immunoreactivity is found in synaptic neuropil regions throughout the brain, with the strongest expression in the hippocampus, the cerebellar cortex, and the olfactory bulb. Immunoelectron microscopy showed that Bassoon immunoreactivity is found in both asymmetric type 1 and symmetric type 2 synapses. Immunopositive asymmetric synapses include mossy fiber boutons and various spine and shaft synapses in the hippocampus and mossy fiber terminals and parallel fiber terminals in the cerebellum. Bassoon‐containing symmetric synapses are observed, e.g., between basket and granule cells in the hippocampus, between Golgi cells and granule cells, and between basket cells and Purkinje cells in the cerebellum. Within synaptic terminals, Bassoon appears highly concentrated at sites opposite to postsynaptic densities. In cultured hippocampal neurons, Bassoon was found to colocalize with GABAA and glutamate (GluR1) receptors. These data indicate that Bassoon is a component of the presynaptic apparatus of both excitatory glutamatergic and inhibitory GABAergic synapses.  1999.

title: The Presynaptic Active Zone Protein Bassoon Is Essential for Photoreceptor Ribbon Synapse Formation in the Retina
abstract: The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.

title: NMDA Receptor GluN2B (GluRε2/NR2B) Subunit Is Crucial for Channel Function, Postsynaptic Macromolecular Organization, and Actin Cytoskeleton at Hippocampal CA3 Synapses
abstract: GluN2B (GluRε2/NR2B) subunit is involved in synapse development, synaptic plasticity, and cognitive function. However, its roles in synaptic expression and function of NMDA receptors (NMDARs) in the brain remain mostly unknown because of the neonatal lethality of global knock-out mice. To address this, we generated conditional knock-out mice, in which GluN2B was ablated exclusively in hippocampal CA3 pyramidal cells. By immunohistochemistry, GluN2B disappeared and GluN1 (GluRζ1/NR1) was moderately reduced, whereas GluN2A (GluRε1/NR2A) and postsynaptic density-95 (PSD-95) were unaltered in the mutant CA3. This was consistent with protein contents in the CA3 crude fraction: 9.6% of control level for GluN2B, 47.7% for GluN1, 90.6% for GluN2A, and 98.0% for PSD-95. Despite the remaining NMDARs, NMDAR-mediated currents and long-term potentiation were virtually lost at various CA3 synapses. Then, we compared synaptic NMDARs by postembedding immunogold electron microscopy and immunoblot using the PSD fraction. In the mutant CA3, GluN1 was severely reduced in both immunogold (20.6-23.6%) and immunoblot (24.6%), whereas GluN2A and PSD-95 were unchanged in immunogold but markedly reduced in the PSD fraction (51.4 and 36.5%, respectively), indicating increased detergent solubility of PSD molecules. No such increased solubility was observed for GluN2B in the CA3 of GluN2A-knock-out mice. Furthermore, significant decreases were found in the ratio of filamentous to globular actin (49.5%) and in the density of dendritic spines (76.2%). These findings suggest that GluN2B is critically involved in NMDAR channel function, organization of postsynaptic macromolecular complexes, formation or maintenance of dendritic spines, and regulation of the actin cytoskeleton.

title: Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre‐recipient layer) of the mouse hippocampal CA3 subfield
abstract: Hippocampal synapses express two distinct forms of the long‐term potentiation (LTP), i.e. NMDA receptor‐dependent and ‐independent LTPs. To understand its molecular‐anatomical basis, we produced affinity‐purified antibodies against the GluRε1 (NR2A), GluRε2 (NR2B), and GluRζ1 (NR1) subunits of the N‐methyl‐d‐aspartate (NMDA) receptor channel, and determined their distributions in the mouse hippocampus. Using NMDA receptor subunit‐deficient mice as the specificity controls, section pretreatment with proteases (pepsin and proteinase K) was found to be very effective to detect authentic NMDA receptor subunits. As the result of modified immunohistochemistry, all three subunits were detected at the highest level in the strata oriens and radiatum of the CA1 subfield, and high levels were also seen in most other neuropil layers of the CA1 and CA3 subfields and of the dentate gyrus. However, the stratum lucidum, a mossy fibre‐recipient layer of the CA3 subfield, contained low levels of the GluRε1 and GluRζ1 subunits and almost excluded the GluRε2 subunit. Double immunofluorescence with the AMPA receptor GluRα1 (GluR1 or GluR‐A) subunit further demonstrated that the GluRε1 subunit was colocalized in a subset, not all, of GluRα1‐immunopositive structures in the stratum lucidum. Therefore, the selective scarcity of these NMDA receptor subunits in the stratum lucidum suggests that a different synaptic targeting mechanism exerts within a single CA3 pyramidal neurone in vivo, which would explain contrasting significance of the NMDA receptor channel in LTP induction mechanisms between the mossy fibre‐CA3 synapse and other hippocampal synapses.

title: Input-Specific Intrasynaptic Arrangements of Ionotropic Glutamate Receptors and Their Impact on Postsynaptic Responses
abstract: To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.

title: Quantitative localisation of synaptic and extrasynaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze‐fracture replica immunolabelling
abstract: Hippocampal CA1 pyramidal cells, which receive γ‐aminobutyric acid (GABA)ergic input from at least 18 types of presynaptic neuron, express 14 subunits of the pentameric GABAA receptor. The relative contribution of any subunit to synaptic and extrasynaptic receptors influences the dynamics of GABA and drug actions. Synaptic receptors mediate phasic GABA‐evoked conductance and extrasynaptic receptors contribute to a tonic conductance. We used freeze‐fracture replica‐immunogold labelling, a sensitive quantitative immunocytochemical method, to detect synaptic and extrasynaptic pools of the alpha1, alpha2 and beta3 subunits. Antibodies to the cytoplasmic loop of the subunits showed immunogold particles concentrated on distinct clusters of intramembrane particles (IMPs) on the cytoplasmic face of the plasma membrane on the somata, dendrites and axon initial segments, with an abrupt decrease in labelling at the edge of the IMP cluster. Neuroligin‐2, a GABAergic synapse‐specific adhesion molecule, co‐labels all beta3 subunit‐rich IMP clusters, therefore we considered them synapses. Double‐labelling for two subunits showed that virtually all somatic synapses contain the alpha1, alpha2 and beta3 subunits. The extrasynaptic plasma membrane of the somata, dendrites and dendritic spines showed low‐density immunolabelling. Synaptic labelling densities on somata for the alpha1, alpha2 and beta3 subunits were 78–132, 94 and 79 times higher than on the extrasynaptic membranes, respectively. As GABAergic synapses occupy 0.72% of the soma surface, the fraction of synaptic labelling was 33–48 (alpha1), 40 (alpha2) and 36 (beta3)% of the total somatic surface immunolabelling. Assuming similar antibody access to all receptors, about 60% of these subunits are in extrasynaptic receptors.

title: The metabotropic glutamate receptor (mGluRlα) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction
abstract: An antiserum to mGluR1 alpha labeled a 160 kd protein in immunoblots of membranes derived from rat brain or cells transfected with mGluR1 alpha. Immunoreactivity for mGluR1 alpha was present in discrete subpopulations of neurons. The GABAergic neurons of the cerebellar cortex were strongly immunoreactive; only some Golgi cells were immunonegative. Somatostatin/GABA-immunopositive cells in the neocortex and hippocampus were enriched in mGluR1 alpha. The hippocampal cells had spiny dendrites that were precisely codistributed with the local axon collaterals of pyramidal and granule cells. Electron microscopic immunometal detection of mGluR1 alpha showed a preferential localization at the periphery of the extensive postsynaptic densities of type 1 synapses in both the cerebellum and the hippocampus. The receptor was also present at sites in the dendritic and somatic membrane where synapses were not located.

title: Input‐dependent synaptic targeting of α2‐subunit‐containing GABAA receptors in synapses of hippocampal pyramidal cells of the rat
abstract: Pyramidal cells, expressing at least 14 subunits of the heteropentameric GABAA receptor, receive GABAergic input on their soma and proximal dendrites from basket cells, activating GABAA receptors and containing either parvalbumin or cholecystokinin and vasoactive intestinal polypeptide. The properties of GABAA receptors are determined by the subunit composition, and synaptic receptor content governs the effect of the presynaptic neuron. Using a quantitative electron microscopic immunogold technique, we tested whether the synapses formed by the two types of basket cell show a difference in the subunit composition of GABAA receptors. Terminals of one of the basket cells were identified by antibodies to parvalbumin. Synapses made by parvalbumin‐negative terminals showed five times more immunoreactivity for the α2 subunit than synapses made by parvalbumin‐positive basket cells, whose synapses were frequently immunonegative. This difference is likely to be due to specific GABAA receptor α subunit composition, because neither synaptic size nor immunoreactivity for the β2/3 subunits, indicating total receptor content, was different in these two synapse populations. Synapses established by axo‐axonic cells on axon initial segments showed an intermediate number of immunoparticles for the α2 subunit compared to those made by basket cells but, due to their smaller size, the density of the α2 subunit immunoreactivity was higher in synapses on the axon. Because the two basket cell types innervate the same domain of the pyramidal cell, the results indicate that pyramidal cells have mechanisms to target GABAA receptors, under presynaptic influence, preferentially to distinct synapses. The two basket cell types act via partially distinct GABAA receptor populations.

title: Large variability in synaptic n-methyl-d-aspartate receptor density on interneurons and a comparison with pyramidal-cell spines in the rat hippocampus
abstract: Pyramidal cells receive input from several types of GABA-releasing interneurons and innervate them reciprocally. Glutamatergic activation of interneurons involves both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) type glutamate receptors expressed in type I synapses, mostly on their dendritic shafts. On average, the synaptic AMPA receptor content is several times higher on interneurons than in the spines of pyramidal cells. To compare the NMDA receptor content of synapses, we used a quantitative postembedding immunogold technique on serial electron microscopic sections, and analysed the synapses on interneuron dendrites and pyramidal cell spines in the CA1 area. Because all NMDA receptors contain the obligatory NR1 subunit, receptor localisation was carried out using antibodies recognising all splice variants of the NR1 subunit. Four populations of synapse were examined: i). on spines of pyramidal cells in stratum (str.) radiatum and str. oriens; ii). on parvalbumin-positive interneuronal dendritic shafts in str. radiatum; iii). on randomly found dendritic shafts in str. oriens and iv). on somatostatin-positive interneuronal dendritic shafts and somata in str. oriens. On average, the size of the synapses on spines was about half of those on interneurons. The four populations of synapse significantly differed in labelling for the NR1 subunit. The median density of NR1 subunit labelling was highest on pyramidal cell spines. It was lowest in the synapses on parvalbumin-positive dendrites in str. radiatum, where more than half of these synapses were immunonegative. In str. oriens, synapses on interneurons had a high variability of receptor content; some dendrites were similar to those in str. radiatum, including the proximal synapses of somatostatin-positive cells, whereas others had immunoreactivity for the NR1 subunit similar to or higher than synapses on pyramidal cell spines. These results show that synaptic NMDA receptor density differs between pyramidal cells and interneurons. Some interneurons may have a high NMDA receptor content, whereas others, like some parvalbumin-expressing cells, a particularly low synaptic NMDA receptor content. Consequently, fast glutamatergic activation of interneurons is expected to show cell type-specific time course and state-dependent dynamics.