Verena C. Wimmer

Short- and Long-Term Plasticity at the Axon Initial Segment

The axon initial segment (AIS) is a highly specialized neuronal subregion that is the site of action potential initiation and the boundary between axonal and somatodendritic compartments. In recent years, our understanding of the molecular structure of the AIS, its maturation, and its multiple fundamental roles in neuronal function has seen major advances. We are beginning to appreciate that the AIS is dynamically regulated, both over short timescales via adaptations in ion channel function, and long timescales via activity-dependent structural reorganization. Here, we review results from this emerging field highlighting how structural and functional plasticity relate to the development of the initial segment, and to neuronal disorders linked to AIS dysfunction.

Driver or Coincidence Detector: Modal Switch of a Corticothalamic Giant Synapse Controlled by Spontaneous Activity and Short-Term Depression

Giant synapses between layer 5B (L5B) neurons of somatosensory (barrel) cortex and neurons of the posteromedial nucleus (POm) of thalamus reside in a key position of the cortico-thalamo-cortical (CTC) loop, yet their synaptic properties and contribution to CTC information processing remain poorly understood. Fluorescence-guided local stimulation of terminals were combined with postsynaptic whole-cell recordings in thalamus to study synaptic transmission at an identified giant synapse. We found large EPSCs mediated by Ca2+-permeable AMPA and NMDA receptors. A single presynaptic electrical stimulus evoked a train of postsynaptic action potentials, indicating that a single L5B input can effectively drive the thalamic neuron. Repetitive stimulation caused strong short-term depression (STD) with fast recovery. To examine how these synaptic properties affect information transfer, spontaneous and evoked activity of L5B neurons was recorded in vivo and played back to giant terminals in vitro. We found that suprathreshold synaptic transmission was suppressed because of spontaneous activity causing strong STD of the L5B–POm giant synapse. Thalamic neurons only spiked after intervals of presynaptic silence or when costimulating two giant terminals. Therefore, STD caused by spontaneous activity of L5B neurons can switch the synapse from a “driver mode” to a “coincidence mode.” Mechanisms decreasing spontaneous activity in L5B neurons and inputs synchronized by a sensory stimulus may thus gate the cortico-thalamo-cortical loop.

Axon initial segment dysfunction in epilepsy

The axon initial segment (AIS) contains the site of action potential initiation and plays a major role in neuronal excitability. AIS function relies on high concentrations of different ion channels and complex regulatory mechanisms that orchestrate molecular microarchitecture. We review recent evidence that a large number of ion channels associated with epilepsy are enriched at the AIS, making it a ‘hotspot’ for epileptogenesis. Furthermore, we present novel data on the clustering of GABRγ2 receptors in the AIS of cortical and hippocampal neurons in a knock in mouse model of a human genetic epilepsy. This article highlights the molecular coincidence of epilepsy mutations at the AIS and reviews pathogenic mechanisms converging at the AIS.

Experience-induced plasticity of thalamocortical axons in both juveniles and adults.

We examined the effect of sensory deprivation on thalamocortical (TC) projections to the rat primary somatosensory cortex at different postnatal ages ranging from P0 to P96. Rats had their whiskers clipped off with one or two vibrissae spared. TC axons innervating barrel cortex were specifically labeled by injecting virus expressing fluorescent proteins into the corresponding primary (VPM) and/or secondary (POm) thalamic nuclei. The density of VPM axons in deprived columns was ≈34% lower relative to spared columns with a concomitant decrease in bouton density, suggesting a deprivation‐induced retraction of VPM axons. Axonal changes were reversible upon regrowth of the clipped whiskers and independent of age at deprivation, indicating the absence of a critical period for anatomical plasticity. The POm projection was not obviously altered by sensory deprivation. We suggest that retraction and regrowth of TC axons substantially contribute to long‐term deprivation‐dependent functional plasticity. J. Comp. Neurol. 518:4629–4648, 2010.

Comparative strength and dendritic organization of thalamocortical and corticocortical synapses onto excitatory layer 4 neurons.

Thalamus is a potent driver of cortical activity even though cortical synapses onto excitatory layer 4 neurons outnumber thalamic synapses 10 to 1. Previous in vitro studies have proposed that thalamocortical (TC) synapses are stronger than corticocortical (CC) synapses. Here, we investigated possible anatomical and physiological differences between these inputs in the rat in vivo. We developed a high-throughput light microscopy method, validated by electron microscopy, to completely map the locations of synapses across an entire dendritic tree. This demonstrated that TC synapses are slightly more proximal to the soma than CC synapses, but detailed compartmental modeling predicted that dendritic filtering does not appreciably favor one synaptic class over another. Measurements of synaptic strength in intact animals confirmed that both TC and CC synapses are weak and approximately equivalent. We conclude that thalamic effectiveness does not rely on enhanced TC strength, but rather on coincident activation of converging inputs.

Axon initial segment structural plasticity in animal models of genetic and acquired epilepsy

A novel form of neuronal plasticity, occurring at the axon initial segment (AIS), has recently been described. Lengthening of the AIS and movement away from the soma are consequences of changes in neuronal input and result in alterations in neuronal excitability. We hypothesised that AIS plasticity may play a role in epilepsy, due to chronic changes in neuronal activity. Immunohistochemistry and confocal microscopy were used to analyse AIS length and position in pyramidal neurons in deep layer 5 of the somatosensory cortex from 5 mice with genetic epilepsy and 4 controls, and from 3 rats subjected to amygdala kindling and 3 controls. The effect of a subtle alteration of AIS position was modelled computationally. We identified a difference in the position of the AIS in animals with seizures: in mice the AIS was positioned 0.2 μm further away from the soma, and in rats the AIS was positioned 0.6 μm closer to the soma compared with controls. Computational modelling indicated that a subtle alteration in AIS position could result in a change in action potential firing threshold. The identification of AIS plasticity in animal models of epilepsy is significant in furthering our understanding of the pathophysiological mechanisms involved in this disorder.

Reduced dendritic arborization and hyperexcitability of pyramidal neurons in a Scn1b-based model of Dravet syndrome

Epileptic encephalopathies, including Dravet syndrome, are severe treatment-resistant epilepsies with developmental regression. We examined a mouse model based on a human β1 sodium channel subunit (Scn1b) mutation. Homozygous mutant mice shared phenotypic features and pharmaco-sensitivity with Dravet syndrome. Patch-clamp analysis showed that mutant subicular and layer 2/3 pyramidal neurons had increased action potential firing rates, presumably as a consequence of their increased input resistance. These changes were not seen in L5 or CA1 pyramidal neurons. This raised the concept of a regional seizure mechanism that was supported by data showing increased spontaneous synaptic activity in the subiculum but not CA1. Importantly, no changes in firing or synaptic properties of gamma-aminobutyric acidergic interneurons from mutant mice were observed, which is in contrast with Scn1a-based models of Dravet syndrome. Morphological analysis of subicular pyramidal neurons revealed reduced dendritic arborization. The antiepileptic drug retigabine, a K+ channel opener that reduces input resistance, dampened action potential firing and protected mutant mice from thermal seizures. These results suggest a novel mechanism of disease genesis in genetic epilepsy and demonstrate an effective mechanism-based treatment of the disease.

Silencing relaxin-3 in nucleus incertus of adult rodents: a viral vector-based approach to investigate neuropeptide function.

Relaxin-3, the most recently identified member of the relaxin peptide family, is produced by GABAergic projection neurons in the nucleus incertus (NI), in the pontine periventricular gray. Previous studies suggest relaxin-3 is a modulator of stress responses, metabolism, arousal and behavioural activation. Knockout mice and peptide infusions in vivo have significantly contributed to understanding the function of this conserved neuropeptide. Yet, a definitive role remains elusive due to discrepancies between models and a propensity to investigate pharmacological effects over endogenous function. To investigate the endogenous function of relaxin-3, we generated a recombinant adeno-associated viral (rAAV) vector expressing microRNA against relaxin-3 and validated its use to knock down relaxin-3 in adult rats. Bilateral stereotaxic infusion of rAAV1/2 EmGFP miR499 into the NI resulted in significant reductions in relaxin-3 expression as demonstrated by ablation of relaxin-3-like immunoreactivity at 3, 6 and 9 weeks and by qRT-PCR at 12 weeks. Neuronal health was unaffected as transduced neurons in all groups retained expression of NeuN and stained for Nissl bodies. Importantly, qRT-PCR confirmed that relaxin-3 receptor expression levels were not altered to compensate for reduced relaxin-3. Behavioural experiments confirmed no detrimental effects on general health or well-being and therefore several behavioural modalities previously associated with relaxin-3 function were investigated. The validation of this viral vector-based model provides a valuable alternative to existing in vivo approaches and promotes a shift towards more physiologically relevant investigations of endogenous neuropeptide function.

Visualization of mouse barrel cortex using ex-vivo track density imaging

We describe the visualization of the barrel cortex of the primary somatosensory area (S1) of ex vivo adult mouse brain with short-tracks track density imaging (stTDI). stTDI produced much higher definition of barrel structures than conventional fractional anisotropy (FA), directionally-encoded color FA maps, spin-echo T1- and T2-weighted imaging and gradient echo T1/T2*-weighted imaging. 3D high angular resolution diffusion imaging (HARDI) data were acquired at 48 micron isotropic resolution for a (3mm)(3) block of cortex containing the barrel field and reconstructed using stTDI at 10 micron isotropic resolution. HARDI data were also acquired at 100 micron isotropic resolution to image the whole brain and reconstructed using stTDI at 20 micron isotropic resolution. The 10 micron resolution stTDI maps showed exceptionally clear delineation of barrel structures. Individual barrels could also be distinguished in the 20 micron stTDI maps but the septa separating the individual barrels appeared thicker compared to the 10 micron maps, indicating that the ability of stTDI to produce high quality structural delineation is dependent upon acquisition resolution. Close homology was observed between the barrel structure delineated using stTDI and reconstructed histological data from the same samples. stTDI also detects barrel deletions in the posterior medial barrel sub-field in mice with infraorbital nerve cuts. The results demonstrate that stTDI is a novel imaging technique that enables three-dimensional characterization of complex structures such as the barrels in S1 and provides an important complementary non-invasive imaging tool for studying synaptic connectivity, development and plasticity of the sensory system.

Loss of synaptic Zn2+ transporter function increases risk of febrile seizures.

Febrile seizures (FS) are the most common seizure syndrome and are potentially a prelude to more severe epilepsy. Although zinc (Zn2+) metabolism has previously been implicated in FS, whether or not variation in proteins essential for Zn2+ homeostasis contributes to susceptibility is unknown. Synaptic Zn2+ is co-released with glutamate and modulates neuronal excitability. SLC30A3 encodes the zinc transporter 3 (ZNT3), which is primarily responsible for moving Zn2+ into synaptic vesicles. Here we sequenced SLC30A3 and discovered a rare variant (c.892C > T; p.R298C) enriched in FS populations but absent in population-matched controls. Functional analysis revealed a significant loss-of-function of the mutated protein resulting from a trafficking deficit. Furthermore, mice null for ZnT3 were more sensitive than wild-type to hyperthermia-induced seizures that model FS. Together our data suggest that reduced synaptic Zn2+ increases the risk of FS and more broadly support the idea that impaired synaptic Zn2+ homeostasis can contribute to neuronal hyperexcitability.