SYNAPSE 10:16-24 (1992)
Compartmental Distribution of Cytochrome Oxidase in the Striatum of the Rat SARAH J. AUGOOD AND PIERS C. EMSON MRC Group, Department of Neuroendocrinology, AFRC Institute of Animal Physiology & Genetics Research, Babraham, Cambridge, CB2 4AT, England
KEY WORDS
Patchhtriosome, Matrix compartment, Calbindin, Tyrosine hydroxylase, Basal ganglia
ABSTRACT Endogenous cytochrome oxidase activity was investigated in the adult rat striatum at the light microscope level to see if it was distributed in accordance with the established striatal patcwmatrix compartmentalisation. Striatal sections stained to visualise cytochrome oxidase activity were compared with serial sections stained to visualise tyrosine hydroxylase and calbindin,,,k-like immunoreactivity, established markers of the matrix compartment. The distribution of endogenous cytochrome oxidase activity was found to coincide with the immunocytochemical staining pattern seen for tyrosine hydroxylase and calbindinD2,kwhereby areas of intense tyrosine hydroxylase and calbindinDZek-likeimmunoreactivity (termed the matrix) corresponded to areas of intense cytochrome oxidase activity. Conversely, areas of less intense tyrosine hydroxylase and calbindinDz,,-like immunoreactivity (termed patches) corresponded t o areas of low cytochrome oxidase activity. In addition, the distribution of two other oxidative enzymes involved in the regulation of mitochondria1respiration, succinic dehydrogenase and NADH-diaphorase, was examined in the striatum and substantia nigra by using histochemical techniques. Both NADH-diaphorase and succinic dehydrogenase histochemistry showed an uneven pattern of neuropil staining in the striatum. In the substantia nigra a few intensely stained cell bodies were seen in the dorsal-lateral tip of the pars reticulata with both histochemical techniques. By using an anti-cytochrome oxidase antibody an abundance of immunoreactive cell bodies and processes were seen in the substantia nigra, particularly in the dorso-medial rim and dorsal tip of the pars reticulata. The substantia nigra pars lateralis contained many intensely stained cytochrome oxidase-like immunoreactive cell bodies and processes. Our results demonstrate that the neurochemically distinct compartments of the adult rat striatum contain differential amounts of endogenous cytochrome oxidase activity suggesting that the matrix compartment is more metabolically active than the patch compartment. This difference in metabolic activity may reflect a difference in synaptic activity between the two striatal compartments. INTRODUCTION The heterogeneous organisation of the mammalian striatum has been studied widely in terms of its cellular morphology, neurochemistry, and basic circuitry. The initial observation that the rat striatum was not a homogeneous structure (Olson et al., 1972)has led to an overwhelming number of studies investigating the extent of this heterogeneity. To date two main striatal compartments have been reported in the rat which differ in their neurochemical composition (Gerfen et al., 1985; Graybiel and Ragsdale, 1978; Graybiel et al., 1987; Herkenham and Pert, 19811, origin of inputs (Donoghue and Herkenham, 1986; Gerfen, 1984,1985, 19891, and their afferent projections (Gerfen, 1984).The two striatal compartments have been termed “patch” 0 1992 WILEY-LISS,INC.
and “matrix,”with the latter compartment constituting approximately 80% of the striatum. Since these two striatal compartments show a differential concentration of neurotransmitters, neuropeptides, receptor binding sites, endogenous enzymes, and neuropeptide mRNA’s (for review see Graybiel, 1990) they may be visualised by using neurochemical parameters. Although the neurochemical composition of these striatal compartments has been well documented, to date their precise functional significance remains unclear. One marker for the striatal matrix compartment is the calcium binding protein calbindinD2,k(CaBP: Gerfen et al., 19851,first isolated from chick small intestine Received January 11,1991; accepted in revised form May 13,1991
DISTRIBUTION OF CO IN RAT STRIATUM
(Wasserman and Taylor, 1966). A number of reports have suggested that expression of calcium binding proteins such as CaBP or parvalbumin (PV) is related to neuronal activity (Braun et al., 1985; Kawaguchi et al., 19891, with fast-firing neurones expressing more calcium binding protein than slow-firing neurones. These observations suggest that in vivo striatal matrix neurones may be faster-firing and metabolically more active than slower-firing patch neurones. A number of studies have demonstrated a positive correlation between high levels of spontaneous and synaptic activity and high levels of endogenous CO activity (Braun et al., 1985; Dimlich et al., 1990; Wong-Riley 1979,19891,and it has been suggested that relative amounts of endogenous CO activity may serve as a marker of neuronal functionaYmetabolic activity (Hevner and Wong-Riley, 1990). Therefore, further to the work of DiFiglia and colleagues (1987) we undertook a study of the distribution of CO activity in the rat striatum in relation to the patch/matrix organisation as demonstrated by tyrosine hydroxylase (TH) and CaBP-like immunoreactivity (-LI). The compartmental distribution of two other enzymes involed in mitochondria1 respiration and oxidative phosphorylation, NADH-diaphorase and succinic dehydrogenase (SDH), was also examined. Preliminary results of these experiments have been published previously (Augood and Emson, 1989).
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through the striatum and collected into ice-cold PBS. Every third serial tissue section was then allowed to warm to room temperature (RT) and immediately incubated in freshly prepared and filtered 0.1 M phosphate buffer (PB) containing 3-3’ diaminobenzidine hydrochloride (DAB: 1.5 mg/ml) for approximately 45 min at RT and then extensively rinsed in PBS, mounted onto gelatinised slides, and coverslipped by using a synthetic mountant (Ralmount). The remaining two serial sections were processed to visualise TH and CaBP-LI as described below. 2. Metal chelation histochemical technique (Burnstone, 1959). Fresh frozen coronal cryostat sections of striatum were cut (15 pm), allowed to dry at RT for 2 hr, and then incubated in 0.2 M Tris-HC1 buffer (pH 7.4) containing 1-hydroxy-2-napthoic acid (0.002%), 4aminodiphenylamine (0.002%), and EtOH (0.01%) at RT. Finally, the napthol compound formed was chelated to Co2+[1%Co(HOAc),] for 1 hr. Sections were then coverslipped by using glycerin jelly. Endogenous CO activity was visualised as an insoluble blue-black granular deposit. Control sections were incubated in reaction medium containing the specific CO inhibitor sodium cyanide (50 mM). 3. Modified peroxidase technique (Carr et al., 1989). Fixed striatal sections (25 p.m) were incubated in the dark a t 37°C for 3 hr in PB containing 0.025% DAB/4% sucrose/0.03% cytochrome c (Sigma, type 111) as subMATERIALS AND METHODS strate. The reaction was terminated when the greatest Endogenous CO activity was investigated in the stri- contrast between highly active and less active areas of atum of adult female rats (Wistar) by using four differ- neuropil staining was seen. Sections were then mounted ent histochemical techniques in an attempt to compare onto gelatinised slides and coverslipped. the relative sensitivities of the methods: 1) a direct 4.Technique using a polyclonal anti-bovine brain CO peroxidase method, 2) a metal chelation histochemical antibody. This antibody has been characterised previmethod, 3) a modified peroxidase technique which uti- ously (Hevner and Wong-Riley, 1989) and has been lises reduced cytochrome c as the substrate, and 4) a shown to react predominantly with subunit IV of CO. method using a polyclonal anti-bovine brain CO anti- Immunocytochemical staining of fixed rat sections was body (gift of Prof. M.T.T. Wong-Riley).TH-LI was visu- as described below. alised by using a polyclonal anti-rat adrenal TH antiImmunocytochemistry body (gift of Prof. Nagatsu) and CaBP-LI was visualised Free-floating fixed sections (25 pm) were incubated by using a polyclonal anti-chick intestine CaBP antibody (gift of Dr. D.E.M. Lawson). Both NADH-diapho- in primary antibody (anti-CO 1/1,000, anti-TH 1/500, rase activity and SDH activity were visualised by using and anti-CaBP 1/400) diluted in PBS/O.2% Triton Xhistochemical methods previously described (Bancroft, 100/1% normal goat serum overnight at 4°C. Sections were then rinsed (3 X 15 min) in PBS/0.2%Triton X-100 1975). and incubated in HRP-conjugated goat anti Rb IgG Cytochrome oxidase histochemistry (1/500: Vector Labs) for several hours a t RT. Finally Details of the four techniques used to demonstrate CO sections were rinsed extensively as before and then activity in the adult rat striatum are given below: incubated in DAB (0.5 mg/ml) activated with HzOz (0.025%).Reacted sections were washed in an excess of 1. Direct peroxidase method (DiFiglia et al., 1987). PBS, mounted onto gelatinised slides, and coverslipped Rats were anaesthetised with pentobarbitone (Sagatal) as above. For control experiments, the specificity of the and perfused transcardially with heparinised saline secondary antibody was tested by omitting the primary (0.1%)followed by ice-cold fixative (2% neutral-buffered antibody from the first incubation. paraformaldehyde/l.25% glutaraldehyde). Exactly 20 NADH-diaphorase histochemistry min followingfixation brains were removed onto ice and NADH-diaphorase activity was demonstrated in immediately sectioned on a freezing microtome. Freefresh-frozen sections by using an established hisfloating coronal serial sections (40 p.m) were cut
DISTRIBUTION OF CO IN RAT STRIATUM
tochemical procedure (Bancroft, 1975).Briefly, cryostat sections were cut (15 pm), air dried, incubated in reaction medium containing the tetrazolium salt 3(4:5-dimethyl thiazolyl-2)5-diphenyltetrazolium bromide (MTT: 0.6 mM), CoCl,, and NADH (nicotinamide adenine dinucleotide reduced: 2.8 mM) as substrate at 37°C for 30-40 min, fixed in formal saline, and finally coverslipped using glycerin jelly. Hydrogen liberated during the catalysed oxidation of NADH to NAD reduces the MTT to form a formazan compound which then chelates with Co2+ to form a black insoluble product. Consequently, sites of enzyme activity are labelled by fine discrete granular deposits. Control sections were incubated in either reaction medium containing the CO inhibitor sodium cyanide (50 mM) or reaction medium omitting the substrate.
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cleus were found to be intensely stained (Fig. 1A-D). In agreement with DiFiglia and co-workers (19871,we also found that the most intensely stained area of the striatum was the dorso-medial rim just ventral to the corpus callosum (Fig. 1E). Adjacent sections processed to visualise TH-LI and CaBP-LI also showed a heterogeneous pattern of staining essentially the same as seen for CO (Fig. 2A-D). However, whilst CO activity and TH-LI were seen concentrated in the neuropil, CaBP-LI was also seen concentrated in the cytoplasm of mediumsized cells. It was noted that, whilst TH-LI was seen heterogeneously distributed throughout the coronal extent of the rat striatum, the dorso-medial part of the striatum was consistently found to be relatively devoid of CaBP-LI (cf. Fig. 2A). It is interesting to note that the boundary between the patch and matrix striatal compartments appears more discrete when defined by SDH histochemistry CaBP-LI than it does when visualised with other striThe activity of this oxidative dehydrogenase was atal markers-for example, AChE staining (data not demonstrated in fresh-frozen cryostat sections (15 pm) shown). Comparisons of the distribution of areas of by using an established histochemical procedure (Ban- intense TH-LI and CaBP-LI (matrix) were found to croft, 1975).This method is essentially the same as used correspond to areas of intense CO reaction product. by Marshall and co-workers (1981) except a higher Conversely, areas of less intense TH-LI and CaBP-LI concentration of substrate was used in accordance with (patches) were found to correspond to areas with less the study by Shimizu and co-workers (1957). The reac- intense CO reaction product. Endogenous CO activity tion conditions are similar to those described for NADH- was also visualised with other techniques as sumdiaphorase histochemistry except the tetrazolium salt marised below. employed was nitro-blue tetrazolium (NBT: 1.2 mM) Metal chelation histochemical technique and the specific substrate was sodium succinate (0.5M). Sites of CO activity were labelled with blue-black Control sections were incubated in reaction medium not granular deposits which were seen concentrated in the containing the substrate. striatal neuropil. Labelled soma were not obvious but RESULTS may have been obscured by the neuropil staining. To compare the distribution of endogenous CO activ- Whilst deposits were found in all parts of the striatum, ity with the pattern of immunocytochemical staining for areas containing relatively less staining were seen. The TH and CaBP, the peroxidase method was used as this uneven distribution of granular deposits was found to be was the method used by DiFiglia and co-workers (19871, unrelated to the distribution of myelinated fibre bunwho first reported an uneven distribution of CO activity dles. Sections incubated with sodium cyanide did not in the striatum. The additional three methods of visu- show any staining. alising CO activity were carried out to compare the The modified peroxidase technique of relative sensitivities of the different methods. Carr et al. (1989) Distribution of CO activity, TH-LI, and CaBP-LI Sites of CO activity were visualised by a concentraSites of endogenous CO activity were labelled with a tion of dark brown reaction product within striatal dark brown DAB reaction product concentrated within sections. Whilst an uneven distribution of reaction prodstriatal sections. Reaction product was seen localised in uct was detected, the contrast between heavily labelled the neuropil. In addition to the heterogeneous distribu- and weakly labelled areas obtained with this method tion of reaction product in the striatum and nucleus was poor (data not shown). accumbens, the cingulate cortex and lateral septa1 nuImmunocytochemistry using a n anti-CO antibody CO-LI was visualised by the localisation of a dark brown reaction product concentrated in the striatal Fig. 1. Comparison of the distribution of CaEiP-LI (A), endogenous CO activity (B), and TH-LI (C) in serial sections of rat nucleus neuropil. Weakly stained cell soma may have been accumbens (NAc).Note the intense patchy distribution of DAB reaction obscured by the neuropil staining. While an uneven product depicting sites of CO activity (B) and TH-LI in (C) in the NAc. Fingers of less intense staining (patches) may be seen emanating from pattern of CO-LI was seen, the contrast between the ventricular edge; this is particularly evident in (A). CO activity in strongly stained and weakly stained areas of the striathe (D) cingulate cortex (Acg) and (E)dorsal rim of rat striatum; note tum was poor (Fig. 3B). On the same sagittal sections an the intensity of DAB reaction product localised just ventral to the abundance of CO-LI pyramidal cells were visible in corpus callosum (cc). AC, anterior commissure. Magnification x 50.
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S.J. AUGOOD AND P.C. EMSON
Fig, 2. Comparison of the distribution of reaction product depicting sites of endogenous CO activity (A,D),TH-LI (B), and CaBP-LI (C) in coronal sections of rat striatum. (A and B) and (C and D) are pairs of serial sections. Sites of activity of endogenous CO were visualized by using the direct peroxidase method. Note the heterogeneous distribu-
tion of reaction product and the coincidence of patches (stars) in A and B and C and D. As noted in the text, the dorsal striatum is always found to be devoid of CaBP-LI using this antibody (C). Abbreviations: claustrum (CL), lateral ventricle (LV), corpus callosum (cc), and anterior commissure (AC). Magnification x 30.
DISTRIBUTION OF CO IN RAT STRIATUM
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Fig. 3. Visualisation of (A) CaBP-LI and (B) CO-LI in sagittal sections of rat striatum (CPu). Note the patchy distribution of reaction product in both A and B. In A strongly stained CaBP-LI cell bodies can be seen localised within areas of strongly stained neuropil (matrix). Conversely, in B only neuropil staining can be seen with no CO-LI cell bodies beingvisualised readily. The boundary between the CPu and GP is marked by a dotted line. In A and B asterisks denote the patch compartment. (C) Coronal section showing the localisation of CO-LI
within cell soma, processes, and the neuropil of the SN. Note the intensely stained cell bodies of the SNR and SNL (arrowhead). (D) Sagittal section of rat frontal cortex (Frl) showing the localisation of CO-LI within pyramidal cells (arrowheads) concentrated in layer V. A few CO-LI cells are also seen in layer 111. Abbreviations: internal capsule (ic), globus pallidus (GP), substantia nigra pars lateralis (SNL), and pars reticulata (SNR).
layer V of the frontal cortex, with a few smaller cells being seen in layer I11 (Fig. 3D). In the SN an abundance of intensely stained CO-LI cell bodies and processes were seen concentrated in the dorso-medial rim and dorsal tip of the pars reticulata (SNR: Fig. 3C). A few CO-LI cell bodies and processes were also visible in the pars compacta (SNC); the pars lateralis (SNL) contained numerous intensely stained CO-LI cell bodies and processes, an observation previously reported in the primate (Ma, 1989). Sections processed without the antibody showed no staining. The differences in contrast between CO active areas (matrix) and less active areas (patches) seen with the four different techniques used here presumably reflects differences in sensitivity of the methods. Interestingly, the best contrast between the CO active matrix and less active patch compartments was seen by using a direct histochemical technique, suggesting that indirect methods, which include several amplification steps for the development of the reaction product, are not so closely related to tissue CO activity.
NADH-diaphorase histochemistry Sites of NADH-diaphorase activity were labelled with granular deposits found concentrated in the striatal neuropil. No staining of cell bodies was seen. While deposits were detected in all parts of the striatum, areas of weak staining were seen which were surrounded by larger areas of intense staining (Fig. 4A). These weakly stained areas did not correspond to areas of fibre bundles. In the SN, granular deposits were detected throughout the nucleus with a few heavily labelled cell bodies being seen in the dorso-lateral and dorsal tip of the SNR (Fig. 4B,C). The pattern and intensity of labelling were unaffected by sodium cyanide, the selective CO inhibitor. However, when the specific substrate (NADH) was omitted from the reaction medium no staining was visible. It is important to note that this histochemical technique does not visualise NADPHdiaphorase activity which has been demonstrated within striatal interneurones containing somatostatinLI (Vincent and Johansson, 1983; Vincent et al., 1983).
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S.J. AUGOOD AND P.C. EMSON
Figure 4.
DISTRIBUTION OF CO IN RAT STRIATUM
SDH histochemistry Sites of SDH activity were labelled with purple granular deposits. In striatal sections, deposits were seen in all parts of the nucleus and these were localised to the neuropil. No obvious labelling of cell bodies were seen. Striatal sections had a patchy appearance; however, the contrast between strongly and weakly stained areas was poor (data not shown), not as clear as the contrast seen with NADH-diaphorase histochemistry. In the SN, the neuropil was heavily labelled; occasional cell bodies were detected usually localised in the lateral extent of the SNR; the SNC contained very few labelled cell bodies. Control sections processed without the substrate showed no staining.
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gesting that CO may well be a marker for neuronal functional activity. Consistent with these observations, our findings therefore suggest a difference in metabolic activity between the matrix and patch compartments in the rat striatum; that is, the matrix compartment displays a greater amount of endogenous CO activity than the striatal patch compartment. This would be consistent with the suggestion that matrix neurones may be more metabolically active than patch neurones. It may be of some physiological importance that the striatal matrix compartment, enriched in endogenous CO activity, is also relatively enriched in a calcium binding protein, CaBP. It is unlikely that the difference in CO activity in the patch and matrix compartments is due to differDISCUSSION ences in the electrophysiological properties of cells in the two compartments as 95% of striatal neurones are Our findings demonstrate that the uneven distribumedium-sized spiny cells (Kemp and Powell, 1971) and tion of endogenous CO activity in the adult rat striatum Kawaguchi and co-workers (1989) have shown that coincides with the distribution of TH- and CaBP-LI, established markers of the matrix compartment; that is, identified neostriatal patch and matrix spiny cells have areas of intense TH- and CaBP-LI coincided with areas similar axonal and dendritic morphology, passive memof intense CO activity. Conversely, areas of weak TH- brane properties, and repetitive firing characteristics. and CaBP-LI (termed patches) coincided with areas of These findings suggest that in the absence of an exterless intense CO activity. Our results therefore, extend nal stimulus, patch and matrix spiny neurones have the findings of DiFiglia and co-workers (19871, who similar electrophysiological properties in vitro. reported that dendritic mitochondria were responsible Whether this is true in vivo when spiny cells are under for the majority of CO activity in the rat striatum. In the influence of all their synaptic inputs remains to be addition, they showed that the differential distribution established. Therefore, it has still to be determined between CO active and less active areas was not a whether the difference in synaptic activity observed reflection of a difference in the density of dendrites or here between the striatal patch and matrix compartsynapses but was due to the presence of a greater ments is due to the neurochemical nature of the various number of reactive mitochondria localised within den- synaptic inputs or to the amount of transmitter/peptide drites and axon terminals. Interestingly, they also released. It is also of interest to note that the activities of two noted that highly reactive axonal terminals usually formed synaptic contact with unreactive cell soma. In additional oxidative enzymes, SDH and NADH-diapholight of these data and the compartmentation of CO rase, involved in mitochondria1 respiration are also activity reported here, it would appear that the ob- unevenly distributed in the rat striatum. However, as served compartmentation of endogenous CO activity the contrast between areas of intense and weak staining seen in the rat striatum is a reflection of relative was poor, it was not possible to compare the heterogedifferences in synaptic activity as opposed to the rela- neous distribution of enzyme activity with adjacent tive number of synaptic contacts in each of the two sections stained to visualise CaBP-LI. Whether NADHcompartments. Indeed, numerous studies have re- diaphorase activity and SDH activity are also distribported a correlation between high levels of spontaneous/ uted in accordance with the patch-matrix subdivision of synaptic activity and high levels of endogenous CO the rat striatum remains unclear. However, it has been activity(Braun et al., 1985; Dimlich et al., 1990;Hevner reported that sites of SDH and CO activity have a nearly and Wong-Riley, 1990; Wong-Riley, 1979, 19891, sug- identical distribution in the rabbit brain (Shimizu et al., 1957).Indeed, from our observations it appears that the distributions of CO-LI, SDH and NADH-diaphorase Fig. 4. Localisation of reaction product denoting sites of NADH- activities within the rat SN are similar as a cluster of a diaphorase activity in (A) a coronal section of rat C P low-power few intensely labelled cell bodies can be found localised photograph showing the uneven distribution of reaction product within the neuropil. The contrast between active and less active areas of in the dorso-lateral and dorsal tip of the SNR. However, neuropil is poor. B: Rat SN: the reaction product is Iocalised within the the number of SN cell bodies labelled with the anti-CO neuropil a s well as cell bodies within the pars reticulata (SN,). Note the clustering of intensely labelled cell bodies within the dorsal lateral and antibody exceeded the number of SDH- and NADHdorsal tip of the SN,. C : High-power magnification of the boxed area in diaphorase-labelled cells. In addition, these CO-LI niB showing intensely labelled cell bodies (arrows; arrowheads in B). gral cell bodies were more evenly distributed throughNote the granular appearance of reaction product denoting sites of enzyme activity. Within cell bodies the reaction product is concen- out the medio-lateral extent of the SN and were not restricted to the dorsal region of the SNR. Indeed, our trated within the cytoplasm. ~~
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S.J. AUGOOD AND P.C. EMSON
observations are consistent with electrophysiological data which reported that SNR neurones were more spontaneously active than SNC cells (Deniau et al., 1978). In conclusion,we have demonstrated that the striatal matrix compartment is enriched in endogenous CO activity relative to the patch compartment. Indeed, studies of the human striatum also indicate that the matrix compartment is enriched in endogenous CO activity relative to the patch compartment (Ferrante et al., 1988).These data suggest that the striatal matrix compartment is metabolically more active than the patch compartment. To our knowledge this is the first functionaUmetabolic difference to be reported between the patch and matrix striatal compartments in the rat. ACKNOWLEDGMENTS
S.J.A.was supported by Bayer-Tropon. We are grateful to Mr. I. King for histological advice and assistance and to Mr. T. Buss €or photographic expertise. REFERENCES Augood, S.J.,and Emson, P.C. (19891 Cytochrome c oxidase: a striatal matrix marker. SOC.Neurosci. Abstr., 15:909. Bancroft, J.D. (1975) Histochemical Techniques, 2nd Ed. Butterworths, London. Braun, K., Scheich, H., Schachner, M., and Heizmann, C.W. (1985) Distribution of parvalbumin, cytochrome oxidase activity and 14C2-deoxyglucose uptake in the brain of the zebra finch 11. Visual system. Cell Tissue Res., 240:117-127. Burstone, M.S. (1959) New histochemical techniques for the demonstration of tissue oxidase (cytochrome oxidasel. J. Histochem. Cytochem., 7:112-122. Carr, P.A., Yamamoto, T., Karmy, G., Baimbridge, K.G., and Nagy, J.I. (1989) Analysis of parvalbumin and calbindin,,,,-immunoreactive neurons in dorsal root ganglia of rat in relation to their cytochrome oxidase and carbonic anhydrase content. Neuroscience, 33:363-371. Deniau, J.M., Hammond, C., Riszk, A,, and Feger, J. (1978) Electrophysiological properties of identified output neurons of the rat substantia nigra (pars compacta and pars reticulata): evidence for the existence of branched neurons. Exp. Brain Res., 32:409-422. 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. Dimlich, R.V.W.,Showers, M.J., and Shipley, M.T. (1990) Densitometric analysis of cytochrome oxidase in ischemic rat brain. Brain Res., 516381-191. Donoghue, J.P., and Herkenham, M. (1986) Neostriatal projections from individual cortical fields conform to histochemically distinct striatal compartments in the rat. Brain Res., 365:397-403. Ferrante, R.J., Kowall, N.W., and Richardson, E.P. (1988) Patchmatrix distribution of cholecystokinin and cytochrome oxidase activity in normal and Huntington’s disease striatum. SOC.Neurosci. Abstr. 14:1046. Gerfen, b.R. (1984) The neostriatal mosaic: compartmentalization of
corticostriatal input and striatonigral output systems. Nature, 311 :461-464. Gerfen, C.R. (1985) The neostriatal mosaic I. Compartmental organisation of projections of the striatonigral system in the rat. J . Comp. Neurol., 236:454476. Gerfen, C.R., Baimbridge, K.G., and Miller, J.J.(19851The neostriatal mosaic: compartmental distribution of calcium-binding protein and parvalbumin in the basal ganglia of the rat and monkey. Proc. Natl. Acad. Sci. USA, 828780-8784. Gerfen, C.R. (1989) The neostriatal mosaic: striatal patch-matrix organization is related to cortical lamination. Science, 246:385-388. Graybiel, A.M. (1990) Neurotransmitters and neuromodulators in the basal ganglia. Trends Neurosci., 13:244-254. Graybiel, A.M., Hirsch, E.C., and Agid, Y.A. (1987) Differences in tyrosine hydroxylase-like immunoreactivity characterize the mesostriatal innervation of striosomes and extrastriosomal matrix at maturity. Proc. Natl. Acad. Sci. USA, 84:303-307. Graybiel, A.M., and Ragsdale, C.W. (1978) Histochemically distinct comDartments in the striatum of human, monkev and cat demonstrated by acetylthiocholinesterase staining. Pro;. Natl. Acad. Sci. USA. 75:5723-5726. . -~ Herkenham, M., and Pert, C.B. (1981) Mosaic distribution of opiate receptors, parafascicular projections and acetylcholinesterase in the rat striatum. Nature, 291:415-418. Hevner, R.F., and Wong-Riley, M.T. (1989)Brain cytochrome oxidase: purification, antibody production, and immunohistochemicaY histochemical correlations in the CNS. J. Neurosci., 9:3884-3989. Hevner, R.F., and Wong-Riley,M.T.T. (1990) Regulation of cytochrome oxidase protein levels by functional activity in the macaque monkey visual system. J. Neurosci., 10:1331-1340. Kawaguchi, Y., Wilson, C.J., and Emson, P.C. (1989) Intracellular recording of identified neostriatal patch and matrix spiny cells in a splice preparation preserving cortical inputs. J . Neurophysiol., 6~(5):1052-1068. Kemp, J.M., and Powell, T.P.S. (1971) The synaptic organisation of the Lond. [Biol.),262403-412. caudate nucleus. Philos. Trans. R. SOC. Ma, T.P. (1989) Identification of the substantia nigra pars lateralis in the macaque using cytochrome oxidase and fiber stains. Brain Res., 480:305-311. Marshall, J.F., Critchfield, J.W., and Kozlowski, M.R. (1981) Altered succinate dehydrogenase activity of basal ganglia following damage to mesotelencephalic dopaminergic projection. Brain Res., 212:367377. Olson, L., Seiger, A,, and Fuxe, K. (1972) Heterogeneity of striatal and limbic dopamine innervation: Highly fluorescent islands in developing and adult rats. Brain Res., 44:283-288. Shimizu, N., Morikawa, N., and Ishi,Y. (1957)Histochemical studies of succinic dehydrogenase and cytochrome oxidase of the rabbit brain, with special reference to the results in the paraventricular structures. J. Comp. Neurol., 108:l-21. Vincent, S.R., Johansson, O., Hokfelt, T.,Skirboll, L., Elde, R.P., Terenius, L., Kimmel, J.,andGoldstein,M. (1983)NADPH-diaphorase:A selective histochemical marker for striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APPI-like immunoreactivities. J . Comp. Neurol., 217:252-263. Vincent, S.R., and Johansson, 0. (1983) Striatal neurons containing both somatostatin- and avian pancreatic polypeptide (UP)-like immunoreactivities and NADPH-diaphorase activity: a light and electron microscopic study. J . Comp. Neurol., 217:264-270. Wasserman, R.H., and Taylor, A.N. (1966) Vitamin D, induced calcium-binding protein in chick intestinal mucosa. Science, 152:791. Wong-Riley, M.T.T. (1979) Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res., 171:ll-28. Wong-Riley, M.T.T. (1989) Cytochrome oxidase: an endogenous metabolic marker for neuronal activity. Trends Neurosci., 12:94-101. ~
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Compartmental Distribution of Cytochrome Oxidase in the Striatum of the Rat SARAH J. AUGOOD AND PIERS C. EMSON MRC Group, Department of Neuroendocrinology, AFRC Institute of Animal Physiology & Genetics Research, Babraham, Cambridge, CB2 4AT, England
KEY WORDS
Patchhtriosome, Matrix compartment, Calbindin, Tyrosine hydroxylase, Basal ganglia
ABSTRACT Endogenous cytochrome oxidase activity was investigated in the adult rat striatum at the light microscope level to see if it was distributed in accordance with the established striatal patcwmatrix compartmentalisation. Striatal sections stained to visualise cytochrome oxidase activity were compared with serial sections stained to visualise tyrosine hydroxylase and calbindin,,,k-like immunoreactivity, established markers of the matrix compartment. The distribution of endogenous cytochrome oxidase activity was found to coincide with the immunocytochemical staining pattern seen for tyrosine hydroxylase and calbindinD2,kwhereby areas of intense tyrosine hydroxylase and calbindinDZek-likeimmunoreactivity (termed the matrix) corresponded to areas of intense cytochrome oxidase activity. Conversely, areas of less intense tyrosine hydroxylase and calbindinDz,,-like immunoreactivity (termed patches) corresponded t o areas of low cytochrome oxidase activity. In addition, the distribution of two other oxidative enzymes involved in the regulation of mitochondria1respiration, succinic dehydrogenase and NADH-diaphorase, was examined in the striatum and substantia nigra by using histochemical techniques. Both NADH-diaphorase and succinic dehydrogenase histochemistry showed an uneven pattern of neuropil staining in the striatum. In the substantia nigra a few intensely stained cell bodies were seen in the dorsal-lateral tip of the pars reticulata with both histochemical techniques. By using an anti-cytochrome oxidase antibody an abundance of immunoreactive cell bodies and processes were seen in the substantia nigra, particularly in the dorso-medial rim and dorsal tip of the pars reticulata. The substantia nigra pars lateralis contained many intensely stained cytochrome oxidase-like immunoreactive cell bodies and processes. Our results demonstrate that the neurochemically distinct compartments of the adult rat striatum contain differential amounts of endogenous cytochrome oxidase activity suggesting that the matrix compartment is more metabolically active than the patch compartment. This difference in metabolic activity may reflect a difference in synaptic activity between the two striatal compartments. INTRODUCTION The heterogeneous organisation of the mammalian striatum has been studied widely in terms of its cellular morphology, neurochemistry, and basic circuitry. The initial observation that the rat striatum was not a homogeneous structure (Olson et al., 1972)has led to an overwhelming number of studies investigating the extent of this heterogeneity. To date two main striatal compartments have been reported in the rat which differ in their neurochemical composition (Gerfen et al., 1985; Graybiel and Ragsdale, 1978; Graybiel et al., 1987; Herkenham and Pert, 19811, origin of inputs (Donoghue and Herkenham, 1986; Gerfen, 1984,1985, 19891, and their afferent projections (Gerfen, 1984).The two striatal compartments have been termed “patch” 0 1992 WILEY-LISS,INC.
and “matrix,”with the latter compartment constituting approximately 80% of the striatum. Since these two striatal compartments show a differential concentration of neurotransmitters, neuropeptides, receptor binding sites, endogenous enzymes, and neuropeptide mRNA’s (for review see Graybiel, 1990) they may be visualised by using neurochemical parameters. Although the neurochemical composition of these striatal compartments has been well documented, to date their precise functional significance remains unclear. One marker for the striatal matrix compartment is the calcium binding protein calbindinD2,k(CaBP: Gerfen et al., 19851,first isolated from chick small intestine Received January 11,1991; accepted in revised form May 13,1991
DISTRIBUTION OF CO IN RAT STRIATUM
(Wasserman and Taylor, 1966). A number of reports have suggested that expression of calcium binding proteins such as CaBP or parvalbumin (PV) is related to neuronal activity (Braun et al., 1985; Kawaguchi et al., 19891, with fast-firing neurones expressing more calcium binding protein than slow-firing neurones. These observations suggest that in vivo striatal matrix neurones may be faster-firing and metabolically more active than slower-firing patch neurones. A number of studies have demonstrated a positive correlation between high levels of spontaneous and synaptic activity and high levels of endogenous CO activity (Braun et al., 1985; Dimlich et al., 1990; Wong-Riley 1979,19891,and it has been suggested that relative amounts of endogenous CO activity may serve as a marker of neuronal functionaYmetabolic activity (Hevner and Wong-Riley, 1990). Therefore, further to the work of DiFiglia and colleagues (1987) we undertook a study of the distribution of CO activity in the rat striatum in relation to the patch/matrix organisation as demonstrated by tyrosine hydroxylase (TH) and CaBP-like immunoreactivity (-LI). The compartmental distribution of two other enzymes involed in mitochondria1 respiration and oxidative phosphorylation, NADH-diaphorase and succinic dehydrogenase (SDH), was also examined. Preliminary results of these experiments have been published previously (Augood and Emson, 1989).
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through the striatum and collected into ice-cold PBS. Every third serial tissue section was then allowed to warm to room temperature (RT) and immediately incubated in freshly prepared and filtered 0.1 M phosphate buffer (PB) containing 3-3’ diaminobenzidine hydrochloride (DAB: 1.5 mg/ml) for approximately 45 min at RT and then extensively rinsed in PBS, mounted onto gelatinised slides, and coverslipped by using a synthetic mountant (Ralmount). The remaining two serial sections were processed to visualise TH and CaBP-LI as described below. 2. Metal chelation histochemical technique (Burnstone, 1959). Fresh frozen coronal cryostat sections of striatum were cut (15 pm), allowed to dry at RT for 2 hr, and then incubated in 0.2 M Tris-HC1 buffer (pH 7.4) containing 1-hydroxy-2-napthoic acid (0.002%), 4aminodiphenylamine (0.002%), and EtOH (0.01%) at RT. Finally, the napthol compound formed was chelated to Co2+[1%Co(HOAc),] for 1 hr. Sections were then coverslipped by using glycerin jelly. Endogenous CO activity was visualised as an insoluble blue-black granular deposit. Control sections were incubated in reaction medium containing the specific CO inhibitor sodium cyanide (50 mM). 3. Modified peroxidase technique (Carr et al., 1989). Fixed striatal sections (25 p.m) were incubated in the dark a t 37°C for 3 hr in PB containing 0.025% DAB/4% sucrose/0.03% cytochrome c (Sigma, type 111) as subMATERIALS AND METHODS strate. The reaction was terminated when the greatest Endogenous CO activity was investigated in the stri- contrast between highly active and less active areas of atum of adult female rats (Wistar) by using four differ- neuropil staining was seen. Sections were then mounted ent histochemical techniques in an attempt to compare onto gelatinised slides and coverslipped. the relative sensitivities of the methods: 1) a direct 4.Technique using a polyclonal anti-bovine brain CO peroxidase method, 2) a metal chelation histochemical antibody. This antibody has been characterised previmethod, 3) a modified peroxidase technique which uti- ously (Hevner and Wong-Riley, 1989) and has been lises reduced cytochrome c as the substrate, and 4) a shown to react predominantly with subunit IV of CO. method using a polyclonal anti-bovine brain CO anti- Immunocytochemical staining of fixed rat sections was body (gift of Prof. M.T.T. Wong-Riley).TH-LI was visu- as described below. alised by using a polyclonal anti-rat adrenal TH antiImmunocytochemistry body (gift of Prof. Nagatsu) and CaBP-LI was visualised Free-floating fixed sections (25 pm) were incubated by using a polyclonal anti-chick intestine CaBP antibody (gift of Dr. D.E.M. Lawson). Both NADH-diapho- in primary antibody (anti-CO 1/1,000, anti-TH 1/500, rase activity and SDH activity were visualised by using and anti-CaBP 1/400) diluted in PBS/O.2% Triton Xhistochemical methods previously described (Bancroft, 100/1% normal goat serum overnight at 4°C. Sections were then rinsed (3 X 15 min) in PBS/0.2%Triton X-100 1975). and incubated in HRP-conjugated goat anti Rb IgG Cytochrome oxidase histochemistry (1/500: Vector Labs) for several hours a t RT. Finally Details of the four techniques used to demonstrate CO sections were rinsed extensively as before and then activity in the adult rat striatum are given below: incubated in DAB (0.5 mg/ml) activated with HzOz (0.025%).Reacted sections were washed in an excess of 1. Direct peroxidase method (DiFiglia et al., 1987). PBS, mounted onto gelatinised slides, and coverslipped Rats were anaesthetised with pentobarbitone (Sagatal) as above. For control experiments, the specificity of the and perfused transcardially with heparinised saline secondary antibody was tested by omitting the primary (0.1%)followed by ice-cold fixative (2% neutral-buffered antibody from the first incubation. paraformaldehyde/l.25% glutaraldehyde). Exactly 20 NADH-diaphorase histochemistry min followingfixation brains were removed onto ice and NADH-diaphorase activity was demonstrated in immediately sectioned on a freezing microtome. Freefresh-frozen sections by using an established hisfloating coronal serial sections (40 p.m) were cut
DISTRIBUTION OF CO IN RAT STRIATUM
tochemical procedure (Bancroft, 1975).Briefly, cryostat sections were cut (15 pm), air dried, incubated in reaction medium containing the tetrazolium salt 3(4:5-dimethyl thiazolyl-2)5-diphenyltetrazolium bromide (MTT: 0.6 mM), CoCl,, and NADH (nicotinamide adenine dinucleotide reduced: 2.8 mM) as substrate at 37°C for 30-40 min, fixed in formal saline, and finally coverslipped using glycerin jelly. Hydrogen liberated during the catalysed oxidation of NADH to NAD reduces the MTT to form a formazan compound which then chelates with Co2+ to form a black insoluble product. Consequently, sites of enzyme activity are labelled by fine discrete granular deposits. Control sections were incubated in either reaction medium containing the CO inhibitor sodium cyanide (50 mM) or reaction medium omitting the substrate.
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cleus were found to be intensely stained (Fig. 1A-D). In agreement with DiFiglia and co-workers (19871,we also found that the most intensely stained area of the striatum was the dorso-medial rim just ventral to the corpus callosum (Fig. 1E). Adjacent sections processed to visualise TH-LI and CaBP-LI also showed a heterogeneous pattern of staining essentially the same as seen for CO (Fig. 2A-D). However, whilst CO activity and TH-LI were seen concentrated in the neuropil, CaBP-LI was also seen concentrated in the cytoplasm of mediumsized cells. It was noted that, whilst TH-LI was seen heterogeneously distributed throughout the coronal extent of the rat striatum, the dorso-medial part of the striatum was consistently found to be relatively devoid of CaBP-LI (cf. Fig. 2A). It is interesting to note that the boundary between the patch and matrix striatal compartments appears more discrete when defined by SDH histochemistry CaBP-LI than it does when visualised with other striThe activity of this oxidative dehydrogenase was atal markers-for example, AChE staining (data not demonstrated in fresh-frozen cryostat sections (15 pm) shown). Comparisons of the distribution of areas of by using an established histochemical procedure (Ban- intense TH-LI and CaBP-LI (matrix) were found to croft, 1975).This method is essentially the same as used correspond to areas of intense CO reaction product. by Marshall and co-workers (1981) except a higher Conversely, areas of less intense TH-LI and CaBP-LI concentration of substrate was used in accordance with (patches) were found to correspond to areas with less the study by Shimizu and co-workers (1957). The reac- intense CO reaction product. Endogenous CO activity tion conditions are similar to those described for NADH- was also visualised with other techniques as sumdiaphorase histochemistry except the tetrazolium salt marised below. employed was nitro-blue tetrazolium (NBT: 1.2 mM) Metal chelation histochemical technique and the specific substrate was sodium succinate (0.5M). Sites of CO activity were labelled with blue-black Control sections were incubated in reaction medium not granular deposits which were seen concentrated in the containing the substrate. striatal neuropil. Labelled soma were not obvious but RESULTS may have been obscured by the neuropil staining. To compare the distribution of endogenous CO activ- Whilst deposits were found in all parts of the striatum, ity with the pattern of immunocytochemical staining for areas containing relatively less staining were seen. The TH and CaBP, the peroxidase method was used as this uneven distribution of granular deposits was found to be was the method used by DiFiglia and co-workers (19871, unrelated to the distribution of myelinated fibre bunwho first reported an uneven distribution of CO activity dles. Sections incubated with sodium cyanide did not in the striatum. The additional three methods of visu- show any staining. alising CO activity were carried out to compare the The modified peroxidase technique of relative sensitivities of the different methods. Carr et al. (1989) Distribution of CO activity, TH-LI, and CaBP-LI Sites of CO activity were visualised by a concentraSites of endogenous CO activity were labelled with a tion of dark brown reaction product within striatal dark brown DAB reaction product concentrated within sections. Whilst an uneven distribution of reaction prodstriatal sections. Reaction product was seen localised in uct was detected, the contrast between heavily labelled the neuropil. In addition to the heterogeneous distribu- and weakly labelled areas obtained with this method tion of reaction product in the striatum and nucleus was poor (data not shown). accumbens, the cingulate cortex and lateral septa1 nuImmunocytochemistry using a n anti-CO antibody CO-LI was visualised by the localisation of a dark brown reaction product concentrated in the striatal Fig. 1. Comparison of the distribution of CaEiP-LI (A), endogenous CO activity (B), and TH-LI (C) in serial sections of rat nucleus neuropil. Weakly stained cell soma may have been accumbens (NAc).Note the intense patchy distribution of DAB reaction obscured by the neuropil staining. While an uneven product depicting sites of CO activity (B) and TH-LI in (C) in the NAc. Fingers of less intense staining (patches) may be seen emanating from pattern of CO-LI was seen, the contrast between the ventricular edge; this is particularly evident in (A). CO activity in strongly stained and weakly stained areas of the striathe (D) cingulate cortex (Acg) and (E)dorsal rim of rat striatum; note tum was poor (Fig. 3B). On the same sagittal sections an the intensity of DAB reaction product localised just ventral to the abundance of CO-LI pyramidal cells were visible in corpus callosum (cc). AC, anterior commissure. Magnification x 50.
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S.J. AUGOOD AND P.C. EMSON
Fig, 2. Comparison of the distribution of reaction product depicting sites of endogenous CO activity (A,D),TH-LI (B), and CaBP-LI (C) in coronal sections of rat striatum. (A and B) and (C and D) are pairs of serial sections. Sites of activity of endogenous CO were visualized by using the direct peroxidase method. Note the heterogeneous distribu-
tion of reaction product and the coincidence of patches (stars) in A and B and C and D. As noted in the text, the dorsal striatum is always found to be devoid of CaBP-LI using this antibody (C). Abbreviations: claustrum (CL), lateral ventricle (LV), corpus callosum (cc), and anterior commissure (AC). Magnification x 30.
DISTRIBUTION OF CO IN RAT STRIATUM
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Fig. 3. Visualisation of (A) CaBP-LI and (B) CO-LI in sagittal sections of rat striatum (CPu). Note the patchy distribution of reaction product in both A and B. In A strongly stained CaBP-LI cell bodies can be seen localised within areas of strongly stained neuropil (matrix). Conversely, in B only neuropil staining can be seen with no CO-LI cell bodies beingvisualised readily. The boundary between the CPu and GP is marked by a dotted line. In A and B asterisks denote the patch compartment. (C) Coronal section showing the localisation of CO-LI
within cell soma, processes, and the neuropil of the SN. Note the intensely stained cell bodies of the SNR and SNL (arrowhead). (D) Sagittal section of rat frontal cortex (Frl) showing the localisation of CO-LI within pyramidal cells (arrowheads) concentrated in layer V. A few CO-LI cells are also seen in layer 111. Abbreviations: internal capsule (ic), globus pallidus (GP), substantia nigra pars lateralis (SNL), and pars reticulata (SNR).
layer V of the frontal cortex, with a few smaller cells being seen in layer I11 (Fig. 3D). In the SN an abundance of intensely stained CO-LI cell bodies and processes were seen concentrated in the dorso-medial rim and dorsal tip of the pars reticulata (SNR: Fig. 3C). A few CO-LI cell bodies and processes were also visible in the pars compacta (SNC); the pars lateralis (SNL) contained numerous intensely stained CO-LI cell bodies and processes, an observation previously reported in the primate (Ma, 1989). Sections processed without the antibody showed no staining. The differences in contrast between CO active areas (matrix) and less active areas (patches) seen with the four different techniques used here presumably reflects differences in sensitivity of the methods. Interestingly, the best contrast between the CO active matrix and less active patch compartments was seen by using a direct histochemical technique, suggesting that indirect methods, which include several amplification steps for the development of the reaction product, are not so closely related to tissue CO activity.
NADH-diaphorase histochemistry Sites of NADH-diaphorase activity were labelled with granular deposits found concentrated in the striatal neuropil. No staining of cell bodies was seen. While deposits were detected in all parts of the striatum, areas of weak staining were seen which were surrounded by larger areas of intense staining (Fig. 4A). These weakly stained areas did not correspond to areas of fibre bundles. In the SN, granular deposits were detected throughout the nucleus with a few heavily labelled cell bodies being seen in the dorso-lateral and dorsal tip of the SNR (Fig. 4B,C). The pattern and intensity of labelling were unaffected by sodium cyanide, the selective CO inhibitor. However, when the specific substrate (NADH) was omitted from the reaction medium no staining was visible. It is important to note that this histochemical technique does not visualise NADPHdiaphorase activity which has been demonstrated within striatal interneurones containing somatostatinLI (Vincent and Johansson, 1983; Vincent et al., 1983).
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S.J. AUGOOD AND P.C. EMSON
Figure 4.
DISTRIBUTION OF CO IN RAT STRIATUM
SDH histochemistry Sites of SDH activity were labelled with purple granular deposits. In striatal sections, deposits were seen in all parts of the nucleus and these were localised to the neuropil. No obvious labelling of cell bodies were seen. Striatal sections had a patchy appearance; however, the contrast between strongly and weakly stained areas was poor (data not shown), not as clear as the contrast seen with NADH-diaphorase histochemistry. In the SN, the neuropil was heavily labelled; occasional cell bodies were detected usually localised in the lateral extent of the SNR; the SNC contained very few labelled cell bodies. Control sections processed without the substrate showed no staining.
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gesting that CO may well be a marker for neuronal functional activity. Consistent with these observations, our findings therefore suggest a difference in metabolic activity between the matrix and patch compartments in the rat striatum; that is, the matrix compartment displays a greater amount of endogenous CO activity than the striatal patch compartment. This would be consistent with the suggestion that matrix neurones may be more metabolically active than patch neurones. It may be of some physiological importance that the striatal matrix compartment, enriched in endogenous CO activity, is also relatively enriched in a calcium binding protein, CaBP. It is unlikely that the difference in CO activity in the patch and matrix compartments is due to differDISCUSSION ences in the electrophysiological properties of cells in the two compartments as 95% of striatal neurones are Our findings demonstrate that the uneven distribumedium-sized spiny cells (Kemp and Powell, 1971) and tion of endogenous CO activity in the adult rat striatum Kawaguchi and co-workers (1989) have shown that coincides with the distribution of TH- and CaBP-LI, established markers of the matrix compartment; that is, identified neostriatal patch and matrix spiny cells have areas of intense TH- and CaBP-LI coincided with areas similar axonal and dendritic morphology, passive memof intense CO activity. Conversely, areas of weak TH- brane properties, and repetitive firing characteristics. and CaBP-LI (termed patches) coincided with areas of These findings suggest that in the absence of an exterless intense CO activity. Our results therefore, extend nal stimulus, patch and matrix spiny neurones have the findings of DiFiglia and co-workers (19871, who similar electrophysiological properties in vitro. reported that dendritic mitochondria were responsible Whether this is true in vivo when spiny cells are under for the majority of CO activity in the rat striatum. In the influence of all their synaptic inputs remains to be addition, they showed that the differential distribution established. Therefore, it has still to be determined between CO active and less active areas was not a whether the difference in synaptic activity observed reflection of a difference in the density of dendrites or here between the striatal patch and matrix compartsynapses but was due to the presence of a greater ments is due to the neurochemical nature of the various number of reactive mitochondria localised within den- synaptic inputs or to the amount of transmitter/peptide drites and axon terminals. Interestingly, they also released. It is also of interest to note that the activities of two noted that highly reactive axonal terminals usually formed synaptic contact with unreactive cell soma. In additional oxidative enzymes, SDH and NADH-diapholight of these data and the compartmentation of CO rase, involved in mitochondria1 respiration are also activity reported here, it would appear that the ob- unevenly distributed in the rat striatum. However, as served compartmentation of endogenous CO activity the contrast between areas of intense and weak staining seen in the rat striatum is a reflection of relative was poor, it was not possible to compare the heterogedifferences in synaptic activity as opposed to the rela- neous distribution of enzyme activity with adjacent tive number of synaptic contacts in each of the two sections stained to visualise CaBP-LI. Whether NADHcompartments. Indeed, numerous studies have re- diaphorase activity and SDH activity are also distribported a correlation between high levels of spontaneous/ uted in accordance with the patch-matrix subdivision of synaptic activity and high levels of endogenous CO the rat striatum remains unclear. However, it has been activity(Braun et al., 1985; Dimlich et al., 1990;Hevner reported that sites of SDH and CO activity have a nearly and Wong-Riley, 1990; Wong-Riley, 1979, 19891, sug- identical distribution in the rabbit brain (Shimizu et al., 1957).Indeed, from our observations it appears that the distributions of CO-LI, SDH and NADH-diaphorase Fig. 4. Localisation of reaction product denoting sites of NADH- activities within the rat SN are similar as a cluster of a diaphorase activity in (A) a coronal section of rat C P low-power few intensely labelled cell bodies can be found localised photograph showing the uneven distribution of reaction product within the neuropil. The contrast between active and less active areas of in the dorso-lateral and dorsal tip of the SNR. However, neuropil is poor. B: Rat SN: the reaction product is Iocalised within the the number of SN cell bodies labelled with the anti-CO neuropil a s well as cell bodies within the pars reticulata (SN,). Note the clustering of intensely labelled cell bodies within the dorsal lateral and antibody exceeded the number of SDH- and NADHdorsal tip of the SN,. C : High-power magnification of the boxed area in diaphorase-labelled cells. In addition, these CO-LI niB showing intensely labelled cell bodies (arrows; arrowheads in B). gral cell bodies were more evenly distributed throughNote the granular appearance of reaction product denoting sites of enzyme activity. Within cell bodies the reaction product is concen- out the medio-lateral extent of the SN and were not restricted to the dorsal region of the SNR. Indeed, our trated within the cytoplasm. ~~
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S.J. AUGOOD AND P.C. EMSON
observations are consistent with electrophysiological data which reported that SNR neurones were more spontaneously active than SNC cells (Deniau et al., 1978). In conclusion,we have demonstrated that the striatal matrix compartment is enriched in endogenous CO activity relative to the patch compartment. Indeed, studies of the human striatum also indicate that the matrix compartment is enriched in endogenous CO activity relative to the patch compartment (Ferrante et al., 1988).These data suggest that the striatal matrix compartment is metabolically more active than the patch compartment. To our knowledge this is the first functionaUmetabolic difference to be reported between the patch and matrix striatal compartments in the rat. ACKNOWLEDGMENTS
S.J.A.was supported by Bayer-Tropon. We are grateful to Mr. I. King for histological advice and assistance and to Mr. T. Buss €or photographic expertise. REFERENCES Augood, S.J.,and Emson, P.C. (19891 Cytochrome c oxidase: a striatal matrix marker. SOC.Neurosci. Abstr., 15:909. Bancroft, J.D. (1975) Histochemical Techniques, 2nd Ed. Butterworths, London. Braun, K., Scheich, H., Schachner, M., and Heizmann, C.W. (1985) Distribution of parvalbumin, cytochrome oxidase activity and 14C2-deoxyglucose uptake in the brain of the zebra finch 11. Visual system. Cell Tissue Res., 240:117-127. Burstone, M.S. (1959) New histochemical techniques for the demonstration of tissue oxidase (cytochrome oxidasel. J. Histochem. Cytochem., 7:112-122. Carr, P.A., Yamamoto, T., Karmy, G., Baimbridge, K.G., and Nagy, J.I. (1989) Analysis of parvalbumin and calbindin,,,,-immunoreactive neurons in dorsal root ganglia of rat in relation to their cytochrome oxidase and carbonic anhydrase content. Neuroscience, 33:363-371. Deniau, J.M., Hammond, C., Riszk, A,, and Feger, J. (1978) Electrophysiological properties of identified output neurons of the rat substantia nigra (pars compacta and pars reticulata): evidence for the existence of branched neurons. Exp. Brain Res., 32:409-422. 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. Dimlich, R.V.W.,Showers, M.J., and Shipley, M.T. (1990) Densitometric analysis of cytochrome oxidase in ischemic rat brain. Brain Res., 516381-191. Donoghue, J.P., and Herkenham, M. (1986) Neostriatal projections from individual cortical fields conform to histochemically distinct striatal compartments in the rat. Brain Res., 365:397-403. Ferrante, R.J., Kowall, N.W., and Richardson, E.P. (1988) Patchmatrix distribution of cholecystokinin and cytochrome oxidase activity in normal and Huntington’s disease striatum. SOC.Neurosci. Abstr. 14:1046. Gerfen, b.R. (1984) The neostriatal mosaic: compartmentalization of
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