Axon Hillock Region Research Articles

Thalamic Neuron Resilience during Osmotic Demyelination Syndrome (ODS) Is Revealed by Primary Cilium Outgrowth and ADP-ribosylation factor-like protein 13B Labeling in Axon Initial Segment.

A murine osmotic demyelinating syndrome (ODS) model was developed through chronic hyponatremia, induced by desmopressin subcutaneous implants, followed by precipitous sodium restoration. The thalamic ventral posterolateral (VPL) and ventral posteromedial (VPM) relay nuclei were the most demyelinated regions where neuroglial damage could be evidenced without immune response. This report showed that following chronic hyponatremia, 12 h and 48 h time lapses after rebalancing osmolarity, amid the ODS-degraded outskirts, some resilient neuronal cell bodies built up primary cilium and axon hillock regions that extended into axon initial segments (AIS) where ADP-ribosylation factor-like protein 13B (ARL13B)-immunolabeled rod-like shape content was revealed. These AIS-labeled shaft lengths appeared proportional with the distance of neuronal cell bodies away from the ODS damaged epicenter and time lapses after correction of hyponatremia. Fine structure examination verified these neuron abundant transcriptions and translation regions marked by the ARL13B labeling associated with cell neurotubules and their complex cytoskeletal macromolecular architecture. This necessitated energetic transport to organize and restore those AIS away from the damaged ODS core demyelinated zone in the murine model. These labeled structures could substantiate how thalamic neuron resilience occurred as possible steps of a healing course out of ODS.

Peripheral Hot Spots for Local Ca 2+ Release after Single Action Potentials in Sympathetic Ganglion Neurons

Ca 2+ release from the endoplasmic reticulum (ER) contributes to Ca 2+ transients in frog sympathetic ganglion neurons. Here we use video-rate confocal fluo-4 fluorescence imaging to show that single action potentials reproducibly trigger rapidly rising Ca 2+ transients at 1–3 local hot spots within the peripheral ER-rich layer in intact neurons in fresh ganglia and in the majority (74%) of cultured neurons. Hot spots were located near the nucleus or the axon hillock region. Other regions exhibited either slower and smaller signals or no response. Ca 2+ signals spread into the cell at constant velocity across the ER in nonnuclear regions, indicating active propagation, but spread with a ( time) 1/2 dependence within the nucleus, consistent with diffusion. 26% of cultured cells exhibited uniform Ca 2+ signals around the periphery, but hot spots were produced by loading the cytosol with EGTA or by bathing such cells in low-Ca 2+ Ringer's solution. Peripheral hot spots for Ca 2+ release within the perinuclear and axon hillock regions provide a mechanism for preferential initiation of nuclear and axonal Ca 2+ signals by single action potentials in sympathetic ganglion neurons.

A Dynamic Dendritic Refractory Period Regulates Burst Discharge in the Electrosensory Lobe of Weakly Electric Fish

Na+-dependent spikes initiate in the soma or axon hillock region and actively backpropagate into the dendritic arbor of many central neurons. Inward currents underlying spike discharge are offset by outward K+ currents that repolarize a spike and establish a refractory period to temporarily prevent spike discharge. We show in a sensory neuron that somatic and dendritic K+ channels differentially control burst discharge by regulating the extent to which backpropagating dendritic spikes can re-excite the soma. During repetitive discharge a progressive broadening of dendritic spikes promotes a dynamic increase in dendritic spike refractory period. A leaky integrate-and-fire model shows that spike bursts are terminated when a decreasing somatic interspike interval and an increasing dendritic spike refractory period synergistically act to block backpropagation. The time required for the somatic interspike interval to intersect with dendritic refractory period determines burst frequency, a time that is regulated by somatic and dendritic spike repolarization. Thus, K+ channels involved in spike repolarization can efficiently control the pattern of spike output by establishing a soma-dendritic interaction that invokes dynamic shifts in dendritic spike properties.

Regulation of axonal neurofilament phosphorylation

Publisher Summary Neurofilaments (NFs)—neuron-specific intermediate filaments—are the major cytoskeletal components of large myelinated axons; over 95% of axonal volume is occupied by NFs. In mature neurons, NFs are composed of three polypeptide subunits identified by their molecular weights as low, NF-L (68,000); medium, NF-M (95,000); and high NF-H (115,000). All three subunits share, with other intermediate filament proteins, a highly conserved α-helical central coiled coil rod domain flanked by an N-terminus head domain and a C-terminus hypervariable tail domain. The central rod domain, because of its highly a-helical coiled-coil structure, is responsible for forming long 10-nm diameter cylindrical polymers. After the biosynthesis of NF proteins in the perikaryon cell body and filament assembly in the axon hillock region, NFs are transported by slow axonal transport in the axon, where the NF-M and NF-H tail domains are extensively phosphorylated. The extent of phosphorylation, particularly of NF-H and the resulting side arm extension, correlates directly with axon caliber and conduction velocity.

Patch-Clamp Recordings from Cerebellar Basket Cell Bodies and Their Presynaptic Terminals Reveal an Asymmetric Distribution of Voltage-Gated Potassium Channels

Cerebellar basket cells form highly specialized inhibitory synaptic contacts with Purkinje cells, namely the pericellular basket and pinceau nerve terminal structures, wrapping around the Purkinje cell somatic and axon hillock regions. These inhibitory synaptic contacts are ideally located to control the ultimate output of the cerebellar cortex. Previous immunohistochemical studies have shown that these synaptic structures possess a very high density of the dendrotoxin (DTX)-sensitive potassium channel subunit, Kv1.2. We have taken advantage of this unique anatomical arrangement offering a high concentration of identified Kv channel subunits by combining whole-cell patch-clamp recording and fluorescence microscopy to establish a novel preparation and perform the first recordings from unambiguously identified mammalian CNS inhibitory presynaptic terminals. We report that DTX-sensitive potassium channels are present in basket cell terminals but not in the basket cell soma. This selective cellular distribution suggests that these channels play an important role in modulating cerebellar inhibitory synaptic transmission.

Phosphorylation at a Single Site in the Rat Brain Sodium Channel Is Necessary and Sufficient for Current Reduction by Protein Kinase A

Voltage-gated sodium channels respond to excitatory inputs in nerve cells, generating spikes of depolarization at axon hillock regions and propagating the initial rising phase of action potentials through axons. It previously has been shown that protein kinase A (PKA) attenuates sodium current amplitude 20-50% by phosphorylating serines located in the I-II linker of the sodium channel. We have tested the individual contributions of five PKA consensus sites in the I-II linker by measuring sodium currents expressed in Xenopus oocytes during conditions of PKA induction. PKA was induced by perfusing oocytes with a cocktail that contained forskolin, chlorophenylthio-cAMP, dibutyryl-cAMP, and 3-isobutyl-1-methylxanthine. Phosphorylation at the second PKA site (serine-573) was necessary and sufficient to diminish sodium current amplitude. Phosphorylation at the third and fourth positions (serine-610 and serine-623) reduced current amplitude, but the effect was considerably smaller at those positions. Introduction of a negative charge at site 2 by substitution of serine-573 with an aspartate constitutively reduced the basal level of sodium current, indicating that the attenuation of sodium current by phosphorylation of site 2 by PKA results from the introduction of a negative charge at this site.

Selective expression of glionexin, a glial glycoprotein, in insect mechanoreceptors

The distribution of a glial cell-associated glycoprotein, glionexin (GX), on sensory receptors of the adult cricket Acheta domesticus is described, using the monoclonal antibody 5B12 as an immunohistochemical probe. GX was previously shown to be widely distributed in the embryo and to persist in the postembryonic to adult central nervous system. Here we demonstrate that it is restricted in the adult periphery to three subclasses of mechano-receptor sensilla: large socketed hair mechanoreceptors, their associated campaniform sensilla, and chordotonal organs. GX was not detected in photoreceptors, chemoreceptors, or other mechanoreceptors. The pattern of distribution differs significantly within the three subclasses of mechanoreceptors. In the hair and campaniform receptors GX is restricted to the extracellular space among glial cells clustered around the axon hillock region, but in chordotonal organs it surrounds the scolopidium at the tip of dendrites. The highly restricted distribution of GX in the periphery suggests possible functions that include mechanical stability of the sensory apparatus and ionic homeostasis in the respective neuronal spike-generating regions. The developmental modulation of GX expression is taken to imply multiple functions for the molecule during the life of the insect.

Cellular and molecular approaches to neural repair in the medicinal leech

Investigation of repair processes and the effects of damage in the leech has the advantage that it is possible to work with single neurons, and that leech neurons can regenerate. The effects of surface topology on neuronal outgrowth in culture were investigated using patterned substrates produced by photolithography. On grooved substrates with a depth, width and separation of 2 μm, the processes of cultured neurons showed significant alignment. In studies on regeneration, different mechanosensory neurons were shown to respond differently to lesions of their axons. High threshold units (N cells) respond by sprouting of additional processes from the axon hillock region within the CNS, whereas low threshold cells respond with a sprouting only at the lesion site in the nerve root and not within the CNS. Sprouted processes are retained for over a year. When a neuron of a particular modality is killed, cells of the same modality (but not the other) expand their receptive fields to cover the denervated skin. Single channel patch clamp recording experiments have been initiated on damaged neurons. Experiments are in progress to clone genes whose expression is regulated after perhipheral nerve injury.

Cytoplasmic segregation and cytoskeletal organization in the electric catfish giant electromotoneuron with special reference to the axon hillock region

The cytoplasm of the highly polarized nerve cell is permanently segregated into domains with differing organellar composition. The mechanisms maintaining this segregation are largely unknown. In order to elucidate the potential role of cytoskeletal elements in this process we compared the cytoplasmic segregation within the giant electromotoneuron of the electric catfish ( Malapterurus electricus) with the distribution of binding sites for antibodies against elements of the cytoskeleton. Most prominent cytoplasmic segregations include the formation of a subplasmalemmal cortical structure free of Nissl bodies and Golgi cisternae, the separation within the soma of domains containing rough endoplasmic reticulum and filament-rich domains, and the soma-axon transition. The cytoplasmic transition at the axon hillock forms a distinct borderline where Nissl bodies, Golgi cisternae and the bulk of lysosomes abruptly terminate and are excluded from the axoplasm. Synaptic vesicles and mitochondria are free to pass compartmental borders. Tropomyosin, spectrin, and α-actinin reveal a rather homogeneous immunofluorescence throughout the neuron. In contrast, neurofilament protein and tubulin display a distinctly increased immunofluorescence in the subplasmalemmal cortical layer, in dendrites as well as in the axon. The increase in immunofluorescence at the axon hillock exactly depicts the small transition zone from the somatic cytoplasm rich in Nissl bodies, Golgi cisternae and lysosomes to the differently structured axoplasm. The picture is similar for β-tubulin, tyrosinylated and detyrosinylated α-tubulin. Detyrosinylated tubulin (glu-tubulin, which is contained in microtubules of increased stability) shows the most prominent enrichment in the axon. The distribution of myosin is comparable to that of neurofilament protein but there is less difference in immunofluorescence between the domains. Our results would be compatible with a role of microtubules together with (the closely associated) neurofilaments in the segregation of neuronal cytoplasmic domains. Active transport as well as stable binding to the somatic cytoskeleton might counteract a homogeneous cytoplasmic distribution of the various classes of organeiles by diffusion.

Ultrastructure and organization of the epineural canal and the nerve cord in sea urchins (Echinodermata, Echinoida)

The radial nerve cord ofMespilia globulus has been examined as an example of echinoid nerve cords. In the radius of echinoids only the ectoneural component of the nerve cord is present which is a derivative of the ectoderm. The nerve cord runs in the interior of the body and is accompanied by the epineural canal. In echinoids, the neuroepithelium makes up the upper and side walls of the epineural canal. Each lateral branch of the nerve cord forms a sort of neural tube. It encloses a branch of the epineural canal which represents an open connection with the sea water. Thus, the epineural canal exhibits numerous openings which probably allow sea water to flow back and forth. This organization is unique in echinoderms. — The neuroepithelium exhibits the organization of an epidermis with well-developed nervous elements. Glial cells are not present. The support cells are the true epithelial cells. Their monociliated cell bodies border the lumen and, by means of cytoplasmic stems that contain a bundle of filaments, they reach up to the basal lamina. The nerve cells and their trunk of nerve fibres fill the spaces between the support cells. — Three types of nerve cells can be distinguished according to their polarity: (1) Primary sensory cells that project a cilium into the epineural canal, the axon hillock region is at the opposite pole. (2) Subluminal cells whose cilium originates in the axon hillock region. (3) Neurones that lie within the trunk of nerve fibres. They are highly stretched in the direction of the nerve cord and are also provided with a cilium. Types 2 and 3 may be homologized with the basal nerve cells of the epidermis. They are possibly multipolar. — The lateral nerve cords make contact with the ampulla and pass the ambulacral plate parallel to the channel that connects the ampulla and the tube foot. The activity of the tube foot-ampulla system is possibly controlled by means of transmitter substances that diffuse through the connective tissue layer between the nerve cord and the myoepithelia of the ampulla and the tube foot respectively.

New growth elicited in adult leech mechanosensory neurones by peripheral axon damage.

1. New growth in cutaneous mechanosensory neurones elicited by axotomy or axon crush was studied using intracellular injection of horseradish peroxidase at different times after the lesion, ranging from a few days to over a year. 2. Cutting or crushing major, large-calibre axon branches of mechanosensory neurones elicits sprouting of new processes, either centrally within the ganglion neuropile or at the site of the lesion in the peripheral nerve. In contrast, cutting or crushing fine-calibre axon branches supplying accessory parts of the receptive field does not elicit sprouting of the main arbor or main axon branches. 3. Different modalities of mechanosensory neurone respond differently to lesions of their axons. Cutting the axons of high-threshold units responding to noxious stimulation of the skin elicits sprouting of additional processes from the axon hillock region within the central nervous system (CNS), whereas cutting or crushing the axons of low-threshold cells responding to light touch of the skin elicits sprouting at the site of the lesion only, and not within the CNS. 4. In addition to the new growth directed into the peripheral nerve, damaged nociceptive neurones also form new processes that wrap the somata of particular cells within the ganglion. 5. Sprouted processes of axotomized neurones are retained for long periods after the lesion (up to 425 days). 6. The electrical properties of touch and nociceptive cells were studied between 1 and 60 days after axotomy, by intracellular recording from the centrally located cell bodies. The amplitude, width and maximum dV/dt of the action potential and after-hyperpolarization, as well as the resting potential and input resistance, did not change significantly after axotomy, despite the considerable process sprouting known to occur during this time.

Functional morphology of insect neuronal cell-surface/glial contacts: the trophospongium.

Ultrastructural studies were carried out on the surfaces of insect nerve cell bodies. Some of the neurons were identified, by using physiological criteria, before filling with dye. Their surface patterns were compared, to provide data needed for understanding dynamic relationships with glial cells, in the trophospongium. The data are also needed in connection with interpretation of electrical signals recorded from the somata and of their roles in integration and in learning and memory. The surfaces were found to be extremely complex and also varied, even for neurons of comparable size and function, as well as for different regions of the same neuron, suggesting that the surface is constantly changing as the neuron receives food and loses waste. There is a variety of cytoplasmic types of invagination of neuron somata by glial processes. The invaginations were classified into four easily recognized types: regular, chunky, filigree, and ridge (present only in axon hillock regions). Motor neurons also make reciprocal invaginations into the glial cells that surround them. Some of these extend for distances up to 40 microns from the surface. The effective surface area is increased, compared with that calculated for a smooth surface, as a result of the invaginations, by from as little as 5% for a small interneuron to as much as 12-fold for a large motor neuron. The axon hillock region of all types of neurons is heavily invaginated.

Scanning electron microscopic studies of bullfrog sympathetic neurons exposed by enzymatic removal of connective tissue elements and satellite cells.

The ninth and tenth abdominal sympathetic ganglia of bullfrogs were studied by light microscopy and transmission and scanning electron microscopy after the removal of the connective tissue elements overlying the neurons. Digestion of tissues with trypsin and subsequent acid hydrolysis exposed the unipolar neurons, which remained covered by their satellite cells. The preganglionic innervation was visible on the proximal segment and axon hillock region of the postganglionic neurite. Clusters of small cells seen at the periphery of ganglia probably corresponded to groups of cells with abundant catecholamine-containing granules (SIF cells). Digestion with collagenase and protease removed some or all of the satellite cells in addition to the connective tissue. The true neuronal surfaces had short finger-like processes, whereas the external surfaces of satellite cells were smooth. Preganglionic nerve varicosities were clearly visible on the proximal segment of the postganglionic neurite, on the axon hillock and on the cell body of neurons. A few axonal varicosities were fractured to reveal the synaptic vesicles within. The possible effects of the distribution and glial ensheathment of nerve varicosities on their function are discussed.

Ectopic dendritogenesis occurs on cortical pyramidal neurons in swainsonine-induced feline α-mannosidosis

Golgi staining has revealed that ectopic neurites sprout from the axon-hillock region of cortical pyramidal neurons in kittens with swainsonine-induced α-mannosidosis. These new growth processes appeared qualitatively identical to those reported in a variety of inherited neuronal storage diseases including the gangliosidoses. In the latter, such processes have been shown to be dendritic-like and postsynaptic to afferents of unknown origin. We believe this to be the first demonstration of induced dendritogenesis in the mammalian CNS and a model system which should prove useful in exploring this remarkable phenomenon occurring in neuronal storage disorders.

Development of the axon cap neuropil of the Mauthner cell in the goldfish.

Development of the axon cap neuropil of the Mauthner neuron in post-hatching larval goldfish brains was observed electron-microscopically. The axonal initial segment of newly hatched (day-4) larvae is completely covered with synaptic terminals containing clear spherical synaptic vesicles. Profiles of thin terminal axons, the spiral fibers, containing similar synaptic vesicles, rapidly increase in number around the initial segment and form glomerular neuropil similar to the central core of the adult axon cap by day 7. Three types of synapses are formed in the core neuropil. Bouton-type synapses contacting the initial segment are most abundant in day-4 to -14 larvae; they decrease thereafter and are rare on the distal half of the initial segment of day-40 larvae. Asymmetric axo-axonic synapses are commonly observed between spiral fibers in the core neuropil of day-7 to -19 larvae, but become fewer by day 40. Unique symmetrical axo-axonic synapses showing accumulation of synaptic vesicles on either side of apposed membrane thickenings first appear in day-14 core neuropil, gradually increase in number, and become the predominant type in day-40 core neuropil. Thick myelinated axons, which lose their myelin sheaths in the glial cap cell layer, start to penetrate into the axon cap on day 10. They gradually increase in number and form the peripheral part of the axon cap together with the cap dendrites, which finally grow into the axon cap from the axon hillock region of the Mauthner cell by day 40.

Synaptic input to the axon hillock and initial segment of inhibitory interneurons in the cerebellar cortex of the rat. An electron microscopic study.

The axon hillock (AH) and initial segment (IS) of 10 Golgi neurons and 6 basket cells in the cerebellar cortex of the rat were investigated by electron microscopy using serial sections. An average of 10.4 and 11.3 synaptic terminals were observed to establish synaptic contact with the axon hillock region of Golgi and basket cells, respectively. Most of these terminals were identified as the varicosities of the ascending parallel fibers. It is suggested that the focal innervation of AH regions represents an excitatory input pattern which is basically different from the randomly distributed, huge, parallel-fiber input onto the dendritic trees of Golgi and basket cells. In contrast to Golgi and basket neurons, no accumulation of parallel-fiber synapses was observed around the AH of stellate cell. The IS proper of the three neuronal types were devoid of true axo-axonal synapses.

Unequal distribution of calcium and magnesium of snail neuron.

Ca and Mg contents of snail neuron differ depending on the cell type. Ca and Mg near the cell membrane are not equally distributed in the isolated neuron. Ca is almost twice as dense in the axonhillock region than in the cell body. The Mg distribution pattern is the reverse of that of Ca.

Fine structure of meganeurites and secondary growth processes in feline G M1-gangliosidosis

Electron microscope studies were carried out on neurons of the hippocampal formation in a feline mutant with β-galactosidase deficiency and G M1-gangliosidosis. Fusiform processes with characteristics similar to meganeurites of Golgi studies 11 were identified between cell bodies and axons of pyramidal and granule cells. The presence of dense material subjacent to the plasma membrane at the meganeurite-axon junction provides evidence that meganeurites form at the axon-hillock region and displace the initial axonal segment distally. Meganeurites of hippocampal neurons exhibited pleomorphic secondary processes with fine structural features of growth cones. Spines and spine-synapses were abundant on perikarya and meganeurites. Numerous membranous cytoplasmic bodies (MCBs) were encountered amongst otherwise normally appearing organelles of the cell body. MCBs were densely packed in meganeurites except near their peripheral area. They were less common in dendrites and rare in synapses of the neuropil. The observations provide further support for the view that meganeurites of mature cortical neurons in ganglioside storage diseases have embryonic growth characteristics.

Postsynaptic specializations at excitatory and inhibitory cholinergic synapses.

In serial sections of neurons in the paravertebral ganglia of the frog (Limmodynastes dumerili), the postsynaptic structures termed 'postsynaptic bar' (PSB) and 'junctional subsurface organ' (JSO) were never observed in the same ganglion cell. Further, PSBs were found mostly in small ganglion cells (less than 22 micrometer), while JSOs were found mostly in large ganglion cells (up to 45 micrometer). Between 10 and 22 PSBs were located at both 'spine' and 'non-spinous' somatic synapses of the smaller ganglion cells; while 8 to 16 JSOs were located largely in the axon hillock region of the larger ganglion cells. Based on these observations, it is suggested that the two ganglion cell populations represent the B and C cell types defined according to electrophysiological data. Further, since the nerve terminals adjacent to both these postsynaptic structures appear to be cholinergic according to their vesicular content, this provides some basis for suggesting that JSOs are associated with slow excitatory synapses, while PSBs are present at slow inhibitory synapses.

Synaptic innervation of sympathetic ganglion cells in the bullfrog

The synaptic innervation of the ganglion cells in the ninth and tenth para-vertebral sympathetic ganglia of the bullfrog was investigated by histochemical and electron microscopic techniques and by intracellular recording. The neurons were unipolar and most ganglion cells were innervated by a single preganglionic axon. The preganglionic fiber stained for acetylcholinesterase and was observed to spiral around the axon hillock of the ganglion cell before arborizing and making synaptic contact with the neuron. Most synapses were located on the soma near the axon hillock region, with features typical for cholinergic junctions. The axosomatic location of the synapses was manifested physiologically by a decrease in membrane resistance (increased conductance) at the peak of the fast EPSP (excitatory postsynaptic potential) and by a demonstrable reversal potential for the fast EPSP.