James Dachtler

The role of nitric oxide in pre-synaptic plasticity and homeostasis

Since the observation that nitric oxide (NO) can act as an intercellular messenger in the brain, the past 25 years have witnessed the steady accumulation of evidence that it acts pre-synaptically at both glutamatergic and GABAergic synapses to alter release-probability in synaptic plasticity. NO does so by acting on the synaptic machinery involved in transmitter release and, in a coordinated fashion, on vesicular recycling mechanisms. In this review, we examine the body of evidence for NO acting as a retrograde factor at synapses, and the evidence from in vivo and in vitro studies that specifically establish NOS1 (neuronal nitric oxide synthase) as the important isoform of NO synthase in this process. The NOS1 isoform is found at two very different locations and at two different spatial scales both in the cortex and hippocampus. On the one hand it is located diffusely in the cytoplasm of a small population of GABAergic neurons and on the other hand the alpha isoform is located discretely at the post-synaptic density (PSD) in spines of pyramidal cells. The present evidence is that the number of NOS1 molecules that exist at the PSD are so low that a spine can only give rise to modest concentrations of NO and therefore only exert a very local action. The NO receptor guanylate cyclase is located both pre- and post-synaptically and this suggests a role for NO in the coordination of local pre- and post-synaptic function during plasticity at individual synapses. Recent evidence shows that NOS1 is also located post-synaptic to GABAergic synapses and plays a pre-synaptic role in GABAergic plasticity as well as glutamatergic plasticity. Studies on the function of NO in plasticity at the cellular level are corroborated by evidence that NO is also involved in experience-dependent plasticity in the cerebral cortex.

Sensory Deprivation Unmasks a PKA-Dependent Synaptic Plasticity Mechanism that Operates in Parallel with CaMKII

Calcium/calmodulin kinase II (CaMKII) is required for LTP and experience-dependent potentiation in the barrel cortex. Here, we find that whisker deprivation increases LTP in the layer IV to II/III pathway and that PKA antagonists block the additional LTP. No LTP was seen in undeprived CaMKII-T286A mice, but whisker deprivation again unmasked PKA-sensitive LTP. Infusion of a PKA agonist potentiated EPSPs in deprived wild-types and deprived CaMKII-T286A point mutants but not in undeprived animals of either genotype. The PKA-dependent potentiation mechanism was not present in GluR1 knockouts. Infusion of a PKA antagonist caused depression of EPSPs in undeprived but not deprived cortex. LTD was occluded by whisker deprivation and blocked by PKA manipulation, but not blocked by cannabinoid antagonists. NMDA receptor currents were unaffected by sensory deprivation. These results suggest that sensory deprivation causes synaptic depression by reversing a PKA-dependent process that may act via GluR1.

Deletion of α-neurexin II results in autism-related behaviors in mice

Autism is a common and frequently disabling neurodevelopmental disorder with a strong genetic basis. Human genetic studies have discovered mutations disrupting exons of the NRXN2 gene, which encodes the synaptic adhesion protein α-neurexin II (Nrxn2α), in two unrelated individuals with autism, but a causal link between NRXN2 and the disorder remains unclear. To begin to test the hypothesis that Nrxn2α deficiency contributes to the symptoms of autism, we employed Nrxn2α knockout (KO) mice that genetically model Nrxn2α deficiency in vivo. We report that Nrxn2α KO mice displayed deficits in sociability and social memory when exposed to novel conspecifics. In tests of exploratory activity, Nrxn2α KO mice displayed an anxiety-like phenotype in comparison with wild-type littermates, with thigmotaxis in an open field, less time spent in the open arms of an elevated plus maze, more time spent in the enclosure of an emergence test and less time spent exploring novel objects. However, Nrxn2α KO mice did not exhibit any obvious changes in prepulse inhibition or in passive avoidance learning. Real-time PCR analysis of the frontal cortex and hippocampus revealed significant decreases in the mRNA levels of genes encoding proteins involved in both excitatory and inhibitory transmission. Quantification of protein expression revealed that Munc18-1, encoded by Stxbp1, was significantly decreased in the hippocampus of Nrxn2α KO mice, which is suggestive of deficiencies in presynaptic vesicular release. Our findings demonstrate a causal role for the loss of Nrxn2α in the genesis of autism-related behaviors in mice.

Experience-Dependent Plasticity Acts via GluR1 and a Novel Neuronal Nitric Oxide Synthase-Dependent Synaptic Mechanism in Adult Cortex

Synaptic plasticity directs development of the nervous system and is thought to underlie memory storage in adult animals. A great deal of our current understanding of the role of AMPA receptors in synaptic plasticity comes from studies on developing cortex and cell cultures. In the present study, we instead focus on plasticity in mature neurons in the neocortex of adult animals. We find that the glutamate receptor 1 (GluR1) subunit of the AMPA receptor is involved in experience-dependent plasticity in adult cortex in vivo and that it acts in addition to neuronal nitric oxide synthase (αNOS1), an enzyme that produces the rapid synaptic signaling molecule nitric oxide (NO). Potentiation of the spared whisker response, following single whisker experience, is ∼33% less in GluR1-null mutants than in wild types. We found that the remaining plasticity depended on αNOS1. Potentiation was reduced by >42% in the single αNOS1-null mutants and completely abolished in GluR1/αNOS1 double-knock-out mice. However, potentiation in GluR1/NOS3 double knock-outs occurred at similar levels to that seen in GluR1 single knock-outs. Synaptic plasticity in the layer IV to II/III pathway in vitro mirrored the results in vivo, in that LTP was present in GluR1/NOS3 double-knock-out mice but not in the GluR1/αNOS1 animals. While basal levels of NO in cortical slices depended on both αNOS1 and NOS3, NMDA receptor-dependent NO release only depended on αNOS1 and not on NOS3. These findings demonstrate that αNOS1 acts in concert with GluR1 to produce experience-dependent plasticity in the neocortex.

Heterozygous deletion of α-neurexin I or α-neurexin II results in behaviors relevant to autism and schizophrenia.

The neurexins are a family of presynaptic cell adhesion molecules. Human genetic studies have found heterozygous deletions affecting NRXN1 and NRXN2, encoding α-neurexin I (Nrxn1α) and α-neurexin II (Nrxn2α), in individuals with autism spectrum disorders and schizophrenia. However, the link between α-neurexin deficiency and the manifestation of psychiatric disorders remain unclear. To assess whether the heterozygous loss of neurexins results in behaviors relevant to autism or schizophrenia, we used mice with heterozygous (HET) deletion of Nrxn1α or Nrxn2α. We found that in a test of social approach, Nrxn1α HET mice show no social memory for familiar versus novel conspecifics. In a passive avoidance test, female Nrxn1α HET mice cross to the conditioned chamber sooner than female wild-type and Nrxn2α HET mice. Nrxn2α HET mice also express a lack of long-term object discrimination, indicating a deficit in cognition. The observed Nrxn1α and Nrxn2α genotypic effects were specific, as neither HET deletion had effects on a wide range of other behavioral measures, including several measures of anxiety. Our findings demonstrate that the heterozygous loss of α-neurexin I and α-neurexin II in mice leads to phenotypes relevant to autism and schizophrenia.

The Role of Nitric Oxide Synthase in Cortical Plasticity Is Sex Specific

Nitric oxide synthase-1 (NOS1) is involved in several forms of plasticity including hippocampal-dependent learning and memory, experience-dependent plasticity in the barrel cortex, and long-term potentiation (LTP) in the hippocampus and neocortex. NOS1 also contributes to ischemic damage during stroke and has a stronger deleterious effect in males than females. We therefore investigated whether the role of NOS1 in plasticity might also be sex specific. We tested LTP in the layer IV–II/III pathway between barrel columns and experience-dependent plasticity in the barrel cortex of αNOS1 knock-out mice and their wild-type littermates. We found that LTP was absent in male αNOS1 knock-out mice but not in females and that the residual LTP in females was not NO dependent. We also found that experience-dependent potentiation due to single whisker experience was significantly reduced in male αNOS1 knockouts but was unaffected in females. The αNOS1 knockout had a small effect on the development of the barrels, which were reduced in size by 20% compared with wild types, but this effect was not sex specific. We therefore conclude that neocortical plasticity mechanisms differ between males and females at the synaptic level, either in their basic plasticity induction pathways or in their ability to compensate for loss of αNOS1.

Gender specific requirement of GluR1 receptors in contextual conditioning but not spatial learning.

The GluR1 subunit of the AMPA receptor is required for hippocampal-dependent memory formation, emotional learning and synaptic plasticity. Recent work has shown that GluR1-independent synaptic plasticity is mediated by nitric oxide. Nitric oxide activity is influenced by estrogen. It is unknown whether this gender-dependent effect conveys a gender dimorphic requirement of GluR1 for learning. This hypothesis was tested in two behavioral paradigms. In Experiment 1, the retention of contextual fear conditioning was impaired in male but not female GluR1 knockout mice. In Experiment 2, GluR1 knockout mice made significantly more arm entry errors during acquisition of a radial-arm watermaze task. This deficit was independent of gender. These results indicate that some forms of learning are gender dimorphic in GluR1 knockout mice. The results are discussed with reference to task and gender-specific interactions between GluR1 receptor intracellular signalling pathways.

Characterization of cognitive deficits in mice with an alternating hemiplegia-linked mutation.

Cognitive impairment is a prominent feature in a range of different movement disorders. Children with Alternating Hemiplegia of Childhood are prone to developmental delay, with deficits in cognitive functioning becoming progressively more evident as they grow older. Heterozygous mutations of the ATP1A3 gene, encoding the Na+,K+-ATPase α3 subunit, have been identified as the primary cause of Alternating Hemiplegia. Heterozygous Myshkin mice have an amino acid change (I810N) in Na+,K+-ATPase α3 that is also found in Alternating Hemiplegia. To investigate whether Myshkin mice exhibit learning and memory deficits resembling the cognitive impairments of patients with Alternating Hemiplegia, we subjected them to a range of behavioral tests that interrogate various cognitive domains. Myshkin mice showed impairments in spatial memory, spatial habituation, locomotor habituation, object recognition, social recognition, and trace fear conditioning, as well as in the visible platform version of the Morris water maze. Increasing the duration of training ameliorated the deficit in social recognition but not in spatial habituation. The deficits of Myshkin mice in all of the learning and memory tests used are consistent with the cognitive impairment of the vast majority of AHC patients. These mice could thus help advance our understanding of the underlying neural mechanisms influencing cognitive impairment in patients with ATP1A3-related disorders.

Missense mutation in DISC1 C-terminal coiled-coil has GSK3β signaling and sex-dependent behavioral effects in mice.

Disrupted-in-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and affective disorders. The full-length DISC1 protein consists of an N-terminal ‘head’ domain and a C-terminal tail domain that contains several predicted coiled-coils, structural motifs involved in protein-protein interactions. To probe the in vivo effects of missense mutation of DISC1’s C-terminal tail, we tested mice carrying mutation D453G within a predicted α-helical coiled-coil region. We report that, relative to wild-type littermates, female DISC1D453G mice exhibited novelty-induced hyperlocomotion, an anxiogenic profile in the elevated plus-maze and open field tests, and reduced social exploration of unfamiliar mice. Male DISC1D453G mice displayed a deficit in passive avoidance, while neither males nor females exhibited any impairment in startle reactivity or prepulse inhibition. Whole brain homogenates showed normal levels of DISC1 protein, but decreased binding of DISC1 to GSK3β, decreased phospho-inhibition of GSK3β at serine 9, and decreased levels of β-catenin in DISC1D453G mice of either sex. Interrupted GSK3β signaling may thus be part of the mechanism underlying the behavioral phenotype associated with D453G, in common with the previously described N-terminal domain mutations Q31L and L100P in mice, and the schizophrenia risk-conferring variant R264Q in humans.

Ventricular myocardium development and the role of connexins in the human fetal heart

The developmental timeline of the human heart remains elusive. The heart takes on its characteristic four chambered appearance by ~56 days gestational age (DGA). However, owing to the complexities (both technical and logistical) of exploring development in utero, we understand little of how the ventricular walls develop. To address this, we employed diffusion tensor magnetic resonance imaging to explore the architecture and tissue organization of the developing heart aged 95–143 DGA. We show that fractional anisotropy increases (from ~0.1 to ~0.5), diffusion coefficients decrease (from ~1 × 10−3mm2/sec to ~0.4 × 10−3mm2/sec), and fiber paths, extracted by tractography, increase linearly with gestation, indicative of the increasing organization of the ventricular myocytes. By 143 DGA, the developing heart has the classical helical organization observed in mature mammalian tissue. This was accompanied by an increase in connexin 43 and connexin 40 expression levels, suggesting their role in the development of the ventricular conduction system and that electrical propagation across the heart is facilitated in later gestation. Our findings highlight a key developmental window for the structural organization of the fetal heart.