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Cytochrome Oxidase activity in neuronal cells

Cytochrome Oxidase activity in neuronal cells


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The abstract of this article says

"… the entire neuron is often not metabolically homogeneous; most of the oxidative activity is usually found in dendrites."

Why would the activity of cytochrome oxidase be localized in the dendrites of the cell? Is it because it has a greater surface area (more channels and such) compared to the rest of the cell?


Abstract

The brain is composed of a heterogeneous population of neurons whose physiological characteristics often elude morphological identification. The tight coupling between neuronal activity and oxidative energy metabolism forms the basis for the use of cytochrome oxidase as an endogenous metabolic marker for neurons. In the past decade, cytochrome oxidase histo- and cytochemistry have provided a window to view the regional, cellular and subcellular functional diversity among neurons. These methods have shown that the entire neuron is often not metabolically homogeneous most of the oxidative activity is usually found in dendrites. They have also revealed the dynamic metabolic responses of developing and mature neurons to altered functional demands.


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In: Brain research , Vol. 578, No. 1-2, 24.04.1992, p. 311-316.

Research output : Contribution to journal › Article › peer-review

T1 - Cytochrome oxidase activity in vagal and glossopharyngeal visceral sensory neurons of the rat

T2 - effect of peripheral axotomy

N1 - Funding Information: This work was supported by NIH Grant R01

N2 - Cytochrome oxidase (CO) activity, an endogenous metabolic marker, was examined in visceral sensory neurons of the rat nodose and petrosal ganglia by using enzyme histochemistry. In the normal nodose and petrosal ganglia, nerve cells showed various degrees of staining intensity. The population of darkly stained neurons in the nodose ganglion was higher than in the petrosal ganglion. Axotomy of the peripheral axons of these bipolar sensory neurons was used to study potential changes in ganglionic cellular metabolism associated with loss of afferent inputs and/or injury. Peripheral axotomy had a significant effect on CO activity in the nodose ganglion. By 3 days after axotomy, darkly stained neurons decreased in number and lightly stained neurons, which were not observed in the normal ganglion, appeared in the nodose ganglion. At 7 days after axotomy, the average population of these lightly stained neurons increased to 29% in the nodose ganglion. Subsequently, the population decreased so that at 14 days and 21 days, 19% and 7% respectively of neurons were stained lightly. Even at 28 days after axotomy, the lightly stained neurons were still observed. In the petrosal ganglion, no remarkable change was observed at any stage after axotomy. These results suggest that metabolic activity decreases in some nodose neurons after peripheral nerve section.

AB - Cytochrome oxidase (CO) activity, an endogenous metabolic marker, was examined in visceral sensory neurons of the rat nodose and petrosal ganglia by using enzyme histochemistry. In the normal nodose and petrosal ganglia, nerve cells showed various degrees of staining intensity. The population of darkly stained neurons in the nodose ganglion was higher than in the petrosal ganglion. Axotomy of the peripheral axons of these bipolar sensory neurons was used to study potential changes in ganglionic cellular metabolism associated with loss of afferent inputs and/or injury. Peripheral axotomy had a significant effect on CO activity in the nodose ganglion. By 3 days after axotomy, darkly stained neurons decreased in number and lightly stained neurons, which were not observed in the normal ganglion, appeared in the nodose ganglion. At 7 days after axotomy, the average population of these lightly stained neurons increased to 29% in the nodose ganglion. Subsequently, the population decreased so that at 14 days and 21 days, 19% and 7% respectively of neurons were stained lightly. Even at 28 days after axotomy, the lightly stained neurons were still observed. In the petrosal ganglion, no remarkable change was observed at any stage after axotomy. These results suggest that metabolic activity decreases in some nodose neurons after peripheral nerve section.


The Hypothalamus–Pituitary–Thyroid (HPT) Axis of Mammals

David O. Norris Ph.D. , James A. Carr Ph.D. , in Vertebrate Endocrinology (Fifth Edition) , 2013

A Metabolic Actions

The effects produced by thyroid hormones on mammalian metabolism include a calorigenic or thermogenic action (heat-generating) as well as specific effects related to carbohydrate, lipid, and protein metabolism. In general, thyroid activity in mammals is greater during prolonged periods of cold stress (winter) than during warmer periods. Thermogenic actions of thyroid hormones become more meaningful when considered together with the actions of other hormones on metabolism (see Chapter 16). Many of these metabolic actions are possibly permissive actions occurring in cooperation with other hormones such as epinephrine and growth hormone (see Chapters 4 and 12 Chapter 4 Chapter 12 ).

Thermogenic actions of thyroid hormones are restricted to certain tissues and are involved in physiological responses to cold stress. They can accelerate the rate at which glucose is oxidized in these tissues and thus increase the amount of metabolic heat produced in a given time. This elevated heat production can be used to warm the body. Accelerated glucose oxidation is reflected in an increased basal metabolic rate (BMR) as measured by an increased in rate of oxygen consumption. In contrast, decreased nutrient intake operates through neural mechanisms that reduce thyroid hormone secretion and lower metabolic rate. As mentioned earlier, there is evidence that thyroid hormone receptors exist in mitochondria, and thyroid hormones induce increased synthesis of several mitochondrial respiratory proteins, especially cytochrome C, cytochrome oxidase, and succinoxidase. In brown adipose tissue (a tissue especially important for thermogenesis), T3 but not T4 stimulates production of a unique mitochondrial protein known as uncoupling protein 1 (UCP-1) in a dose-dependent manner. The high vascularity of brown adipose tissue in rodents, for example, is visibly different from yellow adipose tissue, which primarily stores lipids. Although humans are thought to lack brown adipose tissue, apparently some of the yellow adipose tissue functions as though it were brown.

UCP-1 is one of a group of mitochondrial proteins that are upregulated by thyroid hormones in several tissues, but of these proteins only UCP-1 has been linked to uncoupling oxidative phosphorylation and heat production. This mitochondrial action to augment oxidative metabolism would be advantageous in adapting to chronic cold stress. Long before the discovery of UCP-1, thyroid hormones were postulated to “uncouple” oxidative phosphorylation, which would decrease the efficiency of ATP synthesis in the mitochondria and increase the quantity of heat released per mole of glucose oxidized. It is not clear if the ability of thyroid hormones to increase the total rate of glucose oxidation, or the postulated uncoupling, is more important in heat production, but both would definitely contribute to chronic cold stress adaptation. Rapid cold responses are mediated primarily by epinephrine from the adrenal medulla ( Chapter 8 ) rather than by thyroid hormones. TSH receptors also have been identified in rat brown adipose cells, and treatment of warm acclimated rats with TSH results in an upregulation of mRNA levels for both D2 deiodinase and UCP-1. Although acute cold exposure also caused upregulation of D2 deiodinase and UCP-1, it caused downregulation of TSH receptors, supporting uninvolvement of the HPT axis in the regulation of acute cold stress homeostasis.

In addition to increasing glucose oxidation, thyroid hormones cause hyperglycemia and may secondarily stimulate lipolysis (hydrolysis of fats). These actions may in part be associated with permissive potentiation of the hyperglycemic and lipolytic actions of epinephrine and/or glucocorticoids (see Chapters 8 and 12 Chapter 8 Chapter 12 ). Thyroid hormones may alter nitrogen balance and can be either protein anabolic (through enhancement of GH actions) or catabolic, depending on the tissue being examined and under what experimental conditions it is examined.

In many non-hibernating mammals such as beaver and muskrat, thyroid activity is depressed during the winter months. Hypothyroidism has been described for hibernating ground squirrels and badgers, but there does not appear to be a causal relationship between reduced thyroid function and the onset of hibernation. Increased thyroid activity also is correlated with arousal. Additional field studies employing sophisticated methods for assessing thyroid functions are needed before the endocrine factors related to either onset or termination of hibernation can be established .

As discussed in Chapter 4 , the enzyme aromatic L-amino acid decarboxylase (AADC) is present in many amine precursor uptake and decarboxylase (APUD) cells where it can convert biologically inactive catecholamine (l-DOPA) and indoleamine (5-hydroxytryptophan) precursors to dopamine and serotonin, respectively. Recently it has been shown that decarboxylation of thyroid hormones results in biologically active metabolites called thyronamines (TAM). Two forms of TAM have been detected so far in mammals, 3-iodothyronamine (3-T1AM) and thyronamine (T0AM) ( Box Figure 6D-1 ). Initially, it was suspected that AADC may carry out the conversion of T3 to 3-T1AM, but neither T3 or T4 is a substrate for AADC, and the enzyme (so-called iodothyronine decarboxylase) responsible for decarboxylation of T3 remains a mystery. Administration of exogenous TAMs causes effects that are opposite to those of T3, including reduced metabolic rate and respiratory depression and hypothermia. The effects of TAMs are mediated through an interaction with a G-protein-coupled membrane receptor called the trace amine associated receptor 1 (TAAR1) ( Box Figure 6D-2 ). The physiological role of TAM is unknown.


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Ho, L. H., Read, S. H., Dorstyn, L., Lambrusco, L. & Kumar, S. Caspase-2 is required for cell death induced by cytoskeletal disruption. Oncogene 14 Jan 2008 (doi:10.1038/sj.onc.1211005)


Cytochrome oxidase in the human visual cortex: Distribution in the developing and the adult brain

Cytochrome-oxidase (CO) histochemistry has revealed important functional subdivisions, modules, and processing streams in the macaque visual cortex. The present study is aimed at analyzing the development and characteristics of CO patterns in the human visual cortex by means of histochemistry and immunohistochemistry. At 26 weeks of gestation, both the ventricular and subventricular zones had low levels of CO, while the cortical plate had moderate levels of CO. At birth, supragranular CO-rich zones (puffs) were not clearly organized, indicating that the development of puffs in the neonatal striate cortex lags behind that of the macaque monkey, whose puffs appear weeks before birth. Puffs were more clearly discernible in human cortex at postnatal day 24, and became well organized by the fourth postnatal month. Layer IVc α in the neonate exhibited a higher level of activity and amount of CO than the central portion of IVc β , which contained a dense aggregate of small neurons. The base of IVc β , however, was often as CO reactive as IVc α . In contrast, the majority of specimens available to us from the fourth postnatal month and from adults with no known neurological diseases had significantly greater CO reactivity in layer IVc β than in IVca β . Layer VI was moderately reactive for CO throughout development. In V2, stripes with globular zones of high CO activity were sporadically present at birth, suggesting that their development may parallel or precede that of puffs in VI. These stripes with CO-rich globular zones became more prominent in the adult and radiated orthogonally from the V1/V2 border. They were not, however, clearly organized into alternating thick and thin stripes as they are in the squirrel monkey. Visual cortical areas beyond V2 exhibited high CO activity mainly in layers III and IV and moderate levels in VI, suggesting that sites associated with cortico-cortical pathways may be metabolically most active.


Cytochrome Oxidase Activity in Hippocampal Synaptic Mitochondria during Aging: A Quantitative Cytochemical Investigation

Address for correspondence: Dr. Carlo Bertoni-Freddari, Neurobiology of Aging Laboratory, INRCA Research Department, Via Birarelli 8, 60121 Ancona, Italy. Voice: +39-071-800-4153 fax: +39-071-206791. [email protected] Search for more papers by this author

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Address for correspondence: Dr. Carlo Bertoni-Freddari, Neurobiology of Aging Laboratory, INRCA Research Department, Via Birarelli 8, 60121 Ancona, Italy. Voice: +39-071-800-4153 fax: +39-071-206791. [email protected] Search for more papers by this author

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Neurobiology of Aging Laboratory, INRCA Research Department, 60121, Ancona, Italy

Abstract

A bstract : Synaptic mitochondria, cytochemically positive to cytochrome oxidase (COX) activity, were investigated by morphometric methods in the hippocampal dentate gyrus of adult and old rats. The number of mitochondria/μm 3 of tissue (Nv), the volume fraction occupied by mitochondria/μm 3 of tissue (Vv), the average mitochondrial volume (V), the longer mitochondrial diameter (Fmax), and the ratio R:mitochondrial area/overall area of the cytochemical precipitate due to COX activity were measured on COX-positive organelles. In old animals, Nv, Vv, V, and Fmax increased at a not significant extent R was not significantly decreased. The complement (%) of longer organelles was higher in old animals. COX activity is currently considered an endogenous marker of neuronal oxidative metabolism thus, although our findings refer to the discrete subpopulation of COX-positive organelles located at synaptic terminals, they support that changes of mitochondrial ultrastructure and metabolic competence may contribute to the age-related alterations of neuronal performances.


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In: Neuroscience , Vol. 40, No. 3, 1991, p. 825-839.

Research output : Contribution to journal › Article › peer-review

T1 - Cytochrome oxidase immunohistochemistry in rat brain and dorsal root ganglia

T2 - Visualization of enzyme in neuronal perikarya and in parvalbumin-positive neurons

N2 - Histochemical detection of cytochrome oxidase activity has been widely used to deduce patterns of neuronal electrical activity in the CNS. Here we investigated the utility of cytochrome oxidase localization by immunohistochemistry and compared immunostaining with histochemical staining patterns in dorsal root ganglia of the rat. In addition, a limited survey of cytochrome oxidase immunostaining density within what are thought to be highly active parvalbumin-immunoreactive neurons was conducted. The immunohistochemical approach produced granular cytoplasmic immunolabelling in neuronal cell bodies and allowed identification of individual labelled cells in all brain regions including those within dense immunoreactive networks of neuropil. Neuronal somata exhibited a wide range of staining densities which were particularly evident in the hippocampus and dorsal root ganglia. The distribution of neurons intensely immunoreactive for cytochrome oxidase within various structures was consistent with previous histochemical descriptions of enzyme activity. Densitometric measurements of immunohistochemical reaction product in individual neurons of hippocampus, substantia nigra, cerebellum and dorsal root ganglia showed that the rate of product deposition was linear with time under conditions chosen for comparisons of staining density. Quantitative analysis of cytochrome oxidase immunohistochemical and histochemical staining densities within the same cells in adjacent sections of dorsal root ganglion gave a correlation coefficient of r = 0.75 (P < 0.001). In sections processed immunohistochemically for both cytochrome oxidase and parvalbumin, most but not all parvalbumin-containing cells displayed dense cytochrome oxidase immunolabelling. Conversely, many examples were found of neurons that were densely stained for cytochrome oxidase, but lacked parvalbumin. Immunohistochemistry for cytochrome oxidase reveals the enzyme in neuronal cell bodies with a clarity not usually seen with the histochemical method. Combination of this immunohistochemical approach with simultaneous immunolabelling of other neuronal markers, as shown here in the case of parvalbumin, is expected to assist the elucidation of patterns of activity in neurochemically identified cell types and anatomically defined neural systems.

AB - Histochemical detection of cytochrome oxidase activity has been widely used to deduce patterns of neuronal electrical activity in the CNS. Here we investigated the utility of cytochrome oxidase localization by immunohistochemistry and compared immunostaining with histochemical staining patterns in dorsal root ganglia of the rat. In addition, a limited survey of cytochrome oxidase immunostaining density within what are thought to be highly active parvalbumin-immunoreactive neurons was conducted. The immunohistochemical approach produced granular cytoplasmic immunolabelling in neuronal cell bodies and allowed identification of individual labelled cells in all brain regions including those within dense immunoreactive networks of neuropil. Neuronal somata exhibited a wide range of staining densities which were particularly evident in the hippocampus and dorsal root ganglia. The distribution of neurons intensely immunoreactive for cytochrome oxidase within various structures was consistent with previous histochemical descriptions of enzyme activity. Densitometric measurements of immunohistochemical reaction product in individual neurons of hippocampus, substantia nigra, cerebellum and dorsal root ganglia showed that the rate of product deposition was linear with time under conditions chosen for comparisons of staining density. Quantitative analysis of cytochrome oxidase immunohistochemical and histochemical staining densities within the same cells in adjacent sections of dorsal root ganglion gave a correlation coefficient of r = 0.75 (P < 0.001). In sections processed immunohistochemically for both cytochrome oxidase and parvalbumin, most but not all parvalbumin-containing cells displayed dense cytochrome oxidase immunolabelling. Conversely, many examples were found of neurons that were densely stained for cytochrome oxidase, but lacked parvalbumin. Immunohistochemistry for cytochrome oxidase reveals the enzyme in neuronal cell bodies with a clarity not usually seen with the histochemical method. Combination of this immunohistochemical approach with simultaneous immunolabelling of other neuronal markers, as shown here in the case of parvalbumin, is expected to assist the elucidation of patterns of activity in neurochemically identified cell types and anatomically defined neural systems.


METHODS

Animal preparation and study design.

The study was approved by the Animal Care Committee of the University of Minnesota and its guidelines were followed during the experiments. From gestational d 1 until PND 10, plug-positive pregnant Sprague Dawley rats (Harlan Sprague Dawley, Indianapolis, IN, U.S.A.) received either an iron-fortified diet (Teklad 4% Mouse/Rat Diet 7001, Harlan-Teklad, Madison, WI, U.S.A.) or a low-iron diet (Formula TD 80396, Harlan-Teklad). The iron-fortified diet contained 198 mg elemental Fe/kg and the low-iron diet 3–6 mg elemental Fe/kg chow. The pups born to dams on the iron-fortified diet served as iron-sufficient (IS) controls and those born to dams on the low-iron diet served as the ID experimental group. Animals had free access to food and water and were maintained in a 12-h, day-and-night cycle at room temperature.

Five pups from each group were killed on PND 10 to obtain liver nonheme iron concentrations. Twenty-five animals from each group had their brains removed on PND 10 for iron and CytOx histochemistry.

Animal preparation.

At the time of sacrifice, the pups were deeply anesthetized with sodium pentobarbital (60 mg/kg body weight, i.p.) and then perfusion fixed transcardially with normal saline, followed by 5% neutral buffered formalin solution (Sigma Chemical Co., St. Louis, MO, U.S.A.) and 5% sucrose in 0.1 M phosphate buffer (pH 7.4). The brains were rapidly removed from the skull, postfixed in the same fixative for 4 h at 4°C, and were cryoprotected by serial overnight passage in increasing sucrose concentrations (20% and 30%) in 0.05 M phosphate buffer at 4°C. The brains were mounted in a frozen tissue embedding solution (HISTOPREP™, Fischer Scientific, Fair Lawn, NJ, U.S.A.). Serial 15-μm coronal frozen sections were obtained throughout the brains of the experimental and control animals using a cryostat at −20°C to −25°C (Bright Instruments Co., Ltd., Huntingdon, England). Sections were mounted on poly-l-lysine coated slides and stored at −80°C until histochemical analysis was performed.

Study design.

Based on recently published diagrams of the putative circuits involved in memory processing in humans ( 27 ) and in animals ( 28, 29 ) (Fig. 1), we studied brain structures involved in explicit and implicit forms of memory as well as brain structures without a known function in memory formation. Explicit memory was further subdivided into circuits involved in recognition memory, such as the hippocampus, and those involved in affective or emotional memory, such as the amygdala. Using an atlas of the developing rat brain ( 30 ), we then chose four brain sections that included the brain regions of interest (Table 1).

Schematic diagram representing brain structures involved in memory formation, both implicit and explicit. Information originates at the sensory cortices, travels through the limbic structures into the thalamic relays, and projects to the prefrontal cortex (( 26–28 ).

Tissue iron assay.

Liver iron concentrations were assayed as previously described ( 8, 31 ) and expressed as micrograms of elemental iron per gram dry tissue weight. Briefly, the tissues were thoroughly rinsed in normal saline and lyophilized for 72 h, after which dry weights were obtained. The tissues were digested in 10 mL of a 4:1 nitric:perchloric acid and their iron content was assayed by atomic absorption spectroscopy ( 31, 32 ). Values were compared with stock iron standards (Sigma Chemical Co.) diluted to the values in the expected range of tissue iron concentrations.

Iron histochemistry.

The brain sections were stained for iron by Benkovic and Connor's modification of Perls stain ( 33 ). Briefly, the sections were brought to room temperature and incubated in a 5% solution of DMSO (three changes of 5 min each) followed by 10% potassium ferrocyanide for 5 min. They were then incubated in a freshly made Perls solution (10% potassium ferrocyanide and 10% HCl in a 7:3 ratio with 0.1% Triton X-100) for 20 min at room temperature. For intensification of Perls reaction with 3,3′ diaminobenzidine, sections were thoroughly rinsed in distilled water (twice for 5 min each) and were incubated in a freshly made solution of 3,3′ diaminobenzidine (40 mg in 100 mL of 0.05 M PBS (PBS), to which 0.08 mL of 30% H2O2 was added just before the incubation) for 25 min at room temperature. The reaction was terminated by rinsing the sections in PBS (three changes of 5 min each). The sections were then serially submerged in increasing concentrations of ethyl alcohol (5 min each in 70% and 80%, followed by 10 min each in 95% and 100% ethanol) before being immersed in a clearing agent (HEMO-DE™, Fischer Scientific) for 10 min. They were then air dried and coverslipped using Permount (Fischer Scientific). Control sections were incubated in Perls solution with PBS substituted for potassium ferrocyanide. The control slides did not show any positive staining.

Iron histochemistry analysis.

Brain sections were visualized with light microscopy (Nikon Optiphot, Nippon Kogaku K.K., Tokyo, Japan) at ×400. A 100-mm 2 calibrated grid was placed in an eyepiece and then placed once centrally within each nuclei of interest. All the iron-stained cells within the boundaries of the grid were counted. One observer (K.S.) completed the brain iron quantitation.

CytOx histochemistry.

Brain sections were stained for CytOx activity using the diaminobenzidine method of Hevner and Wong-Riley ( 21 ). Briefly, the tissue sections were incubated in 100 mL of 0.1 M phosphate buffer (pH 7.4) containing 50 mg 3,3′ diaminobenzidine, 25 mg cytochrome c, and 4 g sucrose (all reagents from Sigma Chemical Co.) at 37°C for 2 h in the dark. The reaction was terminated by immersing the slides three times for 5 min each in the 0.1 M phosphate buffer at room temperature. The sections were dehydrated, cleared, and coverslipped as described above for iron histochemistry.

CytOx histochemistry analysis.

The brain sections were visualized through a 4× objective in a light microscope (Model BH-2, Olympus America Inc., Melville, NY, U.S.A.) using a green filter (wave length 510–550 nm) to enhance the contrast and a neutral density filter (Schott Glass Technologies Inc., Duryea, PA, U.S.A.) to attenuate the light intensity.

Digital microscopic images were collected using a Cohu 4915 CCD camera (Cohu, Inc., San Diego, CA, U.S.A.), a Power Macintosh 7100 computer equipped with a model LG-3 frame grabber (Scion Corp., Frederick, MD, U.S.A.) and Scion Corporation's version of the public domain National Institutes of Health Image program (available on the Internet by anonymous FTP from zippy.nimh.nih.gov or on disk from the National Technical Information Service, Springfield, VA, U.S.A., part number PB95–500195GEI).

The intensity of background light was maintained at a constant level among the sections such that the histogram of the gray scale values (range: 1–254) of the overall image showed a normal distribution and did not stack at either the black or white end of the spectrum. Dark areas were assigned a higher value and lighter areas a lower value (white 1 black 254). Hence, a lower gray scale value indicated less staining and subsequently a greater loss of CytOx activity. The intensity of CytOx reactions in the brain structures of interest was measured from the image projected and frozen on the computer screen. Larger nuclei were outlined using a cursor. For very narrow brain regions where the cursor could not be used reliably, an 11 × 11-pixel grid was randomly placed three times within the nuclei and the average OD value obtained.

Nissl histochemistry.

Because iron, as a component of ribonucleotide reductase, may play a role in cell division and cell growth, brain sections were stained for Nissl substance via Vogt's method ( 34 ) to assess for cell density. The brain sections were brought to room temperature for 10 min and subsequently incubated in a working solution of cresyl violet acetate [625 mM cresyl violet acetate (Dye content 70%, Sigma Chemical Co.) in a buffer containing 15 mM sodium acetate buffer and 3 mL/L glacial acetic acid] at room temperature for 60 min. Sections were briefly immersed in 95% ethyl alcohol, dehydrated in absolute alcohol (twice for 10 min each), then cleared and coverslipped as described above for iron histochemistry.

Nissl histochemistry analysis.

Brain sections were visualized with light microscopy (Nikon Optiphot, Nippon Kogaku K.K., Tokyo, Japan) at ×400. A 100-mm 2 calibrated grid was placed in an eyepiece and subsequently placed once within hippocampal subarea CA1 and the caudate putamen of a representative number of sections (n = 10). Using landmarks identified by Sherwood ( 30 ), an attempt was made to place the grid in the same location across the specimens. All the Nissl stained cells within a 10-mm × 3-mm boundary of the grid were counted. One observer (J.W.) completed the brain Nissl quantitation.

Statistical analysis.

The mean ± SEM for the number of iron-positive cells, the number of neurons/30 mm 2 and the CytOx activity, as expressed by OD values, were compared between ID and IS groups by two-tailed unpaired t tests. Given the number of comparisons made, a p value of < 0.01 was considered statistically significant.


CONCLUDING REMARKS

The photosensitivity of respiratory chain enzymes has never gained researchers' attention, as has that of functional photoacceptors, such as chlorophyll and rhodopsin. However, the fragmentary knowledge gathered so far forces one to ask whether the photosensitivity of cytochrome c oxidase may have a physiological significance in spite of the complete adaptation of living systems to photons as a natural external factor. IR-A radiation penetrates rather deep into the tissues (so called “optical window” of the skin or “near IR window” into the body). This circumstance also supports the hypothesis of a possible specific biological role of radiation between ∼600 and 1000 nm.