Sorting of cargos between axons and dendrites: modelling of differences in cargo transport in these two types of neurites

Explaining how intracellular cargos are sorted between axons and dendrites is important for a mechanistic understanding of what happens in many neurodegenerative disorders. A simple model of cargo sorting relies on differences in microtubule (MT) orientation between axons and dendrites: in mammalian neurons all MTs in axons have their plus ends directed outward while in proximal regions of dendrites the MT polarity is mixed. It can therefore be assumed that cargos that need to be driven into axons associate with kinesin motors while cargos that need to be driven into dendrites associate with dynein motors. This paper develops equations of cargo transport in axons and dendrites based on the above assumptions. Propagation of a pulse of radiolabelled cargos entering an axon and dendrite is simulated. The model equations are solved utilising the Laplace transform method. Differences in cargo transport between axons and dendrites are discussed.     

Listening to speech recruits specific tongue motor synergies as revealed by transcranial magnetic stimulation and tissue-Doppler ultrasound imaging

The activation of listener''s motor system during speech processing was first demonstrated by the enhancement of electromyographic tongue potentials as evoked by single-pulse transcranial magnetic stimulation (TMS) over tongue motor cortex. This technique is, however, technically challenging and enables only a rather coarse measurement of this motor mirroring. Here, we applied TMS to listeners’ tongue motor area in association with ultrasound tissue Doppler imaging to describe fine-grained tongue kinematic synergies evoked by passive listening to speech. Subjects listened to syllables requiring different patterns of dorso-ventral and antero-posterior movements (/ki/, /ko/, /ti/, /to/). Results show that passive listening to speech sounds evokes a pattern of motor synergies mirroring those occurring during speech production. Moreover, mirror motor synergies were more evident in those subjects showing good performances in discriminating speech in noise demonstrating a role of the speech-related mirror system in feed-forward processing the speaker''s ongoing motor plan.     

雷特综合征与多巴胺系统功能障碍

雷特综合征(Rett syndrome)属于神经发育障碍类疾病,主要由X性染色体上mecp2基因突变所致,患者多数为女孩。临床症状于出生后6~18个月逐渐显现,主要表现为头部发育缓慢,已获得的语言及手部目的性运动技能消退,智力障碍,呼吸功能障碍及自闭倾向等。多巴胺系统的功能包括运动调节、奖赏学习、情感、内分泌调控以及药物成瘾等多个方面。由于多巴胺系统在运动和精神方面与雷特综合征部分临床症状存在表面相关性,早期有学者根据临床特征提出雷特综合征患者可能存在多巴胺系统功能障碍,但两者之间是否具有实质性的内在联系以及mecp2基因是否会通过影响多巴胺系统导致相关临床症状是目前雷特综合征研究的一个热点。本文将针对雷特综合征与多巴胺系统功能障碍的相关研究进展作一综述。     

Serotonin and the search for the anatomical substrate of aggression

All species of animals display aggression in order to obtain resources such as territories, mates, or food. Appropriate displays of aggression rely on the correct identification of a potential competitor, an evaluation of the environmental signals, and the physiological state of the animal. With a hard-wired circuitry involving fixed numbers of neurons, neuromodulators like serotonin offer adaptive flexibility in behavioral responses without changing the “hard-wiring”. In a recent report, we combined intersectional genetics, quantitative behavioral assays and morphological analyses to identify single serotonergic neurons that modulate the escalation of aggression. We found anatomical target areas within the brain where these neurons appear to form synaptic contacts with 5HT1A receptor-expressing neurons, and then confirmed the likelihood of those connections on a functional level. In this Extra View article, we offer an extended discussion of these recent findings and elaborate on how they can link a cellular and functional mapping of an aggression-regulating circuit at a single-cell resolution level.     

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson’s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.     

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer''s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.     

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABA_A receptors (GABA_ARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABA_ARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABA_AR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABA_AR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABA_ARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.     

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity ^1-3. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations ⁴. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo ¹.The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students’ practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish’s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.     

Odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert & Zhao, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca(2+)-activated Cl(-) channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body. In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors. The suction pipette technique was later adapted to also record from ORNs. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.     

mRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons

mRNA for neuronal Bak (N-Bak), a splice variant of pro-apoptotic Bcl-2 family member Bak is expressed in the neurons. Surprisingly the endogeneous N-Bak protein cannot be demonstrated in the neurons, although the antibodies recognize N-Bak protein from in vitro translation or transiently transfected cells. As N-Bak mRNA contains premature termination codon (PTC) at 89 nucleotides upstream from the last exon–exon junction, it could be degraded by nonsense-mediated decay (NMD) during the pioneer round of translation thus explaining the absence of the protein. We show here that the endogeneous neuronal N-Bak mRNA is not the NMD substrate, as it is not accumulating by cycloheximide treatment, it has a long lifetime, and even prevention of PTC by interfering with the alternative splicing did not lead to translation of the Bak mRNA. N-Bak protein is also not revealed by proteasome inhibitors. Our data suggest strong translational arrest of N-Bak mRNA in the neurons. We show that this arrest is partially mediated by 5′-untranslated region of Bak mRNA and it is not released during mitochondrial apoptosis.