探索GalR1在雌性CUMS C57小鼠海马表达及M617对干细胞增殖的影响

目的:探究GalR1在雌性CUMS C57小鼠海马表达及GalR1激动剂M617对干细胞增殖的影响。方法:(1)将小鼠分为实验组及对照组,对照组不给予任何刺激,实验组给予慢性温和不可预知应激(CUMS)。通过糖水偏好、悬尾实验及血清皮质酮比较两组的抑郁水平;部分小鼠行免疫荧光染色比较两组海马DG的增殖情况;另一部分小鼠行qPCR检测,比较两组海马甘丙肽及其受体的表达。(2)行细胞培养,传代细胞给予不同浓度的M617处理3 d,取每组细胞铺板并加入10μmol/L的Brd U,继续孵育4 h后PFA固定,行Brdu免疫荧光染色并比较各组的阳性细胞数。结果:(1)第2、3周的糖水偏好实验显示两组糖水的消耗量未见差异,第4周CUMS组糖水消耗量少于对照组(P0.05);悬尾实验中,CUMS组静止不动时间延长(P0.05);CUMS组血清皮质酮水平明显高于对照组(P0.01)。(2)CUMS组海马DG区的Brd U+及Ki67+细胞数明显少于对照组(P0.05)。(3)q PCR结果显示CUMS组甘丙肽及GalR1基因表达水平明显增加(P0.01)。(4)不同浓度M617对干细胞增殖无明显组间差异。结论:(1)GalR1在雌性CUMS C57小鼠海马表达增高,可能参与调节抑郁症的发生过程。(2)不同浓度M617对干细胞增殖无明显影响,提示GalR1可能不参与神经干细胞增殖的调节。     

Activation of postsynaptic Ca(2+) stores modulates glutamate receptor cycling in hippocampal neurons

We show that activation of postsynaptic inositol 1,4,5-tris-phosphate receptors (IP(3)Rs) with the IP(3)R agonist adenophostin A (AdA) produces large increases in AMPA receptor (AMPAR) excitatory postsynaptic current (EPSC) amplitudes at hippocampal CA1 synapses. Co-perfusion of the Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid strongly inhibited AdA-enhanced increases in EPSC amplitudes. We examined the role of AMPAR insertion/anchoring in basal synaptic transmission. Perfusion of an inhibitor of synaptotagmin-soluble n-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor SNARE-mediated exocytosis depressed basal EPSC amplitudes, whereas a peptide that inhibits GluR2/3 interactions with postsynaptic density-95 (PDZ) domain proteins glutamate receptor interacting protein (GRIP)/protein interacting with C-kinase-1 (PICK1) enhanced basal synaptic transmission. These results suggest that constitutive trafficking and anchoring of AMPARs help maintain basal synaptic transmission. The regulation of postsynaptic AMPAR trafficking involves synaptotagmin-SNARE-mediated vesicle exocytosis and interactions between AMPARs and the PDZ domains in GRIP/PICK1. We show that inhibitors of synaptotagmin-SNARE-mediated exocytosis, or interactions between AMPARs and GRIP/PICK1, attenuated AdA-enhanced increases in EPSC amplitudes. These results suggest that IP(3)R-mediated Ca(2+) release can enhance AMPAR EPSC amplitudes through mechanisms that involve AMPAR-PDZ interactions and/or synaptotagmin-SNARE-mediated receptor trafficking.     

Bidirectional regulation of stress responses by galanin in mice: Involvement of galanin receptor subtype 1

The neuropeptide galanin has been shown to play a role in psychiatric disorders as well as in other biological processes including regulation of pain threshold through interactions with three G-protein coupled receptors, galanin receptor subtypes 1–3 (GalR1−3). While most of the pharmacological studies on galanin in stress-related disorders have been done with rats, the continuous development of genetically engineered mice involving galanin or its receptor subtype(s) validates the importance of mouse pharmacological studies. The present study on mice examined the homeostatic, endocrinological and neuroanatomical effects of the galanin, injected intracerebroventricularly (i.c.v.), in regulation of stress responses after restraint stress. Furthermore, the roles of GalR1 on these effects were studied using GalR1 knockout (KO) mice. The core body temperature and the locomotor activity were monitored with radio telemetry devices. Galanin (i.c.v.) decreased locomotor activity and exerted a bidirectional effect on the restraint stress–induced hyperthermia; a high dose of galanin significantly attenuated the stress-induced hyperthermic response, while a low dose of galanin moderately enhanced this response. The bidirectional effect of galanin was correlated with changes in stress hormone levels (adrenocorticotropic hormone and corticosterone). To neuroanatomically localize the effects of galanin on stress response, cFos immunoreactivity was assessed in galanin receptor rich areas; paraventricular nucleus (PVN) of the hypothalamus and the locus coeruleus (LC), respectively. A high dose of galanin significantly induced cFos activity in the LC but not in the PVN. In GalR1KO mice, a high dose of galanin failed to induce any of the above effects, suggesting the pivotal role of GalR1 in decreased locomotor activity and stress-resistant effects caused by galanin i.c.v. injection studied here.     

A novel, systemically active, selective galanin receptor type-3 ligand exhibits antidepressant-like activity in preclinical tests

The neuropeptide galanin is widely expressed in limbic nuclei in the brain, and plays an important role in the regulation of homeostatic and affective behaviors, in part through its modulation of central monoamine pathways. Recent evidence suggests that galanin and its receptors may be involved in the efficacy of various modalities of antidepressant treatments. We have previously demonstrated that systemically active, non-peptide galanin receptor type-1/2 agonists exhibit antidepressant-like effects in the rat forced swim test. Here we evaluate a novel galanin receptor type-3 (GalR3) antagonist in preclinical tests of anxiety and depression. At multiple doses, the compound displayed no effects in the elevated plus maze in mice. By contrast, the compound decreased time spent immobile in the tail suspension test by mice. Additionally, the GalR3 drug decreased time spent immobile in the forced swim test in rats, similarly to the effects of desipramine, yet did not increase locomotor activity in an open field test. These combined data from two species indicate that GalR3 receptor antagonists may exhibit antidepressant-like effects.     

The role of galanin receptors in anticonvulsant effects of low-frequency stimulation in perforant path-kindled rats

Low-frequency stimulation (LFS) has antiepileptogenic effects on kindled seizures. In the present study, the role of galanin receptors in the inhibitory effect of LFS on perforant path kindling acquisition was investigated in rats. Animals were kindled by perforant path stimulation in a rapid kindling manner (six stimulations per day). LFS (0.1 ms pulses at 1 Hz, 600 pulses, and 80-150 microA) was applied immediately after termination of each kindling stimulation. M35 (0.5 and 1.0 nM per site), a nonselective galanin receptor antagonist and M871 (1.0 microM per site), a selective galanin receptor type 2 (GalR2) antagonist, were daily microinjected into the dentate gyrus before starting the stimulation protocol. The expression of GalR2 in the dentate gyrus was also investigated using semi-quantitative RT-PCR. Application of LFS significantly retarded the kindling acquisition and delayed the expression of different kindled seizure stages. In addition, LFS significantly reduced the increment of daily afterdischarge duration during kindling development. Intra-dentate gyrus microinjection of both M35 and M871 significantly prevented the inhibitory effects of LFS on kindling parameters. During the focal kindled seizure stages (1-3) M871 had no significant effect. However, during generalized seizure stages (4 and 5), M871 had the same effect as M35. Semi-quantitative RT-PCR also showed that after kindling acquisition, the GalR2 mRNA level decreased in the dentate gyrus but application of LFS prevented this decrease. Obtained results show that activation of galanin receptors by endogenous galanin has a role in mediating the inhibitory effect of LFS on perforant path-kindled seizures. This role is exerted through GalR1 during focal- and through GalR2 during generalized-kindled seizures.     

Mechanism of action of galanin on myenteric neurons

1. Conventional intracellular recording methods were used to investigate the mechanism of action of galanin on electrical behavior of AH/type 2 myenteric neurons in the guinea pig small intestine. 2. The overall action of galanin was inhibitory and consisted of membrane hyperpolarization, decreased input resistance, and suppression of excitability. 3. The action of galanin was on the somatic membrane. There were no effects on spike initiation or propagation velocity in the processes. 4. The reversal potential for the hyperpolarizing action of galanin was near the estimated K+ equilibrium potential and was dependent on the concentration of K+ in the bathing medium. 5. Treatment with tetraethylammonium (TEA) broadened the action-potential and enhanced long-lasting hyperpolarizing after-potentials (AH). Application of galanin or depletion of Ca2+ in the bathing medium offset the effects of TEA on the spike and the AH. Galanin or reduced Ca2+ had the same effect when both TEA and tetrodotoxin (TTX) were present. 6. Simultaneous application of TEA and 4-aminopyridine (4-AP) evoked spontaneous spike discharge with broadened spikes and enhanced AH. This activity was suppressed by galanin. 7. Intrasomatic injection of Cs+ in the presence of TTX appeared to abolish all K+ conductances leaving pure Ca2+ spikes in response to depolarizing current pulse. Galanin abolished these Ca2+ spikes. 8. The results suggest two major mechanisms of action for galanin. One is to open K+ channels, decrease input resistance, and hyperpolarize the membrane toward EK+. The second is blockade of voltage gated Ca2+ channels and suppression of the AH by indirect prevention of opening of Ca2+-dependent K+ channels.     

甘丙肽对GT1-7细胞GnRH释放的调节作用

目的：GT1-7细胞是替代研究GnRH神经元的理想细胞模型。本实验研究甘丙肽1型，2型受体mRNA在GT1-7细胞中的表达及对GnRH的调节作用。方法：（1）采用逆转录-聚合酶链反应（RT-PCR）法观察甘丙肽受体mRNA在GT1-7中的表达；（2）将不同浓度的甘丙肽以不同时间与GT1-7细胞卵育，用RIA法测定细胞上清液中GnRH含量。结果：（1）GT1-7细胞同时表达甘丙肽1型和2型受体mRNA;(2)甘丙肽能刺激GnRH释放，且呈明显的量效关系。结论：甘丙肽可通过其受体直接作用下丘脑GnRH神经元，而对生殖功能起调节作用。     

M(1) and M(2) muscarinic acetylcholine receptor subtypes mediate Ca(2+) channel current inhibition in rat sympathetic stellate ganglion neurons

Muscarinic acetylcholine receptors (mAChRs) are known to mediate the acetylcholine inhibition of Ca(2+) channels in central and peripheral neurons. Stellate ganglion (SG) neurons provide the main sympathetic input to the heart and contribute to the regulation of heart rate and myocardial contractility. Little information is available regarding mAChR regulation of Ca(2+) channels in SG neurons. The purpose of this study was to identify the mAChR subtypes that modulate Ca(2+) channel currents in rat SG neurons innervating heart muscle. Accordingly, the modulation of Ca(2+) channel currents by the muscarinic cholinergic agonist, oxotremorine-methiodide (Oxo-M), and mAChR blockers was examined. Oxo-M-mediated mAChR stimulation led to inhibition of Ca(2+) currents through voltage-dependent (VD) and voltage-independent (VI) pathways. Pre-exposure of SG neurons to the M(1) receptor blocker, M(1)-toxin, resulted in VD inhibition of Ca(2+) currents after Oxo-M application. On the other hand, VI modulation of Ca(2+) currents was observed after pretreatment of cells with methoctramine (M(2) mAChR blocker). The Oxo-M-mediated inhibition was nearly eliminated in the presence of both M(1) and M(2) mAChR blockers but was unaltered when SG neurons were exposed to the M(4) mAChR toxin, M(4)-toxin. Finally, the results from single-cell RT-PCR and immunofluorescence assays indicated that M(1) and M(2) receptors are expressed and located on the surface of SG neurons. Overall, the results indicate that SG neurons that innervate cardiac muscle express M(1) and M(2) mAChR, and activation of these receptors leads to inhibition of Ca(2+) channel currents through VI and VD pathways, respectively.     

Synchronous and asynchronous exocytosis induced by subthreshold high K+ at Cs(+)-loaded terminals of rat hippocampal neurons

Transmitter release at Cs(+)-loaded autaptic terminals was selectively activated by the subthreshold concentration of external K+, and Ca(2+) channel types and transmitter pools involved in synchronous and asynchronous exocytosis were studied. When a neuron was depolarized to +30 mV by applying a current through a pipette containing Cs(+) for >30 s, a rapid external K+ jump to 3.75-10 mM, otherwise ineffective, produced an outward current (K10 response). K10 responses were initially graded (type-1) and then became a spike and plateau-shape with (type-2) or without a latency (type-3). On repolarization to -60 mV, a high K+ jump induced inward currents (called also K10 response) similar to those at +30 mV, whose shape changed from that of type-3, then type-2 and finally type-1 over 30 min. During a period favorable for inducing a type-3 response, a current similar to this response was generated by a voltage pulse (+ 80 or 90 mV, 20 or 30 ms) to the cell soma. Currents similar to K10 responses were rarely induced by a high K+ jump without a conditioning depolarization except for some cells, but consistently produced when 3 mM Cs(+) and 50 microM 4-aminopyridine were externally applied for tens of minutes. Picrotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione with 3-[(RS)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid or Cd(2+) in, or Ca(2+) removal from, a high-K+ solution blocked all the K10 responses, while a plateau remaining after a high K+ jump was not blocked by Ca(2+) removal immediately after the K+ jump. Thus Cs(+) loading and decreased K+ concentration in autaptic terminals by a conditioning depolarizing current selectively sensitize the terminals to a subthreshold high K+ jump for depolarization to activate synchronous or asynchronous transmitter release. Nicardipine (5-10 microM) blocked type-1 and -2 responses but not type-3 responses, while omega-conotoxin (10 microM) blocked all the types of K10 response in the presence of nicardipine. Increasing the interval of high K+ jumps biphasically increased the magnitude of K10 response, preferentially in the postjump fraction reflecting purely the asynchronous activation of exocytotic machinery, and decreased the reduction of miniature postsynaptic current frequency after a K10 response. These results suggest the roles of N(P/Q)-type Ca(2+) channels in synchronous exocytosis at the terminals, L-type Ca(2+) channels in initiating a Ca(2+) action potential at the parent axon and both types in asynchronous exocytosis and also suggest the different releasable pools of transmitter for two modes of exocytosis in cultured hippocampal neurons.     

Influence of Na(+), dicarboxylic amino acids, and pH in modulating the low-calcium response of Yersinia pestis

The virulence of yersiniae is promoted in part by shared approximately 70-kb plasmids (pCD in Yersinia pestis and pYV in enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica) that mediate a low-calcium response. This phenotype is characterized at 37 degrees C by either bacteriostasis in Ca(2+)-deficient medium with expression of pCD/pYV-encoded virulence effectors (Yops and LcrV) or vegetative growth and repression of Yops and LcrV with > or =2.5 mM Ca(2+) (Lcr(+)). Regulation of Yops and LcrV is well defined but little is known about bacteriostasis other than that Na(+) plus l-glutamate promotes prompt restriction of Y. pestis. As shown here, l-aspartate substituted for l-glutamate in this context but only Na(+) exacerbated the nutritional requirement for Ca(2+). Bacteriostasis of Y. pestis (but not enteropathogenic yersiniae) was abrupt in Ca(2+)-deficient medium at neutral to slightly alkaline pH (7.0 to 8.0), although increasing the pH to 8.5 or 9.0, especially with added Na(+) (but not l-glutamate), facilitated full-scale growth. Added l-glutamate (but not Na(+)) favored Ca(2+)-independent growth at acidic pH (5.0 to 6.5). Yops and LcrV were produced in Ca(2+)-deficient media at pH 6.5 to 9.0 regardless of the presence of added Na(+) or l-glutamate, although their expression at alkaline pH was minimal. Resting Ca(2+)-starved Lcr(+) cells of Y. pestis supplied with l-glutamate first excreted and then destroyed l-aspartate. These findings indicate that expression of Yops and LcrV is necessary but not sufficient for bacteriostasis of Ca(2+)-starved yersiniae and suggest that abrupt restriction of Y. pestis requires Na(+) and the known absence of aspartate ammonia-lyase in this species.     

Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN)

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.     

Use of knockout mice reveals involvement of M2-muscarinic receptors in control of the kinetics of acetylcholine release

We have previously suggested that presynaptic M(2)-muscarinic receptors (M(2)R) are involved in the control of the time course of evoked acetylcholine release in the frog neuromuscular junction. The availability of knockout mice lacking functional M(2)R (M(2)-KO) enabled us to address this issue in a more direct way. Using the phrenic diaphragm preparation, we show that in wild-type (WT) mice experimental manipulations known to affect Ca(2+) entry and removal, greatly affected the amount of acetylcholine released (quantal content). However, the time course of release remained unaltered under all these experimental treatments. On the other hand, in the M(2)-KO mice, similar experimental treatments affected both the quantal content and the time course of release. In general, a larger quantal content was accompanied by a longer duration of release. Similarly, the rise time of the postsynaptic current produced by axon stimulation was sensitive to changes in [Ca(2+)](o) or [Mg(2+)](o) in M(2)-KO mice but not in WT mice. Measurements of Ca(2+) currents revealed that the shorter rise time of the postsynaptic current seen in high [Mg(2+)](o) in M(2)-KO mice was not produced by a shorter wave of the presynaptic Ca(2+) current. These results support our earlier findings and provide direct evidence for the major role that presynaptic M(2)-muscarinic receptors play in the control of the time course of evoked acetylcholine release under physiological conditions.     

Antinociceptive effects of intracerebroventricular injection of the galanin receptor 1 agonist M 617 in rats

Previous studies in our laboratory demonstrated that galanin and its receptors play important roles in nociceptive modulation in the central nervous system. The present study was performed to explore the antinociceptive effects of the galanin receptor 1 agonist M 617 in the central nervous system of rats. Intracerebroventricular injection of 0.1nmol, 0.5nmol, 1nmol or 2nmol of M 617 induced dose-dependent increases in hindpaw withdrawal latencies (HWLs) to noxious thermal and mechanical stimulations in rats. Furthermore, both intracerebroventricular injection of M 617 and galanin induced significant increases in HWLs in rats. Interestingly, there were no significant differences between the antinociceptive effects induced by M 617 and galanin, indicating that galanin receptor 1 plays main roles in galanin-induced antinociceptive effects in the brain of rats.     

Role of Ca(2+) in the synchronization of transmitter release at calyceal synapses in the auditory system of rat.

The synchronization of transmitter release in the synapse of the medial nucleus of the trapezoid body (MNTB) is achieved during early postnatal development as a consequence of elimination of delayed asynchronous releases and appears to reflect changes in the dynamics of Ca(2+) entry and clearance. To examine the role of Ca(2+) in regulating synchronization of transmitter release in the mature synapse (after postnatal day 9, P9), we perturbed Ca(2+) dynamics systematically. Replacement of external Ca(2+) (2 mM) with Sr(2+) induced delayed asynchronous release following the major EPSC. We tried to reproduce asynchronous releases without using Sr(2+) and instead by manipulating the time course and the size of Ca(2+) transient in the presynaptic terminal, under the assumption that replacement of external Na(+) with Li(+) or application of eosin-Y would prolong the lifetime of Ca(2+) transient by reducing the rate of Ca(2+) extrusion from the terminal. With application of Li(+), Ca(2+) transient in the terminal was prolonged, the EPSC decay time course was prolonged, and the EPSC amplitude increased. However, these EPSCs were not followed by delayed asynchronous release. When Ca(2+) influx was reduced, either by partial Ca(2+) channel blockade with a low concentration of Cd(2+) or omega-agatoxin IVA, a marked asynchronous release resulted. This was further enhanced by the combined application of Li(+) or eosin-Y. These results suggest that cooperative increases of both Ca(2+) influx and Ca(2+) clearance capacities leading to a sharper Ca(2+) spike in the presynaptic terminal underlie synchronized transmitter release in the presynaptic terminal of the MNTB.