The distribution of monoamine oxidase and acetylcholinesterase in the brain of Xenopus laevis tadpoles

Summary The distribution of monoamine oxidase (MAO) in the brain of Xenopus laevis tadpoles (stage 52–56) was studied histochemically with a modified Glenner's tryptamine-tetrazolium method. A moderate activity was observed in fibre regions of the striatum and septum (including the medial and lateral forebrain bundles), in the neuropil of the nucleus amygdalae, in the commissura anterior and commissura hippocampi, in the fibre regions of the diencephalon (including the optic chiasma), in the fibre regions of the tectum opticum and the tegmentum of the mesencephalon and in the white substance of the ventral half of the medulla oblongata. A greater MAO activity was found in the neuropil of the entire nucleus praeopticus. In the partes anterior and magnocellularis of this nucleus, MAO positive fibres are present in close contact with the perikarya, indicating a monoaminergic innervation of these neurons. The perikarya themselves did not show MAO activity. In the neurons of the nucleus praeopticus epichiasmaticus, the paraventricular organ (PVO) and nucleus infundibularis dorsalis (NID), only a slight MAO activity has been demonstrated in the perikarya, whereas a strong MAO positivity was found in the intraventricular protrusions and the neuropil. These data indicate the aminergic character of the neurons of these nuclei. From the postoptic fibre region a MAO positive tract was observed towards the developing median eminence and pars intermedia of the hypophysis. The pars nervosa and some cells of the pars distalis also contained MAO. Along the border of the aquaeduct of Silvius and the fourth ventricle, MAO positive liquor-containing neurons are also present.The distribution of acetylcholinesterase (AChE) was investigated in the hypothalamohypophysial region. AChE activity was found in the neuropil of the nucleus praeopticus magnocellularis, in the fibres of the optic chiasma and in the postoptic fibre region. The neurons of the PVO and NID were AChE negative. An AChE positive tract could be traced from the postoptic fibre region to the developing median eminence and pars nervosa. The pars distalis did not show AChE activity. However, in tadpoles reaching the metamorphic climax, ChE activity appeared in certain cells of the pars distalis; this might be related to degenerative phenomena in the acidophilic cells. The absence of AChE activity in the pars intermedia indicates a regulation of MSH release by peptidergic nerves to be unlikely.The stimulating interest and helpful advice of Prof. Dr. P. G. W. J. van Oordt is gratefully acknowledged. Thanks are also due to Mr. H. van Kooten and his co-workers for making the photographs.     

Oxidative modifications of cardiac mitochondria and inhibition of cytochrome c oxidase activity by 4-hydroxynonenal

Abstract

4-Hydroxynonenal (HNE) is a highly toxic product of lipid peroxidation (LPO). Its role in the inhibition of cytochrome c oxidase activity and oxidative modifications of mitochondrial lipids and proteins were investigated. The exposure of mitochondria isolated from rat heart to HNE resulted in a time- and concentration-dependent inhibition of cytochrome c oxidase activity with an IC₅₀ value of 8.3 ± 1.0 μM. Immunoprecipitation-Western blot analysis showed the formation of HNE adducts with cytochrome c oxidase subunit I. The loss of cytochrome c oxidase activity was also accompanied by reduced thiol group content and increased HNE-lysine fluorescence. Furthermore, there was a marked increase in conjugated diene formation indicating LPO induction by HNE. Fluorescence measurements revealed the formation of bityrosines and increased surface hydrophobicity of HNE-treated mitochondrial membranes. Superoxide dismutase + catalase and the HO^? radical scavenger mannitol partially prevented inhibition of cytochrome c oxidase activity and formation of bityrosines. These findings suggest that HNE induces formation of reactive oxygen species and its damaging effect on mitochondria involves both formation of HNE–protein adducts and oxidation of membrane lipids and proteins by free radicals.     

Comparison of calbindin D 28K and cytochrome c oxidase in electrosensory nuclei of high- and low-frequency weakly electric fish (Gymnotiformes)

Summary Weakly electric fish (Gymnotiformes) emit quasi-sinusoidal electric organ discharges within speciesspecific frequency ranges. The electrosensory system is organized into 2 parallel pathways which convey either the amplitude or the timing of each electric organ discharge cycle. Two putative metabolic activity markers, calbindin D 28K and cytochrome c oxidase, and their relationship with the electrosensory nuclei of high- and low-frequency species were studied. Calbindin is found in the somata of the spherical neurons in the first-order electrosensory recipient nucleus, the electrosensory lateral-line lobe, and in layer VI of the midbrain's torus semicircularis, in Eigenmannia virescens, an intermediate-frequency species, and Apteronotus leptorhynchus, a high-frequency species. Calbindin immunoreactivity was completely absent in these nuclei in Sternopygus macrurus, a closely related, low-frequency species. Cytochrome c oxidase levels were inversely related to calbindin immunoreactivity since relatively high levels were observed in the electrosensory lateral-line lobe and torus semicircularis of S. macrurus but were absent in these nuclei in A. leptorhynchus. Our studies indicate that calbindin immunoreactivity is present in tonic, repetitively firing neurons with high frequencies.     

Functional activity and ultrastructure of mitochondria isolated from myocardial apoptotic tissue

Apoptosis in myocardial tissue slices was induced by extended incubation under anoxic conditions. Mitochondria were isolated from the studied tissue. A new method of isolation of mitochondria in special conditions by differential centrifugation at 1700, 10,000, and 17,000g resulted in three fractions of mitochondria. According to the data of electron microscopy the heavy mitochondrial fraction (1700g) consisted of mitochondrial clusters only, the middle mitochondrial fraction (10,000g) consisted of mitochondria with typical for isolated mitochondria ultrastructure, and the light fraction consisted of small mitochondria (2 or 3 cristae) of various preservation. The heavy fraction contained unusual structural elements that we detected earlier in apoptotic myocardial tissue—small electron-dense mitochondria incorporated in bigger mitochondria. The structure of small mitochondria from the light fraction corresponded to that of the small mitochondria from these unusual elements—mitochondrion in mitochondrion. The most important functions of isolated mitochondria are strongly inhibited when apoptosis is induced in our model. The detailed study of the activities of the two fractions of the apoptotic mitochondria showed that the system of malate oxidation is completely altered, the activity of cytochrome c as electron carrier is partly inhibited, while succinate oxidase activity is completely preserved (complexes II, III, and IV of the respiration chain). Succinate oxidase activity was accompanied by high permeability of the internal membrane for protons: the addition of uncoupler did not stimulate respiration. ATP synthesis in mitochondria was inhibited. We demonstrated that in our model of apoptosis cytochrome c remains in the intermembrane space, and, consequently, is not involved in the cascade of activation of effector caspases. The possible mechanisms of induction of apoptosis during anoxia are discussed.     

The localisation of succinic dehydrogenase in the muscles of Nereis virens and Homarus gammarus

Summary The localisation of succinic dehydrogenase and cytochrome oxidase in body muscles of Nereis virens and in tail muscles of Homarus gammarus was studied. Pig heart muscle was used for some comparisons.Electron microscopic studies on tissue sections generally showed well developed and independent mitochondria in Homarus gammarus. A lower degree of independence characterised the less developed mitochondria of Nereis virens.Sections were stained with nitro-BT. Light microscopic studies showed a distinct and selective staining of the mitochondria in sections of Homarus gammarus. In addition to the few mitochondria of Nereis virens strings within the cytoplasm became distinctly blue. Electron microscopic studies on Nereis virens showed a higher electron density along the membranes of the vesicular sarcotubular system in incubated than in non-incubated sections.The fractions obtained on centrifugation of the homogenised tissues were used for combined enzyme studies and electron microscopic investigation. Similarly prepared fractions from the two invertebrates showed a similar electron microscopic appearance. The supernatants obtained by centrifugation at 12,000 g for 10 minutes contained vesicles different from the majority of those in the mitochondrial fractions. These supernatants had rather considerable activities of succinate-cytochrome c reductase and of cytochrome c oxidase. The activity of succinate-cytochrome c reductase was most pronounced in the supernatants of Nereis virens and much greater than the cytochrome c oxidase activity in these fractions. The ratio between succinate-cytochrome c reductase activity and cytochrome c oxidase activity in the supernatants of Nereis virens was about three times that in the corresponding fractions of Homarus gammarus.Manometric studies were performed to get the effect of added succinate on the O₂ uptake of the supernatants obtained by centrifugation at 12,000 g for 10 minutes. A distinctly larger increase in oxygen consumption characterised the supernatants of Nereis virens.The results presented indicate the occurrence of an extra-mitochondrial succinic dehydrogenase in Nereis virens. This conclusion was related to the occurrence of alternative oxidative systems in the muscles of this invertebrate.The literature dealing with an extra-mitochondrial localisation of succinic dehydrogenase is briefly reviewed as well as the electron microscopic studies concerning transformations between the membrane structures of cells.     

Mitochondrial polymorphism. III. Heterosis,complementation, and spectral properties of purified cytochrome oxidase of wheat

Cytochrome oxidase was purified twentyfold from mitochondria of seedlings of wheat genotypes 28, 31 MS, and 31 MS/28. The enzyme of the hybrid exceeded in activity the parental enzymes. Mixtures of cytochrome oxidase of the parents exhibited complementation in that they approached the activity of the hybrid cytochrome oxidase. Hybrid mitochondria also exhibited heterosis in NADH: cytochrome c reductase activity. Complementation by parent mitochondria was observed for this enzyme also. The Michaelis constant of cytochrome oxidase and NADH: cytochrome reductase was markedly less in the hybrid and the mixture than in the parents. Difference spectra revealed the following: strain 28 had cytochromes a and b but was deficient in cytochrome c; strain 31 MS had cytochromes b and c but no a; the hybrid had all three cytochromes, as did the mixture. The relationship of cytochromes to heterosis and complementation is considered.This work was supported by DeKalb AgResearch, Inc.     

Oxidative processes in carp liver mitochondria during adaptation to changes of CO2 concentration in water

Two-years-old carps (Cyprinus carpio L.) were kept for 1,3 and 7 days in the medium with different concentrations of CO2. In the fish liver mitochondria the rate of CO2 uptake (state 4p) and phosphorylation (state 3) as well as the value of the respiratory control and the activity of cytochrome oxidase and succinate dehydrogenase undergo significant changes. In fishes which were in the medium with 0.4 mM CO2 for a day the cytochrome oxidase activity in the mitochondria increases sharply, but that of succinate dehydrogenase decreases. The lowest activity of both enzymes is fixed in the carp liver mitochondria during the three-day adaptation to such a medium. In the first day in the medium with 0.4 mM CO2 the rate of O2 uptake by the mitochondria in the states 4p and 3 rises. Seven days later the values of all the studied indices in the mitochondria reach gradually the control level. In the medium with 0.8 mM CO2 the oxidative processes in the mitochondria are more inhibited than in water with 0.4 mM CO2.     

Enhanced formation of methane in plant cell cultures by inhibition of cytochrome c oxidase

The claim of methane (CH₄) formation in plants has caused much controversy and debate within the scientific community over the past 4 years. Here, using both stable isotope and concentration measurements, we demonstrate that CH₄ formation occurs in plant cell cultures that were grown in the dark under sterile conditions. Under non‐stress conditions the plant cell cultures produced trace amounts [0.3–0.6 ng g^?1 dry weight (DW) h^?1] of CH₄ but these could be increased by one to two orders of magnitude (up to 12 ng g^?1 DW h^?1) when sodium azide, a compound known to disrupt electron transport flow at the cytochrome c oxidase (complex IV) in plant mitochondria, was added to the cell cultures. The addition of other electron transport chain (ETC) inhibitors did not result in significant CH₄ formation indicating that a site‐specific disturbance of the ETC at complex IV causes CH₄ formation in plant cells. Our study is an important first step in providing more information on non‐microbial CH₄ formation from living plants particularly under abiotic stress conditions that might affect the electron transport flow at the cytochrome c oxidase in plant mitochondria.     

Alteration of cytochrome C oxidase activity during spermatogenesis in Carcinus maenas

During spermatogenesis in the crab, Carcinus maenas, mitochondria in the developing spermatids degenerate. In close association with mitochondrial fragments in late spermatids, an elaborate lattice-like complex of fused membranes is formed and lies in a position adjacent to the acrosome. Mature sperm possess no mitochondria, but a whorl of membranes is present alongside the acrosome in the diffuse nucleus. To ascertain whether or not cytochrome c oxidase activity is lost as the mitochondria disintegrate, differentiating Carcinus maenas spermatids and mature sperm have been studied cytochemically. Cytochfome c oxidase activity was localised by means of the osmophilic compound 3,3'-diaminobenzidine (DAB). Cytochrome c oxidase activity is confined to the cristae of mitochondria in the testis. As spermatids mature most mitochondria are lost. A few mitochondrial fragments may be caught up within the lamellar complex. While they are recognisalbe as mitochondria they retain cytochrome c oxidase activity. The lamellar complex does not show this enzyme activity. These results therefore suggest that the mature sperm of Carcinus maenas do not contain the enzymes normally incorporated in the mitochondrial membrane, capable of oxidative phosphorylation.     

The Alternative Oxidase of Yarrowia lipolytica Mitochondria Is Unable To Compete with the Cytochrome Pathway for Electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Yarrowia lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

Changes in the Respiratory, Enzymatic, and Swelling and Contraction Properties of Mitochondria from Colytedons of Phaseolus vulgaris L. during Germination

Mitochondria isolated from cotyledons of germinating wax beans (Phaseolus vulgaris L.) showed fairly good respiratory control on days 1 and 2 after planting. The respiratory control was completely lost from days 3 to 5. During this period mitochondria were shown to be very leaky, losing about 88% of their total nicotinamide adenine dinucleotide to the suspending medium in a short time. The respiratory control was partially recovered by day 7, after which it completely disappeared again. By the use of differential centrifugation, the mitochondria were divided into subfractions by sequential centrifugation: 10,000g for 5 minutes, 25,000g for 5 minutes, and 40,000g for 5 minutes. The 10,000g subfraction was responsible for the recovery of mitochondrial activity (respiratory control value, adenosine diphosphate to oxygen ratio, and rate of oxygen utilization), on day 7. Activities of succinate dehydrogenase, cytochrome oxidase, pyruvate dehydrogenase, and isocitrate dehydrogenase from different mitochondrial subfractions of aging cotyledons were determined. In general, the enzyme activities, adenosine diphosphate to oxygen ratios, and the ability of mitochondria to swell and contract followed the same pattern as for respiratory control.     

Galanin-like immunoreactivity in the brain and pituitary of the "four-eyed" fish, Anableps anableps

The distribution of galanin (GAL)-like immunoreactivity was investigated in the brain and pituitary of the "four-eyed" fish, Anableps anableps. GAL-immunoreactive (GAL-ir) perikarya were located in the area ventralis telencephali pars supracommissuralis, nucleus preopticus periventricularis, nucleus preopticus pars parvocellularis, nucleus preopticus pars magnocellularis, nucleus lateralis tuberis ventralis, nucleus lateralis tuberis lateralis, and nucleus lateralis tuberis posterior. A few scattered, GAL-ir neurons were also observed in or adjacent to the nucleus recessus lateralis, nucleus recessus posterioris and lobus facialis (VII). GAL-ir fiber networks were widespread in the brain, with a comparatively higher density in the ventral telencephalic, preoptic and infundibular regions. The neurohypophysis showed GAL-ir innervation and there were GAL-ir cells in the adenohypophysis. The presence of GAL-ir cells in the hypothalamus and in the pituitary is an important asset for the supposed role of GAL-like peptide in neuroendocrine regulation of brain and pituitary functions.     

Effects of running water on brainstem lateral line responses in trout,<Emphasis Type="Italic"> Oncorhynchus mykiss</Emphasis>, to sinusoidal wave stimuli

We investigated how single units in the medial octavolateralis nucleus of the rainbow trout, Oncorhynchus mykiss, respond to a 50-Hz vibrating sphere in still and running water. Four types of units were distinguished. Type MI units (N=16) were flow-sensitive; their ongoing discharge rates either increased or decreased in running water, and as a consequence, responses of these units to the vibrating sphere were masked if the fish was exposed to water flow. Type MII units (N=7) were not flow-sensitive; their ongoing discharge rates were comparable in still and running water, and thus their responses to the vibrating sphere were not masked. Type MIII units (N=7) were also not flow-sensitive; nevertheless, their responses to the vibrating sphere were masked in running water. Type MIV units (N=14) were flow-sensitive, but their responses to the vibrating sphere were not masked. Our data confirm previous findings in the goldfish, Carassius auratus, indicating that the organization of the peripheral lateral line is reflected to a large degree in the medial octavolateralis nucleus. We compare data from goldfish and trout and discuss differences with respect to lateral line morphology, lifestyle and habitat of these species.Abbreviations CN canal neuromast - MON medial octavolateralis nucleus - SN superfical neuromast - a.c. alternating current - d.c. direct current     

STUDIES ON ISOLATED CELL COMPONENTS : XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.     

Oxidative enzyme activities in rat brain subcellular fractions. The effect of deoxycholate, freeze-thaw, and water

(1) The distributions of four oxidative enzymes were studied in crude brain fractions. (2) Freeze-thaw cycle treatment and frozen storage of homogenate fractions gave apparent enhancement of cytochrome oxidase and NADH cytochrome c reductase activities. (3) Deoxycholate released cytochrome oxidase and NADH cytochrome c reductase activities from low-speed precipitates. The NADH diaphorase was enhanced to a small degree while NADPH cytochrome c reductase was not affected by deoxycholate. (4) Distilled water coupled with a single homogenization released trapped soluble enzymes and mitochondria and gave nearly maximal cytochrome oxidase activity as judged by deoxycholate treatment. The total distilled water activity of NADH cytochrome c reductase was much less than that of deoxycholate-stimulated fractions. The activities of other enzymes were not markedly affected by distilled water although their distribution was changed.     

The distribution of synaptosomal oxidative enzymes after exposure to deoxycholate

Abstract— The distributions of NADH₂ dehydrogenase, NADH, cytochrome c reductase and cytochrome oxidase have been determined utilizing synaptosomal isolation techniques. Deoxycholate was used to determine compartmentation and/or ‘latency’ of these activities. NADPH, dehydrogenase proved to be a soluble and mitochondrial enzyme and the activity of this enzyme was not appreciably changed by deoxycholate treatment. NADH_g cytochrome c reductase proved to be a mitochondrial enzyme with considerable activity in microsomal fractions. Deoxycholate treatment increased activity in the synaptosomal fraction 8.3-fold. A bimodal activation pattern was observed with synaptosomal and mitochondrial NADH, cyrochrome c reductase upon exposure to increasing concentrations of deoxycholate, with enhancement of activity at 0.25 % (w/v) and 0.50 % (w/v) deoxycholate. The enzyme was stable at concentrations of deoxycholate less than 0.25% (w/v) but was irreversibly inactivated at concentrations higher than 0.25% (w/v). The mechanism of this activation pattern appeared to be a combination of enzyme release and inactivation. Similar results were not observed in liver mitochondria. Cytochrome oxidase, a known mitochondrial marker, exhibited a 17-fold increase in synaptosomal activity with deoxycholate treatment. The synaptosomal cytochrome oxidase activity after deoxycholate treatment approached the activity in the free mitochondrial fraction. The percentage of mitochondrial protein in synaptosomal fractions was estimated to be about 30 per cent from a comparison of the respective total (deoxycholate-treated) activities. On the basis of these data we suggest that the synaptosomal fraction possesses a relatively sizable energy-producing potential which may be of significance in vivo.     

Cortical Metabolic Deficits in a Rat Model of Cholinergic Basal Forebrain Degeneration

Evidence indicates that the degeneration of basal forebrain cholinergic neurons may represent an important factor underlying the progressive cognitive decline characterizing Alzheimer’s disease (AD). However, the nature of the relationship between cholinergic depletion and AD is not fully elucidated. This study aimed at clarifying some aspects of the relation existing between deficits in cerebral energy metabolism and degeneration of cholinergic system in AD, by investigating the neuronal metabolic activity of several cortical areas after depletion of basal forebrain cholinergic neurons. In cholinergically depleted rats, we evaluated the neuronal metabolic activity by assaying cytochrome oxidase (CO) activity in frontal, parietal and posterior parietal cortices at four different time-points after unilateral injection of 192 IgG-saporin in the nucleus basalis magnocellularis. Unilateral depletion of cholinergic cells in the basal forebrain induced a bilateral decrease of metabolic activity in all the analyzed areas. Frontal and parietal cortices showed decreased metabolic activity even 3 days after the lesion, when the cholinergic degeneration was still incomplete. In posterior parietal cortex metabolic activity decreased only 7 days after the lesion. The possible molecular mechanisms underlying these findings were also investigated. Real-time PCR showed an increase of CO mRNA levels at 3, 7 and 15 days after the lesion both in frontal and parietal cortices, followed by normalization at 30 days. Western Blot analysis did not show any change in CO protein levels at any time-point after the lesion. Our findings support a link between metabolic deficit and cholinergic hypofunctionality characterizing AD pathology. The present model of cholinergic hypofunctionality provides a useful means to study the complex mechanisms linking two fundamental and interrelated phenomena characterizing AD from the early stages.     

Immunocytochemical demonstration of growth hormone,prolactin and somatostatin-like immunoreactivities in the brain of larval,young adult and upstream migrant adult sea lamprey,Petromyzon marinus

Summary Growth hormone, prolactin and somatostatinlike immunoreactivities were demonstrated in the brains of larval, young adult (parasitic) and upstream migrant adult sea lampreys, Petromyzon marinus, by means of immunoperoxidase techniques. Growth hormone (GH) and prolactin (PRL) were observed within separate perikarya in the nucleus praeopticus, within fibers in the commissura praeinfundibularis, and in nerve endings within the neurohypophysis of larval and adult-stage lampreys. Cell bodies demonstrating immunoreactive growth hormone were more numerous than those reactive for prolactin. Unlike in the upstream migrant adult lamprey, no GH or PRL was demonstrated in the adenohypophysis of larval or parasitic lamprey.Somatostatin (SRIF)-like immunoreactive neurons were demonstrated in the nucleus commissurae praeinfundibularis, anterior and posterior pars ventralis hypothalami, pars dorsalis thalami, and the tegmentum motorium rhombencephali of larval, parasitic and upstream migrant adult lampreys. Many of the SRIF containing neurons within the hypothalamus were cerebrospinal fluid (CSF)-contacting cells. SRIF fibers were found throughout most of the brain predominating within the nucleus praeopticus, pars ventralis hypothalami, and the nucleus interpeduncularis. No SRIF immunoreactivity was found within the neurophyophysis. The possible functions of these peptides within the brain of the lamprey are discussed.     

Respiratory enzymes and mitochondrial morphology of HeLa and L cells treated with chloramphenicol and ethidium bromide

Exposure of HeLa and L cells to chloramphenicol causes a progressive dose-dependent decrease in cytochrome oxidase and succinate-cytochrome c reductase activities, concomitant with an increase in the amount of cytochrome c. At 2–3 days, the specific activities of the enzymes have fallen to about one-half of control values; the mitochondria appear swollen. By day 5, enzyme activities are about one-quarter of control values; the mitochondria are more swollen, with disorientation and disintegration of cristae. By day 6–8, after three generations, growth has stopped, enzyme activities are approximately the same as on day 5, and cytochrome c content has reached 170% of control value. Mitochondria show severe changes, cristae being affected more than peripheral inner membrane. The number of profiles continues to be nearly normal. After 30 days, cytochrome oxidase activity remains low but now there are mitochondria in intermediate and condensed configuration. There is a gradual accumulation in the cytoplasm of smooth membrane elements. If chloramphenicol is removed, cells recover. Ethidium bromide treatment for up to 8 days yields results virtually identical to those obtained with chloramphenicol. Cells treated with 10^-4 M KCN show a decrease in cytochrome oxidase activity to about one-third of control value and an elevated amount of cytochrome c. Only a small number of mitochondria appear damaged. Autochthonous mitochondrial syntheses appear to be essential for the organization of the cristae. When cytochrome oxidase activity is impaired, a regulatory mechanism for cytochrome biosynthesis geared to mitochondrial function may be lacking, resulting in an increase in cytochrome c content.     

Development of synaptic transmission at autonomic synapses in vitro revealed by cytochrome oxidase histochemistry

We have studied the development of synaptic transmission by innervating sympathetic neurons in vitro and monitoring synaptic activity with both physiological recording and cytochrome oxidase histochemistry. The onset of synaptic transmission was reflected in increased cytochrome oxidase reaction product within individual neurons. Within 24 hours of co-culture, relatively low frequency suprathreshold potentials were recorded in approximately 20% of the innervated neurons. At this stage the cytochrome oxidase activity of innervated neurons, as assessed by optical density of the histochemical reaction product, was increased twofold compared with uninnervated neurons. Over the next 2-4 days of innervation, changes in the pattern and extent of synaptic activity and superthreshold events were accompanied by a net fourfold increase in cytochrome oxidase activity levels compared with noninnervated neurons. The increase in density of cytochrome oxidase reaction product observed after innervation was reversed completely by blockade of synaptic transmission. Differences in the efficacy of synaptic input provided to the sympathetic neurons by appropriate versus inappropriate presynaptic sources was determined by co-culturing sympathetic neurons with explants that contained either preganglionic neurons or somatic motor neurons. Although sympathetic neurons innervated by motor neuron explants had increased levels of cytochrome oxidase activity compared with noninnervated controls, the density of cytochrome oxidase reaction product was even greater in sympathetic neurons innervated by preganglionic explants. We conclude that both the onset of innervation of sympathetic neurons as well as the subsequent maturation of synaptic function is directly reflected in graded increases in cytochrome oxidase reaction product.