Electrophysiology and 3D-imaging reveal properties of human intracardiac neurons and increased excitability with atrial fibrillation.

Altered autonomic input to the heart plays a major role in atrial fibrillation (AF). Autonomic neurons termed ganglionated plexi (GP) are clustered on the heart surface to provide the last point of neural control of cardiac function. To date the properties of GP neurons in humans are unknown. Here we have addressed this knowledge gap in human GP neuron structure and physiology in patients with and without AF. Human right atrial GP neurons embedded in epicardial adipose tissue were excised during open heart surgery performed on both non-AF and AF patients and then characterised physiologically by whole cell patch clamp techniques. Structural analysis was also performed after fixation at both the single cell and at the entire GP levels via three-dimensional confocal imaging. Human GP neurons were found to exhibit unique properties and structural complexity with branched neurite outgrowth. Significant differences in excitability were revealed between AF and non-AF GP neurons as measured by lower current to induce action potential firing, a reduced occurrence of low action potential firing rates, decreased accommodation and increased synaptic density. Visualisation of entire GPs showed almost all neurons are cholinergic with a small proportion of noradrenergic and dual phenotype neurons. Phenotypic distribution differences occurred with AF including decreased cholinergic and dual phenotype neurons, and increased noradrenergic neurons. These data show both functional and structural differences occur between GP neurons from patients with and without AF, highlighting that cellular plasticity occurs in neural input to the heart that could alter autonomic influence on atrial function. KEY POINTS: The autonomic nervous system plays a critical role in regulating heart rhythm and the initiation of AF; however, the structural and functional properties of human autonomic neurons in the autonomic ganglionated plexi (GP) remain unknown. Here we perform the first whole cell patch clamp electrophysiological and large tissue confocal imaging analysis of these neurons from patients with and without AF. Our data show human GP neurons are functionally and structurally complex. Measurements of action potential kinetics show higher excitability in GP neurons from AF patients as measured by lower current to induce action potential firing, reduced low firing action potential rates, and decreased action potential accommodation. Confocal imaging shows increased synaptic density and noradrenergic phenotypes in patients with AF. Both functional and structural differences occur in GP neurons from patients with AF that could alter autonomic influence on atrial rhythm.

Molecular and cellular neurocardiology in heart disease.

This paper updates and builds on a previous White Paper in this journal that some of us contributed to concerning the molecular and cellular basis of cardiac neurobiology of heart disease. Here we focus on recent findings that underpin cardiac autonomic development, novel intracellular pathways and neuroplasticity. Throughout we highlight unanswered questions and areas of controversy. Whilst some neurochemical pathways are already demonstrating prognostic viability in patients with heart failure, we also discuss the opportunity to better understand sympathetic impairment by using patient specific stem cells that provides pathophysiological contextualization to study 'disease in a dish'. Novel imaging techniques and spatial transcriptomics are also facilitating a road map for target discovery of molecular pathways that may form a therapeutic opportunity to treat cardiac dysautonomia.

Multimodal imaging shows fibrosis architecture and action potential dispersion are predictors of arrhythmic risk in spontaneous hypertensive rats.

Hypertensive heart disease (HHD) increases risk of ventricular tachycardia (VT) and ventricular fibrillation (VF). The roles of structural vs. electrophysiological remodelling and age vs. disease progression are not fully understood. This cross-sectional study of cardiac alterations through HHD investigates mechanistic contributions to VT/VF risk. Risk was electrically assessed in Langendorff-perfused, spontaneously hypertensive rat hearts at 6, 12 and 18 months, and paced optical membrane voltage maps were acquired from the left ventricular (LV) free wall epicardium. Distributions of LV patchy fibrosis and 3D cellular architecture in representative anterior LV mid-wall regions were quantified from macroscopic and microscopic fluorescence images of optically cleared tissue. Imaging showed increased fibrosis from 6 months, particularly in the inner LV free wall. Myocyte cross-section increased at 12 months, while inter-myocyte connections reduced markedly with fibrosis. Conduction velocity decreased from 12 months, especially transverse to the myofibre direction, with rate-dependent anisotropy at 12 and 18 months, but not earlier. Action potential duration (APD) increased when clustered by age, as did APD dispersion at 12 and 18 months. Among 10 structural, functional and age variables, the most reliably linked were VT/VF risk, general LV fibrosis, a measure quantifying patchy fibrosis, and non-age clustered APD dispersion. VT/VF risk related to a quantified measure of patchy fibrosis, but age did not factor strongly. The findings are consistent with the notion that VT/VF risk is associated with rate-dependent repolarization heterogeneity caused by structural remodelling and reduced lateral electrical coupling between LV myocytes, providing a substrate for heterogeneous intramural activation as HHD progresses. KEY POINTS: There is heightened arrhythmic risk with progression of hypertensive heart disease. Risk is related to increasing left ventricular fibrosis, but the nature of this relationship has not been quantified. This study is a novel systematic characterization of changes in active electrical properties and fibrotic remodelling during progression of hypertensive heart disease in a well-established animal disease model. Arrhythmic risk is predicted by several left ventricular measures, in particular fibrosis quantity and structure, and epicardial action potential duration dispersion. Age alone is not a good predictor of risk. An improved understanding of links between arrhythmic risk and fibrotic architectures in progressive hypertensive heart disease aids better interpretation of late gadolinium-enhanced cardiac magnetic resonance imaging and electrical mapping signals.

The role of the tripartite synapse in the heart: how glial cells may contribute to the physiology and pathophysiology of the intracardiac nervous system.

One of the major roles of the intracardiac nervous system (ICNS) is to act as the final site of signal integration for efferent information destined for the myocardium to enable local control of heart rate and rhythm. Multiple subtypes of neurons exist in the ICNS where they are organized into clusters termed ganglionated plexi (GP). The majority of cells in the ICNS are actually glial cells; however, despite this, ICNS glial cells have received little attention to date. In the central nervous system, where glial cell function has been widely studied, glia are no longer viewed simply as supportive cells but rather have been shown to play an active role in modulating neuronal excitability and synaptic plasticity. Pioneering studies have demonstrated that in addition to glia within the brain stem, glial cells within multiple autonomic ganglia in the peripheral nervous system, including the ICNS, can also act to modulate cardiovascular function. Clinically, patients with atrial fibrillation (AF) undergoing catheter ablation show high plasma levels of S100B, a protein produced by cardiac glial cells, correlated with decreased AF recurrence. Interestingly, S100B also alters GP neuron excitability and neurite outgrowth in the ICNS. These studies highlight the importance of understanding how glial cells can affect the heart by modulating GP neuron activity or synaptic inputs. Here, we review studies investigating glia both in the central and peripheral nervous systems to discuss the potential role of glia in controlling cardiac function in health and disease, paying particular attention to the glial cells of the ICNS.

Functional hyperactivity in long QT syndrome type 1 pluripotent stem cell-derived sympathetic neurons.

Sympathetic activation is an established trigger of life-threatening cardiac events in long QT syndrome type 1 (LQT1). KCNQ1 loss-of-function variants, which underlie LQT1, have been associated with both cardiac arrhythmia and neuronal hyperactivity pathologies. However, the LQT1 sympathetic neuronal phenotype is unknown. Here, we aimed to study human induced pluripotent stem cell (hiPSC)-derived sympathetic neurons (SNs) to evaluate neuronal functional phenotype in LQT1. We generated hiPSC-SNs from two patients with LQT1 with a history of sympathetically triggered arrhythmia and KCNQ1 loss-of-function genotypes (c.781_782delinsTC and p.S349W/p.R518X). Characterization of hiPSC-SNs was performed using immunohistochemistry, enzyme-linked immunosorbent assay, and whole cell patch clamp electrophysiology, and functional LQT1 hiPSC-SN phenotypes compared with healthy control (WT) hiPSC-SNs. hiPSC-SNs stained positive for tyrosine hydroxylase, peripherin, KCNQ1, and secreted norepinephrine. hiPSC-SNs at 60 ± 2.2 days in vitro had healthy resting membrane potentials (-60 ± 1.3 mV), and fired rapid action potentials with mature kinetics in response to stimulation. Significant hyperactivity in LQT1 hiPSC-SNs was evident via increased norepinephrine release, increased spontaneous action potential frequency, increased total inward current density, and reduced afterhyperpolarization, compared with age-matched WT hiPSC-SNs. A significantly higher action potential frequency upon current injection and larger synaptic current amplitudes in compound heterozygous p.S349W/p.R518X hiPSC-SNs compared with heterozygous c.781_782delinsTC hiPSC-SNs was also observed, suggesting a potential genotype-phenotype correlation. Together, our data reveal increased neurotransmission and excitability in heterozygous and compound heterozygous patient-derived LQT1 sympathetic neurons, suggesting that the cellular arrhythmogenic potential in LQT1 is not restricted to cardiomyocytes.NEW & NOTEWORTHY Here, we present the first study of patient-derived LQT1 sympathetic neurons that are norepinephrine secreting, and electrophysiologically functional, in vitro. Our data reveal a novel LQT1 sympathetic neuronal phenotype of increased neurotransmission and excitability. The identified sympathetic neuronal hyperactivity phenotype is of particular relevance as it could contribute to the mechanisms underlying sympathetically triggered arrhythmia in LQT1.

The cellular and molecular basis of in vivo synaptic plasticity in rodents.

Plasticity within the neuronal networks of the brain underlies the ability to learn and retain new information. The initial discovery of synaptic plasticity occurred by measuring synaptic strength in vivo, applying external stimulation and observing an increase in synaptic strength termed long-term potentiation (LTP). Many of the molecular pathways involved in LTP and other forms of synaptic plasticity were subsequently uncovered in vitro. Over the last few decades, technological advances in recording and imaging in live animals have seen many of these molecular mechanisms confirmed in vivo, including structural changes both pre- and postsynaptically, changes in synaptic strength, and changes in neuronal excitability. A well-studied aspect of neuronal plasticity is the capacity of the brain to adapt to its environment, gained by comparing the brains of deprived and experienced animals in vivo, and in direct response to sensory stimuli. Multiple in vivo studies have also strongly linked plastic changes to memory by interfering with the expression of plasticity and by manipulating memory engrams. Plasticity in vivo also occurs in the absence of any form of external stimulation, i.e., during spontaneous network activity occurring with brain development. However, there is still much to learn about how plasticity is induced during natural learning and how this is altered in neurological disorders.

Human in vitro systems for examining synaptic function and plasticity in the brain.

The human brain shows remarkable complexity in its cellular makeup and function, which are distinct from nonhuman species, signifying the need for human-based research platforms for the study of human cellular neurophysiology and neuropathology. However, the use of adult human brain tissue for research purposes is hampered by technical, methodological, and accessibility challenges. One of the major problems is the limited number of in vitro systems that, in contrast, are readily available from rodent brain tissue. With recent advances in the optimization of protocols for adult human brain preparations, there is a significant opportunity for neuroscientists to validate their findings in human-based systems. This review addresses the methodological aspects, advantages, and disadvantages of human neuron in vitro systems, focusing on the unique properties of human neurons and synapses in neocortical microcircuits. These in vitro models provide the incomparable advantage of being a direct representation of the neurons that have formed part of the human brain until the point of recording, which cannot be replicated by animal models nor human stem-cell systems. Important distinct cellular mechanisms are observed in human neurons that may underlie the higher order cognitive abilities of the human brain. The use of human brain tissue in neuroscience research also raises important ethical, diversity, and control tissue limitations that need to be considered. Undoubtedly however, these human neuron systems provide critical information to increase the potential of translation of treatments from the laboratory to the clinic in a way animal models are failing to provide.

Functional coculture of sympathetic neurons and cardiomyocytes derived from human-induced pluripotent stem cells.

Sympathetic neurons (SNs) capable of modulating the heart rate of murine cardiomyocytes (CMs) can be differentiated from human stem cells. The electrophysiological properties of human stem cell-derived SNs remain largely uncharacterized, and human neurocardiac cocultures remain to be established. Here, we have adapted previously published differentiation and coculture protocols to develop feeder-free SNs using human-induced pluripotent stem cells (hiPSCs). hiPSC-SNs were characterized in monoculture and coculture with hiPSC-CMs, using antibody labeling, enzyme-linked immunosorbent assay, and whole cell patch-clamp electrophysiology techniques. hiPSC-SNs stained positive for peripherin, tyrosine hydroxylase, and nicotinic acetylcholine receptors, the latter two colocalizing in somas and synaptic varicosities. hiPSC-SNs functionally matured in vitro and exhibited healthy resting membrane potentials (average = -61 ± 0.7 mV), secreted norepinephrine upon activation, and generated synaptic and action currents and inward and outward voltage-dependent currents. All hiPSC-SNs fired action potentials in response to current injection, local application of potassium, or spontaneously, followed by short-medium afterhyperpolarizations. hiPSC-SNs could successfully be maintained in coculture with hiPSC-CMs, and this induced further development of hiPSC-SN action potential kinetics. To test functional coupling between the neurons and cardiomyocytes, the hiPSC-CM beating response to nicotine-induced norepinephrine release was assessed. In neurocardiac cocultures, nicotine exposure significantly increased the hiPSC-CM spontaneous beating rate, but not in hiPSC-CM monocultures, supporting nicotinic neuronal hiPSC-SN stimulation directly influencing hiPSC-CM function. Our data show the development and characterization of electrophysiologically functional hiPSC-SNs capable of modulating the beating rate of hiPSC-CMs in vitro. These human cocultures provide a novel multicellular model to study neurocardiac modulation under physiological and pathological conditions.NEW & NOTEWORTHY We present data on a functional coculture between human-induced pluripotent stem cell-derived sympathetic neurons and cardiomyocytes. Moreover, this study adds significantly to the available data on the electrophysiological function of human-induced pluripotent stem cell-derived sympathetic neurons.

Evidence of structural and functional plasticity occurring within the intracardiac nervous system of spontaneously hypertensive rats.

Plasticity is a fundamental property of neurons in both the central and peripheral nervous systems, enabling rapid changes in neural network function. The intracardiac nervous system (ICNS) is an extensive network of neurons clustered into ganglionated plexi (GP) on the surface of the heart. GP neurons are the final site of neuronal control of heart rhythm, and pathophysiological remodeling of the ICNS is proposed to feature in multiple cardiovascular diseases, including heart failure and atrial fibrillation. To examine the potential role of GP neuron plasticity in atrial arrhythmia and hypertension, we developed whole cell patch clamp recording techniques from GP neurons in isolated ICNS preparations from aged control (Wistar-Kyoto) and spontaneously hypertensive rats (SHRs). Anesthetized SHRs showed frequent premature ventricular contractions and episodes of atrial arrhythmia following carbachol injection, and isolated SHR atrial preparations were susceptible to pacing induced atrial arrhythmia. Whole cell recordings revealed elevated spontaneous postsynaptic current frequency in SHR GP neurons, as well as remodeled electrophysiology, with significant decreases in action potential amplitude and half-width. SHRs also showed a parallel increase in the number of cholinergic neurons and adrenergic glomus cells in cardiac ganglia, a higher proportion of synaptic α7-subunit but not ß2-containing nicotinic receptors, and an elevation in the number of synaptic terminals onto GP neurons. Our data show that significant structural and functional plasticity occurs in the intracardiac nervous system and suggest that enhanced excitability through synaptic plasticity, together with remodeling of cardiac neuron electrophysiology, contributes to the substrate for atrial arrhythmia in hypertensive heart disease.NEW & NOTEWORTHY We have developed intracardiac neuron whole cell recording techniques in atrial preparations from control and spontaneous hypertensive rats. This has enabled the identification of significant synaptic plasticity in the intracardiac nervous system, including enhanced postsynaptic current frequency, increased synaptic terminal density, and altered postsynaptic receptors. This increased synaptic drive together with altered cardiac neuron electrophysiology could increase intracardiac nervous system excitability and contribute to the substrate for atrial arrhythmia in hypertensive heart disease.

Autism-associated Shank3 mutations alter mGluR expression and mGluR-dependent but not NMDA receptor-dependent long-term depression.

SHANK3 is a postsynaptic structural protein localized at excitatory glutamatergic synapses in which deletions and mutations have been implicated in patients with autism spectrum disorders (ASD). The expression of Shank3 ASD mutations causes impairments in ionotropic glutamate receptor-mediated synaptic responses in neurons, which is thought to underlie ASD-related behaviors, thereby indicating glutamatergic synaptopathy as one of the major pathogenic mechanisms. However, little is known about the functional consequences of ASD-associated mutations in Shank3 on another important set of glutamate receptors, group I metabotropic glutamate receptors (mGluRs). Here, we further assessed how Shank3 mutations identified in patients with ASD (one de novo InsG mutation and two inherited point mutations, R87C and R375C) disrupt group I mGluR (mGluR1 and mGluR5) expression and function. To identify potential isoform-specific deficits induced by ASD-associated Shank3 mutations on group I mGluRs, we surface immunolabeled mGluR1 and mGluR5 independently. We also induced mGluR-dependent synaptic plasticity (R,S-3,5-dihydroxyphenylglycine [DHPG]-induced long-term depression [LTD]) as well as N-methyl-D-aspartate receptor (NMDAR)-dependent LTD. ASD-associated mutations in Shank3 differentially interfered with the ability of cultured hippocampal neurons to express mGluR5 and mGluR1 at synapses. Intriguingly, all ASD Shank3 mutations impaired mGluR-dependent LTD without altering NMDAR-dependent LTD. Our data show that the specific perturbation in mGluR-dependent synaptic plasticity occurs in neurons expressing ASD-associated Shank3 mutations, which may underpin synaptic dysfunction and subsequent behavioral deficits in ASD.

SAP97-mediated rescue of NMDA receptor surface distribution in a neuronal model of Huntington's disease.

Huntington's disease (HD) is a genetic neurodegenerative disorder caused by an expansion of the CAG repeat tract in the HTT gene, leading to motor, cognitive, and psychiatric symptoms. At the cellular level, NMDA-type glutamate receptors are upregulated at glutamatergic extrasynaptic sites in HD, triggering cell death signaling pathways and driving HD neurodegeneration. Extrasynaptic and synaptic glutamate receptor trafficking and surface distribution are regulated by the α and ß N-terminal isoforms of SAP97, a postsynaptic density protein localized at glutamatergic synapses. Here we examined the surface distribution of NMDARs and AMPARs in a cellular model of HD, and whether the manipulation of individual SAP97 isoforms can regulate receptor distribution in diseased neurons. Using dSTORM super-resolution imaging, we reveal that mutant HTT drives the elevation of extrasynaptic NMDAR clusters located 100-500 nm from the postsynaptic density. This was accompanied by a decline in synaptic NMDAR-mediated currents while surface NMDAR-mediated currents remained unchanged. These effects were induced within 3 days of mutant HTT expression in rat hippocampal neurons in vitro, and were specific for NMDARs and not observed with AMPARs. Intriguingly, upregulation of either α- or ßSAP97 expression increased synaptic and/or perisynaptic NMDAR localization and prevented the shift of NMDARs to extrasynaptic sites in mutant HTT neurons. This was accompanied by the rescue of normal synaptic NMDAR-mediated currents. Taken together, our high-resolution data reveals plasticity in surface NMDAR localization driven by mutant HTT and identifies the similar but independent roles of SAP97 N-terminal isoforms in maintaining normal synaptic function in pathological states.

Shank3 Is Part of a Zinc-Sensitive Signaling System That Regulates Excitatory Synaptic Strength.

UNLABELLED: Shank3 is a multidomain scaffold protein localized to the postsynaptic density of excitatory synapses. Functional studies in vivo and in vitro support the concept that Shank3 is critical for synaptic plasticity and the trans-synaptic coupling between the reliability of presynaptic neurotransmitter release and postsynaptic responsiveness. However, how Shank3 regulates synaptic strength remains unclear. The C terminus of Shank3 contains a sterile alpha motif (SAM) domain that is essential for its postsynaptic localization and also binds zinc, thus raising the possibility that changing zinc levels modulate Shank3 function in dendritic spines. In support of this hypothesis, we find that zinc is a potent regulator of Shank3 activation and dynamics in rat hippocampal neurons. Moreover, we show that zinc modulation of synaptic transmission is Shank3 dependent. Interestingly, an autism spectrum disorder (ASD)-associated variant of Shank3 (Shank3(R87C)) retains its zinc sensitivity and supports zinc-dependent activation of AMPAR-mediated synaptic transmission. However, elevated zinc was unable to rescue defects in trans-synaptic signaling caused by the R87C mutation, implying that trans-synaptic increases in neurotransmitter release are not necessary for the postsynaptic effects of zinc. Together, these data suggest that Shank3 is a key component of a zinc-sensitive signaling system, regulating synaptic strength that may be impaired in ASD. SIGNIFICANCE STATEMENT: Shank3 is a postsynaptic protein associated with neurodevelopmental disorders such as autism and schizophrenia. In this study, we show that Shank3 is a key component of a zinc-sensitive signaling system that regulates excitatory synaptic transmission. Intriguingly, an autism-associated mutation in Shank3 partially impairs this signaling system. Therefore, perturbation of zinc homeostasis may impair, not only synaptic functionality and plasticity, but also may lead to cognitive and behavioral abnormalities seen in patients with psychiatric disorders.

N-terminal SAP97 isoforms differentially regulate synaptic structure and postsynaptic surface pools of AMPA receptors.

The location and density of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is controlled by scaffolding proteins within the postsynaptic density (PSD). SAP97 is a PSD protein with two N-terminal isoforms, α and ß, that have opposing effects on synaptic strength thought to result from differential targeting of AMPA receptors into distinct synaptic versus extrasynaptic locations, respectively. In this study, we have applied dSTORM super resolution imaging in order to localize the synaptic and extrasynaptic pools of AMPA receptors in neurons expressing α or ßSAP97. Unexpectedly, we observed that both α and ßSAP97 enhanced the localization of AMPA receptors at synapses. However, this occurred via different mechanisms: αSAP97 increased PSD size and consequently the number of receptor binding sites, whilst ßSAP97 increased synaptic receptor cluster size and surface AMPA receptor density at the PSD edge and surrounding perisynaptic sites without changing PSD size. αSAP97 also strongly enlarged presynaptic active zone protein clusters, consistent with both presynaptic and postsynaptic enhancement underlying the previously observed αSAP97-induced increase in AMPA receptor-mediated currents. In contrast, ßSAP97-expressing neurons increased the proportion of immature filopodia that express higher levels of AMPA receptors, decreased the number of functional presynaptic terminals, and also reduced the size of the dendritic tree and delayed the maturation of mushroom spines. Our data reveal that SAP97 isoforms can specifically regulate surface AMPA receptor nanodomain clusters, with ßSAP97 increasing extrasynaptic receptor domains at peri-synaptic and filopodial sites. Moreover, ßSAP97 negatively regulates synaptic maturation both structurally and functionally. These data support diverging presynaptic and postsynaptic roles of SAP97 N-terminal isoforms in synapse maturation and plasticity. As numerous splice isoforms exist in other major PSD proteins (e.g., Shank, PSD95, and SAP102), this alternative splicing may result in individual PSD proteins having divergent functional and structural roles in both physiological and pathophysiological synaptic states.

The anchoring protein SAP97 influences the trafficking and localisation of multiple membrane channels.

SAP97 is a member of the MAGUK family of proteins that play a major role in the trafficking and targeting of membrane ion channels and cytosolic structural proteins in multiple cell types. Within neurons, SAP97 is localised throughout the secretory trafficking pathway and at the postsynaptic density (PSD). SAP97 differs from other MAGUK family members largely in its long N-terminus and in the sequences between the SH3 and GUK domains, where SAP97 undergoes significant alternative splicing to produce multiple SAP97 isoforms. These splice insertions endow SAP97 with differential cellular localisation patterns and functional roles within neurons. With regard to membrane ion channels, SAP97 forms multi-protein complexes with AMPA and NMDA-type glutamate receptors, and Kv1.4, Kv4.2, and Kir2.2 potassium channels, playing a major role in trafficking and anchoring ion channel surface expression. This highlights SAP97 not only as a regulator of neuronal excitability, synaptic function and plasticity in the brain, but also as a target for the pathophysiology of a number of neurological disorders. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Chemicals eluting from disposable plastic syringes and syringe filters alter neurite growth, axogenesis and the microtubule cytoskeleton in cultured hippocampal neurons.

Cultures of dissociated hippocampal neurons are often used to study neuronal cell biology. We report that the development of these neurons is strongly affected by chemicals leaching from commonly used disposable medical-grade syringes and syringe filters. Contamination of culture medium by bioactive substance(s) from syringes and filters occurred with multiple manufacturing lots and filter types under normal use conditions and resulted in changes to neurite growth, axon formation and the neuronal microtubule cytoskeleton. The effects on neuronal morphology were concentration dependent and significant effects were detected even after substantial dilution of the contaminated medium. Gas chromatography-mass spectrometry analyses revealed many chemicals eluting from the syringes and filters. Three of these chemicals (stearic acid, palmitic acid and 1,2-ethanediol monoacetate) were tested but showed no effects on neurite growth. Similar changes in neuronal morphology were seen with high concentrations of bisphenol A and dibutyl phthalate, two hormonally active plasticisers. Although no such compounds were detected by gas chromatography­mass spectrometry, unknown plasticisers in leachates may affect neurites. This is the first study to show that leachates from laboratory consumables can alter the growth of cultured hippocampal neurons. We highlight important considerations to ensure leachate contamination does not compromise cell biology experiments.

Differential effectiveness of dietary zinc supplementation with autism-related behaviours in Shank2 knockout mice.

The family of SHANK proteins have been shown to be critical in regulating glutamatergic synaptic structure, function and plasticity. SHANK variants are also prevalent in autism spectrum disorders (ASDs), where glutamatergic synaptopathology has been shown to occur in multiple ASD mouse models. Our previous work has shown that dietary zinc in Shank3-/- and Tbr1+/- ASD mouse models can reverse or prevent ASD behavioural and synaptic deficits. Here, we have examined whether dietary zinc can influence behavioural and synaptic function in Shank2-/- mice. Our data show that dietary zinc supplementation can reverse hyperactivity and social preference behaviour in Shank2-/- mice, but it does not alter deficits in working memory. Consistent with this, at the synaptic level, deficits in NMDA/AMPA receptor-mediated transmission are also not rescued by dietary zinc. In contrast to other ASD models examined, we observed that SHANK3 protein was highly expressed at the synapses of Shank2-/- mice and that dietary zinc returned these to wild-type levels. Overall, our data show that dietary zinc has differential effectiveness in altering ASD behaviours and synaptic function across ASD mouse models even within the Shank family. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Autism-associated mutations in ProSAP2/Shank3 impair synaptic transmission and neurexin-neuroligin-mediated transsynaptic signaling.

Mutations in several postsynaptic proteins have recently been implicated in the molecular pathogenesis of autism and autism spectrum disorders (ASDs), including Neuroligins, Neurexins, and members of the ProSAP/Shank family, thereby suggesting that these genetic forms of autism may share common synaptic mechanisms. Initial studies of ASD-associated mutations in ProSAP2/Shank3 support a role for this protein in glutamate receptor function and spine morphology, but these synaptic phenotypes are not universally penetrant, indicating that other core facets of ProSAP2/Shank3 function must underlie synaptic deficits in patients with ASDs. In the present study, we have examined whether the ability of ProSAP2/Shank3 to interact with the cytoplasmic tail of Neuroligins functions to coordinate pre/postsynaptic signaling through the Neurexin-Neuroligin signaling complex in hippocampal neurons of Rattus norvegicus. Indeed, we find that synaptic levels of ProSAP2/Shank3 regulate AMPA and NMDA receptor-mediated synaptic transmission and induce widespread changes in the levels of presynaptic and postsynaptic proteins via Neurexin-Neuroligin transsynaptic signaling. ASD-associated mutations in ProSAP2/Shank3 disrupt not only postsynaptic AMPA and NMDA receptor signaling but also interfere with the ability of ProSAP2/Shank3 to signal across the synapse to alter presynaptic structure and function. These data indicate that ASD-associated mutations in a subset of synaptic proteins may target core cellular pathways that coordinate the functional matching and maturation of excitatory synapses in the CNS.

Recording plasticity in neuronal activity in the rodent intrinsic cardiac nervous system using calcium imaging techniques.

The intrinsic cardiac nervous system (ICNS) is composed of interconnected clusters of neurons called ganglionated plexi (GP) which play a major role in controlling heart rate and rhythm. The function of these neurons is particularly important due to their involvement in cardiac arrhythmias such as atrial fibrillation (AF), and previous work has shown that plasticity in GP neural networks could underpin aberrant activity patterns that drive AF. As research in this field increases, developing new techniques to visualize the complex interactions and plasticity in this GP network is essential. In this study we have developed a calcium imaging method enabling the simultaneous recording of plasticity in neuronal activity from multiple neurons in intact atrial GP networks. Calcium imaging was performed with Cal-520 AM labeling in aged spontaneously hypertensive rats (SHRs), which display both spontaneous and induced AF, and age-matched Wistar Kyoto (WKY) controls to determine the relationship between chronic hypertension, arrhythmia and GP calcium dynamics. Our data show that SHR GPs have significantly larger calcium responses to cholinergic stimulation compared to WKY controls, as determined by both higher amplitude and longer duration calcium responses. Responses were significantly but not fully blocked by hexamethonium, indicating multiple cholinergic receptor subtypes are involved in the calcium response. Given that SHRs are susceptible to cardiac arrhythmias, our data provide evidence for a potential link between arrhythmia and plasticity in calcium dynamics that occur not only in cardiomyocytes but also in the GP neurons of the heart.

The serine protease inhibitor neuroserpin regulates the growth and maturation of hippocampal neurons through a non-inhibitory mechanism.

Neuroserpin is a brain-specific serine protease inhibitor that is expressed in the developing and adult nervous system. Its expression profile led to suggestions that it played roles in neuronal growth and connectivity. In this study, we provide direct evidence to support a role for neuroserpin in axon and dendritic growth. We report that axon growth is enhanced while axon and dendrite diameter are reduced following neuroserpin treatment of hippocampal neurons. More complex effects are seen on dendritic growth and branching with neuroserpin-stimulating dendritic growth and branching in young neurons but switching to an inhibitory response in older neurons. The protease inhibitory activity of neuroserpin is not required to activate changes in neuronal morphology and a proportion of responses are modulated by an antagonist to the LRP1 receptor. Collectively, these findings support a key role for neuroserpin as a regulator of neuronal development through a non-inhibitory mechanism and suggest a basis for neuroserpin's effects on complex emotional behaviours and recent link to schizophrenia.

Synaptic integration of newly generated neurons in rat dissociated hippocampal cultures.

In the dentate gyrus of the hippocampus new neurons are born from precursor cells throughout development and into adulthood. These newborn neurons hold significant potential for self-repair of brain damage caused by neurodegenerative disease. However, the mechanism by which newborn neurons integrate into the brain is not understood due to a lack of knowledge of the molecular and functional characteristics of the synapses formed by newborn neurons. Here we report that dissociated hippocampal cultures continue to produce new granule cells in vitro that fire action potentials and become synaptically integrated into the existing network of mature hippocampal neurons. Quantification of the expression of synaptic proteins at newborn and mature granule cell synapses revealed synapse development onto newborn neurons occurs sequentially with initial synaptic contacts evident from 6 days after cell birth. These data also showed that the dendrites of newborn neurons have a high density of Piccolo and Bassoon puncta on them and therefore have a high potential to be integrated into the neuronal network through new synaptic connections. Electrophysiological recordings from newborn neurons reveal these synapses are functional within 10 days of cell birth. GABAergic input synapses were found to mature faster in newborn neurons than glutamatergic synapses where sequential recruitment of postsynaptic glutamate receptors occurred. Group I metabotropic glutamate receptors (mGluR1/5) were present at higher levels compared with ionotropic glutamate receptors (NMDA and AMPA receptors), suggesting that metabotropic and ionotropic receptors play differential roles at glutamatergic synapses in the integration and the maturation of newborn neurons. These data show that dissociated hippocampal cultures can provide a useful model system in which to study the integration of newborn neurons into existing neuronal circuits to increase our understanding of how the function of newborn neuron synapses could contribute to restoring damaged neuronal networks.