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Brain Research, 164 (1979) 328-333 ~ Elsevier/North-Holland Biomedical Press
Complementarity of dopaminergic and noradrenergic innervation in anterior cingulate cortex of the rat
M A R Y S. LEWIS, M A R K E. MOLLIVER*, J O H N H. MORRISON and HART G. W. LIDOV
The Department of Cell Biology and Anatomy, Department of Neurology and Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine, Baltimore, Md. 21205 (U.S.A.) (Accepted November 30th, 1978)
The use of a catecholamine specific histofluorescence technique (the Falck-Hillarp method 7) has revealed a projection from noradrenergic cells in the locus coeruleus to all areas of the rat cerebral cortex 1,s,23. Evidence that dopamine (DA) is a cortical neurotransmitter (independent of norepinephrine) is based on studies showing persistent levels of DA and DA synthesis in rat cortex following bilateral lesions of the ascending noradrenergic bundle 21,22. DA axons have been demonstrated in cortex by the glyoxylic acid (GA) histofluorescence method and are restricted to 4 areas: medial frontal, entorhinal, perirhinal and anterior cingulate cortex3-5,1a,~s. Since DA and NA are not readily distinguished in histofluorescence preparations, both having the same emission wavelength (Emax = 470nm), studies of DA fiber distribution have been based on residual catecholamine fluorescence following lesions of the noradrenergic projections or by selective inhibition of amine uptake or synthesisS,4,13. There has been general agreement that DA axons innervate deep layers (V and VI) of frontal, entorhinal and perirhinal areas4,~,13,~7,ts. There have been conflicting reports concerning the laminar distribution of DA fibers in anterior cingulate cortex. Swedish workers ta,t7,1s describe a dense DA plexus in layers I-llI of this region; but this plexus was not observed by Berger a-5, nor has it been described by others. In an attempt to resolve this disparity and to examine the degree of overlap of cortical DA and NA axon terminals and their spatial relationship we have studied anterior cingulate cortex (ACC) using an approach which entails a comparison of catecholamine (CA) histofluorescence with an immunohistochemical technique which allows the selective visualization of noradrenergic neurons. The latter procedure takes advantage of the fact that dopamine-~-hydroxylase (DBH), the enzyme that catalyzes the conversion of DA to NA, is found in NA but not DA neurons 9,~°. DBH is considered a specific marker for NA neurons and its localization is the only currently available method to distinguish definitively between NA and DA axons~O,2°. * To whom correspondence should be addressed at: Department of Cell Biology and Anatomy.
330 Glyoxylic acid-induced catecholamine histofluorescence was carried out as previously described TM19. Male albino rats (150-250 grams), pretreated with nialamide (100 mg/kg) were perfused with 150 ml ice-cold 0.1 M phosphate buffer (pH 4-5) containing 2 ~ glyoxylic acid, 1.5 ~ paraformaldehyde and MgSOa'7H20 (40 g/150 ml). Cryostat sections were immersed for 30 sec in a cold, buffered 2 % glyoxylic acid solution. Slides, air-dried at 37 °C, were heated in a Coplinjar at 100 °C for 5 min and then covered. In several rats, the NA projection to cortex was interrupted bilaterally by electrolytic lesions of the dorsal CA bundle in the midbrain (coordinates: A 0.6, L 1.3, V 1.4 below aqueduct) and the animals were sacrificed after survivals of 14-30 days. Animals were prepared for immunofluorescence as described by Grzanna et al. 1°. A highly specific antibody, directed against rat DBH, was raised in guinea pigs 9 and criteria for its specificity were previously describedg, 10. Cryostat sections (16 #m) were incubated at 4 °C overnight with a 1:1000 dilution of the first antibody (guinea pig anti-rat DBH) containing 0.05% Triton X-100 (v/v). Immunoreactivity was demonstrated by incubation with FTIC-conjugated anti-globulin from Antibodies Inc., Davis, Calif. (rabbit anti-guinea pig IgG diluted 1:50) at room temperature for 15 min. The anterior medial cortex was examined in a series of coronal sections starting at the genu of the corpus callosum and proceeding caudally, corresponding to the anterior cingulate cortex as described by Krettek and Price 14 (cf. their Fig. 2B; ACd and ACv). Sections were viewed with a Leitz incident (Ploem) fluorescence microscope equipped with standard filters for catecholamine and FITC fluorescence. Fluorescent images were recorded on Kodak Tri-X film with exposures of 60 sec and developed at an ASA of 400. In all cases, sections adjacent to those used for fluorescence were counterstained with cresyl violet for cytoarchitectural analysis. Glyoxylic acid-induced catecholamine-specific fluorescence (Fig. 1A) reveals a dense network of blue-green fluorescent axons throughout all layers of cortex. All of layer I is densely innervated, with tangential fibers prevailing in its inner half, and its outer portion heavily populated with fine, tortuous, fluorescent processes. The highest density of axons is in layer II1 which contains an abundant plexus of tortuous fibers that are of fine caliber with irregularly spaced varicosities. Axons of numerous orientations are scattered through layers V and VI. lmmunofluorescent staining reveals (Fig. 2A, B) numerous DBH-immunoreactive axons parallel to the pial surface primarily in the deep half of layer I, while few fibers are present in the superficial half. Layers II and III contain sparse fluorescent axons, most of those present being straight, relatively unbranched and radial in orientation (Fig. 2B). In layers V and VI are scattered, oblique, DBH-positive axon segments and, especially in layer VI, numerous fluorescent dots that represent transsected axons. The paucity of DBH-positive axons in the outer part of layer I and in layers II and III is in agreement with the results of Morrison et al. z0 in sections through ACd rostral to the genu of the corpus callosum (cf. their Fig. 8). Following lesions of the NA ascending fibers in the midbrain, confirmed by profound loss of histofluorescent axons in lateral neocortex, the rich plexus of DAcontaining axons in ACC remains undiminished (Fig. 2C, D). The highest density of
331
Fig. 2. A and B: immunofluorescence photomontagcs showing DBH-immunoreactiv¢ axons in ACC (ACe). C: catecholamine histofluorescencc in ACe following bilateral lesion of the dorsal CA bundle in midbrain. (Scattered dots in layer V are yellow-brown autofluorescent granules in cortical cell bodies.) D: histofluorescenc¢ photomicrograph (ACd) showing distribution of DA fibers in layers I-III 4 weeks after a bilateral electrolytic lesion of the dorsal CA bundle. Bar, 200 pro.
332 these fibers lies precisely in layer IlI with a second band in the outer half of layer k This field of axons is restricted to the dorsal and ventral divisions of ACC from immediately rostral to the genu of the corpus callosum to the caudal border of this architectonic field 14 at a coronal plane through the columns of the formx a~d the anterior commissure (Fig. 1B, C). The present demonstration - - in zones devoid of DBH immunoreactivity - - of CA-containing fibers which survive lesions of the NA projection confirms the presence of a dopamine innervation to anterior cingulate cortex and supports the fiber distribution described by Lindvatl et al.17, is. The present study reveals a highly specific laminar distribution of DA terminals which is clearly not superimposed on the NA projection in an overlapping fashion. Rather, our principal finding is that the two CA projections are complementary in their respective depths of termination and may therefore contact different postsynaptic elements. In ACC, those layers which receive DA afferents contain the lowest density of NA fibers. This situation differs from that in lateral neocortex where the density of NA fibers is considerably higher and the fibers are distributed more evenly over all cortical layers. The other cortical DA target areas are being examined to determine if the complementarity observed in anterior cingulate cortex is a general property of catecholamine innervation, as suggested by the segregation of NA and DA projections exhibited in several subcortical regions (e.g. caudate, olfactory tubercle and septal nuclei). The region of cingulate cortex which receives the DA innervation corresponds to the area found by Beckstead 2 to receive subcortical afferents from two thalamic nuclei (AM and MD) and from two mesencephalic DA cell groups (A9 and A10). This small cortical field, which lies at the juncture of cingulate and prefrontal cortex, also receives a projection from the serotonin neurons of the raphe nuclei 1 as well as from two other thalamic nuclei (ventromedial n and reuniens 12) which terminate in the molecular layer. Although the functional significance of this confluence of projections is unknown at the present time, this region appears to be a nodal point of convergence of subcortical inputs to both limbic and frontal cortex. The terminals of each set of subcortical afferents to anterior medial cortex are distributed in a distinctive individual laminar pattern. The NA projection (primarily to layers V and VI, and to the inner half of I) overlaps with the terminal field of the anteromedial nucleus~, 14 while the DA projection is in precise registration with terminals from the lateral part of the dorsomedial nucleus (to layers III and the outer half of I14). Hence, the complementarity of NA and DA fibers results in the association of each set of CA terminals with the fibers from a particular thalamic nucleus. Most cortical fields receive a single type of catecholamine input, the noradrenergic projection aa, the terminals of which are coextensive with those from the thalamic relay nucleus to each arealS,2o; in anterior cingulate cortex, the DA fibers have partially assumed that position. Although the function of the CA projections from brain stem to cortex is not yet understood, the present observations along with those on the barrel field of S116 suggest that there is a systematic relationship between catecholamine and thalamic projections to cortex. The widely separated NA and DA cell groups which receive different sets of afferents presumably convey different
333 i n f o r m a t i o n to the cortex. Moreover, the fact that the D A axons are so restricted in their distribution a n d yet u n i q u e l y p r o m i n e n t in the anterior cingulate cortex suggests a u n i q u e role or m e c h a n i s m for D A at the neuroeffector level. This work was supported by U S P H S G r a n t s NS-08153 a n d NS-10920 (to M.E.M.). The a u t h o r s wish to t h a n k Drs. R. G r z a n n a a n d J. T. Coyle for the a n t i s e r u m against D B H a n d for their helpful discussions. 1 And6n, N.-E., Dahlstr6m, A., Fuxe, K., Larsson, K., Olson, L. and Ungerstedt, U., Ascending monoamineneurons to the telencephalonand diencephalon,Actaphysiol. scand., 67 (1966) 313-326. 2 Beckstoad, R. M., Convergent thalamic and mesencephalic projections to the anterior medial cortex in the rat, J. comp. NeuroL, 166 (1976) 403-416. 3 Berger, B., Tassin, J. P., Blanc, G., Moyne, M. and Thierry, A. M., Histochemical confirmation for dopaminergic innervationof the rat cerebral cortex after destruction of the noradrenergic ascending pathways, Brain Research, 81 (1974) 332-337. 4 Berger, B., Thierry, A. M., Tassin, J. P. and Moyne, M. A., Dopaminergic innervation of the rat prefrontal cortex - - a fluorescence histochemical study, Brain Research, 106 (1976) 133-145. 5 Berger, B., Histochemical identification and localization of dopaminergic axons in rat and human cerebral cortex. In E. Costa and G. L. Gessa (Eds.), Advances in BiochemicalPsychopharmacology, Vol. 16, Raven Press, N.Y., 1977, pp. 13-20. 6 Domesick, V. B., Thalamic relationships of the medial cortex in the rat, Brain, Behav. EvoL, 6 (1972) 457-483. 7 Falck, B., Hillarp, N. A., Thieme, G. and Torp, A., Fluorescence of catecholamines and related compounds condensed with formaldehydes, J. Histochem. Cytochem., 10 (1962) 348-354. 8 Fuxe, K., Hamberger, B. and H6kfelt, T., Distribution of noradrenalinenerve terminals in cortical areas of the rat, Brain Research, 8 (1968) 125-131. 9 Grzanna, R. and Coyle, J. T., Rat adrenal dopamine-fl-hydroxylase: purification and immunologic characteristics, J. Neurochem., 27 (1976) 1091-1096. 10 Grzanna, R., Morrison, J. H., Coyle, J. T. and Molliver, M. E., The immunohistochemical demonstration of noradrenergic neurons in the rat brain: the use of homologous antiserum to dopamine-betahydroxylase, Neurosci. Lett., 4 (1977) 127-134. 11 Herkenham, M., The nigro-thalamo-corticalconnection mediated by the nucleus ventralis medialis thalami: evidence for a wide cortical distribution in the rat, Anat. Rec., 184 (1976) 426. 12 Herkenham, M., The connections of the nucleus reuniens thalami: evidence for a direct thalamohippocampal pathway in the rat, J. comp. Neurol., 177 (1978) 589-610. 13 H~kfelt, T., Fuxe, K., Johansson, O. and Ljungdahl, A., Pharmaco-histochemical evidence of the existence of dopamine nerve terminals in the limbiccortex, Europ. J. Pharmacol., 25 (1974) 108-112. 14 Krettek, J. E. and Price, J. L., The cortical projections of the mediodorsal nucleus and adjacent thalamic nuclei in the rat, J. comp. Neurol., 171 (1977) 157-192. 15 Lidov, H., Molliver, M. and Zecevic, N., Characterization of the monoaminergic innervation of immature rat neocortex: a histofluorescence analysis, J. comp. Neurol., 181 (1978) 663-679. 16 Lidov, H., Rice, F. and Molliver, M., The organization of the catecholamineinnervationofsomatosensory cortex: the barrel field of the mouse, Brain Research, 153 (1978) 577-584. 17 Lindvall, O., Bj~rklund, A. and Divac, I., Organization of catecholamine neurons projecting to the frontal cortex in the rat, Brain Research, 142 (1978) 1-24. 18 Lindvall, O., Bj6rklund, A., Moore, R. and Stenevi, U., Mesencephalic dopamine neurons projecting to neocortex, Brain Research, 81 (1974) 325-331. 19 Lor6n, T., Bj~rklund, A. and Lindvall, O., Magnesium ions in catecholamine fluorescence histochemistry, Histochemistry, 52 (1977) 223-239. 20 Morrison, J. H., Grzanna, R., Molliver, M. E. and Coyle, J. T., The pattern ofnoradrenergic innervation in rat neocortex: an immunohistochemicalstudy, J. comp. Neurol., 181 (1978) 17-40. 21 Thierry, A. M., Blanc, G., Sobel, A., Stinus, L. and Glowinski, J., Dopaminergic terminals in the rat cortex, Science, 182 (1973) 499-501. 22 Thierry, A. M., Hirsch, J. C., Tassin, J. P., Blanc, G. and Glowinski, J., Presence of dopaminergic terminals and absence of dopaminergic cell bodies in the cerebral cortex of the cat, Brain Research, 79 (1974) 77-88. 23 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand., Suppl. 367 (1971) 1-48.