Changes in NADPH-diaphorase neurons of the rat laterodorsal and pedunculopontine tegmental nuclei in aging

medemsofag!itq

and-

Mechanisms of Ageing and Development 90 (1996) Ill-128

Changes in NADPH-diaphorase neurons of the rat laterodorsal and pedunculopontine tegmental nuclei in aging Ivanka S. Lolova”,*, Stephan R. Lolovb, Dimitar E. Itzev” “Institute of Physiolog_v, Bulgarian Acadenzy of Sciences, Akad. G. Bonchev str., bl. 23, I 113, Sojia, Bulgaria bInstitute of Biology and Immunology of Reproducrion. Bulgarian Academy Akud. G. Boncheo str.. bl. 2.3, 1113. .%$a. Bulgaria

qf Sciences,

Received II March 1996; revised 20 May 1996; accepted 12 June 1996

Abstract The aim of the present study was to compare the morphological pattern and the quantitative parameters of nitric oxide (NO)-containing neurons in the laterodorsal (LTD) and pedunculopontine (PPN) tegmental nuclei of 3-, 12- and 26-month-old rats. NADPH-diaphorase (NADPH-d) histochemical reaction, as a marker of the cholinergic neurons in the two mesopontine nuclei, and computer-assisted image analysis were used. The relationships between the neurons stained for NADPH-d and choline acetyltransferase (ChAT) were examined using a double-labelling procedure. The results demonstrated only occasional ChAT positive somata that did not exhibit NADPH-d staining. The volume of the LTD and PPN and the number of NADPH-d neurons remained unaltered with advancing age. However, ANOVA demonstrated a significant effect of age and level on the cross-sectional areas, maximum diameters and staining intensity of NADPH-d somata in the LTD and PPN. The three parameters were reduced in 26-month-old rats compared to 3-month-old rats. The changes in the morphological appearance of NADPH-d somata and processes as well as the quantitative analysis pointed to age-related neuronal atrophy. It was accompanied by hypertrophy of some neighbouring neurons, suggesting a compensatory mechanism which

* Corresponding 0047-6374/96/$15.00

author. 0 1996 Elsevier

PII SOO47-6374(96)01767-S

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Ltd. All rights

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IS. Lolova et al. / Mechanisms of Ageing and Development 90 (1996) 11 l-128

would counteract PPN were rather Keywords:

Cholinergic

the degenerative similar.

NADPH-diaphorase; innervation

changes.

Laterodorsal;

The age-dependant

Pedunculopontine;

alterations

Tegmental

in the LTD and

nuclei: Aging;

1. Introduction Nitric oxide (NO) is a neural messenger molecule, whose biological function in the brain is of major importance. The enzyme, synthesizing NO, nitric oxide synthase (NOS), is localized in chemospecific neurons of different type and the localization determines the variety of the NO functions [l]. The NOS-containing neurons change in aging [2-41 and NO may participate in brain pathology [5-71. Recently, it has been demonstrated that the enzyme NADPH-diaphorase (NADPH-d) is a nitric oxide synthase [Xl. One of the most important localizations of NADPH-d is that in the cholinergic neurons where NO stimulates ACh release [9]. Studies indicate that almost all NADPH-d (NOS) neurons in the rat mesopontine nuclei, laterodorsal (LTD) and pedunculopontine (PPN) tegmental nuclei are cholinergic [7,10-121. The NADPH-d neurons in the basal forebrain, the other main source of cholinergic innervation, account for 20-30% of the cholinergic neurons [ll]. This widespread colocalization of NO and ACh in NADPH-d stained neurons could play a role in age-related and neurodegenerative changes. Complex investigations have indicated a parallel between the impairment of memory and learning with advancing age and the specific alterations of the cholinergic neurons in the basal forebrain [13- 151. However, the age-related changes in the cholinergic neurons of the mesopontine nuclei, which also participate in behavioural, motor and autonomic functions have been an object of little research interest [2,10]. An altered distribution of NADPH-d and choline acetyltransferase (ChAT) reactions with severe loss of neurons in the mesopontine nuclei have been observed in neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases and progressive supranuclear palsy [6,16,17]. The aim of the present study was to compare the distribution and morphometric parameters of NO-containing neurons in the LTD and PPN of 3-, 12- and 26-month-old rats. We used the simple histochemical NADPH-d reaction to stain the neurons of the mesopontine nuclei in a Golgi-like manner [2,5,18]. The number, size, diameters and staining intensity of the NADPH-d neurons in the three age groups were examined using computer-assisted image analysis. The relationships between the neurons stained for NADPH-d and ChAT were studied using a double-labelling procedure. The morphological appearance and quantitative analy-

1.S. Lolova et al. ,! Mechanisms of Ageing and Development 90 (1996) ill

sis revealed age-related atrophy of the NADPH-d extent of the LTD and PPN in rats.

1.28

113

neurons along the rostrocaudal

2. Materials and methods 2.1. Histo- and immunohistochemistry Twenty-one male Wistar rats aged 3 months (n = 7) 12 months (n = 7) and 26 months (n = 7) were used. The mean life span of this strain of rat is 22-24 months. Under deep ether anaesthesia the rats were perfused transcardially with phosphate buffered saline (PBS) followed by a fixative containing 4% paraformaldehyde, 0.08% glutaraldehyde and 0.15% picric acid in 0.1 M phosphate buffer (PB) pH 7.4. Slabs containing the mesopontine segment of the brainstem were postfixed in the same fixative without glutaraldehyde for one night at 4°C and soaked in 0.1 M PB containing 20% sucrose for 2 days at 4°C. Coronal sections, 30 pm thick, through the whole rostrocaudal extent of the LTD and PPN were cut serially on a freezing microtome and washed in 0.1 M PBS overnight at 4°C. All sections were collected in subsets of ten. Every first section of the subsets was stained for NADPH-d employing a modification of the procedure of Scherer-Singler et a1.[19]. Free floating sections were rinsed for 30 min in 0.1 M Tris-HCl buffer pH 7.4 with 0.8% Triton X-100, and then incubated for 90 minutes at 37°C in IO ml 0.1 M Tris-HCl buffer pH 7.4 containing 4 mg reduced NADP (Sigma, USA) and 10 mg nitroblue tetrazolium (Sigma, USA). The sections were washed in PBS, mounted on chromegelatin-coated slides, air-dried for 24 h, dehydrated and embedded in Entellan. Control sections were treated identically except for either the omission of the substrate or the omission of the electron acceptor 1201. The specific NADPH-d reaction was eliminated in the control sections. In order to examine the relationship between ChAT and NADPH-d activity, every second section in the subsets was double-labelled for the two enzymes. For ChAT immunostaining, the sections were incubated in rat anti-ChAT bodies (Incstar, USA; diluted 1:200) for 48 h at 4°C then in biotinylated rabbit anti-rat IgG (Immuno Nuclear, diluted 1:200) for 1 h and then in Elite avidin biotin complex (Vector Lab., diluted 1:200) for 1 h. Prior to each incubation the sections were rinsed in PBS. Binding sites were visualized by the standard DAB reaction. Control sections were incubated in 0.1 mg/ml normal rat IgG instead of the monoclonal antibodies. Following this enzyme reaction the sections were stained for NADPH-d. In each experiment, sections from one animal of each age group were processed simultaneously. 2.2. Quantitative analysis

Sections stained for NADPH-d were used for quantitative analysis. To asses the effects of aging on NADPH-d neurons in the LTD and PPN, we compared their number and distribution as well as the somata cross-sectional areas, maximum

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diameters and optical density (OD) along the rostrocaudal extent of the nuclei in 3-, 12- and 26-month-old rats. The quantitative analysis was performed on an image analysis system consisting of an Jenaval photomicroscope, a Hitachi videocamera, an IBM computer and morphometry software developed at the Biophysical Institute, Bulgarian Academy of Sciences (Mr. B. Balev). 2.2.1. Volume of the LTD and PPN For determination of the volume of the nuclei, eighteen rats (6 per age group) were used. The cross-sectional areas of the nucleus were traced bilaterally on all NADPH-d stained sections with a x 4 objective lens starting with the most rostra1 and ending with the most caudal section. The cross-sectional areas and the section thickness were used for volume estimation by a formula based on Simpson’s rule for integration [21]. We also determined the volume at the rostral, middle and caudal subdivisions along the rostrocaudal extent of the LTD and PPN according to the atlas of Paxinos and Watson [22]. 2.22. Cell numbers In the central parts of the LTD and in pars compacta of the PPN, some stained cell bodies and their dendrites were aggregated and overlapped, and thus instead of automatic we used manual cell counting with a Vizopan projection microscope at a magnification x 787. The numbers of cells per section were counted and the total number per nucleus was calculated according to Weibel’s equation cited by Royet [21]. The numbers of NADPH-d neurons were also estimated at the rostral, middle and caudal level since literature data have shown their uneven distribution along the LTD and PPN in mice and rats [2,10]. 22.3. Size, maximum diameter and OD of NADPH-d neurons The cross-sectional areas, maximum diameters and OD of NADPH-d cell bodies were measured bilaterally at rostral, middle and caudal level of the LTD and PPN using an image analyser at magnification x 31. Thirty randomly selected neurons at every level in each rat (n = 21) were measured. Neurons cut only through the nucleus or the stem of the processes were traced by a pointing device. Every cell was measured two times and the averaged values of the cross-sectional area, OD and diameter were used for further calculations. The OD of NADPH-d cell bodies was expressed on an arbitrary gray scale ranging O-l (0, OD of the glass slide; 1, maximal staining intensity). Three recordings were also made of the adjacent background in the fiber tracts and the averaged values were subtracted from the cellular values. 2.2.4. Statistical analysis The means k standard deviation (SD.) are presented. Two-way ANOVA with post hoc Newman-Keuls was used for group analysis and statistical comparison. Statistical significance was accepted at P < 0.05. For the correlation analysis a statistical program for linear regression was used.

I.S. Lokwa et al. / Mechanisms of Ageing und Deoelopment 90 (1996) I1 I --128

115

3. Results 3.1. Distribution

of NADPH-d

and ChAT staining

in the r(lt LTD

and PPN

The topographic distribution and morphological pattern of NADPH-d neurons in the LTD and PPN were similar to those described in rats from other studies [ 1,7, lo- 12,19,23]. The strong diffuse blue reaction product was localized in medium to large nerve cell bodies and their processes (Fig. 1A, Fig. 2A). NADPH-d neurons were multipolar, triangular or bipolar in shape and in the LTD they clustered in the periventricular gray substance of the rostra1 pons. The topography of the PPN was more complicated, due to intermingling with adjacent structures. Briefly, the rostra1 one-third occupied the ventrolateral tegmentum rostra1 to the trochlear nucleus, the middle one-third occupied the central tegmental field caudal to the trochlear nucleus and the caudal one-third was widely determine by the superior cerebellar peduncle. The dense clusters of NADPH-d neurons in the middle one-third of the PPN comprised the pars compacta (PPNc) (Fig. 2) and ventrolateral and most caudal extension of the cells corresponded to the pars dissipata (PPNd). The stained cell bodies emitted approx. two to six straight and thick primary dendrites (Fig. lA, Fig. 2A), oriented perpendicularly to several well-defined fiber tracts. The primary dendrites divided into longer and thinner secondary dendrites. The tertiary dendrites were delicate and varicose. The axons often emitted from a proximal dendritic trunk in every direction, a substantial number traced laterally to superior cerebellar peduncle. In sections double-labelled for visualization of ChAT and NADPH-d almost all ChAT positive cell bodies, besides showing a fine granular reddish-brown reaction product indicative of DAB, exhibited a diffuse blue reaction product indicative of formazan. ChAT positive neurons without NADPH-d reaction were occasionally found (Fig. 3). The pattern of NADPH-d and ChAT staining was similar in the LTD and PPN of the three age groups. The variability in shape, size and staining pattern increased with advancing age. Some NADPH-d cell bodies displayed large focal swellings and spot-like staining (Fig. lB, Fig. 2B). In contrast, other NADPH-d cell bodies appeared hypertrophic and very darkly stained (Fig. lC, Fig. 2C). The dendritic processes in aged rats were short, stunted and had focal multiple swellings that were rarely observed in young rats. The axons in aged rats exhibited irregularly spaced varicosities of different size. 3.1.1. Volume and lerlgth of LTD and PPN und numbers of NADPH-d neurons No significant differences in the volume of the LTD and PPN and the numbers of NADPH-d neurons of the left and right sides were found in 3-month-old rats. Thus, the results included the measurements of the volume and numbers of both sides. The LTD in 3-month-old rats exhibited a length of 1.38 _+0.36 mm (mean I~I SD.), a volume of 0.153 + 0.024 mm3 and contained 1929 f 432 NADPH-d neurons. The largest number of NADPH-d cell bodies was localized at the middle level (73.87X), followed by these at the rostra1 (14.52%) and caudal (13.95%) levels.

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Fig. 1. NADPH-d neurons at the middle level of the laterodorsal tegmental nucleus in 3-month-old (A), 12-month-old (B) and 26-month-old (C) rats. A: The neurons exibit relatively uniform size and staining intensity. The arrows point to straight primary dendrites. B: The small arrows show neurons with irregular outlines and spot-like staining and the thick arrows show very thick, but short primary dendrites. C: Atrophic (small arrows) and hypertrophic (curved arrow) neurons with short and wrinkled primary dendrites. The headarrows show swellings in somata and dendrites. Scale bar = 36 pm.

Fig. 2. NADPH-d neurons in the pedunculopontine tegmental nucleus pars compacta of 3-month-old (A), 12-month-old (B) and 26-month-old (C) rats. A: The arrows point to long regular dendrites. B: The small arrows show unevenly stained neurons. The large arrows show short and thick dendrites. C: The small arrows point to atrophic neurons and large arrows point to hypertrophic neurons. Scale bar = 60 jcm.

The PPN had a length of 1.X8 _+0.43 mm, a volume of 0.372 _+0.104 mm3 and contained 2587 _+284 NADPH-d neurons. Its pars compacta comprised 58.79% of the total population as determined in NADPH-d stained sections.

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ANOVA showed that age did not have a significant influence on the volume and length of the LTD and PPN and numbers of NADPHd neurons at the rostra\, middle and caudal levels (Table 1).

Fig. 3. 1The asterisks show ChAT positive, but NADPH-d (A) and in the pedunculopontine nucl .eus of 3-month-old rats (B). Scale bar = 25 pm. Old

negative neurons in the laterodorsal tegmenta’ tegmental nucleus pars dissipata of 26.month.

(mm2)

(mm)

are means + S.D. Age-related

* Data

3 12 26

26

12

3

26

12

3

Age (months)

of the laterodorsal

Length

Cell (numbers)

Volume

Table 1 Volume and length LTD and PPN and pedunculopontine

tegmental

280 *I82 255 f 142 236 1139

1929

changes

in all parts

*432 1851 ,492 1788 i406

kO.051

are insignificant

(ANOVA:

1.375 * 0.366 1.475 + 0.349 1.450 f 0.352

1425 5390 1261 1357 1322 + 365

0.033 _tO.O18 0.030 kO.016 0.032 +0.017

0.153* + 0.024 0.168 *0.051 0.169

0.100 + 0.026 0.106 + 0.027 0.110 + 0.037

Rostra1

Middle

(PPN)

Total

LTD

(LTD)

P>O.O5).

269 k89 335 +186 230 *91

0.028 *0.011 0.035 kO.016 0.030 _+0.014

Caudal

Rostra1 0.122 * 0.054 0.096 kO.033 0.107 * 0.043 721 f 226 560 + 320 659 +391

0.372 +0.104 0.325 rf;0.088 0.337 _t 0.098 2587 i 284 2208 k631 2396 _+707

1521 * 300 1178 i 382 1383 + 565

0.208 + 0.060 0.169 k 0.058 0.186 * 0.074

Middle

neurons

1.875 ? 0.427 1.850 * 0.509 1.800 k 0.558

of NADPH-d

Total

PPN

nuclei of rats. Numbers

-

f 0.036 0.045 ) 0.028

+0.013 0.059

0.042

Caudal

at the three levels of

i3 0

c P

9

.*

IS.

120

Table 2 Cross-sectional PPN tegmental

Lo/ova et al. 1 Mechanisms

area, maximum nuclei of rats

diameter

Age (months)

Area (pm’)

3 12 26

Diameter

(pm)

3 12 26

OD (arbitrary units)

3

12 26

* Data

are means k S.D. Significant

OJ Age@

and Development

and OD of NADPH-d

90 (1996) 1 I I

neurons

LTD

128

at the three levels in LTD and

PPN

Rostra1

Middle

Caudal

Rostra1

Middle

Caudal

211.9* k73.8 266.3 k88.3 249.9 k87.0

292.3 k75.8 277.0 k81.5 252.4 f 80.8

251.4 i73.1 254.3 k75.0 229. I k69.8

222.4 +55.0 241.1 k79.7 225.5 171.8

218.4 k64.9 215.9 k68.7 200.2 +57.3

228.8 f 57.8 214.3 _+55.3 207.9 k5l.l

26.6 + 6.4 25.0 k5.9 24.8 k6.0

25.3 i5.5 25.0 i7.2 23.1 k5.5

23.0 k5.2 23.6 +7.0 22.3 i 5.2

22.3 1-4.1 22.8 j4.9 22. I +4.6

21.6 k4.0 21.1 i4.4 20.6 i4.2

23.3 k5.3 21.8 k4.3 21.8 k4.0

0.513

0.515

0.470

0.523

0.556

0.548

kO.097 0.489 TO.091 0.511 kO.138

kO.103 0.556 kO.113 0.508 iO.142

+0.104 0.508 io.103 0.473 *0.103

kO.107 0.532 i 0.098 0.494 kO.137

+ 0.098 0.565 to.079 0.538 +0.130

+ 0.090 0.574 2 0.082 0.532 kO.122

differences

are described

in the text

3.1.2. Cross-sectional areas, maximum diameters and OD of NADPH-d 3-month-old rats.

neurons in

Pilot experiments showed that there were no significant differences in cross-sectional areas, maximum diameters and OD of NADPH-d cell bodies of the left and right nuclei. These parameters, however, differed significantly at the three levels of the LTD and PPN in 3-month-old rats (Table 2). In the LTD the cross-sectional areas were largest at the middle and least at the caudal levels (F(2,608) = 9.84; P = 0.000062). The diameters of NADPH-d cell bodies were longest at the rostra1 level and shortest at the caudal level (F(2,608) = 21.23; P < 0.000000). The OD of NADPH-d cell bodies at the rostra1 and middle levels of the LTD were higher than those at the caudal level (F(2,608) = 13.30; P = 0.000002). In the PPN of 3-month-old rats significant differences were also found in the pars dissipata (rostra1 and caudal level) and pars compacta (middle level). Maximum diameters of NADPH-d neurons were smaller in the PPNc compared to the PPNd (F(2528) = 2.27; P = 0.001811). The OD of NADPH-d neurons in the PPNd were lower than that in the PPNc (F(2,528) = 5.69; P = 0.003586).

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3.1.3. Cross-sectional

ureas of NADPH-d

121

cell bodies in aging

The age-related changes in the cross-sectional areas in both the LTD and PPN were identical. ANOVA demonstrated a significant effect of age (F(2,1881) = 27.55; P < 0.000000) and level (F(2,1881) = 19.17; P < 0.000000) on the cross-sectional areas of NADPH-d cell bodies in the LTD. In 26-month-old rats the cross-sectional areas were significantly decreased at the rostra1 (10%; P = 0.0048), middle (14%; P = 0.00003) and caudal (11%; P = 0.0020) levels compared to 3-month-old rats. Significant effects of age (F(2,1603) = 7.52; P = 0.00056) and level (F(2,1603) = 11.60; P = 0.000010) on the cross-sectional areas of NADPH-d neurons in the PPN were shown. In 26-month-old rats significant reduction of the cross-sectional areas occurred in the PPNc (8%; P = 0.0486) and the PPN-caudal level (9%; P = 0.02791) compared to 3-month-old rats. A significant increase of the cross-sectional area at the rostra1 level (8.4%; P = 0.02647) appeared between 3 and 12 months of age. Histograms of the size distribution of NADPH-d cell bodies in the LTD and PPN revealed a shift towards smaller surfaces at the three levels examined (Fig. 4). 3.1.4. Diameters of NADPH-d cell bodies in aging ANOVA revealed a significant effect of age (F(2,1836) = 10.60; P = 0.000026) and level (F(2,1836) = 26.88; P < 0.000000) on the maximum diameters of NADPH-d cell bodies in the LTD. In 12-month-old rats the diameters were significantly decreased (6%); P = 0.01810) only at the rostra1 level of the nucleus compared to 3-month-old rats. In 26-month-old rats the diameters were also decreased at the middle level (9%; P = 0.00341) compared to 3-month-old rats. Age (F(2,1603) = 5.5; P = 0.004177) and level (F(2,1603) = 14.58; P = 0.000001) affected the maximum diameters of NADPH-d neurons in the PPN. In 12-monthold rats the diameters decreased (6%; P = 0.01760) at the PPN-caudal level compared to 3-month-old rats. The changes in the diameters at the rostra1 and middle levels were insignificant. 3.1.5. OD of NADPH-d cell bodies in aging The effect of age (F(2,1881)) = 6.22; P = 0.002031) and level (F(2,1881) = 23.17; P < 0.000000) on the OD of NADPH-d cell bodies in the LTD was significant (Table 2). The OD was increased at the middle (8%; P = 0.00015) and caudal (8%; P = 0.0034) levels in 12-month-old rats compared to 3-month-old rats. Between 12 and 26 months of age the OD significantly decreased at the middle (9%; P = 0.00010) and caudal (7%; P = 0.00426) levels. ANOVA revealed a significant effect of age (F(2,1603) = 15.52; P < 0.000000) and level (F(2,1603) = 20.28; P < 0.000000) on the OD of NADPH-d neurons in the PPN. In 26-month-old rats the OD of the neurons in the PPNd decreased at the rostra1 (7%; P= 0.00414) and caudal (7%; P= 0.00336) levels compared to 12month-old rats, but remained unchanged in the PPNc. Fig. 5 demonstrates a more flat OD distribution in the LTD and PPN with aging, being more pronounced at the rostra1 and middle levels. There was a significant positive correlation between size and OD of NADPH-d neurons in the two mesopontine nuclei examined, being higher in the PPN than in

122

I.S. Lolova et al. 1 Mechanisms

0

100 200 300 400 500 600

of’ Age&g and Development

90 (1996) 11 I

0

0

3

100 200 300 400 500 600

128

100 200 300 400 500 600

12

26

A

AGE (months)

0

100 200 300 400 500 600

3

0

100 200 300 400 500

12

600

0

100

200

300

26

400

500

600

B

AGE (months)

Fig. 4. Histograms of the size distribution (pm’) of NADPH-d neurons at the three levels of the laterodorsal tegmental nucleus (A) and the pedunculopontine tegmental nucleus (B). At each level the cross-sectional area was determined by analysing 210 neurons per age group.

IS.

Lo/ova ef al. 1 Mechanisms

of’ Ageing and Development

0

0.2

0.4 72

0.6

90 (1996) 11 l-128

0.6 0

0.2

123

0.4 26

0.6

0.4 26

0.6

0.6

A

AGE (months)

-0

0.2

04 3

0.6

08

0

0.2

0.4 12

0.6

0.8

0

0.2

0.8 6

AGE (months) Fig. 5. Histograms of the OD (arbitrary units) distribution of NADPH-d neurons at the three levels of the laterodorsal tegmental nucleus (A) and the pedunculopontine tegmental nucleus (B). At each level the OD was determined by analysing 210 neurons per age group.

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the LTD. Therefore, the reduction in the size of NADPH-d accompanied by a reduction in OD (Table 3).

neurons

in aging was

4. Discussion In the present study, the morphological appearance and the quantitative analysis indicated atrophy of NADPH-d neurons in the LTD and PPN of rats during normal aging. The numbers of NADPH-d nerve cells remained unchanged. It is well known that NADPH-d reaction is a selective and excellent marker of the cholinergic neurons in the mesopontine nuclei [10,12,18,23,24]. The present and previous studies on rats [12] have demonstrated that virtually all ChAT positive neurons are NADPH-d stained. Thus, the herein described age-related changes in the morphometric parameters of NADPH-d neurons suggests atrophy of the cholinergic neurons in rat LTD and PPN with advancing age. In this discussion, the term cholinergic references to either ChAT or NADPH-d positive neurons. Together with the basal forebrain nuclei, the mesopontine tegmental nuclei LTD and PPN are the major source of the ascending and descending cholinergic innervation [25-281. The age-related impairment of memory, learning, sleep, motor and autonomic activity in rats correlates with dysfunctions of cholinergic innervation [14.15,29]. Considerable attention has been devoted to the age-related changes in the forebrain nuclei. Reductions in both cholinergic cell number and size in aged rats have been reported [14,15,30,31]. A progressive decline with age in cholinergic cell numbers and projections of the nucleus basalis in humans has also been described [32-351. However, the data on age-related changes in the cholinergic neurons of the mesopontine nuclei are rather scanty. The present results demonstrating an age-related reduction in the neuronal size, but not in the numbers of NADPH-d neurons, are in accordance with the findings of atrophy of NADPH-d and ChAT-positive neurons in the mesopontine nuclei of aged mice and rats [2,10,23]. Moreover, our qualitative and quantitative data indicated similar age-related changes in the LTD and PPN. Besides the same NADPH-d and ChAT-immunochemistry, the two mesopontine nuclei exhibited common cytochemical features [26], which distinguish them from other cholinergic cell groups.

Table 3 Correlation between cross-sectional PPN tegmental nuclei Age (months)

3 I2 26

area and OD of NADPH-d

neurons

at the three levels of LTD and

PPN

LTD Rostra1

Middle

Caudal

Rostra1

Middle

Caudai

0.1 I 0.X3* 0.26*

o.l8* 0.12 0.06

0.29* 0.32* 0.30*

O.l6* 0.44* o-37*

0.41* 0.38* 0.4x*

O.l6* 0.39* 0.19*

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Factors for differential involvement of the cholinergic neurons in the forebrain and mesopontine nuclei in the aging process could be the cytochemical features and the selective vulnerability of the neurons. Unlike the cholinergic neurons in the mesoponine nuclei, the cholinergic neurons in the forebrain have high levels of NGF receptor protein [27]. Mesulam et al. [27] have found ChAT and NADPH-d positive neurons in the nucleus basalis of Meynert. These, however, represent each with different neuronal morphology. The non-overlapping populations, cholinergic neurons in the mesopontine nuclei exhibit a strong NADPH-d reaction. The cholinergic neurons in the forebrain and mesopontine nuclei colocalize with different neuropeptides. The neurons in the basal forebrain and other subcortical areas, which are affected by the degeneration in Alzheimer’s disease are a large part of the same neuronal population damaged during aging and other neurodegenerative diseases [36]. Based on data about the neuronal vulnerability Arendt et al. [36] classified the basal forebrain nuclei as highly vulnerable and the mesopontine nuclei as moderately vulnerable. The causes for the selective vulnerability of the neurons in aging are still unclear. Investigations of the NOS subtypes and the cofactors involved in NO formation could contribute to understanding of the neuronal selective vulnerability in aging [3]. The quantitative analysis in this and other studies on the cholinergic mesopontine nuclei [2,27] has demonstrated that NADPH-d neurons are unevenly distributed along the LTD and PPN of old animals. The age-related changes are not equally manifest along the rostrocaudal extension of the LTD and PPN of rats and mice. A heterogeneous distribution of the changes in the cholinergic neurons has been demonstrated in the forebrain nuclei during aging and in neurodegenerative diseases [35,37-391. A gradient emerges along the rostrocaudal extension of the basal forebrain nuclei suggesting that the degeneration of the cholinergic neurons begins in the most rostra1 parts and then includes all subdivisions of the complex [35,40]. This problem has not yet been clarified. The significance of the neuron size and type, the individual and genetic differences, and differences in the connectivity are discussed [2,35,41]. Different afferent and efferent connections of the cholinergic mesopontine nuclei in rat are described [26,27,42]. The LTD has more extensive projections to the limbic system, whereas the PPN is closely associated with the sensory nuclei and extrapyramidal structures. However, little is now known of the topography of the projections from and to the three parts of the LTD, and PPNc and PPNd. Recently, Gonzalorius et al. [43] have revealed that the LTD and PPN exhibit distinct patterns of the projections into subdivisions of the anterior thalamic nuclear complex. In the present study, the morphological picture as well as OD and histograms of the size distribution of NADPH-d neurons in the LTD and PPN demonstrated the appearance of hypertrophic and darkly stained cells. These results are in accordance with the observations of neuronal hypertrophy in the forebrain nuclei in aged rhesus monkeys [44] and aged humans as well as in neurodegenerative diseases [35,39,44]. Moreover, in the human hippocampal formation, innervated by both forebrain and mesopontine nuclei, an increase in the density of NADPH-d fiber

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staining has been observed in aging [3]. Neuronal hypertrophy accompanied by dendritic reorganization is regarded as a sign of a compensatory mechanism attempting to counteract the degenerative changes of the neighbouring neurons [3,36]. The capacity for lesion-induced sprouting of the cholinergic fibers is retained in aged rats and humans [45]. In addition to the age-related morphological alterations in the dendrites observed in this and previous [2,46] studies, we have also found quantitative changes in the dendritic tree of NADPH-d neurons in the mesopontine nuclei (unpublished data). In conclusion, the NADPN-d neurons in the rat LTD and PPN are atrophied with age which is evidenced from the morphological appearance and the quantitative changes in their size, diameter and OD. These changes are related with dysfunctions of the cholinergic innervation in the aging nervous system.

Acknowledgements This work was supported by Grant No L-530 from the National Science Fund.

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