Synaptic Transmission In Hippocampal Neurons Research Articles

IRAK-M Ablation Promotes Status Epilepticus-Induced Neuroinflammation via Activating M1 Microglia and Impairing Excitatory Synaptic Function.

Epilepsy is one of the most common neurological disorders. The pro-epileptic and antiepileptic roles of microglia have recently garnered significant attention. Interleukin-1 receptor-associated kinase (IRAK)-M, an important kinase in the innate immune response, is mainly expressed in microglia and acts as a negative regulator of the TLR4 signaling pathway that mediates the anti-inflammatory effect. However, whether IRAK-M exerts a protective role in epileptogenesis as well as the molecular and cellular mechanisms underlying these processes are yet to be elucidated. An epilepsy mouse model induced by pilocarpine was used in this study. Real-time quantitative polymerase chain reaction and western blot analysis were used to analyze mRNA and protein expression levels, respectively. Whole-cell voltage-clamp recordings were employed to evaluate the glutamatergic synaptic transmission in hippocampal neurons. Immunofluorescence was utilized to show the glial cell activation and neuronal loss. Furthermore, the proportion of microglia was analyzed using flow cytometry. Seizure dynamics influenced the expression of IRAK-M. Its knockout dramatically exacerbated the seizures and the pathology in epilepsy and increased the N-methyl-d-aspartate receptor (NMDAR) expression, thereby enhancing glutamatergic synaptic transmission in hippocampal CA1 pyramidal neurons in mice. Furthermore, IRAK-M deficiency augmented hippocampal neuronal loss via a possible mechanism of NMDAR-mediated excitotoxicity. IRAK-M deletion promotes microglia toward the M1 phenotype, which resulted in high levels of proinflammatory cytokines and was accompanied by a visible increase in the expressions of key microglial polarization-related proteins, including p-STAT1, TRAF6, and SOCS1. The findings demonstrate that IRAK-M dysfunction contributes to the progression of epilepsy by increasing M1 microglial polarization and glutamatergic synaptic transmission. This is possibly related to NMDARs, particularly Grin2A and Grin2B, which suggests that IRAK-M could serve as a novel therapeutic target for the direct alleviation of epilepsy.

Protein phosphatase-1 inhibitor-2 promotes PP1γ positive regulation of synaptic transmission.

Inhibitor-2 (I-2) is a prototypic inhibitor of protein phosphatase-1 (PP1), a major serine-threonine phosphatase that regulates synaptic plasticity and learning and memory. Although I-2 is a potent inhibitor of PP1 in vitro, our previous work has elucidated that, in vivo, I-2 may act as a positive regulator of PP1. Here we show that I-2 and PP1γ, but not PP1α, positively regulate synaptic transmission in hippocampal neurons. Moreover, we demonstrated that I-2 enhanced PP1γ interaction with its major synaptic scaffold, neurabin, by Förster resonance energy transfer (FRET)/Fluorescence lifetime imaging microscopy (FLIM) studies, while having a limited effect on PP1 auto-inhibitory phosphorylation. Furthermore, our study indicates that the effect of I-2 on PP1 activity in vivo is dictated by I-2 threonine-72 phosphorylation. Our work thus demonstrates a molecular mechanism by which I-2 positively regulates PP1 function in synaptic transmission.

Dysregulation of miRNAs Levels in Glycogen Synthase Kinase-3β Overexpressing Mice and the Role of miR-221-5p in Synaptic Function

Glycogen synthase kinase-3β (GSK-3β) is a highly expressed kinase in the brain, where it has an important role in synaptic plasticity. Aberrant activity of GSK-3β leads to synaptic dysfunction which results in the development of several neuropsychiatric and neurological diseases. Notably, overexpression of constitutively active form of GSK-3β (GSK-3β[S9A]) in mice recapitulates the cognitive and structural defects characteristic for neurological and psychiatric disorders. However, the mechanisms by which GSK-3β regulates synaptic functions are not clearly known. Here, we investigate the effects of GSK-3β overactivity on neuronal miRNA expression in the mouse hippocampus. We found that GSK-3β overactivity downregulates miRNA network with a potent effect on miR-221-5p (miR-221*). Next, characterization of miR-221* function in primary hippocampal cell culture transfected by miR-221* inhibitor, showed no structural changes in dendritic spine shape and density. Using electrophysiological methods, we found that downregulation of miR-221* increases excitatory synaptic transmission in hippocampal neurons, probably via postsynaptic mechanisms. Thus, our data reveal potential mechanism by which GSK-3β and miRNAs might regulate synaptic function and therefore also synaptic plasticity.

Effects of Dexmedetomidine and Oxycodone on Neurocognitive and Inflammatory Response After Tourniquet-Induced Ischemia-Reperfusion Injury.

To evaluate the effects of dexmedetomidine (Dex) and oxycodone (Oxy) on neurocognitive and inflammatory response after tourniquet-induced ischemia-reperfusion (I/R) injury. C57/BL6 mice were used to construct the mouse model of tourniquet-induced I/R injury. Mice (n = 48) were randomly divided into sham, I/R, Dex or Oxy group. Morris water maze test was performed to assess the spatial learning and memory function. The expression of NF-κB, TLR4, NR2B, M1 (CD68 and TNF-α) and M2 (CD206 and IL-10) polarization markers in mice hippocampus were detected by western blot or immunofluorescent staining. Spontaneous excitatory post-synaptic currents (sEPSCs) were recorded by electrophysiology. Dex treatment alleviated I/R-induced declines in learning and memory (p<0.05), while Oxy had no significant effect on it. Compared with I/R group, Dex and Oxy treatment down-regulated the expression of NF-κB, TLR4, TNF-α and CD68 (all p<0.05), while no significantly different was found in CD206 and IL-10. In addition, Dex treatment down-regulated the expression of NR2B and reduced the frequency and amplitude of sEPSCs in I/R model mice (all p<0.05), while Oxy had no significant effect on them. Tourniquet-induced I/R could impair the neurocognitive function of mice. Dex treatment could alleviate I/R-induced neurocognitive disorder by inhibiting abnormal synaptic transmission in hippocampal neurons. Both Dex and Oxy could alleviate the inflammatory response likely by inhibiting the polarization of microglia toward M1 phenotype via TLR4/NF-κB pathway. Future studies are needed to further examine the effects of Dex on neurocognitive disorder after tourniquet-induced I/R injury and investigate the exact mechanism.

Parkinson\u2019s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

BackgroundParkinson’s disease (PD)-associated E3 ubiquitin ligase Parkin is enriched at glutamatergic synapses, where it ubiquitinates multiple substrates, suggesting that its mutation/loss-of-function could contribute to the etiology of PD by disrupting excitatory neurotransmission. Here, we evaluate the impact of four common PD-associated Parkin point mutations (T240M, R275W, R334C, G430D) on glutamatergic synaptic function in hippocampal neurons.ResultsWe find that expression of these point mutants in cultured hippocampal neurons from Parkin-deficient and Parkin-null backgrounds alters NMDA and AMPA receptor-mediated currents and cell-surface levels and prevents the induction of long-term depression. Mechanistically, we demonstrate that Parkin regulates NMDA receptor trafficking through its ubiquitination of GluN1, and that all four mutants are impaired in this ubiquitinating activity. Furthermore, Parkin regulates synaptic AMPA receptor trafficking via its binding and retention of the postsynaptic scaffold Homer1, and all mutants are similarly impaired in this capacity.ConclusionOur findings demonstrate that pathogenic Parkin mutations disrupt glutamatergic synaptic transmission in hippocampal neurons by impeding NMDA and AMPA receptor trafficking. Such effects may contribute to the pathophysiology of PD in PARK2 patients.

Heteromeric channels formed by TRPC1, TRPC4 and TRPC5 define hippocampal synaptic transmission and working memory.

Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high-resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from Trpc1/Trpc4/Trpc5-triple-knockout (Trpc1/4/5-/-) mice, lacking any TRPC1-, TRPC4-, or TRPC5-containing channels, action potential-triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. In vivo, Trpc1/4/5-/- mice displayed impaired cross-frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. Trpc1/4/5-/- animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.

Identifying synapse-specific proteins

Inhibitory Synapses Recently, we have seen enormous progress in our understanding of the protein complexes at excitatory synapses. Much less is known about the molecules at inhibitory synapses. Uezu et al. have discovered that many molecules cluster at postsynaptic inhibitory structures to form a network of proteins with, as yet, mostly unknown functions. However, one novel component, called InSyn1, has been found to colocalize with key scaffolding proteins and regulate inhibitory synaptic transmission in hippocampal neurons. Science , this issue p. [1123][1] [1]: /lookup/doi/10.1126/science.aag0821

Deleting IGF-1 receptor from forebrain neurons confers neuroprotection during stroke and upregulates endocrine somatotropin

Insulin-like growth factors control numerous processes, namely somatic growth, metabolism and stress resistance, connecting this pathway to aging and age-related diseases. Insulin-like growth factor signaling also impacts on neurogenesis, neuronal survival and structural plasticity. Recent reports demonstrated that diminished insulin-like growth factor signaling confers increased stress resistance in brain and other tissues. To better understand the role of neuronal insulin-like growth factor signaling in neuroprotection, we inactivated insulin-like growth factor type-1-receptor in forebrain neurons using conditional Cre-LoxP-mediated gene targeting. We found that brain structure and function, including memory performance, were preserved in insulin-like growth factor receptor mutants, and that certain characteristics improved, notably synaptic transmission in hippocampal neurons. To reveal stress-related roles of insulin-like growth factor signaling, we challenged the brain using a stroke-like insult. Importantly, when charged with hypoxia-ischemia, mutant brains were broadly protected from cell damage, neuroinflammation and cerebral edema. We also found that in mice with insulin-like growth factor receptor knockout specifically in forebrain neurons, a substantial systemic upregulation of growth hormone and insulin-like growth factor-I occurred, which was associated with significant somatic overgrowth. Collectively, we found strong evidence that blocking neuronal insulin-like growth factor signaling increases peripheral somatotropic tone and simultaneously protects the brain against hypoxic–ischemic injury, findings that may contribute to developing new therapeutic concepts preventing the disabling consequences of stroke.

Effect of synthetic cannabinoids on spontaneous neuronal activity: Evaluation using Ca2+ spiking and multi-electrode arrays

Activation of cannabinoid receptor 1 (CB1) inhibits synaptic transmission in hippocampal neurons. The goal of this study was to evaluate the ability of benchmark and emerging synthetic cannabinoids to suppress neuronal activity in vitro using two complementary techniques, Ca2+ spiking and multi-electrode arrays (MEAs). Neuron culture and fluorescence imaging conditions were extensively optimized to provide maximum sensitivity for detection of suppression of neural activity by cannabinoids. The neuronal Ca2+ spiking frequency was significantly suppressed within 10min by the prototypic aminoalkylindole cannabinoid, WIN 55,212-2 (10µM). Suppression by WIN 55,212-2 was not improved by pharmacological intervention with signaling pathways known to interfere with CB1 signaling. The naphthoylindole CB1 agonist, JWH-018 suppressed Ca2+ spiking at a lower concentration (2.5µM), and the CB1 antagonist rimonabant (5µM), reversed this suppression. In the MEA assay, the ability of synthetic CB1 agonists to suppress spontaneous electrical activity of hippocampal neurons was evaluated over 80min sessions. All benchmark (WIN 55,212-2, HU-210, CP 55,940 and JWH-018) and emerging synthetic cannabinoids (XLR-11, JWH-250, 5F-PB-22, AB-PINACA and MAM-2201) suppressed neural activity at a concentration of 10µM; furthermore, several of these compounds also significantly suppressed activity at 1µM concentrations. Rimonabant partially reversed spiking suppression of 5F-PB-22 and, to a lesser extent, of MAM-2201, supporting CB1-mediated involvement, although the inactive WIN 55,212-3 also partially suppressed activity. Taken together, synthetic cannabinoid CB1-mediated suppression of neuronal activity was detected using Ca2+ spiking and MEAs.

Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity.

Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1(T112A)), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca(2+) sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect paired-pulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1(Δ109-116)), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.

The phospholipid-binding protein SESTD1 negatively regulates dendritic spine density by interfering with Rac1-Trio8 signaling pathway.

Dendritic spines are actin-rich protrusions from neuronal dendrites that harbor the majority of excitatory synapses. The balance of spine formation and retraction may influence dendritic integrity. While knowledge of the molecular mechanisms that promote dendritic spine formation has accumulated, little is known about the factors that limit spine formation. Here, we show that SESTD1, a phospholipid-binding protein containing a lipid-binding SEC14-like domain and two spectrin-repeat cytoskeleton interaction domains, negatively regulates dendritic spine density in cultured hippocampal neurons. Overexpression of SESTD1 decreases dendritic spine density in neurons by interfering with the interaction between Rac1 and its guanine nucleotide exchange factor (GEF) Trio8. Conversely, knockdown of SESTD1 increases dendritic spine density. Further analysis reveals that the SPEC1 domain-mediated interaction with Rac1 is required for SESTD1 activity toward a decrease in dendritic spine density. Transfection of GEF domain of Trio8 into neurons rescues SESTD1-mediated decrease in dendritic spine density. More importantly, overexpression of SESTD1 results in a decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs), whereas SESTD1 knockdown increases the mEPSC frequency. These results suggest that SESTD1 may act as a negative regulator of the Rac1-Trio8 signaling pathway to reduce dendritic spine density and lower excitatory synaptic transmission in hippocampal neurons.

Inhibition of Excitatory Synaptic Transmission in Hippocampal Neurons by Levetiracetam Involves Zn2+-Dependent GABA Type A Receptor–Mediated Presynaptic Modulation

Levetiracetam (LEV) is an antiepileptic drug with a unique but as yet not fully resolved mechanism of action. Therefore, by use of a simplified rat-isolated nerve-bouton preparation, we have investigated how LEV modulates glutamatergic transmission from mossy fiber terminals to hippocampal CA3 neurons. Action potential-evoked excitatory postsynaptic currents (eEPSCs) were recorded using a conventional whole-cell patch-clamp recording configuration in voltage-clamp mode. The antiepileptic drug phenytoin decreased glutamatergic eEPSCs in a concentration-dependent fashion by inhibiting voltage-dependent Na⁺ and Ca²⁺ channel currents. In contrast, LEV had no effect on eEPSCs or voltage-dependent Na⁺ or Ca²⁺ channel currents. Activation of presynaptic GABA type A (GABA(A)) receptors by muscimol induced presynaptic inhibition of eEPSCs, resulting from depolarization block. Low concentrations of Zn²⁺, which had no effect on eEPSCs, voltage-dependent Na⁺ or Ca²⁺ channel currents, or glutamate receptor-mediated whole cell currents, reduced the muscimol-induced presynaptic inhibition. LEV applied in the continuous presence of 1 µM muscimol and 1 µM Zn²⁺ reversed this Zn²⁺ modulation on eEPSCs. The antagonizing effect of LEV on Zn²⁺-induced presynaptic GABA(A) receptor inhibition was also observed with the Zn²⁺ chelators Ca-EDTA and RhodZin-3. Our results clearly show that LEV removes the Zn²⁺-induced suppression of GABA(A)-mediated presynaptic inhibition, resulting in a presynaptic decrease in glutamate-mediated excitatory transmission. Our results provide a novel mechanism by which LEV may inhibit neuronal activity.

Pharmacological and Genetic Accumulation of Hypoxia-Inducible Factor-1α Enhances Excitatory Synaptic Transmission in Hippocampal Neurons through the Production of Vascular Endothelial Growth Factor

Hypoxia-inducible factor-1 (HIF-1) is an important transcriptional factor in mammalian cells for coordination of adaptive responses to hypoxia. It consists of a regulatory subunit HIF-1alpha, which accumulates under hypoxic conditions, and a constitutively expressed subunit HIF-1beta. In addition to the well characterized oxygen-dependent mode of action of HIF-1, recent work has shown that various growth factors and cytokines stimulate HIF-1alpha expression, thereby triggering transcription of numerous hypoxia-inducible genes by oxygen-independent mechanisms. In this study, we examined whether accumulation of HIF-1alpha induced by insulin-like growth factor-1 (IGF-1) has a regulatory role in excitatory synaptic transmission in hippocampal neuron cultures. Our results show that IGF-1 induced a time- and dose-dependent increase in HIF-1alpha expression that was blocked by pretreatment with selective IGF-1 receptor antagonist, transcriptional inhibitor, and translational inhibitors. In addition, pharmacological blockade of the phosphatidylinositol 3-kinase/Akt/mammalian target of the rapamycin signaling pathway, but not extracellular signal-regulated kinase, inhibited IGF-1-induced HIF-1alpha expression. More importantly, the increase in HIF-1alpha expression induced by IGF-1 was accompanied by increasing levels of vascular endothelial growth factor (VEGF) mRNA and protein, which enhanced excitatory synaptic transmission. In parallel, blockade of HIF-1alpha activity by echinomycin or lentiviral infection with dominant-negative mutant HIF-1alpha or short hairpin RNA targeting HIF-1alpha inhibited the increase in expression of VEGF and the enhancement of synaptic transmission induced by IGF-1. Conversely, transfection of constitutively active HIF-1alpha into neurons mimicked the effects of IGF-1 treatment. Together, these results suggest that HIF-1alpha accumulation can enhance excitatory synaptic transmission in hippocampal neurons by regulating production of VEGF.

Syntaxin1a Dispersion and Assessment of cis-Snare-Complex Formation-Disassembly during Synaptic Transmission in Hippocampal Neurons

Soluble-N-ethylmaleimide-sensitive-factor-attachment-protein-receptor(SNARE)-complex formation and dissociation are biochemical prerequisites of synaptic transmission that occur during calcium-triggered synaptic vesicle (SV) fusion and subsequent recycling, which prepares cellular machinery for the following responses to action potentials (AP). The exact sequence of molecular events remains unknown, and it's a technological challenge to demonstrate SNARE-protein interactions in-situ, during AP-induced synaptic transmission. In this study, C-termini-labeled fluorescent constructs of Syntaxin1a-Cerulean and VAMP2-Citrine were transiently expressed either alone or together in cultured hippocampal neurons. A Syn1a-Cer-probe revealed a more uniform distribution of fluorescence in the plasma-membrane, with slightly higher intensity in synaptic boutons, while VAMP2-Cit-fluorescence was predominantly in boutons, suggesting that trafficking of heterologously-expressed constructs is similar to endogenous SNAREs. Efficiently transmitting boutons were identified by FM4-64-destaining during electrical-stimulation. These boutons revealed both faster transient increase of VAMP2-Cit-fluorescence (synaptopHluorine-effect), and a slower decrease of Syn1a-Cer-fluorescence. The latter effect started with a 3-6 s delay after beginning 30 Hz-stimulation, and developed as stimulation continued for 10-30 s. The largest reduction for averaged data was seen 5-10 s after stimulation completed, and recovery occurred in the following 20-40s. The decrease of Cer-fluorescence that occurred in regions of intensive FM-destaining was often accompanied by a transient increase of fluorescence in adjacent areas. In transmitting boutons co-expressing both Syn1a-Cer and VAMP2-Cit probes, the reduction was slightly stronger (up to 3-4%, although not significant yet, P<0.15) compared to the Syn1a-Cer alone (1-2%), even though the latter boutons showed on average larger FM-destaining. The decrease remains if measured in larger areas that presumably include dispersed probe. We hypothesize that a fraction of the Cer-fluorescence reduction can be attributed to FRET between Syn1a-Cer probe as a fluorescence-donor and VAMP2-Cit as an acceptor, which come in close proximity upon cis-SNARE-complex formation after SV-fusion.

Synaptic transmission in hippocampal neurons lacking DNA methyltransferase

326 DNA methylation is an epigenetic mechanism that plays a critical role in the maintenance of gene silencing and promotes genome stability in various species. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), which add a methyl group to the 5' position of the cytosine residue at CpG dinucleotides. At present, three DNA methyltransferase (DNMT1-3) genes have been identified in mammals. Previous work has demonstrated that DNMT1 maintains DNA methylation patterns, whereas DNMT3a and DNMT3b are involved in de novo methylation. The DNMTs are highly expressed in neurons in the adult brain, suggesting that they may be crucial for postmitotic neurons. However, few details are known about how alterations in DNA methylation may affect neuronal function, in particular synaptic transmission. Recent studies have implicated misregulation of DNA methylation in cognitive disorders such as schizophrenia and Fragile X Syndrome. To examine whether the loss of DNMTs influences synaptic function, we have obtained floxed DNMT1 and floxed DNMT3 mice. Using a lentiviral approach we have infected disassociated hippocampal floxed neurons with either Cre recombinase tagged with GFP or GFP alone and then performed whole-cell voltage-clamp recordings on mature neurons. We examined the properties of evoked excitatory transmission in DNMT1 or DNMT3a knockout neurons and found that there were no changes in excitatory postsynaptic response depression during 10and 20-Hz stimulation and no changes in synaptic responses mediated by NMDA and AMPA receptors. In contrast, we found that the deletion of either DNMT1 or DNMT3a produced robust changes in spontaneous synaptic activity. The frequency of miniature excitatory postsynaptic currents (mEPSCs) was significantly decreased in both DNMT1 and DNMT3a knockout cultures, while there was no change in mEPSC amplitude, suggestive of a potential presynaptic deficit. To test whether this effect on spontaneous activity is specific for excitatory synapses, we recorded miniature inhibitory postsynaptic currents (mIPSCs). We found no changes in mIPSC frequencies or amplitudes following the loss of either DNMT1 or DNMT3, suggesting that DNMT inhibition is affecting only excitatory synaptic activity.

Activity of Adenosine Receptors Type 1 Is Required for CX3CL1-Mediated Neuroprotection and Neuromodulation in Hippocampal Neurons

The chemokine fractalkine (CX(3)CL1) is constitutively expressed by central neurons, regulating microglial responses including chemotaxis, activation, and toxicity. Through the activation of its own specific receptor, CX(3)CR1, CX(3)CL1 exerts both neuroprotection against glutamate (Glu) toxicity and neuromodulation of the glutamatergic synaptic transmission in hippocampal neurons. Using cultured hippocampal neuronal cell preparations, obtained from CX(3)CR1(-/-) (CX(3)CR1(GFP/GFP)) mice, we report that these same effects are mimicked by exposing neurons to a medium conditioned with CX(3)CL1-treated mouse microglial cell line BV2 (BV2-st medium). Furthermore, CX(3)CL1-induced neuroprotection from Glu toxicity is mediated through the adenosine receptor 1 (AR(1)), being blocked by neuronal cell preparations treatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a specific inhibitor of AR(1), and mimicked by both adenosine and the specific AR(1) agonist 2-chloro-N(6)-cyclopentyladenosine. Similarly, experiments from whole-cell patch-clamped hippocampal neurons in culture, obtained from CX(3)CR1(+/+) mice, show that CX(3)CL1-induced depression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA-) type Glu receptor-mediated current (AMPA-current), is associated with AR(1) activity being blocked by DPCPX and mimicked by adenosine. Furthermore, BV2-st medium induced a similar AMPA-current depression in CX(3)CR1(GFP/GFP) hippocampal neurons and this depression was again blocked by DPCPX. We also report that CX(3)CL1 induced a significant release of adenosine from microglial BV2 cells, as measured by HPLC analysis. We demonstrate that (i) CX(3)CL1, along with AR(1), are critical players for counteracting Glu-mediated neurotoxicity in the brain and (ii) AR(1) mediates neuromodulatory action of CX(3)CL1 on hippocampal neurons.

Activity-dependent regulation of inhibitory synaptic transmission in hippocampal neurons

Neural activity regulates the number and properties of GABAergic synapses in the brain, but the mechanisms underlying these changes are unclear. We found that blocking spike activity globally in developing hippocampal neurons from rats reduced the density of GABAergic terminals as well as the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Chronic inactivity later in development led to a reduction in the mIPSC amplitude, without any change in GABAergic synapse density. By contrast, hyperpolarizing or abolishing spike activity in single neurons did not alter GABAergic synaptic inputs. Suppressing activity in individual presynaptic GABAergic neurons also failed to decrease synaptic output. Our results indicate that GABAergic synapses are regulated by the level of activity in surrounding neurons. Notably, we found that the expression of GABAergic plasticity involves changes in the amount of neurotransmitter in individual vesicles.

Postsynaptic IP 3 receptor-mediated Ca 2+ release modulates synaptic transmission in hippocampal neurons

Ca 2+-dependent mechanisms are important in regulating synaptic transmission. The results herein indicate that whole-cell perfusion of inositol 1,4,5-trisphosphate receptor (IP 3R) agonists greatly enhanced excitatory postsynaptic current (EPSC) amplitudes in postsynaptic hippocampal CA1 neurons. IP 3R agonist-mediated increases in synaptic transmission changed during development and paralleled age-dependent increases in hippocampal type-1 IP 3Rs. IP 3R agonist-mediated increases in EPSC amplitudes were inhibited by postsynaptic perfusion of inhibitors of Ca 2+/calmodulin, PKC and Ca 2+/calmodulin-dependent protein kinase II. Postsynaptic perfusion of inhibitors of smooth endoplasmic reticulum (SER) Ca 2+-ATPases, which deplete intracellular Ca 2+ stores, also enhanced EPSC amplitudes. Postsynaptic perfusion of the IP 3R agonist adenophostin (AdA) during subthreshold stimulation appeared to convert silent to active synapses; synaptic transmission at these active synapses was completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Postsynaptic IP 3R-mediated Ca 2+ release also produced a significant increase in spontaneous EPSC frequency. These results indicate that Ca 2+ release from intracellular stores play a key role in regulating the function of postsynaptic AMPARs.

Dopaminergic Stimulation of Local Protein Synthesis Enhances Surface Expression of GluR1 and Synaptic Transmission in Hippocampal Neurons

The use-dependent modification of synapses is strongly influenced by dopamine, a transmitter that participates in both the physiology and pathophysiology of animal behavior. In the hippocampus, dopaminergic signaling is thought to play a key role in protein synthesis-dependent forms of synaptic plasticity. The molecular mechanisms by which dopamine influences synaptic function, however, are not well understood. Using a GFP-based reporter, as well as a small-molecule reporter of endogenous protein synthesis, we show that dopamine D1/D5 receptor activation stimulates local protein synthesis in the dendrites of hippocampal neurons. We also identify the GluR1 subunit of AMPA receptors as one protein upregulated by dopamine receptor activation, with increased incorporation of surface GluR1 at synaptic sites. The insertion of new GluRs is accompanied by an increase in the frequency of miniature synaptic events. Together, these data suggest a local protein synthesis-dependent activation of previously silent synapses as a result of dopamine receptor stimulation.

Thymosin alpha-1 modulate excitatory synaptic transmission in cultured hippocampal neurons in rats

Thymosin alpha-1 (Tα1) not only possesses immunoregulatory properties in periphery but also is expressed in the central nervous system (CNS) and affects the function of the CNS. To further elucidate the role of Tα1 in the CNS, the whole-cell recording technique was used to observe the effect of Tα1 on the spontaneous excitatory synaptic transmission in cultured rat hippocampal neurons. The results showed that acute treatment with Tα1 significantly enhanced the frequency of AMPA-mediated spontaneous excitatory postsynaptic current (sEPSC) at the concentrations of 1 and 10 μg/ml and also enhanced the frequency of AMPA-mediated miniature excitatory postsynaptic current (mEPSC) at 10 μg/ml. However, the amplitude of both sEPSC and mEPSC were not changed by Tα1. Those results suggested that Tα1 involves in the regulation of excitatory synaptic transmission in hippocampal neurons, which contribute to its neurophysiological function in the CNS.