Proc. Natl Acad. Sci. USA Vol. 79, pp. 2401-2405, April 1982 Neurobiology Noradrenergic and serotonergic fibers innervate complementary layers in monkey primary visual cortex: An immunohistochemical study (cortical organization/visual system/norepinephrine/serotonin/locus ceruleus) J. H. MORRISON*, S. L. FOOTE*, M. E. MOLLIVERt, F. E. BLOOM*, AND H. G. W. LIDOVt *The Salk Institute, La Jolla, California 92037; and tThe Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Contributed by Floyd E. Bloom, December 7, 1981 ABSTRACT Antisera directed against human dopamine 13- MATERIALS AND METHODS hydroxylase or serotonin were used to characterize the noradre- Animals. nergic and serotonergic innervation patterns within the primary Adult Guyanan squirrel monkeys (Saimiri sciureus) visual cortex of the squirrel monkey. The noradrenergic and ser- of either sex were the subjects of this study. otonergic projections exhibit a high degree of laminar comple- Preparation ofAntisera. The antiserum directed against do- mentarity: layers V and VI receive a dense noradrenergic projec- pamine 3-hydroxylase (DBH) was prepared by D. O'Connor tion and a very sparse serotonergic projection, whereas layer IV and his colleagues (Department ofMedicine, University ofCal- receives a very dense serotonergic projection and is largely devoid ifornia at San Diego, School of Medicine) from human pheo- of noradrenergic fibers. In addition, the noradrenergic fibers chromocytoma as described (15, 22). manifest a geometric order that is not so readily apparent in the The antiserum directed against serotonin was that prepared distribution of serotonergic fibers. These patterns of innervation by Lidov et al. (11) as described (8, 11). imply that the two transmitter systems affect different stages of Tissue Preparation and Immunohistochemical Procedure. cortical information processing-the raphe-cortical serotonergic Ten to fifteen minutes prior to perfusion, animals were deeply projection preferentially innervates the spiny stellate cells of lay- anesthetized with ketamine hydrochloride (25 mg/kg, intra- ers IVa and IVc, whereas the ceruleo-cortical noradrenergic pro- muscularly) and pentobarbital sodium (10 mg/kg, intraperito- jection innervates pyramidal cells. neally). Some animals were treatedwith the monoamine oxidase (MAO)-inhibitor pargyline (25 mg/kg, intramuscularly) 24 hr The noradrenergic (NA) and serotonergic (5-HT) projections to prior to perfusion. Animals were perfused transcardially with neocortex arise from brainstem nuclei that project monosynap- either ice-cold phosphate buffer or 1%. paraformaldehyde in tically to all major regions of neocortex (1, 2) in a highly diver- phosphate buffer for 0.5-1 min followed by perfusion with cold gent manner (3), without apparent topographic order (4, 5). Im- 4% paraformaldehyde in phosphate buffer (0.15 M) for 8 min munohistochemical (IHC) techniques for selective visualization at 100 ml/min. After one to six additional hours in fixative, tis- of monoamine fibers (6-8) demonstrate that the monoamine sue blocks were washed in a series of cold, graded sucrose so- innervation ofrat cortex has a characteristic uniform laminar and lutions and stored in 18% sucrose overnight. tangential organization (3, 9-11), with significant regional vari- The IHC has been described in detail in a separate report ation only in the cingulate cortex (11-13). Although the general (15). Briefly, the tissue blocks were frozen and sectioned in a laminar and tangential features ofNA axons are similar to those cryostat at 40 or 50 Am. Freely floating sections in cold phos- in the rat (3, 9, 10), the primate cortex exhibits far greater re- phate-buffered saline were then processed for IHC with either gional specificity in that many diverse neocortical areas possess anti-DBH or anti-serotonin as the primary antiserum. The anti- specific patterns oflaminar distribution and density ofNA fibers DBH and anti-serotonin were commonly used at dilutions (14-16). of 1:4,000 and 1:2,000, respectively. After incubation in the Previously, we observed a distinct laminar pattern of inner- secondary antiserum, peroxidase-conjugated anti-rabbit IgG vation in the primary visual cortex (area 17) ofsquirrel monkey (1:1,000), the sections were developed for peroxidase reactivity (15). The NA innervation ofthis cortical area is ofspecial interest with 3,3'-diaminobenzidine, mounted on gelatin-coated slides, because area 17 is one ofthe most intensively studied and best and dried in a stream of warm air. To enhance visualization of understood neocortical regions with regard to function and to the fine 5-HT fibers, some ofthe mounted sections were briefly neuronal circuitry. Additionally, norepinephrine has been hy- exposed to osmium tetroxide vapor. Several ofthe sections that pothesized to be essential for the "plasticity" seen in the de- were processed for DBH or 5-HT IHC and several unincubated velopment of ocular dominance (17, 18), although the possible sections were counterstained with cresyl violet. anatomical substrate for this plasticity has been studied in only a limited way (19). There is evidence from biochemical studies that 5-HT levels may be higher in visual cortex and norepi- RESULTS nephrine levels relatively low compared with more anterior NA Innervation. As is the case in other regions of primate areas of cortex (20, 21). Given previous examples of comple- neocortex, the DBH-positive fibers in area 17 are highly vari- mentarity in the laminar innervation ofcortical regions by mon- cose and ofrelatively small caliber, although the overall density amines (12, 13), we felt it would be of interest to determine of NA innervation is low in area 17 compared to other primary whether the NA and 5-HT projections to primate visual cortex sensory regions of squirrel monkey cortex. There are very few exhibit distinctive organizational patterns. DBH-positive fibers present in the subcortical white matter. The publication costs ofthis article were defrayed in part by page charge Abbreviations: NA, noradrenergic; 5-HT, serotonergic (5-hydroxytryp- payment. This article must therefore be hereby marked "advertise- taminergic); IHC, immunohistochemical procedure; DBH, dopamine ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. f-hydroxylase; MAO, monoamine oxidase. 2401 Downloaded by guest on September 26, 2021 2402 Neurobiology: Morrison et aL Proc. Natl. Acad. Sci. USA 79 (1982) I ~~~.1T .~~~~~~~~~~~~~~~~W Bi : w '9~~~~~~~~~~~~~~~~~~~~~~~~~ ,l * 4 6't}..# + *: !twX' * ~~~~Zi'~~~~~~ C K1fF til -; '1. f;v* ;: A* 'ept I 4 ~; 4 > I i. I ., 9*' s_ /,t 8 ;'(, *'.r tdt IAr V -wtsI-.... -/¢ _# , * $4fv . t wn* £' 4 4'-b t IVIEr 5 A WM I B FIG. 1. NA innervation of area 17. (A) Cresyl violet-stained section throughprimary visual cortex. Section adjacent toB. (B) DBH-positive fibers in primary visual cortex. Roman numerals refer to cortical layers. WM denotes white matter. Bar = 100 ,um. Downloaded by guest on September 26, 2021 Neurobiology: Morrison et aL Proc. Natl. Acad. Sci. USA 79 (1982) 2403 --A imm w un- -- - , M.,ir, bowaiwow- 0:000",. - -1 . I'.. I &I, - I --- -I.- I I VI .1 r m C-.r I IVa I I !z .I - fib 'I .. .. I FIG. 2. 5-HT innervation of area 17. Serotonin-positive fibers in primary visual cortex. Roman numerals refer to cortical layers. Bar = 100 gm. A photomontage depicting the pattern of NA innervation char- layer IV. The laminae receiving the densest NA innervation are acteristic of area 17 is shown in Fig. 1. There is a moderately deep III, V, and VI. Layer VI contains many long fibers that dense horizontal plexus of NA axons deep and superficial to are oriented parallel to the subcortical white matter as well as Downloaded by guest on September 26, 2021 2404 Neurobiology: Morrison et aL Proc. Nad Acad. Sci. USA 79 (1982) oblique and radial fibers. Layer V also contains long tangential striking complementary relationship to each other. The char- fibers, but there is more arborization in layer V and a slightly acteristic density and geometric pattern oflaminar innervation higher density than in layer VI. At the border between layers for each monoamine are summarized schematically in Fig. 3. V and IVc, the pattern changes abruptly, such that layer IVc The NA innervation of primary visual cortex is most dense in contains extremely few DBH-positive fibers.Thefibers that are layers V, VI, and the deep half of layer III and is extremely present in IVc generally extend across the layer radially, with sparse throughout layer IV. In contrast, the 5-HT innervation a minimal degree ofarborization. Layers IVb and IVa also have is extremely dense in layer IV, particularly in sublaminae IVa a very low density of NA innervation but slightly higher than and IVc, and is nearly absent from layers V and VI. that present in IVc. The density of NA axons increases abruptly In addition, the geometric orientations offibers differ for the at the upper border of layer IVa, and fibers arborize copiously two monoamine systems. This difference is most readily evident in the deep half of layer III. The deep portion of III contains in layer II and the superficial halfoflayer III, where NA fibers fibers of all orientations and is more densely innervated than are predominantly radial and serotonergic fibers are mostly tan- the superficial portion of layer III or of layer II, where the gential or oblique. Both monoamine projections thus exhibit a DBH-positive fibers have a primarily radial orientation. The NA high degree ofspecificity in their laminar and geometric pattern innervation of layer I is relatively sparse, consisting largely of of termination. occasional tangential fibers traveling within layer I and radial The laminar characteristics of the NA innervation ofprimary fibers that enter from layer II. visual cortex differ from those of other regions ofprimate neo- 5-HT Innervation. The laminar pattern of5-HT innervation cortex (15) in several ways. For example, primate primary so- ofarea 17 differs fundamentally from that ofthe NA innervation. A photomontage depicting the pattern of 5-HT innervation SEROTONIN NOREPINEPHRINE characteristic of area 17 is shown in Fig.