Astrocytes Are the Source of TNF Mediating Homeostatic Synaptic Plasticity.

Tumor necrosis factor α (TNF) mediates homeostatic synaptic plasticity (HSP) in response to chronic activity blockade, and prior work has established that it is released from glia. Here we demonstrate that astrocytes are the necessary source of TNF during HSP. Hippocampal cultures from rats of both sexes depleted of microglia still will increase TNF levels following activity deprivation and still express TTX-driven HSP. Slice cultures from mice of either sex with a conditional deletion of TNF from microglia also express HSP, but critically, slice cultures with a conditional deletion of TNF from astrocytes do not. In astrocytes, glutamate signaling is sufficient to reduce NFκB signaling and TNF mRNA levels. Further, chronic TTX treatment increases TNF in an NFκB-dependent manner, although NFκB signaling is dispensable for the neuronal response to TTX-driven HSP. Thus, astrocytes can sense neuronal activity through glutamate spillover and increase TNF production when activity falls, to drive HSP through the production of TNF.

Sustained TNF signaling is required for the synaptic and anxiety-like behavioral response to acute stress.

Acute stress triggers plasticity of forebrain synapses as well as behavioral changes. Here we reveal that Tumor Necrosis Factor α (TNF) is a required downstream mediator of the stress response in mice, necessary for stress-induced synaptic potentiation in the ventral hippocampus and for an increase in anxiety-like behaviour. Acute stress is sufficient to activate microglia, triggering the long-term release of TNF. Critically, on-going TNF signaling specifically in the ventral hippocampus is necessary to sustain both the stress-induced synaptic and behavioral changes, as these could be reversed hours after induction by antagonizing TNF signaling. This demonstrates that TNF maintains the synaptic and behavioral stress response in vivo, making TNF a potential novel therapeutic target for stress disorders.

Divergent Synaptic Scaling of Miniature EPSCs following Activity Blockade in Dissociated Neuronal Cultures.

Neurons can respond to decreased network activity with a homeostatic increase in the amplitudes of miniature EPSCs (mEPSCs). The prevailing view is that mEPSC amplitudes are uniformly multiplied by a single factor, termed "synaptic scaling." Deviations from purely multiplicative scaling have been attributed to biological differences, or to a distortion imposed by a detection threshold limit. Here, we demonstrate in neurons dissociated from cortices of male and female mice that the shift in mEPSC amplitudes observed in the experimental data cannot be reproduced by simulation of uniform multiplicative scaling, with or without the distortion caused by applying a detection threshold. Furthermore, we demonstrate explicitly that the scaling factor is not uniform but is close to 1 for small mEPSCs, and increases with increasing mEPSC amplitude across a substantial portion of the data. This pattern was also observed for previously published data from dissociated mouse hippocampal neurons and dissociated rat cortical neurons. The finding of "divergent scaling" shifts the current view of homeostatic plasticity as a process that alters all synapses on a neuron equally to one that must accommodate the differential effect observed for small versus large mEPSCs. Divergent scaling still accomplishes the essential homeostatic task of modifying synaptic strengths in the opposite direction of the activity change, but the consequences are greatest for those synapses which individually are more likely to bring a neuron to threshold.SIGNIFICANCE STATEMENT In homeostatic plasticity, the responses to chronic increases or decreases in network activity act in the opposite direction to restore normal activity levels. Homeostatic plasticity is likely to play a role in diseases associated with long-term changes in brain function, such as epilepsy and neuropsychiatric illnesses. One homeostatic response is the increase in synaptic strength following a chronic block of activity. Research is focused on finding a globally expressed signaling pathway, because it has been proposed that the plasticity is uniformly expressed across all synapses. Here, we show that the plasticity is not uniform. Our work suggests that homeostatic signaling molecules are likely to be differentially expressed across synapses.

Astrocytic TNFα regulates the behavioral response to antidepressants.

Recent studies have suggested that cytokines, and in particular tumor necrosis factor alpha (TNFα), have a role in modulating antidepressant efficacy. To directly test this idea, we compared the response of TNFα(-/-) mice and astrocyte-specific TNFα(-/-) mice to the antidepressants fluoxetine and desipramine. Using standard behavior models for measuring antidepressant efficacy, the forced swim test (FST) and tail suspension test (TST), we determined that TNFα(-/-) mice were essentially normal in basal behavior in the FST and TST. However, TNFα(-/-) mice showed no behavioral response to a standard dose of chronic antidepressant treatment, in sharp contrast to wildtype mice. Similar results were seen with acute antidepressant treatment, but TNFα(-/-) mice did respond to a very high-dose acute antidepressant treatment. We also assessed in vitro and in vivo effects of fluoxetine on TNFα expression. Glia responded to serotonin in vitro and fluoxetine in vivo by upregulating TNFα mRNA. Consistent with this source of TNFα, mice with an astrocyte-specific deletion of TNFα also did not respond to standard chronic antidepressant treatment. These data suggest that astrocytic TNFα is important to the sensitivity of the behavioral response to administration of antidepressants.

FXR1P limits long-term memory, long-lasting synaptic potentiation, and de novo GluA2 translation.

Translational control of mRNAs allows for rapid and selective changes in synaptic protein expression that are required for long-lasting plasticity and memory formation in the brain. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein that controls mRNA translation in nonneuronal cells and colocalizes with translational machinery in neurons. However, its neuronal mRNA targets and role in the brain are unknown. Here, we demonstrate that removal of FXR1P from the forebrain of postnatal mice selectively enhances long-term storage of spatial memories, hippocampal late-phase long-term potentiation (L-LTP), and de novo GluA2 synthesis. Furthermore, FXR1P binds specifically to the 5' UTR of GluA2 mRNA to repress translation and limit the amount of GluA2 that is incorporated at potentiated synapses. This study uncovers a mechanism for regulating long-lasting synaptic plasticity and spatial memory formation and reveals an unexpected divergent role of FXR1P among Fragile X proteins in brain plasticity.

Persistent synaptic scaling independent of AMPA receptor subunit composition.

Despite long-standing evidence that the specific intracellular domains of AMPA-type glutamate receptor (AMPAR) subunits are critical for trafficking, it has recently been demonstrated that there is no absolute requirement for any AMPAR subunit for the receptor insertion underlying LTP. It is unclear whether this holds true to other forms of plasticity. Homeostatic synaptic plasticity (HSP) is an important form of negative feedback that provides stability to neuronal networks, and results at least in part from the insertion of AMPARs into glutamatergic synapses following chronic reductions in neuronal activity. Similar to LTP, the GluA1 subunit has been suggested to be the requisite subunit for HSP-induced AMPAR insertion and acute treatment with signaling molecules that underlie some forms of HSP results in the preferential incorporation of GluA2-lacking receptors. However, knockdown experiments have instead implicated a requirement for the GluA2 subunit. Here we re-examined the requirement for specific AMPAR subunit during chronic tetrodotoxin-induced HSP using hippocampal cultures derived from AMPAR subunit knock-out mice. We observed HSP in cultures from GluA1⁻/⁻, GluA2⁻/⁻, and GluA2⁻/⁻ GluA3⁻/⁻ mice, and conclude that, as with LTP, there is no subunit requirement for HSP.

An essential role for inhibitor-2 regulation of protein phosphatase-1 in synaptic scaling.

Protein phosphatase-1 (PP1) activity is important for many calcium-dependent neuronal functions including Hebbian synaptic plasticity and learning and memory. PP1 activity is necessary for the induction of long-term depression, whereas downregulation of PP1 activity is required for the normal induction of long-term potentiation. However, how PP1 is activated is not clear. Moreover, it is not known whether PP1 plays a role in homeostatic synaptic scaling, another form of synaptic plasticity which functions to reset the neuronal firing rate in response to chronic neuronal activity perturbations. In this study, we found that PP1 inhibitor-2 (I-2) is phosphorylated at serine 43 (S43) in rat and mouse cortical neurons in response to bicuculine application. Expression of I-2 phosphorylation-blocking mutant I-2 (S43A) blocked the dephosphorylation of GluA2 at serine 880, AMPA receptor trafficking, and synaptic downscaling induced by bicuculline application. Our data suggest that the phosphorylation of I-2 at S43 appears to be mediated by L-type calcium channels and calcium/calmodulin-dependent myosin light-chain kinase. Our work thus reveals a novel calcium-induced PP1 activation pathway critical for homeostatic synaptic plasticity.

Functional and structural properties of the NCKX2 Na(+)-Ca (2+)/K (+) exchanger: a comparison with the NCX1 Na (+)/Ca (2+) exchanger.

Na(+)/Ca(2+)-K(+) exchangers (NCKX), alongside the more widely known Na(+)/Ca(2+) exchangers (NCX), are important players in the cellular Ca(2+) toolkit. But, unlike NCX, much less is known about the physiological roles of NCKX, while emergent evidence indicates that NCKX has highly specialized functions in cells and tissues where it is expressed. As their name implies, there are functional similarities in the properties of the two Ca(2+) exchanger families, but there are specific differences as well. Here, we compare and contrast their key functional properties of ionic dependence and affinities, as well as report on the effects of KB-R7943 - a compound that is widely used to differentiate the two exchangers. We also review structural similarities and differences between the two exchangers. The aim is to draw attention to key differences that will aid in differentiating the two exchangers in physiological contexts where both exist but perhaps play distinct roles.

Residues contributing to the Na(+)-binding pocket of the SLC24 Na(+)/Ca(2+)-K(+) Exchanger NCKX2.

Na(+)/Ca(2+)-K(+) exchangers (NCKX; gene family SLC24) are plasma membrane Ca(2+) transporters that mediate the extrusion of one Ca(2+) ion and one K(+) ion in exchange for four Na(+) ions. NCKX is modeled to have two sets of five transmembrane segments separated by a large cytosolic loop; within each set of transmembrane segments are regions of internal symmetry termed alpha(1) and alpha(2) repeats. The central residues that are important for Ca(2+) and K(+) liganding and transport have been identified in NCKX2, and they comprise three central acidic residues, Glu(188) in alpha(1) and Asp(548) and Asp(575) in alpha(2), as well as Ser/Thr residues one-helical turn away from these residues. In this study, we have scanned through more than 100 single-residue substitutions of NCKX2 for shifts in Na(+) affinity using a fluorescence assay to monitor changes in free Ca(2+) in HEK293 cells treated with gramicidin to control intracellular Na(+). We have identified 31 residues that, when substituted, result in shifts in Na(+) affinity, either toward higher or lower K(m) values when compared with wild type NCKX2 (K(m) for Na(+) 58 mm). These residues include the central acidic residues Glu(188), Asp(548), and Asp(575), and their neighboring residues in alpha(1) and alpha(2), in addition to a number of newly investigated residues in transmembrane segment 3. Our results relate the identification of residues important for Na(+) transport in this study to those previously identified as important in the counter-transport of Ca(2+) and K(+), lending support to the alternating access model of transmembrane transport.

Examining Ca2+ extrusion of Na+/Ca2+-K+ exchangers.

Na+/Ca2+-K+ exchangers (NCKX) are plasma membrane transporters that are thought to mainly mediate Ca2+ extrusion (along with K+) at the expense of the Na+ electrochemical gradient. However, because they are bidirectional, most assays have relied on measuring their activity in the reverse (Ca2+ import) mode. Herein we describe a method to control intracellular ionic conditions, and examine the forward (Ca2+ extrusion) mode of exchange of NCKX2.

Na+/Ca2+-K+ exchangers (NCKX): functional properties and physiological roles.

The most numerous Ca2+ extrusion protein family, in terms of distinct genes, is the SLC24 gene family of Na+/Ca2+-K+ exchangers (NCKX). Five distinct gene products have been identified, mostly from specific animal excitable tissues such as neurons and smooth muscle, but also in places like skin pigment epithelium, signifying that NCKX proteins may play very specific roles, related to Ca2+ homeostasis, in these tissues. However, progress in elucidating the specific physiological roles of NCKX proteins has been slow in coming, largely because of challenges relating to isolating the activity of these proteins in their native tissues. Herein, we provide an overview of NCKX protein functional characteristics, highlighting properties that are unique and useful as distinguishing features over other Ca2+ handling mechanisms. We also present the first comprehensive review of the literature concerning physiological roles of NCKX proteins.

Na+-dependent inactivation of the retinal cone/brain Na+/Ca2+-K+ exchanger NCKX2.

The SLC24 gene family Na+/Ca2+-K+ exchangers (NCKX) are bidirectional plasma membrane transporters whose main function is the extrusion of Ca2+ from the cytosol. In this study, we used human embryonic kidney 293 cells expressing human retinal cone/brain NCKX2 to examine its Na+ affinity and kinetic parameters of Ca2+ transport. With the use of the ionophore gramicidin to control alkali cation concentrations across the plasma membrane, application of high intracellular Na+ promoted large NCKX2-mediated increases in intracellular free Ca2+ in the 15-20 microm range; this also resulted in inactivation of NCKX2 transport, the first description of this novel kinetic state. The affinity of NCKX2 for internal Na+ was found to be sigmoidal, with a Hill coefficient of 2.6 and Kd = 50 mm. The time-dependent (t(1/2) approximately 40s) inactivation of NCKX2 required high intracellular Na+ levels (Kd > 50 mm) as well as high occupancy of the extracellular Ca2+-binding site. Also reported are two residues whose substitution resulted in an increase in internal Na+ affinity to values of approximately 19 mm; these mutants also displayed enhanced inactivation, suggesting that inactivation requires binding of Na+ to its intracellular transport sites. These findings are the first report of a regulatory kinetic state of Ca2+ transport via NCKX2 Na+/Ca2+-K+ exchangers that may play a prominent role in regulation of Ca2+ extrusion in cellular environments such as neuronal synapses that experience frequent and dynamic Ca2+ fluxes.