Changes In Synaptic Activity Research Articles

Activity-Dependent Dendritic Spine Structural Plasticity Is Regulated by Small GTPase Rap1 and Its Target AF-6

Activity-dependent remodeling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the mechanisms that coordinate synaptic structural and functional plasticity are not well understood. Here we investigate the signaling pathways that enable excitatory synapses to undergo activity-dependent structural modifications. We report that activation of NMDA receptors in cultured cortical neurons induces spine morphogenesis and activation of the small GTPase Rap1. Rap1 bimodally regulates spine morphology: activated Rap1 recruits the PDZ domain-containing protein AF-6 to the plasma membrane and induces spine neck elongation, while inactive Rap1 dissociates AF-6 from the membrane and induces spine enlargement. Rap1 also regulates spine content of AMPA receptors: thin spines induced by Rap1 activation have reduced GluR1-containing AMPA receptor content, while large spines induced by Rap1 inactivation are rich in AMPA receptors. These results identify a signaling pathway that regulates activity-dependent synaptic structural plasticity and coordinates it with functional plasticity.

Divergent effects of Aβ1–42 on ionotropic glutamate receptor-mediated responses in CA1 neurons in vivo

Aggregated Aβ1–42 is hypothesized to be the central cause of Alzheimer's disease. However, early changes in synaptic activity may be detected in the disease long before a significant cell loss is manifested. Despite the fact that Aβ1–42 interference with long-term potentiation (LTP) and the field excitatory postsynaptic potential (fEPSP) is well documented, the exact mechanism of these events remains to be clarified. Here we studied the effects of iontophoretically applied Aβ1–42 on the neuronal firing evoked in vivo on the CA1 hippocampal neurons of Wistar rats by different agonists of the ionotropic glutamate receptors: N-methyl- d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainic acid (KA). NMDA elicited firing enhanced in all of the measured cells; in contrast, the AMPA-mediated responses decreased significantly after Aβ1–42 ejection. The changes in KA-evoked responses to Aβ1–42 revealed two types of cells. In the first type, the KA-mediated firing remained at the control level, while in the second type, Aβ1–42 attenuated the KA-evoked responses. A protective pentapeptide, Leu–Pro–Tyr–Phe–Asp–amide, was used to verify the specificity of these β-amyloid-elicited effects. The pentapeptide protected against the modulatory effects of Aβ1–42 on the NMDA and AMPA responses. In conclusion, we have shown that Aβ1–42 exerts divergent effects on the activity of the ionotropic glutamate receptors in vivo. These results suggest that the LTP disruption and fEPSP attenuation seen after Aβ1–42 application are in part due to the altered function of these receptors.

Activity-induced tissue oxygenation changes in rat cerebellar cortex: Interplay of postsynaptic activity and cerebral blood flow

Neuronal activity, cerebral blood flow, and oxygen consumption are all strongly coupled, although the mechanisms behind remain unclear. We examined this coupling in rat cerebellar cortex by simultaneously recording local field potentials, tissue oxygen partial pressure (tpO2), and CBF using laser-Doppler flowmetry. Stimulation of the monosynaptic, excitatory climbing fiber system evoked activity-dependent increases in synaptic activity and blood flow, and biphasic tissue pO2 responses. Relating parameters of the initial negative tpO2 response to changes in synaptic activity revealed that the disappearance rate of oxygen in the tissue varied as a linear function of synaptic activity, while the area of the negative tpO2 response saturated at high levels of synaptic activity. Following the initial oxygen dip, tpO2 increased as a non-linear function of synaptic activity, paralleling the steep rise in blood flow at high levels of synaptic activity. Attenuation of the activity-dependent rise in blood flow by nNOS inhibition enlarged the negative tpO2 response and decreased the positive tpO2 response. These findings imply that the activity-induced reduction in tpO2 is counteracted by an increase in oxygen supply due to the rise in CBF. They also support the hypothesis that the positive tpO2 response is an overshoot caused by the evoked increase in CBF. Blockade of glutamatergic AMPA receptors with CNQX abolished synaptic activity, and CBF and tpO2 responses. We suggest, that in the cerebellum activity-dependent increaes in oxygen consumption can be recorded as the disapperance rate of oxygen in the tissue, and that oxygen consumption increases as a linear function of activity in postsynaptic cellular elements without evidence for a threshold. Our results provide new fundamental insights into the regulation of oxygen consumption during activation, showing direct coupling of activity in postsynaptic AMPA receptors to oxygen consumption.

Molecular Mechanisms of Cytokine-Induced Neuroprotection: NFκB and Neuroplasticity

Since the first attempts to understand the mechanisms of learning, memory, development, and other instances of neuroplasticity, gene expression has been an attractive explanation for the persistence of such processes. It has been hypothesized that changes in the levels of expression of a gene, or a coordinated set of genes, would be necessary for dramatic structural changes like the growth of new neurites. And more subtle biochemical changes at existing synapses might also result from an alteration in the array of gene products being manufactured in the relevant cells. However, a great deal of what is classified as neuroplasticity is dependent on primary changes in electrophysiological activity or other conditions at synapses. Therefore, a seminal question to those interested in the molecular underpinnings of neuroplasticity is that of signal transduction: How do changes in synaptic activity get communicated to the nucleus? To many who learn about the regulation of the transcription factor NFkappaB with this question in mind, its utility seems clear. Furthermore, NFkappaB is an important signaling factor for cytokines that appear to participate in several pathological conditions (e.g., Parkinson's disease, multiple sclerosis, and depression), so understanding its mechanisms of action and its relationship to other elements of cytokine signaling may be fundamental to determining the role of the inflammatory system in psychiatric and neurodegenerative conditions. For these reasons, NFkappaB has garnered considerable attention in various aspects of neuroplasticity, from long-term potentiation to the most dramatic forms of plasticity: cell birth and death. In a few cases, elegant experimental design has resulted in convincing evidence for the involvement of NFkappaB in specific phenomena. However, the complexity of this transcription factor-including confusion over what exactly is meant by "NFkappaB"-has led to some misleading conclusions, as well. This chapter highlights some of the potential red herrings to be encountered in the study of NFkappaB and will summarize the data and interpretations in which some degree of confidence can be placed. The final answers will depend on the application of models and tools only now in development.

High Affinity Transport of Taurine by the Drosophila Aspartate Transporter dEAAT2

Excitatory amino acid transporters (EAATs) are structurally related plasma membrane proteins known to mediate the Na(+)/K(+)-dependent uptake of the amino acids l-glutamate and dl-aspartate. In the nervous system, these proteins contribute to the clearance of glutamate from the synaptic cleft and maintain excitatory amino acid concentrations below excitotoxic levels. Two homologues exist in Drosophila melanogaster, dEAAT1 and dEAAT2, which are specifically expressed in the nervous tissue. We previously reported that dEAAT2 shows unique substrate discrimination as it mediates high affinity transport of aspartate but not glutamate. We now show that dEAAT2 can also transport the amino acid taurine with high affinity, a property that is not shared by two other transporters of the same family, Drosophila dEAAT1 and human hEAAT2. Taurine transport by dEAAT2 was efficiently blocked by an EAAT antagonist but not by inhibitors of the structurally unrelated mammalian taurine transporters. Taurine and aspartate are transported with similar K(m) and relative efficacy and behave as mutually competitive inhibitors. dEAAT2 can mediate either net uptake or the heteroexchange of its two substrates, both being dependent on the presence of Na(+) ions in the external medium. Interestingly, heteroexchange only occurs in one preferred substrate orientation, i.e. with taurine transported inwards and aspartate outwards, suggesting a mechanism of transinhibition of aspartate uptake by intracellular taurine. Therefore, dEAAT2 is actually an aspartate/taurine transporter. Further studies of this protein are expected to shed light on the role of taurine as a candidate neuromodulator and cell survival factor in the Drosophila nervous system.

Activity-Dependent Presynaptic Regulation of Quantal Size at the Mammalian Neuromuscular JunctionIn Vivo

Changes in synaptic activity alter quantal size, but the relative roles of presynaptic and postsynaptic cells in these changes are only beginning to be understood. We examined the mechanism underlying increased quantal size after block of synaptic activity at the mammalian neuromuscular junction in vivo. We found that changes in neither acetylcholinesterase activity nor acetylcholine receptor density could account for the increase. By elimination, it appears likely that the site of increased quantal size after chronic block of activity is presynaptic and involves increased release of acetylcholine. We used mice with muscle hyperexcitability caused by mutation of the ClC-1 muscle chloride channel to examine the role of postsynaptic activity in controlling quantal size. Surprisingly, quantal size was increased in ClC mice before block of synaptic activity. We examined the mechanism underlying increased quantal size in ClC mice and found that it also appeared to be located presynaptically. When presynaptic activity was completely blocked in both control and ClC mice, quantal size was large in both groups despite the higher level of postsynaptic activity in ClC mice. This suggests that postsynaptic activity does not regulate quantal size at the neuromuscular junction. We propose that presynaptic activity modulates quantal size at the neuromuscular junction by modulating the amount of acetylcholine released from vesicles.

Activity-dependent endocytic sorting of kainate receptors to recycling or degradation pathways.

Kainate receptors (KARs) play important roles in the modulation of neurotransmission and plasticity, but the mechanisms that regulate their surface expression and endocytic sorting remain largely unknown. Here, we show that in cultured hippocampal neurons the surface expression of GluR6-containing KARs is dynamically regulated. Furthermore, internalized KARs are sorted into recycling or degradative pathways depending on the endocytotic stimulus. Kainate activation causes a Ca2+- and PKA-independent but PKC-dependent internalization of KARs that are targeted to lysosomes for degradation. In contrast, NMDAR activation evokes a Ca2+-, PKA- and PKC-dependent endocytosis of KARs to early endosomes with subsequent reinsertion back into the plasma membrane. These results demonstrate that GluR6-containing KARs are subject to activity-dependent endocytic sorting, a process that provides a mechanism for both rapid and chronic changes in the number of functional receptors.

Exposure to interferon‐γ during synaptogenesis increases inhibitory activity after a latent period in cultured rat hippocampal neurons

Certain disorders of the nervous system may have their origin in disturbances in the development of synaptic connections and network structure that may not become overt until later in life. As inflammatory cytokines can influence synaptic activity in neuronal cultures, we analysed whether cytokine exposure during synaptogenesis can lead to imbalances in a neuronal network. Short-term application of interferon-gamma (IFN-gamma), but not tumour necrosis factor-alpha, during peak synaptogenesis (but not before or after) in Sprague-Dawley rat hippocampal cultures, caused both a decrease in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) and an increase in the frequency of spontaneous inhibitory postsynaptic currents (IPSCs). These effects were only detected in recordings made weeks later. This was not due to a depression of glutamatergic synapses or to a change in the relative number of neurons containing glutamic acid decarboxylase (GAD). There was an increase in the average amplitude of miniature IPSCs, and in GAD-expressing neurons the amplitude of miniature EPSCs were larger as well as the responses to glutamate. This indicates that IFN-gamma-treatment induced increased inhibition via postsynaptic changes. These effects of IFN-gamma treatment were not observed when neuronal nitric oxide synthase was inhibited. Our study therefore shows that exposure to IFN-gamma during a restricted period of development, which coincides with the peak of excitatory synaptogenesis, can cause progressive changes in synaptic activity in the network. Thus, cytokine exposure at a critical period of development may constitute a 'hit-and-run' mechanism for certain nervous system disorders that become manifest after a latency period.

Subtypes of α1‐ and α2‐Adrenoceptors Mediating Noradrenergic Modulation of Spontaneous Inhibitory Postsynaptic Currents in the Hypothalamic Paraventricular Nucleus

2-6 of the above paper contained a number of errors in the previously published version. The corrected figures are reproduced below. Blockade of the noradrenaline-induced spontaneous inhibitory postsynaptic current (sIPSC) frequency decrease by BRL44408, an α2A-AR antagonist, but not by prazosin, an α2B/2C-AR antagonist. (a) and (b) Time course histograms showing the effects of noradrenaline on sIPSC frequency of paraventricular nucleus (PVN) neurones in the presence of (a) BRL44408 (3 µM) or (b) prazosin (100 µM). (c,d) Current records of the same neurones at the times shown by the arrows in (a) and (b), respectively. (e,f) Mean effects of noradrenaline (30–100 µM) on sIPSC of types I and II PVN neurones in the presence of (e) BRL44408 (1–10 µM) or (f) prazosin (20–100 µM). BRL, BRL44408; Pra, prazosin. Values are presented as means ± SEM from six type I and five type II neurones for BRL44408 and from three type I and five type II PVN neurones for prazosin. Significantly different from the controls at *P < 0.05, **P < 0.01 and ***P < 0.001 by Student's t-test, respectively. Noradrenaline-induced increases in spontaneous inhibitory postsynaptic current (sIPSC) frequency in the presence of RS17053, an α1A-AR antagonist, or BMY7378, an α1D-AR antagonist in type II paraventricular nucleus (PVN) neurones. (a,b) Time-course histograms showing the noradrenaline-induced sIPSC frequency increase in the presence of (a) RS17053 (10 µM) or (b) BMY 7378 (2 µM). (c,d) Current records of the same neurones at the times shown by the arrows in (a) and (b), respectively. (e,f) Mean effects of noradrenaline in the presence of (e) RS17053 (10 µM, n = 5) or (f) BMY7378 (2–20 µM, n = 4). RS, RS17053; BMY, BMY7378. Significantly different from the control at *P < 0.05 by t-test (noradrenaline) or the Mann–Whitney U-test (BMY + noradrenaline), or **P < 0.01 by t-test. Concentration dependent block of noradrenaline-induced spontaneous inhibitory postsynaptic current (sIPSC) frequency increase by prazosin in type II paraventricular nucleus (PVN) neurones. (a) Time-course histogram showing the effect of noradrenaline on sIPSC frequency in the presence of prazosin at 0.1 and 1 µM. (b) Current records showing changes in synaptic activity at the times shown by the arrows in (a). (c) Mean effects of noradrenaline on sIPSC frequency the presence of prazosin at 0.1 µM (n = 5) and 1 µM (n = 6). *P < 0.05 by Student's t-test.

Homeostatic scaling of neuronal excitability by synaptic modulation of somatic hyperpolarization-activated Ih channels.

The hyperpolarization-activated cation current (Ih) plays an important role in determining membrane potential and firing characteristics of neurons and therefore is a potential target for regulation of intrinsic excitability. Here we show that an increase in AMPA-receptor-dependent synaptic activity induced by alpha-latrotoxin or glutamate application as well as direct depolarization results in an increase in Ih recorded from cell-attached patches in hippocampal CA1 pyramidal neurons. This mechanism requires Ca2+ influx but not increased levels of cAMP. Artificially increasing Ih by using a dynamic clamp during whole-cell current clamp recordings results in reduced firing rates in response to depolarizing current injections. We conclude that modulation of somatic Ih represents a previously uncharacterized mechanism of homeostatic plasticity, allowing a neuron to control its excitability in response to changes in synaptic activity on a relatively short-term time scale.

Short- and long-term modulation of synaptic inputs to brain reward areas by nicotine.

Dopamine signaling in brain reward areas is a key element in the development of drug abuse and dependence. Recent anatomical and electrophysiological research has begun to elucidate both complexity and specificity in synaptic connections between ventral tegmental neurons and their inputs. Specifically, the activity of dopamine neurons in the ventral tegmental area relies on the combination of both excitatory and inhibitory inputs. Controlling endogenous neurotransmission to dopamine neurons is one mechanism by which drugs of abuse affect both transient and long-term changes in synaptic activity. Here, we review recent findings concerning glutamatergic, GABAergic, and cholinergic inputs to dopamine neurons, and their roles in the reinforcement associated with drug abuse. Importantly, several studies support that a single drug exposure can lead to changes in synaptic strength that are associated with learning and memory. Ultimately, these cellular changes could underlie the long-lasting effects of drugs. Furthermore, nicotinic acetylcholine receptors in the ventral tegmental area emerge as a possible common target for the behavioral and cellular actions not only of nicotine, but also of several other drugs of abuse. Finally, we explore age-related differences in nicotine sensitivity in order to understand both human epidemiological data, and laboratory animal behavioral findings that suggest adolescents are more susceptible to developing nicotine dependence.

Memories of Degradation

Activity-dependent changes in structure and function of neuronal synapses are thought to provide the molecular basis for learning and memory in the brain. Attention has focused on requirements of transcription and translation for such synaptic changes, but Ehlers describes experiments indicating that ubiquitin-dependent protein degradation also contributes to remodeling at the postsynaptic density (PSD) (where neurotransmitter receptors are clustered with various signaling proteins). Pharmacological treatments of cultured rat cortical neurons that increased synaptic activity caused increases in the abundance of some proteins at isolated PSDs but also caused decreases in the abundance of others. Pharmacological inhibition of synaptic activity caused reciprocal changes (increasing, for example, the abundance of proteins that were lost at activated synapses). Pulse-chase analysis of protein turnover showed that changes in turnover accounted for some of the alterations noted above. Protein degradation can be acutely controlled by ubiquitination, which targets a protein for degradation by the proteasome. Ehlers found that three postsynaptic scaffold proteins (proteins that organize clusters of signaling molecules at synapses) underwent increased ubiquitination in cultured neurons in which synaptic activity was stimulated and showed decreased ubiquitination when isolated from neurons in which synaptic activity was blocked. These changes were correlated with a reciprocal switch in coupling of NMDA-type glutamate neurotransmitter receptors to stimulation of CREB (the cyclic AMP response element-binding protein) or mitogen-activated protein kinases, respectively. These alterations in receptor coupling to downstream signaling pathways were prevented when protein degradation was blocked by treatment of cells with inhibitors of the proteasome. Together the results suggest that long-term changes in synaptic activity can be brought about through regulated degradation of key organizing molecules in the synapse. M. D. Ehlers, Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system. Nature Neurosci . 6 , 231-242 (2003). [Online Journal]

Cross Synaptic Communication

Two groups report how changes in the cadherin-catenin-cytoskeleton interactions alter presynaptic and postsynaptic structure and synaptic activity. Cadherins are calcium-dependent homophilic adhesion proteins that bind to β-catenin, which in turn binds to α-catenin, which interacts with the cytoskeleton. The interaction between cadherin and β-catenin is regulated by phosphorylation with a decreased interaction between the two when β-catenin is phosphorylated. Togashi et al. disrupted cadherin interactions in cultured hippocampal neurons labeled for presynaptic and postsynaptic marker proteins. In cultures transfected with a dominant negative form of N-cadherin (DN-cad) that could not form homophilic interactions, but could still bind β-catenin, the postsynaptic dendritic spine morphology was altered to an elongated structure lacking the typical mushroom-shaped ending. Presynaptic activity was decreased (measured by uptake of the dye FM-64 as an indicator of vesicle cycling), and postsynaptic marker proteins were more diffusely located than in control cultures. The same types of changes were observed in cultured hippocampal neurons from αN-catenin (a neuronal-specific isoform) knockout mice, which die shortly after birth. Murase et al. observed that depolarization or treatment of hippocampal slices with a tyrosine kinase inhibitor increased the intensity of punctate β-catenin staining. In cultured hippocampal neurons, depolarization increased β-catenin in the spine and decreased β-catenin in the dendritic shaft. Treatments that pharmacologically manipulate kinase and phosphatase activities also altered β-catenin distrubution. The changes in β-catenin distribution were mimicked by tyrosine mutants of β-catenin: Y654E mimicked the phosphorylated state of β-catenin, with decreased cadherin binding and decreased accumulation in the spine; and Y654F mimicked the nonphosphorylated state of β-catenin, with increased cadherin binding and increased accumulation in dendritic spines. Changes in synaptic activity also correlated with β-catenin distribution. Synapses with the Y654 mutation had increased miniature excitatory postsynaptic currents and increased FM-64 dye uptake, indicating enhanced synaptic activity. Thus, changes in the dendritic morphology and postsynaptic cadherin-catenin complex can transmit information across the synapse to regulate presynaptic activity and morphology (see Goda for more discussion).H. Togashi, K. Abe, A. Mizoguchi, K. Takaoka, O. Chisaka, M. Takeichi, Cadherin regulates dendritic spine morphogenesis. Neuron 35, 77-89 (2002). [Online Journal]S. Murase, E. Mosser, E. M. Schuman, Depolarization drives β-catenin into neuronal spines promoting changes in synaptic structure and function. Neuron 35, 91-105 (2002). [Online Journal]Y. Goda, Cadherins communicate structural plasticity of presynaptic and postsynaptic terminals. Neuron 35, 1-7 (2002). [Online Journal]

Cross Synaptic Communication

Two groups report how changes in the cadherin-catenin-cytoskeleton interactions alter presynaptic and postsynaptic structure and synaptic activity. Cadherins are calcium-dependent homophilic adhesion proteins that bind to β-catenin, which in turn binds to α-catenin, which interacts with the cytoskeleton. The interaction between cadherin and β-catenin is regulated by phosphorylation with a decreased interaction between the two when β-catenin is phosphorylated. Togashi et al. disrupted cadherin interactions in cultured hippocampal neurons labeled for presynaptic and postsynaptic marker proteins. In cultures transfected with a dominant negative form of N-cadherin (DN-cad) that could not form homophilic interactions, but could still bind β-catenin, the postsynaptic dendritic spine morphology was altered to an elongated structure lacking the typical mushroom-shaped ending. Presynaptic activity was decreased (measured by uptake of the dye FM-64 as an indicator of vesicle cycling), and postsynaptic marker proteins were more diffusely located than in control cultures. The same types of changes were observed in cultured hippocampal neurons from αN-catenin (a neuronal-specific isoform) knockout mice, which die shortly after birth. Murase et al. observed that depolarization or treatment of hippocampal slices with a tyrosine kinase inhibitor increased the intensity of punctate β-catenin staining. In cultured hippocampal neurons, depolarization increased β-catenin in the spine and decreased β-catenin in the dendritic shaft. Treatments that pharmacologically manipulate kinase and phosphatase activities also altered β-catenin distrubution. The changes in β-catenin distribution were mimicked by tyrosine mutants of β-catenin: Y654E mimicked the phosphorylated state of β-catenin, with decreased cadherin binding and decreased accumulation in the spine; and Y654F mimicked the nonphosphorylated state of β-catenin, with increased cadherin binding and increased accumulation in dendritic spines. Changes in synaptic activity also correlated with β-catenin distribution. Synapses with the Y654 mutation had increased miniature excitatory postsynaptic currents and increased FM-64 dye uptake, indicating enhanced synaptic activity. Thus, changes in the dendritic morphology and postsynaptic cadherin-catenin complex can transmit information across the synapse to regulate presynaptic activity and morphology (see Goda for more discussion). H. Togashi, K. Abe, A. Mizoguchi, K. Takaoka, O. Chisaka, M. Takeichi, Cadherin regulates dendritic spine morphogenesis. Neuron 35 , 77-89 (2002). [Online Journal] S. Murase, E. Mosser, E. M. Schuman, Depolarization drives β-catenin into neuronal spines promoting changes in synaptic structure and function. Neuron 35 , 91-105 (2002). [Online Journal] Y. Goda, Cadherins communicate structural plasticity of presynaptic and postsynaptic terminals. Neuron 35 , 1-7 (2002). [Online Journal]

Inhibition and functional magnetic resonance imaging

This review summarizes our current knowledge on how inhibitory phenomena are reflected in the functional magnetic resonance imaging (fMRI) signal. It is well-established that activity-related changes of brain metabolism and blood flow are dominated by changes in synaptic activity. Both excitatory and inhibitory synaptic activities are associated with increased metabolic demands. The amount of energy consumption associated with inhibition vs. excitation is reflected in metabolism- and blood flow-related neuroimaging signals such as the blood oxygen level-dependent (BOLD) contrast; the relationship between the different “signals”, however, may not be linear. The influence on these signals depends on the number of active inhibiting synapses, the duration of inhibition and the degree of propagation within subsequent neuronal circuits. The relative influence of inhibition as compared to excitation on the metabolism/blood flow may also vary in different neuronal circuits. Inhibition leads to locally decreased discharge activity, which does not have a significant effect on the cerebral blood flow (CBF)/BOLD images. However, inhibition may also result in suppression of complex neuronal circuits, leading to a decrease in excitatory as well as inhibitory synaptic activity, and therewith, to a decreased metabolism and blood flow within those complex neuronal networks. The available fMRI data indicate that, depending on the abovementioned factors, inhibition may be reflected in positive, negative or no BOLD-signal at all. Thus, the BOLD-signal is ambiguous with respect to the underlying electrophysiological event. In the future, combining fMRI with electrophysiological methods will strengthen neuroimaging studies.

Interferon-γ-induced changes in synaptic activity and AMPA receptor clustering in hippocampal cultures

Extended release of interferon-γ (IFN-γ) in the nervous system during immunological and infectious conditions may trigger demyelinating disorders and cause disturbances in brain function. The aim of this study was to examine the effects of IFN-γ on neuronal function in rat hippocampal cell cultures by using whole cell patch clamp analysis together with quantitative immunocytochemistry. Acute application of IFN-γ to differentiated neurons in culture caused no immediate neurophysiological responses, but recordings after 48 h of incubation displayed an increase in frequency of AMPA receptor (AMPAR)-mediated spontaneous excitatory postsynaptic currents (EPSCs). Quantitative immunocytochemistry for the AMPAR subunit GluR1 showed no alteration in receptor clustering at this time point. However, prolonged treatment with IFN-γ for 2 weeks resulted in a significant reduction in AMPAR clustering on dendrites but no marked differences in EPSC frequency between treated neurons and controls could be observed. On the other hand, treatment of hippocampal neurons for 4 weeks, instituted at an immature stage (1 day in culture), caused a significant reduction in spontaneous EPSC frequency. These neurons developed with no overt alterations in dendritic arborization or in the appearance of dendritic spines as visualized by α-actinin immunocytochemistry. Nonetheless, there was a marked reduction in AMPAR clustering on dendrites. These observations show that a key immunomodulatory molecule, IFN-γ, can cause long-term modifications of synaptic activity and perturb glutamate receptor clustering.

Optical measurements of presynaptic nicotinic effects

We have used the styryl dye FM 2-10 to monitor changes in synaptic activity mediated by nicotinic acetylcholine receptors (AChRs) on cultured hippocampal neurons. We show that both 100 μM ACh and nicotine at 20 μM causes a significant increase in staining intensities of presynaptic boutons in the presence of 0.5 μM tetrodotoxin (TTX). This effect of nicotine is blocked by d-tubocurarine. Interestingly, nicotine also had a long-lasting effect on high potassium-induced staining of boutons. These results suggest that nicotine can have significant and sustained effects on synaptic efficacy in cultured hippocampal neurons.

The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses.

Neuroplastin-65 and -55 (previously known as gp65 and gp55) are glycoproteins of the Ig superfamily that are enriched in rat forebrain synaptic membrane preparations. Whereas the two-Ig domain isoform neuroplastin-55 is expressed in many tissues, the three-Ig domain isoform neuroplastin-65 is brain-specific and enriched in postsynaptic density (PSD) protein preparations. Here, we have assessed the function of neuroplastin in long-term synaptic plasticity. Immunocytochemical studies with neuroplastin-65-specific antibodies differentially stain distinct synaptic neuropil regions of the rat hippocampus with most prominent immunoreactivity in the CA1 region and the proximal molecular layer of the dentate gyrus. Kainate-induced seizures cause a significant enhancement of neuroplastin-65 association with PSDs. Similarly, long-term potentiation (LTP) of CA1 synapses in hippocampal slices enhanced the association of neuroplastin-65 with a detergent-insoluble PSD-enriched protein fraction. Several antibodies against the neuroplastins, including one specific for neuroplastin-65, inhibited the maintenance of LTP. A similar effect was observed when recombinant fusion protein containing the three extracellular Ig domains of neuroplastin-65 was applied to hippocampal slices before LTP induction. Microsphere binding experiments using neuroplastin-F(c) chimeric proteins show that constructs containing Ig1-3 or Ig1 domains, but not Ig2-3 domains mediate homophilic adhesion. These data suggest that neuroplastin plays an essential role in implementing long-term changes in synaptic activity, possibly by means of a homophilic adhesion mechanism.

Synaptic Activity Modulates the Induction of Bidirectional Synaptic Changes in Adult Mouse Hippocampus

Activity-dependent synaptic plasticity is critical for learning and memory. Considerable attention has been paid to mechanisms that increase or decrease synaptic efficacy, referred to as long-term potentiation (LTP) and long-term depression (LTD), respectively. It is becoming apparent that synaptic activity also modulates the ability to elicit subsequent synaptic changes. We provide direct experimental evidence that this modulation is attributable, at least in part, to variations in the level of postsynaptic depolarization required for inducing plasticity. In slices from adult hippocampal CA1, a brief pairing protocol known to produce LTP can also induce LTD. The voltage-response function for the induction of LTD and LTP in naive synapses exhibits three parts: at a postsynaptic membrane potential during pairing (V(m)) </= -40 mV, no synaptic modification is obtained; at V(m) between -40 and -20 mV, LTD is induced; and, finally, at V(m) > -20 mV, LTP is generated. This function varies with initial synaptic efficacy. In depressed synapses, Theta(-), the V(m) above which LTD is generated, is shifted toward more depolarized V(ms) and Theta(+), the LTD-LTP crossover point or, equivalently, the V(m) above which LTP is induced, toward more polarized V(ms). Conversely in potentiated synapses, Theta(-) is shifted toward more polarized V(ms). Therefore synaptic activity changes synaptic efficacy and accordingly adjusts the voltages for eliciting subsequent synaptic modifications. The concomitant shifts in the voltages for inducing LTD and LTP in opposite directions promote synaptic potentiation and inhibit synaptic depression in depressed synapses and vice versa in potentiated synapses.

Calcium regulation of the brain-derived neurotrophic factor gene.

Results from several laboratories have suggested that peptide factors known as neurotrophins may play roles coupling changes in synaptic activity to lasting changes in synaptic function. Consistent with this idea, increases in synaptic activity and intracellular calcium induce the expression of the gene that encodes the neurotrophin, brain-derived neurotrophic factor. Recently, a pathway has been elucidated in neurons by which the influx of extracellular calcium evokes brain-derived neurotrophic factor transcription (BDNF). Calcium activates BDNF transcription through two adjacent calcium response elements within one of the promoters of the BDNF gene. One of the two elements binds to the cyclic adenosine monophosphate (AMP) response element binding protein (CREB) transcription factor, and interfering with CREB or related family members inhibits calcium-dependent BDNF transcription. This review focuses on the mechanisms by which calcium influx regulates brain-derived neurotrophic factor expression and the implications that these results have for potential roles of neurotrophins in synaptic function.