Morphometrical and microdensitometrical studies on phenylethanolamine-N-methyltransferase- and...

Neuroscience Vol. 26, No. 2, pp. 461-478, 1988 Printed in Great Britain

0306-4522/88 $3.00 + 0.00 Pergamon Press plc

0 1988 IBRO

MORPHOMETRICAL AND MICRODENSITOMETRICAL STUDIES ON PHENYLETHANOLAMINE-N-METHYLTRANSFERASE- AND NEUROPEPTIDE Y-IMMUNOREACTIVE NEURONS

IN THE ROSTRAL MEDULLA OBLONGATA OF THE ADULT AND OLD MALE RAT*

L. F. AGNATI, K. FuxE,~$ M. ZOLI, I. ZINI, A. HKRFSTRANDJ G. TOFFANO$ and M. GOLDSTEINII

Department of Human Physiology, University of Modena, 41100 Modena, Italy, IDepartment of Histology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden, §Fidia Research Laboratories, 35031 Abano Terme, Italy and IIDepartment of Psychiatry, New York University, Medical Center, New

York, NY 10016, U.S.A.

Ahstrati-In the present paper the neuronal systems of the medulla oblongata containing phenylethanolamine-N-methyltransferase- and neuropeptide Y-like immunoreactivity have been characterized in adult (3-month-old) and old (24-month-old) male rats. The phenylethanolamine-N- methyltransferase and neuropeptide Y-immunoreactive neurons have been visualized by means of immunocytochemistry (peroxidase-antiperoxidase technique) and analysed in a quantitative fashion by means of morphometrical (phenylethanolamine-N-methyltransferase- and neuropeptide Y- immunoreactive cell groups) and microdensitometrical (phenylethanolamine-N-methyltransferase- immunoreactive cell groups) approaches developed on the IBAS II image analyser (Zeiss-Kontron). During aging there is (a) a reduction in the area covered by the phenylethanolamine-N-methyltransferase- immunoreactive neuropil for both the Cl and C2 adrenaline cell groups; (b) a reduction in the area covered by the phenylethanolamine-N-methyltransferase-immunoreactive cell bodies, which is highly significant only for the C2 cell group; (c) a decrease in the area covered by the phenylethanolamine-N- methyltransferase-positive cell cluster for both Cl and C2 cell groups; (d) a decrease in the degree of phenylethanolamine-N-methyltransferase immunoreactivity present in the Cl and C2 cell groups; (e) a decay of neuropeptide Y immunoreactivity in the Cl and C2 groups, while the C3 group is unaffected by aging as evaluated by number of phenylethanolamine-N-methyltransferase- and neuropeptide Y- immunoreactive cell body profiles.

These results indicate heterogeneities in the responses of the adrenaline-neuropeptide Y cell groups to the aging process. The possible functional consequences of aging-induced changes in the cardiovascular adrenergic neurons are discussed, especially in relation to development of hypertension.

In recent years adrenaline neurons located in the paragigantocellular reticular region (Cl cell group), in the rostra1 part of the dorsal motor nucleus of the vagus nerve (dmnX) (C2 cell group) and in the medial longitudinal fasciculus area (C3 cell group) in the dorsal and rostra1 medulla oblongata14.15~38~~4W6 have been shown to co-store neuropeptide Y (NPY). 23~30*4’,42 This peptide was first discovered in brain by Tatemoto et al. 56,57 The functional role of

*Dedicated to Professor Sergio Lenzi, chairman of De- partment of Internal Medicine, University of Bologna, Italy, on the occasion of his 70th birthday.

tTo whom correspondence should be addressed. Abbreuiarions: BSA, bovine serum albumin; CCK, chole-

cystokinin; DA, dopamine; DAB, diaminobenzidine; dmnX, dorsal motor nucleus of the vagus; FAc, field area of cell bodies; FAn, field area of neuropil; S-HT, S-hydroxytryptamine; i.c., intracistemal; i.v.t., intra- ventricular; NC, number of cell bodies; NA, noradrenaline; NPY, neuropeptide Y; PAP, peroxidase- antiperoxidase; PBS, phosphate-buffered saline; PNMT, phenylethanolamine-N-methyltransferase; SN, substantia nigra; SP, substance P, TH, tyrosine hydroxylase; VTA, ventral tegmental area.

these adrenaline/NPY neurons has been intensively investigated in biochemical studies, in electro- encephalographic studies, in studies on feeding behaviour and on cardiovascular, respiratory and neuroendocrine control (see Refs 13, 22 and 49). There is evidence that adrenaline/NPY co-storing neurons of the medulla oblongata are part of a vasodepressor system.‘9*20J’29,32~36

Recently, aging induced changes of transmitter- identified neuronal systems have been investigated. The aged brain is characterized by multiple degener- ative processes affecting anatomical, neurochemical and functional parameters.21*34~37~47~52 In this context a number of studies demonstrate a differential vulnerability of neuronal populations to the aging process.6~7*9JoJ2J6~33~50~53 Furthermore, investigations on the neurochemical mechanisms operating at pre- and postsynaptic levels of specific transmitter-identified neuronal systems suggest the existence of a differential impairment of the different transmission lines within the same neuronal population. In particular, the studies on dopamine (DA)/cholecystokinin (CCK) co-storing neurons of the ventral tegmental

NSC 26,2--D 461

462 L. F. AGNATI et al.

SAMPLED SQUARE

Fig. 1. Schematic representation of the level of the neuraxis analysed. A coronal section (level: Bregma-I 1.8 mm according to Ref. 51), with the sampled regions is also given. AP, area postrema; DS, dorsal strip; ECU, external cuneate nucleus; icp, inferior cerebellar peduncle; LVe, lateral vestibular nucleus; mlf, medial longitudinal fasciculus; MVe, medial vestibular nucleus; Sol, nucleus of solitary tract; PGi, paragigantocellular reticular nucleus; PrH, prepositus hypoglossal nucleus; py. pyramidal tract; Rob, raphe obscurus; sp5, spinal tract of trigeminal nerve; SpSi, nucleus of the spinal tract of the trigeminal

nerve.

area (VTA) and substantia nigra (SN), on DA/neurotensin co-storing neurons of the VTA and on 5-hydroxytryptamine (5-HT)/substance P (SP) co-storing neurons of the nucleus raphe magnus open up the possibility of a preferential decay of the co-stored peptide with respect to tyrosine hydroxylase (TH) or 5-HT.6,7.9.‘0.‘2.‘6

In the present article the aging-induced changes in the adrenaline/NPY neuronal systems of the rostra1 medulla oblongata visualized by phenylethanolamine-N-methyltransferase (PNMT) and NPY immunocytochemistry, will be charaterized by means of morphometrical and microdensitometrical procedures comparing the results obtained in the adult (3-month-old) and old (24-month-old) male rat.

EXPERIMENTAL PROCEDURES

Three-month-old (n = 7) and 24-month-old (n = 5) male Sprague-Dawley rats (Fidia Research Laboratories) have been used. The rats were kept under standardized humidity, temperature and lighting conditions (lights on at 8.00 a.m.; lights off at 8.00 p.m.). They had free access to water and food pellets.

Immunocytochemical procedure

The peroxidase-antiperoxidase (PAP) method% was used. The rabbit antibody against rat PNMT which catalyses the synthesis of adrenaline from noradrenaline (NA) has previously been characterized. wo The NPY antiserum used was raised in rabbits using unconjugated synthetic porcine NPY (Amersham Laboratories, code RPN 1702, U.K.). No significant cross-reactivity was observed with peptide with N- and C-terminal tyrosine. Similar immunohistochemical results have also been obtained with another rabbit NPY

serum4* which also does not cross-react with related com- pounds (avian pancreatic polypeptide, bovine pancreatic polypeptide, /?-melanocyte-stimulating hormone, FMRF- amide or avian pancreatic polypeptide 31-36) in dilutions used for immunocytochemistry. Quantitative results obtained with this antibody are, however, not reported in this paper. To obtain the best possible visualization of the NPY-immunoreactive nerve cell profiles, the rats were treated intraventricularly (i.v.t.) and intracistemally (i.c.) with colchicine (6Opg i.v.t. and 6Opg i.c., 24 h before killing).

The rats were anaesthetized with methohexital natrium (Brietale, Lilly, U.S.A.) and thereafter killed by transcardiac perfusion with 50 (37°C) + 50 (4°C) ml of saline followed by 300 ml of ice cold 4% (w/v) paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (pH 7.4). The brains were rapidly dissected out, left in the fixative for 4 h and then overnight in a phosphate buffer containing 5% sucrose. Eight coronal Vibratome sections (50~pm-thick) per rat ‘were collected at a level I .&2.0 mm rostra1 to obex (see Ref. 51). At this level all the three PNMT- and NPY-positive nerve cell groups Cl, C2 and C3 can be analysed in the same section (see Fig. 1; see also Figs 24).

Adjacent pairs of sections were incubated free floating for 36 h at 4°C with the two primary antibodies diluted (PNMT dilution 1: 1000, NPY dilution 1: 100 after reconstruction in 500~1) in PBS containing 0.3% Triton X-100, and 1% normal porcine serum. After sequential washes (10 min each) in PBS containing 0.2% Triton X-100, PBS containing 0.1% Triton X-100, PBS alone, PBS containing 1% bovine serum albumin (BSA), 0.1% normal porcine serum and PBS alone, the sections were exposed to the secondary antiserum porcine anti-rabbit IgG (American Qualex), diluted 1: 30 in 0.3% Triton X-100 and 1% normal porcine serum in PBS, for 2 h at room temperature. After two PBS rinses, the sections were incubated with the PAP complex raised in rabbit (Stemberger Ltd) (diluted I : 80 in I % normal porcine serum present in PBS), for I h at room temperature. The

Adrenaline, neuropeptide Y and aging 463

Fig. 2. PNMT-motive cell bodies with their processes are seen in the Cl area of the adult (a) and old (b) male rate. Primary antiserum 1:1000. PAP immunocytochemistry. Calibration bar = IOOpm. D,

dorsal; M, medial.


Fig. 3. PNMT-positive cell bodies with their processes arc seen in the C2 and C3 area of the adult (a) and old (b) rat. Primary antiserum I : 1000. PAP immunocyt~hemistry. Calibration bar = 2OO~m.

sections were then preincubated in a saturating concen- tration of diaminobenzidine (DAB; 0.5 mg/ml) (&ma) in 50 mM Tri-HCI buffer (RH 7.4) for 5 min and H,O, was added to give a final conc&tration of 0.03% (w/v). The time of the staining reaction was 5 min. The coded sections from the adult and old rats were always run in parallel under identical reaction conditions. The sections were then rinsed in Tris-HCI, transferred onto geiatin~hromalum coated glass slides, defatted and coverslipped with Eukitt (Zeiss). Normal rabbit serum (PNMT) or -NPY antiserum, which had been absorbed with 50 ue. NPY/ml diluted antiserum (1: IOOO), was used as control-serum.

As previously demonstrated in a morphometrical anal-

ysis, ” adrenaline/NPY cells of the C3 region do not form a cluster, but represent scattered cells. Our analysis will mainly focus on the adrenaline/NPY co-storing neurons of the Cl and C2 regions where clusters are found.” However, some data will be presented also on the adrenaline/NPY neurons of the C3 region. The relationship to cyto- architecture was established by counterstaining with Cresyl Violet.

It should be pointed out that a linear relationship has been observed between optical density of immunocytochemical preparations and the respective antigen content”.5s using the unlabelled antibody method in combi- nation with the PAP procedure. A good correlation between


Fig. 4. Higher magnification of the C2 area shown in Fig. 3a and b. (a) and (b) show the PNMT-positive cell bodies and their processes of the adult and old rat, respectively. dmnX, dorsal motor nucleus of the

vagus. Calibration bar = 100 pm.

immunocytochemical visualization of antigens and antigen et al.” we used saturating concentrations of DAB substrate, contents has been observed also in previous experiments performed in our laboratory. 3’ As recommended by Benno

a constant dilution of antibody and an incubation time adjusted so that the darkest elements in the brain sections


Fig. 5. Higher magnification of the C3 area shown in Fig. 3a and b. (a) and (b) show the PNMT cell bodies and their processes of the adult and old rat, respectively. Calibration bar = 100 pm.

were below saturation. Under these conditions the steric Description qf’ the image analysis systems hindrance of the PAP complex binding (e.g. the Bigbee The systems used for image analysis (MOP AM0 II for effe#) does not appear to disturb the linear relationship the semiautomatic analysis and IBAS system for the fully between TH antigen contents and staining intensity.” automatic analysis, both from Zeiss-Kontron, F.R.G.) have


Fig. 6. Immunocytochemical visualization of NPY-positive cells in the Cl area of the adult (a) and (b) rat. Primary antiserum 1: 1000. PAP immunocytochemistry. Calibration bar = 200 pm. D, dorsal:

medial.

old M,


GVccl,c GVn< I$ B 2 level Cl, ,l,)

I] dmrim;otion

Quantltotive characterlzotion of neuropil

GVccl,<GVn < B I level IL,)

vj dwrynatlon

r______ _____--,

Quontitotlve chorocterlzotlon of cell bodies

Fig. 7. Schematic representation of the automatic procedure developed to quantitatively characterize neuropil, cell bodies and clusters of transmitter-identified neurons. GV, gray value; 1,,2, levels of discrimination; B, background; FA, field area; N, number of profiles; A, mean area. Subscripts: n = neuropil; c = cell body; cg = cell group (i.e. cluster of transmitter-identified neurons). For further

details, see text.

been carefully descibed elsewhere.2,5,‘0 A Zeiss photo- microscope was used. Photographs were taken with Pana- tomic film, developed in Rodinal. The prints were made with Agfa Gaevert Rapitone paper 3-4.

The MOP system consists of a magnetic tablet connected with a computer (in our set-up the MOP system is plugged into an Apple II computer) which allows the superposition of Cartesian axes on the original image (photomontage) and the acquisition of the individual objects. It gives a series of morphometrical parameters of the individual profiles, like area, perimeter, maximum diameter, shape factor and the localization of the profiles in the Cartesian plane. It also gives parameters of the overall cell group, such as number of objects and gravity centres on the X- and Y-axes.

We have developed a special software for the Apple11 computer, which operates with these parameters. Thus, we can obtain scatter diagrams, density maps and three- dimensional reconstructions of a group of transmitter- identified neurons.4,5.30,58

The IBAS system consists of a unit which allows the acquisition, storage and management of the image (IBAS II) and of a host computer (IBAS I) which has direct access to the memories of the IBAS II. In our set up the IBAS system is linked to the microscope by means of a TV camera (TYK 92D, Bosch, F.R.G.).

The first step of the automatic analysis is the “acquisition function” which resolves the original image in 512 x 512 elementary units, called pixels, and gives to each pixel a grey tone intensity value ranging from 0 (complete absorption) to 255 (complete transmittance). Different elaborations of the

image can then be performed. Of particular relevance for the present analysis is the “discrimination function”, the “dilatation function”, the “close function” and the “boolean operation ‘and”’ (for further details, see Ref. 8).

The “discrimination function” consists of the exclusion of a chosen range of grey tones, thus allowing, for example, the separation of the specific profiles (darker grey tones) from the background (lighter grey tones).

The “dilatation function” increases the size of the objects by 2 pixels at the boundaries.

The “close function” consists of a “dilatation” of 2 pixels followed by “erosion” of 2 pixels of the newly formed objects.

The “boolean operation ‘and”’ allows a comparison between two discriminated images, giving a new image containing only the pixels which are positive in both the original images.

Morphometrical methods

By means of the IBAS analyser it is possible to develop an automatic procedure to quantitatively characterize neuropil, cell bodies and clusters of transmitter identified neurons (see Fig. 7). The present analysis was carried out on the original sections. This procedure is examplified in Fig. 8 (original image), Fig. 9a (selective visualization of the neuropil by means of interactive discrimination), Fig. 9b (selective visualization of the cell bodies after discrimination) and Fig. lOa,b [cluster demonstration by means of the close function (a), and the cell bodies lying within


Fig. 8. PN~T-~sitive cell bodies with their processes are seen in the Cl region of an adult male rat. Primary antiserum I: 1000. PAP immunocytochemistry. Calibration bar = 100 pm. D, dorsal; L, lateral.

the cluster are shown by means of tlte boolean operation uL,~ as exemplified in Fig. 11 (semiautomatic procedure) to “and” (b)]. be compared with Fig. IOa (automatic procedure).

Neuropil und ceII body characterization Dendrites and cell bodies have been characterized by

evaluating the respective field area (amount of immunoreactive area present in the sampled field) and numbers of profiles. It should be noted that (see, for example, binary image 9b) only profiles having a diameter equal or larger than IO pm have been accepted as cell bodies. This limit has been used also for the evaluation of the number of cell bodies lying within a cell group (see Fig. lob). No limits have been used for the quantitative evaluation of the binary image of the dendrites (Fig. 9a).

After cluster identification (Fig. lOa), it is not only possible to evaluate the area of the cell cluster, but also to selectively visualize the cell bodies lying in the cluster. To this aim the image of the cell cluster is compared with the image of Fig. 9b using the boolean logic operator “and”. In our analysis a cluster has been accepted only when the number of immunoreactive nerve cell bodies (i.e. profiles with a diameter equal or larger than IOpm) within it is equal to or above 5.

Microdensitometrical metho&

Cluster iabtijication

This elaboration is carried out after interactive discrimination to visualize selectively the cell bodies (see Fig. 9b). As stated above and in Fig. 7, cluster identification is based on the close function, which consists of a dilatation of the profiles followed by an erosion of the newly formed profiles. In this way objects lying close together are united to form larger individual objects. Profiles merge together according to the distance among each other, their size and shape and finally the number of times that the step “close” is repeated. We have chosen a number of repetitions of the step “close” so that circular objects of 30pm of diameter lying 60pm apart (i.e. about twice the mean maximal diameter of the profiles), or less are united, while profiles further apart remain as scattered cells. This procedure is well in agreement with the semiautomatic procedure introduced by Agnati et

The procedure introduced by Aguati et al.’ has been modified taking advantage of the discriminative capabilities of the IBAS. Thus, the unspecific background was removed by means of an interactive di~~~nation and the field area measured (FAc), (see Fig. 12). Then, by means of successive discriminations a separation of the gray tones present in the sampled field has been performed. The ratio (Yi) between the field areas measured after each discrimination [(FAc),] and the (FAc), value have been calculated. The summation of the values Wi* Y, has been considerd as a relative quantitative evaluation of the antigen content, where Wi values represent suitable linear weights.

In fact, in ptevous experiments3’ evaluating the SP immunoreactivity in the substantia nigra and comparing this procedure with the radioimmunoassay it has been demonstrated that the best agreement between quantitative immunocytochemical evaluations and biochemistry could be obtained by using this linear weighing procedure.


i

#

‘, - ,

.

Fig. 9. Selective visualization of the neuropil (a) and the nerve cell bodies (b) present in the field of Fig. 8. Binary images are shown. It should be noted that in the binary image 9b only profiles having

a diameter equal or larger than 10 pm have been accepted as cell bodies.


IOa

lob Fig. 10. {a) Demonstration of the existence of a cluster in the Cl cell group, by means of the close function. The elaboration has been carried out on Fig. 9b. (b) Visualization of the cell bodies present in the area of the main cluster. This visualization has been obtained by performing an “and” operation between the

larger cluster present in the image tOa and the cell bodies visualized in image 9b.

L. F. AGNAW et al.

PNMT-IR CELL BCJDIES IN Cl GRCIUP

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Fig, 11. Density distribution of the PNMT-positive cells of the field shown in Fig. 8. The dehmitation of the cell group oF transmitter-identified neurons has been performed according to the semiautamatic procedure introduced by Agnati ez al.” The black squares represent accepted squares (see Ref. 5). The table in the figure gives cell body and cell group parameters (for further details see text and Ref. 5). The origin of the Cartesian axes was defined as the intercept of the midline with the ventral border of the section.

FGi, ~ara~~nt~ll~~ar reticufar nucleus.

I,. -\evel of drsCriminotion FAc=celt body field area

[AQ 3 = antigen rontent

r,= [CFAc,, fiFA~~~jxlO0

Wi i weight

X, - levels of grey tone drxnmlnotlon

Fig. 12. Schematic representation of the procedure followed to obtain a relative quantitative evaluation of the antigen content in a sampled field. On the X-axis the subsequent levels of discrimination are given. On the Y-axis the antigen contents per unit area (Ag), as evaluated by the formula reported in the figure, are given. The panels on the bottom illustrate, in a schematic fashion, the effects of the subsequent steps of discrimination on the original image, where different gray tones (i.e. different Ag contents) are present.

For further details, see text.

Adrenaline, ne~o~ptide Y and aging

RESULTS

Morphometrical analysis of the neuropil of phenylethanolamine - N - methyltransferase - immunoreactive nerve cells of groups Cl and C2

The analysis of the neuropil (Figs 13 and 14) of both the Cl and C2 groups demonstrated a similar pattern of age-induced changes (see also Figs 2-4).

1. A large decrease in the area covered by the neuropil (FAn). This result can be interpreted both as the result of a decrease in the length and thickness of the dendritic arborization as well as the result of a decrease in the antigen contents of the neuropil, a phenomenon confirmed by the microdensitometrical analysis (see below).

2. A maintenance of the number of neuropil profiles. The slight but not signifi~nt increase of the neuropil profiles observed in the Cl group of the old animals may be ascribed to the decrease in the antigen contents, which could cause a fragmentation of the immunoreactive objects.

~orphometrieal analysis of the ~henylethanolamine- N-methyltransferase-immunorea~tive cell bodies of groups CI and C2

The analysis of the cell bodies (Figs 13-l 5) showed different effects of aging on the Cl and C2 cell groups (see also Figs 2-6).

For the PNMT-positive cells of the Cl group the following observations were made:

1. a trend for a decrease of the area covered by the cell bodies (FAG);

2. a trend for a decrease of the number of the cell bodies.

For the PNMT-positive cells of the C2 cell group the following observations were made:

1. a highly significant decrease of the FAc; 2. a significant decrease of the cell bodies.

No effect of aging was observed on the mean area of the PNMT-positive cell bodies in the two regions (data not shown).

A significant reduction in the number of NPY- immunoreactive nerve cell bodies was observed in groups Cl and C2 but not in group C3 of old rats (Fig. 15). In the Cl group only the number of NPY- but not of PNMT-immunoreactive nerve cell bodies was significantly reduced (Fig. 15).

Morphometrical analysis of the phenylethanolamine- N-methyltransferase-immunoreactive cell ChSterS Of

groups C 1 and C2

In a previous paper” a detailed analysis of the PNMT-immunoreactive cell clusters in the medulla oblongata of the adult rat demonstrated the existence of two large PNMT-positive clusters, one located lateral to the paragigantocellular nucleus (part of Cl cell group) and another one located in the rostra1 pole

PNMT lmmunwewtivlty in W are0

FAn

I a_ d

ri

~ NC FAcg Nw

q J-month-old rat q 24-month-old rat

Fig. 13. Morphometrical evaluation of PNMT-positive neurons {neuropil, cell bodies and clusters) in the Cl area of the medulla oblongata of the rat. For abbreviations, see legend to Fig. 7. The statistical analysis was carried out by means of a Mann-Whitney U-test. Means & S.E.M. (n = 7

for adult and n = 5 for old rats). NS, not significant.

of the dmnX(C2 cell group), and one small cluster, located in the dorsal subnuclei of the nucleus tractus solitarius. The cell bodies present in the medial longitudinal fasciculus region (C3 cell group), and also the majority of the medially located cell bodies of the ventral reticular formation (part of group Cl) mainly represent scattered cells.

The present analysis shows for the above men- tioned Cl and C2 cell clusters a highly significant decrease in the total field area of the cluster and in the number of PNMT-immunoreactive cells within the cluster during aging (Figs 13 and 14).

~i~ro~~itometrica~ analysis of the phenyl- eth~olamine - N - methyltransferase - immunoreact~e nerve cells of groups Cl and C2

The microdensitometrical evaluation of the PNMT immunoreactivity has been performed according to the procedure described above. In Fig. 16a,b the plots of the field areas of immunoreactivity at subsequent disc~mination level are represented for the Cl and C2 groups, respectively. The faster decay of the percent cumulative field areas in the old animal indicates a reduced antigen content in the PNMT- immunoreactive neurons. The A adrenaline% values

474 L. F. AGNATI et af.

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Gray tone discr~mi~i~n (arbitrary units)

(a)

A area=AA%B,18.4% NC FAcg

n 3-month-old rat tzl

24-month-aid rat

Fig. 14. Morphometrical evaluation of PNMT-positive neurons (neuropil, cell bodies and clusters) in the C2 area of the medulla oblongata of the rat. Means fS.E.M. Mann-Whitney U-test. For further details, see legend to

Fig. 13. NS, not significant.

Gray tone discrimination (arbitrary units1

(b)

Fig. 16. Microdensitometrical evaluation of PNMT immunoreactivity in the Cl (a) and C2 (b) areas of the medulla oblongata of the male rat. On the X-axis subsequent levels of ~~~~nation (moving further away from the background, thus from low to high gray tones) are given. On the Y-axis the Y, values (percent cumulative field areas of immunoreactivity, see text and Fig. 12) are given. Means & S.E.M. [n = 5 (old) and 7 (adult)]. The ED% values as well as the AA% for the curves observed in the adult and the old animal are reported. The AA% value is the percent ratio of the difference between the areas below each curve

and the area below the upper curve.

express the difference between the areas below the two curves in percent of the area below the upper curve. The main results obtained by means of this analysis are summarized in Table 1. These results demonstrate a clear-cut decay of PNMT immunoreactivity in both Cl and C2 cell groups during aging.

D PNMT-positive neurons

m NPY-positive neurons

Hawever, a more marked drop may take place in the C2 group.

DISCUSSION Fig. 15. Age-induced changes in the number of FNMT- positive and NPY-positive nerve cell bodies in the rostra1 part of the medulla oblongata of cell groups CL, C2 and C3. Means f S.E.M. are given in per cent of adult group mean value. Mann-Whitney U-test. *P < 0.05. For further details

see legend to Fig. 13.

The present study shows that the aging process affects the adrenaline/NPY neurons of the medulla obiongata, which may in part represent vasodepressor systems controlling arterial blood pressure,


Table 1. Microdensitometrical analysis of phenylethanolamine-N-methyltransferase immunoreactivity in

groups Cl and C2 of the adult and old male rat

Antigen EDSO AA% content

Cl Adult 54.3 + 1.9 12.7 4995 f 119 Old 46.3 f 1.0 4110+ 129

P < 0.01 P < 0.01

c2 Adult 51.4+ 1.6 18.4 4610& 111 Old 40.5 * 1.5 3972 k 98

P < 0.01 P < 0.01

Summary of the main results reported in Fig. 16. For further details, see text and Figs 12 and 16a,b. Means + S.E.M. ]n = 7 (adult) and 5 (old)]. Statistical significances have been obtained according to the Mann-Whitney U-test. Antigen content = Z( IVY. Y,).

possibly by taking part in the regulation of the gain of the baroreceptor reflex arc.25,27*36,42 The relevance of this vasodepressor system is underlined by the evidence for an alteration in the adrenaline/NPY transmission and in the interplay between these two lines of transmission in spontaneously hypertensive rats.)*32 It must be underscored that in the present analysis the NPY and PNMT immunoreactivity measured in the old animal can only be compared with the corre- sponding NPY and PNMT immunoreactivity values obtained in the adult animal, since changes in NPY immunoreactivity cannot be compared with changes in PNMT immunoreactivity. Thus, it is conceivable that PNMT as an antigen produces the generation of more clones of antibodies recognizing different determinants than would NPY, which is a smaller antigen than PNMT. Therefore, in immunocytochemical studies it seems possible that PNMT may be more detectable than NPY, since PNMT may possess more antibody generating determinants. It should also be considered that a low titre antiserum compared with a high titre antiserum may be less efficient at detecting a minimum antigen level. Therefore, in view of the absence of control experiments it is possible that the preferential effects observed in the old brain on NPY versus PNMT immunoreactivity in group Cl may be caused not by a differential reduction of NPY immunoreactivity but simply by differential efficacies of the PNMT and NPY antisera used. This limitation should be kept in mind when discussing the present findings.

Within the adrenaline cells of the Cl group there occurs a reduction in the NPY- but not in the PNMT-immunoreactive profiles, when considering the entire group in the sampled area, while a corre- sponding analysis of the C2 group reveals a reduction of both PNMT- and NPY-immunoreactive profiles. The age-induced changes in the number of immunoreactive profiles may therefore depend on different processes in the three cell groups (Cl, C2, C3) of the

medulla oblongata. Thus, the disappearance of NPY-

positive profiles in the Cl group seems to be caused mainly by a decrease of NPY synthesisis and not by a cell body degeneration. In fact, the overall number of cell bodies, evaluated as number of PNMT- positive profiles, most of which probably co-store NPY immunoreactivity, are only slightly altered by aging. However, aging induces a highly significant and marked reduction in the field area of the PNMT cell cluster of the Cl group. Thus, with age the PNMT nerve cells bodies may become more scattered and thus may no longer interact with each other in the same way as in adult life. One consequence may be reduced trophic interactions leading to reduced metabolic capabilities, which may contribute to a reduction of NPY synthesis.

In the C2 cell group the generalized disappearance of both types of profiles points to a reduction of the effectiveness of the biochemical machineries for both PNMT and NPY synthesis or to a real disappearance of the nerve cell bodies. Also in the C2 cell group there was a marked disappearance of the cell cluster. The results again underline the view that the aging cell bodies may become more scattered with a loss of trophic interactions. In contrast, the adrenaline and NPY synthesizing mechanisms appear to be fairly unaltered in the C3 cell group of the aged rats.

Also the PNMT immunoreactivity per neuron seems to decrease with aging and again by comparing the two main cell groups of adrenaline cells (Cl and C2 cell groups) of the medulla oblongata it seems as if the adrenaline cells of the C2 area are slightly more affected than the adrenaline cells of the Cl area.

With regard to the age-induced changes observed in cell group Cl it may be pointed out that also for some 5-HT/SP, neurotensin/DA and CCK/DA co- storing neurons a decay of the peptide co-transmitter has been observed, indicating a vulnerability of the peptide transmission line to the aging processes.6,9,10,12J6 It is likely that different compensatory adjustments can take place to save the integrity of the transfer of information at synaptic level, but the range of information handling by neuronal networks involved in integrative processes may then be reduced.12 If reductions in the NPY immunoreactivity of the cardiovascular adrenergic neurons also take place in old hypertensive patients, it may be relevant to try to develop drugs for treatment of essential hypertension in aging, which act preferentially on the NPY co-transmission line of central NPY/adrenaline synapses.

Acknowledgements-This work has been supported by a grant (MH25504) from NIH, by a grant (1762) from the Council of Tobacco Research, the Italian group for studies on the aging brain, the CNR-I grant and the L. Osterman’s Foundation.

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(Accepted 8 December 1987)

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