The role of cell adhesion molecules in synaptic plasticity and memory.

Studies in the past few years suggest that cell adhesion molecules may play signaling as well as structural roles at adult synapses during plasticity. The observation that many adhesion molecules are expressed both pre-synaptically and post-synaptically raises the possibility that information about synaptic activity might simultaneously be communicated to both sides of the synapse, circumventing the need for distinct anterograde and retrograde messengers.

The integrated stress response contributes to tRNA synthetase-associated peripheral neuropathy.

Dominant mutations in ubiquitously expressed transfer RNA (tRNA) synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth (CMT) disease. Genetic evidence in mouse and Drosophila models suggests a gain-of-function mechanism. In this study, we used in vivo, cell type­specific transcriptional and translational profiling to show that mutant tRNA synthetases activate the integrated stress response (ISR) through the sensor kinase GCN2 (general control nonderepressible 2). The chronic activation of the ISR contributed to the pathophysiology, and genetic deletion or pharmacological inhibition of Gcn2 alleviated the peripheral neuropathy. The activation of GCN2 suggests that the aberrant activity of the mutant tRNA synthetases is still related to translation and that inhibiting GCN2 or the ISR may represent a therapeutic strategy in CMT.

Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus.

The neurotrophins are signaling factors important for the differentiation and survival of distinct neuronal populations during development. To test whether the neurotrophins also function in the mature nervous system, the effects of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophic factor 3 (NT-3) on the strength of synaptic transmission in hippocampal slices were determined. Application of BDNF or NT-3 produced a dramatic and sustained (2 to 3 hours) enhancement of synaptic strength at the Schaffer collateral-CA1 synapses; NGF was without significant effect. The enhancement was blocked by K252a, an inhibitor of receptor tyrosine kinases. BDNF and NT-3 decreased paired-pulse facilitation, which is consistent with a possible presynaptic modification. Long-term potentiation could still be elicited in slices previously potentiated by exposure to the neurotrophic factors, which implies that these two forms of plasticity may use at least partially independent cellular mechanisms.

A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity.

Two neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are able to produce a long-lasting enhancement of synaptic transmission in the hippocampus. Unlike other forms of plasticity, neurotrophin-induced plasticity exhibited an immediate requirement for protein synthesis. Plasticity in rat hippocampal slices in which the synaptic neuropil was isolated from the principal cell bodies also required early protein synthesis. Thus, the neurotrophins may stimulate the synthesis of proteins in either axonal or dendritic compartments, allowing synapses to exert local control over the complement of proteins expressed at individual synaptic sites.

A requirement for the intercellular messenger nitric oxide in long-term potentiation.

Long-term potentiation (LTP) of synaptic transmission is a widely studied model of neuronal plasticity. The induction of LTP is known to require processes in the postsynaptic neuron, while experimental evidence suggests that the expression of LTP may occur in the presynaptic terminal. This has led to speculation that a retrograde messenger travels from the post- to the presynaptic cell during induction of LTP. Extracellular application or postsynaptic injection of two inhibitors of nitric oxide synthase, N-nitro-L-arginine or NG-methyl-L-arginine, blocks LTP. Extracellular application of hemoglobin, which binds nitric oxide, also attenuates LTP. These findings suggest that nitric oxide liberated from postsynaptic neurons may travel back to presynaptic terminals to cause LTP expression.

Locally distributed synaptic potentiation in the hippocampus.

The long-lasting increase in synaptic strength known as long-term potentiation has been advanced as a potential physiological mechanism for many forms of both developmental and adult neuronal plasticity. In many models of plasticity, intercellular communication has been proposed to account for observations in which simultaneously active neurons are strengthened together. The data presented here indicate that long-term potentiation can be communicated between synapses on neighboring neurons by means of a diffusible messenger. This distributed potentiation provides a mechanism for the cooperative strengthening of proximal synapses and may underlie a variety of plastic processes in the nervous system.

A role for endothelial NO synthase in LTP revealed by adenovirus-mediated inhibition and rescue.

Pharmacological studies support the idea that nitric oxide (NO) serves as a retrograde messenger during long-term potentiation (LTP) in area CA1 of the hippocampus. Mice with a defective form of the gene for neuronal NO synthase (nNOS), however, exhibit normal LTP. The myristoyl protein endothelial NOS (eNOS) is present in the dendrites of CA1 neurons. Recombinant adenovirus vectors containing either a truncated eNOS (a putative dominant negative) or an eNOS fused to a transmembrane protein were used to demonstrate that membrane-targeted eNOS is required for LTP. The membrane localization of eNOS may optimally position the enzyme both to respond to Ca2+ influx and to release NO into the extracellular space during LTP induction.

Neurotrophins and time: different roles for TrkB signaling in hippocampal long-term potentiation.

We examined the role of TrkB ligands in hippocampal long-term potentiation (LTP) using function-blocking TrkB antiserum (Ab) and Trk-IgG fusion proteins. Incubation of hippocampal slices with TrkB Ab had no effect on basal synaptic transmission, short-term plasticity, or LTP induced by several trains of tetanic stimulation. The TrkB Ab-treated slices, however, showed significant deficits in LTP induced by either theta-burst stimulation (TBS) or "pairing." Slices exposed to the same number of inducing stimuli, delivered either as TBS or as a single 100 Hz epoch, only exhibited TrkB-sensitive LTP when TBS was used, indicating that the temporal pattern of stimulation determines the neurotrophin dependence. The late phase of LTP (2-3 hr) was also significantly impaired in slices pretreated with TrkB Ab or a TrkB-IgG. The application of a TrkB-IgG 30 min after LTP induction caused previously potentiated synaptic transmission to return to baseline levels, indicating that TrkB ligands are required to maintain LTP for up to 1 hr after induction. Taken together, these results indicate that both the temporal patterns of synaptic activity and the different temporal phases of synaptic enhancement are important in determining the neurotrophin dependence of plasticity in the hippocampus.

A role for the cadherin family of cell adhesion molecules in hippocampal long-term potentiation.

The cadherins are a family of cell-cell adhesion molecules that mediate Ca2+-dependent homophilic interactions between cells and transduce signals by interacting with cytoplasmic proteins. In the hippocampus, immunostaining combined with confocal microscopy revealed that both neural- (N-) and epithelial- (E-) cadherin are present at synaptic sites, implying a role in synaptic function. Pretreatment of hippocampal slices with antibodies (Abs) raised against the extracellular domain of either N-cad or E-cad had no effect on basal synaptic properties but significantly reduced long-term potentiation (LTP). Infusion of antagonistic peptides containing the His-Ala-Val (HAV) consensus sequence for cadherin dimerization also attenuated LTP induction without affecting previously established LTP. Because the intense synaptic stimulation associated with LTP induction might transiently deplete extracellular Ca2+ and hence potentially destabilize cadherin-cadherin interactions, we examined whether slices could be protected from inhibition by N-cad Abs or HAV peptides by raising the extracellular Ca2+ concentration. Indeed, we found that high extracellular Ca2+ prevented the block of LTP by these agents. Taken together, these results indicate that cadherins are involved in synaptic plasticity, and the stability of cadherin-cadherin bonds may be regulated by synaptic stimulation.

Inhibition of hippocampal heme oxygenase, nitric oxide synthase, and long-term potentiation by metalloporphyrins.

Four potent metalloporphyrin inhibitors of heme oxygenase were used to assess whether carbon monoxide production was required for induction of LTP in the CA1 region of the hippocampus. Although the metalloporphyrins produced a similar and substantial inhibition of heme oxygenase activity in hippocampal slices, only two compounds reduced the amount of LTP elicited by tetanic stimulation (chromium mesoporphyrin IX and zinc protoporphyrin IX). Both chromium mesoporphyrin IX and zinc protoporphyrin IX inhibited nitric oxide synthase in the hippocampus; tin mesoporphyrin IX and zinc deuteroporphyrin IX bis glycol neither reduced LTP induction nor inhibited NOS activity, although they did inhibit heme oxygenase. None of these metalloporphyrins reversed established LTP. Thus, together these data do not support carbon monoxide as a mediator in either LTP induction or expression/maintenance and emphasize further the nonselectivity of some metalloporphyrins.

Dynamic visualization of local protein synthesis in hippocampal neurons.

Using pharmacological approaches, several recent studies suggest that local protein synthesis is required for synaptic plasticity. Convincing demonstrations of bona fide dendritic protein synthesis in mammalian neurons are rare, however. We developed a protein synthesis reporter in which the coding sequence of green fluorescent protein is flanked by the 5' and 3' untranslated regions from CAMKII-alpha, conferring both dendritic mRNA localization and translational regulation. In cultured hippocampal neurons, we show that BDNF, a growth factor involved in synaptic plasticity, stimulates protein synthesis of the reporter in intact, mechanically, or "optically" isolated dendrites. The stimulation of protein synthesis is blocked by anisomycin and not observed in untreated neurons. In addition, dendrites appear to possess translational hot spots, regions near synapses where protein synthesis consistently occurs over time.

Neurotrophin regulation of synaptic transmission.

Examples of signaling molecules that are devoted to neuronal development at the exclusion of other functions are scarce. It may then come as no surprise to learn that a family of molecules that promote neuronal survival, differentiation and outgrowth also regulate synaptic transmission at both developing and mature synapses. Indeed, many studies over the past five years have shown that neurotrophins, including nerve growth factor (NGF), neurotrophin-3 (NT-3), NT-4/5 and brain-derived neurotrophic factor (BDNF), have both rapid and long-latency influences on synaptic strength. New research has highlighted the enormous range of neurotrophin actions at both developing and mature synapses, demonstrating that transmission can be enhanced or reduced at excitatory and inhibitory synapses by either pre- or postsynaptic mechanisms.

Tetanic stimulation leads to increased accumulation of Ca(2+)/calmodulin-dependent protein kinase II via dendritic protein synthesis in hippocampal neurons.

mRNA for the alpha-subunit of CaMKII is abundant in dendrites of neurons in the forebrain (Steward, 1997). Here we show that tetanic stimulation of the Schaffer collateral pathway causes an increase in the concentration of alpha-CaMKII in the dendrites of postsynaptic neurons. The increase is blocked by anisomycin and is detected by both quantitative immunoblot and semiquantitative immunocytochemistry. The increase in dendritic alpha-CaMKII can be measured 100-200 micrometer away from the neuronal cell bodies as early as 5 min after a tetanus. Transport mechanisms for macromolecules from neuronal cell bodies are not fast enough to account for this rapid increase in distal portions of the dendrites. Therefore, we conclude that dendritic protein synthesis must produce a portion of the newly accumulated CaMKII. The increase in concentration of dendritic CaMKII after tetanus, together with the previously demonstrated increase in autophosphorylated CaMKII (Ouyang et al., 1997), will produce a prolonged increase in steady-state kinase activity in the dendrites, potentially influencing mechanisms of synaptic plasticity that are controlled through phosphorylation by CaMKII.

Long-range temporal correlations in the spontaneous spiking of neurons in the hippocampal-amygdala complex of humans.

The spontaneous or background discharge patterns of in vivo single neuron is mostly considered as neuronal noise, which is assumed to be devoid of any correlation between successive inter-spike-intervals (ISI). Such random fluctuations are modeled only statistically by stochastic point process, lacking any temporal correlation. In this study, we have investigated the nature of spontaneous irregular fluctuations of single neurons from human hippocampus-amygdala complex by three different methods: (i) detrended fluctuation analysis (DFA), (ii) multiscale entropy (MSE), (iii) rate estimate convergence. Both the DFA and MSE analysis showed the presence of long-range power-law correlation over time in the ISI sequences. Moreover, we observed that the individual spike trains presented non-random structure on longer time-scales and showed slow convergence of rate estimates with increasing counting time. This power-law correlation and the slow convergence of statistical moments were eliminated by randomly shuffling the ISIs even though the distributions of ISIs were preserved. Thus the power-law relationship arose from long-term correlations among ISIs that were destroyed by shuffling the data. Further, we found that neurons which showed long-range correlations also showed statistically significant correlated firing as measured by correlation coefficient or mutual information function. The presence of long-range correlations indicates the history-effect or memory in the firing pattern by the associative formation of a neuronal assembly.

Lateral assembly of N-cadherin drives tissue integrity by stabilizing adherens junctions.

Cadherin interactions ensure the correct registry and anchorage of cells during tissue formation. Along the plasma membrane, cadherins form inter-junctional lattices via cis- and trans-dimerization. While structural studies have provided models for cadherin interactions, the molecular nature of cadherin binding in vivo remains unexplored. We undertook a multi-disciplinary approach combining live cell imaging of three-dimensional cell assemblies (spheroids) with a computational model to study the dynamics of N-cadherin interactions. Using a loss-of-function strategy, we demonstrate that each N-cadherin interface plays a distinct role in spheroid formation. We found that cis-dimerization is not a prerequisite for trans-interactions, but rather modulates trans-interfaces to ensure tissue stability. Using a model of N-cadherin junction dynamics, we show that the absence of cis-interactions results in low junction stability and loss of tissue integrity. By quantifying the binding and unbinding dynamics of the N-cadherin binding interfaces, we determined that mutating either interface results in a 10-fold increase in the dissociation constant. These findings provide new quantitative information on the steps driving cadherin intercellular adhesion and demonstrate the role of cis-interactions in junction stability.

The aspirin metabolite sodium salicylate causes focal cerebral hemorrhage and cell death in rats with kainic acid-induced seizures.

Aspirin (acetylsalicylic acid), and its main metabolite sodium salicylate, have been shown to protect neurons from excitotoxic cell death in vitro. The objective of our study was to investigate the possible neuroprotective effects of sodium salicylate in vivo in rats with kainic acid-induced seizures, a model for temporal lobe epilepsy in human patients. Male Sprague-Dawley rats received intraperitoneal injections of kainic acid either alone, or with sodium salicylate given before and for 40h after kainic acid injections. The control group received either phosphate-buffered saline or sodium salicylate without co-administration of kainic acid. Animals developed status epilepticus, which was aborted 1.5-2h later with diazepam. On day 3 following kainic acid-induced seizures, animals received bromodeoxyuridine to measure cellular proliferation, and were killed under anesthesia 24h later. Brains were removed, sectioned, and analysed for gross histological changes, evidence of hemorrhage, DNA fragmentation, cellular proliferation, and microglial immunohistochemistry. We report that sodium salicylate did not protect neurons from seizure-induced cell death, and to the contrary, it caused focal hemorrhage and cell death in the hippocampal formation and the entorhinal/piriform cortex of rats with kainic acid-induced seizures. Hemorrhage was never observed in animals that received vehicle, kainic acid or sodium salicylate only, which indicated that sodium salicylate exerted its effect only in animals with seizures, and was confined to select regions of the brain that undergo seizure activity. Large numbers of cells displaying DNA fragmentation were detected in the hippocampal formation, entorhinal/piriform cortex and the dorsomedial thalamic nucleus of rats that received kainic acid or kainic acid in combination with sodium salicylate. Bromodeoxyuridine immunohistochemistry revealed large numbers of proliferating cells in and around the areas with most severe neural injury induced by kainic acid or kainic acid co-administered with sodium salicylate. These same brain regions displayed intense staining with a microglia-specific marker, an indication of microglial activation in response to brain damage. In all cases, the degree of cell death, cell proliferation and microglia staining was more severe in animals that received the combination of kainic acid and sodium salicylate when compared to animals that received kainic acid alone. We hypothesize that our findings are attributable to sodium salicylate-induced blockade of cellular mechanisms that protect cells from calcium-mediated injury. These initial observations may have important clinical implications for patients with epilepsy who take aspirin while affected by these conditions, and should promote further investigation of this relationship.

Neurotrophin-induced modulation of synaptic transmission in the adult hippocampus.

The NGF-family of neurotrophic factors including NGF, BDNF and NT-3,4/5 is known to be crucial for neuronal survival and differentiation during development. However, recent studies suggest that the neurotrophins are also widely expressed and play a dynamic role in the mature nervous system. One of the major sites of expression of the neurotrophins in the adult brain is the hippocampus which has been also popular as an important structure for the adult plasticity. Moreover, the level of expression of the neurotrophins in the hippocampus can be regulated by a variety of neuronal inputs, such as experimentally-induced seizures, injection of glutamate receptor agonists, and LTP-inducing stimulation. The possibility that the neurotrophins modulate synaptic transmission in the mature brain has been investigated at the Schaffer collateral-CA1 synapses in the adult rat hippocampus. We report that transient application of BDNF and NT-3, but not NGF induces a long-lasting increase of synaptic transmission, which is likely to be mediated by Trk family of receptor tyrosine kinases. Both BDNF and NT-3 decrease paired pulse facilitation, suggesting a possible presynaptic modification. Interestingly, previous potentiation of synaptic activity by the application of neurotrophic factors does not occlude the induction of long-term potentiation. These results suggest that the neurotrophins may locally regulate synaptic plasticity in the adult nervous system.

Intracellular Ca(2+) signaling is required for neurotrophin-induced potentiation in the adult rat hippocampus.

Recent studies have demonstrated the importance of neurotrophin function in adult synaptic plasticity. In an effort to characterize the intracellular signaling pathways that couple Trk receptor activation to the final physiological effects of neurotrophins, we have examined the role of intracellular calcium rises in neurotrophin-induced synaptic enhancement in hippocampal slices. Using pharmacological blockers to two different calcium ion (Ca(2+)) sources, voltage-gated Ca(2+) channels and intracellular Ca(2+) stores, we show that the potentiating effects of neurotrophins in hippocampal slices are mediated by intracellular Ca(2+) signaling. Although basal synaptic transmission between hippocampal CA3 and CA1 neurons was not affected by nifedipine or thapsigargin, both drugs significantly attenuated brain-derived neurotrophic factor or neurotrophin-3-induced synaptic enhancement. The pharmacological blockade of Ca(2+) signaling is effective only during the initial period of neurotrophin-induced potentiation. These data suggest that the minimal requirements for inducing potentiation by neurotrophins involve a transient increase in intracellular Ca(2+) concentration, via voltage-gated Ca(2+) channels and/or intracellular Ca(2+) stores.