Effects of chemokine MCP-1 on NMDA-mediated exciatory postsynaptic current in hippocampal slice of rats

Objective To evaluate the relationship between the mechanism of spinal monocyte chemoattractant protein-1 (MCP-1)-mediated maintenance of chronic pathological pain and synaptic transmission in spinal dorsal horns of rats. Methods Female Sprague-Dawley rats, aged 2-3 weeks after birth, weighing 150-210 g, were studied.The experiment was performed in 2 parts.Experiment Ⅰ Eighteen Sprague-Dawley rats were randomly divided into 2 groups (n=9 each) on 7 days after intrathecal catheters were inserted: phosphate buffer solution(PBS)group and MCP-1 group.PBS 10 μl was intrathecally injected in group PBS, and PBS 10 μl containing 100 ng MCP-1 was intrathecally injected in group MCP-1.The mechanical pain threshold was measured at 30 and 60 min before intrathecal injection, and 30, 60, 90, 120, 150 and 180 min and 1, 2 and 3 days after intrathecal injection.Experiment Ⅱ The transverse spinal cord slices were prepared, and substantia gelatinosa neurons were selected for whole-cell patch-clamp recording.Electrophysiological recording was performed at 1 h of incubation with artificial cerebrospinal fluid (ACSF) and immediately after adding MCP-1: for excitatory synaptic transmission recording, MCP-1 (final concentration 100 nmol/L), N-methyl-D-aspartate (NMDA, final concentration 100 μmol/L) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA, final concentration 20 μmol/L) were added to ACSF, and spontaneous excitatory postsynaptic currents (sEPSCs), AMPA receptors-mediated currents and NMDA receptors-mediated currents were recorded; for inhibitory synaptic transmission recording, MCP-1 (final concentration 100 nmol/L) and γ-aminobutyric acid (GABA, final concentration 1 mmol/L) were added to ACSF, and spontaneous inhibitory postsynaptic currents (sIPSCs) and GABA receptors-mediated currents were recorded. Results Compared with group PBS, the mechanical pain threshold was significantly decreased at 30 min-2 days after intrathecal injection in group MCP-1 (P<0.01). Compared with those at 1 h of incubation with ACSF, the frequency and amplitude of sEPSCs were significantly increased, the amplitude of NMDA receptors- and AMPA receptors-mediated currents were increased, the frequency and amplitude of sIPSCs were decreased, and the amplitude of GABA receptors-mediated currents was decreased immediately after adding MCP-1 (P<0.05). Conclusion MCP-1 enhances excitatory synaptic transmission through enhancing the function of NMDA and AMPA receptors in the posterior substantia gelatinosa neurons of the spinal cord; MCP-1 weakens inhibitory synaptic transmission through inhibiting GABA receptor function, which may be involved in MCP-1-mediated maintenance of chronic pathological pain in rats. Key words: Chemokine CCL2; Pain; Synaptic transmission; Spinal cord

1. Presynaptic neurons B4 and B5 of Aplysia buccal ganglia produce similar inhibitory postsynaptic currents (PSCs) in several postsynaptic follower cells. Two previous papers have characterized the variability of synaptic current amplitude and decay time both for individual PSCs and also for mean values characterizing synapses and have compared PSC amplitude and time course at different synapses sharing a common presynaptic or postsynaptic neuron. 2. To distinguish similarity in synaptic current amplitude or decay introduced by a common pre- or postsynaptic neuron from similarity because of factors common to the particular ganglion or animal, paired synapses were analyzed in four-cell networks in which each of two identified presynaptic neurons produces similar PSCs in each of two postsynaptic cells. Pairing the same synaptic data by common presynaptic or postsynaptic neuron tests if the presynaptic or postsynaptic element partially specifies a parameter; cross-pairing controls for more global factors. Paired values of peak conductance gpeak and decay time constant tau were compared for both individual sequential PSCs and for averages characterizing synapses. Analyses of individual PSCs examine processes affecting synaptic plasticity on a time scale of seconds to minutes, while average values compare more slowly varying factors. 3. Peak amplitudes were compared between individual PSCs in each of 24 paired sets. Correlations of gpeak fluctuations were significantly larger for PSCs produced by the same presynaptic neuron than for postsynaptic or cross pairings (P less than 0.05), consistent with partially correlated fluctuations in transmitter release at different presynaptic terminals. 4. Firing rates of individual presynaptic neurons were modulated to induce variability of test PSCs. These manipulations altered synaptic peak amplitudes in paired postsynaptic neurons, although not to the same degree. Manipulation of a single presynaptic neuron modulated input from that neuron alone to common postsynaptic cells without any effect on input from the paired presynaptic neuron. When fluctuations in the amplitude of gpeak were examined in runs incorporating presynaptic modulation, correlations were strong for sets of PSCs sharing a common presynaptic neuron (R = 0.87), significantly greater (P less than 0.001) than for other pairings. 5. In contrast to the partial presynaptic specification of fluctuations of individual PSCs, values of synaptic amplitude and time course averaged over 21-132 PSCs at a given synapse reflect postsynaptic determinants. Mean values of gpeak characterizing synapses paired by common postsynaptic cell are highly similar (P = 0.0001), in contrast to the lack of similarity seen when the same data are presynaptically (P = 0.11) or cross (P = 0.36) paired.(ABSTRACT TRUNCATED AT 400 WORDS)

Objective To investigate the effects of GABAergic inhibition medicated by the interneurons on the induction of long-term potentiation （LPT）, and determine the concentration of bicuculline required to block GABAA receptor-mediated inhibition and affect LTP induction at CA1 hippocampal synapse. Methods Using whole-cell recording technique, the mini-inhibitory postsynaptic current （mlPSC） and evoked feedforward inhibitory post synaptic current （IPSC） were measured from the CA1 neurons in the hippocampal slices. Extracellular recording of field excitatory postsynaptic potential （fEPSP） by Schaffer collateral stimulation was used to study the synaptic plasticity in adult mice hippocampal slices, and the effects of bicuculline at different concentrations on mlPSC, IPSC, fEPSP and LTP induction were evaluated. Results Bicuuclline at 10 and 20 μmol/L abolished mlPSC and IPSC currents. The action of 20 μmol/L bicuucUine were more obviously. The slope of fEPSPs were significantly enhanced by application of bicuculline at 20 μmol/L, but not at 5 or 10 μmol/L. In the presence of 5, 10, 20 and 50 μmol/L bicuculline, LTP of the fEPSP slope following 100 Hz tetanization was enhanced, but the increment was significant only for 10 and 20 μmol/L bicuculline （P〈0.05）. Conclusion Bicuculline can abolish GABAA receptor-mediated inhibition and affect the excitatory postsynaptic potential in a dose-dependent manner, and at a critical level, bicuculline induces GABAergic disinhibition to result in enhanced LTP in hippocampal slices. Key words: Hippocampus; Synaptic plasticity; GABAA receptor; Long-term potentiation

The return of the spike: postsynaptic action potentials and the induction of LTP and LTD.

Objective To evaluate the relationship between hippocampal monocyte chemotactic protein-1(MCP-1)and its receptor C-C chemokine receptor 2(CCR2)and postoperative cognitive dysfunction(POCD)in aged rats. Methods Forty-eight male Sprague-Dawley rats, aged 20-22 months, weighing 480-550 g, were randomly divided into 2 groups(n=24 each)using a random number table: control group(group C)and POCD group.POCD group inhaled 2.0% isoflurane and underwent splenectomy.Before surgery and at 1, 3 and 7 days after surgery, Morris water maze test was performed to evaluate the spatial learning and memory ability.The escape latency and swimming distance were recorded.Eight rats were sacrificed after the end of Morris water maze test performed at 1, 3 and 7 days after surgery.Then the brains were removed, and the hippocampi were isolated for detection of the expression of MCP-1 and CCR2 by Western blot. Results Compared with group C, the escape latency and swimming distance were significantly prolonged, and the expression of MCP-1 and CCR2 in hippocampi was up-regulated at 1, 3 and 7 days after surgery in POCD group. Conclusion Up-regulation of hippocampal MCP-1 and CCR2 expression may be involved in the mechanism of POCD in aged rats. Key words: Chemokine CCL2; Receptors, CCR2; Hippocampus; Aged; Cognition disorders; Postoperative complications

：Objective To investigatethe effect of propofol pretreatment on hippocampal monocyte chemotactic protein-1 ( MCP-1) and CC-chemokine receptor type 2 (CCR2) expression following forebrainischemiarepcrfusion (I/R) in rats. Methods Twenty-four male SD rats weighing 250-300 gwere randomly divided into 3 groups ( n = 8 each): group Ⅰ control; group Ⅱ I/R and group Ⅲ propofol pretreatment. Cerebral I/R was induced by clampingbilateral common carotid arteries for 10 min combined with hypotension ( MAP wasmaintained at 35-45 mm Hg) induced by exsanguinations in group Ⅱ and Ⅲ. In group Ⅲ propofol 50 mg/kg was injectedinto femoral vein immediately before cerebral ischemia. The animals were sacrificed at 6 hof reperfusion. Hippocampal tissue was obtained for detection of MCP-1 mRNA and CCR2 mRNAand their protein expression by RT-PCR and Western blot technique. Results I/Rsignificantly increased the expression of MCP-1 and CCR2 in hippoeampal tissue as comparedwith control group. Propofol pretreatment significantly attenuated cerebral I/R inducedincrease in MCP-1 and CCR2 expression. Conclusion Propofol pretreatment can significantlyinhibit forebrain I/R-induced hippocampal MCP-1 and CCP2 expression. Key words: Propofol; Chemokine CCL2; Receptors, CCR2; Reperfasioninjury; Brain

Genes Involved in Osteoclastogenesis

Interaural time difference (ITD) is a major cue for localizing a sound source and is processed in the nucleus laminaris (NL) in birds. Coincidence detection (CD) is a crucial step for processing ITD and critically depends on the size and time course of excitatory postsynaptic potentials (EPSPs). Here, we investigated a role of metabotropic glutamate receptors (mGluRs) in the regulation of EPSP amplitude and CD in the NL of chicks. A non-specific agonist of mGluRs ((±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) reduced the amplitude and extent of depression of excitatory postsynaptic currents (EPSCs) during a stimulus train, while the paired pulse ratio and coefficient of variation of EPSC amplitude were increased. In contrast, the amplitudes of spontaneous EPSCs were not affected, but the frequency was reduced. Thus, the effects of t-ACPD were presynaptic and reduced the release of neurotransmitter from terminals in the NL. Expression of group II mGluRs was graded along the tonotopic axis and was stronger towards the low frequency region in the NL. Both group II (DCG-IV) and group III (l-AP4) specific agonists reduced EPSC amplitude by presynaptic mechanisms, and the reduction was larger in the low frequency region; however, we could not find any effects of group I-specific agonists on EPSCs. The reduced EPSP amplitude in DCG-IV improved CD. A specific antagonist of group II mGluRs (LY341495) increased the amplitude of both EPSCs and EPSPs and enhanced the depression during a stimulus train, indicating constitutive activation of mGluRs in the NL. These observations indicate that mGluRs may work as autoreceptors and regulate EPSP size to improve CD in the NL.

The variation of individual synaptic transmission impacts the dynamics of complex neural circuits. We performed whole-cell recordings from monosynaptically connected hippocampal neurons in rat organotypic slice cultures using a synapse mapping method. The amplitude of unitary excitatory postsynaptic current (uEPSC) varied from trial to trial and was independent of the physical distance between cell pairs. To investigate the source of the transmission variability, we obtained patch-clamp recordings from intact axons. Axonal action potentials (APs) were reliably transmitted throughout the axonal arbour and showed modest changes in width. In contrast, calcium imaging from presynaptic boutons revealed that the amplitude of AP-evoked calcium transients exhibited large variations both among different boutons at a given trial and among trials in a given bouton. These results suggest that a factor contributing to the uEPSC fluctuations is the variability in calcium dynamics at presynaptic terminals. Finally, we acquired triple whole-cell recordings from divergent circuit motifs with one presynaptic neuron projecting to two postsynaptic neurons. Consistent with the independency of calcium dynamics among axonal boutons, a series of uEPSC fluctuations was not correlated between the two postsynaptic cells, indicating that different synapses even from the same neuron act independently.We conclude that the intra-bouton and inter-bouton variability in AP-induced calcium dynamics determine the heterogeneity and independency of uEPSCs.

The present thesis describes functional and morphological properties of interneurons and granule cell in developing mouse dentate gyrus, with special focus on inhibitory GABAergic currents. The dentate gyrus is the main hippocampal input structure receiving strong excitatory cortical afferents via the perforant path. Therefore, inhibition at the ‘hippocampal gate’ is important, particularly during postnatal development, when the hippocampal network is prone to seizures. During this critical period of development the intrinsic and synaptic properties of developing inhibitory interneurons were monitored in the molecular layer of mouse hippocampal slices. In this region, mainly calretinin‐positive cells of multipolar appearance were found. These GABAergic interneurons showed maturational changes of their intrinsic and synaptic properties after the first postnatal week. The maturation of molecular layer interneurons went along with faster and larger action potentials, increased repetitive firing, and increased frequency of spontaneous postsynaptic inhibitory currents. All developmental changes in intrinsic and synaptic properties occurred between postnatal day 6‐8 and postnatal day 9‐11, indicating a rapid functional maturation at the end of the first postnatal week. Age‐dependent changes of intrinsic and synaptic properties were also found in developing dentate gyrus granule cells. Similar to interneurons, mature dentate gyrus granule cells exhibited faster and larger action potentials and showed an increased frequency of spontaneous postsynaptic inhibitory currents. Thus, the integration of granule cells in the inhibitory synaptic network of dentate gyrus took place after the second postnatal week. The data shows a rapid functional maturation of intrinsic and synaptic properties of interneurons and granule cells in the dentate gyrus and an early integration into the synaptic networks. However, stratum molecular interneurons were integrated prior to granule cells in the dentate gyrus network, which is corresponding to their subsequent developmental appearance. Besides the influence of phasic synaptic inhibitory currents, throughout postnatal development the dentate gyrus network was also shown to be inhibited by tonic GABAergic currents. In dentate gyrus granule cells, tonic inhibitory currents were mediated by GABAA‐receptors containing α5‐ and δ‐ subunits. These extrasynaptic receptors were activated through the GABA in the extracellular space. The ambient transmitter was delivered by synaptic GABA release and regulated through GABA uptake by the GABA transporter‐1. The contribution of the main components to tonic inhibition was surprisingly stable during granule cell maturation. Throughout postnatal development, tonic inhibition reduced excitability of dentate gyrus granule cells by increasing action potential threshold. It further regulated hippocampal network excitability by preventing overexcitation of the dentate gyrus upon stimulation of entorhinal cortex. Functionally, tonic inhibiton was shown to influence the excitation/inhibition balance of both, the adult and the maturing dentate gyrus.

Objective To evaluate the effect of bupivacaine on miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) of neurons in hippocampal CA1 region of rats. Methods Twenty SPF healthy male Sprague-Dawley rats, aged 4-6 weeks, weighing 300-350 g, were divided into control group (C group, n=10) and bupivacaine model group (BPV group, n=10) using a random number table method.The central nervous system toxicity model was established by infusing 0.75% bupivacaine at a rate of 1 mg·kg-1·min-1 via the tail vein in BPV group, while normal saline was infused for 7.5 min at a rate of 1 mg·kg-1·min-1 via the tail vein in C group.Rats were sacrificed at 6 h after successful establishment of the model, the brains was removed and hippocampal slices were prepared.The whole-cell patch-clamp technique was used to record the frequency and amplitude of mEPSCs and mIPSCs. Results Compared with C group, the frequency of mIPSCs was significantly decreased (P 0.05). Conclusion The mechanism of bupivacaine-induced central nervous system toxicity is related to decreasing the frequency of mIPSCs in hippocampal neurons of rats. Key words: Bupivacaine; Drug toxicity; Central nervous system; Hippocampus; Neurons; Electrophysiology; Synaptic transmission

The formation of new or the remodeling of pre-existing synapses is thought to provide the cellular correlate of learning and memory processes. In the mammalian forebrain, the postsynaptic site of most excitatory synapses is located on dendritic spines. Even after their formation, dendritic spines remain plastic and undergo experience-dependent remodeling that correlates with adaptations in the synaptic strength. Hereby, changes in neuronal activity have to be translated into long- and short-term modifications of dendritic spines. Here, we identify Copine 6 as a novel activity sensor critically involved in these processes.&#13;\nThis project started with an initial characterization of Copine 6 in vitro. A shRNA-mediated knockdown of Copine 6 in primary hippocampal culture increases the number of dendritic spines and influences their maintenance upon changes in neuronal activity. The cytosolic Copine 6 is recruited into postsynaptic sites upon NMDA receptor activation. This translocation of Copine 6 upon increase in the intracellular calcium concentration influences the localization of its binding partner, the actin cytoskeleton modulator Rac1. We demonstrate that presence of Copine 6 affects not only the localization but also the activation state of Rac1. These data indicate that in vitro Copine 6 translates activity-induced calcium signals into morphological changes of the postsynapse through translocation and promotion of Rac1 activity in activated spines. &#13;\nBy the generation of mice deficient for Copine 6 we aimed to identify the role of Copine 6 in vivo. We found that Copine 6 expression is strongest in the hippocampus and starts postnatally when synapses are formed. In the hippocampus, Copine 6 expression is restricted to excitatory neurons. In line with its expression pattern, Copine 6 is dispensable for development. Copine 6 knockout mice thrive indistinguishable from their littermate controls and do not show an overt phenotype. In the hippocampus of adult Copine 6 knockout mice the spine density and morphology, and overall synaptic function is not changed, consistent with an unaffected Rac1 signaling. In contrast, loss of Copine 6 in vivo strongly affects synaptic plasticity. Copine 6 knockout mice are deficient in hippocampal long-term potentiation, suggesting that Copine 6 is dispensable for spine formation but essential for synaptic plasticity. &#13;\nIn a yeast-two hybrid screen we identified SIMPL as a novel Copine 6 interacting partner. We provide evidence that presence of Copine 6 anchors the NF-kappaB co-activator SIMPL in the cytoplasm and prevents its translocation into the nucleus. In consequence, absence of Copine 6 increases the transcriptional activity of NF-kappaB. These data indicate that Copine 6 may regulate long-term adaptations in neuronal functions that involve transcriptional regulations.&#13;\nTaken together, this thesis identifies Copine 6 as an important player in the regulation of synaptic plasticity in vitro and in vivo.

：Objective To investigatethe effect of midazolam on synaptic transmission in stratum radiatum of CA1 area of rathippocampus and the underlying mechanism. Methods Thirty-five adult male Wistar ratsweighing 190-220 g were randomly divided into 7 groups ( n = 5 each) . Two types ofstimulation were used: single stimulation and paired pulse stimulation. Four groups undersingle stimulation: control group (group C_1), bicuculline group (group B_1 ), midazolamgroup (group M_1) and bicuculline + midazolam group (group BM). Three groups under pairedpulse stimulation: control group ( group C_2 ), bicuculline group (group B_2 ) andmidazolam group (group M_2 ) . The animals were anesthetized with intraperitoneal (IP)pentobarbital 50 mg/kg. The stimulating and recording electrodes were inserted into thestratum radiatum of CA1 area of the hippocampus. The interval between the 2 pulsestimulation was 30 ms. Excitatory post-synaptic potential (EPSP) was elicited before(baseline) and after IP bicuculline (2 mg/kg) or/and midazolam (3 mg/kg) administration.The EPSP amplitude was recorded during single stimulation. EPSP_2 /EPSP_1 ( E_2 /E_1 )ratio was recorded during paired pulse stimulation. Results Compared with group C, , theEPSP amplitude was significantly decreased in group M, ( P 0.05) . Compared with group C_2 ,E_2 and E_2/E_1, ratio were significantly increased in group B_2 , while E_1 , E_ 2, andE_2/E_1, ratio were significantly decreased in group M2 ( P < 0.05). E_1, E_2 and E_2/E_1, ratio were significantly lower in group M_2 than in group B_2 (P < 0.05) .Conclusion Midazolam can reversibly depress the excitatory synaptic transmission efficacyin the CA1 area of the rat hippocampus through presynaptic inhibitory circuit. CABA_Areceptor is not directly disturbed.

<strong>Objective</strong> In the present experiment we investigate the behavior of 4-month-old transgenic <em>APP/PS-1/tau </em>mice model with Alzheimer's disease (3 × Tg-AD mice) to evaluate their abilities of spatial learning and memory. We observe the changes of synaptic plasticity and soluble amyloid-β protein 42 (Aβ42) expression in the CA1 region of hippocampus to explore the mechanism of early cognitive impairment of 3 × Tg-AD mice. <strong>Methods</strong> Ten 4-month-old male 3 × Tg-AD mice and matched ten 129/C57BL/6 hybrid wild type (WT) mice were enrolled. The open field test and Morris water maze test were conducted to observe emotion disorder and ability of spatial learning and memory. Field excitatory postsynaptic potential (fEPSP) and theta burst stimulation (TBS)-induced long-term potentiation (LTP) were recorded in CA1 region of hippocampus. The expression changes of soluble Aβ42 in hippocampus were measured by enzyme-linked immunosorbent assay (ELISA). <strong>Results</strong> The open field test showed that there was no significant differences between 3 × Tg-AD group and control group, which indicated that there was no obvious anxiety tendency in 4-month-old 3 × Tg-AD mice. Compared with control group, 3 × Tg-AD group mice had significantly longer escape latency from the 3rd to 5th day (P = 0.001, 0.003, 0.001) and lower percentage of time through the platform area (P = 0.000). LTP induced by TBS in CA1 region of hippocampus of 3 × Tg-AD group decreased significantly (P &lt; 0.01, for all) compared with that of control group. In contrast to control group, the expression of soluble Aβ42 in the hippocampus of 3 × Tg-AD mice group increased significantly (P = 0.000). <strong>Conclusions</strong> The expression of soluble Aβ42 in the hippocampus of 4-month-old 3 × Tg-AD mice increased significantly, which impaired synaptic plasticity in CA1 region of hippocampus and led to a significant decline in spatial learning and memory ability. <strong>DOI: </strong>10.3969/j.issn.1672-6731.2015.05.012

Objective To investigate the correlation of hippocampal synaptic plasticity with spatial learning and memory under normal and pathological condition, and provide experimental evidence for the coincidence of hippocampal late-phase long-term potentiation (L-LTP) and behavioral experiments. Methods 38 SD rats were randomly divided into two groups, control and AD model. First, Morris water maze was used to test the ability of spatial learning and memory of rats. The escape latencies for rats to search for an underwater platform in 5 days of navigation tests and the swimming time percentage in target quadrant on the 6th day after withdrawing the platform in probe trails were recorded. Then, in vivo hippocampus L-LTP of field excitatory postsynaptic potential (fEPSP) in CA1 region was recorded after delivering high frequency stimulation (HFS). Results Bilateral intrahippocampal injection of 4 nmol amyloid β peptide (Aβ 25-35) significantly impaired spatial learning and memory of rats in water maze tests, as well as in vivo hippocampal L-LTP. In control group, there was a significant negative correlation between the amplitude of fEPSP and the escape latency (r=-0.8306, P<0.01) and a significant positive correlation between the amplitude of fEPSP and the swimming time percentage in target quadrant (r=0.7709, P<0.01). In AD model group, similar correlations were found, with a correlation coefficient of r=-0.7675 (P<0.01) and r=0.8049 (P<0.01), respectively. When putting all data from the two groups together, the hippocampal L-LTP was more correlated with escape latency (r=-0.9124, P<0.01) and swimming time percentage (r=0.9745, P<0.01). Conclusion There is very close correlation between the hippocampal L-LTP and the spatial learning and memory behavior in rats, suggesting that the hippocampal L-LTP may be involved in the electrophysiological mechanism of spatial learning and memory in rats, and the impairment of L-LTP could partly represent the deficits in cognitive function of animals. Key words: Spatial learning and memory; Morris water maze; Late-phase long term potentiation; Amyloid β protein