A quantitative analysis of isotope concentration profiles and rapid transport velocities in the C-fibers of the garfish olfactory nerve

In the olfactory nerve of the long-nosed garfish (Lepisosteus osseus), unusually well-defined isotope concentration distributions can be established with the rapid transport process. Transport velocities of two profile loci can be accurately described and a quantitative profile analysis is possible after profile normalization. Results from such studies indicate that: (1) peak amplitudes decrease exponentially as a function of distance from the olfactory mucosa according to the equation p = 2130 exp (– 0.109x); (2) the wavefront base and the peak apex loci move at rates of 221 ± 2 and 201 ± 4 mm/day, respectively (at 23°C), revealing a peak dispersion or broadening during transport; (3) the broadening is asymmetric with material shifting to the rear of the peak; (4) plateau regions are established behind the peak with material deposited by the peak; (5) only 20% of the total radioactivity in a cut nerve reaches the nerve terminals in the rapid transport peak while 80% is deposited along the axon; (6) profile areas from cut nerves decrease and lose 15% of their activity in 20 hr, while intact nerve profiles increase 10% in 16 hr due to continued somal contribution to the profile; (7) the displacement of the wavefront base (WFB) and peak apex (PA) profile loci can be described by the functions (8) transport velocities are linear functions of temperature between 10 and 25°C and increase 370% in that range. A linear extrapolation of the WFB and PA functions to 37°C yields 410 and 377 mm/day, respectively.     

INFLUENCE OF TEMPERATURE ON THE VELOCITY AND ON THE ISOTOPE PROFILE OF SLOWLY TRANSPORTED LABELED PROTEINS

Abstract— Slow intra-axonal flow of [³H]leucine labeled proteins has been studied in the garfish olfactory nerve. Because of the homogeneity of the nerve a very well defined peak of slowly transported radioactivity is observed. The velocity of slow flow increases linearly with temperature. Between 14 and 28°C, the rate of the peak apex increases from 0.26 to 1.57 mm/day and the rate of the leading edge of the wavefront from 0.54 to 2.75 mm/day. Extrapolation of the rate-temperature function indicates that slow flow should stop at 11°C. However, a velocity of 0.1 mm/day was determined for experiments conducted at 10°C. Between 15 and 25°C a Q ₁₀ of 3.7 was determined for the peak apex and of 3.3 for the leading edge of the wavefront. The Q₁₀'s are significantly larger than the value of 2.2 found for fast transport (G ross & B eidler , 1975) and support the possibility of at least partial differences between the mechanisms of fast and slow transport. A very small peak was found to migrate in front of the main peak. The positioning of this peak seems to be similar to one found by L asek & H offman (1976) in rat ventral motor neurons.
A temperature dependent exponential decrease of the slow moving peak height was measured and it can be estimated that only 1% of the slowly transported radioactivity reaches the synapses. Most of the slow radioactivity appears to remain in the axon behind the peak. The plateau height was also found to decrease exponentially with time. The rate of disappearance greatly affects the profile determined by the slowly transported labeled proteins along the nerve.     

AXONAL TRANSPORT OF SULFATED GLYCOPROTEINS AND MUCOPOLYSACCHARIDES IN THE GARFISH OLFACTORY NERVE

—Application of ³⁵SO₄ to the olfactory mucosa of the long-nosed garfish is found to label sulfated macromolecules which are transported down the olfactory nerve. The transported molecules pass along the nerve as a discrete peak whose leading edge has a transport velocity of 206 ± 6 mm/day. A large portion of the radioactivity from the peak is deposited along the axon. At 2 days after isotope application 83% of the total nerve radioactivity is in the axons and the remaining 17% has accumulated at the terminals in the olfactory bulb. Characterization of sulfated material in the migrating peak indicates that both sulfated glycoproteins (isolated as glycopeptides) and mucopolysaccharides, including chondroitin sulfate and heparan sulfate, are undergoing transport.     

Stop-flow: a new technique for measuring axonal transport, and its application to the transport of dopamine-beta-hydroxylase.

An apparatus was devised which utilizes local cooling to reversibly interrupt the axonal transport of dopamine-beta-hydroxylase (DBH) in rabbit sciatic nerves in vitro. Lowering the temperature of a short region of nerve to between 1 and 3 degrees C, while keeping the remainder at 37 degrees C, caused DBH activity to accumulate in and proximal to the cooled region. This accumulation was evident after 0.5 hr of cooling and increased in a nearly linear fashion with time for about 3 hr. The cooling-induced interruption in transport was rapidly reversed when nerves were rewarmed to 37 degrees C. Upon rewarming after local cooling for 1.5 hr, a peak of accumulated DBH activity migrated toward the distal end of the nerve at a velocity of 300 +/- 17 mm/day. This velocity was maintained for as long as the peak could be followed and was four times greater than the average velocity estimated from the rate of accumulation of DBH activity above a ligature at the distal end of these same nerves. It is concluded that ligation experiments grossly underestimate the true velocity of axonal transport of DBH and that the present technique offers great advantages in permitting direct study of the migration of separate axonal compartments of transported materials.     

Axonal Transport in the Garfish Optic Nerve: Comparison with the Olfactory System

Fast and slow axonal transports were studied in the optic nerve of the garfish and compared with previous studies on the olfactory nerve. The composition of fast-transport proteins was very similar in the two nerves. Although the velocity of fast transport was slightly lower in the optic nerve, there was a linear increase in velocity with temperature in both nerves. As in the olfactory nerve, only a single wave of slow-transport protein radioactivity moves along the nerve. The velocity of slow transport also increased linearly with temperature, but the coefficient was less than in the olfactory system. The composition of slow transport in the optic nerve was significantly different from that in the olfactory nerve, a finding reflecting the different cytoskeletal constituents of the two types of axons. The slow wave could be differentiated into several subcomponents, with the order of velocities being a 105-kilodalton protein and actin greater than tubulins and clathrin greater than fodrin much greater than neurofilaments. It can be concluded that the temperature dependence of fast and slow axonal transport in different nerves reflects the influence of temperature on the individual polypeptides constituting the various transport phases. The garfish optic nerve preparation may be advantageous for studies of axonal transport in retinal ganglion cell axons, because its great length avoids the complications of having to study transport in the optic tract or in material accumulating at the tectum.     

Rapid mechanical and thermal changes in the garfish olfactory nerve associated with a propagated impulse.

Mechanical and thermal changes associated with a propagated nerve impulse were determined using the garfish olfactory nerve. Production of an action potential was found to be accompanied by swelling of the nerve fibers. The swelling starts nearly at the onset of the action potential and reaches its peak at the peak of the action potential. There is a decrease in the length of the fibers while an impulse travels along the fibers. The time-course of the initial heat was determined at room temperature using heat-sensors with a response-time of 2-3 ms. Positive heat production was found to start and reach its peak nearly simultaneously with the action potential. The rise in temperature of the nerve was shown to be 23 (+/- 4) mu degrees C. In the range between 10 degrees and 20 degrees C, the temperature coefficient of heat production is negative, primarily due to prolongation of the period of positive heat production at low temperatures. The amount of heat absorbed during the negative phase varies widely between 45 and 85% of the heat evolved during the positive phase. It is suggested that both mechanical and thermal changes in the nerve fibers are associated with the release and re-binding of Ca-ions in the nerve associated with action potential production.     

Destinations of Some Fast-Transported Proteins in Sensory Neurons of Bullfrog Sciatic Nerve

Many characteristics of proteins that are fast axonally transported have been described, but the destinations of most within the neuron remain unknown. We have studied the destinations of some fast-transported proteins in sensory neurons of the bullfrog sciatic nerve, specifically to determine which may be deposited in axons and which may be destined for more distal, possibly terminal, areas. Dorsal root ganglia were pulse-labeled with [35S]methionine in vitro, following which they were separated from the sciatic nerve. After additional periods of transport, radioactive proteins from two areas of the nerve were separated by two-dimensional polyacrylamide gel electrophoresis and used to develop x-ray film. The first area contained the wavefront of transported radioactivity (wavefront region), whereas the second area was taken from nerve through which the wavefront had already passed (plateau region). The amount of radioactivity in certain fast-transported protein species from each area was determined by computer analysis of digitized video images of fluorographs. Certain proteins were preferentially left behind the wavefront and, therefore, may supply axon and possibly other nerve components, whereas other proteins were found almost exclusively in the wavefront and, hence, may supply more distal, possibly terminal, areas.     

Odor detection ability and thallium-201 transport in the olfactory nerve of traumatic olfactory-impaired mice

Although olfactory nerve damage is a contributing factor in the diagnosis of posttraumatic olfactory loss, at present, there are no methods to directly assess injury to these nerves. We have shown that following olfactory nerve injury in mice, thallium-201 (201 Tl) transport from the nasal cavity to the olfactory bulb decreases. To determine if olfactory function after nerve injury could be assessed with nasal administration of 201 Tl, we measured the correlation between odor detection ability (ODA) and the rate of transport of 201 Tl in olfactory nerves. Both ODA and 201 Tl transport were measured after bilateral olfactory nerve transection for a 4-week period. Cycloheximide solution was used for ODA against tap water. 201 Tl transport was measured as the ratio of radioactivity in the nasal cavity and olfactory bulb with gamma spectrometry. There was a significant correlation between ODA and the rate of 201 Tl transport in the olfactory nerve. These findings suggest that olfactory function after nerve injury can be objectively evaluated with the nasal administration of 201 Tl.     

The intra-axonal transport of acetylcholine and cholinergic enzymes in rat sciatic nerve during regeneration after various types of axonal trauma

The proximo-distal intra-axonal transport of acetylcholine (ACh) and cholinergic enzymes (choline acetyltransferase, CAT, and ACh-esterase, AChE) in rat regenerating sciatic nerve was studied by accumulation technique. Four types of axonal trauma were performed: freezing with solid CO₂, crushing, ligating the nerve with remaining tight silk ligature, and cutting the nerve. Normal and sham-operated rats were used as controls. One to twenty-nine days later, the nerves were crushed about 15 mm proximal to the trauma. The nerve segment proximal to this crush was dissected out 12 hr later and assayed for ACh-content and enzyme activities. The increase in this segment 12 hr after crushing was taken as an indication of proximo-distal transport in the regenerating nerves. ACh transport did not seem to vary during regeneration as compared to controls. In contrast, the transport of both CAT and AChE was initially markedly depressed. Towards the end of the observation period (29 days), a recovery of CAT-transport occurred in all groups. Recovery of AChE-transport was marked in the freeze and crush groups. In the cut group no recovery was seen and in the ligated group only a small recovery occurred. Thus, in the nerves where regeneration was facilitated by the presence of intact connective tissue sheaths (freezing and crushing) recovery of transport occurred earlier than in cut or ligated nerves.     

Thallium transport and the evaluation of olfactory nerve connectivity between the nasal cavity and olfactory bulb

Little is known regarding how alkali metal ions are transported in the olfactory nerve following their intranasal administration. In this study, we show that an alkali metal ion, thallium is transported in the olfactory nerve fibers to the olfactory bulb in mice. The olfactory nerve fibers of mice were transected on both sides of the body under anesthesia. A double tracer solution (thallium-201, (201)Tl; manganese-54, (54)Mn) was administered into the nasal cavity the following day. Radioactivity in the olfactory bulb and nasal turbinate was analyzed with gamma spectrometry. Auto radiographic images were obtained from coronal slices of frozen heads of mice administered with (201)Tl or (54)Mn. The transection of the olfactory nerve fibers was confirmed with a neuronal tracer. The transport of intranasal administered (201)Tl/(54)Mn to the olfactory bulb was significantly reduced by the transection of olfactory nerve fibers. The olfactory nerve transection also significantly inhibited the accumulation of fluoro-ruby in the olfactory bulb. Findings indicate that thallium is transported by the olfactory nerve fibers to the olfactory bulb in mice. The assessment of thallium transport following head injury may provide a new diagnostic method for the evaluation of olfactory nerve injury.     

Autoinhibition of indoleacetic acid transport in the shoots of two-branched pea (Pisum sativum) plants and its relationship to correlative dominance

Two-branched pea plants ( Pisum sativum L. cv. Lisa ZS) with different dominance degrees, obtained by removing the epicotyl shortly after germination, were used to study the interaction between the polar transport of indoleacetic acid (IAA) in both branches of the plants and its relationship to correlative dominance. The dominant shoot had higher transport capacity for 3H-IAA, exported more IAA out of its apex and possessed more endogenous IAA in apex and the first internode than the dominated one. Decapitation of the dominant shoot resulted in a rapid resumption of growth in the dominated shoot, accompanied by a considerable increase in its capacity to export endogenous IAA and to transport 3H-IAA. Parallel experiments with intact two-branched plants and Y-formed explants showed that the 3H-IAA transport on one side was inhibited by the other branch apex or by pre-application of 12C-IAA to the cut stump of the decapitated side. The higher the concentration of 12C-IAA applied to the cut stump of one side of the Y-form explant was used, the stronger the 3H-IAA transport was inhibited and the more the transported IAA was conjugated above the junction on the other side. The results of these experiments support the autoinhibition hypothesis at junctions. The relationship between elongation growth and IAA export/transport in the two-branch pea plants is considered.     

Bidirectional axonal transport of 16S acetylcholinesterase in rat sciatic nerve

Axonal transport of the 16S Molecular form of acetylcholinesterase (16S-AChE) in doubly ligated rat sciatic nerves was studied by means of velocity sedimentation analysis on sucrose gradients. This form of AChE was selectively confined to motor, and not to sensory, fibers in the sciatic nerve, where it represented 3--4% of total AChE. Its activity increased linearly with time (4--20 hr) in nerve segments (7 mm) proximal to the central ligature (4.5 mU/24hr) and distal to the peripheral ligature (2.0 mU/24 hr). From the linear rates of accumulation of 16S-AChE, we conclude that the enzyme is conveyed by anterograde and retrograde axonal transport at velocities close to those previously defined for the movement of total AChE (410 mm/day, anterograde; 220 mm/day, retrograde). The transport of AChE molecular forms, other than the 16S form, could not be resolved presumably due to their presence in blood as well as at extraaxonal sites. The present findings are consistent with the view that in rat sciatic nerve most, if not all, of the small portion of total AChE (approximately 3%) which is transported may be accounted for by 16S-AChE.     

植酸对月季切花瓶插寿命的影响

用0.13％和0.013％植酸对月季切花进行处理,探讨了植酸对月季切花瓶插寿命及衰老过程中一些生理生化指标的影响.结果表明,013％和0.013％植酸处理的切花,瓶插寿命分别延长了2.3 d和1.4d.植酸处理抑制了O-2含量的增加和POD活性的提高,从而减轻了O-2对植物细胞的伤害即抑制丙二醛含量增加,同时抑制了可溶...     

Rapid axoplasmic transport of free leucine

Axoplasmic transport of free 3H-leucine has been studied in vivo in the pike olfactory nerve following application of labeled leucine to the olfactory mucosa. A considerable amount of free 3H-leucine is transported at constant velocity along the axon in the form of a distinct peak. The maximum transport velocity for free 3H-leucine is the same as for rapidly transported 3H-protein (130 and 135 mm/day, respectively, at 19 degrees C). Microtubule inhibitors block or significantly reduce the amount of free 3H-leucine transported, but do not influence the transport velocity. Disruption of the oxygen supply abolishes free 3H-leucine transport, so that this phenomenon cannot be explained by diffusion. The amount of free leucine in the rapidly moving peak decreases with time and distance along the axon and is not detectable after 5 h or more. The transported 3H-leucine is not derived from the circulation or from proteolysis of rapidly transported proteins. This study may help to resolve the controversy over the axoplasmic transport of free amino acids since it shows that free 3H-leucine is transported rapidly but does not travel by rapid axoplasmic transport to the end of axons longer than about 30 mm.     

Transbilayer transport of phosphatidic acid in response to transmembrane pH gradients

Preliminary studies have shown that asymmetric transbilayer distributions of phosphatidic acid (PA) can be induced by transmembrane pH gradients (delta pH) in large unilamellar vesicles [Hope et al. (1989) Biochemistry 28, 4181-4187]. Here the mechanism of PA transport is examined employing TNS as a fluorescent probe of lipid asymmetry. It is shown that the kinetics of PA transport are consistent with the transport of the uncharged (protonated) form. Transport of the neutral form can be rapid, exhibiting half-times for transbilayer transport of approximately 25 s at 45 degrees C. It is also shown that PA transport is associated with a large activation energy (28 kcal/mol) similar to that observed for phosphatidylglycerol. The maximum induced transbilayer asymmetry of PA corresponded to approximately 95% on the inner monolayer for vesicles containing 5 mol % PA.     

Direct comparison of the rapid axonal transport of norepinephrine and dopamine-β-hydroxylase activity

Stop-flow techniques were used to examine the rapid axonal transport of norepinephrine in rabbit sciatic nerves. When the midpoint of a nerve incubated in vitro was cooled to 2°C while the remainder was kept at 37°C, norepinephrine accumulated proximal to the cooled region at a rate corresponding to an average transport velocity between 5 and 6 mm/hr in a distal direction. Since only about half of the norepinephrine appeared to be free to move, the mean velocity of the moving fraction was probably twice as great. No norepinephrine accumulated distal to a broad cooled region under conditions in which there would have been a significant accumulation of dopamine-β-hydroxylase activity. Therefore, unlike dopamine-β-hydroxylase, norepinephrine may not be subject to rapid retrograde transport. When nerves that had been locally cooled for 1.5 hr were rewarmed uniformly to 37°C, a wave of norepinephrine moved exclusively in a distal direction. The peak of this wave moved at a velocity of 12.2 ± 0.5 mm/hr or 293 ± 12 mm/day; the front of the wave moved at about 18 mm/hr. or 430 mm/day; and the tail probably moved faster than 6 mm/hr. This spectrum of velocities was virtually identical to the one displayed by the wave of dopamine-β-hydroxylase activity that was generated under the same conditions. Our results are consistent with the conclusion that all axonal structures containing norepinephrine also contain dopamine-β-hydroxylase, but they are not consistent with the converse.     

Facilitated Transport of Glucose from Blood into Peripheral Nerve

D-Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood-nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D-[14C]glucose, L-[14C]glucose, and [14C]3-O-methyl-D-glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D-glucose. The permeability-surface area product (PA) for D-[14C]glucose and [14C]3-O-methyl-D-glucose decreased, whereas the PA for L-[14C]glucose remained constant, as the perfusate concentration of D-glucose was increased. In the presence of no added unlabelled D-glucose in the perfusate, the PA for L-[14C]glucose equaled one-fifth the PA for D-[14C]glucose. These results demonstrate self-saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D-monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half-saturation constant and maximal rate of transport for the saturable component of D-glucose influx equaled 23 +/- 11 mumol X ml-1 and 6.6 +/- 3.2 X 10(-3) mumol X s-1 X g-1, respectively. The constant of nonsaturable glucose influx equaled 0.5 +/- 0.1 X 10(-4) s-1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D-glucose influx into nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

A light microscopy study of the migration of Naegleria fowleri from the nasal submucosa to the central nervous system during the early stage of primary amebic meningoencephalitis in mice

The migratory pathway of Naegleria fowleri from the nasal submucosa to the central nervous system (CNS) during the early stage of primary amebic meningoencephalitis (PAM) was investigated in mice. Twenty-one-day-old CD-1 mice were inoculated by intranasal instillation of 1 x 10(6) amebas. Animals were divided into 3 groups of 5 and, after being anesthetized, were killed at intervals of 24, 32, and 48 hr postinoculation by transcardial perfusion with formaldehyde, acetic acid, and methanol. The heads were decalcified, divided in the midsagittal plane, and the area of the cribriform plate removed and embedded in paraffin. Serial sections were cut at 8 microm and stained with a combination of celestin blue, Harris' hematoxylin, and acid fuchsin for light microscopy. Focal inflammation and amebas were observed in the submucosal nerve plexus, olfactory nerves penetrating the cribriform plate, and the olfactory bulb of the brain as early as 24 hr postinoculation. The time periods selected assured that the disease process would not obliterate soft tissue structures. Earlier studies used moribund mice in which the inflammation and the number of amebas were overwhelming. The present study provides convincing evidence that amebas gain initial access to the CNS through olfactory nerves within the cribriform plate during the early stages of PAM.     

Postsystolic shortening of ischemic myocardium: a mechanism of abnormal intraventricular filling

Acute myocardial ischemia has been associated with abnormal filling patterns in the left ventricular (LV) apex. We hypothesized that this may in part be due to postsystolic shortening of ischemic apical segments, which leads to reversal of early diastolic apical flow. Fourteen open-chest anesthetized dogs were instrumented with micromanometers in the LV apex and left atrium and myocardial sonomicrometers in the anterior apical LV wall. Intraventricular filling by color Doppler and wall motion by strain Doppler echocardiography (SDE) were assessed from an apical view. Measurements were taken before and after 5 min of left anterior descending coronary artery (LAD) occlusion. In four dogs, we measured the pressure difference between the LV apex and outflow tract. At baseline, peak early diastolic flow velocities in the distal one-third of the LV were directed toward apex (9.2 +/- 1.6 cm/s). After LAD occlusion, the velocities reversed (-2.3 +/- 0.4 cm/s, P < 0.01), indicating that blood was ejected from the apex toward the base during early filling. This interpretation was confirmed by wall motion analysis, which showed postsystolic shortening of apical myocardial segments. The postsystolic shortening represented 9.7 +/- 1.7% (P < 0.01) and 14.2 +/- 2.4% (P < 0.01) of end-diastolic segment length by SDE and sonomicrometry, respectively. Consistent with the velocity changes, we found reversal of the early diastolic pressure gradient from the LV apex to outflow tract. In the present model, acute LAD occlusion resulted in reversal of early diastolic apical flow, and this was attributed to postsystolic shortening of dyskinetic apical segments. The clinical diagnostic importance of this finding remains to be determined.