Enzyme Cytochrome Oxidase Research Articles

Role of Hyperforin in the Pharmacological Activities of St. John's Wort

The phloroglucinol derivative hyperforin has been recently shown to be a major antidepressant component in the extract of Hypericum perforatum. Experimental studies clearly demonstrated its activity in different behavioral models of depression. Moreover clinical studies linked the therapeutic efficacy of Hypericum extracts to their hyperforin content, in a dose-dependent manner. The molecular mechanism of action of hyperforin is still under investigation. Hyperforin has been shown to inhibit, like conventional antidepressants, the neuronal uptake of serotonin, norepinephrine and dopamine. However, hyperforin inhibits also the uptake of gamma-aminobutyric acid (GABA) and L-glutamate. The uptake inhibition by hyperforin does not involve specific binding sites at the transporter molecules; its mechanism of action seems to be related to sodium conductive pathways, leading to an elevation in intracellular Na(+) concentration. Other additional mechanisms of action of hyperforin, involving ionic conductances as well synaptosomal and vesicular function, have been suggested. In addition to its antidepressant activity, hyperforin has many other pharmacological effects in vivo (anxiolytic-like, cognition-enhancing effects) and in vitro (antioxidant, anticyclooxygenase-1, and anticarcinogenic effects). These effects could be of clinical importance. On the other hand, the role of hyperforin in the pharmacological interactions occurring during Hypericum extract therapy must be fully investigated. Hyperforin seems to be responsible for the induction of liver cytochrome oxidase enzymes and intestinal P-glycoprotein. Several pharmacokinetic studies performed in rats and humans demonstrated oral bioavailability of hyperforin from Hypericum extract. Only recently a new chromatographic method for detection of hyperforin in the brain tissue has been developed and validated. Taking into account the chemical instability of hyperforin, current efforts are directed to the synthesis of new neuroactive derivatives.

Leptin and the control of respiratory gene expression in muscle.

Leptin plays a central role in the regulation of fatty acid homeostasis, promoting lipid storage in adipose tissue and fatty acid oxidation in peripheral tissues. Loss of leptin signaling leads to accumulation of lipids in muscle and loss of insulin sensitivity secondary to obesity. In this study, we examined the direct and indirect effects of leptin signaling on mitochondrial enzymes including those essential for peripheral fatty acid oxidation. We assessed the impact of leptin using the JCR:LA-cp rat, which lacks functional leptin receptors. The activities of marker mitochondrial enzymes citrate synthase (CS) and cytochrome oxidase (COX) were similar between wild-type (+/?) and corpulent (cp/cp) rats. In contrast, several tissues showed variations in the fatty acid oxidizing enzymes carnitine palmitoyltransferase II (CPT II), long-chain acyl-CoA dehydrogenase (LCAD) and 3-hydroxyacyl-CoA dehydrogenase (HOAD). It was not clear if these changes were due to loss of leptin signaling or to insulin insensitivity. Consequently, we examined the effects of leptin on cultured C(2)C(12) and Sol8 cells. Leptin (3 days at 0, 0.2, or 2.0 nM) had no direct effect on the activities of CS, COX, or fatty acid oxidizing enzymes. Leptin treatment did not affect luciferase-based reporter genes under the control of transcription factors involved in mitochondrial biogenesis (nuclear respiratory factor-1 (NRF-1), nuclear respiratory factor-2 (NRF-2)) or fatty acid enzyme expression (peroxisome proliferator-activated receptors (PPARs)). These studies suggest that leptin exerts only indirect effects on mitochondrial gene expression in muscle, possibly arising from insulin resistance.

ELECTRON TRANSFER REACTIONS COUPLED TO PROTON TRANSLOCATION: CYTOCHROME OXIDASE, PROTON PUMPS, AND BIOLOGICAL ENERGY TRANSDUCTION

Cytochrome oxidase (COX) is the terminal component of electron transport chain of the respiratory system in mitochondria, and one of the key enzymes responsible for energy generation in cells. COX functions as a proton pump that utilizes free energy of oxygen reduction for translocation of protons across the mitochondrion membrane. The proton gradient created in the process is later utilized to drive synthesis of ATP. Although the structure of COX has been recently resolved, the molecular mechanism of proton pumping remains unknown. In this paper, general principles and possible molecular mechanisms of energy transformations in this enzyme will be discussed. The main question is how exactly chemical energy of oxygen reduction and water formation is transformed into a proton gradient; or, how exactly electron transfer reactions are utilized to translocate protons across the mitochondrion membrane against the electrochemical gradient. A key to the solution of this problem is in understanding correlated transport of electrons and protons. Here, theoretical models are discussed for coupled electron and proton transfer reactions in which an electron is tunneling over long distance between two redox cofactors, and a coupled proton is moving along a proton conducting channel in a classical, diffusion-like random walk fashion. Such reactions are typical for COX and other enzymes involved in biological energy transformations.

Manganese, copper, and zinc in cerebrospinal fluid from patients with multiple sclerosis.

The concentrations of manganese, copper, and zinc in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and patients with no known neurological disease (control group) were measured. Manganese and copper levels were determined by two different analytical methods: atomic absorption spectrometry (AAS) and high-resolution inductively coupled plasma-mass spectrometry (HR-ICP-MS), whereas zinc levels were determined by HR-ICP-MS only. Manganese levels (mean+/-SEM) were significantly decreased in the CSF of MS patients (1.07+/-0.13 microg/L, ICP-MS; 1.08+/-0.11 microg/L, AAS) compared to the levels in the control group (1.78+/-0.26 microg/L, ICP-MS; 1.51+/-0.17 microg/L, AAS). Copper levels were significantly elevated in the CSF of MS patients (10.90+/-1.11 microg/L; ICP-MS, 11.53+/-0.83 microg/L, AAS) compared to the levels in the control group (8.67+/-0.49 microg/L, ICP-MS; 9.10+/-0.62 microg/L, AAS). There were no significant differences between the CSF zinc levels of MS and control patients. The physiological basis for the differences in manganese and copper concentrations between MS patients and controls is unknown, but could be related to alterations in the manganese- containing enzyme glutamine synthetase and the copper-containing enzyme cytochrome oxidase.

Long-term administration of l-carnitine to humans: effect on skeletal muscle carnitine content and physical performance

Background: Long-term administration of high oral doses of l-carnitine on the skeletal muscle composition and the physical performance has not been studied in humans. Methods: Eight healthy male adults were treated with 2×2 g of l-carnitine per day for 3 months. Muscle biopsies and exercise tests were performed before, immediately after, and 2 months after the treatment. Exercise tests were performed using a bicycle ergometer for 10 min at 20%, 40%, and 60% of the individual maximal workload ( P max), respectively, until exhaustion. Results: There were no significant differences between V O 2max , RER max, and P max between the three time points investigated. At submaximal intensities, the only difference to the pretreatment values was a 5% increase in V O 2 at 20% and 40% of P max 2 months after the cessation of the treatment. The total carnitine content in the skeletal muscle was 4.10±0.82 μmol/g before, 4.79±1.19 μmol/g immediately after, and 4.19±0.61 μmol/g wet weight 2 months after the treatment (no significant difference). Activities of the two mitochondrial enzymes citrate synthase and cytochrome oxidase, as well as the skeletal muscle fiber composition also remained unaffected by the administration of l-carnitine. Conclusions: Long-term oral treatment of healthy adults with l-carnitine is not associated with a significant increase in the muscle carnitine content, mitochondrial proliferation, or physical performance. Beneficial effects of the long-term treatment with l-carnitine on the physical performance of healthy adults cannot be explained by an increase in the carnitine muscle stores.

Cortical and thalamic connections of the parietal ventral somatosensory area in marmoset monkeys (Callithrix jacchus).

Microelectrode mapping methods were used to define the parietal ventral somatosensory area (PV) on the upper bank of the lateral sulcus in five marmosets (Callithrix jacchus). In the same animals, neuroanatomical tracers were placed into electrophysiologically identified sites in PV and/or the second somatosensory area (S2). Foci of anterograde and retrograde label were related to electrophysiological maps of cortical areas and cortical and thalamic architecture. The results lead to the following conclusions: (1) Multiunit recordings from cortex on the upper bank of the lateral sulcus demonstrate that PV is somatotopically organized, with the face representation adjoining area 3b and the hindlimb and tail representations away from this border in cortex deep on the upper bank of the lateral sulcus. The forelimb representation is caudal in PV adjacent to the S2 forelimb representation. The body surface representation in PV approximates a mirror image of that in S2; (2) Areas PV and S2 are less myelinated and have less cytochrome oxidase enzyme activity than area 3b; (3) The ventroposterior inferior nucleus (VPI) of the thalamus provides the major somatosensory projections to PV. PV is reciprocally connected with VPI and anterior pulvinar; (4) PV has ipsilateral cortical connections with areas 3a, 3b, 1, and M1 and higher order somatosensory fields, and at least most of these connections are somatotopically matched; and (5) Callosal connections of PV are with S2 and PV of the other cerebral hemisphere. These results further establish PV as one of at least four somatosensory areas of the lateral sulcus of primates.

Mitochondria in Alzheimer's disease

Mitochondrial abnormalities are characteristic of Alzheimer's Disease (AD) and might be etiologically involved in the brain degenerative process. Data detailing AD mitochondrial pathology are described in the chapter, especially those involving the enzyme cytochrome oxidase, as most mitochondrial studies in AD have involved investigation of this enzyme. In future studies, examination of large numbers of patients with AD will probably reveal a modest overall reduction of brain cytochrome-oxidase activity. However, high scatter between the range of AD and control values will indicate that the change is not a robust defining feature of the disorder and therefore unlikely to be etiologically important for at least many patients with AD. The strength of a particular AD paradigm should be determined by its ability to explain all aspects of the disease. Comprehensive hypotheses of AD pathogenesis must take into account cerebral and extracerebral mitochondrial dysfunction.

Quantitative analysis of the frequency spectrum of the radiation emitted by cytochrome oxidase enzymes.

A physical model is proposed that provides a quantitative analysis of the energy emitted by proton flows through mitochondrial walls. The model developed is based on biochemical and biophysical properties of the enzyme cytochrome oxidase and in particular the embedded heme groups that are involved in the electron ferrying mechanism. The estimates of the energies at approximately 1.1 eV and corresponding wavelengths of the near infrared radiation generated, with a peak close to 900 nm, agree extremely well with experimental values. The basic idea in the mechanism proposed is that the passage of a proton through the mitochondrial wall's gate is linked with the creation of a virtual proton-electron pair in an excited state of a hydrogen atom. The electron is temporarily removed from the enzyme when the proton arrives at the gate and is subsequently deposited back at the enzyme's acceptor site when the proton leaves the gate.

Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo

Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing histotoxic hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels of extracellular Ca 2+ and was attenuated by Zn 2+ (modulator of Ca 2+-dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca 2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342 staining), a phenomenon that characterises the ‘apoptotic’ type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5–3 h) of various pharmacological agents viz., Trolox® (antioxidant), EGTA (Ca 2+ modulator) and aurintricarboxylic acid (ATA; Ca 2+/Mg 2+-dependent endonuclease inhibitor). In addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox® was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an early event preceding DNA damage. Both EGTA and ATA could not prevent this damage. Trolox® also increased the LD 50 of KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.

Metabolic changes in the nucleus of the optic tract after monocular enucleation as revealed by cytochrome oxidase histochemistry.

The histochemistry for the mitochondrial enzyme cytochrome oxidase (CO) was used to evaluate the levels of metabolic activity in neurons of the nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN) in the opossum (Didelphis aurita). The observations were performed in four groups: normal juveniles (4 months old), monocularly enucleated juveniles analysed when adults, normal adults (8 to 18 months old) and monocularly enucleated adults. CO labeled cells were observed to have a similar distribution along the NOT-DTN anteroposterior axis in both juvenile and adult normal animals. Monocular enucleation performed in adults produced a significant reduction of the reactive neuropil but not of the number of CO labeled cells in the deafferented NOT-DTN: the number of labeled neurons per section in the deafferented side matched those of the ipsilateral complex. In juveniles, however, this procedure caused a systematic reduction of the number of CO labeled cells in the contralateral NOT-DTN in comparison to the spared complex. The lack of reduction in the number of neurons found on the deafferented side of the NOT-DTN of monocularly enucleated adult opossums compared with the ipsilateral side might result from the presence of compensatory inputs to maintain their metabolic equivalence. However, when the monocular enucleation was performed in juvenile opossums, a statistically significant asymmetry of CO neurons in the NOT-DTN was observed. In other words, the compensatory mechanisms proposed for the adults were either absent or insufficient to achieve symmetry in juveniles, suggesting a more heavily reliance in the retinal input.

Quantitative cytochemical mapping of mitochondrial enzymes in rat cerebella

Mitochondrial metabolic competence, defined as the organelle's capacity to provide adequate amounts of ATP in due time, appears to constitute an important determinant in several biological processes and pathological conditions. Thus, the assessment of the metabolic efficiency of the mitochondrial population in a given tissue area or cellular compartment may provide clues to identifying alterations of the cellular bioenergetic machinery, which may constitute a predisposing condition leading to impaired organ and system functions.In the cerebellar cortex of adult rats, the activities of the enzymes cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were, respectively, evidenced by means of the diaminobenzidine and copper ferrocyanide preferential cytochemical techniques. At the electron microscope, the activities of these two key molecules of the respiratory chain were clearly visualised as dark precipitates at the inner mitochondrial membrane sites where COX and SDH are located. By means of the disector method, unbiased mitochondrial samplings were carried out to measure: the number of mitochondria/μm3 of tissue (numeric density: Nv); the mitochondrial volume fraction/μm3 of tissue (volume density: Vv) and the average mitochondrial volume (V) both on COX- and SDH-positive organelles in the cerebellar glomeruli and Purkinje cells, respectively. The ratio R (total area of the precipitates due either to COX or SDH activity within the single mitochondrion/area of the same organelle) was also evaluated to get information on the enzyme activity related to mitochondrial size.The documented accumulation of mutant mitochondrial DNA particularly in postmitotic cells results in a marked heteroplasmy (mixtures of normal and mutated genomes) at mitochondrial and cellular levels, thus the cellular potential for energy production is demanded to a mosaic of organelles with different functional capabilities. Assessment of the mitochondrial mosaic outline by means of quantitative cytochemistry of key enzymes of the respiratory chain, such as COX and SDH, may allow for the morphofunctional metabolic mapping of mitochondrial efficiency in discrete cellular or tissue compartments.

In vivo near-infrared spectroscopy.

Interrogation of tissue with light offers the potential for noninvasive chemical measurement, and penetration with near-infrared wavelengths (750-1000 nm) is greater than with visible light. Specific absorption by clinically relevant compounds such as oxy- and deoxyhemoglobin and the intracellular respiratory enzyme cytochrome oxidase enable in vivo measurement of these to be performed safely and conveniently. This is the basis of in vivo near-infrared spectroscopy (ivNIRS). Multiple scattering of the interrogating beam by tissues leads to an optical path that is considerably longer than the simple physical pathlength and this complicates the analysis. Modeling of photon propagation through tissues with, for example, finite element and Monte Carlo methods, is assisting in improving the ivNIRS methodology. Instrumentation has advanced from simple continuous wave approaches, through time-resolved methods based on either time-domain or frequency-domain approaches, to spatially resolved measurement based on diffuse reflectance. Initial clinical applications were for monitoring the brain in the neonate and fetus and muscle in adults. Currently, use in adults and children for neurological assessments are of growing interest.

Antioxidant adaptive response of human mononuclear cells to UV-B: effect of lipoic acid

Supplementation of human mononuclear cells with 3 and 6 mM of lipoic acid produces an inhibition of the antioxidant adaptive response triggered by treatment with UV-B light (0.30 W/m 2 for 15 min). Supplementation with 1.5 mM of lipoic acid gives no conclusive results. The adaptive response is characterized by an increase in the activities of superoxide dismutase, catalase, glutathione peroxidase and DT-diaphorase. Catalase (5.5±0.6 pmol/mg prot) increases its activity by up to 22±3 pmol/mg prot, after irradiation with UV-B. Supplementation with 3 and 6 mM of lipoic acid completely inhibits the adaptive response. The activities of the membrane-bound mitochondrial enzymes succinate dehydrogenase and cytochrome oxidase do not increase after UV-B exposure. Moreover, their activities are found to decrease and the addition of lipoic acid does not prevent this effect. The inhibition of the antioxidant response by lipoic acid in human cells appears as indirect evidence of the existence of oxidative stress in the development of this response. As lipoic acid behaves as an effective antioxidant, it seems that its action decreases the intracellular oxidative signals necessary to develop the adaptive response in human mononuclear cells.

The effects of cell ageing on metabolism in rainbow trout (Oncorhynchus mykiss) red blood cells.

The effects of cell age on metabolism in the nucleated red blood cells of rainbow trout (Oncorhynchus mykiss) were examined. Red blood cells were separated according to age using fixed-angle centrifugation. The mean erythrocyte haemoglobin concentration in old red blood cells was found to be 120 % of that in young red blood cells. In young red blood cells, the activities of the mitochondrial enzymes citrate synthase and cytochrome oxidase were 135-200 %, respectively, of those measured in old red blood cells. The activity of the glycolytic enzyme lactate dehydrogenase in young red blood cells was 170 % of that in old red blood cells, whereas the activity of the glycolytic enzyme pyruvate kinase was not significantly affected by cell age. In addition, young red blood cells consumed over twice as much O(2) and devoted 50 % more O(2) to protein synthesis and the activity of Na(+)/K(+)-ATPase than old red blood cells. Red blood cell age did not significantly affect the rate of lactate production. This study shows that ageing in rainbow trout nucleated red blood cells is accompanied by a significant decline in aerobic energy production and the processes it supports, as well as a corresponding increase in the glycolytic contribution to metabolism.

MITOCHONDRIAL KINETICS DURING AMPHIBIAN ERYTHROPOIESIS RELATED TO HAEME SYNTHESIS

Flow cytometry, light and epifluorescence microscopies and transmission electron microscopy were used to follow the mitochondrial kinetics during amphibian erythropoiesis. A similar behaviour in response to the induction of anaemia was observed in the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. A high cellular activity was observed ten days after haemolytic anaemia induced by phenylhydrazine, based on the higher Rhodamine 123 uptake by the erythroid cells. In addition, the more intense expression of the mitochondrial enzyme cytochrome oxidase, isocitrate and succinic dehydrogenases were cytochemically detected at this stage. This suggests that erythroid cell mitochondria, at this time, could be in a more active functional state than at other stages. In both species, mitochondrial plasticity was observed during cell maturation. A progressive loss of oxidation–reduction enzyme expression seemed to follow changes at the mitochondrial cristae morphology, from transverse to longitudinal form, mainly at the 20th day of recovery from anaemia, possibly related to a natural loss of function. The presence of these mitochondrial enzymes in mitochondrion-like organelles also favours their participation in the haeme synthesis, although with a reduced expression, since this suggests the presence of a complete and active enzymatic complex in these modified organelles. This also supports the idea that all these organelles are mitochondria in distinct metabolic stages, and not mitochondrion-like organelles or haemosomes, as proposed by some authors.

A potential role for sterol carrier protein-2 in cholesterol transfer to mitochondria

Mitochondrial cholesterol oxidation rapidly depletes cholesterol from the relatively cholesterol-poor mitochondrial membranes. However, almost nothing is known regarding potential mechanism(s) whereby the mitochondrial cholesterol pool is restored. Since most exogenous cholesterol enters the cell via the lysosomal pathway, this could be a source of mitochondrial cholesterol. In the present study, an in vitro fluorescent sterol transfer assay was used to examine whether the lysosomal membrane could be a putative cholesterol donor to mitochondria. First, it was shown that spontaneous sterol transfer from lysosomal to mitochondrial membranes was very slow (initial rate, 0.316±0.032 pmol/min). This was due, in part, to the fact that 90% of the lysosomal membrane sterol was not exchangeable, while the remaining 10% also had a relatively long half-time of exchange t 1/2=202±19 min. Second, the intracellular sterol carrier protein-2 (SCP-2) and its precursor (pro-SCP-2) increased the initial rate of sterol transfer from the lysosomal to mitochondrial membrane by 5.2- and 2.0-fold, respectively, but not in the reverse direction. The enhanced sterol transfer was due to a 3.5-fold increase in exchangeable sterol pool size and to induction of a very rapidly ( t 1/2=4.1±0.6 min) exchangeable sterol pool. Confocal fluorescence imaging and indirect immunocytochemistry colocalized significant amounts of SCP-2 with the mitochondrial marker enzyme cytochrome oxidase in transfected L-cells overexpressing SCP-2. In summary, SCP-2 and pro-SCP-2 both stimulated molecular sterol transfer from lysosomal to mitochondrial membranes, suggesting a potential mechanism for replenishing mitochondrial cholesterol pools depleted by cholesterol oxidation.

Aging Selectively Decreases Oxidative Capacity in Rat Heart Interfibrillar Mitochondria

Mitochondrial-derived oxidative injury contributes to cellular aging as well as to reperfusion-induced tissue damage. While the aging-heart suffers greater tissue damage following ischemia and reperfusion than the adult heart, the occurrence of aging-related alterations in mitochondrial oxidative metabolism in the elderly heart has remained uncertain. We determined if aging altered oxidative metabolism in either of the two populations of cardiac mitochondria, subsarcolemmal mitochondria (SSM) that reside beneath the plasma membrane or interfibrillar mitochondria (IFM) located between the myofibrils. SSM and IFM were isolated from 6-month adult and 24- and 28-month elderly Fischer 344 rat hearts. Aging-related alterations were limited to IFM, while SSM remained unaffected. Aging decreased the rate of oxidative phosphorylation in IFM, including when stimulated by electron donors specific for cytochrome oxidase. Cytochrome oxidase enzyme activity was decreased in IFM from aging hearts, while activity in SSM remained similar to adult controls. These findings allow future studies of aging-related decrements in oxidative function to focus upon IFM, while SSM provide an inherent control group of mitochondria that are free of aging-related alterations in oxidative function. The selective alteration of IFM during aging raises the possibility that the consequences of aging-induced mitochondrial dysfunction will be enhanced in specific subcellular regions of the senescent myocyte.

Effects of alpha-isoproterenol on denervation atrophy in orbicularis oculi muscle fibers.

The effect of the beta-adrenoceptor agonist isoproterenol on the denervated orbicularis oculi muscle was studied. We cut the facial nerve on the right side of eight rats and injected isoproterenol (5 mg/kg) into four rats every day. The animals were sacrificed after three weeks, and small pieces of the orbicularis oculi muscle were removed. Muscle fibers were separated into single fibers. Cytochrome oxidase enzyme staining was applied, and density of cytochrome oxidase enzyme product and diameter of individual muscle fibers were analyzed by computer. Some of the muscle fibers were observed under electron microscopy. The results showed that diameter and cytochrome oxidase enzyme activity of the orbicularis oculi muscle decreased by denervation, otherwise, it increased to near the normal level by administration of isoproterenol. Electron microscopic observation showed that the myosion filaments became thin and their arrangement were disordered by denervation and their findings recovered to be almost normal by isoproterenol administration. We concluded that isoproterenol could prevent the orbicularis oculi muscle from atrophying which caused by denervation.

Cytochrome oxidase and NADPH‐diaphorase on the afferent relay branch of the optokinetic reflex in the opossum

In the present study, histochemical techniques combined with more conventional anatomical methods were used to refine the identification of the nucleus of the optic tract and the nuclei of the accessory optic system in the opossum. The distribution of the enzyme cytochrome oxidase (CO)<0B> <0R>was examined in the cells and the neuropil of the opossum's mesodiencephalic region. Strong CO labeling was present in the nucleus of the optic tract (NOT)-dorsal terminal nucleus (DTN). Alternate sections, taken from animals that had received bilateral injections of horseradish peroxidase centered in the region of the inferior olive, were subjected to assays for CO and horseradish peroxidase. The region occupied by CO-labeled cells in the NOT-DTN superimposed with the one defined by retrogradely labeled cells. Cell counts along the NOT-DTN anteroposterior axis revealed that although the olivary and CO-positive cells were confined within similar boundaries, the latter are up to twofold more numerous than the former. As revealed by cytochrome oxidase histochemistry, the outlines of the NOT-DTN, the other pretectal nuclei and the nuclei belonging to the accessory optic system coincided with those revealed by the histochemistry for nicotinamide dinucleotide phosphate diaphorase (NADPH-d). After an intraocular injection of cholera toxin beta subunit and alternate sections processing for NADPH-d and CO, the distribution of labeled retinal terminal fields in the mesodiencephalic region was shown to be coincident with regions of high levels of histochemical labeling. These results are discussed in the light of previous anatomofunctional assessments of the pretectum and accessory optic system. J. Comp. Neurol. 398:206–224, 1998. © 1998 Wiley-Liss, Inc.

Cytochrome oxidase and NADPH-diaphorase on the afferent relay branch of the optokinetic reflex in the opossum

In the present study, histochemical techniques combined with more conventional anatomical methods were used to refine the identification of the nucleus of the optic tract and the nuclei of the accessory optic system in the opossum. The distribution of the enzyme cytochrome oxidase (CO) was examined in the cells and the neuropil of the opossum's mesodiencephalic region. Strong CO labeling was present in the nucleus of the optic tract (NOT)-dorsal terminal nucleus (DTN). Alternate sections, taken from animals that had received bilateral injections of horseradish peroxidase centered in the region of the inferior olive, were subjected to assays for CO and horseradish peroxidase. The region occupied by CO-labeled cells in the NOT-DTN superimposed with the one defined by retrogradely labeled cells. Cell counts along the NOT-DTN anteroposterior axis revealed that although the olivary and CO-positive cells were confined within similar boundaries, the latter are up to twofold more numerous than the former. As revealed by cytochrome oxidase histochemistry, the outlines of the NOT-DTN, the other pretectal nuclei and the nuclei belonging to the accessory optic system coincided with those revealed by the histochemistry for nicotinamide dinucleotide phosphate diaphorase (NADPH-d). After an intraocular injection of cholera toxin beta subunit and alternate sections processing for NADPH-d and CO, the distribution of labeled retinal terminal fields in the mesodiencephalic region was shown to be coincident with regions of high levels of histochemical labeling. These results are discussed in the light of previous anatomofunctional assessments of the pretectum and accessory optic system.