Prenatal and postnatal methamphetamine exposure alters prefrontal cortical gene expression and behavior in mice.

Methamphetamine is a highly abused psychostimulant that substantially impacts public health. Prenatal and postnatal methamphetamine exposure alters gene expression, brain development, and behavior in the offspring, although the underlying mechanisms are not fully defined. To assess these adverse outcomes in the offspring, we employed a mouse model of prenatal and postnatal methamphetamine exposure. Juvenile offspring were behaviorally assessed on the open field, novel object recognition, Y-maze, and forced swim tests. In addition, RNA sequencing was used to explore potential alterations in prefrontal cortical gene expression. We found that methamphetamine-exposed mice exhibited decreased locomotor activity and impaired cognitive performance. In addition, differential expression of genes involved in neurotransmission, synaptic plasticity, and neuroinflammation were found with notable changes in dopaminergic signaling pathways. These data suggest potential neural and molecular mechanisms underlying methamphetamine-exposed behavioral changes. The altered expression of genes involved in dopaminergic signaling and synaptic plasticity highlights potential targets for therapeutic interventions for substance abuse disorders and related psychiatric complications.

Fast resupply of synaptic vesicles requires synaptotagmin-3.

Sustained neuronal activity demands a rapid resupply of synaptic vesicles to maintain reliable synaptic transmission. Such vesicle replenishment is accelerated by submicromolar presynaptic Ca2+ signals by an as-yet unidentified high-affinity Ca2+ sensor1,2. Here we identify synaptotagmin-3 (SYT3)3,4 as that presynaptic high-affinity Ca2+ sensor, which drives vesicle replenishment and short-term synaptic plasticity. Synapses in Syt3 knockout mice exhibited enhanced short-term depression, and recovery from depression was slower and insensitive to presynaptic residual Ca2+. During sustained neuronal firing, SYT3 accelerated vesicle replenishment and increased the size of the readily releasable pool. SYT3 also mediated short-term facilitation under conditions of low release probability and promoted synaptic enhancement together with another high-affinity synaptotagmin, SYT7 (ref. 5). Biophysical modelling predicted that SYT3 mediates both replenishment and facilitation by promoting the transition of loosely docked vesicles to tightly docked, primed states. Our results reveal a crucial role for presynaptic SYT3 in the maintenance of reliable high-frequency synaptic transmission. Moreover, multiple forms of short-term plasticity may converge on a mechanism of reversible, Ca2+-dependent vesicle docking.

CA1 Spike Timing is Impaired in the 129S Inbred Strain During Cognitive Tasks.

A spontaneous mutation of the disrupted in schizophrenia 1 (Disc1) gene is carried by the 129S inbred mouse strain. Truncated DISC1 protein in 129S mouse synapses impairs the scaffolding of excitatory postsynaptic receptors and leads to progressive spine dysgenesis. In contrast, C57BL/6 inbred mice carry the wild-type Disc1 gene and exhibit more typical cognitive performance in spatial exploration and executive behavioral tests. Because of the innate Disc1 mutation, adult 129S inbred mice exhibit the behavioral phenotypes of outbred B6 Disc1 knockdown (Disc1-/-) or Disc1-L-100P mutant strains. Recent studies in Disc1-/- and L-100P mice have shown that impaired excitation-driven interneuron activity and low hippocampal theta power underlie the behavioral phenotypes that resemble human depression and schizophrenia. The current study compared the firing rate and connectivity profile of putative neurons in the CA1 of freely behaving inbred 129S and B6 mice, which have mutant and wild-type Disc1 genes, respectively. In cognitive behavioral tests, 129S mice had lower exploration scores than B6 mice. Furthermore, the mean firing rate for 129S putative pyramidal (pyr) cells and interneurons (int) was significantly lower than that for B6 CA1 neurons sampled during similar tasks. Analysis of pyr/int connectivity revealed a significant delay in synaptic transmission for 129S putative pairs. Sampled 129S pyr/int pairs also had lower detectability index scores than B6 putative pairs. Therefore, the spontaneous Disc1 mutation in the 129S strain attenuates the firing of putative pyr CA1 neurons and impairs spike timing fidelity during cognitive tasks.

Perineuronal Nets in the Prefrontal Cortex of a Schizophrenia Mouse Model: Assessment of Neuroanatomical, Electrophysiological, and Behavioral Contributions.

Schizophrenia is a neurodevelopmental disorder whose etiopathogenesis includes changes in cellular as well as extracellular structures. Perineuronal nets (PNNs) associated with parvalbumin-positive interneurons (PVs) in the prefrontal cortex (PFC) are dysregulated in schizophrenia. However, the postnatal development of these structures along with their associated neurons in the PFC is unexplored, as is their effects on behavior and neural activity. Therefore, in this study, we employed a DISC1 (Disruption in Schizophrenia) mutation mouse model of schizophrenia to assess these developmental changes and tested whether enzymatic digestion of PNNs in the PFC affected schizophrenia-like behaviors and neural activity. Developmentally, we found that the normal formation of PNNs, PVs, and colocalization of these two in the PFC, peaked around PND 22 (postnatal day 22). However, in DISC1, mutation animals from PND 0 to PND 60, both PNNs and PVs were significantly reduced. After enzymatic digestion of PNNs with chondroitinase in adult animals, the behavioral pattern of control animals mimicked that of DISC1 mutation animals, exhibiting reduced sociability, novelty and increased ultrasonic vocalizations, while there was very little change in other behaviors, such as working memory (Y-maze task involving medial temporal lobe) or depression-like behavior (tail-suspension test involving processing via the hypothalamic pituitary adrenal (HPA) axis). Moreover, following chondroitinase treatment, electrophysiological recordings from the PFC exhibited a reduced proportion of spontaneous, high-frequency firing neurons, and an increased proportion of irregularly firing neurons, with increased spike count and reduced inter-spike intervals in control animals. These results support the proposition that the aberrant development of PNNs and PVs affects normal neural operations in the PFC and contributes to the emergence of some of the behavioral phenotypes observed in the DISC1 mutation model of schizophrenia.

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus.

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

Distribution of VTA Glutamate and Dopamine Terminals, and their Significance in CA1 Neural Network Activity.

Reciprocal connection between the ventral tegmental area (VTA) and the hippocampus forms a loop that controls information entry into long-term memory. Compared with the widely studied VTA dopamine system, VTA glutamate terminals are anatomically dominant in the hippocampus and less understood. The current study employs anterograde and retrograde labeling of VTA dopamine and glutamate neurons to map the distribution of their terminals within the layers of the hippocampus. Also, functional tracing of VTA dopamine and glutamate projections to the hippocampus was performed by photostimulation of VTA cell bodies during CA1 extracellular voltage sampling in vivo. VTA dopamine terminals predominantly innervate the CA1 basal dendrite layer and modulate the firing rate of active putative neurons. In contrast, anatomical dominance of VTA glutamate terminals in the CA1 pyramidal cell and apical dendrite layers suggests the possible involvement of these terminals in excitability regulation. In support of these outcomes, photostimulation of VTA dopamine neurons increased the firing rate but not intrinsic excitability parameters for putative pyramidal units. Conversely, activation of VTA glutamate neurons increased CA1 network firing rate and burst rate. In addition, VTA glutamate inputs reduced the interspike and interburst intervals for putative CA1 neurons. Taken together, we deduced that layer-specific distribution of presynaptic dopamine and glutamate terminals in the hippocampus determinines VTA modulation (dopamine) or regulation (glutamate) of excitability in the CA1 neural network.

Positive Modulation of SK Channel Impedes Neuron-Specific Cytoskeletal Organization and Maturation.

N-methyl-D-aspartate receptor (NMDAR) modulates the structural plasticity of dendritic spines by impacting cytoskeletal organization and kinase signaling. In the developing nervous system, activation of NMDAR is pertinent for neuronal migration, neurite differentiation, and cellular organization. Given that small conductance potassium channels (SK2/3) repress NMDAR ionotropic signaling, this study highlights the impact of neonatal SK channel potentiation on adult cortical and hippocampal organization. Neonatal SK channel potentiation was performed by one injection of SK2/3 agonist (CyPPA) into the pallium of mice on postnatal day 2 (P2). When the animals reached adulthood (P55), the hippocampus and cortex were examined to assess neuronal maturation, lamination, and the distribution of synaptic cytoskeletal proteins. Immunodetection of neuronal markers in the brain of P2-treated P55 mice revealed the presence of immature neurons in the upper cortical layers (layers II-IV) and CA1 (hippocampus). Also, layer-dependent cortical-cell density was attenuated due to the ectopic localization of mature (NeuN+) and immature (Doublecortin+ [DCX+]) neurons in cortical layers II-IV. Similarly, the decreased count of NeuN+ neurons in the CA1 is accompanied by an increase in the number of immature DCX+ neurons. Ectopic localization of neurons in the upper cortex and CA1 caused the dramatic expression of neuron-specific cytoskeletal proteins. In line with this, structural deformity of neuronal projections and the loss of postsynaptic densities suggests that postsynaptic integrity is compromised in the SK2/3+ brain. From these results, we deduced that SK channel activity in the developing brain likely impacts neuronal maturation through its effects on cytoskeletal formation.

Disc1 Carrier Mice Exhibit Alterations in Neural pIGF-1Rß and Related Kinase Expression.

Mutation of the disc1 gene underlies a broad range of developmental neuropsychiatric defects, including schizophrenia, depression, and bipolar disorder. The pathophysiological phenotypes linked with disc1 mutation are due to the truncation of the DISC1 primary protein structure. This leads to a defective post-synaptic scaffolding and kinase-GSK3ß and Erk1/2-signaling. As a result, synaptic function and maintenance are significantly impaired in the disc1 mutant brain. Among several other pathways, GSK3ß and Erk1/2 are involved in insulin-like growth factor 1 receptor (IGF-1Rß) kinase signaling. Although disc1 mutation alters these kinases, it is unclear if the mutation impacts IGF-1R expression and activity in the brain. Here, we demonstrate that the expression of active IGF-1Rß (pIGF-1Rß) is altered in the hippocampus and prefrontal cortex (PFC) of disc1 mutant mice and vary with the dose of the mutation (homozygous and heterozygous). The expression of pIGF-1Rß decreased significantly in 129S (hom, disc1 -/-) brains. In contrast, 129S:B6 (het, disc1 +/-) brains were characterized by an increase in pIGF-1Rß when compared with the C57BL/6 (disc1 +/+) level. The decrease in pIGF-1Rß level for the 129S brains was accompanied by the loss of Akt activity (S473 pAkt) and decreased Ser9 phosphorylation of GSK3ß (increased basal GSK3ß). Additionally, hippocampal and cortical pErk1/2 activity increased in the 129S hippocampus and cortex. Although 129S:B6 recorded alterations in pIGF-1Rß-pAkt-GSK3ß (like 129S), there was no observable change in pErk1/2 activity for the heterozygote (disc1 +/-) mutant. In addition to GSK3ß inhibition, we conclude that pIGF-1R, pAkt, and pErk1/2 are potential targets in disc1 -/- mutant brain. On the other hand, pIGF-1R and pAkt can be further explored in disc1 +/- brain.

SK Channel Modulates Synaptic Plasticity by Tuning CaMKIIα/ß Dynamics.

N-Methyl-D-Aspartate Receptor 1 (NMDAR)-linked Ca++ current represents a significant percentage of post-synaptic transient that modulates synaptic strength and is pertinent to dendritic spine plasticity. In the hippocampus, Ca++ transient produced by glutamatergic ionotropic neurotransmission facilitates Ca++-Calmodulin-dependent kinase 2 (CaMKII) Thr286 phosphorylation and promote long-term potentiation (LTP) expression. At CA1 post-synaptic densities, Ca++ transients equally activate small conductance (SK2) channel which regulates excitability by suppressing Ca++ movement. Here, we demonstrate that upstream attenuation of GluN1 function in the hippocampus led to a decrease in Thr286 CaMKIIα phosphorylation, and increased SK2 expression. Consistent with the loss of GluN1 function, potentiation of SK channel in wild type hippocampus reduced CaMKIIα expression and abrogate synaptic localization of T286 pCaMKIIα. Our results demonstrate that positive modulation of SK channel at hippocampal synapses likely refine GluN1-linked plasticity by tuning dendritic localization of CaMKIIα.

RARß2-dependent signaling represses neuronal differentiation in mouse ES cells.

Embryonic Stem (ES) cells are pluripotent cells that can be induced to differentiate into cells of all three lineages: mesoderm, endoderm, and ectoderm. In culture, ES cells can be differentiated into mature neurons by treatment with Retinoic Acid (RA) and this effect is mediated mainly through the activation of the RA nuclear receptors (RAR α, ß, and γ), and their isoforms. However, little is known about the role played by specific RAR types on ES cell differentiation. Here, we found that treatment of ES cells with AC55649, an RARß2 agonist, increased endodermal marker gene expression. On the other hand, we found that the inhibition of RARß with 5µM LE135, together with RA treatment, increased the efficiency of mouse ES cell differentiation into neurons by more than 4-fold as compared to cells treated with RA only. Finally, we performed proteomic analyses on ES cells treated with RA vs RA plus AC55649 in order to identify the signaling pathways activated by the RARß agonist. Our proteomic analyses using antibody microarrays indicated that proteins such as p38 and AKT were upregulated in cells treated with RA plus the agonist, as compared to cells treated with RA alone. Our results indicate that RARß may function as a repressor of neuronal differentiation through the activation of major cell signaling pathways, and that the pharmacological inhibition of this nuclear receptor may constitute a novel method to increase the efficiency of ES to neuronal differentiation in culture.