Functional Microstructure of CaV-Mediated Calcium Signaling in the Axon Initial Segment

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential (AP) output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (Ca_V). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we use conventional two-photon laser scanning and diffraction-limited, high-speed spot two-photon imaging to resolve AP-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of Ca_V2 and Ca_V3 channels that differentially localized to discrete regions. Ca_V3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas Ca_V2 localized to non-hotspot regions. Thus, different pools of Ca_Vs appear to play distinct roles in AIS function.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is the site where synaptic input is transformed into action potential (AP) output. It achieves this function through a diverse complement of sodium, potassium, and calcium channels (Ca_V). While the localization and function of sodium channels and potassium channels at the AIS is well described, less is known about the functional distribution of Ca_Vs. We used high-speed two-photon imaging to understand activity-dependent calcium dynamics in the AIS of mouse neocortical pyramidal neurons. Surprisingly, we found that calcium influx occurred in two distinct domains: Ca_V3 generates hotspot regions of calcium influx coupled to calcium stores, whereas Ca_V2 channels underlie diffuse calcium influx between hotspots. Therefore, different Ca_V classes localize to distinct AIS subdomains, possibly regulating distinct cellular processes.     

Cortical Interneurons Upregulate Neurotrophinsin Vivoin Response to Targeted Apoptotic Degeneration of Neighboring Pyramidal Neurons

Intercellular signals provided by growth and neurotrophic factors play a critical role during neurogenesis and as part of cellular repopulation strategies directed toward reconstruction of complex CNS circuitry. Local signals influence the differentiation of transplanted and endogenous neurons and neural precursors, but the cellular sources and control over expression of these molecules remain unclear. We have previously examined microenvironmental control in neocortex over neuron and neural precursor migration and differentiation following transplantation, using an approach of targeted apoptotic neuronal degeneration to specific neuronal populationsin vivo.Prior results suggested the hypothesis that upregulated or reexpressed developmental signal molecules, produced by degenerating pyramidal neurons and/or by neighboring neurons or nonneuronal cells, may be responsible for observed events of directed migration, differentiation, and connectivity by transplanted immature neurons and precursors. To directly investigate this hypothesis, we analyzed the gene expression of candidate and control neurotrophins, growth factors, and receptors within regions of targeted neuronal cell death, first by quantitative Northern blot analysis and then byin situhybridization combined with immunocytochemical analysis. The genes for BDNF, NT-4/5, trkB receptors, and to a lesser extent NT-3 were upregulated specifically within the regions of neocortex undergoing targeted neuronal degeneration and specifically during the period of ongoing pyramidal neuron apoptosis. Upregulation occurred during the same 3-week period as the previously investigated cellular events of directed migration, differentiation, and integration. No upregulation was seen in panels of control neurotrophins, growth factors, and receptors that are not as developmentally regulated in cortex or that are thought to have primary actions in other CNS regions.In situhybridization and immunocytochemistry revealed that BDNF mRNA expression was upregulated specifically by local interneurons adjacent to degenerating pyramidal neurons. These findings suggest specific effects of targeted apoptosis on neurotrophin and other gene expression via mechanisms, including intercellular signaling between degenerating pyramidal neurons and surrounding interneurons. Further understanding of these and other controls over neocortical projection neuron differentiation may provide insight regarding normal neocortical development, intercellular signaling induced by apoptosis, and toward reconstruction and cellular repopulation of complex neocortical and other CNS circuitry.     

Ferric ion-ferrocyanide staining in ganglioside storage disease establishes that meganeurites are of axon hillock origin and distinct from axonal spheroids

Ferric ion-ferrocyanide staining and safranin-0-counterstaining of neocortical tissue from cats with GM1 gangliosidosis have established that pyramidal neuron meganeurites occur proximal to axonal initial segments and that they are distinct from axonal spheroids. The latter, which were found to be widely distributed throughout cerebral cortex, were located distal to axonal initial segments and could be differentiated from meganeurites at both light and electron microscopic levels. This report confirms an earlier electron microscopic study which suggested that meganeurites are of axon hillock origin, and illustrates the striking distinction between abnormalities in the soma-dendritic and axonal domains of neurons in a lysosomal storage disease.     

FADD在脑缺血鼠大脑皮质锥体细胞中表达

目的 :探讨在Fas死亡区内的相关蛋白 (Fas associatedproteinwithdeathdomain ,FADD)在大鼠局灶性缺血损伤中的表达及其与缺血性神经细胞损伤的关系。方法 :用免疫组化法观察大鼠局灶性脑缺血损伤后脑组织中的FADD蛋白免疫反应阳性细胞分布。结果 :脑缺血组大鼠脑内发现FADD的免疫阳性表达细胞以大脑皮质锥体细胞、神经轴突、神经胞体较明显 ,分布于患侧大脑皮质、基底节、海马等部位。结论 :FADD的活化及超量表达可能介导缺血神经细胞信号传导过程 ,并参与了脑缺血神经细胞损伤与修复的病理生理过程     

阿魏酸钠对培养的皮质神经细胞内游离Ca2+的影响

目的 :研究阿魏酸钠对谷氨酸诱导培养的皮质神经细胞损伤的作用。方法 :采用新生大鼠皮质神经细胞原代培养建立谷氨酸神经细胞损伤模型 ,用Ca2 +指示剂Fura 2 /AM检测神经细胞内游离钙浓度 ( [Ca2 +] i)的变化 ,并观察反映神经细胞受损程度的培养液中乳酸脱氢酶 (LDH)的释放量的变化。结果 :阿魏酸钠 40～ 10 0 μmol·L-1能剂量依赖性抑制谷氨酸钠所引起的 [Ca2 +] i 升高及LDH释放。结论 :阿魏酸钠通过抑制谷氨酸钠所引起的 [Ca2 +] i 升高可能是其抗氧化性神经损伤作用的重要机制     

Brief Sensory Deprivation Triggers Cell Type-Specific Structural and Functional Plasticity in Olfactory Bulb Neurons

Can alterations in experience trigger different plastic modifications in neuronal structure and function, and if so, how do they integrate at the cellular level? To address this question, we interrogated circuitry in the mouse olfactory bulb responsible for the earliest steps in odor processing. We induced experience-dependent plasticity in mice of either sex by blocking one nostril for one day, a minimally invasive manipulation that leaves the sensory organ undamaged and is akin to the natural transient blockage suffered during common mild rhinal infections. We found that such brief sensory deprivation produced structural and functional plasticity in one highly specialized bulbar cell type: axon-bearing dopaminergic neurons in the glomerular layer. After 24 h naris occlusion, the axon initial segment (AIS) in bulbar dopaminergic neurons became significantly shorter, a structural modification that was also associated with a decrease in intrinsic excitability. These effects were specific to the AIS-positive dopaminergic subpopulation because no experience-dependent alterations in intrinsic excitability were observed in AIS-negative dopaminergic cells. Moreover, 24 h naris occlusion produced no structural changes at the AIS of bulbar excitatory neurons, mitral/tufted and external tufted cells, nor did it alter their intrinsic excitability. By targeting excitability in one specialized dopaminergic subpopulation, experience-dependent plasticity in early olfactory networks might act to fine-tune sensory processing in the face of continually fluctuating inputs.SIGNIFICANCE STATEMENT Sensory networks need to be plastic so they can adapt to changes in incoming stimuli. To see how cells in mouse olfactory circuits can change in response to sensory challenges, we blocked a nostril for just one day, a naturally relevant manipulation akin to the deprivation that occurs with a mild cold. We found that this brief deprivation induces forms of axonal and intrinsic functional plasticity in one specific olfactory bulb cell subtype: axon-bearing dopaminergic interneurons. In contrast, intrinsic properties of axon-lacking bulbar dopaminergic neurons and neighboring excitatory neurons remained unchanged. Within the same sensory circuits, specific cell types can therefore make distinct plastic changes in response to an ever-changing external landscape.     

A new type of specific interneuron in the monkey hippocampus forming synapses exclusively with the axon initial segments of pyramidal cells

By means of Golgi staining and gold-toning, we have found an interneuron in the pyramidal cell layer of the hippocampus which forms synapses exclusively on the axon initial segments of pyramidal neurons. An individual initial segment receives up to 30 symmetrical synapses from one axo-axonic cell. Each axo-axonic cell is in synaptic contact with the axon initial segments of several hundred pyramidal neurons. This interneuron is thus ideally situated to synchronise the output of a large population of pyramidal cells and so might be involved in the generation of rhythmic activity and in epileptogenesis.     

Intracortical distribution of axonal collaterals of pyramidal tract cells in the cat motor cortex

Slow and fast pyramidal tract cells (Pt cells) from the cat motor cortex were identified antidromically and injected with horseradish peroxidase (HRP). The axonal collaterals of these cells were mapped following HRP histochemistry with benzidine di-hydrochloride. All cells, slow or fast, show a similar arrangement of their collaterals. A proximal axonal network of 0.5–0.8 mm in diameter delimits a local field of action for collaterals in layers V and VI. The tangential expansion of this local field corresponds to that of the basal dendritic domain of Pt neurons. Much longer collaterals running for millimeters in the lower gray or white matter were observed in all cells. They form at a cortical level a distal field of action for Pt neurons. Many of these long branches were traced to other regions of area 4 or toward other cytoarchitectonic areas. In one case a collateral was seen entering and dividing in area 3a. Due to limitations of the HRP technique most of these long branches could not be followed to their terminals. On the basis of the laminar distribution of Pt cell collaterals (mostly in layers V and VI) synaptic sites where recurrent excitation and inhibition are produced on Pt neurons are discussed.     

Conduction-block induced by capsaicin in crayfish giant axon

The effects of capsaicin on the crayfish giant axon were examined by using microelectrode and double sucrose gap-voltage clamp methods. Capsaicin (1–3 × 10⁻⁴ M), when applied externally, had no effect on the resting membrane potential but gradually suppressed the action potential. The rate of rise of the action potential was simultaneously decreased. Voltage clamp experiments revealed the following: capsaicin (10⁻⁴ M) reduced more effectively the transient sodium current than the steady-state potassium current. Inhibition of the sodium current was derived from a decrease in maximum sodium conductance, as the equilibrium potential remained much the same. The inhibitory effects of capsaicin on action potentials and membrane ionic currents were slowly reversible after removal of capsaicin. These results indicate that capsaicin seems to produce a conduction block in the crayfish giant axon due to an inhibition of sodium channels. The significance of these findings is discussed in relation to the sensory neurone-blocking action of capsaicin.     

Distribution of phosphate-activated glutaminase in the human cerebral cortex

Phosphate-activated glutaminase (PAG), which catalyses conversion of glutamine to glutamate, is a potential marker for glutamatergic, and possibly GABA, neurons in the central nervous system. A polyclonal antibody, raised in rabbits against rat brain PAG, was applied to postmortem human brain tissue to reveal the distribution of PAG in the cerebral cortex. PAG immunoreactivity was observed in pyramidal and non-pyramidal neurons but not in glial cells. In the neocortex, large to medium-sized pyramidal neurons in layers III and V were stained most intensely, while the majority of smaller pyramidal cells were labeled either lightly or moderately. Such modified pyramids as the giant Betz cells, the large pyramidal cells of Meynert, and the solitary cells of Ramón y Cajal were also stained intensely. Fusiform cells in layer VI showed moderate to intense labeling. A number of cortical non-pyramidal neurons of various sizes stained moderately to intensely. These included large basket cells which were identified by their characteristic morphology and size in primary cortical areas. Pyramidal cells in the hippocampal formation as well as basket cells of the stratum oriens stained moderately to intensely. Since pyramidal cells are believed to be glutamatergic and large basket cells GABAergic, these results suggest that PAG plays a role in generating not only transmitter glutamate, but also GABA precursor glutamate.     

Pyramidal cell axons show a local specialization for GABA and 5-HT inputs in monkey and human cerebral cortex

Various mechanisms are thought to control excitation of pyramidal cells of the cerebral cortex. With immunocytochemical methods, we found that the proximal portions of numerous pyramidal cell axons (Pyr-axons) in the human and monkey neocortex are immunoreactive for the serotonin (5-HT) receptor 5-HT-(1A). With double-labeling experiments and confocal laser microscopy, we found that most (93.4%) of the 5-HT(1A)-immunoreactive Pyr-axons present in layers II and III were innervated by parvalbumin-immunoreactive chandelier cell axon terminals. In addition, Pyr-axons were compartmentalized: 5-HT-(1A) receptors were found proximal to inputs from chandelier cells. Although we found close appositions between GABAergic chandelier cell axon terminals and Pyr-axons, suggesting synaptic connections, we did not observe 5-HT-immunoreactive fibers in close proximity to the Pyr-axons. These results suggested that Pyr-axons are under the influence of 5-HT in a paracrine manner (via 5-HT-(1A) receptors) and, more distally, are under the influence of gamma-aminobutyric acid (GABA) in a synaptic manner (through the axons of chandelier cells). The local axonal specialization might represent a powerful inhibitory mechanism by which the responses of large populations of pyramidal cells can be globally controlled by subcortical serotonin afferents, in addition to local inputs from GABAergic interneurons.     

Conduction Block in Acute and Chronic Spinal Cord Injury: Different Dose–Response Characteristics for Reversal by 4-Aminopyridine

The effect of the potassium channel blocker, 4-aminopyridine (4-AP), on conduction of action potentials in injured guinea pig spinal cord axons was measured using isolated tracts in oxygenated Krebs' solution at 37°C. The dose–response characteristics of acutely and chronically injured axons were compared. The maximal improvement of conduction occurred in acutely injured axons at a concentration of 100 μM 4-AP, but in chronically injured spinal cord at 10 μM. The threshold for significant response to 4-AP was between 0.5 and 1 μM in chronically injured cords, and between 1 and 10 μM following acute compression injury. The difference in susceptibility to potassium channel blockade may be related to underlying differences in the mechanism of conduction block at the two stages of injury. Initially, junctions between axons and myelin are acutely disrupted, altering primarily the leakage resistance of the myelin sheath and periaxonal space. In chronically injured cords, there is widespread but incomplete process of repair in the lesion site, which leaves many axons partially myelinated. The difference in sensitivity to 4-AP suggests there is also some modification of the accessibility of axonal potassium channel or a change in their affinity for the drug.     

Effects of adrenalectomy, propranolol and methylatropine on the increase in heart rate induced by injection of dermorphin in the rat anterior hypothalamic nucleus

We have examined, at the light microscope level, putative direct synaptic interconnections, via motor axon collaterals, between type-identified triceps surae alpha-motoneurons labeled by intracellular injection of HRP. The results indicate that monosynaptic recurrent contacts can occur between synergist motoneurons (in this case, medial gastrocnemius to soleus, and vice versa), irrespective of motor unit type (type FF to type S, and vice versa).     

Effects of phenytoin on pyramidal neurons of the rat hippocampus

The effectsof phenytoin (35 μg/ml) on membrane properties and inhibitory postsynaptic potentials (IPSPs) in CA1 and CA3 pyramidal neurons of the in vitro rat hippocampus were examined. No significant change was observed on input resistance or resting membrane potential. Action potential amplitude, overshoot, rate of rise and rate of decay were decreased. IPSP conductance increase and reversal potential, evoked in CA3 cells through mossy fiber stimulation and in CA1 cells through recurrent and Schaffer's collateral stimulation, were unaffected.     

Hyperexcitability and Pharmacological Responsiveness of Cortical Neurons Derived from Human iPSCs Carrying Epilepsy-Associated Sodium Channel Nav1.2-L1342P Genetic Variant

With the wide adoption of genomic sequencing in children having seizures, an increasing number of SCN2A genetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrent SCN2A genetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.SIGNIFICANCE STATEMENT A mounting number of SCN2A genetic variants have been identified from patients with epilepsy, but how SCN2A variants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurring SCN2A variant (L1342P) using human iPSC-derived neurons and revealed both intrinsic and network hyperexcitability of neurons carrying a mutant Nav1.2 channel. Importantly, this study recapitulated elements of clinical observations of drug-resistant features of the L1342P variant, and provided a platform for in vitro drug testing. Our study sheds light on cellular mechanism of seizures resulting from a recurring Nav1.2 variant, and helps to advance personalized drug discovery to treat patients carrying pathogenic SCN2A variant.     

The SP1 Antigen in Subplate Neurons of the Developing Cat Cortex is an Immunoglobulin-like Molecule

Monoclonal antibody subplate-l (mAb SP1) specifically stains somata, dendrites and axons of spiny inverted pyramidal neurons in the subplate zone in the early postnatal kitten neocortex. The SP1 antigen has been previously identified as a cytosolic protein of apparent molecular weight 56 kDa. We have now employed immune-affinity chromatography to further characterize this antigen. An antigen with SP1-like immunoreactivity (ir) is present in various organs, and is particularly enriched in blood plasma. Exsanguination of the organs prior to protein extraction reduces the SP1-ir band dramatically, indicative of a blood-borne molecule. The 56 kDa SP1-ir antigen was purified from plasma by affinity chromatography and subjected to Edman degradation. The first 20 N-terminal amino acids show 80% homology to the N-terminus of immunoglobulin heavy chain of man, the mouse and the dog. If the 56 kDa SP1-ir antigen in plasma is an immunoglobulin, and if an immunoglobulin-like molecule is present in the subplate, then antisera against cat immunoglobulins should stain subplate neurons. A polyclonal antiserum against cat IgG intensely stains the somata and dendrites of subplate neurons. On protein blots, this antiserum recognizes the 56 kDa band, and an additional band of ∼27 kDa, corresponding in size to immunoglobulin light chains. Preabsorbing mAb SP1 with cat immunoglobulin G abolishes the immunoreactivity in sections of kitten cortex. Further, it dramatically reduces the reactivity on protein blots. The results suggest that the 56 kDa SP1-ir antigen in cortical subplate neurons belongs to the immunoglobulin superfamily.     

Patterns of intrinsic and associational circuitry in monkey prefrontal cortex

Both local and long-range connections are critical mediators of information processing in the cerebral cortex, but little is known about the relationships among these types of connections, especially in higher-order cortical regions. We used quantitative reconstructions of the label arising from discrete (approximately 350 μm diameter) injections of biotinylated dextran amine and cholera toxin B to determine the spatial organization of the axon collaterals and principal axon projections furnished by pyramidal neurons in the supragranular layers of monkey prefrontal cortex (areas 9 and 46). Both terminals and cell bodies labeled by transport along axon collaterals in the gray matter formed intrinsic clusters which were arrayed as a series of discontinuous stripes of similar size and shape. The co-registration of anterograde and retrograde transport confirmed that these convergent and divergent intrinsic connections also were reciprocal. Transport from the same injection sites along principal axons through the white matter formed associational clusters which were also arrayed as a series of discontinuous stripes. The dimensions of the anterogradely- and retrogradely-labeled associational stripes were very similar to each other and to the intrinsic stripes. These findings demonstrate that divergence, convergence, and reciprocity characterize both the intrinsic and associational excitatory connections in the prefrontal cortex. These patterns of connections provide an anatomical substrate by which activation of a discrete group of neurons would lead to the recruitment of a specific neuronal network comprised of both local and distant groups of cells. Furthermore, the consistent size of the intrinsic and associational stripes (approximately 275 by 1,800 μm) suggests that they may represent basic functional units in the primate prefrontal cortex. © 1996 Wiley-Liss, Inc.     

Reduced excitability in the dentate gyrus network of βIV‐spectrin mutant mice in vivo

The submembrane cytoskeletal meshwork of the axon contains the scaffolding protein βIV‐spectrin. It provides mechanical support for the axon and anchors membrane proteins. Quivering (qv^3j) mice lack functional βIV‐spectrin and have reduced voltage‐gated sodium channel (VGSC) immunoreactivity at the axon initial segment and nodes of Ranvier. Because VGSCs are critically involved in action potential generation and conduction, we hypothesized that qv^3j mice should also show functional deficits at the network level. To test this hypothesis, we investigated granule cell function in the dentate gyrus of anesthetized qv^3j mice after electrical stimulation of the perforant path in vivo. This revealed an impaired input‐output relationship between stimulus intensity and granule cell population spikes and an enhanced paired‐pulse inhibition of population spikes, indicating a reduced ability of granule cells to generate action potentials and decreased network excitability. In contrast, the input‐output curve for evoked field excitatory postsynaptic potentials (fEPSPs) and paired‐pulse facilitation of fEPSPs were unchanged, suggesting normal excitatory synaptic transmission at perforant path‐granule cell synapses in qv^3j mutants. To corroborate our findings, we analyzed the influence of VGSC density reduction on dentate network activity using an established computational model of the dentate gyrus network. This in silico approach confirmed that the loss of VGSCs is sufficient to explain the electrophysiological changes observed in qv^3j mice. Taken together, our findings demonstrate that βIV‐spectrin is required for normal granule cell firing and for physiological levels of network excitability in the mouse dentate gyrus in vivo. © 2009 Wiley‐Liss, Inc.