Neuropeptide Y-containing neurons in the human infant hippocampus

Brain Research, 478 (1989) 211-226 Elsevier

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Neuropeptide Y-containing neurons in the human infant hippocampus F. Lotstra, S.N. Schiffmann and J.-J. Vanderhaeghen Laboratories of Neuropathology and Neuropeptide Research and of Pathology and Electron Microscopy, Facultyof Medicine, Erasme and Brugmann Hospitals, Universit~Libre de Bn~xelles, Brussels (Belgium) (Accepted 12 July 1988)

Key words: Human; Infant; Hippocampus; Ontogeny; Neuropeptide Y; Immunohistochemistry

Using immunohistochemistry, high concentrations and widespread distribution of neuropeptide Y-immunoreactive (NPY-IR) neurons were found and examined in each region of the hippocampal formation from birth to 42 years. NPY interneurons are particularly numerous in the stratum oriens of the CA 1 subfield, in the deep layers of the subicular complex and entorhinai cortex. They are multipolar round, ovoid or triangular or bipolar and fusiform. There is a dense network of NPY-IR nerve fibers in the subicular complex and the entorhinal cortex. In addition, numerous NPY-IR nerve cell bodies and fibers are observed ill the angular bundle and the adjacent white matter and this contrasts with the absence of NPY immunoreactivity in the fiber tracts of the alveus. These NPY-IR neurons which correspond to the interstitial neurons of the white matter, have the morphology and the size of the interneurons detected in the cortex. During the postnatal brain growth spurt which corresponds to the phase of rapid myelination, there is no decline in total number of NPY-IR neurons but there is a decrease in density. They have been spread apart by the growth of the rest of the tissue. So in humans, the total number of NPY nerve cell bodies iL the hippocampal system, firmly established at birth, is not modified during consequent brain growth which continues until ages 3-4 years and stays stable at least until age 42 years.

INTRODUCTION The compelling problems of human developmental diseases and the increasing prospects of fetal diagnosis demand that ontogenic mechanisms in the human brain be viewed and understood. The hippocampus or hippocampal formation is a brain structure well suited to study development considering its lamellar organization and its stratified structure as well as the large body of information available about its synaptic connections 39. It is generally considered to be comprised of several distinct regions including the area dentata, the Ammon's horn, the subicular complex and the entorhinal cortex 42. Neuropeptide Y (NPY) is one of the most abundant peptides in the human adult hippocampus as detected by radioimmunoassay LI4 and immunohistochemistry 7'9. In all species studied, the highest den-

sities of NPY-specific binding sites have been observed in the hippocampus 6"26"33. In addition to its abundance, NPY is of interest because it appears to be involved in the excitatory transmission of the hippocampus 4.11.12'2°. NPY has also been shown to be a modulator of memory processes 17. Numerous other peptidergic neurons have been previously reported as being found in the human hippocampus both in infant and adult 7"9"15"3°-32"37 and marked age-related changes in their distribution have been noted during postnatal development. It would be expected that any alterations in peptidergic hippocampal pathways and functions in the developing human would be manifested in later behavior. In this respect, this work reports the morphology and the distribution of NPY-immunoreactive (NPY-

Correspondence: F. Lotstra. Laboratory of Neuropathology and Neuropeptide Research. Universit6 Libre de Bruxelles. Campus Anderlecht, CP 601. 808 route de Lennik, B-1070 Brussels. Belgium. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

212 IR) neurons in the infant as compared to adults using the peroxidase-antiperoxidase (PAP) technique of Sternberger 4~modified for floating sections.

7.6, for a duration of I h before starting the immunohistochemical staining procedure.

Immunohistochemical staining procedure MATERIALSAND METHODS

Human material Brains belonging to persons of both sexes, without obvious neurological or psychiatric diseases, were used in this study. The postmortem delay was 8 + 5.8 h (mean + S.E.M.). Brains were subsequently examined by classic~ neuropathological macroscopic and microscopic methods and no evidence of pathologicaI changes was observed. There were 1 male patient aged 42 years and 9 infants: 5 males (respectively 2 and 3 days; 4 and 10 months and 4 years old) and 4 females (4 days; 3 and 6 months and 1 year of age).

Antiserum The anti-NPY antiserum was developed in New Zealand rabbits after multiple subcutaneous injections of a mixture of synthetic porcine NPY, methylated bovine serum albumin (BSA, Sigma, St. Louis, U.S.A.) and complete Freund's adjuvant. Using radioimmunoassay procedures 35, this antiserum was found to cross-react at less than 0.1% with avian pancreatic polypeptide (APP), 0.01% with peptide YY (PYY), 0.0001% with gamma-MSH and VIP and not with a-MSH, 1-39 ACTH, B-LPH, SRIF-14, CCK-8, CRF, Leu-enkephalin, Met-enkephalin, TRH, arginin-vasopressin and neurotensin.

Preparation of tissue sections The hippocampal formation was dissected and cut in the rostrocaudal orientation in 3 coronal blocks (anterior, middle and posterior hippocampus). Blocks were immediately transferred into a 4% w/v paraformaldehyde solution diluted by 0.1 M phosphate-buffered saline (PBS), pH 7.4, and then fixed for 24 h at 4 °C. Specimens were then placed successively in 10%, 20%, 30% w/v sucrose solutions in PBS at 4 °C, each for a period ranging from 24 to 36 h. They were then frozen in 2-methylbutane (Merck, Darmstadt, F.R.G.) which had been cooled in dry ice, and finally stored at -80 °C until use. Serial coronal slices (35/zm in thickness) were cut with a cryostat (Dittes, Heidelberg, F.R.G.), and floating sections were rinsed in 0.01 M Tris-buffered saline (TBS), pH

The PAP technique of Sternberger 41 was used. Each step was executed at room-temperature except for the overnight incubation with NPY antiserum. Prior to the PAP procedure, the sections were treated for 15 min with 2% v/v hydrogen peroxide solution to inhibit red cells eadogenous peroxidase activity. After 3 washing steps in TBS, they were preincubated for 60 min in a 20% v/v mixture containing swine normal serum (Dakopatts, Glostrup, Denmark) diluted in TBS containing 0.1% v/v Triton X100 (TBS-TX) (Merck, Darmstadt, F.R.G.). They were then incubated overnight at 4 °C in the NPY antiserum diluted with TBS-TX in decreasing assay dilutions, ranging from 1:2,000 to 1:10,000. Dilution of 1:4000 gave optimal immunostaining and was used as a standard. After 5 washings in TBS-TX, the sections were further incubated for 30 min in a 1:30 v/v mixture containing swine anti-rabbit antiserum (Dakopatts, Glostrup, Denmark) diluted in TBS-TX. They were again washed 3 times in TBS-TX, and then incubated for a 30-rain duration in a 1:300 v/v mixture containing rabbit PAP complex (gift from F. Vandesande, Leuven, Belgium) diluted in TBS-TX. After 3 final washings in TBS-TX, they were incubated for 15-25 min in a solution containing 0.0125% w/v 3,3-diaminobenzidine tetrahydrochloride (Sigma, U.S.A.), and 0.003% v/v hydrogen peroxide diluted in 0.05 M Tris buffer. Reactions were stopped by immersion of sections in distilled water.

Immunohistochemical control procedures Controls were made by exchanging the primary antiserum with a rabbit normal serum. Immunohistochemical cross-reaction experiments were also performed by using NPY antiserum diluted at 1:4000 and absorbed with an excess (10 -6 M) of PYY, APP, ACTH, endorphins, VIP, CCK-8, SRIF and LHRH. Soluble immunoabsorption with these various peptides did not affect the staining intensity 35, whereas synthetic NPY (10 -6 M) completely blocked the NPY-I R nerve cell bodies and fibers from staining. Since ~t is impossible to exclude the possibility that this antiserum could cross-react with unknown peptides with similar antigen'ic sites, in this study 'NPY

213 immunoreactivity' is synonymus with the more correct 'NPY-li~e immunoreactivity'.

Image analysis Section area, number of NPY-IR nerve cell bodies per section and their density expressed as the number of NPY-IR nerve cell bodies per cm 2, were determined on transversal sections through the hippocampal formation at the middle level. These sections include the Ammon's horn~ the area dentata, the subicular complex, the entorhinal cortex as well as the angular bundle and the adjacent white matter (see Fig. 9). Two newborns (2 and 4 days), 4 infants (3, 4, 10 and 12 months), 1 child (4 years) and 1 adult (42 years) were studied. For this analysis a Zeiss-Kontron IBAS image analyzer (Zeiss-Kontron, Munich, F.R.G.) was used in combination with a Nikon microscope equiped with an MTI video camera for cell count or directly with the MTI video camera for section area. The analysis was performed on interactively discriminated images and subsequently performed on binary images showing exclusively the NPY-IR nerve ceil bodies.

Mapping For identification of hippocampal cytoarchitecture, some preparations were counterstained either with Cresyl violet or hematoxylin eosin, or impregnated with reduced silver nitrate. Nomenclature used in this report follows those used by Cajal 5, Lorente de N o 2~'29 and Blackstadt 3.

RESULTS

General comments NPY-IR neurons will first be described as found in the infant and then compared with results obtained in the adult. The description covers the area dentata, the Ammon's horn, the subicular complex, which includes the prosubiculum, the subiculum, the presubiculum and the parasubiculum, as well as the entorhiha! cortex and the white matter. No appreciable rostrocaudal difference in distribution of NPY-IR cell bodies and fibers was found. NPY immunoreactivity was detected in several types of neurons but never observed in identifiable pyramidal or granular cells. The NPY-IR cell bodies most frequently observed were multipolar, ovoid or round. Bipolar and fusi-

form NPY-IR cell bodies were also detected. The diameter of the round cells is between 12 and 25 #m. Bipolar fusiform and ovoid cell bodies may measure between 20 and 40 um in length. More rarely observed were NPY-IR triangular cell bodies measuring between 12 and 20 #m high and between 20 and 40~m in breadth. Most of the NPY-IR cell bodies are situated in the hilus, the stratum oriens of the CA1 subfield, the deep parts of the prosubiculum, subicuium and the entorhinal cortex. In addition they were found to be numerous in ~he angular bundle and the adjacent white matter. Most of the NPY-IR cells are vertically oriented between the pyramidal cells of the Ammon's horn, subicular complex and entorhinai cortex. NPY-IR horizontally oriented cells were observed in the stratum oriens, and in the deepest layers of the subicular complex and entorhinal cortex. Some NPY-IR cells of the subicular complex and entorhinal area are distinguished by their long, densely branching dendrites. NPY-IR nerve fibers are present in all areas of the hippocampal formation but are far more prominent in the stratum oriens of the CA1 subfield, in the subicular complex, entorhinal cortex as well as in the angular bundle and the adjacent white matter.

Area dentata The term area dentata comprises the granular layer, its associated molecular layer and the subjacent polymorphic zone or hilus of fascia dentata 3. A few NPY-IR cell bodies are seen in the outer two-thirds of the molecular layer (Fig. 1A). They are multipolar, ovoid and horizontally oriented. Their horizontal processes are parallel to the granular layer. They are fairly long and may measure up to I mm. Numerous NPY-IR cell bodies are observed in the polymorphic zone of the hilus (Fig. 1C). Most of them are mu~tipolar and ovoid (Fig. 1E) or bipolar and fusiform (Fig. 1D) but multipolar and round (Fig. 1B) were also detected. No NPY-IR cell body was detected in the inner part of the molecular layer or in the granular layer. Numerous NPY-IR scattered beaded fibers run,rag in a crisscross fashion were detected in the outer two-thirds of the molecular layer. In the inner one-third of the molecular layer only rare beaded NPY-IR fibers were seen and most of them were perpendicular or parallel to the granular layer. Large beaded fibers, vertically oriented, cross the subgranular zone, granular and

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Fig. 1• NPY-IR neurons in the area dentata. Infant of 10 months of age. A: multipolar and ovoid nerve cell body parallel to the granular layer in the outer two-thirds of the molecular layer. C: NPY-IR fibers in a meshwork arrangement and NPY-IR cell bodies in the polymorphic zone of the hilus where small round multipolar (B), fusiform bipolar (D) and ovoid multipolar (E) nerve cell bodies are observed. PAP technique. Bar = 50~um.

i n n e r molecular layers• Some of t h e m are processes of N P Y - I R p o l y m o r p h cells located in the hilus.

cept in the stratum m o l e c u l a r e w h e r e only fibers w e r e be d e t e c t e d .

T h r o u g h o u t the hilus, scattered b e a d e d N P Y - I R fibers in a m e s h w o r k a r r a n g e m e n t w e r e observed.

A l v e u s . N P Y - I R cell bodies are rare in this stratum, they are bipolar, fusiform and horizontal (Fig.

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2B). T h e y are essentially d e t e c t e d in the CA1 subfield of the A m m o n ' s horn. N P Y - I R b e a d e d fibers

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are also rarely found in the alveus, and if so they are mostly parallel to the pyramidal layer. Some of t h e m

Fig. 2. NPY-IR neurons in the Ammon's horn. Infant of 10 months of age. A: numerous nerve fibers and cell bodies of the stratum oriens (so) in the CA1 subfield contrast with the poverty of NPY-IR material in the stratum pyramidale (sp). B: bipolar fusiform nerve cell body in the alveus, parallel to the pyramidal layer. C: CA2 subfield. Note the difference in the content of nerve fibers and cell bodies in the stratum oriens (so) as compared to the CA1 region in A. Fibers of the pyramidal layer (sp) are particularly numerous in its deep part. Stratum radiatum (sr) is distinguished by the limited number of nerve fibers and cell bodies. D: multipolar round or ovoid neurons in the stratum oriens (so) with dendrites reaching the alveus and the stratum pyramidale (sp). E: high magnification of NPYIR fibers around the non-immunoreactivepyramidal neurons (arrowheads) in the deep part of the stratum pyramidale (sp) in the CA2 subfield. F: multipolar round neuron in the stratum pyramidale of the CA2 subfield. G: thick horizontal fibers (arrow) and thin vertical fibers (arrowhead) in the stratum moleculare within the subfield CA2. H: multipolar ovoid neuron in the stratum pyramidale of the CAI subfield. PAP technique. Bar = 50lim.

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are processes of the rare positive cell bodies detected in the alveus or processes of the NP~'-IR cells in the stratum oriens. Stratum oriens. The two NPY-IR cell types most frequently observed are multipolar, round or ovoid and horizontally oriented with dendrites in the stratum oriens and dendrites reaching the alveus and the stratum pyramidale (Fig. 2D). Bipolar, fusiform NPY-IR cell bodies, horizontally oriented are rarely seen in this layer. There is a clear gradient in the distribution of NPY-IR nerve cell bodies and fibers of the stratum oriens, more abundant in the CA1 subfield (Fig. 2A) than in the CA2 and CA3 regions (Fig. 2C) and more abundant at the limit between CA1 and prosubiculum than at the limit between CA1 and CA2. Most of the NPY-IR beaded fibers in the stratum oriens are processes from positive cell bodies observed in this layer and are horizontally oriented. These fibers are fairly long, they may measure up to 1.5 mm in length in the section. Stratum pyramidale. NPY-IR cell bodies are seen in the superficial part of the subfield of the pyramidal layer (Fig. 2C). They are more numerous in the CA1 region. They are multipolar and ovoid or round with vertically oriented dendrites (Fig. 2F,H). Large beaded NPY-IR fibers, most of which are vertical, are present in the stratum pyramidale. A few can be identified as processes from NPY-IR cell bodies observed in this layer. Fibers are particularly abundant in the deep part of the CA3 and CA2 subfields of the stratum pyramidale, giving rise to a plexus which envelops non-immunoreactive pyramidal cell bodies (Fig. 2E). Stratum radiatum. Positive cell bodies identical to those present in the stratum pyramidale are rarely observed. Scattered large beaded NPY-IR fibers similar to those observed in the stratum pyramidale were detected in the stratum radiatum (Fig. 2C). Stratum moleculare. No NPY-IR cell body is observed in this stratum• Thin and thick short beaded NPY-IR fibers appear to emerge from layer I of the prosubiculum. They are parallel to the stratum pyramidale and, in the CA2 and CA3 subfields, they tend to become more numerous and perpendicular to the pyramidal layer (Fig. 2G).

The subicular complex A large number of NPY-IR cell bodies and fibers

are present throughout all subfields of the subicular complex without variation from anterior to posterior levels. Each part of the subicular complex is distinguished by its content and its distribution of NPY-IR cell bodies and beaded fibers. While NPY-IR fibers are present in all layers of the subicular complex and mostly in the superficial layers, NPY-IR cell bodies

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217 are essentially found in the deep layers and sometimes they are located in islands. Their general morphology is the same in all subfields of the subicular complex as in the Ammon's horn except that their processes are often more tufted, longer and more

ramified. Most of the NPY-IR cell bodies are multipolar ovoid or round. More rarely observed are NPY-IR bipolar and fusiform cells. Most of these NPY-IR cell bodies and their ramified dendrites are vertically oriented between the non-immunoreactive

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Fig. 4. NPY-IR nerve fibers and cell bodies in the subiculum of a 3-day-old infant. Nerve cell bodies are observed in layers II, III, IV and V and in the white matter of the angular bundle (WM). In layer III, they are located in islands• Most of them are multipolar ovoid or round and vertically oriented in layers II, III and in the superficial part of layer IV. They are horizontally oriented in the deep part of layer IV and layer V. PAP technique• Bar = 100/im.

218 pyramidal cells, although in the deepest layer of the subicular complex most of them are horizontally oriented. Prosubiculum. The limit between CA1 and the prosubiculum is determined by the sudden disappearance of the stratum radiatum 29. Numerous NPYIR cell bodies are seen in layers III and IV (Fig. 3). They are vertically oriented in layer III and horizontally oriented in layer IV. NPY-IR beaded fibers are numerous in layer I. They may be thick or thin and most of them are short and horizontally oriented. In layers ii and II1, NPY-IR beaded fibers are scattered. Most of them are long, thin and vertically oriented. In the deep part of the layer III, fibers become more numerous and are arranged in a crisscross fashion. The limit between layer 111 and IV is determined by a distinct decrease of NPY-IR beaded fibers in layer IV which are usually horizontally oriented. Subiculum. The subiculum is the region of the subicular complex where the most numerous NPY-IR cell bodies are found (Fig. 4). They are essentially located in layer I11 but they are also detected in layers II and V. In layer III NPY-IR cell bodies are often distributed in islands of 5 or 6. They are multipolar, round, ovoid or triangular. Bipolar and fusiform NPY-IR cell bodies are rarely detected in these islands. All the NPY-IR cell bodies are vertically oriented except in layer V where they are horizontal. Layer I of the subiculum is exceptionally rich in NPY-IR beaded fibers. Most of them are vertically oriented but large, beaded, horizontally oriented fibers are numerous in the most superficial part of layer I. NPY-IR beaded fibers become progressively less numerous in layers II and III. Some of them are long and vertically oriented. They are homogenously distributed in these two layers, such that the distribution in islands of small non-immunoreactive pyramidal cells in layer II is not distinct. In layer IV, NPYIR fibers become much less abundant. Layer V is distinguished by its density of large horizontally oriented beaded fibers. Presubiculum. Islands of NPY-IR cell bodies similar to these observed in the subiculum are observed in layers III and IV (Fig. 6C). Horizontally oriented cell bodies are numerous in layer V (Fig. 5A). A dense network of thin and short NPY-IR beaded fibers oriented in all directions is present in layers I,

II, III and V of the presubiculum. Layer I is distinguished by the presence of numerous horizontally oriented beaded fibers in its superficial part. In layer IV, the density of the NPY-IR fibers is less intense than in the other layers. Long and ramified fibers, vertically oriented, in layers III and IV, are processes from NPY-stained cell bodies seen in these two layers. Parasubiculum. Horizontal cell bodies were rarely detected in layer I but large triangular cell bodies measuring 40/~m in breadth and 20/~m high have been observed in this layer (Fig. 6G). Multipolar, round or ovoid NPY-IR cell bodies with vertical long processes are usually detected in layer III (Fig. 5B). They are often located in islands of 3 or more cells. In layer VI, horizontal NPY-IR cell bodies are less numerous than in layer V of the subiculum and presubiculum. NPY-IR fibers in layer I of the parasubiculum are similar to those of the presubiculum. Short beaded NPY-IR fibers oriented in all directions are relatively evenly distributed throughout the remaining layers with a slightly higher density in layers II and VI. Some of the NPY-IR fibers are very long and vertically oriented in all layers except layer I. The entorhinal cortex As in the subicular complex, most NPY-IR cell bodies are located in the deep layers V and VI and in layer III (Fig. 7A). The entorhinal cortex contains fewer NPY-IR cell bodies than the subicular complex and the arrangement of islands is never observed. Most of the NPY-IR cells are multipolar ovoid or round (Fig. 6B). They often have 3 dendritic axes (Fig. 6D,F). Round cells can be large measuring up to 20-25/~m in diameter. Bipolar and fusiform cell bodies are detected in all layers (Fig. 6A,E). Horizontal cell bodies with horizontal ramified dendrites are essentially located in layers I, i V and VI (Figs. 61, 7A). The entorhinal cortex harbors as many NPY-IR beaded fibers as the subicular complex. Each layer is distinguished by its content and its distribution patterns of fibers. Layer I, characterized by short, thin fibers, horizontally oriented is identical to layer I of the pre- and parasubiculum. A dense network of thin beaded fibers oriented in all directions is present in layer II where long ramified fibers, horizontal or ver-

219 tical, are also detected. In the superficial part of layer III, this network of N P Y - I R fibers becomes more dense. In layers IV and V, N P Y - I R fibers appear to be m o r e scattered and have no predominant orientation. A higher density of N P Y - I R fibers is present in layer VI where long horizontal fibers can also be detected. Long, vertical fibers cross the entire depth of the cortex and reach the subcortical white matter. S o m e of these are ramified processes of NPY-IR cell bodies situated in the immediately adjacent subcortical white matter.

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T h e w h i t e m a t t e r ( f i m b r i a , a n g u l a r b u n d l e o.nd a d j a cent white matter)

Numerous N P Y - I R cell bodies are present in the angular bundle and the adjacent white matter although none were detected in the fimbria. There is a high density in the subcortical white m a t t e r all along the entorhinal cortex (Fig. 7A). U n d e r layer VI of the lateral part of the e n t o r h i ~ l cortex N P Y - I R cell bodies and their processes run parallel to the cortex although they are perpendicular to the parasubiculum and to the median part of ti~e entorhinal cortex.

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Fig. 9. Schematic diagrams of NPY-IR nerve cell body distribution in 35 jum transversal section through the hippocampal formation at the middle level of infant (4 months) and adult (42 years) brain• Figures include the area dentata, the Ammon's horn (CA1, CA2, CA3 subfields), the prosubiculum (Prosub), the subiculum (Sub), the presubiculum (Presub), the parasubiculum (Parasub), the entorhinai cortex (EA) as well as the angular bundle and the adjacent white matter (WM). Arrows indicate the limits between these regions• NPY-IR nerve cell bodies are essentially found in the deep layers and the underlying white matter of the subicular complex and the entorhinal cortex but also in the Ammon's horn and the polymorphic zone or hilus. The total number of NPY-IR nerve cell bodies is similar in infant and adult, while the density, essentially in the white matter, is greater in infant than in adult.

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Near the alveus and under the subicular complex, NPY-IR cell bodies are less numerous and they seem to have no particular orientation (Fig. 7B). Their size and shape are similar to those of the NPY-IR inter-

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Fig. 8. NPY-IR nerve cell bodies and fibers in layers V and Vi of the entorhinal cortex and in the subcortical white matter (WM). Adult of 42 years of age. Compared with the infant (Fig. 7A), the density of nerve cell bodies and fiber networks is decreased, particularly in the subcortical white matter. PAP technique• Bar = 1001~m.

223 neurons of the cortex. They are multipolar fusiform, round, ovoid or triangular. Some of them are bipolar and fusiform. Most of the fibers detected in the white matter are processes of these NPY-IR cell bodies. These processes are fairly long, they may measure up to 1 mm in the section.

per section. The total number of NPY-IR nerve cell bodies per section, 196 + 14 (mean + S.E.M., n = 8), is remarkably constant from birth to adulthood with a variation coefficient of 7.3%. However, the density of these NPY-IR nerve cell bodies decreases from birth to adulthood, parallel to the brain growth (Table I).

Comparison with the adult hippocampal formation

The general distribution of NPY-IR neurons and networks in the infant hippocampal formation reported here is basically in accordance with that observed in the adult (Fig. 9). No difference can be detected in the types of NPY-IR neurons in the different cortical areas examined in adulthood. However, two importar~ differences are recognized in infants of a year or less, compared to adults (Fig. 8): the exuberance of the neuror.ai processes and the density of the neuronal population and of the fiber networks in each region of the hippocampal formation and particularly in the angular bundle and the adjacent whi~e matter. In addition, the laminar distribution of the NPY-IR in the subicular complex and the entorhinai cortex is more distinct in the infants than in the adult. Quantitative data

The number of NPY-IR nerve cell bodies is determined in the hippocampal formation including the Ammon's horn, the area dentata, the subicular complex, the entorhinal cortex and the white matter. In all analyzed cases, the larger number of these cell bodies was found in the subicular complex and the entorhinal cortex as well as in the angular bundle and the adjacent white matter where they represented 30-40% of the total number of NPY-IR cell bodies TABLE I Compar, Ttive evaluation at several ages of total number and density (number per cm:) of NPY nerve cell bodies in hippocampal formation 3~-~lm thick sections (see Fig. 9) Age

Number per section (total number)

Number per cm 2 (density)

2 days 4days 3months 4months 10months 12months 4 years 42 years

189 216 193 174 205 201 211 181

205 174 130 156 105 150 75 86

DISCUSSION All the regions of the human infant hippocampal formation are exceptionally rich in NPY-IR nerve cell bodies and fibers. The shape and size of the NPY-IR neurons reported here resemble the population of non-pyramidal cells described in these regions by Cajal 5 and Lorente de No 28"29. Similar NPY interneurons have been seen in the hippocampus and the neocortex of the cat, monkey 22"24and adult human s'9. Throughout the hippocampal formation and the neocortex, such peptidergic interneurons have already been reported including those that contain somatostatin 2"7, enkephalin 1s'37, cholecystokinin 19"3°'3~ and substance pl3.15,38 both in man and other mammals. In the Ammon's horn and hilus of the rat, a new class of hippocampal neurons was recently found. They combine the morphological characteristics of interneurons and neurons projecting to the septum and contralateral hippocampus, respectively3s. There is little probability of non-pyramidal NPY-IR neurons with similar projections in view of the absence of NPY-IR fibers in the fimbria. The remarkable comparability of high densities of NPY-IR neuronal population and of NPY-IR fiber populations in these particular regions suggests the likelihood that the NPYIR interneurons give rise to much of the dense axonal network in their immediate subfield. This would mean that the NPY-IR interneurons and their fibers are part of the intrahippocampal circuitry. This does not rule out the possibility that extrahippocampal sources of NPY-IR fibers exist. Unfortunately, the present material provides little information on NPY afferent fiber systems or their sources. In rats, intrinsic as well as extrinsic NPY-IR afferent systems have been shown to contribute to the NPY innervation of the retrohippocampal region 2s. These are, to a large extent, neurons with long association projections situated predominantly in the piriform and perirhinal cortex. However, important species differences are

224 reported in the distribution and the origin of neuropeptides in the hippocampus. The NPY-IR neurons of the angular bundle and the adjacent white matter correspond to the interstitial neurons or white matter neurons originally described by Cajai 5. These interstitial cells have the typically neuronal morphology and the ultrastructural characteristics of neurons and they receive synaptic contacts. They also have long projections and 20% of them show acetylcholinesterase-positive reaction. Their highest density and the most extensgve distribution is found during the neonatal period both in human and monkey 27. In the visual cortex both in human and other mammals, NPY-IR neurons are also embedded in the white matter 1°'22"43"44. NPY-IR neurons in the angular bundle and adjacent white matter reach a large distribution in the neonatal period. The spreading out of this NPY-IR interstitial system into the subcortical white matter all along the entorhinal cortex and its connection with all the layers suggest f,,~nctional relationships with the overlying cortical neurons. It is known that the density of the interstitial neurons decreases during infancy but numerous cells remain in the adult. Although no quantitative study has been done, Kostovic and Rakic27 suggested that these interstitial neurons degenerate during infancy. In our study, the decrease of the density of NPY-IR interstitial neurons is essentially the result of the increase in volume of the wk;te matter. Although the possibility of changes in their location has not been verified quantitatively, on!y a very limited number of NPY interstitial neurons could enter into layer VI of the cortex. Indeed, since at birth these neurons are already morpho[ogicai!y well-developed with profusely branched ~endritic and axonal processes, it is probable that their positions are fixed prenatally 27.4°. In the gray matter of the Ammon's horn, subicular complex and entorhinal cortex, there is also an apparent decrease of NPY-immunoreactivity during development with the cells becoming more scattered. Our quantitative morphological studies show no decline in the total number of NPY-IR neurons but there is a decrease in density; they seem to become spread out by the growth of the rest of the tissue. In humans, the total number of NPY nerve cell bodies in the hippocampal system established at birth is not modified during consequent brain growth which continues until age 3-4 years and this number

stays stable at least to age 42 years. Our study, however, does not permit us to evaluate the growth of its fibers and its capacity to synthesize during this period. Thus it appears that the postnatal NPY system in humans is different from that of the rat 34.45and the cat 44 according to the maturity of the species at birth 16. In the human newborn, but not in the adult, high concentrations of neurotensin neurons are observed in the subiculum 36. In addition, cholecystokinin 3°.31 and substance P neurons 32 are particularly apparent in the entorhinai cortex during the postnatal period. It is probable that like the NPY neurons, the cholecystokinin, substance P and neurotensin neurons present after birth do not disappear during postnatal development. Age-related changes in different peptidergic neurons could result from variations in their peptide concentratioi~s. The decline in immunostaining during postnatal development might also be affected by myelination interfering with antibody penetration. In addition to their roles as neurotransmitters or neuromodulators, neuropeptides are candidates to trophic and plastic functions in the central nervous system 21"23. It is therefore of great importance to understand their effects on the immature brain. Accordingly it is of interest to investigate the normal development of peptidergic pathways in the human brain. Up to now there has been no report of a specific developmental effect of NPY on the hippocampal formation. The extent of NPY involvement in normal and abnormal brain development has yet to be investigated. ACKNOWLEDGEMENTS We thank Drs G. Pelletier and H. Vaudry for the generous gift of NPY antiserum. We thank G. Vierendeels for fine technical assistance in various aspects of this study, for drawings and for typing the manuscript and Mr. J.-L. Conreur for help with the photographs. This work was supported by grants from the Belgian Queen Elisabeth Medical Foundation (86-88), National Fund for Scientific Research (FNRS 85-86), the Medical Scientific Research (FRSM 34523.86-89) and the Belgian National Lotery. S.N.S. is a Research Assistant of the Belgian National Fund for Scientific Research.

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