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Distribution of neuropeptide Y immunoreactivity in human visual cortex and underlying white matter
Peptides, Vol. 8, pp. 1107-1117.©PergamonJournals Ltd., 1987. Printedin the U.S.A.
01%-9781/87 $3.00 + .00
Distribution of Neuropeptide Y Immunoreactivity in Human Visual Cortex and Underlying White Matter O. V A N R E E T H , * ? 1 S. G O L D M A N , * ? S. S C H I F F M A N N , * ? A. V E R S T A P P E N , * G. P E L L E T I E R , $ H. V A U D R Y § A N D J. J. V A N D E R H A E G H E N *
*Neuropathology and Neuropeptides Research Laboratory, Erasme and Brugman Hospitals Universit~ libre de Bruxelles, Campus Anderleeht-Erasme Batiment C, 808 route de Lennik, B-1070 Bruxelles ?Aspirants du FAIRS ~Groupe de Recherche M~dicale, Endocrinologie Mol~culaire CHU Laval, Qu~Sbec, Canada and §Laboratoire d'Endocrinologie Mol~culaire, CNRS-INSERM Mont-Saint-Aignan, France R e c e i v e d 2 M a r c h 1987 VAN REETH, O., S. GOLDMAN, S. SCHIFFMANN, A. VERSTAPPEN, G. PELLETIER, H. VAUDRY AND J. J. VANDERHAEGHEN. Distribution of neuropeptide Y immunoreaetivity in human visual cortex and underlying white matter. PEPTIDES 8(6) 1107-1117, 1987.--Immunocytochemical techniques have been used to study neuropeptide Y (NPY) distribution in the human visual cortex (Brodman's areas 17, 18 and 19) NPY cell bodies belong mostly to inhibitory (multipolar and bitufted) but also to excitatory (bipolar and some pyramidal) neuronal types. Their distribution is similar in the three cortical areas studied: 20 to 40% of the NPY perikarya are located in the cortical gray matter, mostly in the deep layers, while the remaining 60 to 80% are located in the underlying white matter. Immunoreactive NPY processes form a rich network of intersecting fibers throughout the entire visual cortex. A superficial plexus (layers I and II) and a deep plexus (deep layer V and layer VI) of NPY fibers are present in areas 17, 18 and 19. In area 17, an additional well developed plexus is present in layers IVb and IVc. These plexuses receive branches from long parallel fibers arising from deep cortical layers or underlying white matter and terminating in superficial layers. Local or extrinsic NPY terminals wind around vessels in the cortex as well as in the white matter, and either penetrate them or form clusters of club endings on their walls. Our results suggest a role for NPY in human visual circuitry and in cortical blood flow regulation. Neuropeptide Y
Immunocytochemistry
Human
NPY, a 36-amino acid residue peptide, was first isolated from porcine brain extracts [47,48] and then described in rat and human brain [1,2]. Subsequently, both immunohistochemistry and radioimmunoassay have revealed its large distribution within rat [5, 6, 32, 33], cat [51], monkey [19,20], guinea pig [12] and human cerebral cortex [11]. More recently, its detailed anatomical distribution was reported in the human frontal, parietal and temporal cortices [4]. Visual information processing in the rat, cat and monkey occipital cortex has been recently reviewed both anatomically and physiologically [30, 34, 38--40]. Immunohistochemistry combined with the Golgi technique indicated that some corticovisual cells, e.g., "Chandelier" and "basket" cells, use gamma aminobutyric acid (GABA) as neurotransmitter [22,23]. Microinjections of GABA into short axon cortical cells pointed to an inhibitory role of GABA in cat visual processing [43,44]. Only a few percent of
Visual cortex
Vision
these short axon cortical neurons are GABA-ergic and little is known about the role of other interneurons and their putative transmitters in visual processing, especially in man. An important step towards this goal is the identification of their transmitter(s). Although NPY has already fulfilled many of the criteria for a neurotransmitter in the peripheral nervous system, its role in the cortex is still unclear [3,33]. The large distribution of NPY in cortical interneurons and its coexistence either with GABA [18-20] or somatostatin [5, 6, 20] in many of these neurons suggest that NPY may play a role in the regulation of cortical activities. Evidence suggesting a role for NPY in vertebrate vision processing includes the presence of NPY in some retinal amacrine cells [35,50], the thalamic lateral geniculate bodies and the superior colliculus [5,6], the visual cortex [5, 6, 18-20, 51] and the more recent demonstration of NPY binding sites in the rat lateral geniculate bodies,
JRequests for reprints should be addressed to Dr. O. Van Reeth, Laboratory of Neuropathology and Neuropeptides Research, Universit6 Libre de Bruxelles, Campus Andedecht-Erasme, CP 601, Brit. C, Local C3-131, Route de Lennick, 808, B-1070 Brussels, Belgium.
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VAN REETH ET AL.
1108 TABLE 1 CLINICALDATAON CASESUSEDFORIMMUNOHISTOCHEMISTRY
Case
Age
Sex
Hours Post Mortem
1 2 3 4 5 6 7 8 9
55Y 64Y 77Y 60Y 51Y 63Y 65Y 70Y 21m
F M M F M F M M F
10 5 7 22 7 3 13 4 6
Cause of Death Cirrhosis Renal Failure Bronchic Neoplasm Pulmonar Embolism Abdominal Abcess Subphrenic Abcess Prostatic Neoplasm Cardiopathy X Congenital Cardiopathy
F, female; M, male; m, months, Y, years. mamillary nuclei and cortical areas [28,49]. We have examined the existence of NPY immunoreactivity in the human visual cortex in order to compare its distribution with that of neurons and fibers implicated in cortical circuitry and which have been described using other techniques [36]. METHOD
Preparation of Tissue Nine human brains were obtained at post mortem from five males and four females without any history of neurological or psychiatric disease (Table 1). The anterior part of Brodman's area 17, area 18 and area 19 of the visual cortex were dissected and cut into several small blocks (0.4-1.5 cm thick), immediately transferred to a 4% paraformaldehyde solution in 0.1 M phosphate buffer saline (PBS) at pH 7.4 and fixed for 24-48 hours at 4°C. The post mortem delay prior to fixation ranged between 3 to 22 hours. After fixation, the blocks were progressively placed in a 10%, 20% and finally 30% sucrose in 0.1 M PBS solution (pH 7.4) for 24-72 hours at 4°C and then frozen in 2-methylbutan (Merck, Darmstadt, Germany) cooled in dry ice. Serial coronal sections (25-100 t~m) were cut on a cryostat (Dittes, Heidelberg, Germany) and rinsed in ice cold PBS for 15 minutes before staining. Some sections were stained either with cresyl violet, hematoxylin eosin or silver nitrate (Holmes) for identification of cytoarchitecture and examination of possible pathological conditions. No evidence of pathology was observed in any of the brains.
Antisera The anti-NPY antiserum was raised in New Zealand rab-
bits after multiple subcutaneous injections of a mixture of synthetic porcine NPY, methylated bovine serum albumin (BSA, Sigma, St. Louis, MO) and complete Freund's adjuvant. Radioimmunoassay procedures were used to verify the possibility of cross reactivity of the NPY antiserum with closely related peptides including APP, PYY, and some unrelated peptides such as alpha-MSH, 1-39 ACTH,/3-LPH, CRF, SRIF-14, CCK-8, CRF, Leu-enkephalin, Met-enkephalin, TRH, arginin-vasopressin and neurotensin. This antiserum was found to cross react less than 0.1% with APP and less than 0.01% with PYY. Cross reactivities with synthetic gamma-MSH and VIP were lower than 0.0001% and absent with the other peptides. Immunohistochemical cross reactivity experiments were also performed by using the NPY antiserum diluted 1/500 and absorbed with an excess (10 -7 M) of PYY, APP, ACTH, endorphins, VIP, CCK-8, SRIF and LHRH. Immunoabsorption with these various peptides did not affect the intensity of staining [37]. Immunoabsorption with synthetic NPY completely blocked the staining of NPY neurons and fibers. Since it is impossible to exclude the possibility that this antiserum is cross reacting with unknown peptides, in this study " N P Y " is synonymous with the more correct "NPY-like immunoreactivity."
Histochemical Procedures Both the peroxidase-antiperoxidase (PAP) technique of Sternberger [46] and the immunofluorescent technique of Coons [10] were used in this study. Some sections, prior to the PAP reaction, were incubated in hydrogen peroxide 2% for 15 rain to inhibit the endogenous peroxidase of the red cells contained in blood vessels. After rinsing, sections were preincubated in 10% swine nor-
FACING PAGE FIG. 1. Various NPY immunoreactive neurons in the human visual cortex. (A) Bipolar positive neuron in area 18: a primary dendritic trunk emanates from each pole of the cell and a very thin axon emerges from the upper dendritic trunk (arrow). (B) NPY immunoreactive small pyramidal cell in area 17, showing its descending axon (arrow head), its basal and apical beaded dendritic trunks (arrows). An ascendant NPY fiber crosses the cortical field close to the neuron (asterisks). (C) A multipolar NPY immunoreactive neuron in area 19 displaying two types of processes: on the left, unbeaded processes branch in the vicinity of the cell (arrows) and on the right, highly circonvoluted beaded processes end up non immunoreactive cells (arrow heads). (D) NPY immunoreactive vertical bitufted neuron in area 18 with a thin axon emerging directly from the soma (arrow); the network of large puncta around the cell is due to the fluorescence of the neuronal lipofuschine, the thin puncta represent processes derived from other cells. (E) In area 18, a poorly beaded process (arrow) emerges directly from the perikarya of this multipolar cell, surrounds it and branches or crosses over the other cell processes (arrow heads). Rabbit antiserum raised against NPY (A-E). PAP technique of Sternberger (A-C, E). FITC technique of Coons (D). Interferential contrast (B,C), Cresyl violet (A,E). Cortical layers: I-VI. Bar=25/zm.
NPY IN HUMAN
VISUAL
CORTEX
1110
VAN R E E T H E T AL.
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FIG. 2. Composite camera lucida drawings of various NPY immunoreactive neurons in cortical layers I-VI and underlying white matter in the human primary visual cortex, demonstrated on 100/zm thick slices with the PAP technique. Axons are indicated by black arrows. Cortical layers: I-VI. White matter: WM. Bar= 100 p.m. mal serum (SNS, Dako, Santa Barbara, CA) diluted in 0.1 M PBS containing 0.3% Triton X-100 (Tx, Merck, Darmstadt, Germany) for 60 min at room temperature and then incubated with NPY antiserum in dilutions from 1/5,000 to 1/20,000 in PBS-Tx for 16-30 hours at 4°C. The dilution 1/10,000 gave optimal staining of the sections and was used as a standard. After thorough washing in 0.1 M PBS-Tx 0.3% at room temperature, sections were further incubated for 30 min at room temperature in a 1/30 swine antirabbit antiserum (SAR, Dako, Santa Barbara, CA) diluted with 10% SNS in 0.1 M PBS-Tx 0.3%. After a 30 min washing in 0.1 M PBS-Tx 0.3% at room temperature, sections were incubated in a rabbit peroxidase-antiperoxidase complex (PAP, Pr. Vandesande, Leuven, Belgium) diluted 1/200 in 0.1 M PBS-Tx 0.3% for 30 min at room temperature. They were then, after thorough washing, reacted for 5-10 min with 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, MO) at 12.5 mg/100 ml in 0.1 M Tris saline buffer to which 1000 microliters of 0.3% hydrogen peroxide per 100 ml had been added. Some of these sections were further counterstained with cresyl violet or hematoxylin eosin. All sections were mounted on albumincoated slides, dehydrated, cleared and coverslipped. The slides were observed with a Nikon Microphot optical microscope equipped with an interferential contrast system and photographed with a Nikon FX-35A apparatus. Control sections were processed in the same way except for the replacement of the primary, secondary or tertiary antibody with swine normal serum. Other control sections were incubated with NPY
antiserum at the dilution of 1/10,000 previously absorbed with 3.8 micrograrn/ml of synthetic NPY. Other sections of the same cortical regions were processed for NPY labelling using the immunofluorescent technique of Coons: after 15 rain rinsing in 0.1 M PBS, sections were incubated at 4°C for 15-24 hours with NPY antiserum diluted 1/1000 in 0.1 M PBS-Tx 0.3%. After 30 min of multiple washing in PBS-Tx 0.3%, sections were finally incubated for 30 min at room temperature with fluoresceine isothiocyanate conjugated with swine antirabbit immunoglobulines (Fitc, Sigma, St. Louis, MO) diluted 1/100 with 0.3% Triton X-100. Sections were then rinsed three times for 10 min each in 0.1 M PBS, dried and mounted in a glycerol-water solution containing 25 g/l of 1,4-Diazabicyclo(2,2,2)octane (Dabco, Aldrich, Steinheim, Germany). Slides were examined with a Leitz Wetzlar fluorescence microscope equipped with a special Leitz L3 filter mirror, reducing the yellow interference fluorescence of neuronal lipofushine. In order to facilitate the study of the distribution of NPY immunoreactivity, composite drawings of NPY cells, processes and fibers plexuses were made with a Nikon camera lucida. NPY positive cells were counted and a semi quantitative estimate of their distribution was made. The nomenclature used to designate the cortical cells was based on their axonal and dendritic arborization patterns, and characteristics of the cell bodies examined were compared with the Golgi impregnation cells described elsewhere [36].
NPY IN H U M A N VISUAL CORTEX
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FIG. 3. Composite camera lucida drawings of NPY cells and fibers immunoreactivity in the human primary visual cortex and underlying white matter, demonstrated with PAP and FITC techniques. In A, all the subtypes of NPY cells are visible: a pyramidal in layer III (a), a bipolar in layer IV (b), bitufted cells in layers I to VI (c, d, e, f), multipolar cells in layer IV and V which give highly branched processes (g, h, i). Note the bitufted cells in layer VI (f) sending long processes towards layer I. In the underlying white matter, many immunoreactive cells are present, either separate (j) or in small groups (k). In B, a superficial plexus of NPY horizontal parallel fibers runs in layers I and II. A second clear cut plexus is located in layer IVb and c while the third plexus runs in deep layer V and in layer VI. These plexuses receive branches from oblique and vertical fibers ascending from the deep cortical layers or from the underlying white matter and distributing through the entire cortex. Note the background rich network of positive processes. Cortical layers: I-VI. White matter: WM. Bar=400 /zm.
RESULTS
N P Y lmmunoreactivity Sections from all the cortical areas examined displayed numerous intensely stained somata, processes and fibers, with an identical distribution among all the cases studied. The intensity of staining was much more higher in the young child (case 9) than in the adult brains (cases 1-8). It diminished when the post mortem delay prior to fixation was increased (cases 4 and 7). Treatment of the sections with hydrogen peroxide resulted in a clearer picture of the NPY distribution due to the attenuation of the staining of the red blood cells peroxidase. None of the control sections displayed any positive staining.
N P Y Cell Somata Both non pyramidal and pyramidal neurons were labelled but a much more intense and frequent staining was seen in the former. Non pyramidal NPY positive cells were bipolar (Fig. IA), bitufted (Fig. 1D) or multipolar (Fig. 1C, E) aspiny neurons. Multipolar neurons were the most prevalent NPY immunoreactive cells, bitufted neurons comprised the second most common morphological group of NPY cells while the bipolar neurons were only sparsly scattered through the cortex (Fig. 2). Some NPY neurons, principally in layer III and V, were typically pyramidal in shape, with a long apical and a few
basal aspiny dendrites and a descending beaded axon (Figs. 1B, 2, 3A). There was no reliable difference in the morphology and distribution of NPY positive cells between cortical areas 17, 18 and 19, except for a relatively poor distribution of NPY neurons in layer IV or area 17 compared to the same layer in area 18 and 19. NPY positive cells were mostly dispersed in a network of non immunoreactive cells, and only a few were aggregated in small groups. NPY neurons were present in cortical layers I to VI (Figs. 1, 2, 3A) but their distribution varied from one layer to another. Only a few cells were demonstrable in layer I where they were typically small (5-10 p.m) and mostly bitufted (Fig. 2). The few positive bipolar cells were located in layer III and IV (Figs. 1A, 2, 3A). Many NPY cells were present in deeper layers, especially in layers V and VI, where they were mostly bitufted or mulfipolar (Figs. IC-E, 2, 3A). These cells were usually larger (8-25/zm) than those found in layer I, with a great number of long processes. One or more of these processes always showed a vertical orientation. In deep layer V and layer VI, many horizontal bitufted neurons showing long rectilign processes running parallel to the border with white matter were recognized with the NPY antiserum (Fig. 2). In these layers, some vertical bitufted cells send long ascending processes sometimes ending in layer I and II. Interestingly, NPY positive cells were also visible in the underlying white matter of the three cortical areas studied
1112
VAN REETH
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FIG. 4. N P Y i m m u n o r e a c t i v e cells in the h u m a n corticovisual underlying white matter. (A) The perikarya and the p r o c e s s e s of this N P Y cell are parallel to the cortical junction. Note the long process entering layer VI (arrow), and a cluster of varicosities of the inferior cortical N P Y plexus (arrow head), The regular dark gray structures are red cells in blood vessels colored with the PAP technique (asterisk). (B) A small fusiform N P Y i m m u n o r e a c t i v e cell with two poorly branching beaded processes. (C, D) N P Y cells with two different types of processes arborization: highly beaded (C) and non beaded (D). (E) A squared N P Y cell in the deep white matter, Note the thin axon (arrow) running obliquely in regard to the myelinated fibers orientation, given by the dendritic orientation. Rabbit a n t i s e r u m raised against NPY. P A P technique of Sternberger. Interferential contrast. Cortical layers: I-VI. White matter: WM. B a r = 10 ~ m .
NPY IN HUMAN VISUAL CORTEX
1113
FIG. 5. NPY immunoreactive terminals in the primary visual cortex. (A) Multipolar cell arborizing in layer III and terminating upon a small vessel obliquely cut. Note the fiber entering the vessel wall. Some NPY processes originating from elsewhere also end upon this vessel. (B) Terminal arborization of a NPY bitufted cell; note the palissade aspect of this highly branched ending process. (C) NPY immunoreactive cortical multipolar cell surrounding a small capillary: the small NPY vesicules fulfill the entire perikarya except the cell nucleus (arrow). (D) Detail of the deep cortical fibers plexus: a network of highly circonvoluted and branched processes is crossed over by vertical ascending fibers originating from the underlying white matter (arrows) and sending branches to each other (arrow heads). Rabbit antiserum raised against NPY. PAP technique of Sternberger. Intefferential contrast. Vessel: V. Bar=25/xm.
1114 (Figs. 2, 3A, 4). A semi-quantitative analysis indicated that, according to a surface basis, only 20-40% of the NPY positive cells were present in the cortical layers while the remaining 60-80% were found in the underlying white matter. These cells, often aggregated in groups of three or four, showed a typical distribution in the white matter: a palissade of small cells, mostly bitufted, were located just under or at the junction with layer VI (Figs. 2, 3A, 4A) with their long axis parallel to the myelinated fibers. Many of them gave processes that extended first horizontally and then projected vertically or obliquely into the grey matter (Figs. 2, 4A). Deeper, many small NPY cells with a great variety of shapes (Fig. 4B-E) and orientations (parallel, perpendicular or oblique towards the myelinated fibers) were noted. Their processes were very long, being either linear or circonvoluted, beaded or not (Fig. 4C, D). N P Y Cell Processes
Most of the NPY cells showed numerous and very long processes, often presenting round or irregular beaded varicosities separated by thin or thick intervaricose sections (Fig. 1C). Emerging at various angles of the cell somata, these processes branched at different distances from the cell of origin. Other processes were short, twisted or isolated labelled puncta without any evident cell of origin. NPY processes formed a rich network of vertical, horizontal or oblique tracts present in all the layers of the three regions examined. These processes showed various morphological patterns: rectilignes, twisted, in S or in scale shape. Some NPY processes, often originating from bitufted cells, had vertically oriented branching terminals, showing an aspect "en palissade" (Fig. 5b). Clusters of terminals were observed, forming bulbous swellings which contributed to the formation of pericellular baskets arranged around other NPY immunoreactive or non immunoreactive cells (Fig. 5B, D). In the primary visual cortex (area 17), three clearly separate and well organized plexuses of NPY fibers could be distinguished (Figs. 3B, 6). A superficial plexus, located in layer I and II, consisted of a great number of long fibers running tengantially to the cortical surface. These fibers could be followed for long distances (1-4 mm), going sometimes from one gyrus to another. Cells of origin of these fibers could not be determined and must probably be searched in other cortical gyri. A few of these long parallel fibers send to each other small beaded and not beaded processes. Just beneath the cortical surface, fibers were dense and linear, sometimes pooled into spindles of 3 to 4 fibers, while deeper in layer II they were more distant, twisted and anarchically disposed (Fig. 6). This superficial plexus received long vertical parallel fibers arising from the depth: these fibers were thin and arise either from the various NPY cells of the deep cortical layers or from the underlying white matter, where cells of origin were rarely visible. The origin of many of these vertical fibers could not be established. From this superficial plexus branched off a number of mostly twisted fibers which terminated in layers III and IV (Figs.
VAN REETH ET AL. 3B, 5D, 6). A mid plexus was present in layer IV, principally in the sublayers IVb and IVc, i.e., the external band of Balllarger (Figs. 3B, 6). Fibers of this dense plexus had a strongly vertical or oblique orientation. Terminals were principally made of clusters of highly branched and circumvoluted processes ending on non immunoreactive neurons. Some of these bulbous terminals did not contact to any cell. Cells of origin of all fibers could not be established, this layer being particularly void of NPY cells in this area. This plexus received branches of the long vertical fibers arising from the depth and ending in the superficial plexus. A deep plexus of fibers was located in deep layer V and layer VI of the primary visual cortex (Figs. 3B, 6). It consisted principally of clusters of highly branched and twisted fibers emanating from multipolar or bitufted cells from layers IV, V and VI and from underlying white matter cells. This plexus was crossed both by horizontal processes of the bitufted cells of these layers and by vertical and oblique NPY fibers arising from the underlying white matter. In visual areas 18 and 19, only the superficial plexus (layer I and II) and the deep plexus (deep layer V and layer VI) of NPY fibers were clearly visible, and the mid plexus of layer IV was not demonstrable. In the subcortical white matter of the eight adult brains, NPY processes were less abundant and stained lighter than in the cortical layers. NPY cells located at, or just under the cortical junction gave many processes, one of which at least took a vertical (or oblique) orientation and reached the upperlying gray matter, where it gave branches and terminated. Deeper in the white matter, NPY small cells gave long and thin processes which mostly followed the myelinated fibers, sometimes for several hundred micrometers. Only a few processes showed a perpendicular or oblique disposition towards the myelinated fibers (Fig. 4E). Many long thin and thick fibers could be followed for long distances without showing their cell of origin: they may originate from other cortical areas (Fig. 3B). The subcortical white matter of the juvenile brain showed a more richer network of NPY processes than the adult. N P Y Immunoreactivity and the Vessels
In all cortical and subcortical regions examined, many NPY positive beaded fibers were observed to run along and wind around small blood vessels (Fig. 5A, C). These processes formed clusters of dilatations and terminated with club endings on vessels, sometimes entering their walls (Fig. 5A). Cells of origin of these fibers, mostly bitufted and multipolar, were sometimes seen in the vicinity of the innervated vessels (Fig. 5A), but more often were not visible. DISCUSSION
The need for studies of human neuropeptides distribution is stressed by the well known species variation concerning their distribution and by the uniqueness of the human brain. Since no experimental studies can be performed in humans, a discussion of what is already known in other species will be
FACING PAGE FIG. 6. NPY immunoreactivity in the human primary visual cortex. Photomicrograph of NPY positive processes (arrow) from a section passing through the bank of the calcarine fissure. Main line of Gennari is located in layer IVb. Three main plexuses are distinguishable: the upper one, just under the pial surface, the medium one in layer IVb, c and the deep one located in deep layer V and layer VI. Arrowheads indicate vertical ascending NPY fibers reaching the superficial plexus of fibers. A multipolar cell is clearly seen in layer III. Rabbit antiserum raised against NPY. PAP technique of Sternberger. Cortical layers: I-VI. White matter: WM. Bar=400 tzm.
NPY IN H U M A N V I S U A L CORTEX
1115 reported. Previous studies have shown the existence of NPY in the human cerebral cortex [1, 5, 11] and have studied its distribution in frontal, parietal and temporal cortices [4]. The present immunocytochemical study confirms the presence of a large number of NPY cells and processes in the human visual cortex and further elucidates its distribution in this particular cortical region. The visual cortex of the rat [5,6], cat [51] and monkey [19,20] have been the focus of several studies on distribution and morphology of NPY neurons and processes. In these animal studies, it has been reported that NPY immunoreactivity is located exclusively in non pyramidal neurons. Our results reveal that NPY is also present in some medium-sized pyramidal neurons in layers III and V of the human visual cortex. These findings raise the possibility that NPY may play a role in visual efferent pathways, as already suggested for somatostatin in the rat visual cortex [24]. This hypothesis could also be supported by our findings of many positive, presumably associative, NPY fibers running from the subadjacent white matter through the corpus callosum (unpublished data). Similar findings have indeed been recently reported in the rat [31] and the cat [51] cortex. Numerous NPY neurons are small, spineless, multipolar and bitufted neurons. Electron microscopic and immunocytochemical studies performed in cat and monkey visual cortex have clearly shown that axons of these small and aspiny multipolar cells form G r a y ' s type II synapses [26, 36, 39] and use G A B A as a neurotransmitter [22,40]. Both findings suggest that these neurons exert a local inhibitory role. The large (15-25/xm) multipolar neurons with an abundant axonal arbor which forms one or more, either " e n passant" or terminal, baskets around pyramidal or nonpyramidal perikarya could be "Basket cells" well described in the cat [29, 39, 41] and considered as one of the major inhibitory neurons in the cat visual cortex [43,44]. A positive NPY staining of cortical "Basket cells" has recently been reported in rat and cat by some [21,51] but not in cat and monkey by others [20]. Some cortical bipolar neurons are also recognized with our NPY antiserum. These cells have been proven to form G r a y ' s I synapses with pyramidal and non pyramidal cells in rat and cat visual cortex [36, 38, 39] and to not contain GABA. Based on these observations, it is assumed that bipolar cells exert a local excitatory effect in the visual cortex [36]. These results are of particular interest since they suggest that in the human visual cortex, NPY is located both in inhibitory (bitufted and multipolar) and excitatory neurons (bipolar and some pyramidal), the former being predominant. It implies that NPY may coexist with other neurotransmitters or neuromodulators in the same neurons. A coexistence of NPY and somatostatin has already been reported in various rat and human cortical areas [5]. Immunofluorescence studies performed in the cat and monkey visual cortex indicate that all the NPY positive neurons in the cat and a majority in the monkey also stain positively for GAD, suggesting that these neurons could release G A B A as an inhibitory transmitter together with N P Y as a neuromodulator [19]. In the human visual cortex, NPY stained perikarya tend to be seen predominantly in deeper layers of the cortex, as described in the rat [5,6], cat [51] and monkey [20] visual cortex. Our results are in accordance with previous reports on NPY cell distribution in other areas of the human
1116
VAN REETH E T A L .
cortex [1, 4, 11], except for the particularly poor distribution of NPY cells in the well developed layer IV of area 17. NPY cells found in the underlying white matter are comparable in size and shape to those in the cortex. These cells are described in rat, cat and monkey and considered as neurons, on morphological basis [5, 20, 51]. Large (50-120 /zm) somatostatin cells have also been described in the human subcortical white matter [45]. Functional properties of NPY white matter cells is unclear: the superficial ones, which are probably neurons that have not migrated enough during the cortical development, send processes to the upperlying gray matter and participate to the elaboration of the deep NPY cortical plexus. The deep white matter cells, which can be located as far as the corpus callosum, may participate to intra- and interhemispheric associative cortical pathways. NPY fibers distribution shows a typical laminar organization. We describe three clearly separated plexuses in area 17, while only two can be distinguished in area 18 and 19. The superficial and deep plexuses have been described in other cortical areas of the human brain [4, 5, 11], while the mid plexus of layers IVb and IVc seems to be characteristic of the primary visual cortex. It has also been described in layer IV of cat and monkey primary visual cortex, being predominant in sublayers IVa and IVb in these species [20]. Laminar distribution of NPY fibers seems to correspond to the distribution of afferent and efferent pathways or cells within the visual cortex: the upper plexus of layers I and II and its extent to the underlying layers is located in the ending region of most of the corticocortical associative fibers [9, 25, 41, 42]. Cells of origin of many of these superficial fibers may arise from other cortical or subcortical regions. The mid plexus of layers IVb and IVc (i.e., the outer band of Bail-
larger) is in the ending region of thalamocortical fibers and some associative corticocortical fibers [7,8], while the deepest plexus of deep layer V (i.e., the inner band of Baillarger), layer VI and underlying white matter is located in the infragranular cortical region involved in the efferent pathways of the visual cortex where also terminate thalamacortical fibers. Many NPY fibers present terminals around blood vessels both in gray and white matter. This localization suggests that, together with VIP and Substance P, NPY may participate to the regulation of cortical blood flow [13--15, 27]. Some of these fibers originate from neurons located in the vicinity of the innervated vessels, suggesting that neurons involved in the inner cortical circuitry may also regulate the cortical blood flow. However, more often, cells of origin are not visible and are probably located in other areas. Coexistence of NPY and noradrenaline in the same nerve fibers, well established in the peripheral nervous system, has also been reported in dense nervous plexuses surrounding cerebral and pial vessels in feline [ 16]. The functional interaction between NPY and central adrenergic control of blood pressure, together with the rich network of NPY fibers innervating the cortical vessels and originating out of the cortex, suggest the possibility of a coexistence of NPY and noradrenaline in the human locus coeruleus, as reported in the rat [17]. ACKNOWLEDGEMENTS This work was supported by grants from the Belgian National Fund for Scientific Research (FNRS 85-86) and Medical Scientific Research (FRSM 34521.82-86 and 34523.86-89), the Belgian National Lotery (1983 and 1986) and the Queen Etisabeth Medical Foundation (FMRE 81-84 and 86-89).
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11. Dawbarn, D., S. P. Hunt and P. C. Emson. Neuropeptide Y: Regional distribution, chromatographic characterisation and immunohistochemical demonstration in post mortem human brains. Brain Res 296: 168-173, 1984. 12. De Quidt, M. E., P. J. Richarson and P. C. Emson. Subcellular distribution of neuropeptide Y immunoreactivity in guinea pig neocortex. Brain Res 335: 354-359, 1985. 13. Edvinsson, J. M. and J. Scarby. Interaction of peptides present in perivascular nerves on the smooth muscle of cat cerebral arteries and on the stimulation-evoked effiux of ~H noradrenaline. J Auton Pharmacol 4: 193-198, 1984. 14. Edvinsson, L., P. Emson, J. McCulloch, K. Tatemoto and R. Uddman. Neuropeptide Y: cerebrovascular innervation and vasomotor effects in the cat. Neurosci Lett 43: 7%84, 1983. 15. Edvinsson, J. M., P. Emson, J. McCulloch, K. Tatemoto and R. Uddman. Neuropeptide Y: immunocytochemical localization to and effect upon feline pial arteries and veins in vitro and in situ. Acta Physiol Scand 122: 155-163, 1984. 16. Ekblad, E., J. M. Edvinsson, C. Wahlestedt, R. Uddman, R. Hakanson and F. Sundler. Neuropeptide Y coexists and cooperates with noradrenaline in perivascular nerve fibers. Regul Pept 8: 225-235, 1984. 17. Everitt, B. J., T. Hokfelt, L. Terenius, K. Tatemoto, V. Mutt and M. Goldstein. Differential coexistence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat. Neuroscience 11: 443-462, 1984. 18. Hendry, S. H. C., C. R. Houser, E. G. Jones and J. E. Vaughn. Synaptic organization of immunocytocbemically identified G A B A neurons in the monkey sensory-motor cortex. J Neurocytol 12: 63%660, 1983.
NPY IN HUMAN VISUAL CORTEX
19. Hendry, S. H. C., E. G. Jones, J. Defelipe, D. Schmechel, C. Brandon and P. C. Emson. Neuropeptide-containing neurons of the cerebral cortex are also GABAergic. Proc Natl Acad Sci USA 81: 6526--6530, 1984. 20. Hendry, S. H. C., E. G. Jones and P. C. Emson. Morphology, distribution, and synaptic relations of somatostatin- and neuropeptide Y-immunoreactive neurons in rat and monkey neocortex. J Neurosci 4: 2497-2517, 1984. 21. Hodgon, A. J. and J. Olivier. Neurochemical markers for subsets of inhibitory neurons in cerebral cortex. Is NPY a marker for a small subset of basket cells? Soc Neurosci Abstr 11: 502, 1985. 22. Houser, C. R., G. D. Crawford, P. M. Salvaterra and J. E. Vaughn. Cholinergic neurons in rat cerebral cortex identified by immunocytochemical localization of choline acetyltransferase. Soc Neurosci Abstr 9: 80, 1983. 23. Houser, C. R., G. D. Crawford, P. M. Savaterra and J. E. Vaughn. Morphological diversity of immunocytochemically identified GABA neurons in the monkey sensory-motor cortex. J Neurocytol 12: 617-638, 1983. 24. Laemle, L. K. and S. C. Feldman. Somatostatin (SRIF)-like immunoreactivity in subcortical and cortical visual centers of the rat. J Comp Neurol 233: 452-462, 1985. 25. Legros Clarck, W. E. The visual centers of the brain and their connections. Physiol Rev 22: 205-232, 1942. 26. Levay, S. Synaptic patterns in the visual cortex of the cat and monkey: Electron microscopy of Golgi preparations. J Comp Neurol 150: 53-86, 1973. 27. McCuiloch, J. and L. Edvinsson. Cerebral circulatory and metabolic effects of vasoactive intestinal polypeptide. Am J Physiol 231: H449-H456, 1976. 28. Martel, J. C., S. St-Pierre and R. Quirion. Neuropeptide Y receptors in rat brain: autoradiography localization. Peptides 7" 55-60, 1986. 29. Martin, K. A. C., P. Somogyi and D. Whitteridge. Physiological and morphological properties of identified basket cells in the cat's visual cortex. Exp Brain Res 50: 193-200, 1983. 30. Meyer, G. Axonal patterns and topography of short-axon neurons in visual areas 17, 18 and 19 of the cat. J Comp Neurol 220: 405-438, 1983. 31. Nakagawa, Y., S. Shiosaka, P. C. Emson and M. Tohyama. Distribution of neuropeptide Y in the forebrain and diencephalon: an immunohistochemical analysis. Brain Res 361: 52-60, 1985. 32. O'Donohue, T. L., B. M. Chronwall and D. A. Di Maggio. Distribution of neuropeptide Y immunoreactivity in the rat brain. Soc Neurosci Abstr 9: 290, 1983. 33. O'Donohue, T. L., B. M. Chronwall, R. M. Pruss, E. Mezey, J. Z. Kiss, L. E. Eiden, V. J. Massari, R. E. Tessel, V. M. Pickel, D. A. Di Maggio, A. J. Hotchkiss, W. R. Crowley and Z. Zukowska-Grojec. Neuropeptide Y and peptide YY neuronal and endocrine systems. Peptides 6: 755-768, 1985. 34. Orban, G. A. Neuronal operations in the visual cortex. In: Studies o f Brain Function, vol 11, edited by H. B. Barlow, T. H. Bullock, E. Florey, O. J. Grosser and A. Peters. Bedim Springer-Verlag, 1984, pp. 5-367.
1117
35. Osborne, N. N., S. Patel, G. Terenghi, J. M. Allen, J. M. Polak and S. R. Bloom. Neuropeptide Y (NPY)-like immunoreactive amacrine cells in retinas of frog and goldfish. Cell Tissue Res 241: 651-656, 1985. 36. Parnavelas, J. B. Physiological properties of identified neurons. In: Cerebral Cortex, Vol 2, Functional Properties o f Cortical Cells, edited by E. B. Jones and A. Peters. New York: Plenum Press, 1984, pp. 205-236. 37. Pelletier, G., L. Desy, L. Kerkerian and J. Cote. Immunocytochemical localization of neuropeptide Y (NPY) in the human hypothalamus. Cell Tissue Res 238: 203-205, 1984. 38. Peters, A. and L. M. Kimerer. Bipolar neurons in rat visual cortex: A combined Golgi-electron microscope study. J Neurocytol 10: 921-946, 1981. 39. Peters, A. and J. Redigor. A reassessment of the form of non pyramidal neurons in area 17 of cat visual cortex. J Comp Neurol 203: 685-716, 1981. 40. Peters, A., C. C. Proskauer and C. E. Ribak. Chandelier cells in the rat visual cortex. J Comp Neurol 206: 397-416, 1982. 41. Ramon y Cajal, S. Histalogie du Systbme Nerveux de l'Homme et des Vertdbr~s, vol 2. Paris: Maloine, 1911. 42. Schade, J. P. and W. B. Van Groeningen. Structural organization of the human cerebral cortex. Acta Anat 47:74-111, 1961. 43. Sillito, A. M. The effectiveness of bicuculline as an antagonist of GABA and visually evoked inhibition in the cat's striate cortex. J Physiol 250: 287-304, 1975. 44. Sillito, A. M. Inhibitory mechanisms underlying the directional specificity of simple, complex and hypercomplex cells in the cat's visual cortex. J Physiol 271: 690-720, 1977. 45. Sorensen, K. V. Somatostatin: localization and distribution in the cortex and the subcortical white matter of human brain. Neuroscience 7: 1227-1232, 1982. 46. Sternberger, L. A. The unlabelled antibody peroxidaseantiperoxidase (PAP) method. In: Immunocytochemistry, 2nd edition. New York: John Wiley, 1979, pp. 104-109. 47. Tatemoto, K., M. Carlquist and V. Mutt. Neuropeptide Y - - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296: 659-660, 1982. 48. Tatemoto, K. Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc Natl Acad Sci USA 79: 5485-5489, 1982. 49. Unden, A., K. Tatemoto, V. Mutt and T. Bartfai. Neuropeptide Y receptor in rat brain. Eur J Biochem 145: 525-530, 1985. 50. Verstappen, A., O. Van Reeth, H. Vaudry, G. Pelletier and J.-J. Vanderhaeghen. Demonstration of Neuropeptide Y (NPY) immunoreactivity in the pigeon retina. Neurosci Lett 70: 193-197, 1986. 51. Whale, P., G. Meyer and K. Albus. Localization of NPY-like immunoreactivity in the cat's visual cortex. Exp Brain Res 61: 364-374, 1986.
01%-9781/87 $3.00 + .00
Distribution of Neuropeptide Y Immunoreactivity in Human Visual Cortex and Underlying White Matter O. V A N R E E T H , * ? 1 S. G O L D M A N , * ? S. S C H I F F M A N N , * ? A. V E R S T A P P E N , * G. P E L L E T I E R , $ H. V A U D R Y § A N D J. J. V A N D E R H A E G H E N *
*Neuropathology and Neuropeptides Research Laboratory, Erasme and Brugman Hospitals Universit~ libre de Bruxelles, Campus Anderleeht-Erasme Batiment C, 808 route de Lennik, B-1070 Bruxelles ?Aspirants du FAIRS ~Groupe de Recherche M~dicale, Endocrinologie Mol~culaire CHU Laval, Qu~Sbec, Canada and §Laboratoire d'Endocrinologie Mol~culaire, CNRS-INSERM Mont-Saint-Aignan, France R e c e i v e d 2 M a r c h 1987 VAN REETH, O., S. GOLDMAN, S. SCHIFFMANN, A. VERSTAPPEN, G. PELLETIER, H. VAUDRY AND J. J. VANDERHAEGHEN. Distribution of neuropeptide Y immunoreaetivity in human visual cortex and underlying white matter. PEPTIDES 8(6) 1107-1117, 1987.--Immunocytochemical techniques have been used to study neuropeptide Y (NPY) distribution in the human visual cortex (Brodman's areas 17, 18 and 19) NPY cell bodies belong mostly to inhibitory (multipolar and bitufted) but also to excitatory (bipolar and some pyramidal) neuronal types. Their distribution is similar in the three cortical areas studied: 20 to 40% of the NPY perikarya are located in the cortical gray matter, mostly in the deep layers, while the remaining 60 to 80% are located in the underlying white matter. Immunoreactive NPY processes form a rich network of intersecting fibers throughout the entire visual cortex. A superficial plexus (layers I and II) and a deep plexus (deep layer V and layer VI) of NPY fibers are present in areas 17, 18 and 19. In area 17, an additional well developed plexus is present in layers IVb and IVc. These plexuses receive branches from long parallel fibers arising from deep cortical layers or underlying white matter and terminating in superficial layers. Local or extrinsic NPY terminals wind around vessels in the cortex as well as in the white matter, and either penetrate them or form clusters of club endings on their walls. Our results suggest a role for NPY in human visual circuitry and in cortical blood flow regulation. Neuropeptide Y
Immunocytochemistry
Human
NPY, a 36-amino acid residue peptide, was first isolated from porcine brain extracts [47,48] and then described in rat and human brain [1,2]. Subsequently, both immunohistochemistry and radioimmunoassay have revealed its large distribution within rat [5, 6, 32, 33], cat [51], monkey [19,20], guinea pig [12] and human cerebral cortex [11]. More recently, its detailed anatomical distribution was reported in the human frontal, parietal and temporal cortices [4]. Visual information processing in the rat, cat and monkey occipital cortex has been recently reviewed both anatomically and physiologically [30, 34, 38--40]. Immunohistochemistry combined with the Golgi technique indicated that some corticovisual cells, e.g., "Chandelier" and "basket" cells, use gamma aminobutyric acid (GABA) as neurotransmitter [22,23]. Microinjections of GABA into short axon cortical cells pointed to an inhibitory role of GABA in cat visual processing [43,44]. Only a few percent of
Visual cortex
Vision
these short axon cortical neurons are GABA-ergic and little is known about the role of other interneurons and their putative transmitters in visual processing, especially in man. An important step towards this goal is the identification of their transmitter(s). Although NPY has already fulfilled many of the criteria for a neurotransmitter in the peripheral nervous system, its role in the cortex is still unclear [3,33]. The large distribution of NPY in cortical interneurons and its coexistence either with GABA [18-20] or somatostatin [5, 6, 20] in many of these neurons suggest that NPY may play a role in the regulation of cortical activities. Evidence suggesting a role for NPY in vertebrate vision processing includes the presence of NPY in some retinal amacrine cells [35,50], the thalamic lateral geniculate bodies and the superior colliculus [5,6], the visual cortex [5, 6, 18-20, 51] and the more recent demonstration of NPY binding sites in the rat lateral geniculate bodies,
JRequests for reprints should be addressed to Dr. O. Van Reeth, Laboratory of Neuropathology and Neuropeptides Research, Universit6 Libre de Bruxelles, Campus Andedecht-Erasme, CP 601, Brit. C, Local C3-131, Route de Lennick, 808, B-1070 Brussels, Belgium.
1107
VAN REETH ET AL.
1108 TABLE 1 CLINICALDATAON CASESUSEDFORIMMUNOHISTOCHEMISTRY
Case
Age
Sex
Hours Post Mortem
1 2 3 4 5 6 7 8 9
55Y 64Y 77Y 60Y 51Y 63Y 65Y 70Y 21m
F M M F M F M M F
10 5 7 22 7 3 13 4 6
Cause of Death Cirrhosis Renal Failure Bronchic Neoplasm Pulmonar Embolism Abdominal Abcess Subphrenic Abcess Prostatic Neoplasm Cardiopathy X Congenital Cardiopathy
F, female; M, male; m, months, Y, years. mamillary nuclei and cortical areas [28,49]. We have examined the existence of NPY immunoreactivity in the human visual cortex in order to compare its distribution with that of neurons and fibers implicated in cortical circuitry and which have been described using other techniques [36]. METHOD
Preparation of Tissue Nine human brains were obtained at post mortem from five males and four females without any history of neurological or psychiatric disease (Table 1). The anterior part of Brodman's area 17, area 18 and area 19 of the visual cortex were dissected and cut into several small blocks (0.4-1.5 cm thick), immediately transferred to a 4% paraformaldehyde solution in 0.1 M phosphate buffer saline (PBS) at pH 7.4 and fixed for 24-48 hours at 4°C. The post mortem delay prior to fixation ranged between 3 to 22 hours. After fixation, the blocks were progressively placed in a 10%, 20% and finally 30% sucrose in 0.1 M PBS solution (pH 7.4) for 24-72 hours at 4°C and then frozen in 2-methylbutan (Merck, Darmstadt, Germany) cooled in dry ice. Serial coronal sections (25-100 t~m) were cut on a cryostat (Dittes, Heidelberg, Germany) and rinsed in ice cold PBS for 15 minutes before staining. Some sections were stained either with cresyl violet, hematoxylin eosin or silver nitrate (Holmes) for identification of cytoarchitecture and examination of possible pathological conditions. No evidence of pathology was observed in any of the brains.
Antisera The anti-NPY antiserum was raised in New Zealand rab-
bits after multiple subcutaneous injections of a mixture of synthetic porcine NPY, methylated bovine serum albumin (BSA, Sigma, St. Louis, MO) and complete Freund's adjuvant. Radioimmunoassay procedures were used to verify the possibility of cross reactivity of the NPY antiserum with closely related peptides including APP, PYY, and some unrelated peptides such as alpha-MSH, 1-39 ACTH,/3-LPH, CRF, SRIF-14, CCK-8, CRF, Leu-enkephalin, Met-enkephalin, TRH, arginin-vasopressin and neurotensin. This antiserum was found to cross react less than 0.1% with APP and less than 0.01% with PYY. Cross reactivities with synthetic gamma-MSH and VIP were lower than 0.0001% and absent with the other peptides. Immunohistochemical cross reactivity experiments were also performed by using the NPY antiserum diluted 1/500 and absorbed with an excess (10 -7 M) of PYY, APP, ACTH, endorphins, VIP, CCK-8, SRIF and LHRH. Immunoabsorption with these various peptides did not affect the intensity of staining [37]. Immunoabsorption with synthetic NPY completely blocked the staining of NPY neurons and fibers. Since it is impossible to exclude the possibility that this antiserum is cross reacting with unknown peptides, in this study " N P Y " is synonymous with the more correct "NPY-like immunoreactivity."
Histochemical Procedures Both the peroxidase-antiperoxidase (PAP) technique of Sternberger [46] and the immunofluorescent technique of Coons [10] were used in this study. Some sections, prior to the PAP reaction, were incubated in hydrogen peroxide 2% for 15 rain to inhibit the endogenous peroxidase of the red cells contained in blood vessels. After rinsing, sections were preincubated in 10% swine nor-
FACING PAGE FIG. 1. Various NPY immunoreactive neurons in the human visual cortex. (A) Bipolar positive neuron in area 18: a primary dendritic trunk emanates from each pole of the cell and a very thin axon emerges from the upper dendritic trunk (arrow). (B) NPY immunoreactive small pyramidal cell in area 17, showing its descending axon (arrow head), its basal and apical beaded dendritic trunks (arrows). An ascendant NPY fiber crosses the cortical field close to the neuron (asterisks). (C) A multipolar NPY immunoreactive neuron in area 19 displaying two types of processes: on the left, unbeaded processes branch in the vicinity of the cell (arrows) and on the right, highly circonvoluted beaded processes end up non immunoreactive cells (arrow heads). (D) NPY immunoreactive vertical bitufted neuron in area 18 with a thin axon emerging directly from the soma (arrow); the network of large puncta around the cell is due to the fluorescence of the neuronal lipofuschine, the thin puncta represent processes derived from other cells. (E) In area 18, a poorly beaded process (arrow) emerges directly from the perikarya of this multipolar cell, surrounds it and branches or crosses over the other cell processes (arrow heads). Rabbit antiserum raised against NPY (A-E). PAP technique of Sternberger (A-C, E). FITC technique of Coons (D). Interferential contrast (B,C), Cresyl violet (A,E). Cortical layers: I-VI. Bar=25/zm.
NPY IN HUMAN
VISUAL
CORTEX
1110
VAN R E E T H E T AL.
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FIG. 2. Composite camera lucida drawings of various NPY immunoreactive neurons in cortical layers I-VI and underlying white matter in the human primary visual cortex, demonstrated on 100/zm thick slices with the PAP technique. Axons are indicated by black arrows. Cortical layers: I-VI. White matter: WM. Bar= 100 p.m. mal serum (SNS, Dako, Santa Barbara, CA) diluted in 0.1 M PBS containing 0.3% Triton X-100 (Tx, Merck, Darmstadt, Germany) for 60 min at room temperature and then incubated with NPY antiserum in dilutions from 1/5,000 to 1/20,000 in PBS-Tx for 16-30 hours at 4°C. The dilution 1/10,000 gave optimal staining of the sections and was used as a standard. After thorough washing in 0.1 M PBS-Tx 0.3% at room temperature, sections were further incubated for 30 min at room temperature in a 1/30 swine antirabbit antiserum (SAR, Dako, Santa Barbara, CA) diluted with 10% SNS in 0.1 M PBS-Tx 0.3%. After a 30 min washing in 0.1 M PBS-Tx 0.3% at room temperature, sections were incubated in a rabbit peroxidase-antiperoxidase complex (PAP, Pr. Vandesande, Leuven, Belgium) diluted 1/200 in 0.1 M PBS-Tx 0.3% for 30 min at room temperature. They were then, after thorough washing, reacted for 5-10 min with 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, MO) at 12.5 mg/100 ml in 0.1 M Tris saline buffer to which 1000 microliters of 0.3% hydrogen peroxide per 100 ml had been added. Some of these sections were further counterstained with cresyl violet or hematoxylin eosin. All sections were mounted on albumincoated slides, dehydrated, cleared and coverslipped. The slides were observed with a Nikon Microphot optical microscope equipped with an interferential contrast system and photographed with a Nikon FX-35A apparatus. Control sections were processed in the same way except for the replacement of the primary, secondary or tertiary antibody with swine normal serum. Other control sections were incubated with NPY
antiserum at the dilution of 1/10,000 previously absorbed with 3.8 micrograrn/ml of synthetic NPY. Other sections of the same cortical regions were processed for NPY labelling using the immunofluorescent technique of Coons: after 15 rain rinsing in 0.1 M PBS, sections were incubated at 4°C for 15-24 hours with NPY antiserum diluted 1/1000 in 0.1 M PBS-Tx 0.3%. After 30 min of multiple washing in PBS-Tx 0.3%, sections were finally incubated for 30 min at room temperature with fluoresceine isothiocyanate conjugated with swine antirabbit immunoglobulines (Fitc, Sigma, St. Louis, MO) diluted 1/100 with 0.3% Triton X-100. Sections were then rinsed three times for 10 min each in 0.1 M PBS, dried and mounted in a glycerol-water solution containing 25 g/l of 1,4-Diazabicyclo(2,2,2)octane (Dabco, Aldrich, Steinheim, Germany). Slides were examined with a Leitz Wetzlar fluorescence microscope equipped with a special Leitz L3 filter mirror, reducing the yellow interference fluorescence of neuronal lipofushine. In order to facilitate the study of the distribution of NPY immunoreactivity, composite drawings of NPY cells, processes and fibers plexuses were made with a Nikon camera lucida. NPY positive cells were counted and a semi quantitative estimate of their distribution was made. The nomenclature used to designate the cortical cells was based on their axonal and dendritic arborization patterns, and characteristics of the cell bodies examined were compared with the Golgi impregnation cells described elsewhere [36].
NPY IN H U M A N VISUAL CORTEX
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FIG. 3. Composite camera lucida drawings of NPY cells and fibers immunoreactivity in the human primary visual cortex and underlying white matter, demonstrated with PAP and FITC techniques. In A, all the subtypes of NPY cells are visible: a pyramidal in layer III (a), a bipolar in layer IV (b), bitufted cells in layers I to VI (c, d, e, f), multipolar cells in layer IV and V which give highly branched processes (g, h, i). Note the bitufted cells in layer VI (f) sending long processes towards layer I. In the underlying white matter, many immunoreactive cells are present, either separate (j) or in small groups (k). In B, a superficial plexus of NPY horizontal parallel fibers runs in layers I and II. A second clear cut plexus is located in layer IVb and c while the third plexus runs in deep layer V and in layer VI. These plexuses receive branches from oblique and vertical fibers ascending from the deep cortical layers or from the underlying white matter and distributing through the entire cortex. Note the background rich network of positive processes. Cortical layers: I-VI. White matter: WM. Bar=400 /zm.
RESULTS
N P Y lmmunoreactivity Sections from all the cortical areas examined displayed numerous intensely stained somata, processes and fibers, with an identical distribution among all the cases studied. The intensity of staining was much more higher in the young child (case 9) than in the adult brains (cases 1-8). It diminished when the post mortem delay prior to fixation was increased (cases 4 and 7). Treatment of the sections with hydrogen peroxide resulted in a clearer picture of the NPY distribution due to the attenuation of the staining of the red blood cells peroxidase. None of the control sections displayed any positive staining.
N P Y Cell Somata Both non pyramidal and pyramidal neurons were labelled but a much more intense and frequent staining was seen in the former. Non pyramidal NPY positive cells were bipolar (Fig. IA), bitufted (Fig. 1D) or multipolar (Fig. 1C, E) aspiny neurons. Multipolar neurons were the most prevalent NPY immunoreactive cells, bitufted neurons comprised the second most common morphological group of NPY cells while the bipolar neurons were only sparsly scattered through the cortex (Fig. 2). Some NPY neurons, principally in layer III and V, were typically pyramidal in shape, with a long apical and a few
basal aspiny dendrites and a descending beaded axon (Figs. 1B, 2, 3A). There was no reliable difference in the morphology and distribution of NPY positive cells between cortical areas 17, 18 and 19, except for a relatively poor distribution of NPY neurons in layer IV or area 17 compared to the same layer in area 18 and 19. NPY positive cells were mostly dispersed in a network of non immunoreactive cells, and only a few were aggregated in small groups. NPY neurons were present in cortical layers I to VI (Figs. 1, 2, 3A) but their distribution varied from one layer to another. Only a few cells were demonstrable in layer I where they were typically small (5-10 p.m) and mostly bitufted (Fig. 2). The few positive bipolar cells were located in layer III and IV (Figs. 1A, 2, 3A). Many NPY cells were present in deeper layers, especially in layers V and VI, where they were mostly bitufted or mulfipolar (Figs. IC-E, 2, 3A). These cells were usually larger (8-25/zm) than those found in layer I, with a great number of long processes. One or more of these processes always showed a vertical orientation. In deep layer V and layer VI, many horizontal bitufted neurons showing long rectilign processes running parallel to the border with white matter were recognized with the NPY antiserum (Fig. 2). In these layers, some vertical bitufted cells send long ascending processes sometimes ending in layer I and II. Interestingly, NPY positive cells were also visible in the underlying white matter of the three cortical areas studied
1112
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FIG. 4. N P Y i m m u n o r e a c t i v e cells in the h u m a n corticovisual underlying white matter. (A) The perikarya and the p r o c e s s e s of this N P Y cell are parallel to the cortical junction. Note the long process entering layer VI (arrow), and a cluster of varicosities of the inferior cortical N P Y plexus (arrow head), The regular dark gray structures are red cells in blood vessels colored with the PAP technique (asterisk). (B) A small fusiform N P Y i m m u n o r e a c t i v e cell with two poorly branching beaded processes. (C, D) N P Y cells with two different types of processes arborization: highly beaded (C) and non beaded (D). (E) A squared N P Y cell in the deep white matter, Note the thin axon (arrow) running obliquely in regard to the myelinated fibers orientation, given by the dendritic orientation. Rabbit a n t i s e r u m raised against NPY. P A P technique of Sternberger. Interferential contrast. Cortical layers: I-VI. White matter: WM. B a r = 10 ~ m .
NPY IN HUMAN VISUAL CORTEX
1113
FIG. 5. NPY immunoreactive terminals in the primary visual cortex. (A) Multipolar cell arborizing in layer III and terminating upon a small vessel obliquely cut. Note the fiber entering the vessel wall. Some NPY processes originating from elsewhere also end upon this vessel. (B) Terminal arborization of a NPY bitufted cell; note the palissade aspect of this highly branched ending process. (C) NPY immunoreactive cortical multipolar cell surrounding a small capillary: the small NPY vesicules fulfill the entire perikarya except the cell nucleus (arrow). (D) Detail of the deep cortical fibers plexus: a network of highly circonvoluted and branched processes is crossed over by vertical ascending fibers originating from the underlying white matter (arrows) and sending branches to each other (arrow heads). Rabbit antiserum raised against NPY. PAP technique of Sternberger. Intefferential contrast. Vessel: V. Bar=25/xm.
1114 (Figs. 2, 3A, 4). A semi-quantitative analysis indicated that, according to a surface basis, only 20-40% of the NPY positive cells were present in the cortical layers while the remaining 60-80% were found in the underlying white matter. These cells, often aggregated in groups of three or four, showed a typical distribution in the white matter: a palissade of small cells, mostly bitufted, were located just under or at the junction with layer VI (Figs. 2, 3A, 4A) with their long axis parallel to the myelinated fibers. Many of them gave processes that extended first horizontally and then projected vertically or obliquely into the grey matter (Figs. 2, 4A). Deeper, many small NPY cells with a great variety of shapes (Fig. 4B-E) and orientations (parallel, perpendicular or oblique towards the myelinated fibers) were noted. Their processes were very long, being either linear or circonvoluted, beaded or not (Fig. 4C, D). N P Y Cell Processes
Most of the NPY cells showed numerous and very long processes, often presenting round or irregular beaded varicosities separated by thin or thick intervaricose sections (Fig. 1C). Emerging at various angles of the cell somata, these processes branched at different distances from the cell of origin. Other processes were short, twisted or isolated labelled puncta without any evident cell of origin. NPY processes formed a rich network of vertical, horizontal or oblique tracts present in all the layers of the three regions examined. These processes showed various morphological patterns: rectilignes, twisted, in S or in scale shape. Some NPY processes, often originating from bitufted cells, had vertically oriented branching terminals, showing an aspect "en palissade" (Fig. 5b). Clusters of terminals were observed, forming bulbous swellings which contributed to the formation of pericellular baskets arranged around other NPY immunoreactive or non immunoreactive cells (Fig. 5B, D). In the primary visual cortex (area 17), three clearly separate and well organized plexuses of NPY fibers could be distinguished (Figs. 3B, 6). A superficial plexus, located in layer I and II, consisted of a great number of long fibers running tengantially to the cortical surface. These fibers could be followed for long distances (1-4 mm), going sometimes from one gyrus to another. Cells of origin of these fibers could not be determined and must probably be searched in other cortical gyri. A few of these long parallel fibers send to each other small beaded and not beaded processes. Just beneath the cortical surface, fibers were dense and linear, sometimes pooled into spindles of 3 to 4 fibers, while deeper in layer II they were more distant, twisted and anarchically disposed (Fig. 6). This superficial plexus received long vertical parallel fibers arising from the depth: these fibers were thin and arise either from the various NPY cells of the deep cortical layers or from the underlying white matter, where cells of origin were rarely visible. The origin of many of these vertical fibers could not be established. From this superficial plexus branched off a number of mostly twisted fibers which terminated in layers III and IV (Figs.
VAN REETH ET AL. 3B, 5D, 6). A mid plexus was present in layer IV, principally in the sublayers IVb and IVc, i.e., the external band of Balllarger (Figs. 3B, 6). Fibers of this dense plexus had a strongly vertical or oblique orientation. Terminals were principally made of clusters of highly branched and circumvoluted processes ending on non immunoreactive neurons. Some of these bulbous terminals did not contact to any cell. Cells of origin of all fibers could not be established, this layer being particularly void of NPY cells in this area. This plexus received branches of the long vertical fibers arising from the depth and ending in the superficial plexus. A deep plexus of fibers was located in deep layer V and layer VI of the primary visual cortex (Figs. 3B, 6). It consisted principally of clusters of highly branched and twisted fibers emanating from multipolar or bitufted cells from layers IV, V and VI and from underlying white matter cells. This plexus was crossed both by horizontal processes of the bitufted cells of these layers and by vertical and oblique NPY fibers arising from the underlying white matter. In visual areas 18 and 19, only the superficial plexus (layer I and II) and the deep plexus (deep layer V and layer VI) of NPY fibers were clearly visible, and the mid plexus of layer IV was not demonstrable. In the subcortical white matter of the eight adult brains, NPY processes were less abundant and stained lighter than in the cortical layers. NPY cells located at, or just under the cortical junction gave many processes, one of which at least took a vertical (or oblique) orientation and reached the upperlying gray matter, where it gave branches and terminated. Deeper in the white matter, NPY small cells gave long and thin processes which mostly followed the myelinated fibers, sometimes for several hundred micrometers. Only a few processes showed a perpendicular or oblique disposition towards the myelinated fibers (Fig. 4E). Many long thin and thick fibers could be followed for long distances without showing their cell of origin: they may originate from other cortical areas (Fig. 3B). The subcortical white matter of the juvenile brain showed a more richer network of NPY processes than the adult. N P Y Immunoreactivity and the Vessels
In all cortical and subcortical regions examined, many NPY positive beaded fibers were observed to run along and wind around small blood vessels (Fig. 5A, C). These processes formed clusters of dilatations and terminated with club endings on vessels, sometimes entering their walls (Fig. 5A). Cells of origin of these fibers, mostly bitufted and multipolar, were sometimes seen in the vicinity of the innervated vessels (Fig. 5A), but more often were not visible. DISCUSSION
The need for studies of human neuropeptides distribution is stressed by the well known species variation concerning their distribution and by the uniqueness of the human brain. Since no experimental studies can be performed in humans, a discussion of what is already known in other species will be
FACING PAGE FIG. 6. NPY immunoreactivity in the human primary visual cortex. Photomicrograph of NPY positive processes (arrow) from a section passing through the bank of the calcarine fissure. Main line of Gennari is located in layer IVb. Three main plexuses are distinguishable: the upper one, just under the pial surface, the medium one in layer IVb, c and the deep one located in deep layer V and layer VI. Arrowheads indicate vertical ascending NPY fibers reaching the superficial plexus of fibers. A multipolar cell is clearly seen in layer III. Rabbit antiserum raised against NPY. PAP technique of Sternberger. Cortical layers: I-VI. White matter: WM. Bar=400 tzm.
NPY IN H U M A N V I S U A L CORTEX
1115 reported. Previous studies have shown the existence of NPY in the human cerebral cortex [1, 5, 11] and have studied its distribution in frontal, parietal and temporal cortices [4]. The present immunocytochemical study confirms the presence of a large number of NPY cells and processes in the human visual cortex and further elucidates its distribution in this particular cortical region. The visual cortex of the rat [5,6], cat [51] and monkey [19,20] have been the focus of several studies on distribution and morphology of NPY neurons and processes. In these animal studies, it has been reported that NPY immunoreactivity is located exclusively in non pyramidal neurons. Our results reveal that NPY is also present in some medium-sized pyramidal neurons in layers III and V of the human visual cortex. These findings raise the possibility that NPY may play a role in visual efferent pathways, as already suggested for somatostatin in the rat visual cortex [24]. This hypothesis could also be supported by our findings of many positive, presumably associative, NPY fibers running from the subadjacent white matter through the corpus callosum (unpublished data). Similar findings have indeed been recently reported in the rat [31] and the cat [51] cortex. Numerous NPY neurons are small, spineless, multipolar and bitufted neurons. Electron microscopic and immunocytochemical studies performed in cat and monkey visual cortex have clearly shown that axons of these small and aspiny multipolar cells form G r a y ' s type II synapses [26, 36, 39] and use G A B A as a neurotransmitter [22,40]. Both findings suggest that these neurons exert a local inhibitory role. The large (15-25/xm) multipolar neurons with an abundant axonal arbor which forms one or more, either " e n passant" or terminal, baskets around pyramidal or nonpyramidal perikarya could be "Basket cells" well described in the cat [29, 39, 41] and considered as one of the major inhibitory neurons in the cat visual cortex [43,44]. A positive NPY staining of cortical "Basket cells" has recently been reported in rat and cat by some [21,51] but not in cat and monkey by others [20]. Some cortical bipolar neurons are also recognized with our NPY antiserum. These cells have been proven to form G r a y ' s I synapses with pyramidal and non pyramidal cells in rat and cat visual cortex [36, 38, 39] and to not contain GABA. Based on these observations, it is assumed that bipolar cells exert a local excitatory effect in the visual cortex [36]. These results are of particular interest since they suggest that in the human visual cortex, NPY is located both in inhibitory (bitufted and multipolar) and excitatory neurons (bipolar and some pyramidal), the former being predominant. It implies that NPY may coexist with other neurotransmitters or neuromodulators in the same neurons. A coexistence of NPY and somatostatin has already been reported in various rat and human cortical areas [5]. Immunofluorescence studies performed in the cat and monkey visual cortex indicate that all the NPY positive neurons in the cat and a majority in the monkey also stain positively for GAD, suggesting that these neurons could release G A B A as an inhibitory transmitter together with N P Y as a neuromodulator [19]. In the human visual cortex, NPY stained perikarya tend to be seen predominantly in deeper layers of the cortex, as described in the rat [5,6], cat [51] and monkey [20] visual cortex. Our results are in accordance with previous reports on NPY cell distribution in other areas of the human
1116
VAN REETH E T A L .
cortex [1, 4, 11], except for the particularly poor distribution of NPY cells in the well developed layer IV of area 17. NPY cells found in the underlying white matter are comparable in size and shape to those in the cortex. These cells are described in rat, cat and monkey and considered as neurons, on morphological basis [5, 20, 51]. Large (50-120 /zm) somatostatin cells have also been described in the human subcortical white matter [45]. Functional properties of NPY white matter cells is unclear: the superficial ones, which are probably neurons that have not migrated enough during the cortical development, send processes to the upperlying gray matter and participate to the elaboration of the deep NPY cortical plexus. The deep white matter cells, which can be located as far as the corpus callosum, may participate to intra- and interhemispheric associative cortical pathways. NPY fibers distribution shows a typical laminar organization. We describe three clearly separated plexuses in area 17, while only two can be distinguished in area 18 and 19. The superficial and deep plexuses have been described in other cortical areas of the human brain [4, 5, 11], while the mid plexus of layers IVb and IVc seems to be characteristic of the primary visual cortex. It has also been described in layer IV of cat and monkey primary visual cortex, being predominant in sublayers IVa and IVb in these species [20]. Laminar distribution of NPY fibers seems to correspond to the distribution of afferent and efferent pathways or cells within the visual cortex: the upper plexus of layers I and II and its extent to the underlying layers is located in the ending region of most of the corticocortical associative fibers [9, 25, 41, 42]. Cells of origin of many of these superficial fibers may arise from other cortical or subcortical regions. The mid plexus of layers IVb and IVc (i.e., the outer band of Bail-
larger) is in the ending region of thalamocortical fibers and some associative corticocortical fibers [7,8], while the deepest plexus of deep layer V (i.e., the inner band of Baillarger), layer VI and underlying white matter is located in the infragranular cortical region involved in the efferent pathways of the visual cortex where also terminate thalamacortical fibers. Many NPY fibers present terminals around blood vessels both in gray and white matter. This localization suggests that, together with VIP and Substance P, NPY may participate to the regulation of cortical blood flow [13--15, 27]. Some of these fibers originate from neurons located in the vicinity of the innervated vessels, suggesting that neurons involved in the inner cortical circuitry may also regulate the cortical blood flow. However, more often, cells of origin are not visible and are probably located in other areas. Coexistence of NPY and noradrenaline in the same nerve fibers, well established in the peripheral nervous system, has also been reported in dense nervous plexuses surrounding cerebral and pial vessels in feline [ 16]. The functional interaction between NPY and central adrenergic control of blood pressure, together with the rich network of NPY fibers innervating the cortical vessels and originating out of the cortex, suggest the possibility of a coexistence of NPY and noradrenaline in the human locus coeruleus, as reported in the rat [17]. ACKNOWLEDGEMENTS This work was supported by grants from the Belgian National Fund for Scientific Research (FNRS 85-86) and Medical Scientific Research (FRSM 34521.82-86 and 34523.86-89), the Belgian National Lotery (1983 and 1986) and the Queen Etisabeth Medical Foundation (FMRE 81-84 and 86-89).
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