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Mapping cerebral functional activity with radioactive deoxyglucose
TINS- September 1978
75
Mapping cerebral functional activ,ty with radioactive deoxyglucose Louis Sokoloff ,,
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Energy metabolism is closely coupled to functional activity in most tissues, including those of the central nerrous system. In this article we find out how [t'CJdeoxyglucose can be used to trace glucose metabolism and to measure the rates of glucose utilization simuhaneously in all anatomical and functional components of the central nervous system. When combined with autoradiography of serial sections of the brain under conditions in which the optical density of the autoradiographs reflects the rate of glucose utilization, pictorial representation of the relative rates of glucose utilization throughout the brain are obtained. Because of the coupling between functional activity and energy metabolism, these autoradiographs are like a stain for functional activity in nervous tissues and can be used to identify regions with altered functional activity in varying physiological, pharmacological, and pathological states.
The brain is a complex, heterogeneous organ composed of many anatomical and functional componeets with markedly different levels of functional activity that vary independe,3tly with time and function. Other tissues are generally far more homogeneous with most of their cells fJnctioning similarly and synchronously in response to a common stimulus or regulatory influence. The central nervous system, however, consists of innumerable subunits, each integrated into its own set of functional pathways and networks and subserving only one or a few of the many activities in which the nervous system participates. Understanding how the nervous system functions requires knowledge not only of the mechanisms of excitation and inhibition, but even more so of their precise localization in the nervous system and the relationships of neural subunits to specific functions. Historically, studies of the central nervous system have concentrated heavily on localization of function and mapping of pathways related to specific functions. These have been carried out neuroanatomieally and histologically with staining and degeneration techniques, behaviourally with ablation and stimulation techniques, electrophysiologh:ally with electrical recording and evoked electrical respoeses, and histochemically with a variety of techniques, including fluorescent and immunofluorescent methods and autor~lio~aphy of orthograde and retrograde axopla~mic flow. These methods
have provided a fairly detailed map of the roadways of the net~'ous system, but m view of its complexity there may well yet be as many to be discovered. Many of the conventional methods suffer from a sampling problem. They generally permit examination of only one potential pathway at a time, :lnd only positive results are interpretable. Furthermore, the ~iemon. stration of a pathway reveals only a potential for function; it does not reveal
its significance i~ normal function. Tissues that do physical and;or ~:hemical work, such as i~eart, kidney, and skeletal muscle, exhibit a close relationshl.p between energy metabolism and functional activity. From measurement of energy. ~aetabolism it is then possible to estimate the level of functional activity. This relationship has recently been utilized to develop a method that maps functional activity simultaneously in all components of the central nervous .system in the normal conscious state and during physiological, pharmacological, or pathological alterations of functional activity. The method employs radioactive deoxyglucose (DG), an analogue of glucose, to trace glucose metaboli:,m in the brain. The procedure is so designed that the concentration of radioactivity in the tissue is more or h.,ss proportional to the rate of glucose utilization. The concentrations of radioactivity in the local cerebral tissues are measmed by a quantitative autoradiographic technique. The method not only allows quantification of the actual rates
of glucose utilization in the individual cerebral tissues, but the autoradiograph~ obtained with it pro,~'ide pictorial representations of the re;ative rates of glucose utilization in all the cerebral structures seen in autoradiographs of 20 #m serial sections of the entire brain. It is, therefore, now possible to obtain not an anatomical map but a functional map of the entire central nervous system in normal and experimental states. Theoretical basis of the radioactive deoxyglucose method The radioactive deoxyglucose method was developed to measure the local rates of energy metabolism simultaneousl) in all components of the brain in conscious laboratory animals. It was designed specifically to take advantage of the extraordinary spatial resolution made possible by quantitative autoradiography. The dependence on autoradiography prescribed the use of radioactive substrates for ener~" metabolism, the labelled products of which could be assayed in the tissues by the autoradiographic technique. Although oxygen consumption is the most direct measure of energ) metabohsm, the ~olatility of oxygen and its metabolic products and the short physical half-life of its radioactive isotopes precluded measurement of oxidative metabolism by the autoradiographic technique in most cir cumstances glucose i.~ almost the sole substrate for cerebral oxidative metabolism, and tts utihzation ,s stotchtmetrically related to oxygen consumption. Radioactive glucose ts, however, not fully satisfactory because ,ts labelled products are lost too rapidly from the cerebral tissues. The labelled analogue of glucose, 2-deoxy-o-[tq~lglucose. was, therefore, selected because Its biochemical properties make tt parttcularly appropriate to trace glucose metabohsm and to measure local cerebral glucose utihzatton by the autoradiographic technique. The method was derived by analysis of a model based on the biochemtcai properttes of 2-deoxyglucose (Fig. !/,) t~. 2-Deoxygluco,~ is tt~msported I:,i-directionall) between blood and brain by the same carrier that transports glucose across the blood-brain barrier. In the cerebral tissues it is phcsphcrylated by hexokinase to 2-deoxygl~cose 6-phosphate (DG-6-P). Deox~glu~:ose and glucose are, therefore, competitive svbstrates for both blood-brain transport and hexokinase-catalyzed phosphorylatic.n. Unlike glucose 6-phosphate, however, which is metabolized further eventv~lly to CO= and water,
76
T I N S - September 1978
DG-6-P gm :'tot be converted to fructo~ 6-phosphatt and it is also not a substrate for glucose 6-phosphate dehydro~enase. The.4~ is vmy little glucose 6-phosphatase activity in brain, and even less deoxyglucose
A PLASMA
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6-pbosphatase activity. Dcoxy~ucose 6phosphate, once formed, remains, therefore, essentially trapped in the cerebral tissues duri,~ the experimental period. If the ir, i~rval of brae is kept short enouBh, for example less than ! h, to allow ~ :,~gumption of negligible loss of ['~]DG-6-P from the tissues, then ehe quantity of [t~']DG-6-P accmnulated in any c~-rebr~J tissue at any given time following the introduction of [UC]D~ into the circulation is equal to the integral of the rate of [ttC]DG phosphorylation by hexokinase in tluit tissue during that interval of ",Jme. This integral is in turn ,elated to the amount of glucose that has be~n phosphorylated over the same int=rva;, depending on the time courses of the relative concentrations of [UCJDG and glucose in the precursor pools and the Michaelis-Menten kinetic constants for hexokinase with respect to both p~C]DG and glucose.. With cerebral glucose cons.umption i,, a steady state, the amount of glucose phosphorylated during the interval of time equals the steady state flex of glucose through the hexokinase-catalyzed step, times the duration of the interval;and the net rate of flux of glucose through this step equalg the rate of glucose utilization. These relationships can be rigorously combined into a model (Fig. IA) which can be mathematically analyzed to derive an operational equation (Fig. I B), provide~ that the following assumptions are made: (!) there is a steady state for glucose (i.e. constant plasma glucose concentration and constant ra~ of glucose consumption) throughout the experimental period; (2) there is a homoBeneous tissue compartment within which the conc=ntrations of [uC]DG and glucose are uniform and exchange directly ":,.?,h the plasma; and (3) tracer concentratic~s of [ ~ ] D G (i.e. ~tolecular concentrations of f~e [sq~]DG csscndally equal to zero) are m~d. The operational equation which defines Rt, the rate of glucose utiim~ion per unit mass of tissue, i, in terms of ~e~sun.Me variables is presented in Fig IE. The rate constants, k;, k~, a~d ~, are determined in a separate group (,~ animals by a non-I/near, iterative W,~.,-: which provides the least squares ~esg-~.~ of ;m equation which defines the time course of total tissue uC concen'~tioa in terms of the time, the history ¢f the plasma concentration, and the rate con-
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Integrated &recursor spemfic activity in tissue Fi#. !. Theoretical basis o f re~liouetive dcoxy~glucose meshed/or measurement of local cerebral glucose utilizatio~*. A. Diagrammatic representation o[ the tl~retical model. C~trepresents the total t4C conceatration in a siawie Aomogenema tLtsue of the braise. C~rand Cp represent the concentraiiont o f ["C]deoxyglucose and glucose in the arterial plasma, respectively; ~ and CL represent their respectipe concentrations M the tis.~ pools that serve ag substrates /or hexokMase. C u represents the concentration o f ["Cldeoxy. glucose 6 - ~ t e in tile #issue. The cortstants k;, k~ atJd k; represent the rate constants for carriermediated transport of[t4C]deoxyglucose from plasma to tLttue, for carrier-mediated transport back from fix~e to plasm& a~l for p&~phorylation by hexokiaase, respectively. The comtants kb ks, and ks are the ~ m l e n t rate cott~tants for glucose. ["C]Deoxylrlocose and glucose share wtd compete for the carrier ~ tra~portt them bath between plasma o~[ tittite and/or hexokinase whi~'kpko.*phorylotes them to d,~, respective hexose 6-pbaspAetes. 7~e dashed arrow represents the possibility o f £1ucose 6-phoaplmle l~f~iysia by glucose 6-phosphataseactivity, if atty. B. Operatiomt/equationo f radioactive deox)wlucose metkod and its functio2ml anatomy. T represents the time at the termination o f tit2 experimental period; A equals tile ratio of the diltributim, tImee o f deox2gl,~L ~se in ~ e tissue to tAat o f glucose: ~ eqt~r the fraction o f glucose w,~it~,once i~asphorylated, rnntt~ues down the ~lycoLqi¢ pathway; and IC'- and ~"- and ICm and I"m represent the familiar ~4~c~eih.~Jenten kh:et;¢ constants of hexokiaase for deoxyglucose and glucow, respectively. The other ~v,~d~oL, ore d~e mune as :hose ~
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TINS- September 1978 slants to the experimentally determined time courses o f tissue and plasma concentrations o f " C n. The ~, ~, and the enzyme kinetic constants are grouped together to constitute a single, lumped constant (see equation). This lumped constant can be shown mathematically to be equal to the asymptotic value of the product o f the ratio of the cerebral extraction ratios of [t%"]DG and glucose and the ratio of the arterial blood to plasma specific activities when the arterial plasma ["C]DG concentration is maintained constant. The lumped constant is also determined in a separate group of animals from arterial and cerebral venot,s blood samples drawn d u n n g a programreed intravenous infusion which produces and maintains a constant arterial plasma [tq~]DG concentrationt'. Thus far it has been determined only in the albino rat and the monkey. Values are 0.483 (S.E.M. = +0.022) in the rat tg and 0.344 (S.E.M. = -_k0.036) in the monkeys. The lumped constant appears to i;e characteristic o f the species and does n¢~t appear to change significantly in a wide range o f conditionsTM. Despite its complex appearance, the operational equation is really nothing more than a general statement of the standard relationship by which rates of enzymecatalyzed reactions :sre determined from measurements mad¢: with radioactive tracers {Fig, I B). Tl;e numerator o f the equation represents the amount of radioactive product formed in a given interval of time; it is equal to C~, the combined concentrations of [uCJDG and ["C]DG6-P in the tissue at time, T, measured by the quantitative autoradiographic technique, less a term ~.hat represents the free unmetabolized [ug..IDG still ,-emaining in the tissue. The denominator eO~esents the integrated specific activity o f the precursor pool, times a factor, the lumped constant, which is equivalent to a correction factor for an isotope effect. The term with the exponential factor in the denominator takes into account the lag in the equilibration o f the tissue precursor pool with the plasma.
Pmeedmre The operational equation dictates the variables to be measured to determine the local rates o f cerebral glucose utilization. The specific procedure employed is designed to evaluate these variables and to minimize potential errors that might occur in the actual application of the method. If the rate constants, k;, k~, and k~, are known precisely, then the equation is
77 generally applicable with any mode of administration of ["C]DG and for a wide range of time intervals. At the present time the rate constants have been determined only in the conscious rat and monkey. These rate constants can he expected to vary with the condition of the animal, however, and for most accurate results should be redetermined for each condition studied. The structure c:f the operational equation suggests a more practicable alternative. All the terms in the equation that contain the rate constants approach zero with increasing time if the ["C]DG is so administered that the plasma [tq~]DG concentration also approaches zero. From the values of the rate constants determined in normal animals and the usual time course of the clearance of ptC]DG from the arterial plasma following a single intravenous pulse at zero time, it has been determined that an interval of 3 0 4 5 min after a pulse is adequate for these terms to become sufficiently small that considerable latitude in inaccuracies of the rate const:,nts is permissible without appreciable error in the estimates of local glucose consumption n. An additional advantage derived from the use of a single pulse of [I~"]DG followed by ~ relauvely long interval before killing the animal for measurement of local Ussue " C concentration is that by then most of the free ["C]DG in the tissues has been either converted to [ttC]DG-6-P or transported back to the plasma; the optical densities in the autoradiograph~ then represent mainly the conc,entratio~s of pq~]DG-6-P and, therefore, reflect dicectly the relative rates of glucose utilization in the various cerebral tissues. The experimental procedure-"~ ._ t~ inject a pulll~ of ["C~DG i n t r a v e n o ~ ' :~t zero time and t o ~.~:apitate the ac, ~al and freeze the brai.~ at a measured tt.ae, T, 30-45 win later: in the interval, t~med arterial samples are taken for the measurement of plasma [t~"llX3 and glucose concentrations. Tissue " C concentrations, C: ,,re measured at time, T, by quantitat,,e autoradiography of 20pro frozen dried sections prepared serially from the entire brain. Local cerebral glucose utilization is then calculated by the operational equation. Rates of g l E m e utilization ia anatoad¢~ ¢ e m p e a e m of Im~a The rates of local cerebral glucose utilization have been measured in the normal conscious and anaesthetized albino rat and in the conscious Macaque monkey. The rates in the conscious rat vary widely
throughout the brain with the values in white matter distributed in a narrow low range and the ,,alues in grey structures broadly distributed around an average which is about three Umes greater than that of white matter 1Table I). The highest values are in stractures of the auditor' system with the inferior colliculus clearly the most metabolically active structure in the brain (Table i). The rates of local cerebral utdization in the conscious monkey exhibit similar heterogeneity, bug they are generally one-third to one-half of the values in corresponding structures of the rat brain (Table i). ihe" differences in rates in the rat and monke~ brain are consistent wtth the different ce!lular packing denstues in the brains of these two species. General anaesthesia induced by thtopental reduces the rates of glucose utihzation in all sVucture~ .,i the rat brain n. The effects are not uniform, however. The greatest reductions occur m the grey structures, par~.tcularly those of the primary sensory pathways. The effects in white matter, though definitely present, are relatively smal, compared with tho~e in grey matted'. TABLE !. Reptesentattve values for Io~al cerebral glucose utilizanon in the normal conscious albino rag and monkey (~mol,100 ~mm) Struct..,c
Albino tat" Monk~'~" (n I0) {n ~ ~)
(3rey matter
45.2 107 6 79 • 4 162 : 5 47 - 4 Parietal cortez 112 - 5 44 - l Sensory-mot ~,,~¢ex 120 ,. .< 54 2 Thalamus: ~ : ~, aucleu~ 116 ~* 5 ~.3 : -" T h a l a m t : ~r~:~ nucleus 109 - 5 65 3 Medial gcni~-~,~.~c oody 131 : 5 39 • I Lateral Ig~ni~at¢ ~ody 96 : 5 25 • I Hypothalamus 54 ± 2 5 57 • 3 Mamillary I~,dy 12l 39:2 Hippoc, tmpus 79 = 3 25-2 Amygdala 52 : 2 52- ~, Caudate-putameq ! 10 : 4 3b-: 2 Nucleus accumbens 82 3 26:2 Globus palhdus 58 .~ 2 29:2 Substantm mgra 58 : 3 66 _.~ ? Vestibular nucleus 128 _~ 5 51-.3 Cochlear nuclcu> i 13 = 7 63~ 4 Superior olivary ~mcleus 133 = 7 Inferior colhculu~. 197 ± 10 103 ~ 6 .'5:4 Superior collicu!u~ 95 = 5 28 "i. ! Pontine grey ma::et 62 : 3 ?i ~-2 Ccrcbellar cortex 57 + 2 45~. 2 Cerebellar nuclet 100 *- 4 Whtte matter ll-kl Corpus callosum 40 ! 2 134-1 Internal capsule 33 + 2 12__1 Cerebdiar white ma~.lcr 37 ± 2 Visual cortes Audflory cortex
The values are the roans ± standard errors from measurements mack in the.. number of animals indicated (n).
78 MeUdmlic mappieg el" fmetiewl acttdty in central nerveus syMem The results of a va;iety of studies on the effects of experimentally-induced focal alterations of functional activity on local glucose utilization have demonstrated a close coupling between local functional activity and eneqD" metabolism in the brain". The effects are often so prono,raced that they can be visualized dir;ctl) on the autoradiographs which pr,vide pictorial reprt~entations of the rehtti~e rates of glucose utilizatton throughout the brain. ,'D),is technique of autoradiographic visualization of evokcti metabolic responses offers a powerful tool to map functional neur~;! pathways simultaneously in all anatomical compor.ents of ~he central nervous system~.n. The use of the technique for metabolic mapping has been growing rapidly, and only a few examples can be cited Jn the present report. More comprehenswe re~,,¢ws of these apphcations have recently been. pubhshed:, n. The results thus far clearly demonstrate tae effectivenes~ of cerebral metabolic res?onses, either positive or negative, in identifying regions of the central nervous system involved zn si~'cific functions. For example, elect~ cal stimulation of one sciatic nerve in ,he anaesthetized rat evokes increased glucose utilization, seen as increased density ;n the autoradiographs, in the ipsllateral lumoar spinal cord grey matter, especiall~ ~n he dorsal horn s. The distribution of ~he spread of activation -~uring focal seizares induced by local urilaterai injectiov, of penicillin into the ~.totor cortex in the monkey can be vist:alized by ipsilateral areas of increased ~ens[~) in the autoradiographs of the m~tor cortex, putat~en, globus pallidus, thr.lamic nuclei, ~abstantla nigra, etc. (Fig 2). The distribution o.t" changes in activity associated ~'ith normal or spontaneous alterattons in physiological function can be studzed. For example, the suprachiasmatic nuclei of the rat exhibit diurnal rhythmicity in metabolic activity. They are active during the day and inactive at night; t':ey are visualized as distinct, relatively dark areas on the autoradiographs of ani~.ais studied in the morning and are essentially invisible in the auto,adiographs of enimals studied at nighP. Olfactory stimul~.~on with specific odours has been shown to produce metabolic activation of .,,pccific discrete regions of the olfactory bulb t°. Obstruction of both external auditory canals of the conscious rat r,.arkedly diminishes glucose utilization bilaterally in all components of the auditory pathway,
T I N S . S e p t e m b e r 1978
e.g. the cochlear nuclei, superior olives, nuclei of the lateral lemnisci, medial geniculate ganglia, and the auditory cortex. With unilateral auditory obstruction, some of the structures are affected only ipsilaterally, some only contralaterally, and some bilaterally: with quantification it becomes possible to estimate the percentage crossing of the various pathways involvedn. it is known that in the rat the visual pathways from the retinae are 80-85 ~,, crossed to the opposite hemisphere: unilateral enucleation results in approximately 80% reductions in glucose utilization of the contralateral lateral geniculate ganglion, superior colliculus, and visual cortex, effects which are strikingly visible in the autoradiographs4. The most dramatic demonstrations of the ability of the deoxyglucose method to map functional ac:~'.'ity in the central nervous system have been in the primary vtsuai system of the monkey. In lhis species, as in all animals with binocular vision, the visual pathways are approximately 50°0 crossed at the optic chiasma, and the structures of the visual system on each side of the brain receive equal inputs from both retinae. The projections from the two retinae remain segregated and terminate in six well-defipcd laminae in each of the two lateral geniculate Ianglia, three each for the ipsilateral and
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contralateral eyes. The .¢,egregation is preserved in the geniculocalcarine pathways which terminate in the striate cortex in alternating columns receiving input from the ipsilaterai and contralateral eyes. These are the ocular dominance columns originally dcscfi,~.-d by Hubei and Wiesel on the basis of electrophysiological studiesz. The P%."IDG method has been used to map all the components of the binocular visual system of the monkey simultaneously in the same animals, and to provide a complete pictorial representation of the nature, extent, and distribution of the ocuiar dominance columns (Figs. 3 and 4p. it has also identified the loci of the visual cortical representation of the blind spots of the retinae {Fig. 4). The ocular dominance columns exhibit functional plasticity in the immature striate cortex. The pq:::]DG method has demonstrated their presenc: in the new-born monkey and their disappearance by 3 months of age if one eye is kept patched from the first day of lifeI. The (~cular dominance columns that represent t.the functional eye broaden and appear to take o~er the adjacent areas of cortex that contain the ocular dominance columns normally representing the occluded eyet. The striate cortex of the monkey is also charactertzed by a different type of columnar organization. Cells in the striate
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Fig. 2. "E-~'ectxof focal~ei'.ures produce",l" 'b)-'"~ca'l app,'tcation-of-penicillin to motor cortex on local cerebral glucose utili2ation in the Rhesus monkey 4. The penicillin was applied to the hand and face area of the left motor cortex. The left side of the brain is on the left in each of the autoradiographs in the figure. The numbers are the rates of local cerebral giucose utilization in t~moillO0 g tissue/rain. Note the following: Upper left. motor coitex in region of penicillin application and corresponding region of contralateral motor cortex: Lower left, ipsilateral and contralateral motor cortical regions remote from area of penicillin applications; Upper right, ipsilateral and contralaterai putamen and globus pallidus ; Lower right, ipsilateral and contralaterai thalamic nuclei and substantia nigra.
79
T I N S - September 1978
Future developments Two major extensions of the DG method that will unquestionably greatly expand its usefulness in basic and clinical research are currently under development. The theoretical basis of the method is fully consistent ~,ith its application at the lightmicroscopic and electron-microscopic levels. Only some technical problems associated with the autoradiography need to be sohed. The labelled metabolic product, deoxyglucose 6-phosphate is water-soluble, and high resolution autoradiograph) requires fixation, dehydration,
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Fig. 3. Autoradiographs of coronal brain sections from Rhesus monkeys at the ievel of the lateral geniculate bodiese. Large arrows point to the lateral geniculate bodies; small arrows point to oculo. motor nuclear complex. ( A ) Animal with intact binocular vision. Note the bilateral ~ygmetry and relative homogeneity of the lateral geniculate bodies and oculomotor nuclei. (B) Animal with bilateral vLeualocclusion. Note the reduced relative densities, the relative homogeneity, and the bilateral symmetr) of the lateral geniculate bodies and oculomotor nuclei. ( C) Animal with right eye occluded. The left side of the brain is on the left side of the photograph• Note the laminae and the inverse order of ~e dark and light bands in the two laterm gcni~ulate bodies. Note also the lesser density of the oculomotor nuclear complex on the side contralateral to the occluded eye.
cortex that respond to specifically oriented lines in the visual field are also arranged in columns, designated the orientation columnsa. Hubel, Wiesel, and Strykera have recently employed the [t~]DG method to demonstrate pictorially the spatial relations of the orientation columns. Pharmacological ai~li~lions
The deoxyglucose mcthcd may offer an additional tool with which to identify the sites of action of centrally active pharmacological agents. This method has been used in studies of the effects of anaesthetic agents; y-hydror,'butyrolactone; morphine and its antagonist, naIoxone; and agonists and am~gonists of putative neurotransmittersn. T ,'zese :;tudies have generally demonstrated local effects which are consisteat with known actions of these drugs. Foz" example, ~-amphetamine, an agonist ~f the neurotransmitter, dopamine, stimulated glucose utilization in the zona compacta and zona reticulata of the substantia nigra, the caudate nucleus, the subthalamic nucleus, and other areas known to be involved in dopaminergi¢ pathwaysn.
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1.0mm" Fig. 4. Autorad,ographs of coronal brain sections from Rhesus moMceys at the level of the striate cortex t. (A) A~imal with normal binocular vision. Note the laminar distributwn of the density; the dark band corresponds to layer IV. I B) Animal with bilateral v,sual deprivation. Note e.~e almost uniform and redaced relative density, especially the virtual disappearance of the dark band corresponding to Layer IV. (C) Animal with right eye occluded. The imlf.brain on the left side of the photograph represents the eft hemisphere contralateral to the oc:luded eye. Note dw alternate dark and light striations, each approximately 0.3-0.4 mm in width, representing the ocular dominance colanms. These columns are most apparent in the dark lamina corresponding to ga)~er IV but extend through the en'ire thickness of the cortex. The arrows point to regions of bdateral asymmetry where the oc,~r dominance columns are absent. These are presumably areas with normally only monocular inpu . The one on the left, contralaterai to the occluded eye, has a continuous dark lamina corresponding :o Layer 1V which is completely absent on the side ipsilateral to the occluded eye. These regions are believed to be the loci of the cortical repre~entation.¢ of the blind spots.
embedding, and staining, all of which may wash out or displace the tracer from it: original sites in the tissue. These problems arc currently under study, and sigmficant progress toward their solution is being made (M. H. Des Rosiers and L. Deccarries, personal communication). Recen: developments in computerized emission tomography have provided the means to apply the DG method to man. Emission tomography requires, howev, ~-radiation, preferably annihilation ~-ra~ deri,,ed from positron emission. A positronemitting derivative of deoxyglucose, 2-[lSFlfluoro-2-deoxy-D-glucose, has been synthesized and found to retain the necessary biochemical properties of 2-deoxyglucoses. it is currently being used in studies in man, and although the resolution is thus far considerably below that obtained with autoradiography, the technique does provide a fair c~egree of imaging of the structural components of the human brain s it is likely that this modification will prove immensely useful, not only in chnical research but also in medical diagnosis. Reading list !. Des Rosier. M. H., Sakurada. O., Jehle, J., Shinohara. M., Kennedy.C. and Sokoloff.L. (19781 Science, N. Y. (m press). 2. Hubel, D H. and W.es¢l, T. N. (19681 Z Physiol. ( Lond.) 195, .~15-243 3. Hubel, D. H., Wiesel, T. N. and Stryker, M. P. (19771 Nature. Lond. 269, 328-3.t0. 4. Kennedy, C., Des Roslers, M. H., Jehle, J, W. Reivl,:h. M., Sharp. F. and Sokoloff, L. (1975) Science, N Y. 187, 850 853. 5. Kennedy.C, D¢~ Roslet~, M. H., Sakurada, O., Shinohara, M, Reivsch, M., Jehle, J W. and Sokoloff.L. (19761Proc. hath. Acad. Sea. U.S.A. 73. 4230-4234. 6. Kennedy, C., Sakurada. O., Shmohara, M, Jehl¢, J. and Sokoloff, L. (19781 Ann. Neurol. (in press). 7. Plum, F.. Gjedde, A. and Samson, F. E. (19761Neurosci. Res Prog. Bull. 14, 457-518. 8. Reivi,:h,M., Kuhl, D., Wolf, A., Greenberg, J., Phelps. M., ido. T.0 C,asella, V., Fowler. J., C.ddlagher, B., Hoffman. E., Alavi, A. drtcl C-Jokoloff, L. (1977) In: D. lngvar and N Lasscn (Eds), Cerebral Function, Metabohsm, and Circulation, Munksgaard, Copenhagen,
pp. 190-191. 9. ~hwzrtz. W. J. and Gainer, H. (19771 Scien'e, N. It. 197. !089-1091.
10. Sharp, F. R., Kaues. J. S. and Shepherd, G. M. (1975) Brain Res. 98. 596-600. Ii. Sokoloff, L. (1977)J. Ne~,.rochem. 29, 13-26. 12. Souo~off, L., geivich, M., Kennedy, C., Des Ro'ier% M. H., Pazlak, C. S.. Pet~igrew, K. D., Saku.-ada. O. and Shinohar. M. (19771 J. Nearochen'. 28. 879-916. 13. Wech~kr, L. R., Savaks, H., Kennedy, C. and Sokoloff, L. (19771 Neurosci. Abstr. 111, 325. |
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L. Sokolo~'is Chief of the Laboratory of Cerebral Aletabolism, National lnslitute of Mental Health, U.S. Department of Heallh, Education, and Welfare, Public Health ~ervice. Bethesda, MD 20014. U.S.A.
75
Mapping cerebral functional activ,ty with radioactive deoxyglucose Louis Sokoloff ,,
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Energy metabolism is closely coupled to functional activity in most tissues, including those of the central nerrous system. In this article we find out how [t'CJdeoxyglucose can be used to trace glucose metabolism and to measure the rates of glucose utilization simuhaneously in all anatomical and functional components of the central nervous system. When combined with autoradiography of serial sections of the brain under conditions in which the optical density of the autoradiographs reflects the rate of glucose utilization, pictorial representation of the relative rates of glucose utilization throughout the brain are obtained. Because of the coupling between functional activity and energy metabolism, these autoradiographs are like a stain for functional activity in nervous tissues and can be used to identify regions with altered functional activity in varying physiological, pharmacological, and pathological states.
The brain is a complex, heterogeneous organ composed of many anatomical and functional componeets with markedly different levels of functional activity that vary independe,3tly with time and function. Other tissues are generally far more homogeneous with most of their cells fJnctioning similarly and synchronously in response to a common stimulus or regulatory influence. The central nervous system, however, consists of innumerable subunits, each integrated into its own set of functional pathways and networks and subserving only one or a few of the many activities in which the nervous system participates. Understanding how the nervous system functions requires knowledge not only of the mechanisms of excitation and inhibition, but even more so of their precise localization in the nervous system and the relationships of neural subunits to specific functions. Historically, studies of the central nervous system have concentrated heavily on localization of function and mapping of pathways related to specific functions. These have been carried out neuroanatomieally and histologically with staining and degeneration techniques, behaviourally with ablation and stimulation techniques, electrophysiologh:ally with electrical recording and evoked electrical respoeses, and histochemically with a variety of techniques, including fluorescent and immunofluorescent methods and autor~lio~aphy of orthograde and retrograde axopla~mic flow. These methods
have provided a fairly detailed map of the roadways of the net~'ous system, but m view of its complexity there may well yet be as many to be discovered. Many of the conventional methods suffer from a sampling problem. They generally permit examination of only one potential pathway at a time, :lnd only positive results are interpretable. Furthermore, the ~iemon. stration of a pathway reveals only a potential for function; it does not reveal
its significance i~ normal function. Tissues that do physical and;or ~:hemical work, such as i~eart, kidney, and skeletal muscle, exhibit a close relationshl.p between energy metabolism and functional activity. From measurement of energy. ~aetabolism it is then possible to estimate the level of functional activity. This relationship has recently been utilized to develop a method that maps functional activity simultaneously in all components of the central nervous .system in the normal conscious state and during physiological, pharmacological, or pathological alterations of functional activity. The method employs radioactive deoxyglucose (DG), an analogue of glucose, to trace glucose metaboli:,m in the brain. The procedure is so designed that the concentration of radioactivity in the tissue is more or h.,ss proportional to the rate of glucose utilization. The concentrations of radioactivity in the local cerebral tissues are measmed by a quantitative autoradiographic technique. The method not only allows quantification of the actual rates
of glucose utilization in the individual cerebral tissues, but the autoradiograph~ obtained with it pro,~'ide pictorial representations of the re;ative rates of glucose utilization in all the cerebral structures seen in autoradiographs of 20 #m serial sections of the entire brain. It is, therefore, now possible to obtain not an anatomical map but a functional map of the entire central nervous system in normal and experimental states. Theoretical basis of the radioactive deoxyglucose method The radioactive deoxyglucose method was developed to measure the local rates of energy metabolism simultaneousl) in all components of the brain in conscious laboratory animals. It was designed specifically to take advantage of the extraordinary spatial resolution made possible by quantitative autoradiography. The dependence on autoradiography prescribed the use of radioactive substrates for ener~" metabolism, the labelled products of which could be assayed in the tissues by the autoradiographic technique. Although oxygen consumption is the most direct measure of energ) metabohsm, the ~olatility of oxygen and its metabolic products and the short physical half-life of its radioactive isotopes precluded measurement of oxidative metabolism by the autoradiographic technique in most cir cumstances glucose i.~ almost the sole substrate for cerebral oxidative metabolism, and tts utihzation ,s stotchtmetrically related to oxygen consumption. Radioactive glucose ts, however, not fully satisfactory because ,ts labelled products are lost too rapidly from the cerebral tissues. The labelled analogue of glucose, 2-deoxy-o-[tq~lglucose. was, therefore, selected because Its biochemical properties make tt parttcularly appropriate to trace glucose metabohsm and to measure local cerebral glucose utihzatton by the autoradiographic technique. The method was derived by analysis of a model based on the biochemtcai properttes of 2-deoxyglucose (Fig. !/,) t~. 2-Deoxygluco,~ is tt~msported I:,i-directionall) between blood and brain by the same carrier that transports glucose across the blood-brain barrier. In the cerebral tissues it is phcsphcrylated by hexokinase to 2-deoxygl~cose 6-phosphate (DG-6-P). Deox~glu~:ose and glucose are, therefore, competitive svbstrates for both blood-brain transport and hexokinase-catalyzed phosphorylatic.n. Unlike glucose 6-phosphate, however, which is metabolized further eventv~lly to CO= and water,
76
T I N S - September 1978
DG-6-P gm :'tot be converted to fructo~ 6-phosphatt and it is also not a substrate for glucose 6-phosphate dehydro~enase. The.4~ is vmy little glucose 6-phosphatase activity in brain, and even less deoxyglucose
A PLASMA
BRAIN TISSUE
i I
6-pbosphatase activity. Dcoxy~ucose 6phosphate, once formed, remains, therefore, essentially trapped in the cerebral tissues duri,~ the experimental period. If the ir, i~rval of brae is kept short enouBh, for example less than ! h, to allow ~ :,~gumption of negligible loss of ['~]DG-6-P from the tissues, then ehe quantity of [t~']DG-6-P accmnulated in any c~-rebr~J tissue at any given time following the introduction of [UC]D~ into the circulation is equal to the integral of the rate of [ttC]DG phosphorylation by hexokinase in tluit tissue during that interval of ",Jme. This integral is in turn ,elated to the amount of glucose that has be~n phosphorylated over the same int=rva;, depending on the time courses of the relative concentrations of [UCJDG and glucose in the precursor pools and the Michaelis-Menten kinetic constants for hexokinase with respect to both p~C]DG and glucose.. With cerebral glucose cons.umption i,, a steady state, the amount of glucose phosphorylated during the interval of time equals the steady state flex of glucose through the hexokinase-catalyzed step, times the duration of the interval;and the net rate of flux of glucose through this step equalg the rate of glucose utilization. These relationships can be rigorously combined into a model (Fig. IA) which can be mathematically analyzed to derive an operational equation (Fig. I B), provide~ that the following assumptions are made: (!) there is a steady state for glucose (i.e. constant plasma glucose concentration and constant ra~ of glucose consumption) throughout the experimental period; (2) there is a homoBeneous tissue compartment within which the conc=ntrations of [uC]DG and glucose are uniform and exchange directly ":,.?,h the plasma; and (3) tracer concentratic~s of [ ~ ] D G (i.e. ~tolecular concentrations of f~e [sq~]DG csscndally equal to zero) are m~d. The operational equation which defines Rt, the rate of glucose utiim~ion per unit mass of tissue, i, in terms of ~e~sun.Me variables is presented in Fig IE. The rate constants, k;, k~, a~d ~, are determined in a separate group (,~ animals by a non-I/near, iterative W,~.,-: which provides the least squares ~esg-~.~ of ;m equation which defines the time course of total tissue uC concen'~tioa in terms of the time, the history ¢f the plasma concentration, and the rate con-
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Integrated &recursor spemfic activity in tissue Fi#. !. Theoretical basis o f re~liouetive dcoxy~glucose meshed/or measurement of local cerebral glucose utilizatio~*. A. Diagrammatic representation o[ the tl~retical model. C~trepresents the total t4C conceatration in a siawie Aomogenema tLtsue of the braise. C~rand Cp represent the concentraiiont o f ["C]deoxyglucose and glucose in the arterial plasma, respectively; ~ and CL represent their respectipe concentrations M the tis.~ pools that serve ag substrates /or hexokMase. C u represents the concentration o f ["Cldeoxy. glucose 6 - ~ t e in tile #issue. The cortstants k;, k~ atJd k; represent the rate constants for carriermediated transport of[t4C]deoxyglucose from plasma to tLttue, for carrier-mediated transport back from fix~e to plasm& a~l for p&~phorylation by hexokiaase, respectively. The comtants kb ks, and ks are the ~ m l e n t rate cott~tants for glucose. ["C]Deoxylrlocose and glucose share wtd compete for the carrier ~ tra~portt them bath between plasma o~[ tittite and/or hexokinase whi~'kpko.*phorylotes them to d,~, respective hexose 6-pbaspAetes. 7~e dashed arrow represents the possibility o f £1ucose 6-phoaplmle l~f~iysia by glucose 6-phosphataseactivity, if atty. B. Operatiomt/equationo f radioactive deox)wlucose metkod and its functio2ml anatomy. T represents the time at the termination o f tit2 experimental period; A equals tile ratio of the diltributim, tImee o f deox2gl,~L ~se in ~ e tissue to tAat o f glucose: ~ eqt~r the fraction o f glucose w,~it~,once i~asphorylated, rnntt~ues down the ~lycoLqi¢ pathway; and IC'- and ~"- and ICm and I"m represent the familiar ~4~c~eih.~Jenten kh:et;¢ constants of hexokiaase for deoxyglucose and glucow, respectively. The other ~v,~d~oL, ore d~e mune as :hose ~
ia A.
TINS- September 1978 slants to the experimentally determined time courses o f tissue and plasma concentrations o f " C n. The ~, ~, and the enzyme kinetic constants are grouped together to constitute a single, lumped constant (see equation). This lumped constant can be shown mathematically to be equal to the asymptotic value of the product o f the ratio of the cerebral extraction ratios of [t%"]DG and glucose and the ratio of the arterial blood to plasma specific activities when the arterial plasma ["C]DG concentration is maintained constant. The lumped constant is also determined in a separate group of animals from arterial and cerebral venot,s blood samples drawn d u n n g a programreed intravenous infusion which produces and maintains a constant arterial plasma [tq~]DG concentrationt'. Thus far it has been determined only in the albino rat and the monkey. Values are 0.483 (S.E.M. = +0.022) in the rat tg and 0.344 (S.E.M. = -_k0.036) in the monkeys. The lumped constant appears to i;e characteristic o f the species and does n¢~t appear to change significantly in a wide range o f conditionsTM. Despite its complex appearance, the operational equation is really nothing more than a general statement of the standard relationship by which rates of enzymecatalyzed reactions :sre determined from measurements mad¢: with radioactive tracers {Fig, I B). Tl;e numerator o f the equation represents the amount of radioactive product formed in a given interval of time; it is equal to C~, the combined concentrations of [uCJDG and ["C]DG6-P in the tissue at time, T, measured by the quantitative autoradiographic technique, less a term ~.hat represents the free unmetabolized [ug..IDG still ,-emaining in the tissue. The denominator eO~esents the integrated specific activity o f the precursor pool, times a factor, the lumped constant, which is equivalent to a correction factor for an isotope effect. The term with the exponential factor in the denominator takes into account the lag in the equilibration o f the tissue precursor pool with the plasma.
Pmeedmre The operational equation dictates the variables to be measured to determine the local rates o f cerebral glucose utilization. The specific procedure employed is designed to evaluate these variables and to minimize potential errors that might occur in the actual application of the method. If the rate constants, k;, k~, and k~, are known precisely, then the equation is
77 generally applicable with any mode of administration of ["C]DG and for a wide range of time intervals. At the present time the rate constants have been determined only in the conscious rat and monkey. These rate constants can he expected to vary with the condition of the animal, however, and for most accurate results should be redetermined for each condition studied. The structure c:f the operational equation suggests a more practicable alternative. All the terms in the equation that contain the rate constants approach zero with increasing time if the ["C]DG is so administered that the plasma [tq~]DG concentration also approaches zero. From the values of the rate constants determined in normal animals and the usual time course of the clearance of ptC]DG from the arterial plasma following a single intravenous pulse at zero time, it has been determined that an interval of 3 0 4 5 min after a pulse is adequate for these terms to become sufficiently small that considerable latitude in inaccuracies of the rate const:,nts is permissible without appreciable error in the estimates of local glucose consumption n. An additional advantage derived from the use of a single pulse of [I~"]DG followed by ~ relauvely long interval before killing the animal for measurement of local Ussue " C concentration is that by then most of the free ["C]DG in the tissues has been either converted to [ttC]DG-6-P or transported back to the plasma; the optical densities in the autoradiograph~ then represent mainly the conc,entratio~s of pq~]DG-6-P and, therefore, reflect dicectly the relative rates of glucose utilization in the various cerebral tissues. The experimental procedure-"~ ._ t~ inject a pulll~ of ["C~DG i n t r a v e n o ~ ' :~t zero time and t o ~.~:apitate the ac, ~al and freeze the brai.~ at a measured tt.ae, T, 30-45 win later: in the interval, t~med arterial samples are taken for the measurement of plasma [t~"llX3 and glucose concentrations. Tissue " C concentrations, C: ,,re measured at time, T, by quantitat,,e autoradiography of 20pro frozen dried sections prepared serially from the entire brain. Local cerebral glucose utilization is then calculated by the operational equation. Rates of g l E m e utilization ia anatoad¢~ ¢ e m p e a e m of Im~a The rates of local cerebral glucose utilization have been measured in the normal conscious and anaesthetized albino rat and in the conscious Macaque monkey. The rates in the conscious rat vary widely
throughout the brain with the values in white matter distributed in a narrow low range and the ,,alues in grey structures broadly distributed around an average which is about three Umes greater than that of white matter 1Table I). The highest values are in stractures of the auditor' system with the inferior colliculus clearly the most metabolically active structure in the brain (Table i). The rates of local cerebral utdization in the conscious monkey exhibit similar heterogeneity, bug they are generally one-third to one-half of the values in corresponding structures of the rat brain (Table i). ihe" differences in rates in the rat and monke~ brain are consistent wtth the different ce!lular packing denstues in the brains of these two species. General anaesthesia induced by thtopental reduces the rates of glucose utihzation in all sVucture~ .,i the rat brain n. The effects are not uniform, however. The greatest reductions occur m the grey structures, par~.tcularly those of the primary sensory pathways. The effects in white matter, though definitely present, are relatively smal, compared with tho~e in grey matted'. TABLE !. Reptesentattve values for Io~al cerebral glucose utilizanon in the normal conscious albino rag and monkey (~mol,100 ~mm) Struct..,c
Albino tat" Monk~'~" (n I0) {n ~ ~)
(3rey matter
45.2 107 6 79 • 4 162 : 5 47 - 4 Parietal cortez 112 - 5 44 - l Sensory-mot ~,,~¢ex 120 ,. .< 54 2 Thalamus: ~ : ~, aucleu~ 116 ~* 5 ~.3 : -" T h a l a m t : ~r~:~ nucleus 109 - 5 65 3 Medial gcni~-~,~.~c oody 131 : 5 39 • I Lateral Ig~ni~at¢ ~ody 96 : 5 25 • I Hypothalamus 54 ± 2 5 57 • 3 Mamillary I~,dy 12l 39:2 Hippoc, tmpus 79 = 3 25-2 Amygdala 52 : 2 52- ~, Caudate-putameq ! 10 : 4 3b-: 2 Nucleus accumbens 82 3 26:2 Globus palhdus 58 .~ 2 29:2 Substantm mgra 58 : 3 66 _.~ ? Vestibular nucleus 128 _~ 5 51-.3 Cochlear nuclcu> i 13 = 7 63~ 4 Superior olivary ~mcleus 133 = 7 Inferior colhculu~. 197 ± 10 103 ~ 6 .'5:4 Superior collicu!u~ 95 = 5 28 "i. ! Pontine grey ma::et 62 : 3 ?i ~-2 Ccrcbellar cortex 57 + 2 45~. 2 Cerebellar nuclet 100 *- 4 Whtte matter ll-kl Corpus callosum 40 ! 2 134-1 Internal capsule 33 + 2 12__1 Cerebdiar white ma~.lcr 37 ± 2 Visual cortes Audflory cortex
The values are the roans ± standard errors from measurements mack in the.. number of animals indicated (n).
78 MeUdmlic mappieg el" fmetiewl acttdty in central nerveus syMem The results of a va;iety of studies on the effects of experimentally-induced focal alterations of functional activity on local glucose utilization have demonstrated a close coupling between local functional activity and eneqD" metabolism in the brain". The effects are often so prono,raced that they can be visualized dir;ctl) on the autoradiographs which pr,vide pictorial reprt~entations of the rehtti~e rates of glucose utilizatton throughout the brain. ,'D),is technique of autoradiographic visualization of evokcti metabolic responses offers a powerful tool to map functional neur~;! pathways simultaneously in all anatomical compor.ents of ~he central nervous system~.n. The use of the technique for metabolic mapping has been growing rapidly, and only a few examples can be cited Jn the present report. More comprehenswe re~,,¢ws of these apphcations have recently been. pubhshed:, n. The results thus far clearly demonstrate tae effectivenes~ of cerebral metabolic res?onses, either positive or negative, in identifying regions of the central nervous system involved zn si~'cific functions. For example, elect~ cal stimulation of one sciatic nerve in ,he anaesthetized rat evokes increased glucose utilization, seen as increased density ;n the autoradiographs, in the ipsllateral lumoar spinal cord grey matter, especiall~ ~n he dorsal horn s. The distribution of ~he spread of activation -~uring focal seizares induced by local urilaterai injectiov, of penicillin into the ~.totor cortex in the monkey can be vist:alized by ipsilateral areas of increased ~ens[~) in the autoradiographs of the m~tor cortex, putat~en, globus pallidus, thr.lamic nuclei, ~abstantla nigra, etc. (Fig 2). The distribution o.t" changes in activity associated ~'ith normal or spontaneous alterattons in physiological function can be studzed. For example, the suprachiasmatic nuclei of the rat exhibit diurnal rhythmicity in metabolic activity. They are active during the day and inactive at night; t':ey are visualized as distinct, relatively dark areas on the autoradiographs of ani~.ais studied in the morning and are essentially invisible in the auto,adiographs of enimals studied at nighP. Olfactory stimul~.~on with specific odours has been shown to produce metabolic activation of .,,pccific discrete regions of the olfactory bulb t°. Obstruction of both external auditory canals of the conscious rat r,.arkedly diminishes glucose utilization bilaterally in all components of the auditory pathway,
T I N S . S e p t e m b e r 1978
e.g. the cochlear nuclei, superior olives, nuclei of the lateral lemnisci, medial geniculate ganglia, and the auditory cortex. With unilateral auditory obstruction, some of the structures are affected only ipsilaterally, some only contralaterally, and some bilaterally: with quantification it becomes possible to estimate the percentage crossing of the various pathways involvedn. it is known that in the rat the visual pathways from the retinae are 80-85 ~,, crossed to the opposite hemisphere: unilateral enucleation results in approximately 80% reductions in glucose utilization of the contralateral lateral geniculate ganglion, superior colliculus, and visual cortex, effects which are strikingly visible in the autoradiographs4. The most dramatic demonstrations of the ability of the deoxyglucose method to map functional ac:~'.'ity in the central nervous system have been in the primary vtsuai system of the monkey. In lhis species, as in all animals with binocular vision, the visual pathways are approximately 50°0 crossed at the optic chiasma, and the structures of the visual system on each side of the brain receive equal inputs from both retinae. The projections from the two retinae remain segregated and terminate in six well-defipcd laminae in each of the two lateral geniculate Ianglia, three each for the ipsilateral and
"~108 ~,,~
contralateral eyes. The .¢,egregation is preserved in the geniculocalcarine pathways which terminate in the striate cortex in alternating columns receiving input from the ipsilaterai and contralateral eyes. These are the ocular dominance columns originally dcscfi,~.-d by Hubei and Wiesel on the basis of electrophysiological studiesz. The P%."IDG method has been used to map all the components of the binocular visual system of the monkey simultaneously in the same animals, and to provide a complete pictorial representation of the nature, extent, and distribution of the ocuiar dominance columns (Figs. 3 and 4p. it has also identified the loci of the visual cortical representation of the blind spots of the retinae {Fig. 4). The ocular dominance columns exhibit functional plasticity in the immature striate cortex. The pq:::]DG method has demonstrated their presenc: in the new-born monkey and their disappearance by 3 months of age if one eye is kept patched from the first day of lifeI. The (~cular dominance columns that represent t.the functional eye broaden and appear to take o~er the adjacent areas of cortex that contain the ocular dominance columns normally representing the occluded eyet. The striate cortex of the monkey is also charactertzed by a different type of columnar organization. Cells in the striate
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Fig. 2. "E-~'ectxof focal~ei'.ures produce",l" 'b)-'"~ca'l app,'tcation-of-penicillin to motor cortex on local cerebral glucose utili2ation in the Rhesus monkey 4. The penicillin was applied to the hand and face area of the left motor cortex. The left side of the brain is on the left in each of the autoradiographs in the figure. The numbers are the rates of local cerebral giucose utilization in t~moillO0 g tissue/rain. Note the following: Upper left. motor coitex in region of penicillin application and corresponding region of contralateral motor cortex: Lower left, ipsilateral and contralateral motor cortical regions remote from area of penicillin applications; Upper right, ipsilateral and contralaterai putamen and globus pallidus ; Lower right, ipsilateral and contralaterai thalamic nuclei and substantia nigra.
79
T I N S - September 1978
Future developments Two major extensions of the DG method that will unquestionably greatly expand its usefulness in basic and clinical research are currently under development. The theoretical basis of the method is fully consistent ~,ith its application at the lightmicroscopic and electron-microscopic levels. Only some technical problems associated with the autoradiography need to be sohed. The labelled metabolic product, deoxyglucose 6-phosphate is water-soluble, and high resolution autoradiograph) requires fixation, dehydration,
5.0m m
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Fig. 3. Autoradiographs of coronal brain sections from Rhesus monkeys at the ievel of the lateral geniculate bodiese. Large arrows point to the lateral geniculate bodies; small arrows point to oculo. motor nuclear complex. ( A ) Animal with intact binocular vision. Note the bilateral ~ygmetry and relative homogeneity of the lateral geniculate bodies and oculomotor nuclei. (B) Animal with bilateral vLeualocclusion. Note the reduced relative densities, the relative homogeneity, and the bilateral symmetr) of the lateral geniculate bodies and oculomotor nuclei. ( C) Animal with right eye occluded. The left side of the brain is on the left side of the photograph• Note the laminae and the inverse order of ~e dark and light bands in the two laterm gcni~ulate bodies. Note also the lesser density of the oculomotor nuclear complex on the side contralateral to the occluded eye.
cortex that respond to specifically oriented lines in the visual field are also arranged in columns, designated the orientation columnsa. Hubel, Wiesel, and Strykera have recently employed the [t~]DG method to demonstrate pictorially the spatial relations of the orientation columns. Pharmacological ai~li~lions
The deoxyglucose mcthcd may offer an additional tool with which to identify the sites of action of centrally active pharmacological agents. This method has been used in studies of the effects of anaesthetic agents; y-hydror,'butyrolactone; morphine and its antagonist, naIoxone; and agonists and am~gonists of putative neurotransmittersn. T ,'zese :;tudies have generally demonstrated local effects which are consisteat with known actions of these drugs. Foz" example, ~-amphetamine, an agonist ~f the neurotransmitter, dopamine, stimulated glucose utilization in the zona compacta and zona reticulata of the substantia nigra, the caudate nucleus, the subthalamic nucleus, and other areas known to be involved in dopaminergi¢ pathwaysn.
A
B
C
1.0mm" Fig. 4. Autorad,ographs of coronal brain sections from Rhesus moMceys at the level of the striate cortex t. (A) A~imal with normal binocular vision. Note the laminar distributwn of the density; the dark band corresponds to layer IV. I B) Animal with bilateral v,sual deprivation. Note e.~e almost uniform and redaced relative density, especially the virtual disappearance of the dark band corresponding to Layer IV. (C) Animal with right eye occluded. The imlf.brain on the left side of the photograph represents the eft hemisphere contralateral to the oc:luded eye. Note dw alternate dark and light striations, each approximately 0.3-0.4 mm in width, representing the ocular dominance colanms. These columns are most apparent in the dark lamina corresponding to ga)~er IV but extend through the en'ire thickness of the cortex. The arrows point to regions of bdateral asymmetry where the oc,~r dominance columns are absent. These are presumably areas with normally only monocular inpu . The one on the left, contralaterai to the occluded eye, has a continuous dark lamina corresponding :o Layer 1V which is completely absent on the side ipsilateral to the occluded eye. These regions are believed to be the loci of the cortical repre~entation.¢ of the blind spots.
embedding, and staining, all of which may wash out or displace the tracer from it: original sites in the tissue. These problems arc currently under study, and sigmficant progress toward their solution is being made (M. H. Des Rosiers and L. Deccarries, personal communication). Recen: developments in computerized emission tomography have provided the means to apply the DG method to man. Emission tomography requires, howev, ~-radiation, preferably annihilation ~-ra~ deri,,ed from positron emission. A positronemitting derivative of deoxyglucose, 2-[lSFlfluoro-2-deoxy-D-glucose, has been synthesized and found to retain the necessary biochemical properties of 2-deoxyglucoses. it is currently being used in studies in man, and although the resolution is thus far considerably below that obtained with autoradiography, the technique does provide a fair c~egree of imaging of the structural components of the human brain s it is likely that this modification will prove immensely useful, not only in chnical research but also in medical diagnosis. Reading list !. Des Rosier. M. H., Sakurada. O., Jehle, J., Shinohara. M., Kennedy.C. and Sokoloff.L. (19781 Science, N. Y. (m press). 2. Hubel, D H. and W.es¢l, T. N. (19681 Z Physiol. ( Lond.) 195, .~15-243 3. Hubel, D. H., Wiesel, T. N. and Stryker, M. P. (19771 Nature. Lond. 269, 328-3.t0. 4. Kennedy, C., Des Roslers, M. H., Jehle, J, W. Reivl,:h. M., Sharp. F. and Sokoloff, L. (1975) Science, N Y. 187, 850 853. 5. Kennedy.C, D¢~ Roslet~, M. H., Sakurada, O., Shinohara, M, Reivsch, M., Jehle, J W. and Sokoloff.L. (19761Proc. hath. Acad. Sea. U.S.A. 73. 4230-4234. 6. Kennedy, C., Sakurada. O., Shmohara, M, Jehl¢, J. and Sokoloff, L. (19781 Ann. Neurol. (in press). 7. Plum, F.. Gjedde, A. and Samson, F. E. (19761Neurosci. Res Prog. Bull. 14, 457-518. 8. Reivi,:h,M., Kuhl, D., Wolf, A., Greenberg, J., Phelps. M., ido. T.0 C,asella, V., Fowler. J., C.ddlagher, B., Hoffman. E., Alavi, A. drtcl C-Jokoloff, L. (1977) In: D. lngvar and N Lasscn (Eds), Cerebral Function, Metabohsm, and Circulation, Munksgaard, Copenhagen,
pp. 190-191. 9. ~hwzrtz. W. J. and Gainer, H. (19771 Scien'e, N. It. 197. !089-1091.
10. Sharp, F. R., Kaues. J. S. and Shepherd, G. M. (1975) Brain Res. 98. 596-600. Ii. Sokoloff, L. (1977)J. Ne~,.rochem. 29, 13-26. 12. Souo~off, L., geivich, M., Kennedy, C., Des Ro'ier% M. H., Pazlak, C. S.. Pet~igrew, K. D., Saku.-ada. O. and Shinohar. M. (19771 J. Nearochen'. 28. 879-916. 13. Wech~kr, L. R., Savaks, H., Kennedy, C. and Sokoloff, L. (19771 Neurosci. Abstr. 111, 325. |
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L. Sokolo~'is Chief of the Laboratory of Cerebral Aletabolism, National lnslitute of Mental Health, U.S. Department of Heallh, Education, and Welfare, Public Health ~ervice. Bethesda, MD 20014. U.S.A.