Assessing the role of Ih channels in synaptic transmission and mossy fiber LTP

Hyperpolarization-activated nonselective cation channels (Ih channels) play an important role in the control of membrane excitability and rhythmic neuronal activity. The functional relevance of presynaptic Ih channels in regulating synaptic function, however, is not well established. Recently, it has been proposed [Mellor, J., Nicoll, R. A. & Schmitz, D. (2002) Science 295, 143-147] that presynaptic Ih channels are necessary for hippocampal mossy fiber long-term potentiation (LTP). This observation challenges an alternative model that suggests presynaptic forms of LTP are caused by a direct modification of the transmitter release machinery. Here, we assess the role of Ih in hippocampal mossy fiber LTP as well as cerebellar parallel fiber LTP, forms of potentiation that share common mechanisms. Our results show that after Ih blockade neither mossy fiber LTP nor parallel fiber LTP are affected. Furthermore, Ih does not significantly modify basal excitatory synaptic transmission in the hippocampus, whereas the organic Ih blockers ZD7288 and DK-AH 269 induce a large Ih-independent depression of synaptic transmission. In summary, our results indicate that Ih-mediated persistent changes in presynaptic excitability do not underlie presynaptic forms of LTP.     

Effects of ethanol on excitatory and inhibitory synaptic transmission in rat cortical neurons

BACKGROUND: The gamma-aminobutyric acid-A (GABA(A)) receptor and glutamate receptors are among the most important target sites for the behavioral effects of ethanol. However, data in the literature concerning the ethanol modulation of the GABA(A) and glutamate receptors have been controversial. The activity of the neuronal nicotinic acetylcholine (ACh) receptors (nAChRs) has recently been reported to be potently augmented by ethanol. The activation of nAChRs is also known to cause the release of various neurotransmitters including GABA and glutamate. Thus, ethanol potentiation of nAChRs is expected to stimulate the GABAergic and glutamatergic systems. METHODS: Whole-cell patch clamp experiments were performed using rat cortical neurons in primary culture to record spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and spontaneous miniature excitatory postsynaptic currents (mEPSCs). RESULTS: Two types of neurons were distinguished: bipolar neurons possessed alpha4beta2 nAChRs generating a steady current in response to 30 nM ACh, and multipolar neurons that did not generate a current by ACh application. Acetylcholine greatly increased the frequency of mEPSCs and mIPSCs in bipolar neurons but not in multipolar neurons. The amplitude of neither type of neuron was affected by ACh. Ethanol at 10 to 100 mM suppressed the amplitude of mEPSCs while augmenting the amplitude of mIPSCs in both bipolar and multipolar neurons, indicating the direct action on the respective receptors. In bipolar neurons, ACh plus 100 mM ethanol greatly increased the frequency of mIPSCs beyond the levels achieved by ACh alone, while no such increases were observed in multipolar neurons. CONCLUSIONS: It is concluded that ethanol stimulation of nAChRs modulates the activity of both glutamate and GABA receptors in rat cortical bipolar neurons.     

Ethanol modulation of excitatory and inhibitory synaptic transmission in rat and monkey dentate granule neurons

BACKGROUND: The physiological mechanisms underlying the behavioral and cognitive effects of ethanol are not fully understood. However, there is now compelling evidence that ethanol acts, at least in part, by modulating the function of a small group of proteins that mediate excitatory and inhibitory synaptic transmission. For example, intoxicating concentrations of ethanol have been shown to enhance GABAergic synaptic inhibition and depress glutamatergic excitatory neurotransmission in a number of brain regions. Because all of these electrophysiological studies have been performed in rodent brain slice or neuronal culture preparations, direct evidence that ethanol exerts similar effects on synaptic transmission in the primate central nervous system is lacking. METHODS: We have therefore developed methods to perform patch-clamp electrophysiological recordings from neurons in acutely prepared monkey (Macaca fascicularis) hippocampal slices. We have used these methods to compare the acute effects of ethanol on excitatory and inhibitory synaptic transmission in rat and monkey dentate granule neurons. RESULTS: Under our recording conditions, ethanol significantly potentiated gamma-aminobutyric acid type A inhibitory postsynaptic currents in both rat and monkey neurons. In addition, ethanol significantly inhibited NMDA, but not AMPA, excitatory postsynaptic currents in dentate granule neurons from both species. Notably, no significant differences were observed in any of the pharmacological properties of inhibitory or excitatory synaptic responses recorded from rat and monkey neurons. CONCLUSIONS: These data suggest that the differences in the behavioral effects of ethanol that have been observed between rats and higher-order mammals, such as monkeys and humans, may not reflect differences in the sensitivity of some of the major synaptic sites of ethanol action. Moreover, our results provide empirical evidence for the use of rodent brain slice preparations in elucidating synaptic mechanisms of ethanol action in the primate central nervous system.     

Dissociation of experience-dependent and -independent changes in excitatory synaptic transmission during development of barrel cortex

A fundamental problem in the study of cortical development is the extent to which the formation and refinement of synaptic circuitry depends upon sensory experience. The barrel cortex is a useful model system to study experience-dependent cortical development because there is a simple mapping of individual whiskers to the corresponding barrel columns in the cortex. We investigated experience-dependent and -independent changes in glutamatergic synaptic transmission in the barrel cortex during the second postnatal week by comparing synaptic responses from whisker-intact mice at postnatal day (P) 7 and P14 with those from whisker-deprived mice at P14. alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA)-receptor-mediated excitatory synaptic responses were recorded from layer 2/3 pyramidal cells in vitro during voltage-clamp in response to stimulation in layer 4. We observed that the ratio of synaptic AMPA- to NMDA-receptor-mediated current (A/N ratio) increased with developmental age. The development of the A/N ratio was unchanged by deprivation of the whisker input throughout the second postnatal week. In contrast, the NMDA-receptor current decay and sensitivity to the NMDA receptor 2B subunit-selective antagonist ifenprodil was affected strongly by such deprivation. These results demonstrate a concurrent dissociation between sensory experience-dependent and -independent changes of glutamatergic transmission in the barrel cortex during the second postnatal week. Furthermore, they suggest that the development of subunit composition of synaptic receptors is dependent on sensory experience, whereas maturation of the synaptic A/N ratio is independent of such experience. Thus, different components of synaptic development may be governed by different developmental rules.     

Growth hormone and insulin-like growth factor-I alter hippocampal excitatory synaptic transmission in young and old rats

In rats, as in humans, normal aging is characterized by a decline in hippocampal-dependent learning and memory, as well as in glutamatergic function. Both growth hormone (GH) and insulin-like growth factor-I (IGF-I) levels have been reported to decrease with age, and treatment with either GH or IGF-I can ameliorate age-related cognitive decline. Interestingly, acute GH and IGF-I treatments enhance glutamatergic synaptic transmission in the rat hippocampus of juvenile animals. However, whether this enhancement also occurs in old rats, when cognitive impairment is ameliorated by GH and IGF-I (des-IGF-I), remains to be determined. To address this issue, we used an in vitro CA1 hippocampal slice preparation and extracellular recording techniques to study the effects of acute application of GH and IGF-I on compound field excitatory postsynaptic potentials (fEPSPs), as well as AMPA- and NMDA-dependent fEPSPs, in young adult (10 months) and old (28 months) rats. The results indicated that both GH and IGF-I increased compound-, AMPA-, and NMDA-dependent fEPSPs to a similar extent in slices from both age groups and that this augmentation was likely mediated via a postsynaptic mechanism. Initial characterization of the signaling cascades underlying these effects revealed that the GH-induced enhancement was not mediated by the JAK2 signaling element in either young adult or old rats but that the IGF-I-induced enhancement involved a PI3K-mediated mechanism in old, but not young adults. The present findings are consistent with a role for a GH- or IGF-I-induced enhancement of glutamatergic transmission in mitigating age-related cognitive impairment in old rats.     

Critical roles for fast synaptic transmission in mediating estradiol negative and positive feedback in the neural control of ovulation

A switch in the balance of estradiol feedback actions from negative to positive initiates the GnRH surge, triggering the LH surge that causes ovulation. Using an ovariectomized, estradiol-treated (OVX+E) mouse model that exhibits daily switches between negative in the morning and positive feedback in the evening, we investigated the roles of fast synaptic transmission in regulating GnRH neuron firing during negative and positive feedback. Targeted extracellular recordings were used to monitor activity of GnRH neurons from OVX+E and OVX mice in control solution or solution with antagonists to both ionotropic glutamate and gamma-aminobutyric acid receptors (blockade). Blockade had no effect on activity of OVX cells. In contrast, in OVX+E cells in the morning, blockade increased activity compared with control cells, whereas in the evening, blockade decreased activity. In vivo barbiturate sedation of OVX+E mice that blocked LH surge induction prevented the in vitro evening changes in firing rate and response to blockade. These observations suggest at least partial inversion of the negative-to-positive switch in estradiol feedback action and indicate that changes in fast synaptic transmission to GnRH neurons and within the network of cells presynaptic to GnRH neurons are critical for mediating estradiol negative and positive feedback actions on GnRH neurons. Fast synaptic transmission may also affect GnRH neuron activity indirectly through altering release of excitatory and inhibitory neuromodulators onto GnRH neurons at specific times of day. Fast synaptic transmission is thus critical for proper generation and timing of the GnRH surge.     

Effect of insulin on excitatory synaptic transmission onto dopamine neurons of the ventral tegmental area in a mouse model of hyperinsulinemia

Obesity has drastically increased over the last few decades. Obesity is associated with elevated insulin levels, which can gain access to the brain, including into dopamine neurons of the ventral tegmental area (VTA), a brain region critical for mediating reward-seeking behavior. Synaptic plasticity of VTA dopamine neurons is associated with altered motivation to obtain reinforcing substances such as food and drugs of abuse. Under physiological circumstances, insulin in the VTA can suppress excitatory synaptic transmission onto VTA dopamine neurons and reduce aspects of palatable feeding behavior. However, it is unknown how insulin modulates excitatory synaptic transmission in pathological circumstances such as hyperinsulinemia. Using patch-clamp electrophysiology, we demonstrate that, in a hyperinsulinemic mouse model, insulin has reduced capacity to cause a synaptic depression of VTA dopamine neurons, although both low-frequency stimulation-induced long-term depression and cannabinoid-induced depression were normal. These results suggest that insulin action in the VTA during pathological hyperinsulinemia is disrupted and may lead to increased feeding behavior.     

Folliculin-interacting proteins Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn

Folliculin (FLCN)-interacting proteins 1 and 2 (FNIP1, FNIP2) are homologous binding partners of FLCN, a tumor suppressor for kidney cancer. Recent studies have revealed potential functions for Flcn in kidney; however, kidney-specific functions for Fnip1 and Fnip2 are unknown. Here we demonstrate that Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn. We observed no detectable phenotype in Fnip2 knockout mice, whereas Fnip1 deficiency produced phenotypes similar to those seen in Flcn-deficient mice in multiple organs, but not in kidneys. We found that absolute Fnip2 mRNA copy number was low relative to Fnip1 in organs that showed phenotypes under Fnip1 deficiency but was comparable to Fnip1 mRNA copy number in mouse kidney. Strikingly, kidney-targeted Fnip1/Fnip2 double inactivation produced enlarged polycystic kidneys, as was previously reported in Flcn-deficient kidneys. Kidney-specific Flcn inactivation did not further augment kidney size or cystic histology of Fnip1/Fnip2 double-deficient kidneys, suggesting pathways dysregulated in Flcn-deficient kidneys and Fnip1/Fnip2 double-deficient kidneys are convergent. Heterozygous Fnip1/homozygous Fnip2 double-knockout mice developed kidney cancer at 24 mo of age, analogous to the heterozygous Flcn knockout mouse model, further supporting the concept that Fnip1 and Fnip2 are essential for the tumor-suppressive function of Flcn and that kidney tumorigenesis in human Birt–Hogg–Dubé syndrome may be triggered by loss of interactions among Flcn, Fnip1, and Fnip2. Our findings uncover important roles for Fnip1 and Fnip2 in kidney tumor suppression and may provide molecular targets for the development of novel therapeutics for kidney cancer.Germline alteration of the folliculin (FLCN) gene, a novel tumor suppressor, is responsible for Birt–Hogg–Dubé (BHD) syndrome, an inherited kidney cancer syndrome characterized by cutaneous fibrofolliculomas, pulmonary cysts, and an increased risk for the development of kidney cancer (1–4). Genetic studies using Flcn knockout mice have defined important roles for Flcn in metabolism. Kidney-targeted Flcn knockout mice developed enlarged polycystic kidneys with elevated mechanistic target of rapamycin complex 1 (mTORC1) activity (5) and increased mitochondrial biogenesis through up-regulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a) (6). Muscle-targeted Flcn knockout mice displayed both red-colored muscle with increased mitochondrial biogenesis caused by elevated Ppargc1, and cardiac hypertrophy with up-regulated mTORC1, which were ameliorated by Ppargc1a inactivation, suggesting that Flcn might serve as a critical link connecting mTORC1 with Ppargc1a-driven mitochondrial biogenesis (5–7). Importantly, mice heterozygous for Flcn inactivation develop renal tumors at 24 mo of age with increased mTORC1/2 activity and Ppargc1a expression, which mimics the human BHD tumor phenotype (6, 8–10), suggesting potential therapeutic targets in metabolic pathways for BHD-associated kidney cancer.The first FLCN interacting protein FNIP1 was identified through protein–protein interaction studies of the FLCN protein (11). FNIP1 binds to the C terminus of FLCN and to AMP-activated protein kinase (AMPK) (11), a critical molecule for energy sensing, further underscoring a central role for the FLCN/FNIP1 pathway in cellular metabolism. A second folliculin-interacting protein FNIP2 was discovered through bioinformatics searches for sequences similar to FNIP1 (12, 13). Similar to FNIP1, FNIP2 was found to bind to the C terminus of FLCN and to AMPK (12), suggesting a potential functional redundancy with FNIP1. Recent studies with Fnip1 knockout mouse models have demonstrated that Fnip1 is required for B-cell development (14, 15). Interestingly, a Flcn knockout mouse model using the tamoxifen-inducible ER (mutated form of the ligand-binding domain of the estrogen receptor)-Cre system also displayed defects in B-cell development (14), suggesting Fnip1 knockout mice might develop phenotypes similar to those that develop as a consequence of Flcn deficiency.Furthermore, FLCN has been shown to have a variety of functions that might potentially link AMPK, mTOR, and Ppargc1a with other important pathways. Crystallographic studies have shown that the C terminus of FLCN may be distantly related to Differentially Expressed in Normal Cells and Neoplasia (DENN) domain proteins and may possess guanine nucleotide exchange factor activity toward RAB35 (16). FLCN modulates TFE3 localization (17), which may play an important role in the exit of stem cells from pluripotency (18), and interacts with other signaling pathways including the von Hippel-Lindau–hypoxia inducible factor–vascular endothelial growth factor axis (19–21), the TGF-beta pathway (22, 23), Rho A signaling (24, 25), cell cycle regulation (26, 27), Rag-mediated amino acid sensing (28, 29), and autophagy (30, 31). These findings underscore FLCN as an important molecule, inactivation of which affects multiple pathways.To clarify the function of FLCN-interacting proteins Fnip1 and Fnip2, we inactivated Fnip1 and/or Fnip2 in mouse kidneys, muscle, and heart and investigated the effect on the mTOR pathway and mitochondrial metabolism. The absolute mRNA copy number of Fnip1 and Fnip2 was measured using droplet digital PCR (ddPCR) technology. To evaluate functional synergy of Fnip1 and Fnip2 with Flcn, we also inactivated Flcn, Fnip1, and Fnip2 simultaneously in mouse kidneys. Finally, we searched for latent tumor development in Fnip1 and Fnip2 knockout mice.     

Membrane-shaping host reticulon proteins play crucial roles in viral RNA replication compartment formation and function

Positive-strand RNA viruses replicate their genomes on membranes with virus-induced rearrangements such as single- or double-membrane vesicles, but the mechanisms of such rearrangements, including the role of host proteins, are poorly understood. Brome mosaic virus (BMV) RNA synthesis occurs in ≈70 nm, negatively curved endoplasmic reticulum (ER) membrane invaginations induced by multifunctional BMV protein 1a. We show that BMV RNA replication is inhibited 80–90% by deleting the reticulon homology proteins (RHPs), a family of membrane-shaping proteins that normally induce and stabilize positively curved peripheral ER membrane tubules. In RHP-depleted cells, 1a localized normally to perinuclear ER membranes and recruited the BMV 2a^pol polymerase. However, 1a failed to induce ER replication compartments or to recruit viral RNA templates. Partial RHP depletion allowed formation of functional replication vesicles but reduced their diameter by 30–50%. RHPs coimmunoprecipitated with 1a and 1a expression redirected >50% of RHPs from peripheral ER tubules to the interior of BMV-induced RNA replication compartments on perinuclear ER. Moreover, RHP-GFP fusions retained 1a interaction but shifted 1a-induced membrane rearrangements from normal vesicles to double membrane layers, a phenotype also induced by excess 1a-interacting 2a^pol. Thus, RHPs interact with 1a, are incorporated into RNA replication compartments, and are required for multiple 1a functions in replication compartment formation and function. The results suggest possible RHP roles in the bodies and necks of replication vesicles.     

The synaptic proteins neurexins and neuroligins are widely expressed in the vascular system and contribute to its functions

Unlike other neuronal counterparts, primary synaptic proteins are not known to be involved in vascular physiology. Here, we demonstrate that neurexins and neuroligins, which constitute large and complex families of fundamental players in synaptic activity, are produced and processed by endothelial and vascular smooth muscle cells throughout the vasculature. Moreover, they are dynamically regulated during vessel remodeling and form endogenous complexes in large vessels as well as in the brain. We used the chicken chorioallantoic membrane as a system to pursue functional studies and demonstrate that a monoclonal recombinant antibody against β-neurexin inhibits angiogenesis, whereas exogenous neuroligin has a role in promoting angiogenesis. Finally, as an insight into the mechanism of action of β-neurexin, we show that the anti-β-neurexin antibody influences vessel tone in isolated chicken arteries. Our finding strongly supports the idea that even the most complex and plastic events taking place in the nervous system (i.e., synaptic activity) share molecular cues with the vascular system.     

白细胞介素5和嗜酸粒细胞趋化因子在支气管哮喘大鼠肺和骨髓信号传导中的作用

目的探讨支气管哮喘(简称哮喘)大鼠模型中白细胞介素5(IL-5)和嗜酸粒细胞趋化因子(eotaxin)是否参与肺和骨髓间信号传导,观察不同干预措施的影响。方法清洁级雄性Wistar大鼠40只,按随机数字表法分为正常对照组和哮喘组,每组20只。用卵白蛋白(OVA)致敏和激发制作哮喘模型,于激发后30 min及6、12、24、48 h处死大鼠,分别取外周血涂片、骨髓细胞涂片。肺组织以10％甲醛固定做石蜡切片。将血涂片、骨髓涂片、肺组织切片行苏木精-伊红(HE)染色,观察细胞总数和嗜酸粒细胞(EOS)比例。取不同时间点肺组织匀浆,用酶联免疫吸附测定(ELISA)法测定肺组织匀浆IL-5和eotaxin浓度。将不同时间点的肺组织匀浆与正常对照组和哮喘组大鼠骨髓细胞共同孵育,并分别加入地塞米松(DXM)、半乳凝素3(Galectin-3)、孟鲁司特钠、抗IL-5抗体进行干预,采用抗IL-5、抗IL-SRα、抗eotaxin、抗CD34和抗CC趋化因子受体3(CCR3)多克隆抗体进行免疫组化染色,检测两组骨髓细胞中EOS计数和IL-5+细胞、IL-5Rα+细胞、CCR3+细胞、eotaxin+细胞、CD34+细胞的百分比。结果哮喘大鼠外周血、骨髓、肺组织中EOS数分别为0．020 0±0．002 0、0．023±0．003、0．025 0±0．009 0,正常对照组分别为0．010 0±0．003 0、0．009±0．003、0．009 0±0．002 0,两组比较差异有统计学意义(t值分别为2．547、2．718、2．718,P均<0．05);哮喘大鼠肺组织匀浆于激发6 h的IL-5为(89．3±2．4)pg/ml,12 h的eotaxin水平为(4．9±0．5)pg／ml,48 h均回到基线[(1．45±0．23) pg／ml]水平;除30 min外,各时间点哮喘大鼠肺组织匀浆均能诱导正常对照组或哮喘组大鼠骨髓细胞中CD34+、EOS、IL-5+、IL-SRα+、CCR3+、eotaxin+细胞表达增加,Galectin-3干预哮喘大鼠骨髓细胞后,骨髓EOS、IL-5+、IL-5Rα+、CCR3+、eotaxin+和CD34+细胞表达减少(分别为0．021±0．005、0．074±0．007、0．138±0．014、0．067±0．010、0．040±0．005、0．087±0．012)。结论IL-5 mRNA转录选择性抑制物(Galectin-3)能抑制EOS炎症,有可能成为一种特异性的抗哮喘药物。     

SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice

How the nuclei in mammalian skeletal muscle fibers properly position themselves relative to the cell body is an interesting and important cell biology question. In the syncytial skeletal muscle cells, more than 100 nuclei are evenly distributed at the periphery of each cell, with 3–8 nuclei anchored beneath the neuromuscular junction (NMJ). Our previous studies revealed that the KASH domain–containing Syne-1/Nesprin-1 protein plays an essential role in anchoring both synaptic and nonsynaptic myonuclei in mice. SUN domain–containing proteins (SUN proteins) have been shown to interact with KASH domain–containing proteins (KASH proteins) at the nuclear envelope (NE), but their roles in nuclear positioning in mice are unknown. Here we show that the synaptic nuclear anchorage is partially perturbed in Sun1, but not in Sun2, knockout mice. Disruption of 3 or all 4 Sun1/2 wild-type alleles revealed a gene dosage effect on synaptic nuclear anchorage. The organization of nonsynaptic nuclei is disrupted in Sun1/2 double-knockout (DKO) mice as well. We further show that the localization of Syne-1 to the NE of muscle cells is disrupted in Sun1/2 DKO mice. These results clearly indicate that SUN1 and SUN2 function critically in skeletal muscle cells for Syne-1 localization at the NE, which is essential for proper myonuclear positioning.     

Small G proteins exhibit pattern sensitivity in MAPK activation during the induction of memory and synaptic facilitation in Aplysia

Memory formation is highly sensitive to specific patterns of training, but the cellular and molecular mechanisms underlying pattern sensitivity are not well understood. We explored this general question by using Aplysia californica as a model system. We examined the regulation of MAPK (ERK1/2) activation by small G proteins in the CNS by using different patterns of analog stimuli that mimic different patterns of behavioral training for memory induction. We first cloned and characterized the Aplysia homologs of the small G proteins, Ras and Rap1 (ApRas and ApRap, respectively). We next examined changes in ApRas and ApRap activity that accompany MAPK activation. Last, by delivering recombinant ApRas and ApRap into the CNS, we directly manipulated their activity and examined the resultant MAPK activation. We found that MAPK activation induced by analog training depends on the combined activity of ApRas and ApRap, rather than the individual activity of either one alone. Also, ApRas and ApRap have a complex role in MAPK activation: they can act as activators or inhibitors, depending on the specific pattern of the training. The pattern-sensitive regulation of MAPK by interactive ApRas and ApRap activity that we have identified could contribute to the molecular routing of different downstream effects of spatially localized MAPK required for the induction of specific pattern-sensitive forms of synaptic facilitation and memory.