Molecular Neuroscience seminars

Survival is predicated on the ability of an organism to respond to stress. The reliability of this response is ensured by a synaptic architecture that is relatively inflexible (i.e. hard-wired). Our work has shown that in naive animals, synapses on CRH neurons in the paraventricular nucleus of the hypothalamus are very reluctant to modification. If animals are stressed, however, these synapses become willing to learn. This seminar will focus on mechanisms linking acute stress to metaplastic changes at glutamate synapses, and also show how stress, and these synaptic changes can be transmitted from one individual to another.

Developmental epileptic encephalopathies are early-onset epilepsies, often refractory to therapy, with developmental delay or regression. These disorders carry poor neurodevelopmental prognosis, with long-term refractory epilepsy and persistent cognitive, behavioral and motor deficits. Mutations in the CACNA1A gene, encoding the pore-forming α1 subunit of CaV2.1 voltage-gated calcium channels, result in a spectrum of neurological disorders, including severe, early-onset epileptic encephalopathies. Recent work from the Rossignol lab helped characterize the phenotypic spectrum of CACNA1A-related epilepsies in humans. Using conditional genetics and novel animal models, the Rossignol lab unveiled some of the underlying pathophysiological mechanisms, including critical deficits in cortical inhibition, resulting in seizures and a range of cognitive-behavioral deficits. Importantly, Dr. Rossignol’s team demonstrated that the targeted activation of specific GABAergic interneuron populations in selected cortical regions prevents motor seizures and reverts attention deficits and cognitive rigidity in mouse models of the disorder. These recent findings open novel avenues for the treatment of these severe CACNA1A-associated neurodevelopmental disorders.

The long-term goal of our research is to understand the mechanisms of SUDEP, defined as Sudden, Unexpected, witnessed or unwitnessed, nontraumatic and non-drowning Death in patients with EPilepsy, excluding cases of documented status epilepticus. The majority of SUDEP patients die during sleep. SUDEP is the most devastating consequence of epilepsy, yet little is understood about its causes and no biomarkers exist to identify at risk patients. While SUDEP accounts for 7.5-20% of all epilepsy deaths, SUDEP risk in the genetic epilepsies varies with affected genes. Patients with ion channel gene variants have the highest SUDEP risk. Indirect evidence variably links SUDEP to seizure-induced apnea, pulmonary edema, dysregulation of cerebral circulation, autonomic dysfunction, and cardiac arrhythmias. Arrhythmias may be primary or secondary to hormonal or metabolic changes, or autonomic discharges. When SUDEP is compared to Sudden Cardiac Death secondary to Long QT Syndrome, especially to LQT3 linked to variants in the voltage-gated sodium channel (VGSC) gene SCN5A, there are parallels in the circumstances of death. To gain insight into SUDEP mechanisms, our approach has focused on channelopathies with high SUDEP incidence. One such disorder is Dravet syndrome (DS), a devastating form of developmental and epileptic encephalopathy (DEE) characterized by multiple pharmacoresistant seizure types, intellectual disability, ataxia, and increased mortality. While all patients with epilepsy are at risk for SUDEP, DS patients may have the highest risk, up to 20%, with a mean age at SUDEP of 4.6 years. Over 80% of DS is caused by de novo heterozygous loss-of-function (LOF) variants in SCN1A, encoding the VGSC Nav1.1  subunit, resulting in haploinsufficiency. A smaller cohort of patients with DS or a more severe DEE have inherited, homozygous LOF variants in SCN1B, encoding the VGSC 1/1B non-pore-forming subunits. A related DEE, Early Infantile EE (EIEE) type 13, is linked to de novo heterozygous gain-of-function variants in SCN8A, encoding the VGSC Nav1.6. VGSCs underlie the rising phase and propagation of action potentials in neurons and cardiac myocytes. SCN1A, SCN8A, and SCN1B are expressed in both the heart and brain of humans and mice. Because of this, we proposed that cardiac arrhythmias contribute to the mechanism of SUDEP in DEE. We have taken a novel approach to the development of therapeutics for DS in collaboration with Stoke Therapeutics. We employed Targeted Augmentation of Nuclear Gene Output (TANGO) technology, which modulates naturally occurring, non-productive splicing events to increase target gene and protein expression and ameliorate disease phenotype in a mouse model. We identified antisense oligonucleotides (ASOs) that specifically increase the expression of productive Scn1a transcript in human and mouse cell lines, as well as in mouse brain. We showed that a single intracerebroventricular dose of a lead ASO at postnatal day 2 or 14 reduced the incidence of electrographic seizures and SUDEP in the F1:129S-Scn1a+/- x C57BL/6J mouse model of DS. Increased expression of productive Scn1a transcript and NaV1.1 protein were confirmed in brains of treated mice. Our results suggest that TANGO may provide a unique, gene-specific approach for the treatment of DS.

Neuronal alternative splicing is a key gene regulatory mechanism in the brain. However, the spliceosome machinery is insufficient to fully specify splicing complexity. In considering the role of the epigenome in activity-dependent alternative splicing, we and others find the histone modification H3K36me3 to be a putative splicing regulator. In this study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with the enrichment of H3K36me3 at differentially spliced junctions. Importantly, only targeted epigenetic editing can distinguish between a direct role of H3K36me3 in splicing and an indirect role via regulation of splice factor expression elsewhere on the genome. We targeted Srsf11, which was both alternatively spliced and H3K36me3 enriched in the brain following cocaine self-administration. Epigenetic editing of H3K36me3 at Srsf11 was sufficient to drive its alternative splicing and enhanced cocaine self-administration, establishing the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior.

Pathogenic variants in HCN1 are associated with severe developmental and epileptic encephalopathies (DEE). We have engineered the Hcn1 M294L heterozygous knock-in (Hcn1M294L) mouse which is a homolog of the de novo HCN1 M305L recurrent pathogenic variant. The mouse recapitulates the phenotypic features of patients including having spontaneous seizures and a learning deficit. In this talk I will present experimental work that probes the molecular and cellular mechanisms underlying hyper-excitability in the mouse model. This will include testing the efficacy of currently available antiepileptic drugs and a novel precision medicine approach. I will also briefly touch on how disease biology can give insights into the biophysical properties of HCN channels.

CA3-CA1 synapses in the hippocampus are the initial locus of episodic memory. The action of acetylcholine alters cellular excitability, modifies neuronal networks, and triggers secondary signaling that directly affects long-term plasticity (LTP) (the cellular underpinning of memory). It is therefore considered a critical regulator of learning and memory in the brain. Its action via M4 metabotropic receptors in the presynaptic terminal of the CA3 neurons and M1 metabotropic receptors in the postsynaptic spines of CA1 neurons produce rich dynamics across multiple timescales. We developed a model to describe the activation of postsynaptic M1 receptors that leads to IP3 production from membrane PIP2 molecules. The binding of IP3 to IP3 receptors in the endoplasmic reticulum (ER) ultimately causes calcium release. This calcium release from the ER activates potassium channels like the calcium-activated SK channels and alters different aspects of synaptic signaling. In an independent signaling cascade, M1 receptors also directly suppress SK channels and the voltage-activated KCNQ2/3 channels, enhancing post-synaptic excitability. In the CA3 presynaptic terminal, we model the reduction of the voltage sensitivity of voltage-gated calcium channels (VGCCs) and the resulting suppression of neurotransmitter release by the action of the M4 receptors. Our results show that the reduced initial release probability because of acetylcholine alters short-term plasticity (STP) dynamics. We characterize the dichotomy of suppressing neurotransmitter release from CA3 neurons and the enhanced excitability of the postsynaptic CA1 spine. Mechanisms underlying STP operate over a few seconds, while those responsible for LTP last for hours, and both forms of plasticity have been linked with very distinct functions in the brain. We show that the concurrent suppression of neurotransmitter release and increased sensitivity conserves neurotransmitter vesicles and enhances the reliability in plasticity. Our work establishes a relationship between STP and LTP coordinated by neuromodulation with acetylcholine.

Neuromodulators control information processing in cortical microcircuits by regulating the cellular and synaptic physiology of neurons. Computational models and detailed simulations of neocortical microcircuitry offer a unifying framework to analyze the role of neuromodulators on network activity. In the present study, to get a deeper insight in the organization of the cortical neuropil for modeling purposes, we quantify the fiber length per cortical volume and the density of varicosities for catecholaminergic, serotonergic and cholinergic systems using immunocytochemical staining and stereological techniques. The data obtained are integrated into a biologically detailed digital reconstruction of the rodent neocortex (Markram et al, 2015) in order to model the influence of modulatory systems on the activity of the somatosensory cortex neocortical column. Simulations of ascending modulation of network activity in our model predict the effects of increasing levels of neuromodulators on diverse neuron types and synapses and reveal a spectrum of activity states. Low levels of neuromodulation drive microcircuit activity into slow oscillations and network synchrony, whereas high neuromodulator concentrations govern fast oscillations and network asynchrony. The models and simulations thus provide a unifying in silico framework to study the role of neuromodulators in reconfiguring network activity.

Repeat-rich sequence blocks are considered major determinants for 3D folding and structural genome organization in the cell nucleus in all higher eukaryotes. Here, we discuss how megabase-scale chromatin domain and chromosomal compartment organization in adult mouse cerebral cortex is linked, in highly cell type-specific fashion, to multiple retrotransposon superfamilies which comprise the vast majority of mobile DNA elements in the murine genome. We show that neuronal megadomain architectures include an evolutionarily adaptive heterochromatic organization which, upon perturbation, unleashes proviruses from the Long Terminal Repeat (LTR) Endogenous Retrovirus family that exhibit strong tropism in mature neurons. Furthermore, we mapped, in the human brain, cell type-specific genomic integration patterns of the human pathogen and exogenous retrovirus, HIV, together with changes in genome organization and function of the HIV infected brain. Our work highlights the critical importance of chromosomal conformations and the ‘spatial genome’ for neuron- and glia-specific regulatory mechanisms and defenses aimed at exogenous and endogenous retrotransposons in the brain

Three complementary lectures explain the development and applications of optogenetics. Gero Miesenböck traces optical control of neuronal activity from early experiments to studies of sleep, including neurons that respond to accumulated sleep need and molecular mechanisms governing sleep pressure. Peter Hegemann connects microbial photobiology to channelrhodopsins that control neuronal membrane voltage. He discusses their use in studying development and learning, and prospects for extending optical control to enzymes, transcription and translation. Georg Nagel follows the discovery and expression of microbial rhodopsins with light-sensitive transport and enzymatic functions. His examples include Channelrhodopsin-2 and its H134R variant, halorhodopsin, the more light-sensitive ChR2/XXL, the rhodopsin guanylyl cyclase Cyclop, and anion-channelrhodopsins in tobacco plants. Together, the talks connect molecular mechanisms with experimental control of neural and plant systems.

Biological neural networks can represent information in the collective action potential discharge of neurons, and store that information amongst the synaptic connections between the neurons that both comprise the network and govern its function. The strength and organization of synaptic connections adjust during learning, but many cognitive neural systems are multifunctional, making it unclear how continuous activity alternates between the transient and discrete cognitive functions like encoding current information and recollecting past information, without changing the connections amongst the neurons. This lecture will first summarize our investigations of the molecular and biochemical mechanisms that change synaptic function to persistently store spatial memory in the rodent hippocampus. I will then report on how entorhinal cortex-hippocampus circuit function changes during cognitive training that creates memory, as well as learning to learn in mice. I will then describe how the hippocampus system operates like a competitive winner-take-all network, that, based on the dominance of its current inputs, self organizes into either the encoding or recollection information processing modes. We find no evidence that distinct cells are dedicated to those two distinct functions, rather activation of the hippocampus information processing mode is controlled by a subset of dentate spike events within the network of learning-modified, entorhinal-hippocampus excitatory and inhibitory synapses.

Tuberous sclerosis complex (TSC) and focal cortical dysplasia type II (FCDII) are caused by mutations in mTOR pathway genes leading to mTOR hyperactivity, focal malformations of cortical development (fMCD), and seizures in 80-90% of the patients. The current definitive treatments for epilepsy are surgical resection or treatment with everolimus, which inhibits mTOR activity (only approved for TSC). Because both options have severe limitations, there is a major need to better understand the mechanisms leading to seizures to improve life-long epilepsy treatment in TSC and FCDII. To investigate such mechanisms, we recently developed a murine model of fMCD-associated epilepsy that recapitulates the human TSC and FCDII disorders. fMCD are defined by the presence of misplaced, dysmorphic cortical neurons expressing hyperactive mTOR – for simplicity we will refer to these as “mutant” neurons. In our model and in human TSC tissue, we made a surprising finding that mutant neurons express HCN4 channels, which are not normally functionally expressed in cortical neurons, and increased levels of filamin A (FLNA). FLNA is an actin-crossing linking molecule that has also multiple binding partners inside cells. These data led us to ask several important questions: (1) As HCN4 channels are responsible for the pacemaking activity of the heart, can HCN4 channel expression lead to repetitive firing of mutant neurons resulting in seizures? (2) HCN4 is the most cAMP-sensitive of the four HCN isoforms. Does increase in cAMP lead to the firing of mutant neurons? (3) Does increase in FLNA contribute to neuronal alterations and seizures? (4) Is the abnormal HCN4 and FLNA expression in mutant neurons due to mTOR? These questions will be discussed and addressed in the lecture.

What are the things that draw us to a particular field of science and what is it that keeps us there? For Dr. Bahia, there was a particular attraction to sensory nerves; the monitors of the worlds inside and outside of our bodies. In this talk, Dr. Bahia will outline her career path as a neuroscientist resulting in the title of Research Associate. She will also talk about the longest project she has participated in, 'exploring the role of ion channels in sensory nerves' (rupress.org/jgp/article/147/6/451/43495/The-exceptionally-high-reactivity-of-Cys-621-is)