Immunoelectron microscopic localization of the neural recognition molecules L1, NCAM, and its isoform NCAM180, the NCAM-associated polysialic acid, beta1 integrin and the extracellular matrix molecule tenascin-R in synapses of the adult rat hippocampus

We have investigated the possibility that morphologically different excitatory glutamatergic synapses of the "trisynaptic circuit" in the adult rodent hippocampus, which display different types of long-term potentiation (LTP), may express the immunoglobulin superfamily recognition molecules L1 and NCAM, the extracellular matrix molecule tenascin-R, and the extracellular matrix receptor constituent beta1 integrin in a differential manner. The neural cell adhesion molecules L1, NCAM (all three major isoforms), NCAM180 (the largest major isoform with the longest cytoplasmic domain), beta1 integrin, polysialic acid (PSA) associated with NCAM, and tenascin-R were localized by pre-embedding immunostaining procedures in the CA3/CA4 region (mossy fiber synapses) and in the dentate gyrus (spine synapses) of the adult rat hippocampus. Synaptic membranes of mossy fiber synapses where LTP is expressed presynaptically did not show detectable levels of immunoreactivity for any of the molecules/epitopes studied. L1, NCAM, and PSA, but not NCAM180 or beta1 integrin, were detectable on axonal membranes of fasciculating mossy fibers. In contrast to mossy fiber synapses, spine synapses in the outer third of the molecular layer of the dentate gyrus, which display postsynaptic expression mechanisms of LTP, were both immunopositive and immunonegative for NCAM, NCAM180, beta1 integrin, and PSA. Those spine synapses postsynaptically immunoreactive for NCAM or PSA also showed immunoreactivity on their presynaptic membranes. NCAM180 was not detectable presynaptically in spine synapses. L1 could not be found in spine synapses either pre- or postsynaptically. Also, the extracellular matrix molecule tenascin-R was not detectable in synaptic clefts of all synapses tested, but was amply present between fasciculating axons, axon-astrocyte contact areas, and astrocytic gap junctions. Differences in expression of the membrane-bound adhesion molecules at both types of synapses may reflect the different mechanisms for induction and/or maintenance of synaptic plasticity.     

Immunoelectron microscopic localization of the neural recognition molecules L1, NCAM,and its isoform NCAM180, the NCAM‐associated polysialic acid,beta1 integrin and the extracellular matrix molecule tenascin‐R in synapses of the adult rat hippocampus

We have investigated the possibility that morphologically different excitatory glutamatergic synapses of the “trisynaptic circuit” in the adult rodent hippocampus, which display different types of long‐term potentiation (LTP), may express the immunoglobulin superfamily recognition molecules L1 and NCAM, the extracellular matrix molecule tenascin‐R, and the extracellular matrix receptor constituent beta1 integrin in a differential manner. The neural cell adhesion molecules L1, NCAM (all three major isoforms), NCAM180 (the largest major isoform with the longest cytoplasmic domain), beta1 integrin, polysialic acid (PSA) associated with NCAM, and tenascin‐R were localized by pre‐embedding immunostaining procedures in the CA3/CA4 region (mossy fiber synapses) and in the dentate gyrus (spine synapses) of the adult rat hippocampus. Synaptic membranes of mossy fiber synapses where LTP is expressed presynaptically did not show detectable levels of immunoreactivity for any of the molecules/epitopes studied. L1, NCAM, and PSA, but not NCAM180 or beta1 integrin, were detectable on axonal membranes of fasciculating mossy fibers. In contrast to mossy fiber synapses, spine synapses in the outer third of the molecular layer of the dentate gyrus, which display postsynaptic expression mechanisms of LTP, were both immunopositive and immunonegative for NCAM, NCAM180, beta1 integrin, and PSA. Those spine synapses postsynaptically immunoreactive for NCAM or PSA also showed immunoreactivity on their presynaptic membranes. NCAM180 was not detectable presynaptically in spine synapses. L1 could not be found in spine synapses either pre‐ or postsynaptically. Also, the extracellular matrix molecule tenascin‐R was not detectable in synaptic clefts of all synapses tested, but was amply present between fasciculating axons, axon‐astrocyte contact areas, and astrocytic gap junctions. Differences in expression of the membrane‐bound adhesion molecules at both types of synapses may reflect the different mechanisms for induction and/or maintenance of synaptic plasticity. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 142–158, 2001     

The immunoglobulin superfamily of neuronal cell adhesion molecules: Lessons from animal models and correlation with human disease

Neuronal cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) play a crucial role in the formation of neural circuits at different levels: cell migration, axonal and dendritic targeting as well as synapse formation. Furthermore, in perinatal and adult life, neuronal IgCAMs are required for the formation and maintenance of specialized axonal membrane domains, synaptic plasticity and neurogenesis. Mutations in the corresponding human genes have been correlated to several human neuronal disorders. Perturbing neuronal IgCAMs in animal models provides powerful means to understand the molecular and cellular basis of such human disorders. In this review, we concentrate on the NCAM, L1 and contactin subfamilies of neuronal IgCAMs summarizing recent functional studies from model systems and highlighting their links to disease pathogenesis.     

Increase in proportion of hippocampal spine synapses expressing neural cell adhesion molecule NCAM180 following long-term potentiation

Neural recognition molecules such as the neural cell adhesion molecule (NCAM) have been implicated in synaptic plasticity, including long-term potentiation (LTP), sensitization, and learning and memory. The major isoform of NCAM carrying the longest cytoplasmic domain of all NCAM isoforms (NCAM180) is predominantly localized in postsynaptic membranes and postsynaptic densities of hippocampal neurons, with only a proportion of synapses carrying detectable levels of NCAM180. To investigate whether this differential expression of NCAM180 may correlate with distinct states of synaptic activity, LTP was induced by high-frequency stimulation of the perforant path and the percentage of NCAM180 immunopositive spine synapses determined in the outer third of the dentate molecular layer of the dentate gyrus by immunoelectron microscopy. Twenty-four hours following induction of LTP by high-frequency stimulation, the percentage of spine synapses expressing NCAM180 increases from 37% (passive control) to 70%. This increase was inhibited by the noncompetitive N-methyl-D -aspartate receptor antagonist MK801. Following repeated LTP induction at 10 consecutive days with one tetanization each day, 60% of all spine synapses were NCAM180 immunoreactive. Compared to passive control animals, the percentage of NCAM180 expressing synapses in low-frequency stimulated animals decreased from 37% to 28%. Spine synapses in the inner part of the dentate molecular layer not contacted by the afferents of the perforant path did not change the percentage of NCAM180-expressing synapses. The results obtained by the postembedding immunogold staining technique confirmed the difference in NCAM180 expression of spine synapses between passive control and potentiated animals. These observations suggest a role for NCAM180 in synaptic remodeling accompanying LTP. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 359–372, 1998     

Facilitation of AMPA receptor synaptic delivery as a molecular mechanism for cognitive enhancement

Cell adhesion molecules and downstream growth factor-dependent signaling are critical for brain development and synaptic plasticity, and they have been linked to cognitive function in adult animals. We have previously developed a mimetic peptide (FGL) from the neural cell adhesion molecule (NCAM) that enhances spatial learning and memory in rats. We have now investigated the cellular and molecular basis of this cognitive enhancement, using biochemical, morphological, electrophysiological, and behavioral analyses. We have found that FGL triggers a long-lasting enhancement of synaptic transmission in hippocampal CA1 neurons. This effect is mediated by a facilitated synaptic delivery of AMPA receptors, which is accompanied by enhanced NMDA receptor-dependent long-term potentiation (LTP). Both LTP and cognitive enhancement are mediated by an initial PKC activation, which is followed by persistent CaMKII activation. These results provide a mechanistic link between facilitation of AMPA receptor synaptic delivery and improved hippocampal-dependent learning, induced by a pharmacological cognitive enhancer.     

Impaired learning with enhanced hippocampal long-term potentiation in PTPdelta-deficient mice

Protein tyrosine phosphatase delta (PTPdelta) is a receptor-type PTP expressed in the specialized regions of the brain including the hippocampal CA2 and CA3, B lymphocytes and thymic medulla. To elucidate the physiological roles of PTPdelta, PTPdelta-deficient mice were produced by gene targeting. It was found that PTPdelta-deficient mice were semi-lethal due to insufficient food intake. They also exhibited learning impairment in the Morris water maze, reinforced T-maze and radial arm maze tasks. Interestingly, although the histology of the hippocampus appeared normal, the magnitudes of long-term potentiation (LTP) induced at hippocampal CA1 and CA3 synapses were significantly enhanced in PTPdelta-deficient mice, with augmented paired-pulse facilitation in the CA1 region. Thus, it was shown that PTPdelta plays important roles in regulating hippocampal LTP and learning processes, and that hippocampal LTP does not necessarily positively correlate with spatial learning ability. To our knowledge, this is the first report of a specific PTP involved in the regulation of synaptic plasticity or in the processes regulating learning and memory.     

Enhanced hippocampal long-term potentiation and learning by increased neuronal expression of tissue-type plasminogen activator in transgenic mice.

Adult cortical neurons can produce tissue-type plasminogen activator (tPA), an extracellular protease that plays a critical role in fibrinolysis and tissue remodelling processes. There is growing evidence that extracellular proteolysis may be involved in synaptic plasticity, axonal remodelling and neurotoxicity in the adult central nervous system. Here we show that transgenic mice overexpressing tPA in post-natal neurons have increased and prolonged hippocampal long-term potentiation (LTP), and improved performance in spatial orientation learning tasks. Extracellular proteolysis catalysed by tPA may facilitate synaptic micro-remodelling, and thereby play a role in activity-dependent neuronal plasticity and learning.     

The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function.

Members of the Eph family of receptor tyrosine kinases control many aspects of cellular interactions during development, including axon guidance. Here, we demonstrate that EphB2 also regulates postnatal synaptic function in the mammalian CNS. Mice lacking the EphB2 intracellular kinase domain showed wild-type levels of LTP, whereas mice lacking the entire EphB2 receptor had reduced LTP at hippocampal CA1 and dentate gyrus synapses. Synaptic NMDA-mediated current was reduced in dentate granule neurons in EphB2 null mice, as was synaptically localized NR1 as revealed by immunogold localization. Finally, we show that EphB2 is upregulated in hippocampal pyramidal neurons in vitro and in vivo by stimuli known to induce changes in synaptic structure. Together, these data demonstrate that EphB2 plays an important role in regulating synaptic function.     

Altered synaptic plasticity and behavioral abnormalities in CNGA3‐deficient mice

The role of the cyclic nucleotide‐gated (CNG) channel CNGA3 is well established in cone photoreceptors and guanylyl cyclase‐D‐expressing olfactory neurons. To assess a potential function of CNGA3 in the mouse amygdala and hippocampus, we examined synaptic plasticity and performed a comparative analysis of spatial learning, fear conditioning and step‐down avoidance in wild‐type mice and CNGA3 null mutants (CNGA3^?/?). CNGA3^?/? mice showed normal basal synaptic transmission in the amygdala and the hippocampus. However, cornu Ammonis (CA1) hippocampal long‐term potentiation (LTP) induced by a strong tetanus was significantly enhanced in CNGA3^?/? mice as compared with their wild‐type littermates. Unlike in the hippocampus, LTP was not significantly altered in the amygdala of CNGA3^?/? mice. Enhanced hippocampal LTP did not coincide with changes in hippocampus‐dependent learning, as both wild‐type and mutant mice showed a similar performance in water maze tasks and contextual fear conditioning, except for a trend toward higher step‐down latencies in a passive avoidance task. In contrast, CNGA3^?/? mice showed markedly reduced freezing to the conditioned tone in the amygdala‐dependent cued fear conditioning task. In conclusion, our study adds a new entry on the list of physiological functions of the CNGA3 channel. Despite the dissociation between physiological and behavioral parameters, our data describe a so far unrecognized role of CNGA3 in modulation of hippocampal plasticity and amydgala‐dependent fear memory.     

Reelin and ApoE receptors cooperate to enhance hippocampal synaptic plasticity and learning

Two apolipoprotein E (apoE) receptors, the very low density lipoprotein (VLDL) receptor and apoE receptor 2 (apoER2), are also receptors for Reelin, a signaling protein that regulates neuronal migration during brain development. In the adult brain, Reelin is expressed by GABA-ergic interneurons, suggesting a potential function as a modulator of neurotransmission. ApoE receptors have been indirectly implicated in memory and neurodegenerative disorders because their ligand, apoE, is genetically associated with Alzheimer disease. We have used knockout mice to investigate the role of Reelin and its receptors in cognition and synaptic plasticity. Mice lacking either the VLDL receptor or the apoER2 show contextual fear conditioning deficits. VLDL receptor-deficient mice also have a moderate defect in long term potentiation (LTP), and apoER2 knockouts have a pronounced one. The perfusion of mouse hippocampal slices with Reelin has no effect on baseline synaptic transmission but significantly enhances LTP in area CA1. This Reelin-dependent augmentation of LTP is abolished in VLDL receptor and apoER2 knockout mice. Our results reveal a role for Reelin in controlling synaptic plasticity in the adult brain and suggest that both of its receptors are necessary for Reelin-dependent enhancement of synaptic transmission in the hippocampus. Thus, the impairment of apoE receptor-dependent neuromodulation may contribute to cognitive impairment and synaptic loss in Alzheimer disease.     

Impaired hippocampal long-term potentiation and failure of learning in β1,4-N-acetylgalactosaminyltransferase gene transgenic mice

Gangliosides (sialic acid-containing glycosphingolipids) play important roles in many physiological functions, including synaptic plasticity in the hippocampus, which is considered as a cellular mechanism of learning and memory. In the present study, three types of synaptic plasticity, long-term potentiation (LTP), long-term depression (LTD) and reversal of LTP (depotentiation, DP), in the field excitatory post-synaptic potential in CA1 hippocampal neurons and learning behavior were examined in β1,4-N-acetylgalactosaminyltransferase (β1,4 GalNAc-T; GM2/GD2 synthase) gene transgenic (TG) mice, which showed a marked decrease in b-pathway gangliosides (GQ1b, GT1b and GD1b) in the brain and isolated hippocampus compared with wild-type (WT) mice. The magnitude of the LTP induced by tetanus (100 pulses at 100?Hz) in TG mice was significantly smaller than that in control WT mice, whereas there was no difference in the magnitude of the LTD induced by three short trains of low-frequency stimulation (LFS) (200 pulses at 1?Hz) at 20?min intervals between the two groups of mice. The reduction in the LTP produced by delivering three trains of LFS (200 pulses at 1?Hz, 20?min intervals) was significantly greater in the TG mice than in the WT mice. Learning was impaired in the four-pellet taking test (4PTT) in TG mice, with no significant difference in daily activity or activity during the 4PTT between TG and WT mice. These results suggest that the overexpression of β1,4 GalNAc-T resulted in altered synaptic plasticity of LTP and DP in hippocampal CA1 neurons and learning in the 4PTT, and this is attributable to the shift from b-pathway gangliosides to a-pathway gangliosides.     

Impaired long-term potentiation and long-term memory deficits in xCT-deficient sut mice

xCT is the functional subunit of the cystine/glutamate antiporter system xc-, which exchanges intracellular glutamate with extracellular cystine. xCT has been reported to play roles in the maintenance of intracellular redox and ambient extracellular glutamate, which may affect neuronal function. To assess a potential role of xCT in the mouse hippocampus, we performed fear conditioning and passive avoidance for long-term memories and examined hippocampal synaptic plasticity in wild-type mice and xCT-null mutants, sut mice. Long-term memory was impaired in sut mice. Normal basal synaptic transmission and short-term presynaptic plasticity at hippocampal Schaffer collateral-CA1 synapses were observed in sut mice. However, LTP (long-term potentiation) was significantly reduced in sut mice compared with their wild-type counterparts. Supplementation of extracellular glutamate did not reverse the reduction in LTP. Taken together, our results suggest that xCT plays a role in the modulation of hippocampal long-term plasticity.     

Structure–function–behavior relationship in estrogen-induced synaptic plasticity

This article is part of a Special Issue “Estradiol and Cognition”.In estrogen-induced synaptic plasticity, a correlation of structure, function and behavior in the hippocampus has been widely established. 17ß-estradiol has been shown to increase dendritic spine density on hippocampal neurons and is accompanied by enhanced long-term potentiation and improved performance of animals in hippocampus-dependent memory tests. After inhibition of aromatase, the final enzyme of estradiol synthesis, with letrozole we consistently found a strong and significant impairment of long-term potentiation (LTP) in female mice as early as after six hours of treatment. LTP impairment was followed by loss of hippocampal spine synapses in the hippocampal CA1 area. Interestingly, these effects were not found in male animals. In the Morris water maze test, chronic administration of letrozole did not alter spatial learning and memory in either female or male mice. In humans, analogous effects of estradiol on hippocampal morphology and physiology were observed using neuroimaging techniques. However, similar to our findings in mice, an effect of estradiol on memory performance has not been consistently observed.     

Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I

We have generated mice lacking synaptogyrin I and synaptophysin I to explore the functions of these abundant tyrosine-phosphorylated proteins of synaptic vesicles. Single and double knockout mice were alive and fertile without significant morphological or biochemical changes. Electrophysiological recordings in the hippocampal CA1 region revealed that short-term and long-term synaptic plasticity were severely reduced in the synaptophysin/synaptogyrin double knockout mice. LTP was decreased independent of the induction protocol, suggesting that the defect in LTP was not caused by insufficient induction. Our data show that synaptogyrin I and synaptophysin I perform redundant and essential functions in synaptic plasticity without being required for neurotransmitter release itself.     

Stress downregulates hippocampal expression of the adhesion molecules NCAM and CHL1 in mice by mechanisms independent of DNA methylation of their promoters

Stress is an important physiological regulator of brain function in young and adult mammals. The mechanisms underlying regulation of the consequences of stress, and in particular severe chronic stress, are thus important to investigate. These consequences most likely involve changes in synaptic function of brain areas being part of neural networks that regulate responses to stress. Cell adhesion molecules have been shown to regulate synaptic function in the adult and we were thus interested to investigate a regulatory mechanism that could influence expression of three adhesion molecules of the immunoglobulin superfamily (NCAM, L1 and CHL1) after exposure of early postnatal and adult mice to repeated stress. We hypothesized that reduction of adhesion molecule expression after chronic stress, as observed previously in vivo, could be due to gene silencing of the three molecules by DNA methylation. Although adhesion molecule expression was reduced after exposure of C57BL/6 mice to stress, thus validating our stress paradigm as imposing changes in adhesion molecule expression, we did not observe differences in methylation of CpG islands in the promoter regions of NCAM, L1 and CHL1, nor in the promoter region of the glucocorticoid receptor in the hippocampus, the expression of which at the protein level was also reduced after stress. We must therefore infer that severe stress in mice of the C57BL/6 strain downregulates adhesion molecule levels by mechanisms that do not relate to DNA methylation.Key words: stress, immunoglobulin superfamily, adhesion molecules, promoter, DNA methylation, hippocampus, glucocorticoid receptor     

Modulation of learning and hippocampal, neuronal plasticity by repetitive transcranial magnetic stimulation (rTMS)

The influence of high-frequency repetitive transcranial magnetic stimulation (rTMS) on learning process in mice and on neuronal excitability of the hippocampal tissue obtained from stimulated animals were investigated. While the stimulation with rTMS at higher frequency (15 Hz) improved animals' performance in novel object recognition test (NOR), lower frequency (1 and 8 Hz) impaired the memory. The effect was observed when evaluated immediately after rTMS exposure and declined with time. In parallel to the results of behavioral test, there was a significant enhancement of the synaptic efficiency expressed as of the long-term potentiation (LTP) recorded from hippocampal slices prepared from the animals exposed to 15 Hz rTMS. The stimulation with 1 and 8 Hz had no influence on the magnitude of LTP. Our results demonstrate that rTMS modifies mechanisms involved in memory formation. The effects of rTMS in vivo are preserved and expressed in the hippocampus tested in vitro.     

AMPA receptor regulation during synaptic plasticity in hippocampus and neocortex

Discovery of long-term potentiation (LTP) in the dentate gyrus of the rabbit hippocampus by Bliss and L?mo opened up a whole new field to study activity-dependent long-term synaptic modifications in the brain. Since then hippocampal synapses have been a key model system to study the mechanisms of different forms of synaptic plasticity. At least for the postsynaptic forms of LTP and long-term depression (LTD), regulation of AMPA receptors (AMPARs) has emerged as a key mechanism. While many of the synaptic plasticity mechanisms uncovered in at the hippocampal synapses apply to synapses across diverse brain regions, there are differences in the mechanisms that often reveal the specific functional requirements of the brain area under study. Here we will review AMPAR regulation underlying synaptic plasticity in hippocampus and neocortex. The main focus of this review will be placed on postsynaptic forms of synaptic plasticity that impinge on the regulation of AMPARs using hippocampal CA1 and primary sensory cortices as examples. And through the comparison, we will highlight the key similarities and functional differences between the two synapses.     

Stress downregulates hippocampal expression of the adhesion molecules NCAM and CHL1 in mice by mechanisms independent of DNA methylation of their promoters

Stress is an important physiological regulator of brain function in young and adult mammals. The mechanisms underlying regulation of the consequences of stress, and in particular severe chronic stress, are thus important to investigate. These consequences most likely involve changes in synaptic function of brain areas being part of neural networks that regulate responses to stress. Cell adhesion molecules have been shown to regulate synaptic function in the adult and we were thus interested to investigate a regulatory mechanism that could influence expression of three adhesion molecules of the immunoglobulin superfamily (NCAM, L1 and CHL1) after exposure of early postnatal and adult mice to repeated stress. We hypothesized that reduction of adhesion molecule expression after chronic stress, as observed previously in vivo, could be due to gene silencing of the three molecules by DNA methylation. Although adhesion molecule expression was reduced after exposure of C57BL/6 mice to stress, thus validating our stress paradigm as imposing changes in adhesion molecule expression, we did not observe differences in methylation of CpG islands in the promoter regions of NCAM, L1 and CHL1, nor in the promoter region of the glucocorticoid receptor in the hippocampus, the expression of which at the protein level was also reduced after stress. We must therefore infer that severe stress in mice of the C57BL/6 strain downregulates adhesion molecule levels by mechanisms that do not relate to DNA methylation.     

Melatonin is Involved in Regulation of the Expression of Neural Cell Adhesion Molecules in the Rat Brain

Cell adhesion molecules play a diverse role in neural development, signal transduction, structural linkage to extracellular and intracellular proteins, synaptic stabilization, neurogenesis, and learning. Neural cell adhesion molecules (NCAM) are members of the immunoglobulin superfamily and are involved in synaptic rearrangements in the mature brain. There are three major NCAM isoforms: NCAM 180, NCAM 140, and NCAM 120. Several studies reported that NCAM play a central role in memory formation. We investigated the effects of melatonin on the expression of NCAM in the hippocampus, cortex, and cerebellum of rats. The levels of NCAM isoforms were determined by Western blotting. After administration of melatonin for 7 days, the expression of NCAM 180 increased both in the hippocampus and in the cortex, as compared with the control. In contrast, in rats exposed to constant illumination for 7 days (a procedure that inhibits endogenous production of melatonin), levels of NCAM 180 dropped in the hippocampus and became undetectable in the cortex and cerebellum. Levels of NCAM 140 in the hippocampus of light-exposed rats also decreased. There was no change in the expression of NCAM 120 in any brain region. This is the first report indicating that melatonin exerts a modulatory effect on the expression of NCAM in brain areas related to realization of cognitive functions. Melatonin may be involved in structural remodeling of synaptic connections during memory and learning processes.