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 GABA(A)- 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 GABA(A)R-NMDAR cooperation during early development. We suggest that during SSA, synaptic GABA(A)R-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 Ca(2+)-influx through the NMDA receptors.     

Long-term potentiation selectively expressed by NMDA receptors at hippocampal mossy fiber synapses

The mossy fiber to CA3 pyramidal cell synapse (mf-CA3) provides a major source of excitation to the hippocampus. Thus far, these glutamatergic synapses are well recognized for showing a presynaptic, NMDA receptor-independent form of LTP that is expressed as a long-lasting increase of transmitter release. Here, we show that in addition to this "classical" LTP, mf-CA3 synapses can undergo a form of LTP characterized by a selective enhancement of NMDA receptor-mediated transmission. This potentiation requires coactivation of NMDA and mGlu5 receptors and a postsynaptic calcium rise. Unlike classical LTP, expression of this mossy fiber LTP is due to a PKC-dependent recruitment of NMDA receptors specifically to the mf-CA3 synapse via a SNARE-dependent process. Having two mechanistically different forms of LTP may allow mf-CA3 synapses to respond with more flexibility to the changing demands of the hippocampal network.     

Mobile NMDA receptors at hippocampal synapses

Glutamate receptors are concentrated in the postsynaptic complex of central synapses. This implies a highly organized and stable postsynaptic membrane with tightly anchored receptors. Recent reports of rapid AMPA receptor insertion and removal at synapses have challenged this view. We examined the stability of synaptic NMDA receptors on cultured hippocampal neurons using the open-channel blockers (+)-MK-801 and ketamine to tag synaptic NMDA receptors. NMDA receptor-mediated EPSCs showed an anomalous recovery following "irreversible" MK-801 block. The recovery could not be attributed to MK-801 unbinding or insertion of new receptors, suggesting that membrane receptors had moved laterally into the synapse. At least 65% of synaptic NMDA receptors were mobile. Our results indicate that NMDA receptors can move laterally between synaptic and extrasynaptic pools, providing evidence for a dynamic organization of synaptic NMDA receptors in the postsynaptic complex.     

Glutamate decarboxylase in developing rat neocortex: Does it correlate with the differentiation of GABAergic neurons and synapses?

Postnatal development of glutamate decarboxylase was studied in the rat cerebral cortex. Two methods were used: estimation of the enzymatic activity of glutamate decarboxylase in homogenates of developing cortical tissue and visualization of structures containing glutamate decarboxylase-like immunoreactivity. Glutamate decarboxylase-like immunoreactivity appeared first in perikarya and dendrites and only later in axons and axon varicosities. The most rapid increase in the glutamate decarboxylase activity took place during the second postnatal week and this coincided with a rapid increase in the density of axon varicosities containing glutamate decarboxylase-like immunoreactivity but preceded the most rapid phase in the formation of GABAergic synapses by several days. However, there was a change in the characteristics of glutamate decarboxylase which correlated with GABA synaptogenesis: two fractions of glutamate decarboxylase with different sensitivities to the activating effects of Triton X-100 could be distinguished as from about the time when most of the GABAergic synapses are formed.     

Olfactory bulb glomerular NMDA receptors mediate olfactory nerve potentiation and odor preference learning in the neonate rat

Rat pup odor preference learning follows pairing of bulbar beta-adrenoceptor activation with olfactory input. We hypothesize that NMDA receptor (NMDAR)-mediated olfactory input to mitral cells is enhanced during training, such that increased calcium facilitates and shapes the critical cAMP pattern. Here, we demonstrate, in vitro, that olfactory nerve stimulation, at sniffing frequencies, paired with beta-adrenoceptor activation, potentiates olfactory nerve-evoked mitral cell firing. This potentiation is blocked by a NMDAR antagonist and by increased inhibition. Glomerular disinhibition also induces NMDAR-sensitive potentiation. In vivo, in parallel, behavioral learning is prevented by glomerular infusion of an NMDAR antagonist or a GABA(A) receptor agonist. A glomerular GABA(A) receptor antagonist paired with odor can induce NMDAR-dependent learning. The NMDA GluN1 subunit is phosphorylated in odor-specific glomeruli within 5 min of training suggesting early activation, and enhanced calcium entry, during acquisition. The GluN1 subunit is down-regulated 3 h after learning; and at 24 h post-training the GluN2B subunit is down-regulated. These events may assist memory stability. Ex vivo experiments using bulbs from trained rat pups reveal an increase in the AMPA/NMDA EPSC ratio post-training, consistent with an increase in AMPA receptor insertion and/or the decrease in NMDAR subunits. These results support a model of a cAMP/NMDA interaction in generating rat pup odor preference learning.     

Presynaptic kainate receptors are localized close to release sites in rat hippocampal synapses

The subsynaptic distribution of kainate receptors is still a matter of much debate given its importance to understand the way they influence neuronal communication. Here, we show that, in synapses of the rat hippocampus, presynaptic kainate receptors are localized within the presynaptic active zone close to neurotransmitter release sites. The activation of these receptors with low concentrations of agonists induces the release of [(3)H]glutamate in the absence of a depolarizing stimulus. Furthermore, this modulation of [(3)H]glutamate release by kainate is more efficient when compared with a KCl-evoked depolarization that causes a more than two-fold increase in the intra-terminal calcium concentration but no apparent release of [(3)H]glutamate, suggesting a direct receptor-mediated process. Using a selective synaptic fractionation technique that allows for a highly efficient separation of presynaptic, postsynaptic and non-synaptic proteins we confirmed that, presynaptically, kainate receptors are mainly localized within the active zone of hippocampal synapses where they are expected to be in a privileged position to modulate synaptic phenomena.     

NMDA receptors mediate peripheral chemoreceptor afferent input in the conscious rat

N-methyl-D-aspartate(NMDA) glutamate receptors mediate critical components ofcardiorespiratory control in anesthetized animals. The role of NMDAreceptors in the ventilatory responses to peripheral and centralchemoreceptor stimulation was investigated in conscious, freelybehaving rats. Minute ventilation(E)responses to 10% O₂, 5%CO₂, and increasing intravenousdoses of sodium cyanide were measured in intact rats before and afterintravenous administration of the NMDA receptor antagonist MK-801 (3 mg/kg). After MK-801, eupcapnic tidal volume(VT) decreased while frequencyincreased, resulting in a modest reduction inE.Inspiratory time (TI) decreased, whereas expiratory time remained unchanged. TheE responsesto hypercapnia were qualitatively similar in control and MK-801conditions, with slight reductions in respiratory drive (VT/TI)after MK-801. In contrast, responses to hypoxia were markedly attenuated after MK-801 and were primarily due to reduced frequency changes, whereas VT wasunaffected. Sodium cyanide doses associated with significantEincreases were 5 and 50 µg/kg before and after MK-801,respectively. Thus 1-log shift to the right of individual dose-responsecurves occurred with MK-801. Selective carotid body denervation reducedE duringhypoxia by 70%, and residual hypoxic ventilatory responses wereabolished after MK-801. These findings suggest that, in conscious rats,carotid and other peripheral chemoreceptor-mediated hypoxic ventilatoryresponses are critically dependent on NMDA receptor activation and thatNMDA receptor mechanisms are only modestly involved during hypercapnia.

     

Distinct NMDA receptors provide differential modes of transmission at mossy fiber-interneuron synapses

Dentate gyrus granule cells innervate inhibitory interneurons via a continuum of synapses comprised of either Ca(2+)-impermeable (CI) or Ca(2+)-permeable (CP) AMPA receptors. Synapses at the extreme ends of this continuum engage distinct postsynaptic responses, with activity at CI synapses being strongly influenced by NMDA receptor activation. NMDARs at CI synapses have a lower NR2B subunit composition and a higher open probability, which generate larger amplitude and more rapid EPSCs than their CP counterparts. A novel form of NMDAR-dependent long-term depression (iLTD) is associated with CI-mossy fiber synapses, whereas iLTD at CP synapses is dependent on Ca(2+)-permeable AMPA receptor activation. Induction of both forms of iLTD required elevation of postsynaptic calcium. Thus mossy fibers engage CA3 interneurons via multiple synapse types that will act to expand the computational repertoire of the mossy fiber-CA3 network.     

NMDA receptors in afferent synapses of frog semicircular canals

The effects of competitive (2-amino-phosphonovaleric acid) and noncompetitive (Mg²⁺, ketamine, kynurenic acid) antagonists of N-methyl-D-aspartate (NMDA) receptors on synaptic transmission were studied in afferent synapses of the frog semicircular canals. All of these antagonists reduced the rate of background activity in the nerve of posterior semicircular canal by 30–50%, which confirms the presence of glutamate NMDA receptors in the hair cell synapses in the frog semicircular canals.Neurofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 168–169, May–June, 1993.     

Incorporation of inwardly rectifying AMPA receptors at silent synapses during hippocampal long-term potentiation

Despite decades of study, the mechanisms by which synapses express the increase in strength during long-term potentiation (LTP) remain an area of intense interest. Here, we have studied how AMPA receptor subunit composition changes during the early phases of hippocampal LTP in CA1 pyramidal neurons. We studied LTP at silent synapses that initially lack AMPA receptors, but contain NMDA receptors. We show that strongly inwardly rectifying AMPA receptors are initially incorporated at silent synapses during LTP and are then subsequently replaced by non-rectifying AMPA receptors. These findings suggest that silent synapses initially incorporate GluA2-lacking, calcium-permeable AMPA receptors during LTP that are then replaced by GluA2-containing calcium-impermeable receptors. We also show that LTP consolidation at CA1 synapses requires a rise in intracellular calcium concentration during the early phase of expression, indicating that calcium influx through the GluA2-lacking AMPA receptors drives their replacement by GluA2-containing receptors during LTP consolidation. Taken together with previous studies in hippocampus and in other brain regions, these findings suggest that a common mechanism for the expression of activity-dependent glutamatergic synaptic plasticity involves the regulation of GluA2-subunit composition and highlights a critical role for silent synapses in this process.     

Presynaptic NMDA receptors act as local high‐gain glutamate detector in developing cerebellar molecular layer interneurons

In the classical view, NMDA receptors (NMDARs) are located postsynaptically and play a pivotal role in excitatory transmission and synaptic plasticity. In developing cerebellar molecular layer interneurons (MLIs) however, NMDARs are known to be solely extra‐ or presynaptic and somewhat poorly expressed. Somatodendritic NMDARs are exclusively activated by glutamate spillover from adjacent synapses, but the mode of activation of axonal NMDARs remains unclear. Our data suggest that a volume transmission is likely to stimulate presynaptic NMDARs (preNMDARs) since NMDA puffs directed to the axon led to inward currents and Ca²⁺ transients restricted to axonal varicosities. Using local glutamate photoliberation, we show that pre‐ and post‐synaptic NMDARs share the same voltage dependence indicating their containing NR2A/B subunits. Ca²⁺ transients elicited by NMDA puffs are eventually followed by delayed events reminding of the spontaneous Ca²⁺ transients (ScaTs) described at the basket cell/Purkinje cell terminals. Moreover, the presence of Ca²⁺ transients at varicosities located more than 5 μm away from the uncaging site indicates that the activation of preNMDARs sensitizes the Ca²⁺ stores in adjacent varicosities, a process that is abolished in the presence of a high concentration of ryanodine. Altogether, the data demonstrate that preNMDARs act as high‐gain glutamate detectors.     

Synaptic drive at developing synapses: Transient upregulation of kainate receptors

At the onset of a period of intense synaptic refinement initiated by synchronized eye opening (EO), rapid changes in postsynaptic NMDA receptor and AMPA receptor currents (NMDARcs and AMPARcs) occur within the superficial visual layers of the rodent superior colliculus (sSC; Lu and Constantine‐Paton [ 2004 ]: Neuron 43:237–249). Subsequently, evoked non‐NMDARc amplitudes increase, but by 2 weeks after EO (AEO) they decrease significantly. Here, using whole‐cell patch‐clamp recording, we demonstrate that small, slowly desensitizing excitatory kainate receptor currents (KARcs) are responsible for the rise and subsequent fall in non‐NMDARcs. The increase in KAR transmission parallels inhibitory GABA_A responses that plateau at 7 days AEO. By 2 weeks AEO, KARcs are gone. AMPARcs remain unchanged during the appearance and disappearance of the KARcs, despite increases in sSC neuropil activity and continued refinement of inputs to individual sSC neurons. We suggest that in the interval of heightened activity, before SC inhibition matures, many AMPARcs desensitize and are relatively ineffective at relieving the Mg²⁺ block on NMDARs. This transient appearance of slowly desensitizing, long‐duration KARcs may provide increased membrane depolarization necessary for NMDAR function and continuation of synaptic refinement. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 737–750, 2010     

Plasticity of GABAergic synapses in the neonatal rat hippocampus

While the development and plasticity of excitatory synaptic connections have been studied into detail, little is known about the development of inhibitory synapses. As proposed for excitatory synapses, recent studies have indicated that activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, may play a role in the establishment of functional inhibitory synaptic connections. Here, I review these different forms of plasticity and focus on their possible role in the developing neuronal network.     

NMDA receptors mediate olfactory learning and memory in Drosophila

BACKGROUND: Molecular and electrophysiological properties of NMDARs suggest that they may be the Hebbian "coincidence detectors" hypothesized to underlie associative learning. Because of the nonspecificity of drugs that modulate NMDAR function or the relatively chronic genetic manipulations of various NMDAR subunits from mammalian studies, conclusive evidence for such an acute role for NMDARs in adult behavioral plasticity, however, is lacking. Moreover, a role for NMDARs in memory consolidation remains controversial. RESULTS: The Drosophila genome encodes two NMDAR homologs, dNR1 and dNR2. When coexpressed in Xenopus oocytes or Drosophila S2 cells, dNR1 and dNR2 form functional NMDARs with several of the distinguishing molecular properties observed for vertebrate NMDARs, including voltage/Mg(2+)-dependent activation by glutamate. Both proteins are weakly expressed throughout the entire brain but show preferential expression in several neurons surrounding the dendritic region of the mushroom bodies. Hypomorphic mutations of the essential dNR1 gene disrupt olfactory learning, and this learning defect is rescued with wild-type transgenes. Importantly, we show that Pavlovian learning is disrupted in adults within 15 hr after transient induction of a dNR1 antisense RNA transgene. Extended training is sufficient to overcome this initial learning defect, but long-term memory (LTM) specifically is abolished under these training conditions. CONCLUSIONS: Our study uses a combination of molecular-genetic tools to (1) generate genomic mutations of the dNR1 gene, (2) rescue the accompanying learning deficit with a dNR1+ transgene, and (3) rapidly and transiently knockdown dNR1+ expression in adults, thereby demonstrating an evolutionarily conserved role for the acute involvement of NMDARs in associative learning and memory.     

Analysis of relations between NMDA receptors and GABA release at olfactory bulb reciprocal synapses

In the mammalian olfactory bulb, signal processing is mediated by synaptic interactions between dendrites. Glutamate released from mitral cell dendrites excites dendritic spines of granule cells, which in turn release GABA back onto the mitral cell dendrites, forming a reciprocal synaptic pair. This feedback synaptic circuit was shown to be mediated predominantly by NMDA receptors. We further utilized caged Ca2+ compounds to obtain insight into the mechanism that couples NMDA receptor activation to GABA release. Feedback inhibition elicited by photo-release of caged Ca2+ in mitral cell secondary dendrites persisted when voltage-gated Ca2+ channels were blocked by cadmium (Cd2+) and nickel (Ni2+). These results indicate that Ca2+ influx through NMDA receptors can directly trigger presynaptic GABA release for local dendrodendritic feedback inhibition.     

Endocannabinoids mediate synaptic plasticity at mixed synapses

Endocannabinoids are generally known to suppress excitatory or inhibitory synaptic transmission. Now, in an elegant series of experiments, Cachope et al. reveal a novel signaling pathway whereby endocannabinoids indirectly potentiate mixed chemical and electrical synapses. Gap junction-mediated transmission can thus be potentiated via distinct frequency-dependent mechanisms.     

Determination of the numerical density of perforated synapses in rat neocortex

Summary The numerical density and frequency of perforated synapses in the molecular layer of rat parietal cortex have been determined using 4 procedures in an attempt to overcome problems associated with the size and complex three-dimensional shape of perforated synapses. The following procedures were compared: A, single-section analysis; B, adjacent-section analysis; C, semi-serial-section analysis; and D, complete serial-section analysis. All procedures made use of an unbiased counting rule.Estimates of the numerical density of perforated synapses ranged from 0.06 to 0.27×10⁹ mm^-3, and that of all synapses (non-perforated and perforated) from 1.88 to 2.50×10⁹ mm^-3. The frequency of perforated synapses varied from 4.5 to 18.0%. Procedures B (adjacent-section analysis) and D (complete serial-section analysis), neither of which utilize assumptions regarding the shape of synapses, produced comparable results (numerical density of perforated synapses 0.19–0.27×10⁹ mm^-3, and of all synapses 2.24–2.45×10⁹ mm^-3; frequency of perforated synapses 8.6–10.9%). The frequency of perforated synapses appeared to be underestimated by procedure A (single section analysis; 4.5%) and overestimated by C (semi-serial section analysis; 18%).It is concluded that adjacent-section analysis is the most efficient and effective procedure for determining the numerical density and frequency of complex particles, such as perforated synapses. There is, however, no significant difference in the performance of this procedure compared with that of single-section analysis, for determining the numerical density of synapses in general. Nevertheless, inherent problems of bias within the single-section procedure make the adjacent section method the procedure of choice.     

Long-term potentiation at hippocampal perforant path-dentate astrocyte synapses

Accumulated evidence indicates that astroglial cells actively participate in neuronal synaptic transmission and plasticity. However, it is still not clear whether astrocytes are able to undergo plasticity in response to synaptic inputs. Here we demonstrate that a long-term potentiation (LTP)-like response could be detected at perforant path-dentate astrocyte synapses following high-frequency stimulation (HFS) in hippocampal slices of GFAP-GFP transgenic mice. The potentiation was not dependent on the glutamate transporters nor the group I metabotropic glutamate receptors. However, the induction of LTP requires activation of the NMDA receptor (NMDAR). The presence of functional NMDAR was supported by isolating the NMDAR-gated current and by identifying mRNAs of NMDAR subunits in astrocytes. Our results suggest that astrocytes in the hippocampal dentate gyrus are able to undergo plasticity in response to presynaptic inputs.