Despite the importance of 5-HT1A as a major target for the action of several anxiolytics/antidepressant drugs, little is known about its regulation in central serotonin (5-hydroxytryptamine, 5-HT) neurons.
We report that expression of 5-HT1A and the transcription factor Pet1 was impaired in the rostral raphe nuclei of mice lacking tryptophan hydroxylase 2 (Tph2) after birth. The downregulation of Pet1 was recapitulated in 5-Ht1a
mice. Using an explant culture system, we show that reduction of Pet1 and 5-HT1A was rescued in Tph2
brainstem by exogenous 5-HT. In contrast, 5-HT failed to rescue reduced expression of Pet1 in 5-Ht1a
brainstem explant culture.
These results suggest a causal relationship between 5-HT1A and Pet1, and reveal a potential mechanism by which 5-HT1A-Pet1 autoregulatory loop is maintained by 5-HT in a spatiotemporal-specific manner during postnatal development. Our results are relevant to understanding the pathophysiology of certain psychiatric and developmental disorders.
Mouse; Serotonin; 5-HT1A; Pet1; Tph2; Development; Autoregulatory loop
Retinitis pigmentosa (RP) is an inherited human retinal disorder that causes progressive photoreceptor cell loss, leading to severe vision impairment or blindness. However, no effective therapy has been established to date. Although genetic mutations have been identified, the available clinical data are not always sufficient to elucidate the roles of these mutations in disease pathogenesis, a situation that is partially due to differences in genetic backgrounds.
We generated induced pluripotent stem cells (iPSCs) from an RP patient carrying a rhodopsin mutation (E181K). Using helper-dependent adenoviral vector (HDAdV) gene transfer, the mutation was corrected in the patient’s iPSCs and also introduced into control iPSCs. The cells were then subjected to retinal differentiation; the resulting rod photoreceptor cells were labeled with an Nrl promoter-driven enhanced green fluorescent protein (EGFP)-carrying adenovirus and purified using flow cytometry after 5 weeks of culture. Using this approach, we found a reduced survival rate in the photoreceptor cells with the E181K mutation, which was correlated with the increased expression of endoplasmic reticulum (ER) stress and apoptotic markers. The screening of therapeutic reagents showed that rapamycin, PP242, AICAR, NQDI-1, and salubrinal promoted the survival of the patient’s iPSC-derived photoreceptor cells, with a concomitant reduction in markers of ER stress and apoptosis. Additionally, autophagy markers were found to be correlated with ER stress, suggesting that autophagy was reduced by suppressing ER stress-induced apoptotic changes.
The use of RP patient-derived iPSCs combined with genome editing provided a versatile cellular system with which to define the roles of genetic mutations in isogenic iPSCs with or without mutation and also provided a system that can be used to explore candidate therapeutic approaches.
iPS cells; Retina; Neurodegeneration; Gene delivery; Drug screening; ER stress
Irritable bowel syndrome (IBS) is characterized by recurrent abdominal discomfort, spontaneous pain, colorectal hypersensitivity and bowel dysfunction. Patients with IBS also suffer from emotional anxiety and depression. However, few animal studies have investigated IBS-induced spontaneous pain and behavioral anxiety. In this study, we assessed spontaneous pain and anxiety behaviors in an adult mouse model of IBS induced by zymosan administration. By using Fos protein as a marker, we found that sensory and emotion related brain regions were activated at day 7 after the treatment with zymosan; these regions include the prefrontal cortex, anterior cingulate cortex, insular cortex and amygdala. Behaviorally, zymosan administration triggered spontaneous pain (decreased spontaneous activities in the open field test) and increased anxiety-like behaviors in three different tests (the open field, elevated plus maze and light/dark box tests). Intraperitoneal injection of NB001, an adenylyl cyclase 1 (AC1) inhibitor, reduced spontaneous pain but had no significant effect on behavioral anxiety. In contrast, gabapentin reduced both spontaneous pain and behavioral anxiety. These results indicate that NB001 and gabapentin may inhibit spontaneous pain and anxiety-like behaviors through different mechanisms.
Irritable bowel syndrome; Zymosan; Visceral pain; Spontaneous pain; Anxiety
Huntington’s Disease (HD) is a progressive neurodegenerative disorder with a single causal mutation in the Huntingtin (HTT) gene. MicroRNAs (miRNAs) have recently been implicated as epigenetic regulators of neurological disorders, however, their role in HD pathogenesis is not well defined. Here we study transgenic HD monkeys (HD monkeys) to examine miRNA dysregulation in a primate model of the disease.
In this report, 11 miRNAs were found to be significantly associated (P value < 0.05) with HD in the frontal cortex of the HD monkeys. We further focused on one of those candidates, miR-128a, due to the corresponding disruption in humans and mice with HD as well as its intriguing lists of gene targets. miR-128a was downregulated in our HD monkey model by the time of birth. We then confirmed that miR-128a was also downregulated in the brains of pre-symptomatic and post-symptomatic HD patients. Additionally, our studies confirmed a panel of canonical HD signaling genes regulated by miR-128a, including HTT and Huntingtin Interaction Protein 1 (HIP1).
Our studies found that miR-128a may play a critical role in HD and could be a viable candidate as a therapeutic or biomarker of the disease.
microRNAs; Noncoding RNAs; Huntington’s disease; Brain; miR-128a
Mutant mice have been used successfully as a tool for investigating the mechanisms of memory at multiple levels, from genes to behavior. In most cases, manipulating a gene expressed in the brain impairs cognitive functions such as memory and their underlying cellular mechanisms, including synaptic plasticity. However, a remarkable number of mutations have been shown to enhance memory in mice. Understanding how to improve a system provides valuable insights into how the system works under normal conditions, because this involves understanding what the crucial components are. Therefore, more can be learned about the basic mechanisms of memory by studying mutant mice with enhanced memory. This review will summarize the genes and signaling pathways that are altered in the mutants with enhanced memory, as well as their roles in synaptic plasticity. Finally, I will discuss how knowledge of memory-enhancing mechanisms could be used to develop treatments for cognitive disorders associated with impaired plasticity.
Memory; Synaptic plasticity; Long-term potentiation (LTP); Hippocampus; Treatment
Voltage-dependent block of the NMDA receptor by Mg2+ is thought to be central to the unique involvement of this receptor in higher brain functions. However, the in vivo role of the Mg2+ block in the mammalian brain has not yet been investigated, because brain-wide loss of the Mg2+ block causes perinatal lethality. In this study, we used a brain-region specific knock-in mouse expressing an NMDA receptor that is defective for the Mg2+ block in order to test its role in neural information processing.
We devised a method to induce a single amino acid substitution (N595Q) in the GluN2A subunit of the NMDA receptor, specifically in the hippocampal dentate gyrus in mice. This mutation reduced the Mg2+ block at the medial perforant path–granule cell synapse and facilitated synaptic potentiation induced by high-frequency stimulation. The mutants had more stable hippocampal place fields in the CA1 than the controls did, and place representation showed lower sensitivity to visual differences. In addition, behavioral tests revealed that the mutants had a spatial working memory deficit.
These results suggest that the Mg2+ block in the dentate gyrus regulates hippocampal spatial information processing by attenuating activity-dependent synaptic potentiation in the dentate gyrus.
Mg2+ block; NMDA receptor; Dentate gyrus; Place cell
Inorganic polyphosphate (polyP) is a highly charged polyanion capable of interacting with a number of molecular targets. This signaling molecule is released into the extracellular matrix by central astrocytes and by peripheral platelets during inflammation. While the release of polyP is associated with both induction of blood coagulation and astrocyte extracellular signaling, the role of secreted polyP in regulation of neuronal activity remains undefined. Here we test the hypothesis that polyP is an important participant in neuronal signaling. Specifically, we investigate the ability of neurons to release polyP and to induce neuronal firing, and clarify the underlying molecular mechanisms of this process by studying the action of polyP on voltage gated channels.
Using patch clamp techniques, and primary hippocampal and dorsal root ganglion cell cultures, we demonstrate that polyP directly influences neuronal activity, inducing action potential generation in both PNS and CNS neurons. Mechanistically, this is accomplished by shifting the voltage sensitivity of NaV channel activation toward the neuronal resting membrane potential, the block KV channels, and the activation of CaV channels. Next, using calcium imaging we found that polyP stimulates an increase in neuronal network activity and induces calcium influx in glial cells. Using in situ DAPI localization and live imaging, we demonstrate that polyP is naturally present in synaptic regions and is released from the neurons upon depolarization. Finally, using a biochemical assay we demonstrate that polyP is present in synaptosomes and can be released upon their membrane depolarization by the addition of potassium chloride.
We conclude that polyP release leads to increased excitability of the neuronal membrane through the modulation of voltage gated ion channels. Together, our data establishes that polyP could function as excitatory neuromodulator in both the PNS and CNS.
Polyphosphate; Voltage gated channels; Neuroactive compounds; Synaptic vesicles; Synaptic transmission; Neuronal activity; Pain; Platelets; Inflammation
Alzheimer’s disease (AD) pathology occurs in part as the result of excessive production of β-amyloid (Aβ). Metabotropic glutamate receptor 5 (mGluR5) is now considered a receptor for Aβ and consequently contributes to pathogenic Aβ signaling in AD.
Genetic deletion of mGluR5 rescues the spatial learning deficits observed in APPswe/PS1ΔE9 AD mice. Moreover, both Aβ oligomer formation and Aβ plaque number are reduced in APPswe/PS1ΔE9 mice lacking mGluR5 expression. In addition to the observed increase in Aβ oligomers and plaques in APPswe/PS1ΔE9 mice, we found that both mTOR phosphorylation and fragile X mental retardation protein (FMRP) expression were increased in these mice. Genetic deletion of mGluR5 reduced Aβ oligomers, plaques, mTOR phosphorylation and FMRP expression in APPswe/PS1ΔE9 mice.
Thus, we propose that Aβ activation of mGluR5 appears to initiate a positive feedback loop resulting in increased Aβ formation and AD pathology in APPswe/PS1ΔE9 mice via mechanism that is regulated by FMRP.
Alzheimer’s disease; APPswe/PS1ΔE9; mGluR5; Beta amyloid; FMRP; Learning and memory
Schizophrenia, a severe psychiatric disorder, has a lifetime prevalence of 1%. The exact mechanisms underlying this disorder remain unknown, though theories abound. Recent studies suggest that particular cell types and biological processes in the schizophrenic cortex have a pseudo-immature status in which the molecular properties partially resemble those in the normal immature brain. However, genome-wide gene expression patterns in the brains of patients with schizophrenia and those of normal infants have not been directly compared. Here, we show that the gene expression patterns in the schizophrenic prefrontal cortex (PFC) resemble those in the juvenile PFC.
We conducted a gene expression meta-analysis in which, using microarray data derived from different studies, altered expression patterns in the dorsolateral PFC (DLFC) of patients with schizophrenia were compared with those in the DLFC of developing normal human brains, revealing a striking similarity. The results were replicated in a second DLFC data set and a medial PFC (MFC) data set. We also found that about half of the genes representing the transcriptomic immaturity of the schizophrenic PFC were developmentally regulated in fast-spiking interneurons, astrocytes, and oligodendrocytes. Furthermore, to test whether medications, which often confound the results of postmortem analyses, affect on the juvenile-like gene expressions in the schizophrenic PFC, we compared the gene expression patterns showing transcriptomic immaturity in the schizophrenic PFC with those in the PFC of rodents treated with antipsychotic drugs. The results showed no apparent similarities between the two conditions, suggesting that the juvenile-like gene expression patterns observed in the schizophrenic PFC could not be accounted for by medication effects. Moreover, the developing human PFC showed a gene expression pattern similar to that of the PFC of naive Schnurri-2 knockout mice, an animal model of schizophrenia with good face and construct validity. This result also supports the idea that the transcriptomic immaturity of the schizophrenic PFC is not due to medication effects.
Collectively, our results provide evidence that pseudo-immaturity of the PFC resembling juvenile PFC may be an endophenotype of schizophrenia.
Schizophrenia; Transcriptome; Prefrontal cortex; Immaturity; Parvalbumin; Endophenotype
The gamma-amino-butyric acid (GABA) system is a critical mediator of fear extinction process. GABA can induce “phasic” or “tonic” inhibition in neurons through synaptic or extrasynaptic GABAA receptors, respectively. However, role of the thalamic “tonic GABA inhibition” in cognition has not been explored. We addressed this issue in extinction of conditioned fear in mice.
Here, we show that GABAA receptors in the mediodorsal thalamic nucleus (MD) modulate fear extinction. Microinjection of gabazine, a GABAA receptor antagonist, into the MD decreased freezing behavior in response to the conditioned stimulus and thus facilitated fear extinction. Interestingly, microinjection of THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), a preferential agonist for the δ-subunit of extrasynaptic GABAA receptors, into the MD attenuated fear extinction. In the opposite direction, an MD-specific knock-out of the extrasynaptic GABAA receptors facilitated fear extinction.
Our results suggest that “tonic GABA inhibition” mediated by extrasynaptic GABAA receptors in MD neurons, suppresses fear extinction learning. These results raise a possibility that pharmacological control of tonic mode of GABAA receptor activation may be a target for treatment of anxiety disorders like post-traumatic stress disorder.
Fear extinction; Mediodorsal thalamus; Extrasynaptic GABAA receptor; GABRA4; Tonic GABA inhibition; Anxiety disorders
Cerebral overexcitation needs inhibitory neurons be functionally upregulated to rebalance excitation vs. inhibition. For example, the intensive activities of GABAergic neurons induce spontaneous spikes, i.e., activity-induced spontaneous spikes (AISS). The mechanisms underlying AISS onset remain unclear. We investigated the roles of sodium channels in AISS induction and expression at hippocampal GABAergic neurons by electrophysiological approach.
AISS expression includes additional spike capability above evoked spikes, and the full spikes in AISS comprise early phase (spikelets) and late phase, implying the emergence of new spikelet component. Compared with the late phase, the early phase is characterized as voltage-independent onset, less voltage-dependent upstroke and sensitivity to TTX. AISS expression and induction are independent of membrane potential changes. Therefore, AISS’s spikelets express based on voltage-independent sodium channels. In terms of AISS induction, the facilitation of voltage-gated sodium channel (VGSC) activation accelerates AISS onset, or vice versa.
AISS expression in GABAergic neurons is triggered by the spikelets based on the functional emergence of voltage-independent sodium channels, which is driven by intensive VGSCs’ activities.
Action potential; Spontaneous spikes; Threshold potential; Sodium channel; Hippocampus; GABAergic neurons
HIV-associated neurocognitive disorder (HAND) is characterized by development of cognitive, behavioral and motor abnormalities, and occurs in approximately 50% of HIV infected individuals. In the United States, the prevalence of cigarette smoking ranges from 35-70% in HIV-infected individuals compared to 20% in general population. Cognitive impairment in heavy cigarette smokers has been well reported. However, the synergistic effects of nicotine and HIV infection and the underlying mechanisms in the development of HAND are unknown.
In this study, we explored the role of nicotine in the progression of HAND using SK-N-MC, a neuronal cell line. SK-N-MC cells were infected with HIV-1 in the presence or absence of nicotine for 7 days. We observed significant increase in HIV infectivity in SK-N-MC treated with nicotine compared to untreated HIV-infected neuronal cells. HIV and nicotine synergize to significantly dysregulate the expression of synaptic plasticity genes and spine density; with a concomitant increase of HDAC2 levels in SK-N-MC cells. In addition, inhibition of HDAC2 up-regulation with the use of vorinostat resulted in HIV latency breakdown and recovery of synaptic plasticity genes expression and spine density in nicotine/HIV alone and in co-treated SK-N-MC cells. Furthermore, increased eIF2 alpha phosphorylation, which negatively regulates eukaryotic translational process, was observed in HIV alone and in co-treatment with nicotine compared to untreated control and nicotine alone treated SK-N-MC cells.
These results suggest that nicotine and HIV synergize to negatively regulate the synaptic plasticity gene expression and spine density and this may contribute to the increased risk of HAND in HIV infected smokers. Apart from disrupting latency, vorinostat may be a useful therapeutic to inhibit the negative regulatory effects on synaptic plasticity in HIV infected nicotine abusers.
Human synaptic plasticity; PCR array; Spine density; HIV; Nicotine; HDAC2; eIF2α; Vorinostat; SK-N-MC neuronal cells; Neurocognitive disorder
Low-voltage-activated (T-type) calcium channels play a crucial role in a number of physiological processes, including neuronal and cardiac pacemaker activity and nociception. Therefore, finding specific modulators and/or blockers of T-type channels has become an important field of drug discovery. One characteristic of T-type calcium channels is that they share several structural similarities with voltage-gated sodium channels (VGSCs). We therefore hypothesized that binding sites for certain sodium channel blocking peptide toxins may be present in T-type calcium channels.
The sodium channel blocker ProTx I tonically blocked native and transiently expressed T-type channels in the sub- to low micro molar range with at least a ten-fold selectivity for the T-type calcium channel hCav3.1 over hCav3.3, and more than one hundred fold selectivity over hCav3.2. Using chimeras of hCav3.1 and hCav3.3, we determined that the domain IV region of hCav3.1 is a major determinant of toxin affinity, with a minor contribution from domain II. Further analysis revealed several residues in a highly conserved region between T-type and sodium channels that may correspond to toxin binding sites. Mutagenesis of several of these residues on an individual basis, however, did not alter the blocking effects of the toxin. ProTx II on the other hand preferentially blocked hCav3.2 and significantly shifted the steady state inactivation of this channel.
ProTx I blocks hCav3.1 both selectively and with high affinity. Domain IV appears to play a major role in this selectivity with some contribution from domain II. Given the structural similarities between sodium and T-type calcium channels and the apparent conservation in toxin binding sites, these data could provide insights into the development and synthesis of novel T-type channel antagonists.
Calcium channels; ProTx I; ProTx II; T-type blockers; Electrophysiology
Functional heterologous expression of naturally-expressed and apparently functional mammalian α6*-nicotinic acetylcholine receptors (nAChRs; where ‘*’ indicates presence of additional subunits) has been difficult. Here we wanted to investigate the role of N-terminal domain (NTD) residues of human (h) nAChR α6 subunit in the functional expression of hα6*-nAChRs. To this end, instead of adopting random mutagenesis as a tool, we used 15 NTD rare variations (i.e., Ser43Pro, Asn46Lys, Asp57Asn, Arg87Cys, Asp92Glu, Arg96His, Glu101Lys, Ala112Val, Ser156Arg, Asn171Lys, Ala184Asp, Asp199Tyr, Asn203Thr, Ile226Thr and Ser233Cys) in nAChR hα6 subunit to probe for their effect on the functional expression of hα6*-nAChRs.
N-terminal α-helix (Asp57); complementary face/inner β-fold (Arg87 or Asp92) and principal face/outer β-fold (Ser156 or Asn171) residues in the hα6 subunit are crucial for functional expression of the hα6*-nAChRs as variations in these residues reduce or abrogate the function of hα6hβ2*-, hα6hβ4- and hα6hβ4hβ3-nAChRs. While variations at residues Ser43 or Asn46 (both in N-terminal α-helix) in hα6 subunit reduce hα6hβ2*-nAChRs function those at residues Arg96 (β2-β3 loop), Asp199 (loop F) or Ser233 (β10-strand) increase hα6hβ2*-nAChR function. Similarly substitution of NTD α-helix (Asn46), loop F (Asp199), loop A (Ala112), loop B (Ala184), or loop C (Ile226) residues in hα6 subunit increase the function of hα6hβ4-nAChRs. All other variations in hα6 subunit do not affect the function of hα6hβ2*- and hα6hβ4*-nAChRs. Incorporation of nAChR hβ3 subunits always increase the function of wild-type or variant hα6hβ4-nAChRs except for those of hα6(D57N, S156R, R87C or N171K)hβ4-nAChRs. It appears Asp57Lys, Ser156Arg or Asn171Lys variations in hα6 subunit drive the hα6hβ4hβ3-nAChRs into a nonfunctional state as at spontaneously open hα6(D57N, S156R or N171K)hβ4hβ3V9’S-nAChRs (V9’S; transmembrane II 9’ valine-to-serine mutation) agonists act as antagonists. Agonist sensitivity of hα6hβ4- and/or hα6hβ4hβ3-nAChRs is nominally increased due to Arg96His, Ala184Asp, Asp199Tyr or Ser233Cys variation in hα6 subunit.
Hence investigating functional consequences of natural variations in nAChR hα6 subunit we have discovered additional bases for cell surface functional expression of various subtypes of hα6*-nAChRs. Variations (Asp57Asn, Arg87Cys, Asp92Glu, Ser156Arg or Asn171Lys) in hα6 subunit that compromise hα6*-nAChR function are expected to contribute to individual differences in responses to smoked nicotine.
Electrophysiology; Ion channels; Nicotinic acetylcholine receptor; Receptor structure-function; Single nucleotide variation
The L-type calcium channel Cav1.2 is important for brain and heart function. The ubiquitous calcium sensing protein calmodulin (CaM) regulates calcium dependent gating of Cav1.2 channels by reducing calcium influx, a process known as calcium-dependent inactivation (CDI). Dissecting the calcium-dependence of CaM in this process has benefited greatly from the use of mutant CaM molecules which are unable to bind calcium to their low affinity (N-lobe) and high affinity (C-lobe) binding sites. Unlike CDI, it is unknown whether CaM can modulate the activation gating of Cav1.2 channels.
We examined a Cav1.2 point mutant in the N-terminus region of the channel (A39V) that has been previously linked to Brugada syndrome. Using mutant CaM constructs in which the N- and/or C-lobe calcium binding sites were ablated, we were able to show that this Brugada syndrome mutation disrupts N-lobe CDI of the channel. In the course of these experiments, we discovered that all mutant CaM molecules were able to alter the kinetics of channel activation even in the absence of calcium for WT-Cav1.2, but not A39V-Cav1.2 channels. Moreover, CaM mutants differentially shifted the voltage-dependence of activation for WT and A39V-Cav1.2 channels to hyperpolarized potentials. Our data therefore suggest that structural changes in CaM that arise directly from site directed mutagenesis of calcium binding domains alter activation gating of Cav1.2 channels independently of their effects on calcium binding, and that the N-terminus of the channel contributes to this CaM dependent process.
Our data indicate that caution must be exercised when interpreting the effects of CaM mutants on ion channel gating.
Calcium channel; Calmodulin mutant; CDI; N-terminus; Brugada; Activation; Cav1.2; L-type; IQ; Channelopathy; Voltage; Gating; CACNA1C
Chronic stress is generally known to exacerbate the development of numerous neuropsychiatric diseases such as fear and anxiety disorders, which is at least partially due to the disinhibition of amygdala subsequent to the prolonged stress exposure. GABA receptor A (GABAAR) mediates the primary component of inhibition in brain and its activation produces two forms of inhibition: the phasic and tonic inhibition. While both of them are critically engaged in limiting the activity of amygdala, their roles in the amygdala disinhibition subsequent to chronic stress exposure are largely unknown.
We investigated the possible alterations of phasic and tonic GABAAR currents and their roles in the amygdala disinhibition subsequent to chronic stress. We found that both chronic immobilization and unpredictable stress led to long lasting loss of tonic GABAAR currents in the projection neurons of lateral amygdala. By contrast, the phasic GABAAR currents, as measured by the spontaneous inhibitory postsynaptic currents, were virtually unaltered. The loss of tonic inhibition varied with the duration of daily stress and the total days of stress exposure. It was prevented by pretreatment with metyrapone to block corticosterone synthesis or RU 38486, a glucocorticoid receptor antagonist, suggesting the critical involvement of glucocorticoid receptor activation. Moreover, chronic treatment with corticosterone mimicked the effect of chronic stress and reduced the tonic inhibition in lateral amygdala of control mice. The loss of tonic inhibition resulted in the impaired GABAergic gating on neuronal excitability in amygdala, which was prevented by metyrapone pretreatment.
Our study suggests that enduring loss of tonic but not phasic GABAAR currents critically contributes to the prolonged amygdala disinhibition subsequent to chronic stress. We propose that the preferential loss of tonic inhibition may account for the development of stress-related neuropsychiatric diseases.
Amygdala; Chronic stress; GABA; Electrophysiology; Tonic inhibition; Corticosterone; Glucocorticoid receptor; Neuronal excitability
Neuroinflammation plays a key role in the initiation and progression of neurodegeneration in Alzheimer’s disease (AD). Chronic neuroinflammation results in diminished synaptic plasticity and loss of GluN1 N-methyl-D-aspartate (NMDA) receptors in the hippocampus, leading to the cognitive deficits that are the most common symptoms of AD. Therefore, it is suggested that chronic inflammation may alter expression levels of GluN2A and GluN2B subunits of NMDA receptors and associated intracellular signalling. Chronic neuroinflammation was induced by chronic infusion of lipopolysaccharide (LPS) into the fourth ventricle in Fischer-344 rats. The status of hippocampus-dependent memory was evaluated in control rats and rats chronically infused with LPS. Microglial activation in the hippocampus was examined using immunohistochemical staining. Western blot analysis was used to measure membrane levels of GluN2A and GluN2B subunits of NMDA receptors and mitogen-activated protein kinase (MAPK) in the hippocampi of these rats, and immunofluorescent double labeling was used to assess the cellular location of MAPK. Microglial activation was observed in the hippocampi of rats that showed memory impairments with chronic LPS infusion. Chronic LPS infusion reduced the levels of GluN2A and GluN2B and increased the levels of phosphorylated MAPKs in the hippocampus. MAPK-positive immunoreactivity was observed mostly in the neurons and also in non-neuronal cells. Reductions in GluN2A and GluN2B subunits of NMDA receptors coupled with altered MAPK signaling, in response to inflammatory stimuli may be related to the cognitive deficits observed in AD.
Neuroinflammation; Memory; NMDA; MAPK; Microglia
We previously performed systematic association studies of glutamate receptor gene family members with schizophrenia, and found positive associations of polymorphisms in the GRM3 (a gene of metabotropic glutamate receptor 3: mGluR3) with the disorder. Physiological roles of GRM3 in brain functions and its functional roles in the pathogenesis of schizophrenia remain to be resolved.
We generated mGluR3 knockout (KO) mice and conducted comprehensive behavioral analyses. KO mice showed hyperactivity in the open field, light/dark transition, and 24-hour home cage monitoring tests, impaired reference memory for stressful events in the Porsolt forced swim test, impaired contextual memory in cued and contextual fear conditioning test, and impaired working memory in the T-Maze forced alternation task test. Hyperactivity and impaired working memory are known as endophenotypes of schizophrenia. We examined long-term synaptic plasticity by assessing long-term potentiation (LTP) in the CA1 region in the hippocampi of KO and wild-type (WT) mice. We observed no differences in the amplitude of LTP between the two genotypes, suggesting that mGluR3 is not essential for LTP in the CA1 region of the mouse hippocampus. As hyperactivity is typically associated with increased dopaminergic transmission, we performed in vivo microdialysis measurements of extracellular dopamine in the nucleus accumbens of KO and WT mice. We observed enhancements in the methamphetamine (MAP)-induced release of dopamine in KO mice.
These results demonstrate that a disturbance in the glutamate-dopamine interaction may be involved in the pathophysiology of schizophrenia-like behavior, such as hyperactivity in mGluR3 KO mice.
Metabotropic glutamate receptors; Grm3; Knockout mice; Working memory; Reference memory; Contextual memory; Hyperactivity; LTP; Microdialysis; Schizophrenia
Prolonged re-exposure to a fear-eliciting cue in the absence of an aversive event extinguishes the fear response to the cue, and has been clinically used as an exposure therapy. Arc (also known as Arg3.1) is implicated in synaptic and experience-dependent plasticity. Arc is regulated by the transcription factor cAMP response element binding protein, which is upregulated with and necessary for fear extinction. Because Arc expression is also activated with fear extinction, we hypothesized that Arc expression is required for fear extinction.
Extinction training increased the proportion of Arc-labeled cells in the basolateral amygdala (BLA). Arc was transcribed during latter part of extinction training, which is possibly associated with fear extinction, as well as former part of extinction training. Intra-BLA infusions of Arc antisense oligodeoxynucleotide (ODN) before extinction training impaired long-term but not short-term extinction memory. Intra-BLA infusions of Arc antisense ODN 3 h after extinction training had no effect on fear extinction.
Our findings demonstrate that Arc is required for long-term extinction of conditioned fear and contribute to the understanding of extinction as a therapeutic manner.
Arc/Arg3.1; Fear conditioning; Extinction; Amygdala
Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common cause of dominant and sporadic Parkinson’s disease (PD), a common neurodegenerative disorder. Yeast-two-hybrid screening using human LRRK2 kinase domain as bait identified microtubule associated protein 1B (MAP1B) as a LRRK2 interactor. The interacting domains were LRRK2 kinase and the light chain portion of MAP1B (LC1). LRRK2 + LC1 interaction resulted in LRRK2 kinase inhibition. LRRK2 mutants (R1441C, G2019S and I2020T) exhibited decreased endogenous LC1 expression and its co-expression with LC1 rescued LRRK2 mutant-mediated toxicity. This study presented the first data on the effects of LRRK2 + LC1 interaction and also suggested that LCI possibly rescued LRRK2 mutant-induced cytotoxicity by inhibiting LRRK2 kinase activity. Compounds that upregulate LC1 expression may therefore hold therapeutic potential for LRRK2-linked diseases.
LRRK2; MAP1B; LC1; Phosphorylation; Apoptosis
Inflammatory reaction in blood–spinal cord barrier (BSCB) plays a crucial role in ischemia/reperfusion (I/R) injury. It has been shown that microglia could be activated through Toll-like receptors (TLRs). Therefore, we hypothesize that TLR4 is involved in the microglial activation and BSCB disruption after I/R.
To verify our hypothesis, we analyzed the behavioral data, changes of BSCB permeability, as well as expressions of microglial marker Iba-1 and TLR4 in spinal cord I/R model induced by 14 min aortic occlusion. Double immunostaining reveals that after I/R, Iba-1 immunoreactivity increased gradually 12 h after reperfusion and maintained at a such level throughout 36 h. Such increasing pattern of Iba-1 expression is consistent with the increases in Evan’s Blue (EB) extravasation, spinal water content and mechanical allodynia demonstrated by lowed withdrawal threshold to Von Frey filaments. Moreover, double immunostaining suggested that TLR4 was highly expressed in microglia. Intrathecal infusion of minocycline and TAK-242 (TLR4 inhibitor) treatment attenuated I/R-induced allodynia and BSCB leakage. In contrast, LPS induced TLR4 expression aggregated above-mentioned injuries. Furthermore, the nuclear factor-kappa B (NF-κB) activity has a similar profile as TLR4 activity. It is consisted with the results of NF-κB mRNA and protein expression changes and activation of downstream cytokine, IL-1β. Expectedly, intrathecal infusion of pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, showed similar protective effects as minocycline and TAK-242. In addition, our data show that TLR4 closely involved in I/R-induced inflammatory damage induced neuronal apoptosis. Significantly, neutralizing TLR4 function largely reduced neuronal apoptosis determined by NeuN immunoreactivity in ventral gray matter and increased percentage of double-label cells with cleaved caspase3, whereas LPS reversed these effects. Similarly, inhibitions of microglia and NF-κB with minocycline or PDTC treatment accordingly perform the same protective effects on I/R injury.
The results indicate that compromised BSCB caused by I/R injury lead to spinal microglial activation and TLR4, its membrane-bound receptor, up-regulation, which then initiate neuro-inflammation and neuro-apoptosis via NF-κB/ IL-1β pathway. To inhibit the positive feedback loop of TLR4-microglia-NF-κB/ IL-1β pathway by minocycline, TAK-242 (TLR4 inhibitor) and pyrrolidine dithiocarbamate (PDTC, NF-κB inhibitor) may provide new targets for treating I/R injury in clinic.
Blood–spinal cord barrier; Intrathecal transplantation; Microglia; Minocycline; Toll-like receptor 4; Spinal cord ischemia/reperfusion injury
Action potentials can be initiated at various subcellular compartments, such as axonal hillock, soma and dendrite. Mechanisms and physiological impacts for this relocation remain elusive, which may rely on input signal patterns and intrinsic properties in these subcellular compartments. We examined this hypothesis at the soma and axon of cortical pyramidal neurons by analyzing their spike capability and voltage-gated sodium channel dynamics in response to different input signals.
Electrophysiological recordings were simultaneously conducted at the somata and axons of identical pyramidal neurons in the cortical slices. The somata dominantly produced sequential spikes in response to long-time steady depolarization pulse, and the axons produced more spikes in response to fluctuated pulse. Compared with the axons, the somata possessed lower spike threshold and shorter refractory periods in response to long-time steady depolarization, and somatic voltage-gated sodium channels demonstrated less inactivation and easier reactivation in response to steady depolarization. Based on local VGSC dynamics, computational simulated spike initiation locations were consistent with those from the experiments. In terms of physiological impact, this input-dependent plasticity of spike initiation location made neuronal encoding to be efficient.
Long-time steady depolarization primarily induces somatic spikes and short-time pulses induce axonal spikes. The input signal patterns influence spike initiations at the axon or soma of cortical pyramidal neurons through modulating local voltage-gated sodium channel dynamics.
Action potential; Soma; Axon; Neuron and sodium channel
Itch, chronic itch in particular, can have a significant negative impact on an individual’s quality of life. However, the molecular mechanisms underlying itch processing in the central nervous system remain largely unknown.
We report here that activation of ERK signaling in the spinal cord is required for itch sensation. ERK activation, as revealed by anti-phosphorylated ERK1/2 immunostaining, is observed in the spinal dorsal horn of mice treated with intradermal injections of histamine and compound 48/80 but not chloroquine or SLIGRL-NH2, indicating that ERK activation only occurs in histamine-dependent acute itch. In addition, ERK activation is also observed in 2, 4-dinitrofluorobenzene (DNFB)-induced itch. Consistently, intrathecal administration of the ERK phosphorylation inhibitor U0126 dramatically reduces the scratching behaviors induced by histamine and DNFB, but not by chloroquine. Furthermore, administration of the histamine receptor H1 antagonist chlorpheniramine decreases the scratching behaviors and ERK activation induced by histamine, but has no effect on DNFB-induced itch responses. Finally, the patch-clamp recording shows that in histamine-, chloroquine- and DNFB-treated mice the spontaneous excitatory postsynaptic current (sEPSC) of dorsal horn neurons is increased, and the decrease of action potential threshold is largely prevented by bathing of U0126 in histamine- and DNFB-treated mice but not those treated with chloroquine.
Our results demonstrate a critical role for ERK activation in itch sensation at the spinal level.
Acute itch; Chronic itch; pERK; H1 receptor; Spinal cord; Atopic dermatitis
The common marmoset (Callithrix jacchus) is a New World primate sharing many similarities with humans. Recently developed technology for generating transgenic marmosets has opened new avenues for faithful recapitulation of human diseases, which could not be achieved in rodent models. However, the longer lifespan of common marmosets compared with rodents may result in an extended period for in vivo analysis of common marmoset disease models. Therefore, establishing rapid and efficient techniques for obtaining neuronal cells from transgenic individuals that enable in vitro analysis of molecular mechanisms underlying diseases are required. Recently, several groups have reported on methods, termed direct reprogramming, to generate neuronal cells by defined factors from somatic cells of various kinds of species, including mouse and human. The aim of the present study was to determine whether direct reprogramming technology was applicable to common marmosets.
Common marmoset induced neuronal (cjiN) cells with neuronal morphology were generated from common marmoset embryonic skin fibroblasts (cjF) by overexpressing the neuronal transcription factors: ASCL1, BRN2, MYT1L and NEUROD1. Reverse transcription-polymerase chain reaction of cjiN cells showed upregulation of neuronal genes highly related to neuronal differentiation and function. The presence of neuronal marker proteins was also confirmed by immunocytochemistry. Electrical field stimulation to cjiN cells increased the intracellular calcium level, which was reversibly blocked by the voltage-gated sodium channel blocker, tetrodotoxin, indicating that these cells were functional. The neuronal function of these cells was further confirmed by electrophysiological analyses showing that action potentials could be elicited by membrane depolarization in current-clamp mode while both fast-activating and inactivating sodium currents and outward currents were observed in voltage-clamp mode. The 5-bromodeoxyuridine (BrdU) incorporation assay showed that cjiN cells were directly converted from cjFs without passing a proliferative state.
Functional common marmoset neuronal cells can be obtained directly from embryonic fibroblasts by overexpressing four neuronal transcription factors under in vitro conditions. Overall, direct conversion technology on marmoset somatic cells provides the opportunity to analyze and screen phenotypes of genetically-modified common marmosets.
Common marmoset; Direct reprogramming; Induced neuronal cells; Transcription factor; Regenerative medicine; Disease modeling; Cell-fate plasticity; Transdifferentiation
Different pools and functions have recently been attributed to spontaneous and evoked vesicle release. Despite the well-established function of evoked release, the neuronal information transmission, the origin as well as the function of spontaneously fusing synaptic vesicles have remained elusive. Recently spontaneous release was found to e.g. regulate postsynaptic protein synthesis or has been linked to depressive disorder. Nevertheless the strength and cellular localization of this release form was neglected so far, which are both essential parameters in neuronal information processing.
Here we show that the complete recycling pool can be turned over by spontaneous trafficking and that spontaneous fusion rates critically depend on the neuronal localization of the releasing synapse. Thereby, the distribution equals that of evoked release so that both findings demonstrate a uniform regulation of these fusion modes.
In contrast to recent works, our results strengthen the assumption that identical vesicles are used for evoked and spontaneous release and extended the knowledge about spontaneous fusion with respect to its amount and cellular localization. Therefore our data supported the hypothesis of a regulatory role of spontaneous release in neuronal outgrowth and plasticity as neurites secrete neurotransmitters to initiate process outgrowth of a possible postsynaptic neuron to form a new synaptic connection.