a metabolic map of cytochrome oxidase in the rat brain: histochemical, densitometric and biochemical...

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Neuroscience Vol. 65, No. 2, pp. 313-342, 1995 Elsevier Science Ltd

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A METABOLIC MAP OF CYTOCHROME OXIDASE IN THE RAT BRAIN: HISTOCHEMICAL, DENSITOMETRIC

A N D BIOCHEMICAL STUDIES

R. F. H E V N E R , * S. LIU and M. T. T. W O N G - R I L E Y t

Department of Cellular Biology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, U.S.A.

Abstract--To examine brain patterns of metabolic and functional activity, the distribution of cytochrome oxidase, a mitochondrial enzyme marker for neuronal functional activity, was mapped throughout the rat brain. Mapping was done qualitatively by enzyme histochemistry of brain sections cut in three planes (coronal, sagittal and horizontal), and quantitatively by optical densitometry of stained sections and by biochemical assays of brain tissue homogenates.

Activity of the enzyme was distributed in characteristic patterns and amounts that differed among various neural pathways, brain nuclei, cerebral cortical areas and layers, and neuron types. Gray matter essentially always had higher enzyme activity than did white matter, by a factor of eight- to 12-fold. Among different neural pathways, cytochrome oxidase activity was relatively high in special sensory, somatosensory and motor systems, and was relatively low in associative, limbic, autonomic and visceral regulatory systems (though exceptional areas were present). Among 11 different neuron types, nearly a two-fold range of histochemical staining intensities was observed, with the darkest staining in neurons of the mesencephalic trigeminal nucleus.

The observed patterns of cytochrome oxidase activity were mostly similar to the patterns of 2-deoxyglucose uptake seen previously [Schwartz W. J. and Sharp F. R. (1978) J. comp. Neurol. 177, 335-360; Sokoloff L. et al. (I 977) J. Neurochem. 28, 897-916] in conscious, "resting" animals, though some differences were found. For example, whereas 2-deoxyglucose uptake was about three-fold higher in gray matter than in white matter [Sokoloff L. et al. (1977) J. Neurochem. 28, 897-916], cytochrome oxidase activity was about eight- to 12-fold higher. This and other discrepancies probably reflect basic technical differences between these two methods. Compared to 2-deoxyglucose, cytochrome oxidase is more specific for oxidative rather than glycolytic metabolism, and more reflective of overall neuronal functional activity occurring over longer time periods lasting hours to weeks, rather than minutes. The anatomical resolution of cytochrome oxidase histochemistry is also finer than that of 2-deoxyglucose autoradiography, extending to the electron microscopic level.

The metabolic map of cytochrome oxidase activity reveals patterns of normal brain function, and may be useful as a baseline for comparison in studies of brain disease, development, ageing and plasticity.

To study patterns of brain activity, researchers have frequently turned to metabolic mapping methods, which utilize parameters such as glucose utilization, blood flow and oxidative enzyme activity as con- venient markers for neuronal functional activity. Metabolic mapping is based on the principle that energy utilization in brain tissue is determined by the overall functional activity of neurons (reviewed by Erecinska and Silver m and DiRocco et al.9).

Examples of metabolic mapping methods include 2-deoxyglucose (2-DG) autoradiography, cytochrome oxidase (CO) histochemistry and positron emission tomography. These methods have been used to map brain activity during "norma l" or "rest ing"

*Present address: Department of Pathology, Division of Neuropathology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305, U.S.A.

tTo whom correspondence should be addressed. Abbreviations: 2-DG, 2-deoxyglucose; CO, cytochrome

oxidase; EDTA, ethylenediaminetetra-acetate.

conditions, and during conditions of particular interest such as sensory stimulation, development, ageing and disease.

Our goal in the present study was to generate a map of metabolic and functional activity in the normal rat brain using CO as a marker. In general, CO activity is linked to the level of neuronal functional activity occurring over periods of hours to weeks, as shown by previous studies in which neuronal stimulation was manipulated to induce changes in CO activity (reviewed by Wong-Riley~8). Pre- viously, metabolic maps of functional activity in the rat brain under conscious, "rest ing" (physically restrained) conditions have been generated using the 2 -DG method. 28'3° However, 2 -DG uptake is determined by neuronal activity during relatively short experimental periods, e.g. 40-45 min, and therefore "rest ing" conditions might not reflect overall brain activity throughout the full cycle of normal daily activities. Thus, the picture of brain activity obtained using CO as marker might differ from that obtained

313

314 R . F . Hevner et al.

using 2 - D G . The h i s tochemica l m e t h o d for C O offered the add i t i ona l a d v a n t a g e o f h igh reso lu t ion , ex t end ing to the subcel lu lar (e lec t ron mic roscop ic ) level.

W e m a p p e d C O activi ty qual i ta t ive ly t h r o u g h a series o f C O - s t a i n e d ra t b ra in sect ions, and quan t i - ta t ively by two m e t h o d s : (i) dens i t ome t ry , which indirec t ly m e a su r e s C O activi ty f r o m the in tens i ty o f t issue s ta ining, and (ii) b iochemica l assays , wh ich direct ly m e a su r e e n z y m e act ivi ty in t issue h o m o g e n - ates. W e have p re sen ted these d a t a prev ious ly in ab s t r ac t form. 2°

EXPERIMENTAL PROCEDURES

Materials

Deoxycholate was purchased from Fluka. Bovine serum albumin and cytochrome c (horse heart, type III) were from Sigma. Phiobupabarbital (Inactin) for anesthesia was obtained from Byk Gulben (Konstanz, Germany).

Animals

All animals in this study were used in accordance with NIH and Medical College of Wisconsin regulations. A total of 21 adult albino rats were used; of these, seven were perfused for histochemistry (male, Sprague-Dawley, 300~400 g) and 14 were used for biochemical work (male and female, Sprague-Dawley and Munich Wistar strains, 220-440 g). The rats used for biochemistry (provided cour- tesy of Dr Shanhong Lu, Department of Physiology, Medical College of Wisconsin) received a calcium-channel blocker, diltiazem (20-800 #g/kg, intrarenal infusion over 20 min), as part of a separate study. The diltiazem was used to induce local alterations in renal medullary blood flow, and normal blood pressures were maintained. Some data from these animals were reported in our previous paper on optimization of the CO biochemical assay/6

Histochemistry

Rats for histochemistry were deeply anesthetized with chloral hydrate (4% solution, 1 ml/100 g, i.p.) and perfused transcardially with buffer and 4% paraformaldehyde, as described previously. 23 The brains were removed from the skull and postfixed by immersion for 1 h in fixative at 4°C. The fixed brains were cryoprotected in increasing concen- trations of sucrose (4%, 10%, 20%, 30%) in 0.1 M sodium phosphate buffer at 4°C, frozen on dry ice and sectioned at 30/ tm on a freezing sliding microtome. Two brains were cut in the coronal plane, two in the sagittal plane and three in the horizontal plane. One of the horizontally cut brains reacted unevenly due to poor fixation and was not included in further analyses. Every third section was reacted histochemically for CO activity, as described previously? 7 An adjacent one-in-three series was stained with Cresyl Violet. Sections were photographed using a Wild Makroskop M420 and 4 x 5 inch Kodak Ektapan film. CO-stained sections were photographed using a blue filter (Kodak Wratten No. 47) for contrast enhancement. Areas were identified according to the atlas of Paxinos and Watson. z7

Regional densitometry

Optical density measurements over relatively large brain regions (e.g. thalamus, cerebellum, optic nerve) were obtained using a Zeiss microscope and PI-2 photometer. Readings were taken through a 625/~m diameter circular aperture using a x4 objective. Background density was subtracted by setting optical density at zero over a blank area (containing no tissue) in each slide. White (tungsten) light was used for illumination, and lighting was held

constant between sections and areas. Optical density (arbitrary units) was averaged from readings taken over three different positions within the region of interest in one to six slides from each animal; thus, three to 18 readings were taken per region per animal. The mean optical density in each region was calculated for individual animals, and these values were then averaged across animals (n = 6 rats for most regions, except n = 4 rats for visual cortex).

Cellular densitometry

To measure optical density in individual cell bodies, a Zeiss Zonax MPM 03 photometer system was used with a x 25 objective and a 2/~m diameter circular aperture. White

(tungsten) light was used for illumination. Lighting conditions were held constant between sections and cell types. The background was substracted by setting zero over a blank area (containing no tissue) in each slide. For each cell type, optical density readings (arbitrary units) were taken from perikarya in five different sections from each animal; the mean optical densities of each cell type in individual animals were then averaged across animals (n = 5 rats).

Preparation of brain tissue homogenates

Rats were deeply anesthetized with phiobupabarbital (100mg/kg, i.p.) and decapitated. Barbiturates such as phiobupabarbital do not affect CO activity? The brains were immediately removed, and either weighed and homogenized whole (n = 6) or dissected into regions, which were then weighed and homogenized separately (n = 8; not all regions were assayed in all animals; see legend to Fig. 31 for regional n values). The brain regions were kept frozen on dry ice covered with aluminum foil until they could be weighed and homogenized. Some of the dissected regions were used for preliminary experiments to optimize the brain CO assay. ~6 For the numbers of animals in which each region was assayed biochemically, see the legend to Fig. 31.

Biochemical assays

The brain samples were homogenized, solubilized and assayed for CO activity using a spectrophotometric assay 1~,34 modified as described previously, t6 Briefly, each brain or region was homogenized with isolation buffer (0.32 M sucrose, 1 mM dipotassium EDTA, I0 mM Tris, pH 7.4) to prepare a 20% homogenate, which was subsequently kept on ice. A motor-driven Teflon-glass (Potte~Elvehjem type) homogenizer was used for whole brain, while hand-held 1.5 ml polypropylene microfuge tubes and matched polypropylene pestles (Kimble) were used for homogenizing brain regions. Aliquots of the 20% homogenates were solubilized with deoxycholate, diluted with additional isolation buffer and assayed for CO activity within 30 rain of solubilization. One unit (U) of enzyme activity was defined as 1 #tool cytochrome c oxidized/min. Homogenates (20% or deoxycholate-solubilized) were frozen after CO assay and stored at - 2 0 ° C until needed for protein determination, done by the BCA method (Pierce) using bovine serum albumin as the standard.

RESULTS

Cytochrome oxidase histochemistry

C O activi ty in di f ferent b ra in regions was exami ned in ad jacen t CO- and Niss l - s ta ined sect ions cut in the co rona l , sagi t tal and ho r i zon ta l p lanes . F igures 1 and 2 s h o w one pa i r o f ad jacen t Nissl- and C O - s t a i n e d hor i zon ta l sec t ions for c o m p a r i s o n o f the s ta in ing m e t h o d s . F igures 3 28 s h o w add i t iona l whole b ra in sec t ions s ta ined for CO. H i ghe r magn i f i ca t ion views o f selected areas ( s e n s o r i m o t o r cor tex and midb ra in ) are p resen ted in Figs 29 and 30.

Map of cytochrome oxidase in the rat brain 315

Abbreviations used in the figures

2 n

3 3V 4 4V 6 7 7n 8n 10 12 12n a c

Acb ACo AD AH AHC AHi AM Amb Amg AO AP APT Aq a r

Arc ATg AV bic BL BM bnd

brl bsc BST CI C2 CA1 CA2 CA3 CA4 Cb CC CC

Ce cg Cg CG CGD cic CI CM Co Cop cp CPu CSC

Cu cu DCIC DCo DEn DG dhc Dk DLG DLL

optic nerve principal oculomotor nucleus third ventricle trochlear nucleus fourth ventricle abducens nucleus facial nucleus facial nerve or its root vestibulocochlear nerve dorsal motor nucleus of the vagus hypoglossal nucleus hypoglossal nerve or its root anterior commissure accumbens nucleus anterior cortical amygdaloid nucleus anterodorsal thalamic nucleus anterior hypothalamic area anterior hypothalamic area, central part amygdalohippocampal area anteromedial thalamic nucleus ambiguus nucleus amygdala anterior olfactory nucleus area postrema anterior pretectal area cerebral aqueduct acoustic radiation arcuate hypothalamic nucleus anterior tegmental nucleus anteroventral thalamic nucleus brachium of inferior colliculus basolateral amygdaloid nucleus basomedial amygdaloid nucleus bands of dark CO staining in cerebellar vermis barrels in somatosensory cortex brachium of superior colliculus bed nucleus of stria terminalis crus I of cerebellar ansiform lobule crus 2 of cerebellar ansiform lobule field CA1 of hippocampus field CA2 of hippocampus field CA3 of hippocampus field CA4 of hippocampus cerebellum central canal of spinal cord corpus callosum central amygdaloid nucleus cingulum bundle cingulate cortex central gray central gray, dorsal part commissure of inferior colliculus claustrum central medial thalamic nucleus cortical amygdaloid nucleus copula pyramis of cerebellum cerebral peduncle caudate-putamen commissure of superior colliculus cuneate nucleus cuneate fasciculus dorsal cortex of inferior colliculus dorsal cochlear nucleus dorsal endopiriform nucleus dentate gyrus dorsal hippocampal commissure nucleus of Darkschewitsch dorsal lateral geniculate nucleus dorsal nucleus of lateral lemniscus

DpG DPGi DpMe DR DTg dtgx e c

ECIC ECu Ent EP EPI EW f fi FL fr

Frl Fr2 g7 gcc Gi GI GP Gr gr HC HDB HL ic IC ~cj icp IGr InG

Ins Int InWh

IO IP IPC IP1 IPR

IRt isl

La Lat LD lfp LH LHb LHBL 11 lo LO LP LPO LRt LS LSD LSI LSO LV LVe MB

deep gray layer of superior colliculus dorsal paragigantocellular nucleus deep mesencephalic nucleus dorsal raphe nucleus dorsal tegmental nucleus dorsal tegmental decussation external capsule external cortex of inferior colliculus external cuneate nucleus entorhinal cortex entopeduncular nucleus external plexiform layer of olfactory bulb Edinger-Westphal nucleus fornix fimbria of hippocampus forelimb somatomotor cortex fasciculus retroflexus (habenulopeduncular tract) frontal cortex area 1 frontal cortex area 2 genu of the facial nerve genu of corpus callosum gigantocellular reticular nucleus glomerular layer of the olfactory bulb globus pallidus gracile nucleus gracile fasciculus hippocampus horizontal limb, nucleus of diagonal band hindlimb somatomotor cortex internal capsule inferior colliculus islands of Calleja inferior cerebellar peduncle internal granular layer of olfactory bulb intermediate gray layer of superior colliculus insular cortex interposed cerebellar nucleus intermediate white layer of superior colliculus inferior olivary nucleus interpeduncular nucleus interpeduncular nucleus, caudal subnucleus inner plexiform layer of olfactory bulb interpeduncular nucleus, rostral subnucleus intermediate reticular nucleus islands of dark CO staining in entorhinal cortex lateral amygdaloid nucleus lateral cerebellar nucleus laterodorsal thalamic nucleus longitudinal fasciculus of pons lateral hypothalamic area lateral habenular nucleus lateral habenular nucleus, lateral part lateral lemniscus lateral olfactory tract lateral orbital cortex lateral posterior thalamic nucleus lateral preoptic area lateral reticular nucleus lateral septal nucleus lateral septal nucleus, dorsal part lateral septal nucleus, intermediate part lateral superior olive lateral ventricle lateral vestibular nucleus mammillary body

Abbreviations continued overleaf


mcp MD MdD MdV Me Me5 Med mfb MG MHb Mi ml mlf MnR MO Mo5 mp MPA mt MVe MVPO OB Ocl OclB Oc lM Oc2 o n

OP opt OX

Parl Par2 PaS pat

PaV PB pc PCRt PDTg PF PF1 PH Pin Pir PiRe PM PMR Pn PnC PnO Po Pr5 PrH PRh PrS PV PY pyx

middle cerebellar peduncle mediodorsal thalamic nucleus medullary reticular field, dorsal medullary reticular field, ventral medial amygdaloid nucleus mesencephalic trigeminal nucleus medial cerebellar nucleus medial forebrain bundle medial geniculate nucleus medial habenular nucleus mitral cell layer of olfactory bulb medial lemniscus medial longitudinal fasciculus median raphe nucleus medial orbital cortex motor trigeminal nucleus mammillary peduncle medial preoptic area mammillothalamic tract medial vestibular nucleus medioventral periolivary nucleus olfactory bulb occipital cortex area 1 occipital cortex area 1, binocular part occipital cortex area 1, monocular part occipital cortex area 2 olfactory nerve optic nerve layer of superior colliculus optic tract optic chiasm parietal cortex area 1 parietal cortex area 2 parasubiculum patches of dark CO reactivity in superior colliculus paraventricular hypothalamic nucleus parabrachial nuclei posterior commissure parvocellular reticular nucleus posterodorsal tegmental nucleus parafascicular thalamic nucleus paraflocculus posterior hypothalamic area pineal body piriform cortex pineal recess paramedian lobule of cerebellum paramedian raphe nucleus pontine nuclei pontine reticular nucleus, caudal part pontine reticular nucleus, oral part posterior thalamic nuclear group principal sensory trigeminal nucleus prepositus hypoglossal nucleus perirhinal cortex presubiculum paraventricular thalamic nucleus pyramidal tract pyramidal decussation

Abbreviations (continued)

R red nucleus ReIC recess of inferior colliculus RF rhinal fissure rs rubrospinal tract RS retrosplenial cortex Rt reticular thalamic nucleus RtTg reticulotegmental nucleus of pons S subiculurn s5 sensory root of trigeminal nerve SC superior colliculus SCh suprachiasmatic nucleus scp superior cerebellar peduncle SFi septofimbrial nucleus SG suprageniculate nucleus SI substantia innominata Sim simple lobule of cerebellum sm stria medullaris of thalamus SNC substantia nigra, pars compacta SNR substantia nigra, pars reticulata sol solitary tract Sol solitary nucleus sp5 spinal trigeminal tract Sp5 spinal trigeminal nucleus Sp5C spinal trigeminal nucleus, caudal part Sp5I spinal trigeminal nucleus, interpolar

part Sp50 spinal trigeminal nucleus, oral part SPO superior paraolivary nucleus STh subthalamic nucleus SubG subgeniculate nucleus SuG superficial gray layer of superior

colliculus SuVe superior vestibular nucleus TC tuber cinereum Tel temporal cortex area 1 Te2 temporal cortex area 2 Te3 temporal cortex area 3 TT tenia tecta Tu olfactory tubercle tz trapezoid body VCo ventral cochlear nucleus vhc ventral hippocampal commissure VL ventrolateral thalamic nucleus VLG ventral lateral geniculate nucleus VLL ventral nucleus of lateral lemniscus VM ventromedial thalamic nucleus VMH ventromedial hypothalamic nucleus VP ventral pallidum VPL ventral posterior thalamic nucleus,

lateral part VPM ventral posterior thalamic nucleus,

medial part vsc ventral spinocerebellar tract VTg ventral tegmental nucleus wm white matter xscp decussation of superior cerebellar

peduncle ZI zona incerta ZID zona incerta, dorsal part

Fig. 1-28. CO reactivity in sections through the whole rat brain. Horizontal sections are shown in Figs 1 3, sagittal sections in Figs 4-10 and coronal sections in Figs 11 28. The section in Fig. 1 was stained with Cresyl Violet, for comparison with the adjacent CO-stained section in Fig. 2; all other sections were stained by CO histochemistry. The coordinates given at the bottom of each figure represent estimated stereotactic positions according to the atlas of Paxinos and Watson. 27 Scale bars = 2 mm (Figs 1-10); 1 mm

(Figs 11-28).


OB

LO

Ins

Te3

RF

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Fig. 1

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~3

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Fig. 10

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Infer~rol 15.70ram

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Interaural 14.20mm

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Interaural lO.60mm Fig. 13

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Fig. 29. Adjacent sections of rat somatomotor cortex (border zone of forelimb and hindlimb areas) stained with Cresyl Violet (A) and CO (B). Note that the aggregates of granule cells (granular zones) in layer IV (A, below arrows) stain slightly darker for CO (B, below arrows) than do the intervening dysgranular (or agranular) zones. The border between cortex and white matter is sharply demarcated by Cresyl Violet as well as CO (arrowheads in B) staining. Asterisks indicate corresponding blood vessels. Cortical layers are

indicated at left. Scale bar = 0.3 mm.

General observations. Throughout the brain, the following general properties of CO reactivity were observed. (1) Gray matter essentially always had higher CO activity than did white matter. This obser- vation was further confirmed by densitometric measurements and biochemical assays (see below). (2) Gray matter areas displayed more variations of CO reactivity than did white matter areas. Whereas white matter appeared almost uniformly pale in CO-reacted sections, staining intensity in gray matter areas varied markedly among different structures. These variations usually highlighted particular areas, which conformed to specific anatomical boundaries: e.g. the thalamic anterodorsal nucleus was sharply demarcated as a zone of very dark reactivity, contrasting with the adjacent, moderately reactive anteroventral and anteromedial nuclei (Figs 5, 6, 15). (3) CO reactivity in gray matter did not depend on the density of neuronal cell bodies relative to neuropil, nor were either cell bodies or neuropil consistently high in CO. For example, CO activity in the red nucleus was higher in cell bodies than it was in the neuropil (Fig. 30D), but in the nearby superficial gray of the superior colliculus, CO activity was higher in the neuropil and cell bodies did not stand out (Fig. 30A). Similar variations among neurons and neuropil

have also been demonstrated at the electron microscopic level, e.g. in the hippocampus. ~7

Neural systems. Relatively high CO reactivity was present in most areas belonging to special sensory, somatosensory and somatomotor pathways, while lower reactivity was found in areas linked to associational, limbic, autonomic and visceral regulatory functions. (However, several exceptions were observed, some of which are described below.)

Sensory and motor systems. The auditory system contained several darkly reactive nuclei. Among them, the dorsal cochlear nucleus was particularly dark (especially in the neuropil) and was one of the most intensely stained structures in the brain (Figs 2, 7, 8, 22, 23). Other auditory nuclei with very dark CO reactivity included the ventral cochlear nucleus (Figs 22, 23), the dorsal and ventral nuclei of the lateral lemniscus (Figs 2, 20, 21), and the lateral superior olivary nucleus (Fig. 8). Somewhat lower overall levels of CO activity, focally ranging from low to moderate or high, were present in the medial geniculate nucleus (Figs 3, 9, 18, 19), inferior colliculus (Figs 3-9, 20) and primary auditory cortex (Figs 3, 17).

Examples of other special sensory and somatosensory nuclei with high CO reactivity included the


Fig. 30. CO reactivity in the rat midbrain. A low-magnification view through the upper midbrain at the level of the superior colliculus is shown in A. Boxed areas in A are shown at higher magnification in B (substantia nigra), C (mesencephalic trigeminal nucleus) and D (red nucleus). Sections through the lower midbrain at the level of the inferior colliculus are shown in E and F. Note the dark reactivity of the oculomotor and trochlear nuclei (A, E). The mesencephalic trigeminal nucleus comprises intensely CO-reactive neurons (A, C, E, F). Darkly reactive individual neurons and their proximal processes are also visible in the substantia nigra pars reticulata (A, B) and in the red nucleus (A, D). Other darkly CO-reactive nuclei include the interpeduncular nucleus (A) and the ventral tegmental nucleus (F). Structures such as the central gray (A), the pineal body (A) and neuropil of the substantia nigra (A, B) were moderately reactive. The central gray contained some staining heterogeneities, which have been described previously by Conti e t a l . 6 Scale bars = 0.5 mm (A, F); 50 ~m (B, D); 40 #m (C); 0.3 mm (E).


following: in the olfactory system, the olfactory bulb glomeruli and external plexiform layer (Fig. 11); in the vestibular system, the superior (Fig. 22) and medial (Figs 2, 5, 6, 22-24) vestibular nuclei; in the somatosensory system, neurons of the mesencephalic trigeminal nucleus (Figs 3, 30), portions of the principal (Figs 9, 22) and spinal (Figs 2, 8, 9, 22-28) trigeminal nuclei, cortical barrels (Figs 3, 10, 13-16) and thalamic barreloids in the ventral posterior medial nucleus (Figs 3, 6); and in the visual system, the primary visual cortex including monocular and binocular subdivisions (Figs 9, 10, 19, 20).

Somatomotor areas with high CO reactivity included cranial nerve somatomotor nuclei, as well as components of the basal ganglia and cerebellar systems (extrapyramidal motor pathways). Very intense CO reactivity was present in the oculomotor (Figs 4, 19, 30A), trochlear (Figs 4, 30E), abducens (Figs 5, 22) and facial nuclei (Figs 8, 22, 23). Dark, but slightly less intense, reactivity was also present in the nucleus of Darkschewitz (Figs 4, 18), the nucleus ambiguus (Figs 24-26), the hypoglossal nucleus (Figs 2, 4, 5, 25, 26) and the prepositus hypoglossal nucleus (Fig. 23). The motor trigeminal nucleus was heterogeneous, but mostly had moderate to high CO reactivity (Figs 2, 7).

Basal ganglia. Subdivisions of the basal ganglia with high or very high CO reactivity included the caudate-putamen (Figs 2, 3, 6-10, 13-17) and the nucleus accumbens (Figs 5-8, 13). Neuropil in the caudate-putamen and nucleus accumbens was generally more reactive than were neurons, and in both nuclei there was a distinct patchy pattern of dark and moderate CO reactivity (e.g. Figs 2, 8, 13), as shown by DiFiglia et al. 8 (see their paper for more details). The substantia nigra had moderate overall CO reactivity in the neuropil of both pars compacta and pars reticulata (Figs 7, 8, 18, 19, 30A, B); however, cell bodies were stained much darker in pars reticulata than in pars compacta (Fig. 30A, B), as previously observed by Weiss- Wunder and Chesselet. 33 The globus pallidus (Figs 2, 8, 9, 15, 16) and ventral pallidum (Figs 6-8, 14) had predominantly low CO reactivity, though some moderately reactive cell bodies were noted in the globus pallidus.

Cerebellum. In the cerebellum, the cerebellar cortex (Figs 3-9, 22-26) as well as the medial (Med), interposed (Int) and lateral (Lat) deep nuclei (Figs 3, 6-9, 23) all contained high overall levels of CO reactivity. The cerebellar cortex of each hemivermis contained two parasagittal bands of darker CO reactivity that alternated with bands of lighter reactivity (e.g. Fig. 3), as previously observed by Hess and Voogd 13 and Leclerc et al. 19 Different layers and cell types in the cerebellar cortex had characteristic enzyme reactivities, as described previously by Mjaatvedt and Wong-Riley. 23 Dark CO reactivity was also seen in the red nucleus (Figs 2, 5, 6, 30A, D) and the inferior olivary nucleus (Figs 4-6, 24-26),

both of which have strong direct connections with the cerebellum. In the red nucleus, intense reactivity was localized in cell bodies (Fig. 30D).

Autonomic, associational, limbie and visceral regulatory systems. Most components of these systems had moderate or low CO reactivity, with few exceptions. Among cranial nerve nuclei belonging to these systems, the nucleus solitarius (Figs 2, 4-6, 25, 26), which mediates visceral sensation, and the Edinger-Westphal (Figs 18, 19) and dorsal motor vagal (Fig. 26) nuclei, which comprise preganglionic parasympathetic neurons, all had overall low levels of CO reactivity. By comparison, most nuclei in other types of sensory and motor systems were darkly CO-reactive (as described above). However, the autonomic nuclei were not uniformly low in CO reactivity: e.g. some cell bodies in the nucleus solitarius were darkly CO-reactive (not shown).

Virtually all subdivisions of the hypothalamus had low CO reactivity (Figs 4-6, 14-18), except for the mammillary bodies, which were intensely CO-reactive (Figs 4-6, 18). The anterodorsal nucleus of the thalamus, which receives direct input from the mammillary body, was also darkly CO-reactive (Figs 6, 15). The closely related anteroventral and anteromedial thalamic nuclei (Figs 3, 5-7, 15) had somewhat lower CO reactivity, in the moderate range. Other limbic areas with uncharacteristically dark CO reactivity were the medial and lateral habenula (Figs 4, 5, 16, 17), and the caudal and (somewhat less intense) rostral subdivisions of the interpeduncular nucleus (Figs 4, 19, 30A). The retrosplenial cortex was also relatively dark (Figs 5, 6, 15-20). Nevertheless, the majority of limbic structures contained overall moderate levels of CO reactivity, including the cingulate cortex (Figs 4, 13, 14), the septal nuclei (LS, LSD, LSI, SFi; Figs 3-6, 13, 14), the amygdala (Amg, ACo, BL, BM, Ce, Co, La, Me; Figs 9, 10, 15-18) and the hippocampal formation (HC, PaS, PrS, S, CA1, CA3, DG; Figs 2, 3, 5-9, 15-19; see also densitometric and biochemical results below). Certain subdivisions and cell types in some of these limbic areas (notably the hippocampal formation and the amygdala) displayed considerable heterogeneity of CO reactivity, including some darkly stained structures, but overall reactivity in these major limbic areas was judged to be moderate.

Associational areas of cortex such as parietal cortex area 2 (Figs 3, 14-16), occipital cortex area 2 (Figs 7-10, 17-19) and temporal cortex area 3 (Figs 2, 17-19) were also relatively low in CO reactivity, compared to other cortical areas. Most "non- specific" thalamic nuclei with limbic or associational connections, e.g. the paraventricular, mediodorsal and lateral posterior nuclei (Figs 3 5, 7 9, 15-17), likewise had low CO activity.

Reticular system. The reticular system is difficult to analyse comprehensively because of its diverse and, in some instances, diffuse nuclei. Nevertheless, a few of the better-defined reticular nuclei were quite rich in CO activity. Most remarkable was the


ventral tegmental nucleus (Figs 20, 30), which was one of the most intensely CO-reactive structures in the brain.

Cerebral cortex. In the cerebral cortex, CO activity varied not only among cytoarchitectonic areas (several examples are given above), but also according to cortical layer and, in some areas, according to columnar or modular organization. The highest overall levels of CO activity were seen in the primary sensory and motor areas, including the hindlimb and forelimb sensorimotor cortex (Figs 7-9, 14-16, 29), the primary somatosensory cortex (Figs 3, 10, 13-17), the primary visual cortex (including monocular and binocular subdivisions; Figs 8-10, 19, 20) and the primary auditory cortex (Figs 3, 17, 18). However, the primary olfactory, or piriform cortex (Figs 8-10, 13-17), had only moderate overall CO levels, similar to those in associational and limbic areas. The cortical layers with the darkest CO reactivity were, in most areas, layers I l l - IV (e.g. in forelimb and hindlimb sensorimotor cortex; Figs 7-9, 14-16, 29). One excep- tion to this rule was the entorhinal cortex (Figs 2, 3, 10, 18 21), where CO reactivity was darkest in layer II, and in layer V in the ventral medial portion of this area (Fig. 10), as we have described previously. 14

In some cortical areas, columnar or modular patterns were prominently marked by CO staining. For example, in the limb sensorimotor areas (hindlimb and forelimb), the granular zones of layer IV had somewhat higher CO reactivity than did the dysgranular (or agranular) zones (Fig. 29). In the primary somatosensory cortex, whisker barrels were darkly CO-reactive, and were separated by zones of lighter CO reactivity in the barrel septa (Figs 3, 10, 13-16), as first shown in the mouse by Wong-Riley and Welt. 4° In the lateral entorhinal cortex, the cell clus- ters in layer II formed darkly CO-reactive islands separated by less reactive interstices (Figs 1, 2, 20), as we have recently reported. 14 Each of these patterns was clearly visible by Nissl staining, but was enhanced or complemented by CO histochemistry.

Subcortical modular patterns. Modular patterns of CO activity were not limited to the cerebral cortex, but were found in some subcortical structures as well. The alternating bands of dark and light CO reactivity in the cerebellar cortex of the vermis, the barreloids in the somatosensory thalamus, and the patchy reactivity in the nucleus accumbens and caudate-putamen are examples that were described above. A modular pattern was also seen in the superior colliculus, where patches of dark CO reactivity were observed in the intermediate gray layer (Fig. 19), similar to those reported in the mouse superior colliculus by Wallace 32 and Wiener. 35 These subcortical modular patterns were not visible by routine Nissl staining.

Neurons and neuropil. Major variations of CO reactivity were found on a relatively small size scale, i.e. among neurons and among local zones of neuropil. In some areas, neuronal cell bodies and their

processes were darkly stained against a background of less reactive neuropil, for example in the mesencephalic trigeminal nucleus and substantia nigra pars reticulata (both in Fig. 30). Not all neuronal cell bodies were darkly reactive, though, and various neuron types differed markedly in their CO reactivity. For example, mesencephalic trigeminal and thalamic reticular neurons appeared very dark, while cerebellar granule cells, dentate granule cells and hippocampal CA1 pyramids appeared quite pale. Such variations did not correlate in any consistently observable way with cell size, morphological classification (e.g. pyramidal shape), neurotransmitter type or functional role.

Quantitative measurements of the staining differences among some neuron types were obtained by densitometry of cell bodies (see below). Differences of CO reactivity at the subcellular level, e.g. among different processes and segments of processes belonging to the same neuron, were beyond the scope of the present study, but have been observed previously by electron microscopy. 8,17,23,24,38,41

Cytochrome oxidase biochemistry

Biochemical assays were used to study whole brains, and large brain regions (such as the hippocampus and cerebral cortex) that could be quickly and reliably dissected from the brain. Smaller brain structures (such as neurons or cortical layers) that are not as readily amenable to biochemical analysis were studied by histochemistry and densitometry only (see below).

Whole brain cytochrome oxidase assays. Overall enzyme activity was measured in homogenates of the whole adult rat brain (n = 6). The average brain weight was 1.69 ___ 0.14 g (mean + S.D.) and the average protein content was about 9% of wet weight (89.9___ 6.4mg protein/g tissue). The average CO activity content was 158+ 12mU/mg tissue (wet weight) and the average specific activity was 1760 ___ 100mU/mg protein. The molecular activity, or turnover number, of the rat brain enzyme was calculated to be 479 s -1, based on a cytochrome aa 3 content in the adult rat brain of 5.5 nmol cytochrome aa3/g tissue. 3 Some of these results have been pre- sented previously in our paper describing the CO assay method. ~6

Regional cytochrome oxidase assays. The overall CO activity content (by wet weight) and CO specific activity (by protein) were measured in 11 regions of adult rat brain, including eight gray matter structures and three white matter structures. To control for possible sampling errors related to the dissec- tions, several areas were dissected from both the fight and left hemispheres and assayed separately; no significant right-left differences were found (data not shown).

As shown in Fig. 31A and B, all of the gray matter regions had higher CO activity than did the white

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Fig. 3 I. CO activity in the whole rat brain, and in eight large regions of gray matter and three white matter areas. A and B show CO activity content (by tissue wet weight) and CO specific activity, respectively, as determined by biochemical assays of tissue homogenates (n - 6 for whole brains; n = 3 for corpus callosum; n = 4 for visual cortex and trigeminal nerve; n = 5 for all other regions). C shows the optical density of CO histochemical staining, as determined densitometrically through a 625 #m aperture in the same large regions of brain (n = 4 for visual cortex; n = 6 for all other regions). Significant differences among regions are stated in the text. D and E show the optical density in each area plotted against the CO activity content (by tissue wet weight) and CO specific activity, respectively. D and E demonstrate that CO activity was well correlated with histochemical staining intensity, but there were few differences in overall CO activity or staining among the white matter areas, or among the large gray matter regions. Smaller brain structures such as cortical layers, brain nuclei and cell types differed much more in their CO activity than did the large brain regions analysed in this figure (see text for details). All data shown

as mean _+ S .E .M.N.D. , not determined. Methods are described in the text.

m a t t e r regions. The C O act ivi ty c o n t e n t (by wet weight ) was genera l ly a b o u t 10- to 12-fold h igher in gray m a t t e r t h a n in whi te m a t t e r (Fig. 31A), whi le the specific act ivi ty o f the enzyme was a b o u t eight- to 10-fold h igher in gray t h a n in whi te m a t t e r (Fig. 31B). Dif ferences be tw een gray and whi te m a t t e r were g rea te r for C O act ivi ty c o n t e n t (by wet weight) t h a n for C O specific act ivi ty, because whi te m a t t e r h a d a lower p r o t e in c o n t e n t t han d id gray ma t t e r . Stat- ist ically s ignif icant d i f ferences were f o u n d on all

pa i r -wise c o m p a r i s o n s be tween gray and whi te m a t t e r regions, for b o t h C O activi ty c o n t e n t (by wet weight) and C O specific act ivi ty ( P < 0.001 for each c o m - pa r i son , o n e - f a c t o r A N O V A with least s ignif icant di f ference test).

A m o n g the large b ra in reg ions tested, the h ighes t C O act ivi ty c o n t e n t (by wet weight ) was f o u n d in cerebel la r cor tex (157 -I- 17 m U / m g tissue, m e a n _+ S .E.M.) , while the h ighes t CO specific act ivi ty was f o u n d in visual cor tex (1760 _+ 150 m U / m g prote in) .


However, neither of these measurements was significantly different between these two regions. Indeed, no significant differences of CO specific activity were detected among any gray matter regions, and only two significant differences of CO activity content (by wet weight) were found: between cerebellar cortex and hippocampus, and between cerebellar cortex and olfactory bulb (P < 0.05, one-factor ANOVA with least significant difference test). Likewise, none of the white matter regions differed significantly from one another in their CO activity content (by wet weight) or in their CO specific activity. Thus, CO levels differed little or not at all among major brain regions of the same type (i.e. gray or white matter). We emphasize that these observations concerning gray matter apply only to relatively large brain regions, such as the entire thalamus, hippocampus, etc.; consistent differences of CO histochemical reactivity were observed between smaller structures (e.g. cortical layers, brainstem nuclei and cell types; see below), which would be averaged in the biochemical and densitometric measurements of large _regions. Our results also indicated that the whole rat brain contained an overall level of CO activity that was about the same as that in most of the individual large gray matter regions (Fig. 31A, B).

The lack of major differences of CO activity among large brain regions was evident not only in the statistically averaged data, but also in individual animals. Even in brains from individual rats, most large gray matter regions appeared to have about the same CO activity. Indeed, regional differences of CO activity in individual animals were usually smaller than interanimal differences for the same region (data not shown). Again, we point out that these observations regarding gray matter concern large brain regions only, and do not apply to smaller structures such as cortical laminae, thalamic subnuclei, etc.

Cytochrome oxidase densitometry

Regional analys&. To measure histochemical reactivity in large brain regions, optical readings of CO-stained sections were taken through a large diameter aperture (625/~m). Readings were taken in the same 11 regions that were assayed biochemically (see above).

The findings regarding CO densitometry in large brain regions (Fig. 31C) were similar to those from biochemistry. Gray matter regions stained darker than did white matter regions in all cases (P < 0.001 for all pairwise gray-white comparisons), but within groups of gray or white matter regions, no significant differences of staining intensity were found. (Differences of staining intensity were identified among smaller gray matter structures; see below.) The optical density of CO staining was generally about four to five times greater in gray than in white matter (Fig. 31C). By comparison,

CO activity levels measured biochemically were eight- to 12-fold higher in gray than in white matter (see above). The discrepancy between these ratios as determined by densitometry and biochemistry suggests that the optical density of histochemical staining was partially saturated in areas of high CO biochemical activity. (Conditions for obtaining a linear increase in optical density over a range of CO biochemical activities have been established in other studies. 12'26)

To examine the correlation between CO staining intensity and biochemical activity more quantitatively, linear regression analysis was used. The results showed that CO staining intensity was highly correlated with both the CO activity content (by wet weight) (Fig. 31D; r 2= 0.929) and the CO specific activity (Fig. 31E; r 2 = 0.946) in regions of gray and white matter. However, the graphs in Fig. 31D and E also demonstrate that the gray matter regions were all clustered at high CO enzymatic activity and high optical density, while the white matter regions were all clustered at low enzyme activity and low optical density. Thus, the strength of the histochemical/biochemical correlations depended largely on the major differences of CO activity between gray and white matter; differences among the large gray matter regions, or among the white matter regions, were comparatively small and, in most cases, statistically insignificant. The graphs also reveal that the best fit lines relating staining intensity to CO enzyme activity did not pass through the origins (Fig. 31D, E), again indicating that staining intensity was partially saturated in areas of high enzyme activity.

Cellular analysis. Since individual neurons are extremely difficult to dissect from brain tissue, the densitometric method was particularly useful for studying enzyme activity at the cellular level. CO staining intensity was measured densitometrically in the perikarya of 11 different neuron types, using a small diameter aperture (2 #m). As shown in Fig. 32, staining intensities varied widely, spanning almost a two-fold range of optical density values. As expected, the ceils with the highest optical densities were those that appeared qualitatively darkest. Among the different types examined, neurons of the mesencephalic trigeminal nucleus had the highest optical density. The optical density in these cells was significantly greater than in any other neuron type (P < 0.05 for all pairwise comparisons, one-factor ANOVA with least significant difference test), and was 78% higher than in dentate granule cells (the lightest-stained cell type). Significant differences of optical density were also detected between closely related neuron types in the same brain region: for example, optical density was significantly greater in hippocampal CA3 pyramids (0.495 ___ 0.017, mean _ S.E.M.) than in CA1 pyramids (0.357 _ 0.005; P < 0.05). Other significant differences among neuron types were observed and are listed in the legend to Fig. 32.


Cell type

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Fig. 32. Optical density (OD) of CO histochemical staining in different neuron types in the rat brain (n = 5 rats). The optical density was determined densitometrically through a 2/~m aperture. Significant differences (P < 0.05, one-factor ANOVA with least significant difference test) were found on all pairwise comparisons between cell types, except for the following: reticular nucleus of thalamus vs interpeduncular nucleus; interpeduncular nucleus vs oculomotor nucleus of III; oculomotor nucleus of III vs medial mammillary nucleus; oculomotor nucleus of III vs CA3 hippocampal pyramid; medial mammillary nucleus vs CA3 hippocampal pyramid; Purkinje cell vs motor nucleus of V; motor nucleus of V vs cerebellar granule cell; motor nucleus of V vs CA1 hippocampal pyramid; cerebellar granule cell vs CA1 hippocampal pyramid; cerebellar granule cell vs dentate granule cell; CA1 hippocampal pyramid vs

dentate granule cell. See text for details of the methods.

Comparison o f cytochrome oxidase and 2-deoxyglucose metabolism: linear regression analysis

As a test to determine how well CO activity might be correlated with glucose utilization in major regions of the rat brain, we calculated correlation coefficients between CO activity as determined in the present study and glucose utilization rates as reported by Sokoloff et al. 3° The regional glucose utilization rates were determined by 2 -DG autoradiography and densitometry through a 200 # m aperture, using animals that received 2-DG under conscious, "rest ing" (physically immobilized) conditions. 3° (For comparison, CO densitometric measurements were taken through a larger 625 # m aperture.) Seven regions of gray and white matter examined in both studies were avail- able for analysis. We found that the correlation coefficients were quite low, whether the glucose utilization was compared with the CO activity content (by tissue wet weight) (r2= 0.32), the CO specific activity (r 2= 0.30) or the optical density of CO histochemical staining (r 2= 0.27). The correlations were poor because the values for regional glucose utilization in gray matter varied much more (four- fold range) than did the values for regional CO activity (little or no variation). However, discrepancies between the 2-DG and CO results may have resulted in part from significant differences in densitometric sampling, due to the use of different aperture sizes (200/~m for 2-DG, 625/zm for co).

DISCUSSION

Cytochrome oxidase and neuronal functional activity

In the present study, we mapped the distribution of CO activity comprehensively throughout the rat brain. To interpret this map, the roles of CO in energy metabolism, and of energy metabolism in neuronal functional activity, should be clearly under- stood. (For a more detailed discussion of CO, energy metabolism and neuronal functional activity, refer to the review by Wong-Riley. 38)

A fundamental concept in metabolic mapping is that energy utilization in brain tissue is tightly coupled to neuronal functional activity (reviewed by Erecinska and Silver ~° and DiRocco et al.9). Neurons are extremely dependent on glucose as the primary substrate and on oxidative phosphorylation as the primary pathway for generating ATP. Furthermore, neurons contain few reserves of glycogen or fat, and are highly susceptible to cellular damage if deprived of metabolic substrates for even a few minutes. These biochemical properties, combined with the high metabolic demands of neuronal activity, are responsible for the tight linkage between neuronal activity and oxidative energy metabolism in brain. The neuro- physiological events that require high energy consumption have not been identified conclusively, though strong excitatory input j7'23'~4 and high action potential frequency ~7'2~'3~ have both been associated with high CO levels. Events involving ionic currents


are thought to be of major importance because ~40-60% of the ATP in brain is devoted to ion pumping by Na +, K+-ATPase. l° Furthermore, Na +, K+-ATPase and CO are regulated in parallel by neuronal functional activity. 15

Numerous previous experiments have demonstrated that neuronal functional activity is tightly coupled to both CO activity (reviewed by Wong- Riley 38) and 2-DG uptake (reviewed by Sokoloff29). Since both CO and 2-DG are metabolic markers, it might be expected that both methods would produce identical results; however, they actually reveal information about metabolic activity occurring over different time periods and along metabolic pathways that do not entirely overlap. Uptake of 2-DG, a substrate, is determined by both glycolytic and oxidative activity during the experimental period, lasting some 30-45min. 29'3° In contrast, levels of CO, a protein, are regulated according to oxidative (but not glycolytic) metabolic needs during the hours to weeks before tissue is obtained. 38 Thus, the two methods reveal neuronal activity through different temporal and metabolic windows. Since glycolytic metabolism may be more prominent in some neural pathways than in others, 2 differences between the CO and 2-DG methods may be significant under some conditions.

Cytochrome oxidase activity in rat brain structures: patterns and implications

We encountered heterogeneities of CO activity among brain structures ranging in complexity from neuron types, to functional systems, to categories of brain tissue (gray and white matter). Heterogeneities of CO activity have also been observed at the subcellular level, e.g. among different dendritic segments of the same neuron, by CO cytochemistry at the electron microscopic level. T M While these subcellular-level heterogeneities may be widespread in the brain, they were not examined in the present study. The only scale at which we did not find many differences of CO activity was among large gray matter regions; at this scale, activity in smaller structures was averaged and heterogeneity within regions was obscured. We con- clude that overall energy consumption varies much more widely among relatively small brain structures (e.g. cortical layers, thalamic nuclei and cell types) than it does among larger gray regions (e.g. thalamus and cerebellum as a whole).

Gray versus white matter. All of our results--histochemical, densitometric and biochemical----consistently indicated that gray matter had higher CO activity than did white matter. The ratio of gray matter CO activity to white matter CO activity, determined biochemically, was ~eight- to 12-fold. Other studies of many brain areas have also found that gray matter generally contains more enzyme activity than does white matter. 7'38 Darriet et al. 7 reported the magnitude of this difference to be

approximately 10-fold, in good agreement with the present study. The reason that gray matter has higher oxidative activity is presumably ascribable to differences in ion pumping activity: most of the ATP in brain is utilized for ion pumping, mainly by the enzyme Na ÷, K+-ATPase which, like CO, is more abundant in gray than in white matter (see above). Ion pumping activity, in turn, reflects the overall functional activity of neurons.

Large gray matter regions. On the regional scale, we found no significant differences in CO activity among most major gray matter structures, such as the thalamus, caudate-putamen, cerebellar cortex and superior colliculus. This apparent lack of regional- scale heterogeneities could reflect possible metabolic limitations imposed by blood supplies or, more likely, may be a consequence of averaging across multiple areas of brain tissue having diverse metabolic and functional properties (e.g. the superior colliculus and hippocampus both have subregions of high and low CO activity).

Neural systems. High CO histochemical reactivity was encountered in most nuclei belonging to special sensory, somatic sensory and somatic motor pathways, suggesting that overall neuronal functional activity in these systems is relatively high compared to that in visceral, autonomic, associative and limbic pathways, which generally had lower CO activity. No single pathway predominated as having especially intense or especially weak CO reactivity. Further- more, there were several examples of individual nuclei that had uncharacteristic CO levels for the systems to which they belonged, e.g. the mammillary bodies had intense CO reactivity even though most limbic structures were only moderately reactive.

Modular and columnar patterns. In some areas, CO staining showed patterns of modular or columnar organization that were either less obvious or not visible by routine Nissl staining. Perhaps the best known of these areas was the cortical barrel field (somatosensory area for whiskers). Barrel centers were darkly CO-reactive, while barrel septa were relatively pale. 4° The corresponding barreloids in the ventral posterior medial thalamic nucleus were also recognized, based on their previous identification using succinate dehydrogenase histochemisty.l Another area with modular organization was the lateral entorhinal cortex, where darkly reactive cell islands in layer II were surrounded by moderately reactive neuropil.14 In the cerebellar cortex, parasagittal bands of dark CO reactivity alternating with bands of lighter CO reactivity were seen in the vermis. 13'19 In the superior colliculus, patches of dark CO reactivity were observed in the intermediate gray layer. 32'35 The caudate nucleus contained an irregular mosaic of darker and lighter staining zones, related to the patch and matrix subdivisions of this structure, as described by DiFiglia et al. 8 These examples illustrate the potential of CO histochemistry for revealing subtle patterns of brain organization.


Neuron types. CO reactivity (as measured densitometrically) varied over a wide range among different neuron types, but did not appear to be associated with any particular cell morphology, size, neurotransmitter or functional role. For example, the pyramidal neurons in hippocampal areas CA1 and CA3 differed markedly in their CO reactivity, 17 even though the cells in both hippocampal fields use the same neurotransmitter (glutamate), have similar morphologies (pyramidal) and are directly connected in the same "trisynaptic pathway" of the hippocampusY Like- wise, neurons that use GABA as their neurotransmitter may have high, moderate or low CO activity, depending on their anatomical location; e.g. GABA- ergic neurons in the feline lateral geniculate nucleus have relatively low CO activity, while GABAergic neurons in the perigeniculate nucleus have high CO activity. 22 As noted above, it appears that the best correlates of high CO activity in neurons and their processes are strong excitatory input 17,23,24 and high spontaneous impulse activity. 17'z1'3~ The high CO activity associated with these physiological properties is presumably needed to supply ATP for ion pumping to recharge ionic concentration gradients across the neuronal plasma membrane.

Comparison with previous studies. Our results were consistent with prior findings concerning CO activity in most brain structures (as noted for many examples in the Results), with rare exceptions. For example, Darriet et al. 7 reported that the interpeduncular nucleus had relatively low CO histochemical reactivity, while we found this nucleus to be very rich in CO. Such discrepancies may have been due to differences in CO methodology and/or sampling of nuclei.

Species differences in brain cytochrome oxidase activity

Comparisons with previous results from other mammalian species indicate that levels and patterns of CO activity may vary significantly among species. Levels of CO activity in rat brain, for example, are several-fold higher than in human brain. 16'18 The pattern of enzyme distribution in particular brain areas may also vary among species, for example in the hippocampus. ~7 Likewise, CO activity in the interme- diolateral cell column of the spinal cord appears to be much higher in monkeys than in other mammalian species, such as rats. 39 The globus pallidus in monkeys contains darkly CO-reactive neuronal cell bodies, 36 while no such darkly reactive neurons in the rat globus pallidus were identified in the present study (though some moderately reactive neurons were seen). These species specific variations indicate that our results from the rat cannot be readily general- ized to all mammalian species, including conclusions regarding particular neural systems. As an example at the systems level, autonomic nuclei in the rat brain appear to be generally low in CO reactivity, while preganglionic sympathetic neurons in the monkey spinal cord are clearly high in CO reactivity. 39

Comparison o f patterns observed by cytochrome oxidase and 2-deoxyglucose methods

Gray and white matter. Extensive quantitative and qualitative studies of regional 2-DG uptake in the rat brain under conscious, "resting" conditions have been done by Sokoloff et al. 3° and by Schwartz and Sharp. 28 Those researchers found that uptake of 2-DG was higher in gray matter than in white matter by a factor of approximately three-fold? ° In comparison, we found that CO levels were about eight- to 12-fold higher in gray than in white matter. This discrepancy between the ratios obtained by CO and 2-DG methods could suggest that gray and white matter have somewhat different metabolic properties (e.g different rates of glycolysis vs oxidative phosphorylation) or, more likely, could reflect inherent technical differences between the CO and 2-DG methods. Specifically, 2-DG uptake reflects glucose requirements for both glycolytic and oxidative metabolism to the time of the experiment (lasting some minutes), while CO activity reflects oxidative capacity as regulated by overall neuronal functional activity over the previous hours to weeks.

Neural systems. Sokoloff et al. 3° demonstrated that, under the conditions of their experiments, the highest levels of 2-DG uptake were in auditory structures (i.e. the inferior colliculus, superior olive, medial geniculate body and auditory cortex). We found that, whereas auditory structures such as the dorsal cochlear nucleus were rich in CO, enzyme activity was similarly high in nuclei belonging to other sensory pathways (e.g. the medial vestibular nucleus). Fur- thermore, although we did see zones of very high CO activity in some regions of the inferior colliculus by histochemistry (especially in the caudal nucleus), our quantitative biochemical assays indicated that overall enzyme activity in the inferior colliculus was about the same as in large gray matter regions belonging to other pathways, such as the superior colliculus.

These differences between the CO and 2-DG results could be caused by several factors. With regard to auditory structures, it is possible that the experimental conditions used by Sokoloff et al. 3° may have been associated with selective activation of the auditory pathway, perhaps related to immobilization. A more general contributing factor was probably the use of different aperture sizes for densitometric sampling of brain areas (see below). For example, CO activity in the superior colliculus was highest in the superficial gray at the dorsal edge of the structure, and in the inferior colliculus was highest in caudal and lateral zones; thus, densitometric results could be highly dependent on sampling technique. Finally, it is con- ceivable that the auditory centers might have extra- ordinarily high 2-DG uptake due to a heavy reliance on glycolytic metabolism, though no such reliance has been documented, and oxidative activity is also quite high.


Gray m a t t e r regions. In the 2-DG experiments by Sokoloff e t a / . , 3° gray matter areas displayed a broad (~3.5-fold) range of metabolic activities, whereas large gray matter regions in the present study differed little or not at all in their CO activity as determined biochemically or densitometrically. Furthermore, our linear regression analysis detected very little correlation between regional CO activity and 2-DG uptake rates as reported by Sokoloff et al. 3° However, it is important to note that our quantitative results regarding gray matter regions were obtained from analysis of areas larger than those analysed by Sokoloff et al., 3° because we used a larger densitometric aperture (625 #m diameter in the present study vs 200 ~m in the 2-DG study, 3° with approximately 10-fold more tissue area covered by the larger aperture) and dissected only large regions for biochemical analysis. When we examined smaller regions (such as cortical layers and brainstem nuclei) by CO histochemistry, local variations in enzyme activity were detected.

Whereas sampling differences due to aperture size probably account for much of the discrepancy between the CO and 2-DG results, it is also possible that some brain areas were relatively activated or deactivated under the "resting" conditions used during the 2-DG uptake period. Rats in the 2-DG mapping studies 28"3° were physically restrained and could conceivably have relied on auditory cues as their primary source of sensory information. Finally, it remains possible that local areas of high glycolytic metabolism may have shown more intense metabolic activity by 2-DG than by CO staining.

Despite the evident discrepancies, the majority of brain nuclei had metabolic activity levels that appeared concordant by CO and 2-DG methods. Examples of areas with particularly high metabolic activity by both methods include the habenula, mammillary bodies, interpeduncular nucleus, inferior olive, and cortical layers III and IV. Examples of areas with relatively low or moderate metabolic activity by both methods included the globus pallidus, amygdala, hippocampus and hypothalamus (excluding the mammillary bodies). (These state- ments refer to overall metabolic activity onlyl since heterogeneities were present in many of these

structures.) One area of questionable discrepancy was the anterior nucleus of the thalamus: the anteroventral subdivision appeared dark on 2-DG autoradiography, 28 whereas the anterodorsal nucleus appeared dark by CO histochemistry. However, the relatively small size of these nuclear subdivisions may have made interpretation of the anatomy difficult, particularly with the 2-DG method, which has relatively low anatomical resolution.

Selected areas with discordant results by CO and 2-DG included the nucleus accumbens (high CO, low 2-DG), red nucleus (high CO, moderate 2-DG) and oculomotor nucleus (high CO, moderate 2-DG). However, some of these discrepancies may be more apparent than real, since small areas like the oculomotor nucleus are not well demarcated by 2-DG autoradiography. One specific example is the olfactory bulb glomeruli, which were not resolved at all by 2-DG autoradiography 28'3° but were clearly visible by CO histochemistry as discrete, darkly reactive structures. As these examples demonstrate, CO histochemistry is capable of resolving foci of high or low metabolic activity that are below the resolution of current 2-DG techniques.

The various similarities and differences between CO and 2-DG mapping underscore the necessity of understanding the purposes, advantages and signifi- cance of these two metabolic markers. CO is most useful when high anatomical resolution is needed or when relatively long-term (i.e. hours to weeks) periods of neuronal activity are to be examined, e.g. in relation to plasticity, development, ageing or disease. In contrast, 2-DG is better for mapping neuronal activity under specific conditions imposed over a shorter period of some minutes, e.g. in relation to the performance of a particular task. Dependening on which method is selected, quite different results may be obtained.

A c k n o w l e d g e m e n t s ~ e thank Dr Shanhong Lu for provid- ing many of the rats used in this study and Mr Dang Vang for excellent technical assistance with the optical densitometry. Supported by NIH Grants NS18122 and EY05439 to M. W.-R. and by a fellowship from the Medical Scientist Training Program at the Medical College of Wisconsin to R.F.H.

R E F E R E N C E S

1. Belford G. R. and Killackey H. P, (1979) Vibrissae representation in subcortical trigeminal centers of the neonatal rat. J. comp. Neurol. 183, 305-322.

2. Borowsky I. W. and Collins R. C. (1989) Metabolic anatomy of brain: a comparison of regional capillary density, glucose metabolism, and enzyme activities. J. comp. Neurol. 288, 401-413.

3. Brown G. C., Crompton M. and Wray S. (1991) Cytochrome oxidase content of rat brain during development. Biochim. biophys. Acta 1057, 273-275.

4. Carroll E. W. and Wong-Riley M. T. T. (1984) Quantitative light and electron microscopic analysis of cytochrome oxidase-rich zones in the striate cortex of the squirrel monkey. J. comp. Neurol. 222, 1 17.

5. Cohen P. J. (1973) Effect of anesthetics on mitochondrial function. Anesthesiology 39, 153-164. 6. Conti F., Barbaresi P. and Fabri M. 0988) Cytochrome oxidase histochemistry reveals regional subdivisions in the

rat periaqueductal gray matter. Neuroscience 24, 629~33. NSC 65/2 B


7. Darriet D., Der T. and Collins R. C. (1986) Distribution of cytochrome oxidase in rat brain: studies with diaminobenzidine histochemistry in vitro and [14C]cyanide tissue labeling in vivo. J. cerebr. Blood Flow Metab. 6, 8-14.

8. DiFiglia M., Graveland G. A. and Schiff L. (1987) Cytochrome oxidase activity in the rat caudate nucleus: light and electron microscopic observations. J. comp. Neurol. 255, 137-145.

9. DiRocco R. J., Kageyama G. H. and Wong-Riley M. T. T. (1989) The relationship between CNS metabolism and cytoarchitecture: a review of ~4C-deoxyglucose studies with correlation to cytochrome oxidase histochemistry. Comput. med. Imag. Graphics 13, 81-92.

10. Erecinska M. and Silver I. A. (1989) ATP and brain function. J. cerebr. Blood Flow Metab. 9, 2 19. l 1. Errede B., Kamen M. D. and Hatefi Y. (1978) Preparation and properties of complex IV (ferrocytochrome c:oxygen

oxidoreductase EC 1.9.3.1). Meth. Enzym. 53, 40-47. 12. Gonzalez-Lima F. and Garrosa M. (1991) Quantitative histochemistry of cytochrome oxidase in rat brain. Neurosci.

Lett. 123, 251 253. 13. Hess D. T. and Voogd J. (1986) Chemoarchitectonic zonation of the monkey cerebellum. Brain Res. 369, 383-387. 14. Hevner R. F. and Wong-Riley M. T. T. (1992) Entorhinal cortex of the human, monkey, and rat: metabolic map as

revealed by cytochrome oxidase. J. comp. Neurol. 326, 451-469. 15. Hevner R. F., Duff R. S. and Wong-Riley M. T. T. (1992) Coordination of ATP production and consumption in brain:

parallel regulation of cytochrome oxidase and Na ÷, K÷-ATPase. Neurosci. Lett. 138, 188-192. 16. Hevner R. F., Liu S. and Wong-Riley M. T. T. (1993) An optimized method for determining cytochrome oxidase

activity in brain tissue homogenates. J. Neurosci. Meth. 50, 309 319. 17. Kageyama G. H. and Wong-Riley M. T. T. (1982) Histochemical localization of cytochrome oxidase in the

hippocampus: correlation with specific neuronal types and afferent pathways. Neuroscience 7, 2337 2361. 18. Kish S. J., Bergeron C., Rajput A., Dozic S., Mastrogiacomo F., Chang L.-J., Wilson J. M., DiStefano L. M. and

Nobrega J. N. (1992) Brain cytochrome oxidase in Alzheimer's disease. J. Neurochem. 59, 776 779. 19. Leclerc N., Dor6 L., Parent A. and Hawkes R. (1990) The compartmentalization of the monkey and rat cerebellar

cortex: zebrin I and cytochrome oxidase. Brain Res. 506, 70--78. 20. Liu S., Hevner R. and Wong-Riley M. (1991) Metabolic map of the normal rat brain as revealed by cytochrome oxidase

histochemistry and biochemistry. Soc. Neurosci. Abstr. 17, 864. 21. Livingstone M. S. and Hubel D. H. (1984) Anatomy and physiology of a color system in the primate visual cortex.

J. Neurosci. 4, 309-356. 22. Luo X. G., Hevner R. F. and Wong-Riley M. T. T. (1989) Double labeling of cytochrome oxidase and )'-aminobutyric

acid in central nervous system neurons of adult cats. J. Neurosci. Meth. 30, 189 195. 23. Mjaatvedt A. E. and Wong-Riley M. T. T. (1988) Relationship between synaptogenesis and cytochrome oxidase activity

in Purkinje cells of the developing rat cerebellum. J. comp. Neurol. 277, 155 182. 24. Mjaatvedt A. E. and Wong-Riley M. T. T. (1991) Effects of unilateral climbing fibre deafferentation on cytochrome

oxidase activity in the developing rat cerebellum. J. Neuroeytol. 20, 2 16. 25. Nicoll R. A., Kauer J. A. and Malenka R. C. (1988) The current excitement in long-term potentiation. Neuron 1, 97 103. 26. Nobrega J. N., Raymond R., DiStefano L. and Burnham W. M. (1993) Long-term changes in regional brain cytochrome

oxidase activity induced by electroconvulsive treatment in rats. Brain Res. 605, 1 8. 27. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn. Academic Press, San Diego. 28. Schwartz W. J. and Sharp F. R. (1978) Autoradiographic maps of regional brain glucose consumption in resting, awake

rats using [14C]2-deoxyglucose. J. eomp. Neurol. 177, 335-360. 29. Sokoloff L. (1984) Metabolic Probes of Central Nervous System Activity in Experimental Animals and Man. Sinauer,

Sunderland, MA. 30. Sokoloff L., Reivich M., Kennedy C., Des Rosiers M. H., Patlak C. S., Pettigrew K. D., Sakurada O. and Shinohara

M. (1977) The [~4C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28, 897 916.

31. Trusk T. C., Wong-Riley M. T. T. and DeYoe E. A. (1992) Changes in cytochrome oxidase and neuronal activity in VI induced by monocular TTX blockade in macaque monkeys. Soc. Neurosci. Abstr. 18, 298.

32. Wallace M. N. (1986) Spatial relationship of histochemically demonstrable patches in the mouse superior colliculus. Expl Brain Res. 62, 241-249.

33. Weiss-Wunder L. T. and Chesselet M.-F. (1990) Heterogeneous distribution of cytochrome oxidase activity in the rat substantia nigra: correlation with tyrosine hydroxylase and dynorphin immunoreactivities. Brain Res. 529, 269-276.

34. Wharton D. C. and Tzagoloff A. (1967) Cytochrome oxidase from beef heart mitochondria. Meth. Enzym. 10, 245 250.

35. Wiener S. I. (1986) Laminar distribution and patchiness of cytochrome oxidase in mouse superior colliculus. J. comp. Neurol. 244, 137 148.

36. Wong-Riley M. T. T. (1976) Endogenous peroxidatic activity in brain stem neurons as demonstrated by their staining with diaminobenzidine in normal squirrel monkeys. Brain Res. 108, 257 277.

37. Wong-Riley M. (1979) Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res. 171, 11-28.

38. Wong-Riley M. T. T. (1989) Cytochrome oxidase: an endogenous metabolic marker for neuronal activity. Trends Neurosci. 12, 94-101.

39. Wong-Riley M. T. T. and Kageyama G. H. (1986) Localization of cytochrome oxidase in the mammalian spinal cord and dorsal root ganglia, with quantitative analysis of ventral horn cells in monkey. J. comp. Neurol. 245, 41~51.

40. Wong-Riley M. T. T. and Welt C. (1980) Histochemical changes in cytochrome oxidase of cortical barrels after vibrissal removal in neonatal and adult mice. Proc. natn. Aead. Sei. U.S.A. 77, 2333-2337.

41. Wong-Riley M. T. T., Tripathi S. C., Trusk T. C. and Hoppe D. A. (1989) Effect of retinal impulse blockage on cytochrome oxidase-rich zones in the macaque striate cortex: I. Quantitative electron-microscopic (EM) analysis of neurons. Vis. Neurosci. 2, 483-497.

(Accepted 23 September 1994)

a metabolic map of cytochrome oxidase in the rat brain: histochemical, densitometric and biochemical...

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