Pharmacological characterization of the α2A-adrenergic receptor inhibiting rat hippocampal CA3 epileptiform activity: comparison of ligand efficacy and potency.

The mechanism underlying the antiepileptic actions of norepinephrine (NE) is unclear with conflicting results. Our objectives are to conclusively delineate the specific adrenergic receptor (AR) involved in attenuating hippocampal CA3 epileptiform activity and assess compounds for lead drug development. We utilized the picrotoxin model of seizure generation in rat brain slices using electrophysiological recordings. Epinephrine (EPI) reduced epileptiform burst frequency in a concentration-dependent manner. To identify the specific receptor involved in this response, the equilibrium dissociation constants were determined for a panel of ligands and compared with established binding values for α1, α2, and other receptor subtypes. Correlation and slope of unity were found for the α2A-AR, but not other receptors. Effects of different chemical classes of α-AR agonists at inhibiting epileptiform activity by potency (pEC50) and relative efficacy (RE) were determined. Compared with NE (pEC50, 6.20; RE, 100%), dexmedetomidine, an imidazoline (pEC50, 8.59; RE, 67.1%), and guanabenz, a guanidine (pEC50, 7.94; RE, 37.9%), exhibited the highest potency (pEC50). In contrast, the catecholamines, EPI (pEC50, 6.95; RE, 120%) and α-methyl-NE (pEC50, 6.38; RE, 116%) were the most efficacious. These findings confirm that CA3 epileptiform activity is mediated solely by α2A-ARs without activation of other receptor systems. These findings suggest a pharmacotherapeutic target for treating epilepsy and highlight the need for selective and efficacious α2A-AR agonists that can cross the blood-brain barrier.

Characterization of prostanoids response to Bordetella pertussis antigen BscF and Tdap in LPS-challenged monocytes.

Prostanoids are potent inflammatory mediators that play a regulatory role in the innate immune activation of the adaptive immune response to determine the duration of protection against infection. We aim to quantify the modulation of prostanoids profiles in lipopolysaccharide (LPS)-stimulated THP-1 cells treated with the novel pertussis antigen BscF. We compared the effect with pertussis antigens present in the current Tdap vaccine to understand the immunomodulatory effect that might contribute to the diminished Tdap vaccine effectiveness. The inflammatory challenge with LPS induced a robust elevation of most prostanoid family members compared to the control treatment. Treatment with BscF and Tdap significantly reduced the LPS-stimulated elevation of prostaglandins (PGs) D2, E2, and F2α, as well as thromboxane (TX) A2 levels. An opposite trend was observed for PGI2, as both antigens accelerated the LPS-stimulated upregulation. Further, we quantified cyclooxygenases (COXs) that catalyze the biosynthesis of prostanoids and found that both antigens significantly reduced LPS-stimulated COX-1 and COX-2, demonstrating that the waning of acellular pertussis vaccines' protective immunity may be due to other downstream enzymes not related to COXs. Our present study validates the potential role of BscF as an adjuvant, resulting in the next-generation pertussis vaccine discovery.

Modulation of Inflammatory Signaling Molecules in Bordetella pertussis Antigen-Challenged Human Monocytes in Presence of Adrenergic Agonists.

BscF is a type III secretion system (T3SS) needle protein from Bordetella pertussis and has previously been shown to induce a sufficient Th1 and Th17 response in human monocytes and mice as a prerequisite for long-lasting protective immunity against pertussis infection. In our current study, we aim to compare the modulation of inflammatory signaling molecules as a direct measure of the immune response to the B. pertussis antigens BscF and Tdap in the presence or absence of the adrenergic receptor agonists phenylephrine (PE) or isoproterenol (ISO) to observe differences that may contribute to the diminished protective immunity of the current acellular pertussis (aP) vaccine, Tdap. Stimulation of human monocyte THP-1 cells with LPS, BscF, and Tdap induced a robust elevation of CCL20, CXCL10, PGE2, and PGF2α among most chemokine and prostanoid members when compared with the control treatment. Treatment with the adrenergic agonist PE or ISO significantly enhanced the BscF- and Tdap-stimulated modulation of CCL20 and CXCL10 but not PGE2 and PGF2α, suggesting that adrenergic modulation of pertussis antigen responses might be a new therapeutic strategy to improve the longevity of pertussis immunity. Stimulation of THP-1 cells with BscF alone initiated significant expression of CXCL10 and PGF2α but not when Tdap was used, suggesting that BscF might be an important pertussis antigen for next-generation pertussis vaccines or when combined with the current aP vaccine. Our data offer opportunities for designing new therapeutic approaches against pertussis infection.

Method of Microglial DNA-RNA Purification from a Single Brain of an Adult Mouse.

Microglia, the resident brain immune effectors cells, show dynamic activation level changes for most neuropsychiatric diseases, reflecting their complex regulatory function and potential as a therapeutic target. Emerging single-cell molecular biology studies are used to investigate the genetic modification of individual cells to better understand complex gene regulatory pathways. Although multiple protocols for microglia isolation from adult mice are available, it is always challenging to get sufficient purified microglia from a single brain for simultaneous DNA and RNA extraction for subsequent downstream analysis. Moreover, for data comparison between treated and untreated groups, standardized cell isolation techniques are essential to decrease variability. Here, we present a combined method of microglia isolation from a single adult mouse brain, using a magnetic bead-based column separation technique, and a column-based extraction of purified DNA-RNA from the isolated microglia for downstream application. Our current method provides step-by-step instructions accompanied by visual explanations of important steps for isolating DNA-RNA simultaneously from a highly purified microglia population.

Alpha-Synuclein-induced DNA Methylation and Gene Expression in Microglia.

Synucleinopathy disorders are characterized by aggregates of α-synuclein (α-syn), which engage microglia to elicit a neuroinflammatory response. Here, we determined the gene expression and DNA methylation changes in microglia induced by aggregate α-syn. Transgenic murine Thy-1 promoter (mThy1)-Asyn mice overexpressing human α-syn are a model of synucleinopathy. Microglia from 3 and 13-month-old mice were used to isolate nucleic acids for methylated DNA and RNA-sequencing. α-Syn-regulated changes in gene expression and genomic methylation were determined and examined for functional enrichment followed by network analysis to further elucidate possible connections within the data. Microglial DNA isolated from our 3-month cohort had 5315 differentially methylated gene (DMG) changes, while RNA levels demonstrated a change in 119 differentially expressed genes (DEGs) between mThy1-Asyn mice and wild-type littermate controls. The 3-month DEGs and DMGs were highly associated with adhesion and migration signaling, suggesting a phenotypic transition from resting to active microglia. We observed 3742 DMGs and 3766 DEGs in 13-month mThy1-Asyn mice. These genes were often related to adhesion, migration, cell cycle, cellular metabolism, and immune response. Network analysis also showed increased cell mobility and inflammatory functions at 3 months, shifting to cell cycle, immune response, and metabolism changes at 13 months. We observed significant α-syn-induced methylation and gene expression changes in microglia. Our data suggest that α-syn overexpression initiates microglial activation leading to neuroinflammation and cellular metabolic stresses, which is associated with disease progression.

ZINC40099027 activates human focal adhesion kinase by accelerating the enzymatic activity of the FAK kinase domain.

Focal adhesion kinase (FAK) regulates gastrointestinal epithelial restitution and healing. ZINC40099027 (Zn27) activates cellular FAK and promotes intestinal epithelial wound closure in vitro and in mice. However, whether Zn27 activates FAK directly or indirectly remains unknown. We evaluated Zn27 potential modulation of the key phosphatases, PTP-PEST, PTP1B, and SHP2, that inactivate FAK, and performed in vitro kinase assays with purified FAK to assess direct Zn27-FAK interaction. In human Caco-2 cells, Zn27-stimulated FAK-Tyr-397 phosphorylation despite PTP-PEST inhibition and did not affect PTP1B-FAK interaction or SHP2 activity. Conversely, in vitro kinase assays demonstrated that Zn27 directly activates both full-length 125 kDa FAK and its 35 kDa kinase domain. The ATP-competitive FAK inhibitor PF573228 reduced basal and ZN27-stimulated FAK phosphorylation in Caco-2 cells, but Zn27 increased FAK phosphorylation even in cells treated with PF573228. Increasing PF573228 concentrations completely prevented activation of 35 kDa FAK in vitro by a normally effective Zn27 concentration. Conversely, increasing Zn27 concentrations dose-dependently activated kinase activity and overcame PF573228 inhibition of FAK, suggesting the direct interactions of Zn27 with FAK may be competitive. Zn27 increased the maximal activity (Vmax ) of FAK. The apparent Km of the substrate also increased under laboratory conditions less relevant to intracellular ATP concentrations. These results suggest that Zn27 is highly potent and enhances FAK activity via allosteric interaction with the FAK kinase domain to increase the Vmax of FAK for ATP. Understanding Zn27 enhancement of FAK activity will be important to redesign and develop a clinical drug that can promote mucosal wound healing.

Conserved Transcriptional Signatures in Human and Murine Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes. In this study, we employed a systems biology approach to identify DPN-related transcriptional pathways conserved across human and various murine models. Eight microarray datasets on peripheral nerve samples from murine models of type 1 (streptozotocin-treated) and type 2 (db/db and ob/ob) diabetes of various ages and human subjects with non-progressive and progressive DPN were collected. Differentially expressed genes (DEGs) were identified between non-diabetic and diabetic samples in murine models, and non-progressive and progressive human samples using a unified analysis pipeline. A transcriptional network for each DEG set was constructed based on literature-derived gene-gene interaction information. Seven pairwise human-vs-murine comparisons using a network-comparison program resulted in shared sub-networks including 46 to 396 genes, which were further merged into a single network of 688 genes. Pathway and centrality analyses revealed highly connected genes and pathways including LXR/RXR activation, adipogenesis, glucocorticoid receptor signalling, and multiple cytokine and chemokine pathways. Our systems biology approach identified highly conserved pathways across human and murine models that are likely to play a role in DPN pathogenesis and provide new possible mechanism-based targets for DPN therapy.

A protocol for preparation and transfection of rat entorhinal cortex organotypic cultures for electrophysiological whole-cell recordings.

Understanding how neuromodulators influence synaptic transmission and intrinsic excitability within the entorhinal cortex (EC) is critical to furthering our understanding of the molecular and cellular aspects of this region. Organotypic cultures can provide a cost-effective means to employ selective molecular biological strategies in elucidating cellular mechanisms of neuromodulation in the EC. We therefore adapted our acute slice model for organotypic culture applications and optimized a protocol for the preparation and biolistic transfection of cultured horizontal EC slices. Here, we present our detailed protocol for culturing EC slices. Using an n-methyl-d-glucamine (NMDG)-containing cutting solution, we obtain healthy EC slice cultures for electrophysiological recordings. We also present our protocol for the preparation of "bullets" carrying one or more constructs and demonstrate successful transfection of EC slices. We build upon previous methods and highlight specific aspects in our method that greatly improved the quality of our results. We validate our methods using immunohistochemical, imaging, and electrophysiological techniques. The novelty of this method is that it provides a description of culturing and transfection of EC neurons for specifically addressing their functionality. This method will enable researchers interested in entorhinal function to quickly adopt a similar slice culture transfection system for their own investigations.

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease.

UNLABELLED: Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid ß (Aß)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP(-/-)) microglial cultures, oligomeric Aß was unable to stimulate increased secretion from mAPP(-/-) cells. This was consistent with an ability of oligomeric Aß to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aß produced less microgliosis in mAPP(-/-) mice compared with wild-type mice. The mAPP(-/-) mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aß plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT: A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid ß (Aß) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aß stimulation of microglial activation is one source of brain inflammatory changes during disease. Aß is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aß are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aß production to drive the microgliosis associated with AD brains.

Calcitonin gene-related peptide mediates an inflammatory response in Schwann cells via cAMP-dependent ERK signaling cascade.

AIMS: Calcitonin gene-related peptides (CGRP), an endogenous neuropeptide, play an important role in the development of neuroinflammation by acting upon its receptor. The CGRP receptor immunoreactivity was identified on Schwann cells. However the effects of CGRP on Schwann cells are unknown and the exact signaling mechanisms associated with CGRP receptor activation related to Schwann cells inflammatory responses are not well understood. We investigated the effect of CGRP on CGRP receptor activation mediates a proinflammatory signaling response in Schwann cells. MAIN METHODS: CGRP-induced ERK-MAPK phosphorylation and proinflammatory cytokines, interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) expressions were measured by immune blotting. We also used specific antagonist and inhibitors to confirm the exactly signaling pathway including CGRP (8-37), SQ 22536 and H-89. KEY FINDINGS: Treatment with CGRP demonstrated a significant generation of IL-1ß and IL-6 but not in the level of TNF-α. In addition, there was a temporal increase in the activated form of ERK caused by CGRP that was prevented after pretreatment with CGRP (8-37), SQ 22536 and H-89. Furthermore, use of the CGRP (8-37), ERK inhibitor PD 98059, SQ 22536 or H-89 abolished the CGRP mediated increase in IL-1ß. SIGNIFICANCE: This investigation provides evidence for a novel CGRP activation on Schwann cells that mediates inflammatory response by increasing of IL-1ß and IL-6 expression. CGRP activates the cAMP-PKA-ERK signaling cascade leading to IL-1ß production. These results support the notion that CGRP may play a direct role to initiate inflammatory processes in the peripheral nervous system.

Neurotensinergic Excitation of Dentate Gyrus Granule Cells via Gαq-Coupled Inhibition of TASK-3 Channels.

Neurotensin (NT) is a 13-amino acid peptide and serves as a neuromodulator in the brain. Whereas NT has been implicated in learning and memory, the underlying cellular and molecular mechanisms are ill-defined. Because the dentate gyrus receives profound innervation of fibers containing NT and expresses high density of NT receptors, we examined the effects of NT on the excitability of dentate gyrus granule cells (GCs). Our results showed that NT concentration dependently increased action potential (AP) firing frequency of the GCs by the activation of NTS1 receptors resulting in the depolarization of the GCs. NT-induced enhancement of AP firing frequency was not caused indirectly by releasing glutamate, GABA, acetylcholine, or dopamine, but due to the inhibition of TASK-3 K(+) channels. NT-mediated excitation of the GCs was G protein dependent, but independent of phospholipase C, intracellular Ca(2+) release, and protein kinase C. Immunoprecipitation experiment demonstrates that the activation of NTS1 receptors induced the association of Gαq/11 and TASK-3 channels suggesting a direct coupling of Gαq/11 to TASK-3 channels. Endogenously released NT facilitated the excitability of the GCs contributing to the induction of long-term potentiation at the perforant path-GC synapses. Our results provide a cellular mechanism that helps to explain the roles of NT in learning and memory.

Activation of neurotensin receptor 1 facilitates neuronal excitability and spatial learning and memory in the entorhinal cortex: beneficial actions in an Alzheimer's disease model.

Neurotensin (NT) is a tridecapeptide distributed in the CNS, including the entorhinal cortex (EC), a structure that is crucial for learning and memory and undergoes the earliest pathological alterations in Alzheimer's disease (AD). Whereas NT has been implicated in modulating cognition, the cellular and molecular mechanisms by which NT modifies cognitive processes and the potential therapeutic roles of NT in AD have not been determined. Here we examined the effects of NT on neuronal excitability and spatial learning in the EC, which expresses high density of NT receptors. Brief application of NT induced persistent increases in action potential firing frequency, which could last for at least 1 h. NT-induced facilitation of neuronal excitability was mediated by downregulation of TREK-2 K(+) channels and required the functions of NTS1, phospholipase C, and protein kinase C. Microinjection of NT or NTS1 agonist, PD149163, into the EC increased spatial learning as assessed by the Barnes Maze Test. Activation of NTS1 receptors also induced persistent increases in action potential firing frequency and significantly improved the memory status in APP/PS1 mice, an animal model of AD. Our study identifies a cellular substrate underlying learning and memory and suggests that NTS1 agonists may exert beneficial actions in an animal model of AD.

1-Methyl-4-phenylpyridinium-induced cell death via autophagy through a Bcl-2/Beclin 1 complex-dependent pathway.

Several lines of evidence suggest that the mechanism underlying drug-induced neuronal apoptosis is initiated by the increased production of reactive oxygen species (ROS). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin, has been shown to initiate an apoptotic cascade by increasing ROS in the dopaminergic neurons of the substantia nigra, leading to the morphological and physiological features associated with Parkinson's disease. Recently, it has been reported that autophagy, a type of programmed cell death independent of the apoptotic cascade, also plays a role in neuronal damage. Although autophagy is negatively regulated by the mammalian target of rapamycin receptor (mTOR), there is some evidence showing a novel function for the anti-apoptotic protein Bcl-2. Bcl-2 is proposed to play a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Nevertheless, it is unclear whether autophagy is also correlated with apoptotic signaling in 1-methyl-4-phenylpyridinium (MPP(+)) toxicity. Therefore, we hypothesized that the MPP(+) toxicity generally associated with initiating the apoptotic signaling cascade also increases an autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell lines, we demonstrate that MPP(+) increases the expression of microtubule-associated protein light chain 3 (LC3-II), an autophagosome membrane marker and the mTOR signaling pathway, and Beclin 1 while decreasing the Bcl-2 levels. Moreover, these expressions correlate with a decreased binding ratio between Bcl-2 and Beclin 1, in effect limiting the regulation of the downstream autophagic markers, such as LC3-II. Our results indicate that MPP(+) can induce autophagy in SK-N-SH cells by decreasing the Bcl-2/Beclin 1 complex.

Phospholipase C not protein kinase C is required for the activation of TRPC5 channels by cholecystokinin.

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to G(q/11) proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca(2+). The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP(3) receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca(2+) stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.

Requirement of phospholipase C and protein kinase C in cholecystokinin-mediated facilitation of NMDA channel function and anxiety-like behavior.

Although cholecystokinin (CCK) has long been known to exert anxiogenic effects in both animal anxiety models and humans, the underlying cellular and molecular mechanisms are ill-defined. CCK interacts with CCK-1 and CCK-2 receptors resulting in up-regulation of phospholipase C (PLC) and protein kinase C (PKC). However, the roles of PLC and PKC in CCK-mediated anxiogenic effects have not been determined. We have shown previously that CCK facilitates glutamate release in the hippocampus especially at the synapses formed by the perforant path and dentate gyrus granule cells via activations of PLC and PKC. Here we further demonstrated that CCK enhanced NMDA receptor function in dentate gyrus granule cells via activation of PLC and PKC pathway. At the single-channel level, CCK increased NMDA single-channel open probability and mean open time, reduced the mean close time, and had no effects on the conductance of NMDA channels. Because elevation of glutamatergic functions results in anxiety, we explored the roles of PLC and PKC in CCK-induced anxiogenic actions using the Vogel Conflict Test (VCT). Our results from both pharmacological approach and knockout mice demonstrated that microinjection of CCK into the dentate gyrus concentration-dependently increased anxiety-like behavior via activation of PLC and PKC. Our results provide a novel unidentified signaling mechanism whereby CCK increases anxiety.

α1-adrenergic receptors positively regulate Toll-like receptor cytokine production from human monocytes and macrophages.

Catecholamines released from the sympathetic nervous system in response to stress or injury affect expression of inflammatory cytokines generated by immune cells. α(1)-Adrenergic receptors (ARs) are expressed on innate immune cell populations, but their subtype expression patterns and signaling characteristics are not well characterized. Primary human monocytes, a human monocytic cell line, and monocyte-derived macrophage cells were used to measure expression of the proinflammatory mediator interleukin (IL)-1ß responding to lipopolysaccharide (LPS) in the presence or absence of α(1)-AR activation. Based on our previous findings, we hypothesized that α(1)-AR stimulation on innate immune cells positively regulates LPS-initiated IL-1ß production. IL-1ß production in response to LPS was synergistically higher for both monocytes and macrophages in the presence of the selective α(1)-AR agonist (R)-(-)-phenylephrine hydrochloride (PE). This synergistic IL-1ß response could be blocked with a selective α(1)-AR antagonist as well as inhibitors of protein kinase C (PKC). Radioligand binding studies characterized a homogenous α(1B)-AR subtype population on monocytes, which changed to a heterogeneous receptor subtype expression pattern when differentiated to macrophages. Furthermore, increased p38 mitogen-activated protein kinase (MAPK) activation was observed only with concurrent PE and LPS stimulation, peaking after 120 and 30 min in monocytes and macrophages, respectively. Blocking the PKC/p38 MAPK signaling pathway in both innate immune cell types inhibited the synergistic IL-1ß increase observed with concurrent PE and LPS treatments. This study characterizes α(1)-AR subtype expression on both human monocyte and macrophage cells and illustrates a mechanism by which increased IL-1ß production can be modulated by α(1)-AR input.

The mechanism for the neuroprotective effect of melatonin against methamphetamine-induced autophagy.

Methamphetamine (METH) is a common drug of abuse that induces toxicity in the central nervous system and is connected to neurological disorders such as Parkinson's disease. METH neurotoxicity is induced by reactive oxygen species (ROS) production and apoptosis. Moreover, autophagy is an alternative to cell death and a means for eliminating dysfunctional organelles. In other cases, autophagy can end up in cell death. Nonetheless, it is not clear whether autophagy is also correlated with apoptotic signaling in drug-induced neurotoxicity. Therefore, we hypothesized that METH-generated toxicity associated with initiating the apoptotic signaling cascade can also increase the autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell line as our model system, we found that METH-induced autophagy by inhibiting dissociation of Bcl-2/Beclin 1 complex and its upstream pathway that thereby led to cell death. We uncovered a novel function for the anti-apoptotic protein Bcl-2, as it played a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Furthermore, Bcl-2 was activated by c-Jun N-terminal kinase 1 (JNK 1), which is upstream of Bcl-2 phosphorylation, to induce Bcl-2/Beclin 1 dissociation. Furthermore, we demonstrated a novel role for melatonin in protecting cells from autophagic cell death triggered by the Bcl-2/Beclin 1 pathway by inhibiting the activation of the JNK 1, Bcl-2 upstream pathway. This study provides information regarding the link between apoptosis and autophagy signaling, which could lead to the development of therapeutic strategies that exploit the neurotoxicity of drugs of abuse.

Cholecystokinin facilitates glutamate release by increasing the number of readily releasable vesicles and releasing probability.

Cholecystokinin (CCK), a neuropeptide originally discovered in the gastrointestinal tract, is abundantly distributed in the mammalian brains including the hippocampus. Whereas CCK has been shown to increase glutamate concentration in the perfusate of hippocampal slices and in purified rat hippocampal synaptosomes, the cellular and molecular mechanisms whereby CCK modulates glutamatergic function remain unexplored. Here, we examined the effects of CCK on glutamatergic transmission in the hippocampus using whole-cell recordings from hippocampal slices. Application of CCK increased AMPA receptor-mediated EPSCs at perforant path-dentate gyrus granule cell, CA3-CA3 and Schaffer collateral-CA1 synapses without effects at mossy fiber-CA3 synapses. CCK-induced increases in AMPA EPSCs were mediated by CCK-2 receptors and were not modulated developmentally and transcriptionally. CCK reduced the coefficient of variation and paired-pulse ratio of AMPA EPSCs suggesting that CCK facilitates presynaptic glutamate release. CCK increased the release probability and the number of readily releasable vesicles with no effects on the rate of recovery from vesicle depletion. CCK-mediated increases in glutamate release required the functions of phospholipase C, intracellular Ca(2+) release and protein kinase Cgamma. CCK released endogenously from hippocampal interneurons facilitated glutamatergic transmission. Our results provide a cellular and molecular mechanism to explain the roles of CCK in the brain.