Morphological changes and expression of protein kinase CK2 β subunit in the microglia after hypoglossal nerve transection

Following hypoglossal nerve transection, the microglia of the rat hypoglossal nucleus expressed protein kinase CK2 β subunit immunoreactivity. CK2 β immunostaining occurred on the operated side from postoperative day 3; on day 5 we observed strong immunoreactivity and the immunopositive microglial cell processes surrounded the injured neurones. Thereafter, the immunoreactivity decreased gradually and on day 10 the immunopositive cells surrounded only a few injured neurones. Electron microscopic observations on the hypoglossal nucleus revealed microglia-neuronal contact within 3 hours of nerve injury, and by day 3 all the injured neurones were in contact with microglial cells. These observations indicated that microglia-neuronal contact occurred earlier than the CK2 β subunit immunoreactivity. CK2 may not be implicated during the initial migration of the microglia to the injured neurones; however, it may enhance the growth and elongation of the microglial cell processes around the injured neurones.     

The nerve impulse in the axon — a new theory

The Classical Theory of function in the nervous system postulates that the nerve impulse is the result of a sequential reversal of the membrane potential due to an increased permeability of the membrane, first to sodium ions, then to potassium ions. The new theory presents a bio-physical model which depicts the nerve impulse as an event involving the motions of electrons and waves, and their interactions with sodium and potassium atoms and ions. The velocity of the nerve impulse (the most important parameter of nerve function) is determined by the product of two constants: c = the speed of light, which is a constant for all nerves; k =a constant for each nerve and is believed to be a specific property of nerve matter related in some way to the atomic process. The theory proposes that the nerve impulse in the axon is dualistic in nature (particles and waves play equally significant roles). The dualistic nature accounts for the three most fundamental characteristics of conduction of the nerve impulse: periodicity (conduction of a nerve impulse over long distances with constant velocity and form); non-summing (two nerve impulses cannot be in the same place at the same time); quantum nature of each nerve impulse — i.e., the unit message of the nerve impulse is an indivisible unit.     

How is the cytoplasmic calcium concentration controlled in nerve terminals?

1. The ability of intraterminal organelles to sequester calcium and buffer the cytoplasmic free Ca2+ concentration ([Ca2+]i) has been investigated in isolated mammalian presynaptic nerve terminals (synaptosomes). A combination of biochemical and morphological methods has been used. 2. When the plasmalemma of synaptosomes is disrupted by osmotic shock or saponin, Ca from the medium can be sequestered by two types of intraterminal organelles in the presence of ATP. 2. Typical mitochondrial poisons (e.g., oligomycin, azide and 2,4-dinitrophenol) block the Ca uptake into one type of organelle (mitochondria); the second type of organelle, which has a higher affinity for Ca (half-saturation congruent to 0.35 microM Ca2+) is spared by the mitochondrial poisons. 4. When the "leaky" synaptosomes are incubated in media containing oxalate, and then fixed and prepared for electron microscopy, electron-dense deposits are observed in the intraterminal mitochondria and smooth endoplasmic reticulum (SER). Mitochondrial poisons block the formation of the deposits in the mitochondria, but spare the SER. 5. X-ray microprobe analysis demonstrates that these deposits contain Ca. 6. Experiments with the Ca-sensitive metallochromic indicator, arsenazo III, demonstrate that the intraterminal organelles in the "leaky" synaptosomes can buffer Ca2+ in the medium to below 5 X 10(-7) M. With small (physiological) Ca loads, the Ca2+ is effectively buffered (to < 5 X 10(-7) M) even in the presence of mitochondrial poisons. 7. The data indicate that the SER in presynaptic terminals may play an important role in helping to buffer the Ca that normally enters during neuronal activity.     

Study of neuronal organization of the “gastric” region in the dorsal motor nucleus of the vagus nerve

The peculiarities of neuronal electrical activity in the dorsal motor nucleus (0.5–2.0 mm rostrally to the obex) were investigated in acute experiments on cats, using the microelectrode technique, under conditions of stimulation of the gastric vagal branches. In the gastric region of the nucleus, two groups of cells responding to nerve stimulation were identified: preganglionic parasympathetic neurons antidromically activated by such stimulation, and cells excited orthodromically with the involvement of afferent fibers of the vagus nerve.Neirofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 190–196, May-June, 1993.     

Can the function of the extirpated amygdaloid complex be compensated for by the transplantation of embryonic nerve tissue?

In 20 white rats bilateral coagulation of the amygdalar complex was produced; on the fifth day to one half of them transplantation was performed by introducing stereotaxically on the left side 0.2-0.5 mm3 of the brain embryonal tissue from the corresponding area of the amygdala of 20-days embryo; in control saline was administered. After two months the rats were sacrificed to determine the activity of antiradical defense by superoxide dismutase (SOD) and the level of lipids peroxide oxidation (LPO) in the cerebral cortex. The transplantation decreased LPO even more and increased SOD as compared to amygdalectomy, e. i. caused still greater deviations from the norm (in this meaning--paradoxal effect), what apparently corresponds to intensification of adaptative-compensatory processes caused by amygdalectomy. The transplantation did not reverse the rats behaviour to the initial one and did not eliminate memory defect in the test of conditioned reaction of passive avoidance (like pyrazetam); it had different direction influence on "drinking under current" in conflict situation, only in particular cases approaching it to the norm.     

Cloning and sequence of the cDNA encoding the β4 integrin subunit in rat peripheral nerve.

β4 and α6 integrin subunits dimerize to form an adhesion receptor that is necessary to nucleate hemidesmosomes and to anchor epithelial cells to their basal laminae. β4 is also expressed in Schwann cells (which do not contain hemidesmosomes) in peripheral nerve, where it may function in the formation or maintenance of myelin. The cDNA for β4 integrin has been cloned from epithelia-derived human and mouse tissues. We cloned cDNAs encoding β4 integrin from libraries derived from rat peripheral nerve, and determined the complete nucleotide sequence encoding the signal peptide and mature protein. Comparison of the deduced amino acid (aa) sequence revealed 95.1% and 87.5% identity with the mouse and human epithelia-derived sequences, respectively. The amino acid sequence of postulated signal transduction domains in β4 was 100% identical among rat, mouse, and human. Our cDNA clones included two of the four postulated alternatively spliced variants previously described in epithelial clones. Despite the potentially diverse functions of β4 integrin in Schwann cells and keratinocytes, the cDNAs for nerve-derived β4 integrin are highly similar to those cloned from epithelia.     

Effect of peripheral nerve on the neurite growth from retinal explants in culture

The effect of peripheral nerve (PN) on neurite outgrowth from retinal explants of adult hamsters was examined.Cultures of retinal explants,and co-cultures of retinal explants and PN were performed using chick retinal basement memebrane (BM) as substrate.The presence of PN increases the number and length of neurite outgrowth.In addition,a high proportion of neurites situated close to PN tend to grow towards it.Since there was no contact between retinal explants and PN,we suggest that PN might secete diffusible substances to attract the neurites to grow towards it.     

Evidence for PHI-immunoreactive nerve fibres in the human skin: coexistence with VIP?

Using indirect immunofluorescence methodology, PHI-like immunoreactivity was found in a certain subpopulation of nerve fibres and terminals of the human skin. The immunoreactive fibres were mainly seen close to and around blood vessels and sweat glands, and they were of a fine-calibre type with smooth preterminal axons and a sparse plexus of varicosities at their terminal field. Furthermore, they were also observed around hair follicles, though more rarely around sebaceous glands. Finally, single PHI immunoreactive fibres could be seen in the close vicinity of the erector pili muscles. These fibres in all probability represent peripheral branches of the autonomic nervous system. Single (somatic?) immunoreactive fibers were, however, also found in the apical parts of the dermis, close to the epidermal-dermal junctional zone. The occurrence of VIP was also analysed and found to be similar to that of PHI. Thus, the present data point to a probable coexistence of PHI and VIP, a possibility that should be taken into account when discussing functional effects of VIP in human skin.     

Modulation of taurine uptake in the goldfish retina and axonal transport to the tectum¶Effect of crushing the optic nerve or axotomy

Summary. Although there are a great number of studies concerning the uptake of taurine in several tissues, the regulation of taurine transport has not been studied in the retina after lesioning the optic nerve. In the present study, isolated retinal cells of the goldfish retina were used either immediatly after cell suspension or in culture. The high-affinity transport system of [³H]taurine in these cells was sodium-, temperature- and energy-dependent, and was inhibited by hypotaurine and β-alanine, but not by γ-aminobutyric acid. There was a decrease in the maximal velocity (V_max) without modifications in the substrate affinity (K_m) after optic axotomy. These changes were mantained for up to 15 days after the lesion. The results might be the summation of mechanisms for providing extracellular taurine to be taken up by other retinal cells or eye structures, or regulation by the substrate taurine, which increases after lesioning the optic nerve. The in vivo accumulation of [³H]taurine in the retina after intraocular injection of [³H]taurine was affected by crushing the optic nerve or by axotomy. A progressive retinal decrease in taurine transport was observed after crushing the optic nerve, starting at 7 hours after surgery on the nerve. The uptake of [³H]taurine by the tectum was compensated in the animals that were subjected to crushing of the optic nerve, since the concentration of [³H]taurine was only different from the control value 24 hours after the lesion, indicating an efficient transport by the remaining axons. On the contrary, the low levels of [³H]taurine in the tectum after axotomy might be an index of the non-axonal origin of taurine in the tectum. Axonal transport was illustrated by the differential presence of [³H]taurine in the intact or crushed optic nerve. The uptake of [³H]taurine into retinal cells in culture in the absence or in the presence of taurine might indicate the existence of an adaptive regulation of taurine transport in this tissue, however taurine transport probably differentially occurs in specific populations of retinal cells. The use of a purified preparation of cells might be useful for future studies on the modulation of taurine transport by taurine in the retina and its role during regeneration. Received June 11, 1999/Accepted August 31, 1999     

Is the concentration of γ-aminobutyric acid in the nerve terminal regulated via product inhibition of glutamic acid decarboxylase?

Fractions of synaptosomes were used to study the regulation of -aminobutyric acid (GABA) synthesis. The isolated synaptosomes were superfused in media of various compositions. [³H]GABA and GABA released into the medium or remaining in the synaptosomes were analyzed by liquid scintillation and HPLC techniques. Different conditions, designed to increase the GABA efflux rate were used: the rate of superfusion was varied and the concentrations of K⁺ and Ca²⁺ were altered. Stimulation of GABA efflux was paralleled with an increased synthesis of GABA, since, in spite of the increased GABA efflux, a relatively constant intraterminal level was found. The findings suggest that the intraterminal concentration of GABA and thus also its synthesis is regulated via product inhibition. In addition, [³H]GABA, exogenous, and GABA, endogenous, responded to external stimulae (Ca²⁺, veretradine, various GABA concentrations and the glutaminase inhibitor diazo-nor-leucine) in a way which was compatible with them being localized in and/or released from different compartments.     

A method for the staining of intraosseous nerve fibers using Sihler’s staining technique

Abstract

Understanding nerve fiber distribution in the jaw bone is important when performing invasive surgical treatments. Both microscopic and macroscopic anatomical techniques have been developed to study innervation. Conventional methods of removing and staining these structures, however, often alter structure and lack reproducibility of the resulting specimens. We sought to optimize Sihler’s staining technique to stain intraosseous nerves in mandibles. Four cadaver specimens were used. The best staining of intraosseous nerve fibers was achieved by using the Plank-Rychlo solution. When the Styrene monomer was used, the resulting transparency was better than that obtained with glycerin under the same conditions. No significant differences were found between Sihler's staining procedure performed according to the conventional method and the procedure in which the second decalcification step was omitted. Our results demonstrate that applying Sihler’s staining technique to bones makes them transparent and allows observation of nerves while preserving the external shape of the bone and maintaining the position of intraosseous nerve fibers. Our findings suggest our Sihler staining method for intraosseous nerve fibers can provide an intermediate resolution between macroscopic and microscopic techniques.     

Decreased biosynthesis of saturated C20–C24 fatty acids by the trembler mouse sciatic nerve

Excised Trembler mouse sciatic nerves synthesize, from acetate, only minute amounts of C20 and C22 saturated fatty acids (about $\text{1}\text{10}$ of the normal value) and almost no lignoceric acid. The elongation activity is localized in the microsomal fraction. The microsomes from Trembler sciatic nerves can elongate stearoyl-CoA into C20, C22 and C24 saturated fatty acids. The elongation rate is only $\text{1}\text{3}$ of the normal value, whereas the stearoyl-CoA hydrolysis is 3 times higher than in the control; the malonyl-CoA concentration remains at the same level in microsomes from normal and trembler sciatic nerves. When ATP-Mg²⁺ is added to the Trembler microsomes, the stearoyl-CoA hydrolysis is reduced, the stearoyl-CoA concentration remains nearly normal and the elongation reaches an almost normal level.     

Embryonic somatic nerve destruction with β-bungarotoxin

Summary The effects and time course of a single injection of -bungarotoxin into E14 rat embryos were examined with an electron-microscopic study of development of the internal intercostal somatic nerve. Within 24 h of injection, axons in this nerve became swollen and fused at points along their length. By 48 h after injection no component of the nerve remained in distal segments of ribcage; complete loss of axons and components of the nerve sheath from proximal regions took slightly longer. At later times, no trace of peripheral nerve axons, Schwann cells or elements of the nerve sheath remained. -Bungarotoxin applied on E17 destroyed developing axons in a similar manner, but the perineurium remained in place, and axons regenerated within the original nerve trunk. The study confirms that sensory and motor neurons are much less able to survive axon degeneration on E14 than after the major period of normal cell death (which is nearly over by E18), and that the maintenance and continued development of the perineurium during E14–E16 depends on the presence of peripheral nerve axons.Supported by the New Zealand Medical Research Council