Systematic Quantification of Synapses in Primary Neuronal Culture.

Most neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset for in vitro synapse screens.

High-throughput microscopy exposes a pharmacological window in which dual leucine zipper kinase inhibition preserves neuronal network connectivity.

Therapeutic developments for neurodegenerative disorders are redirecting their focus to the mechanisms that contribute to neuronal connectivity and the loss thereof. Using a high-throughput microscopy pipeline that integrates morphological and functional measurements, we found that inhibition of dual leucine zipper kinase (DLK) increased neuronal connectivity in primary cortical cultures. This neuroprotective effect was not only observed in basal conditions but also in cultures depleted from antioxidants and in cultures in which microtubule stability was genetically perturbed. Based on the morphofunctional connectivity signature, we further showed that the effects were limited to a specific dose and time range. Thus, our results illustrate that profiling microscopy images with deep coverage enables sensitive interrogation of neuronal connectivity and allows exposing a pharmacological window for targeted treatments. In doing so, we revealed a broad-spectrum neuroprotective effect of DLK inhibition, which may have relevance to pathological conditions that ar.e associated with compromised neuronal connectivity.

Projecting future costs to U.S. electric utility customers from power interruptions.

This analysis integrates regional models of power system reliability, output from atmosphere-ocean general circulation models, and results from the Interruption Cost Estimate (ICE) Calculator to project long-run costs to electric utility customers from power interruptions under different future severe weather and electricity system scenarios. We discuss the challenges when attempting to model long-run costs to utility customers including the use of imperfect metrics to measure severe weather. Despite these challenges, initial findings show that discounted cumulative customer costs, through the middle of the century, could range from $1.5-$3.4 trillion ($2015) without aggressive undergrounding of the power system and increased utility operations and maintenance (O&M) spending and $1.5-$2.5 trillion with aggressive undergrounding and increased spending. By the end of the century, cumulative customer costs could range from $1.9-$5.6 trillion (without aggressive undergrounding and increased spending) and $2.0-$3.6 trillion (with aggressive undergrounding and increased spending). We find that, in some scenarios, aggressive undergrounding of distribution lines and increased O&M spending is not always cost-effective. We conclude by identifying important topics for follow-on research, which have the potential to improve the cost estimates of this model.

The multimodal antidepressant vortioxetine may facilitate pyramidal cell firing by inhibition of 5-HT3 receptor expressing interneurons: An in vitro study in rat hippocampus slices.

The multimodal antidepressant vortioxetine is thought to mediate its pharmacological effects via 5-HT1A receptor agonism, 5-HT1B receptor partial agonism, 5-HT1D, 5-HT3, 5-HT7 receptor antagonism and 5-HT transporter inhibition. Here we studied vortioxetine's functional effects across species (canine, mouse, rat, guinea pig and human) in cellular assays with heterologous expression of 5-HT3A receptors (in Xenopus oocytes and HEK-293 cells) and in mouse neuroblastoma N1E-115 cells with endogenous expression of 5-HT3A receptors. Furthermore, we studied the effects of vortioxetine on activity of CA1 Stratum Radiatum interneurons in rat hippocampus slices using current- and voltage-clamping methods. The patched neurons were subsequently filled with biocytin for confirmation of 5-HT3 receptor mRNA expression by in situ hybridization. Whereas, both vortioxetine and the 5-HT3 receptor antagonist ondansetron potently antagonized 5-HT-induced currents in the cellular assays, vortioxetine had a slower off-rate than ondansetron in oocytes expressing 5-HT3A receptors. Furthermore, vortioxetine's but not ondansetron's 5-HT3 receptor antagonistic potency varied considerably across species. Vortioxetine had the highest potency at rat and the lowest potency at guinea pig 5-HT3A receptors. Finally, in 5-HT3 receptor-expressing GABAergic interneurons from the CA1 stratum radiatum, vortioxetine and ondansetron blocked depolarizations induced by superfusion of either 5-HT or the 5-HT3 receptor agonist mCPBG. Taken together, these data add to a growing literature supporting the idea that vortioxetine may inhibit GABAergic neurotransmission in some brain regions via a 5-HT3 receptor antagonism-dependent mechanism and thereby disinhibit pyramidal neurons and enhance glutamatergic signaling.

A mouse model of the schizophrenia-associated 1q21.1 microdeletion syndrome exhibits altered mesolimbic dopamine transmission.

1q21.1 hemizygous microdeletion is a copy number variant leading to eightfold increased risk of schizophrenia. In order to investigate biological alterations induced by this microdeletion, we generated a novel mouse model (Df(h1q21)/+) and characterized it in a broad test battery focusing on schizophrenia-related assays. Df(h1q21)/+ mice displayed increased hyperactivity in response to amphetamine challenge and increased sensitivity to the disruptive effects of amphetamine and phencyclidine hydrochloride (PCP) on prepulse inhibition. Probing of the direct dopamine (DA) pathway using the DA D1 receptor agonist SKF-81297 revealed no differences in induced locomotor activity compared to wild-type mice, but Df(h1q21)/+ mice showed increased sensitivity to the DA D2 receptor agonist quinpirole and the D1/D2 agonist apomorphine. Electrophysiological characterization of DA neuron firing in the ventral tegmental area revealed more spontaneously active DA neurons and increased firing variability in Df(h1q21)/+ mice, and decreased feedback reduction of DA neuron firing in response to amphetamine. In a range of other assays, Df(h1q21)/+ mice showed no difference from wild-type mice: gross brain morphology and basic functions such as reflexes, ASR, thermal pain sensitivity, and motor performance were unaltered. Similarly, anxiety related measures, baseline prepulse inhibition, and seizure threshold were unaltered. In addition to the central nervous system-related phenotypes, Df(h1q21)/+ mice exhibited reduced head-to tail length, which is reminiscent of the short stature reported in humans with 1q21.1 deletion. With aspects of both construct and face validity, the Df(h1q21)/+ model may be used to gain insight into schizophrenia-relevant alterations in dopaminergic transmission.

Optimized CLARITY technique detects reduced parvalbumin density in a genetic model of schizophrenia.

BACKGROUND: Novel tissue clearing technologies have, for the first time, made it possible to study intact tissue samples. This approach provides a tool for further clarifying findings from animal models of schizophrenia by studying parvalbumin-positive (PV+) interneuron density from a 3D perspective. NEW METHOD: This study has developed an optimised CLARITY protocol, including an improved electrophoretic tissue clearing (ETC) chamber, an evaluation of antibody diffusion into cleared tissue slices, and a computational method for detecting PV+ interneurons in 3D. RESULTS: A reduced PV+ interneuron density was found in both prelimbic and motor cortex regions of the Df(h15q13)/+ mice, while no changes were observed in the Df(h22q11)/+ mice. COMPARISON WITH EXISTING METHOD: The developed ETC chamber enables tissue clearing of variable tissue sizes while minimizing the resistance. It was found that a high concentration of primary and secondary antibodies were necessary for sufficient antibody staining of PV+ interneurons. Additionally, the developed computational method showed improved detection rates of interneurons compared to non-processed image stacks. CONCLUSION: Our optimization of the CLARITY technology and automated 3D counting of cells were found to be useful for quantification of PV+ interneuron density. The results may provide insight into understanding the pathophysiology underlying the phenotype observed in Df(h15q13)/+ mice.

Persistent gating deficit and increased sensitivity to NMDA receptor antagonism after puberty in a new mouse model of the human 22q11.2 microdeletion syndrome: a study in male mice.

BACKGROUND: The hemizygous 22q11.2 microdeletion is a common copy number variant in humans. The deletion confers high risk for neurodevelopmental disorders, including autism and schizophrenia. Up to 41% of deletion carriers experience psychotic symptoms. METHODS: We present a new mouse model (Df(h22q11)/+) of the deletion syndrome (22q11.2DS) and report on, to our knowledge, the most comprehensive study undertaken to date in 22q11.2DS models. The study was conducted in male mice. RESULTS: We found elevated postpubertal N-methyl-D-aspartate (NMDA) receptor antagonist-induced hyperlocomotion, age-independent prepulse inhibition (PPI) deficits and increased acoustic startle response (ASR). The PPI deficit and increased ASR were resistant to antipsychotic treatment. The PPI deficit was not a consequence of impaired hearing measured by auditory brain stem responses. The Df(h22q11)/+ mice also displayed increased amplitude of loudness-dependent auditory evoked potentials. Prefrontal cortex and dorsal striatal elevations of the dopamine metabolite DOPAC and increased dorsal striatal expression of the AMPA receptor subunit GluR1 was found. The Df(h22q11)/+ mice did not deviate from wild-type mice in a wide range of other behavioural and biochemical assays. LIMITATIONS: The 22q11.2 microdeletion has incomplete penetrance in humans, and the severity of disease depends on the complete genetic makeup in concert with environmental factors. In order to obtain more marked phenotypes reflecting the severe conditions related to 22q11.2DS it is suggested to expose the Df(h22q11)/+ mice to environmental stressors that may unmask latent psychopathology. CONCLUSION: The Df(h22q11)/+ model will be a valuable tool for increasing our understanding of the etiology of schizophrenia and other psychiatric disorders associated with the 22q11DS.

A mouse model of the 15q13.3 microdeletion syndrome shows prefrontal neurophysiological dysfunctions and attentional impairment.

RATIONALE: A microdeletion at locus 15q13.3 is associated with high incidence rates of psychopathology, including schizophrenia. A mouse model of the 15q13.3 microdeletion syndrome has been generated (Df[h15q13]/+) with translational utility for modelling schizophrenia-like pathology. Among other deficits, schizophrenia is characterised by dysfunctions in prefrontal cortical (PFC) inhibitory circuitry and attention. OBJECTIVES: The objective of this study is to assess PFC-dependent functioning in the Df(h15q13)/+ mouse using electrophysiological, pharmacological, and behavioural assays. METHOD: Experiments 1-2 investigated baseline firing and auditory-evoked responses of PFC interneurons and pyramidal neurons. Experiment 3 measured pyramidal firing in response to intra-PFC GABAA receptor antagonism. Experiments 4-6 assessed PFC-dependent attentional functioning through the touchscreen 5-choice serial reaction time task (5-CSRTT). Experiments 7-12 assessed reversal learning, paired-associate learning, extinction learning, progressive ratio, trial-unique non-match to sample, and object recognition. RESULTS: In experiments 1-3, the Df(h15q13)/+ mouse showed reduced baseline firing rate of fast-spiking interneurons and in the ability of the GABAA receptor antagonist gabazine to increase the firing rate of pyramidal neurons. In assays of auditory-evoked responses, PFC interneurons in the Df(h15q13)/+ mouse had reduced detection amplitudes and increased detection latencies, while pyramidal neurons showed increased detection latencies. In experiments 4-6, the Df(h15q13)/+ mouse showed a stimulus duration-dependent decrease in percent accuracy in the 5-CSRTT. The impairment was insensitive to treatment with the partial α7nAChR agonist EVP-6124. The Df(h15q13)/+ mouse showed no cognitive impairments in experiments 7-12. CONCLUSION: The Df(h15q13)/+ mouse has multiple dysfunctions converging on disrupted PFC processing as measured by several independent assays of inhibitory transmission and attentional function.

Identification and electrophysiological evaluation of 2-methylbenzamide derivatives as Nav1.1 modulators.

Voltage-gated sodium channels (Nav) are crucial to the initiation and propagation of action potentials (APs) in electrically excitable cells, and during the past decades they have received considerable attention due to their therapeutic potential. Here, we report for the first time the synthesis and the electrophysiological evaluation of 16 ligands based on a 2-methylbenzamide scaffold that have been identified as Nav1.1 modulators. Among these compounds, N,N'-(1,3-phenylene)bis(2-methylbenzamide) (3a) has been selected and evaluated in ex-vivo experiments in order to estimate the activation impact of such a compound profile. It appears that 3a increases the Nav1.1 channel activity although its overall impact remains moderate. Altogether, our preliminary results provide new insights into the development of small molecule activators targeting specifically Nav1.1 channels to design potential drugs for treating CNS diseases.

A mouse model that recapitulates cardinal features of the 15q13.3 microdeletion syndrome including schizophrenia- and epilepsy-related alterations.

BACKGROUND: Genome-wide scans have uncovered rare copy number variants conferring high risk of psychiatric disorders. The 15q13.3 microdeletion is associated with a considerably increased risk of idiopathic generalized epilepsy, intellectual disability, and schizophrenia. METHODS: A 15q13.3 microdeletion mouse model (Df[h15q13]/+) was generated by hemizygous deletion of the orthologous region and characterized with focus on schizophrenia- and epilepsy-relevant parameters. RESULTS: Df(h15q13)/+ mice showed marked changes in neuronal excitability in acute seizure assays, with increased propensity to develop myoclonic and absence-like seizures but decreased propensity for clonic and tonic seizures. Furthermore, they had impaired long-term spatial reference memory and a decreased theta frequency in hippocampus and prefrontal cortex. Electroencephalogram characterization revealed auditory processing deficits similar to those observed in schizophrenia. Gamma band power was increased during active state, but evoked gamma power following auditory stimulus (40 Hz) was dramatically reduced, mirroring observations in patients with schizophrenia. In addition, Df(h15q13)/+ mice showed schizophrenia-like decreases in amplitudes of auditory evoked potentials. Although displaying a grossly normal behavior, Df(h15q13)/+ mice are more aggressive following exposure to mild stressors, similar to what is described in human deletion carriers. Furthermore, Df(h15q13)/+ mice have increased body weight, and a similar increase in body weight was subsequently found in a sample of human subjects with 15q13.3 deletion. CONCLUSIONS: The Df(h15q13)/+ mouse shows similarities to several alterations related to the 15q13.3 microdeletion syndrome, epilepsy, and schizophrenia, offering a novel tool for addressing the underlying biology of these diseases.

Negative modulation of GABAA α5 receptors by RO4938581 attenuates discrete sub-chronic and early postnatal phencyclidine (PCP)-induced cognitive deficits in rats.

RATIONALE: A growing body of evidence suggests that negative modulation of γ-aminobutyric acid (GABA) GABA(A) α5 receptors may be a promising strategy for the treatment of certain facets of cognitive impairment; however, selective modulators of GABA(A) α5 receptors have not yet been tested in "schizophrenia-relevant" cognitive assay/model systems in animals. OBJECTIVES: The objectives of this study were to investigate the potential of RO4938581, a negative modulator of GABA(A) α5 receptors, and to attenuate cognitive impairments induced following sub-chronic (sub-PCP) and early postnatal PCP (neo-PCP) administration in the novel object recognition (NOR) and intra-extradimensional shift (ID/ED) paradigms in rats. Complementary in vitro, ex vivo and in vivo studies were performed to confirm negative modulatory activity of RO4938581 and to investigate animal model validity, concept validity and potential side effect issues, respectively. RESULTS: In vitro studies confirmed the reported negative modulatory activity of RO4938581, whilst immunohistochemical analyses revealed significantly reduced parvalbumin-positive cells in the prefrontal cortex of sub-PCP- and neo-PCP-treated rats. RO4938581 (1 mg/kg) ameliorated both sub-PCP- and neo-PCP-induced cognitive deficits in NOR and ID/ED performance, respectively. In contrast, QH-II-066 (1 and 3 mg/kg), a GABA(A) α5 receptor positive modulator, impaired cognitive performance in the NOR task when administered to vehicle-treated animals. Additional studies revealed that both RO4938581 (1 mg/kg) and QH-II-066 (1 and 3 mg/kg) attenuated amphetamine-induced hyperactivity in rats. CONCLUSIONS: Taken together, these novel findings suggest that negative modulation of GABA(A) α5 receptors may represent an attractive treatment option for the cognitive impairments, and potentially positive symptoms, associated with schizophrenia.

Toll-like receptors in brain development and homeostasis.

Toll-like receptors (TLRs) are best known as initiators of the innate immune response to pathogens. Recent reports now reveal intriguing roles for TLRs in the central nervous system (CNS). These include the regulation of neuroinflammation and of neurite outgrowth. The archetypal Toll protein in Drosophila melanogaster was implicated in the development of the nervous system. Now similar functions have been uncovered for the mammalian orthologs, the TLRs. TLRs expressed on CNS glia and neurons may recognize endogenous ligands and participate both in development and in responses associated with CNS injury.

Myelin formation during development of the CNS is delayed in matrix metalloproteinase-9 and -12 null mice.

The matrix metalloproteinases (MMPs) are implicated in several activities within the nervous system. Although many functions of abnormally elevated MMPs are undesirable, the discrete expression of particular MMP members can have beneficial roles. We previously found that MMP-9 expressed locally around a demyelinating lesion of the spinal cord of adult mice facilitated remyelination. In the current study, we have addressed whether and how MMPs might be required for myelin formation in normal ontogeny. Using a probe for multiple MMPs and the developing mouse optic nerve, we found two members, MMP-9 and -12, to be upregulated during the period of myelin formation. These MMPs partake in myelinogenesis because myelination in the corpus callosum of MMP-9 and/or MMP-12 null mice was deficient from postnatal days 7 to 14 compared with that of wild-type mice. The deficient myelination was correlated with fewer mature oligodendrocytes, but similar precursor cell numbers, in MMP null animals compared with wild type. Because an important growth factor for oligodendrocyte maturation is insulin-like growth factor-1 (IGF-1), we addressed whether this was involved in the deficient myelination in MMP null mice. Indeed, the addition of IGF-1 normalized the lack of maturation of oligodendrocytes that occurred in cultures from MMP-12 null mice. Furthermore, we determined that IGF binding protein 6 (IGFBP-6), which sequesters IGF-1, was a substrate for MMP processing. Finally, we found IGFBP-6 levels to remain high in MMP-deficient mice. These results reveal a novel function for MMP-9 and -12 in developmental myelination likely through regulating IGF-1 bioavailability.

Matrix metalloproteinase (MMP)-12 expression has a negative impact on sensorimotor function following intracerebral haemorrhage in mice.

We investigated the role of matrix metalloproteinases (MMPs) in a mouse model of intracerebral haemorrhage (ICH). Transcripts encoding nine of the 23 known mammalian MMPs were measured. MMP-12 levels were the most elevated. To evaluate the role of MMP-12 in ICH, haemorrhages were induced in wild-type (WT) and MMP-12 null mice. The results show that MMP-12 null mice exhibited significant functional recovery of forelimb reaching and reduced dependence on the ipsilateral forelimb compared to WT mice. There was also a trend for improved sensory function in the tape removal test. With respect to single pellet skilled reaching, MMP-12 null mice recovered to a level that was not significantly different from sham at 14 and 28 days post-ICH. In contrast, WT animals demonstrated a persistent impairment relative to sham controls throughout the survival period (P < 0.05). The cylinder task revealed a lesion-induced reliance on the ipsilateral forelimb that was apparent at day 7 in both MMP-12 null and WT mice (P < 0.05), but only persisted in WT mice at 14 days post-ICH (P < 0.05). Differences in functional outcome could not be explained by tissue sparing. However, Iba1 immunostaining indicated that more cells bearing macrophage morphology were recruited to the lesion area in WT mice. This is the first study to profile the expression patterns of a number of the known MMPs following ICH in mice. The data indicate that MMP-12 expression following haemorrhagic stroke is deleterious and contributes to the development of secondary injury in this disease.

The expression of matrix metalloproteinase-12 by oligodendrocytes regulates their maturation and morphological differentiation.

Matrix metalloproteinases (MMPs) have important roles in many processes of the developing CNS requiring proteolytic activity such as the migration of neuronal precursors, axonal outgrowth, and vascularization. Another developmental event involving proteolysis is myelin formation, whereby the extensive processes elaborated from oligodendrocytes (OLs) enwrap axons. Here we find MMP-12 transcripts to be produced by OLs in much higher levels than other MMP members examined. MMP-12 activity correlated with the ability of OLs to extend processes in vitro, suggesting a role for MMP-12 in the morphological differentiation of OLs. This was corroborated by results that OL lineage cells from MMP-12 null mice were retarded in their ability to differentiate morphologically and that this deficiency was overcome by the exogenous addition of active MMP-12. Finally, the maturation of oligodendrocyte precursor cells (OPCs) to OLs was significantly reduced in cultures from MMP-12 null mice compared with wild-type controls. We conclude that OL lineage cells express MMP-12 during their maturation and that MMP-12 activity has functional involvement both in maturation of OPCs and in the ability of OPCs and OLs to extend processes.

Matrix metalloproteinase-9 facilitates remyelination in part by processing the inhibitory NG2 proteoglycan.

Remyelination is a critical repair process that is initiated after a demyelinating insult. The failure to remyelinate contributes to neurological diseases such as multiple sclerosis. Here, we test the hypothesis that proteinase activity is required for the extensive remodeling of the extracellular matrix that occurs during remyelination. We show that mice lacking matrix metalloproteinase (MMP)-9 are impaired in myelin reformation after lysolecithin-induced demyelination. This deficiency may be explained at least in part by the failure to clear the accumulation of NG2, an inhibitory proteoglycan that retards the maturation and differentiation of oligodendrocytes that are needed for remyelination. These results emphasize for the first time that upregulation of MMP activity can be important for facilitating regeneration from some types of CNS injury.

Intracerebral hemorrhage induces macrophage activation and matrix metalloproteinases.

Intracerebral hemorrhage (ICH) is characterized by parenchymal hematoma formation with surrounding inflammation. Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of neurological diseases defined by inflammation and cell death. To investigate the expression profile and pathogenic aspects of MMPs in ICH, we examined MMP expression in vivo using a collagenase-induced rat model of ICH. ICH increased brain MMP-2, -3, -7, and -9 mRNA levels relative to sham-injected (control) animals in the vicinity of the hematoma, but MMP-12 (macrophage metalloelastase) was the most highly induced MMP (>80-fold). Immunohistochemistry showed MMP-12 to be localized in activated monocytoid cells surrounding the hematoma. In vitro studies showed that thrombin, released during ICH, induced MMP-12 expression in monocytoid cells, which was reduced by minocycline application. Similarly, in vivo minocycline treatment significantly reduced MMP-12 levels in brain. Neuropathological studies disclosed marked glial activation and apoptosis after ICH that was reduced by minocycline treatment. Neurobehavioral outcomes also were improved with minocycline treatment compared with untreated ICH controls. Thus, select MMPs exhibit increased expression after ICH, whereas minocycline is neuroprotective after ICH by suppressing monocytoid cell activation and downregulating MMP-12 expression.

CXCR4 is a major chemokine receptor on glioma cells and mediates their survival.

Chemokines were described originally in the context of providing migrational cues for leukocytes. They are now known to have broader activities, including those that favor tumor growth. We addressed whether and which chemokines may be important promoters of the growth of the incurable brain neoplasm, malignant gliomas. Analyses of 16 human glioma lines for the expression of chemokine receptors belonging to the CXCR and CCR series revealed low to negligible levels of all receptors, with the exception of CXCR4 that was expressed by 13 of 16 lines. All six resected human glioma specimens showed similarly high CXCR4 expression. The CXCR4 on glioma lines is a signaling receptor in that its agonist, stromal cell-derived factor-1 (SDF-1; CXCL12), produced rapid phosphorylation of mitogen-activated protein kinases. Furthermore, SDF-1 induced the phosphorylation of Akt (protein kinase B), a kinase associated with survival, and prevented the apoptosis of glioma cells when serum was withdrawn from the culture medium. SDF-1 also mediated glioma chemotaxis, in accordance with this better known role of chemokines. We conclude that glioma cells express a predominant chemokine receptor, CXCR4, and that this functions to regulate survival in part through activating pathways such as Akt.