Corticotropin‐releasing hormone‐binding protein is up‐regulated by brain‐derived neurotrophic factor and is secreted in an activity‐dependent manner in rat cerebral cortical neurons

A recent study revealed that corticotropin‐releasing hormone (CRH) in the cerebral cortex (CTX) plays a regulatory role in emotional behaviors in rodents. Given the functional interaction between brain‐derived neurotrophic factor (BDNF) and the CRH‐signaling pathway in the hypothalamic‐pituitary‐adrenal axis, we hypothesized that BDNF may regulate gene expression of CRH and its related molecules in the CTX. Findings of real‐time quantitative PCR (RT‐qPCR) indicated that stimulation of cultured rat cortical neurons with BDNF led to marked elevations in the mRNA levels of CRH and CRH‐binding protein (CRH‐BP). The BDNF‐induced up‐regulation of CRH‐BP mRNA was attenuated by inhibitors of tropomyosin related kinase (Trk) and MEK, but not by an inhibitor for PI3K and Phospholipase C gamma (PLCγ). The up‐regulation was partially blocked by an inhibitor of lysine‐specific demethylase (KDM) 6B. Fluorescent imaging identified the vesicular pattern of pH‐sensitive green fluorescent protein‐fused CRH‐BP (CRH‐BP‐pHluorin), which co‐localized with mCherry‐tagged BDNF in cortical neurons. In addition, live‐cell imaging detected drastic increases of pHluorin fluorescence in neurites upon membrane depolarization. Finally, we confirmed that tetrodotoxin partially attenuated the BDNF‐induced up‐regulation of CRH‐BP mRNA, but not that of the protein. These observations indicate the following: In cortical neurons, BDNF led to gene expression of CRH‐BP and CRH. TrkB, MEK, presumably ERK, and KDM6B are involved in the BDNF‐induced gene expression of CRH‐BP, and BDNF is able to induce the up‐regulation in a neuronal activity‐independent manner. It is suggested that CRH‐BP is stored into BDNF‐containing secretory granules in cortical neurons, and is secreted in response to membrane depolarization.

     

Single cocaine exposure does not alter striatal pre‐synaptic dopamine function in mice: an [18F]‐FDOPA PET study

Cocaine is a recreational drug of abuse that binds to the dopamine transporter, preventing reuptake of dopamine into pre‐synaptic terminals. The increased presence of synaptic dopamine results in stimulation of both pre‐ and post‐synaptic dopamine receptors, considered an important mechanism by which cocaine elicits its reinforcing properties. However, the effects of acute cocaine administration on pre‐synaptic dopamine function remain unclear. Non‐invasive imaging techniques such as positron emission tomography have revealed impaired pre‐synaptic dopamine function in chronic cocaine users. Similar impairments have been seen in animal studies, with microdialysis experiments indicating decreased basal dopamine release. Here we use micro positron emission tomography imaging techniques in mice to measure dopamine synthesis capacity and determine the effect of acute cocaine administration of pre‐synaptic dopamine function. We show that a dose of 20 mg/kg cocaine is sufficient to elicit hyperlocomotor activity, peaking 15–20 min post treatment (p < 0.001). However, dopamine synthesis capacity in the striatum was not significantly altered by acute cocaine treatment (: 0.0097 per min vs. 0.0112 per min in vehicle controls, p > 0.05). Furthermore, expression levels of two key enzymes related to dopamine synthesis, tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase, within the striatum of scanned mice were not significantly affected by acute cocaine pre‐treatment (p > 0.05). Our findings suggest that while the regulation of dopamine synthesis and release in the striatum have been shown to change with chronic cocaine use, leading to a reduced basal tone, these adaptations to pre‐synaptic dopaminergic neurons are not initiated following a single exposure to the drug.

     

Expression of the purine biosynthetic enzyme phosphoribosyl formylglycinamidine synthase in neurons

Purines are metabolic building blocks essential for all living organisms on earth. De novo purine biosynthesis occurs in the brain and appears to play important roles in neural development. Phosphoribosyl formylglycinamidine synthase (FGAMS , also known as PFAS or FGARAT ), a core enzyme involved in the de novo synthesis of purines, may play alternative roles in viral pathogenesis. To date, no thorough investigation of the endogenous expression and localization of de novo purine biosynthetic enzymes has been conducted in human neurons or in virally infected cells. In this study, we characterized expression of FGAMS using multiple neuronal models. In differentiated human SH ‐SY 5Y neuroblastoma cells, primary rat hippocampal neurons, and in whole‐mouse brain sections, FGAMS immunoreactivity was distributed within the neuronal cytoplasm. FGAMS immunolabeling in vitro demonstrated extensive distribution throughout neuronal processes. To investigate potential changes in FGAMS expression and localization following viral infection, we infected cells with the human pathogen herpes simplex virus 1. In infected fibroblasts, FGAMS immunolabeling shifted from a diffuse cytoplasmic location to a mainly perinuclear localization by 12 h post‐infection. In contrast, in infected neurons, FGAMS localization showed no discernable changes in the localization of FGAMS immunoreactivity. There were no changes in total FGAMS protein levels in either cell type. Together, these data provide insight into potential purine biosynthetic mechanisms utilized within neurons during homeostasis as well as viral infection.

Cover Image for this Issue: doi: 10.1111/jnc.14169 .

     

Increased acetylcholine and glutamate efflux in the prefrontal cortex following intranasal orexin‐A (hypocretin‐1)

Orexin/hypocretin neurons of the lateral hypothalamus and perifornical area are integrators of physiological function. Previous work from our laboratory and others has shown the importance of orexin transmission in cognition. Age‐related reductions in markers of orexin function further suggest that this neuropeptide may be a useful target for the treatment of age‐related cognitive dysfunction. Intranasal administration of orexin‐A (OxA) has shown promise as a therapeutic option for cognitive dysfunction. However, the neurochemical mechanisms of intranasal OxA administration are not fully understood. Here, we use immunohistochemistry and in vivo microdialysis to define the effects of acute intranasal OxA administration on: (i) activation of neuronal populations in the cortex, basal forebrain, and brainstem and (ii) acetylcholine (AC h) and glutamate efflux in the prefrontal cortex (PFC ) of Fischer 344/Brown Norway F1 rats. Acute intranasal administration of OxA significantly increased c‐Fos expression, a marker for neuronal activation, in the PFC and in subpopulations of basal forebrain cholinergic neurons. Subsequently, we investigated the effects of acute intranasal OxA on neurotransmitter efflux in the PFC and found that intranasal OxA significantly increased both AC h and glutamate efflux in this region. These findings were independent from any changes in c‐Fos expression in orexin neurons, suggesting that these effects are not resultant from direct activation of orexin neurons. In total, these data indicate that intranasal OxA may enhance cognition through activation of distinct neuronal populations in the cortex and basal forebrain and through increased neurotransmission of AC h and glutamate in the PFC .

     

Validated multi‐step approach for in vivo recording and analysis of optogenetically evoked glutamate in the mouse globus pallidus

Precise quantification of extracellular glutamate concentrations upon neuronal activation is crucial for the understanding of brain function and neurological disorders. While optogenetics is an outstanding method for the correlation between distinct neurons and their role in circuitry and behavior, the electrochemically inactive nature of glutamate has proven challenging for recording upon optogenetic stimulations. This difficulty is due to the necessity for using enzyme‐coated microelectrodes and the risk for light‐induced artifacts. In this study, we establish a method for the combination of in vivo optogenetic stimulation with selective measurement of glutamate concentrations using enzyme‐coated multielectrode arrays and amperometry. The glutamatergic subthalamic nucleus (STN ), which is the main electrode target site in deep brain stimulation treatment of advanced Parkinson′s disease, has recently proven opotogenetically targetable in Pitx2‐Cre‐transgenic mice and was here used as model system. Upon stereotactic injection of viral Channelrhodopsin2‐eYFP constructs into the STN , amperometric recordings were performed at a range of optogenetic stimulation frequencies in the globus pallidus, the main STN target area, in anesthetized mice. Accurate quantification was enabled through a multi‐step analysis approach based on self‐referencing microelectrodes and repetition of the experimental protocol at two holding potentials, which allowed for the identification, isolation and removal of photoelectric and photoelectrochemical artifacts. This study advances the field of in vivo glutamate detection with combined optogenetics and amperometric recordings by providing a validated analysis framework for application in a wide variety of glutamate‐based approaches in neuroscience.

     

APLP1 and APLP2, members of the APP family of proteins,behave similarly to APP in that they associate with NMDA receptors and enhance NMDA receptor surface expression

The function of amyloid precursor protein (APP) is unknown, although the discovery that it contributes to the regulation of surface expression of N‐methyl‐d ‐aspartate (NMDA) receptors has afforded new insights into its functional significance. Since APP is a member of a gene family that contains two other members, amyloid precursor‐like proteins 1 and 2 (APLP1 and APLP2), it is important to determine if the related APP proteins possess the same properties as APP with respect to their interactions with NMDA receptors. Following expression in mammalian cells, both APLP1 and APLP2 behaved similarly to APP in that they both co‐immunoprecipitated with the two major NMDA receptor subtypes, GluN1/GluN2A and GluN1/GluN2B, via interaction with the obligatory GluN1 subunit. Immunoprecipitations from detergent extracts of adult mammalian brain showed co‐immunoprecipitation of APLP1 and APLP2 with GluN2A‐ and GluN2B‐containing NMDA receptors. Furthermore, similarly to APP, APLP1 and APLP2 both enhanced GluN1/GluN2A and GluN1/GluN2B cell surface expression. Thus, all the three members of the APP gene family behave similarly in that they each contribute to the regulation of cell surface NMDA receptor homoeostasis.

     

Induction of depressive‐like effects by subchronic exposure to cocaine or heroin in laboratory rats

The effect of psychoactive drugs on depression has usually been studied in cases of prolonged drug addiction and/or withdrawal, without much emphasis on the effects of subchronic or recreational drug use. To address this issue, we exposed laboratory rats to subchronic regimens of heroin or cocaine and tested long‐term effects on (i) depressive‐like behaviors, (ii) brain‐derived neurotrophic factor (BDNF) levels in reward‐related brain regions, and (iii) depressive‐like behavior following an additional chronic mild stress procedure. The long‐term effect of subchronic cocaine exposure was a general reduction in locomotor activity whereas heroin exposure induced a more specific increase in immobility during the forced swim test. Both cocaine and heroin exposure induced alterations in BDNF levels that are similar to those observed in several animal models of depression. Finally, both cocaine and heroin exposure significantly enhanced the anhedonic effect of chronic mild stress. These results suggest that subchronic drug exposure induces depressive‐like behavior which is accompanied by modifications in BDNF expression and increases the vulnerability to develop depressive‐like behavior following chronic stress. Implications for recreational and small‐scale drug users are discussed.

     

Urotensin II promotes vagal‐mediated bradycardia by activating cardiac‐projecting parasympathetic neurons of nucleus ambiguus

Urotensin II (U‐II) is a cyclic undecapeptide that regulates cardiovascular function at central and peripheral sites. The functional role of U‐II nucleus ambiguus, a key site controlling cardiac tone, has not been established, despite the identification of U‐II and its receptor at this level. We report here that U‐II produces an increase in cytosolic Ca²⁺ concentration in retrogradely labeled cardiac vagal neurons of nucleus ambiguus via two pathways: (i) Ca²⁺ release from the endoplasmic reticulum via inositol 1,4,5‐trisphosphate receptor; and (ii) Ca²⁺ influx through P/Q‐type Ca²⁺ channels. In addition, U‐II depolarizes cultured cardiac parasympathetic neurons. Microinjection of increasing concentrations of U‐II into nucleus ambiguus elicits dose‐dependent bradycardia in conscious rats, indicating the in vivo activation of the cholinergic pathway controlling the heart rate. Both the in vitro and in vivo effects were abolished by the urotensin receptor antagonist, urantide. Our findings suggest that, in addition, to the previously reported increase in sympathetic outflow, U‐II activates cardiac vagal neurons of nucleus ambiguus, which may contribute to cardioprotection.

     

News & ISN April 2019

We are very sad that the ISN lost its President Kazuhiro Ikenaka, Professor and Chairman at National Institute for Physiological Sciences (NIPS), Director of Okazaki Institute of Integrative Biology. JNeurochem published an Obituary to value his outstanding achievements: Akio Wanaka et al. (2019) OBITUARY Kazuhiro Ikenaka (1952‐2018). https://doi.org/10.1111/jnc.14679

     

AMP‐activated protein kinase counteracts brain‐derived neurotrophic factor‐induced mammalian target of rapamycin complex 1 signaling in neurons

Growth factors and nutrients, such as amino acids and glucose, regulate mammalian target of rapamycin complex 1 (mTORC1) signaling and subsequent translational control in a coordinated manner. Brain‐derived neurotrophic factor (BDNF), the most prominent neurotrophic factor in the brain, activates mTORC1 and induces phosphorylation of its target, p70S6 kinase (p70S6K), at Thr389 in neurons. BDNF also increases mammalian target of rapamycin‐dependent novel protein synthesis in neurons. Here, we report that BDNF‐induced p70S6K activation is dependent on glucose, but not amino acids, sufficiency in cultured cortical neurons. AMP‐activated protein kinase (AMPK) is the molecular background to this specific nutrient dependency. Activation of AMPK, which is induced by glucose deprivation, treatment with pharmacological agents such as 2‐Deoxy‐d ‐glucose, metformin, and 5‐aminoimidazole‐4‐carboxamide ribonucleoside or forced expression of a constitutively active AMPKα subunit, counteracts BDNF‐induced phosphorylation of p70S6K and enhanced protein synthesis in cortical neurons. These results indicate that AMPK inhibits the effects of BDNF on mTORC1‐mediated translation in neurons.

     

Development of mature BDNF‐specific sandwich ELISA

Mature brain‐derived neurotrophic factor (mBDNF) plays a vital role in the nervous system, whereas proBDNF elicits neurodegeneration and neuronal apoptosis. Although current enzyme‐linked immunosorbent assay (ELISA) has been widely used to measure BDNF levels, it cannot differentiate mBDNF from proBDNF. As the function of proBDNF differs from mBDNF, it is necessary to establish an ELISA assay specific for the detection of mBDNF. Therefore, we aimed to establish a new mBDNF‐specific sandwich ELISA. In this study, we have screened and found a combination of antibodies for a sandwich ELISA. A monoclonal antibody and sheep anti‐BDNF were chosen as capture and detection antibody for sandwich ELISA respectively. The new ELISA showed no cross‐reactivity to human recombinant NT‐3, NT‐4, nerve growth factor and negligible cross‐reactivity (0.99–4.99%) for proBDNF compared to commercial ELISA kits (33.18–91.09%). The application of the new mBDNF ELISA was shown through the measurement of mBDNF levels in different brain regions of rats and in the brain of β‐site amyloid precursor protein cleaving enzyme 1 (BACE1)^?/? and WT mice and compared to western blot. Overall, this new ELISA will be useful for the measurement of mBDNF levels with high specificity.

     

Clenbuterol reduces GABAergic transmission in prefrontal cortex layer 5/6 pyramidal neurons of juvenile rat via reducing action potentials firing frequency of GABAergic interneurons

Beta‐adrenoceptors (β₂‐AR s) have beneficial effects on prefrontal cortex (PFC ) working memory, however, the cellular and molecular mechanisms are unclear yet. In this study, we probed the effect of β₂‐AR ‐selective agonist clenbuterol (Clen) on synaptic transmission in layer 5/6 pyramidal neurons of PFC . Bath application of Clen reduced spontaneous IPSC (sIPSC ) frequency without effects on sEPSC s. Clen did not alter the frequency and amplitude of miniature IPSC s (mIPSC s), but exerted heterogeneous effects on evoked IPSC s (eIPSC s) recorded from PFC layer 5/6 pyramidal neurons. Clen decreased the firing rate of action potentials of fast‐spiking GABA ergic interneurons. Clen‐induced hyperpolarization of fast‐spiking GABA ergic interneurons required potentiation of an inward rectifier K⁺ channels. Clen‐induced hyperpolarization of fast‐spiking interneurons was dependent on Gs protein rather than cAMP and protein kinase A. Our findings demonstrate that Clen (10 μM) enhances inward rectifier K⁺ channels via Gs protein to cause membrane hyperpolarization of fast‐spiking GABA ergic interneurons resulting in reduction of action potentials firing rate to reduce GABA ergic transmission.

     

Ghrelin regulates phasic dopamine and nucleus accumbens signaling evoked by food‐predictive stimuli

Environmental stimuli that signal food availability hold powerful sway over motivated behavior and promote feeding, in part, by activating the mesolimbic system. These food‐predictive cues evoke brief (phasic) changes in nucleus accumbens (NAc) dopamine concentration and in the activity of individual NAc neurons. Phasic fluctuations in mesolimbic signaling have been directly linked to goal‐directed behaviors, including behaviors elicited by food‐predictive cues. Food‐seeking behavior is also strongly influenced by physiological state (i.e., hunger vs. satiety). Ghrelin, a stomach hormone that crosses the blood‐brain barrier, is linked to the perception of hunger and drives food intake, including intake potentiated by environmental cues. Notwithstanding, whether ghrelin regulates phasic mesolimbic signaling evoked by food‐predictive stimuli is unknown. Here, rats underwent Pavlovian conditioning in which one cue predicted the delivery of rewarding food (CS+) and a second cue predicted nothing (CS?). After training, we measured the effect of ghrelin infused into the lateral ventricle (LV) on sub‐second fluctuations in NAc dopamine using fast‐scan cyclic voltammetry and individual NAc neuron activity using in vivo electrophysiology in separate groups of rats. LV ghrelin augmented both phasic dopamine and phasic increases in the activity of NAc neurons evoked by the CS+. Importantly, ghrelin did not affect the dopamine nor NAc neuron response to the CS?, suggesting that ghrelin selectively modulated mesolimbic signaling evoked by motivationally significant stimuli. These data demonstrate that ghrelin, a hunger signal linked to physiological state, can regulate cue‐evoked mesolimbic signals that underlie food‐directed behaviors.

     

Sulfatide isoform pattern in cerebrospinal fluid discriminates progressive MS from relapsing‐remitting MS

Multiple sclerosis (MS ) is an inflammatory demyelinating disease of the central nervous system (CNS ). Several biomarkers including proteins and lipids have been reported in MS cerebrospinal fluid (CSF ), reflecting different aspects of the pathophysiology particularly of relapsing‐remitting MS (RRMS ). Sulfatide, abundant in the myelin sheath and a proposed target for autoimmune attack in MS , has been reported altered in MS CSF . Here, we investigated the potential of CSF sulfatide and its isoforms as biomarkers in MS . A highly sensitive and quantitative mass spectrometry method was employed to determine levels of sulfatide isoforms in CSF from RRMS and progressive MS (PMS ) patients, and healthy donors (HD ). We demonstrate that levels of total CSF sulfatide and C24:1, C26:1, and C26:1‐OH isoforms were significantly increased in PMS compared with RRMS patients and HD , while C23:0‐OH was significantly decreased in CSF from PMS patients compared to the other two groups. Multivariate discriminant analysis showed that CSF sulfatide isoform pattern in PMS patients was distinct and non‐overlapping with that of RRMS patients and HD . Sulfatide levels did not correlate with tested biomarkers or clinical parameters. The results suggest that CSF sulfatide isoform levels may be used to discriminate the phenotype of MS and might play a role in the progression of the disease.

     

Engineering,applications, and future perspectives of GPCR-based genetically encoded fluorescent indicators for neuromodulators

This review explores the evolving landscape of G-protein-coupled receptor (GPCR)-based genetically encoded fluorescent indicators (GEFIs), with a focus on their development, structural components, engineering strategies, and applications. We highlight the unique features of this indicator class, emphasizing the importance of both the sensing domain (GPCR structure and activation mechanism) and the reporting domain (circularly permuted fluorescent protein (cpFP) structure and fluorescence modulation). Further, we discuss indicator engineering approaches, including the selection of suitable cpFPs and expression systems. Additionally, we showcase the diversity and flexibility of their application by presenting a summary of studies where such indicators were used. Along with all the advantages, we also focus on the current limitations as well as common misconceptions that arise when using these indicators. Finally, we discuss future directions in indicator engineering, including strategies for screening with increased throughput, optimization of the ligand-binding properties, structural insights, and spectral diversity.

     

HPC‐1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival

Two types of syntaxin 1 isoforms, HPC‐1/syntaxin 1A (STX1A) and syntaxin 1B (STX1B), are thought to have similar functions in exocytosis of synaptic vesicles. STX1A^?/? mice which we generated previously develop normally, possibly because of compensation by STX1B. We produced STX1B^?/? mice using targeted gene disruption and investigated their phenotypes. STX1B^?/? mice were born alive, but died before postnatal day 14, unlike STX1A^?/? mice. Morphologically, brain development in STX1B^?/? mice was impaired. In hippocampal neuronal culture, the cell viability of STX1B^?/? neurons was lower than that of WT or STX1A^?/? neurons after 9 days. Interestingly, STX1B^?/? neurons survived on WT or STX1A^?/? glial feeder layers as well as WT neurons. However, STX1B^?/? glial feeder layers were less effective at promoting survival of STX1B^?/? neurons. Conditioned medium from WT or STX1A^?/? glial cells had a similar effect on survival, but that from STX1B^?/? did not promote survival. Furthermore, brain‐derived neurotrophic factor (BDNF) or neurotrophin‐3 supported survival of STX1B^?/? neurons. BDNF localization in STX1B^?/? glial cells was disrupted, and BDNF secretion from STX1B^?/? glial cells was impaired. These results suggest that STX1A and STX1B may play distinct roles in supporting neuronal survival by glia.

     

Expression of the synaptic exocytosis‐regulating molecule complexin 2 in taste buds and its participation in peripheral taste transduction

Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2‐knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2‐knockout mice were significantly lower than those in wild‐type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons.

     

Rac1‐mediated indentation of resting neurons promotes the chain migration of new neurons in the rostral migratory stream of post‐natal mouse brain

New neurons generated in the ventricular‐subventricular zone in the post‐natal brain travel toward the olfactory bulb by using a collective cell migration process called ‘chain migration.’ These new neurons show a saltatory movement of their soma, suggesting that each neuron cycles through periods of ‘rest’ during migration. Here, we investigated the role of the resting neurons in chain migration using post‐natal mouse brain, and found that they undergo a dynamic morphological change, in which a deep indentation forms in the cell body. Inhibition of Rac1 activity resulted in less indentation of the new neurons in vivo. Live cell imaging using a Förster resonance energy transfer biosensor revealed that Rac1 was activated at the sites of contact between actively migrating and resting new neurons. On the cell surface of resting neurons, Rac1 activation coincided with the formation of the indentation. Furthermore, Rac1 knockdown prevented the indentation from forming and impaired migration along the resting neurons. These results suggest that Rac1 regulates a morphological change in the resting neurons, which allows them to serve as a migratory scaffold, and thereby non‐cell‐autonomously promotes chain migration.