Brain Research, 540 (1991) 195-203 Elsevier

195

BRES 16286

Functional recovery and collateral neuronal sprouting examined in young and aged rats following a partial neural lesion George A. Kuchel 1-3'* and Richard E. Zigmond 3'4 1Division on Aging, Harvard Medical School, Boston, MA 02115 (U.S.A.), 2Department of Medicine, Beth Israel and Brigham and Women's Hospitals, Boston, MA 02215 (U.S.A.), 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School Boston, MA 02115 (U.S.A.) and ~Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) (Accepted 21 August 1990)

Key words: Neural plasticity; Aging; Sympathetic; Pineal gland; Collateral sprouting

The rat pineal gland was chosen as a model system to study how aging affects the capacity of surviving neurons to compensate for partial destruction of a neural pathway. The pineal gland receives bilateral overlapping sympathetic innervation from the two internal carotid nerves, whose activity regulates several aspects of pineal metabolism in a circadian fashion. The most dramatic of these is the marked nighttime increase in the activity of N-acetyltransferase, the rate-limiting enzyme in melatonin synthesis. These features allow for the pineal gland to be used as a model system for studies on neuronal plasticity, since it is possible to create specific partial neural lesions and to evaluate functional recovery subsequently at the cellular level. We examined the activity of N-acetyltransferase and the content of melatonin in the pineal gland as indices of pineal function at various time points after unilateral surgical denervation (lesion of one of the two internal carotid nerves) in 4-month(young) and 25-month-old (aged) rats. At both ages, the nighttime levels of the two parameters were significantly lower 8 h after this lesion than in sham-operated animals of the same age, indicating impaired function. When examined at later time points (i.e., 1.5 and 10 days after this lesion), both young and aged animals exhibited full recovery in these two parameters. Measurement of specific neuronal uptake of [3H]norepinephrine was utilized as an index of the number of sympathetic varicosities innervating the pineal gland. One and a half days after unilateral denervation, [3H]norepinephrine uptake in 4-, 10-, 16- and 25-month-old animals was approximately 50% of that obtained in sham-operated animals of the same age, as expected if half of the innervation of the gland was lesioned. Ten days after surgery, [3H]norepinephrine uptake in 4- and 10-month-old animals was 78% of the value found in sham-operated animals of the same age, suggesting collateral sprouting of the remaining sympathetic nerve fibers. However, in 16- and 25-month-old animals, uptake remained at 50%. These data indicate that recovery of pineal function after unilateral denervation occurs to a similar extent and with a similar time course in young and aged animals. In contrast, the data suggest that collateral sprouting is impaired in aged animals. The fact that functional recovery occurs in young animals sooner after the lesions than does collateral sprouting and that recovery occurs in the aged animals in the apparent absence of collateral sprouting suggests that the relationship between this anatomical form of plasticity and functional recovery may be complex. Among other possibilities, collateral sprouting may play a role in the maintenance of recovered function rather than in the initial recovery process or sprouting may be functionally important after nerve losses greater than those represented by a 50% lesion. INTRODUCTION N e u r o n a l death has traditionally been emphasized as the p r e d o m i n a n t process taking place in the course of aging and n e u r o d e g e n e r a t i v e diseases associated with aging. More recently, a n u m b e r of examples of n e u r o n a l plasticity such as axonal growth, traditionally associated with d e v e l o p m e n t , have been shown to persist into maturity and old age 1°. These p h e n o m e n a are important for an u n d e r s t a n d i n g of the impact of aging on neural function and for the capacity of the nervous system to m a i n t a i n function in spite of n e u r o n a l losses. The sympathetic innervation of the rat pineal gland is an attractive model for studies of neural plasticity since it is possible to make both specific and reproducible

lesions of this innervation and to evaluate the time course and extent of any functional recovery at the cellular level s'27'51'52. The pineal gland receives overlapping sympathetic innervation from fibers in the right and left internal carotid nerves (ICN), whose cell bodies are located in the two superior cervical ganglia (SCG) 9'28. This innervation regulates m e l a t o n i n synthesis in a circadian fashion, with increased synthesis occurring at night. Pineal parenchymal cells synthesize melatonin from serotonin in two steps 3'24. The first step involves the acetylation of serotonin to N-acetylserotonin, catalyzed by serotonin N-acetyltransferase (NAT; arylamine Nacetyltransferase, EC 2.3.1.87). This c o m p o u n d is subsequently O-methylated to produce m e l a t o n i n in a reaction catalyzed by the enzyme hydroxyindole O-

* Present address: Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, U.S.A. Correspondence: R.E. Zigmond, Department of Neurosciences, Case Western Reserve University, School of Medicine, 2119 Abington Rd., Cleveland, OH 44106, U.S.A. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

196 methyltransferase

(N-acetylserotonin

ase, E C 2.1.1.4).

T h e o n s e t of d a r k n e s s

dramatic

O-methyltransferresults in a

(40- to 100-fold) increase in the activity of

N A T 3'24. This diurnal r h y t h m i c i t y a p p e a r s to be g e n e r ated

by changes

in the

activity of the

sympathetic

n o r a d r e n e r g i c fibers i n n e r v a t i n g the pineal gland, which

ficial neck exploration only. without a nerve lesion. Fhe success o! the nerve lesion was evaluated by the development of ptosis, which was found to be present ipsilaterally in all of the lesioned animals studied, with the exception of one which was excluded from analysis. In all of the lesion studies examining NAT activity, pineal melatonin content or [3H]NE uptake, animals were sacrificed under a dim red light 7-9 h into the dark cycle, with the pineal gland removed within 1 min 7

p r o d u c e c h a n g e s in t h e i r release of n o r e p i n e p h r i n e ( N E ) . Part of the e v i d e n c e for this h y p o t h e s i s is that bilateral s u p e r i o r cervical g a n g l i o n e c t o m y 13'e3 o r a d m i n i s t r a t i o n of the f l - a d r e n e r g i c a n t a g o n i s t

propranolo113 abolish this

r h y t h m . In a d d i t i o n , electrical stimulation of the s y m p a thetic i n n e r v a t i o n o f the pineal gland during the d a y t i m e i n c r e a s e pineal N A T activity to levels c o m p a r a b l e nighttime

values 7. T h u s ,

the

level of N A T

to

activity

a p p e a r s to reflect the e x t e n t of s y m p a t h e t i c s t i m u l a t i o n of the pineal gland. In studies with y o u n g animals, we h a v e p r e v i o u s l y d e m o n s t r a t e d i m p a i r e d pineal f u n c t i o n during the first night a f t e r u n i l a t e r a l d e n e r v a t i o n ( p r o d u c e d by a unilateral lesion of t h e I C N or by unilateral s u p e r i o r cervical g a n g l i o n e c t o m y ) , with full r e c o v e r y o f f u n c t i o n by the s e c o n d night after t h e s e lesions 27'51"52. H e r e we h a v e e v a l u a t e d the effect of aging on the capacity of the rat pineal gland to r e c o v e r functionally f o l l o w i n g partial s y m p a t h e t i c d e n e r v a t i o n . Since collateral s p r o u t i n g c o u l d play a role in the f u n c t i o n a l r e c o v e r y d e m o n s t r a t e d in y o u n g a n i m a l s , we also e x a m i n e d the effect of aging on the e x t e n t of collateral s p r o u t i n g f o l l o w i n g this lesion,

NAT activity and melatonin content In all experiments where pineal NAT activity was to be measured. pineal glands were immediately frozen on dry ice and stored at -80 °C until assayed. NAT activity was determined by a modification of a radiochemical assay developed by Deguchi and Axelrod ~4 in which the rate of formation of [14C]N-acetyltryptamine from [IZC]acetyl-CoA (NEC 313; New England Nuclear Corp., Boston, MA) and tryptamine is measured 7. Individual pineal glands werc homogenized in 100 F~I of 0. I M sodium phosphate buffer, pH 6.8, using glass-glass homogenizers and NAT activity was determined in a 40 ~ul aliquot. A second aliquot was used to measure NE and melatonin content of the pineal gland. For this purpose, 125 itl of a 165 mM trichloroacetic acid solution, containing 50 nM epinephrine as an internal standard, were added to a 40 I~1 aliquot of the homogenate. Following protein precipitation, NE and melatonin levels were measured using high performance liquid chromatography with electrochemical detection. NE was eluted from a 25 cm Beckman Ultrasphere ODS 5 /~ column with a mobile phase containing 50 mM H3PO 4 (pH 2.6), 0.1 mM EDTA, 12% methanol and 0.4 mM octylsodium sulfate. Detection was accomplished electrochemically (using an ESA 5100A detector; ESA, Bedford, MA) with three electrodes set in series at +0.40 V, +0.05 V and -0.42 V. Melatonin was eluted from a 7.5 cm Beckman Uhrasphere ODS 5 ,u column with a buffer containing 0.1 M sodium citrate, 0.1 M sodium acetate (pH 3.8), 0.1 mM EDTA, 8% methanol and 8% acetonitrile (a modification of the method of Mefford et al.3~). For melatonin detection, the three electrodes were set in series at +0.71 V. +0.37 V and -0.28 V 27,

using n o r e p i n e p h r i n e u p t a k e as an index of sprouting. If a g e d animals are also able to r e c o v e r pineal f u n c t i o n f o l l o w i n g this lesion, t h e n the c o m p e n s a t o r y m e c h a n i s m s i n v o l v e d in this r e c o v e r y could play a role in e n a b l i n g the a g e d o r g a n i s m to m a i n t a i n n o r m a l f u n c t i o n f o l l o w i n g n e u r o n a l losses, such as occurs in a g e - r e l a t e d n e u r o l o g ical diseases, f o l l o w i n g injury, o r as a result of ' n o r m a l ' aging. A p r e l i m i n a r y r e p o r t of this study was m a d e to the S o c i e t y for N e u r o s c i e n c e 26.

MATERIALS AND METHODS

Animals and surgical procedures Unless noted otherwise, animals were Fischer-344 male rats obtained from a National Institute on Aging supervised colony (Harlan Industries, Indianapolis, IN) and were 4, 10, 16 or 25 months old at the time of sacrifice. They were housed in individual cages for 2-4 weeks prior to the beginning of an experiment, with food (Purina rat chow) and water available ad libitum. Lighting in the room was maintained on a 12 h:12 h light-dark cycle. Surgical procedures were performed in the last 7 h of the light period under chloral hydrate anaesthesia (Sigma Chemical Co., St. Louis, MO), supplemented with ether. An age-adjusted dose of chloral hydrate 34 was given subcutaneously (735 mg/kg weight in 4-month-old; 560 mg/kg in 10-month-old; 420 mg/kg in 16- and 25-month-old rats). Surgery consisted of making a neck incision, exposing the SCG, cutting the ICN near its entry into the carotid canal and again near the SCG, and removing a segment of the nerve trunk. "Sham-operated' animals underwent anaesthesia and super-

ff H]NE uptake [3H]NE uptake has been used as an index of the extent of adrenergic innervation in a number of central and peripheral nervous tissues 21'37. For the [3H]NE uptake studies, each pineal gland was homogenized in 100 ,ul of 0.32 M cold sucrose in individual Teflon-glass Potter-Elvehjem homogenizers. Following a 3 min preincubation period, a 40 #1 aliquot of this homogenate was incubated for 7 min at 37 °C in modified potassium phosphateRinger's buffer j~ containing nialamide (12.5/~M; Sigma Chemical Co., St. Louis, MO) and I-[7-3H]NE (77 nM, spec. act. 10-30 Ci/mmol; NET 377: New England Nuclear, Boston, MA). In the studies of NE uptake done at saturating NE concentrations (Fig. 3), I-NE (Sigma Chemical Co., St. Louis, MO) was added to the [3H]NE to yield a final NE concentration of 2 mM and a specific activity of 1.96 Ci/mmol. The reaction was stopped by the addition of cold buffer, and the contents were filtered through nylon filters (no. 66601 ; Gelman, Ann Arbor, MI) to separate free [3H]NE from that taken up by sympathetic varicosities. The radioactivity retained by the filter was quantified by liquid scintillation counting. This assay was a modification of the centrifugation assay used by Dornay et all 6 to measure NE uptake in pairs of pineal glands. It was necessary to establish that the assay we used measured a specific uptake process into enclosed nerve membranes. Using this assay, NE uptake was linear with time from 0 to 16 min and was also proportional to the amount of tissue used. The K m value obtained (0.17 /~M; performed on 3-month-old Sprague-Dawley rats) was similar to that seen in other peripheral and central noradrenergic nerve fibers 2~. as was the ability of desipramine to block this uptake 21 (Table I). More than 90% of the uptake seen at a NE concentration of 77 nM was dependent on the presence of sympathetic innervation, as it was not seen after bilateral denervation of the pineal gland (Table I). The disruption of cell membranes

197 by the addition the detergent Triton X-100 to a final concentration of 0.1% in the homogenization buffer inhibited over 90% of the uptake process, as did performing the incubation at 0 °C (Table I). These data suggest that the assay is measuring the active transport of NE across cell membranes. No detectable difference between [3H]NE uptake in pineal glands from control animals sacrificed during the day and those sacrificed at night was found (data not shown). The protein content of the pineal gland was determined by the method of Lowry3~. Removal of all sympathetic input by a bilateral denervation did not result in any detectable change in pineal protein content (data not shown), confirming anatomical studies suggesting that nerve terminals contribute a negligible proportion of the mass of this target tissue3°'49. Thus, in studies evaluating biochemical parameters in the nerve terminals (NE uptake and NE content), data were expressed per pineal gland, whereas, in studies of parameters reflective of pinealocyte function (NAT activity and melatonin content), values were expressed per #g of protein.

Statistical analysis Statistical analysis was performed by Student's t-test for two means (two-tailed) or using one- or two-way analysis of variance, as noted. When analysis of variance was significant, it was followed by Tukey's Honesty Sum Difference test, allowing for multiple comparisons of group pairs. Linear regression was used to measure the correlation between aging and [3H]NE uptake.

TABLE I

Characterization of [3H]NE uptake Each group represents three to six 4-month-old rats. Results are expressed as the percentage of the value obtained in control animals (1.5 pmol NE/7 min/pineal). One-way analysis of variance followed by Tukey's Honesty Sum Difference test revealed that NE uptake under each experimental condition studied was significantly lower than under control conditions (P < 0.05).

Condition

[SHINE uptake (% of control values)

Control Bilateral denervation Desipramine (6nM) (10pM) Triton X-100 (0.1%) Incubationat 0 °C

100.0 + 6.9 9.1 + 1.3 45.5 + 14.1 8.3 + 0.5 6.9 + 1.2 1.3 + 1.1

60% of those o b t a i n e d in the young animals (Table II). Similar results were obtained at shorter intervals after a sham operation (data not shown). Nighttime N A T activity and m e l a t o n i n levels in the

RESULTS

pineal gland were measured in rats of different ages

Biochemical indices of recovery o f function after unilateral denervation Nighttime N A T activity in animals that had undergone

during the first, second and tenth night following unilateral sympathetic denervation or after a sham-operation (Figs. 1 and 2). W h e n expressed as the percentage of the

a sham operation 10 days earlier was not significantly different in aged (25-month-old) as compared to young

values obtained in animals of the same age that had u n d e r g o n e a sham-operation, N A T activity was impaired

(4-month-old) animals (Table II). However, nighttime pineal m e l a t o n i n levels in these aged animals were only

during the first night after unilateral denervation in both young and aged animals (Fig. 1). This was followed by a complete recovery of N A T activity by the second night after the lesion in both young and aged animals. Mean N A T activity was also at control levels in young and aged animals 10 days after unilateral denervation. The nocturnal rise in pineal melatonin levels during the first night after surgery was also impaired, though less than the rise in N A T activity, in both young and aged animals (Fig. 2).

175 150 .~-

125

~

100

< "~

75

z~

50

--

- - -."--""" "-%Y'""

-

TABLE II

25 -~ ~** 0 .,z. 0

o.--o 4 monthold : = 25 month old 1

2

3

4

5 6 Days

7

8

9

10

Nighttime NAT activity and melatonin content in pineal glands from sham-operated animals 11

Fig. 1. Recovery in nighttime pineal NAT activity following unilateral sympathetic denervation of the pineal gland. Results are expressed as the mean percentages _+S.E.M. of the values obtained for sham-operated animals of the same age at the same survival times. Animals were sacrificed 7-9 h into the dark cycle under a red light. Each of the two early time points (8 h, 1.5 days) represents at least five animals, with the late time point (10 days) representing at least 10. Statistics were performed using a Student's t-test for two means comparing the actual values for enzyme activity in pmol/pg/20 min obtained in the lesioned animals with those for the shamoperated group (**P < 0.01).

Eleven young and 10 aged sham-operated animals were studied in two separate experiments representing the control animals studied 10 days after surgery in Figs. 1 and 2. Animals were sacrificed 7-9 h into the dark cycle. Results are expressed as the mean + S.E.M. The difference in NAT activity at the two ages was not statistically significant; however, pineal melatonin content was significantly lower in the aged animals (*P < 0.05).

Age (months)

NAT activity Pineal melatonin (pmol/Mgprotein/20 rain) (pmol//zgprotein)

4 25

24.9 _+2.1 20.0 + 4.1

34.6 + 3.3 21.0 _+4.0*

198 125

LtJ

"¢

2.0.

T

n

100 W

7s oc~~.-= 1.0

"6

50"

1,1-6

25.

r.) Ix.

0---0 4 month old z -- 25 month old

1

I,I 13-

0

0.0

0

;

&

Days

1'2

1'6

2'o

2'4

AGE ( m o n t h s )

Fig. 2. Recovery in nighttime pineal melatonin content following unilateral sympathetic denervation of the pineal gland. Results are expressed as the percentages + S.E.M. of the values obtained for sham-operated animals of the same age at the same survival times. Animals were sacrificed 7-9 h into the dark cycle under a red light. Each of the two early time points (8 h, 1.5 days) represents at least 5 animals, and the late time points (10 days) represent at least 10. Statistics were performed using a Student's t-test for two means comparing the actual valaes for melatonin content in pmol//,g protein measured in the lesioned and sham-operated groups (*P < 0.05).

There was an essentially full recovery in this parameter by the second night after surgery in both age groups. Ten days after the lesion, pineal melatonin levels were also not statistically different from sham-operated control groups.

Fig. 4. Changes with age in specific neuronal NE uptake at a subsaturating concentration of NE. Specific NE uptake was defined as the uptake measured in homogenates of pineals from control animals minus the uptake measured in bilaterally denervated pineals from 4-month-old animals. Each point represents the mean _+ S.E.M. for at least 4 animals. The data shown here were measured in the sham-operated animals from the lesion experiments shown in Fig. 5. In each of three experiments both 4-month-old animals and either 10-, 16-, or 25-month-old animals were studied. The results for the 4-month-old sham-operated animals were not significantly different from each other in the 3 experiments (data not shown). A linear regression revealed a significant decline in specific NE uptake with age (P < 0.001; r = -0.74).

spite of technical difficulties posed by the low signal to noise ratio (specific vs non-specific uptake) with saturating NE concentrations, it was possible to observe a decline in the specific neuronal NE uptake between 4 and

[3H]NE uptake before and after unilateral denervation Specific neuronal NE uptake was first measured in pineal glands from control Fischer-344 rats at a saturating concentration (2 ~M or 10-fold greater than the Km). In

LO 5~

1

4.0

T

z~

.T

T

¢0

E

tad

0.0

0

~

~

1'2

1'6

2'o

2'4

AGE ( m o n t h s )

Fig. 3. Changes with age in specific neuronal NE uptake at a saturating concentration of NE. Specific NE uptake was defined as the uptake measured in the absence of the inhibitor desipramine minus that measured in its presence (10 /,M). Animals were sacrificed during the first 6-8 h of the light cycle. Each point represents at least 4 animals. Results are expressed as the mean _+ S.E.M. for each group. A linear regression revealed a significant decline in specific NE uptake with age (P < 0.05; r = -0.43).

TABLE If!

Daytime and nighttime pineal N E and protein content in animals of various ages Daytime pineal NE levels were measured in control animals sacrificed 6-8 h into the light period. The nighttime NE levels and protein contents were measured in pineal glands from the shamoperated animals studied in Fig. 5 and sacrificed 7-9 h into the dark period. Each group represents at least 5 animals. Two-way analysis of variance revealed an age-related decline in NE content and a day/night difference in NE content. Tukey's Honesty Sum Difference (HSD) test was used to compare the rise in NE content between 4 and l0 months of age, as well as the decline between 10 and 16 and between 16 and 25 months of age, which were all significant (P < 0.05). When day and night NE contents were compared in individual age groups, these differences were no longer statistically significant. When protein content was analysed using one-way analysis of variance followed by the HSD test, it was shown to be higher in 10-month-old animals than in 4-month-old (P < 0.05) or 25month-old (P < 0.05) animals.

Age (months)

4 10 16 25

NE content (pmol/pineal)

Protein content (l~g)

Day

Night

23.0 + 3.0 27.5+2.1 23.9+2.5 14.4 _+ 1.9

30.4 + 1.0 34.7+3.7 31.1+4.3 2l .9 _+ 1.9

108 + 4 t53_+9 133_+7 107 _+ 12

199 4 month old

10 month old

2.0'

2.0

1.5'

1.5'

1.0'

1.0"

"~A

0,5,

0.5,

mr-z~

0

O'

:~ ~

2.0

=~.~.

16 month old

25 month old

=~. 1.5

1.0 0.5 0

b~,,~-,~ Dqw~mnnll~n

(10 dlyll)

(1.5 dllys)

(10 c~nl)

~

O0 daym)

U n i ~ d Dw~a~Won (1.5~lys) (10 dill)

Fig. 5. Changes in specific [3H]NE neuronal uptake 1.5 and 10 days after unilateral denervation in animals of different ages. At least 4 animals were included in each group, with the exception of the 16-month-old animals denervated 10 days earlier which included 3 animals. Results were analysed using Student's t-test for comparison of two means (*P < 0.05, **P < 0.001 as compared to shamoperated animals of the same age; +P < 0.05 as compared to animals of the same age 10 days after unilateral denervation).

25 months of age (Fig. 3). Subsequent studies were all performed at a subsaturating NE concentration (77 nM or about one-half the Kin), since this resulted in a higher signal to noise ratio. Specific neuronal NE uptake in sham-operated animals of different ages measured under these conditions is illustrated in Fig. 4. The latter data demonstrate a nearly 60% decline in the specific neuronal uptake, particularly between 10 and 25 months of age.

Pinear

ICST Control

32 hours after Unilateral L.es~on

2 weeks after Uoil01~mlLesion

Fig. 6. Schematic diagram of the distribution and density of innervation of the pineal gland from the internal carotid nerves before and after a unilateral lesion. Diagram is from a histochemical study in young Sprague-Dawley rats (reprinted with permission from Lingappa and Zigmond28). In that study, pineals were studied 1.5 and 14 days after a unilateral lesion. SCG, the superior cervical ganglion; CST, cervical sympathetic trunk; ICN, internal carotid nerve.

At all 4 ages, animals that had undergone a unilateral lesion of the right ICN 1.5 days earlier showed specific [3H]NE uptake that was approximately 50% of that obtained from animals of the same age having undergone a sham operation 10 days earlier (Fig. 5). Animals aged 4 and 10 months that had been lesioned 10 days earlier had specific [3H]NE uptake which in both cases was 78% of that obtained from sham-operated animals of the same age (Fig. 5). The increase in uptake between 1.5 and 10 days after the lesion was statistically significant in studies with 4-month-old animals, but not 10-month-old animals. In 16- and 25-month-old animals, 10 days after the surgery, NE uptake was 49% and 45%, respectively, of the value obtained from sham-operated animals of the same age (Fig. 5), showing no change from the values obtained 1.5 days after the lesion. Pineal NE content tended to be higher in animals sacrificed at night than in those sacrificed during the daytime (Table III). However, there was a 30-50% decline in NE content between 10- and 25-month-old animals, whether they were sacrificed during the day or at night. As shown in Table III, the total pineal protein content increased nearly 50% between 4 and 10 months of age. There was a subsequent decline of approximately 30% between 10 and 25 months of age. DISCUSSION

Neuronal plasticity in aging In the past, studies of the aging nervous system have emphasized generalized losses in nerve cell numbers accompanied by progressive and irreversible functional declines. In recent years, this view has been modified by a number of observations. The first has been the demonstration that some changes previously thought to represent aging are actually the result of disease processes. Also, both animal 44 and human 48 studies have shown that neuronal losses in aging are specific for certain neuronal populations. In addition, these losses, as well as other structural and functional changes can, in some cases, be modified by extrinsic factors such as stress, behavioral experience and nutrition 5. It has been observed that the function of many neural systems in adult animals continues in spite of large cellular losses following surgical lesions 27,5°-52 or injection of a neurot o x i n 32"4°'5°. Similarly, Parkinson's disease does not appear to manifest itself clinically until major nigrostriatal degeneration has taken place 4. The capacity of the animal 1° or human 22 brain to recover from injury appears to be lowered in aging; however, examples of neuronal plasticity, though often diminshed, persist past development into old age 1°. Taken together, all of these findings have led to the hope that studies of the specific cellular

200 processes that enable neural systems to maintain function following partial nerve injury can enhance our ability to understand and, possibly, intervene in the capacity of the nervous system to compensate for neural losses in aging. The rat pineal gland presents a number of attractive features for the study of neural plasticity as a function of age. Among these is the ability to create precise and reproducible partial lesions and to subsequently measure recovery at the cellular level s'27"51'52. Several additonal features of the sympathetic nervous system have led to our use of the rat pineal gland as a model system. In the adult, the peripheral nervous system tends to exhibit more robust plasticity than the central nervous system ~' 17. In addition, specific physiological changes have been described in the aging human sympathetic nervous system, which have clinical implications for cardiac function, the regulation of blood pressure and body temperature and the response to stress in the elderly43. Functional recovery following partial neural lesions in young animals Pineal NAT activity and melatonin tissue content were used as indices of functional recovery following unilateral cutting of the ICN. The nearly 75% decline in NAT activity during the first night after a unilateral denervation in young Fischer-344 rats is in keeping with our previous data obtained in young Sprague-Dawley rats 27 51.5z. It has been previously shown that this transient deficit during the first night after unilateral denervation is likely due to 'heleroneuronai uptake' of NE 52. This term refers to the uptake of the NE released from terminals of neurons whose cell bodies are in the SCG on the unopera'ted side by nerve terminals from the SCG on the operated side, which are electrophysiologically silent, but are, at this early time, still anatomically intact 5~'52 In the present study, a decline in the pineal melatonin content during the first night .after this lesion was also found, suggesting that .the synthesis of the final product of this tissue is impaired. However, this 25-50% decline in pineal melatonin content is considerably smaller than the decline in NAT activity. One possible explanation for this discrepancy is that NAT activity may be the ratelimiting step in melatonin synthesis during the daytime and during the early hours of the night, while later in the night, NAT activity may exceed the activity of hydroxyindole O-methyltransferase (HIOMT), the enzyme that catalyses the final step in melatonin synthesis zT. Thus, 25% remaining NAT activity may be sufficient to maintain 50-75% of the pineal melatonin content 27. On the second and tenth night after the lesion there was complete recovery in both NAT activity and pineal melatonin content.

Collateral sprouting alter unilateral denervation in young animals A previous study has provided evidence for collateral sprouting in the pineal innervation following unilateral denervation of the pineal gland (ICN lesion or superior cervical ganglionectomy) by examining catecholamine fluorescence histochemistry 2s (Fig. 6). Pineals from young Sprague-Dawley rats that had undergone unilateral denervation 1.5 days earlier had approximately 50% of the innervation found in sham-operated animals, distributed equally throughout the pineal gland. In this anatomical study, the innervation reached 80% of the sham value 14 days after the lesion, demonstrating the development of collateral neuronal sprouting (Fig. 6). These findings confirmed those of an earlier study measuring [3H]NE uptake following unilateral ganglionectomy 16. In this study, [3H]NE uptake decreased to 50% 1.5 days after this lesion, with a significant increase detectable by 3-4 days. This level of [3H]NE uptake persisted for at least 52 days after the lesion, the longest time period examined ~6. NE uptake has been used in many studies as a measurement of the sympathetic innervation to tissues ~6' 22.37. In this study, we present evidence that [3H]NE uptake, as measured in pineal homogenates, is an assay of active transport of NE into enclosed nerve membranes. A close correlation has been found between [3H]NE uptake and histochemical determinations of sympathetic innervation in a number of intact 21 and lesioned tissues 37. Nevertheless, it is not known whether the capacity of terminals to take up NE changes in individual newly formed and/or aged nerve terminals in this tissue. Similarly, the increased rigidity described in the course of aging of many cellular membranes 35 could possibly affect the formation of enclosed nerve membranes and, thus, affect [3H]NE uptake as measured in homogenates from aged animals. The uptake of NE in 4-month-old animals was 50% lower in animals lesioned 1.5 days earlier as compared to sham-operated control animals, confirming earlier studies 16"2~. Ten days after the lesions, 4-month-old animals exhibited nearly 80% of the specific uptake compared to sham-operated animals. Taken together with a similar increase in earlier studies 16-2s, these results suggest that collateral sprouting has taken place in these young animals between 1.5 and 10 days after this lesion. Interestingly, there is no evidence for collateral sprouting occurring by 1.5 days after unilateral denervation 16'2~'52, a time at which full recovery is seen in nighttime NAT activity (Fig. 1), pineal melatonin content (Fig. 2), pineal N-acetylserotonin 27, the immediate product of NAT, and urinary excretion of the final metabolic product of melatonin, 6-hydroxymelatonin27. Thus, the role of col-

201 lateral sprouting in this early functional recovery is unclear 52. One possibility is that, while not necessary for the initial recovery of pineal function, sprouting is important for the maintenance of recovered function. Alternatively, sprouting may correlate with a yet unstudied aspect of pineal recovery or it may play a role in recovery from lesions greater than those produced by unilateral denervation.

Functional recovery following unilateral denervation in aging Nighttime pineal content of melatonin was about 40% lower in the sham-operated aged animals as compared to the younger animals. This was in agreement with previous data showing an age-related decrease in nighttime pineal melatonin content in rats 38'41 and of serum melatonin in both rats 38"47and humans 2°. As indicated by our data and that of Dax and Sugden ~2, these age-related declines in nighttime melatonin levels do not appear to be the result of changes in NAT activity, since this enzyme activity did not change with aging. The recently described 18% decrease in pinealocyte numbers between 2-3 and 22-26 month-old rats 42 may contribute to the ageassociated decline seen in pineal protein content (Table III) and perhaps in indole metabolism. Aged animals exhibited a decline in NAT activity and pineal melatonin content during the first night after unilateral denervation, which was proportionally similar to that seen in young animals. In addition, full recovery of both measures to pre-lesion levels was found in both the young and aged animals, two and ten nights after the surgical lesions. Neuronal NE uptake in aging Specific neuronal uptake in sham-operated animals measured at a subsaturating NE concentration demonstrated a nearly 60% decline in NE uptake between 10 and 25 months of age. These findings could be due to decreased affinity of uptake sites for NE, changes in the NE transport rate of each uptake site, or decreased numbers of uptake sites due either to losses of nerve terminals or a decreased density of uptake sites on each nerve terminal. The demonstration of a decline in specific neuronal NE uptake between 4 and 25 months of age measured at a saturating concentration of NE suggests that while an age-related decrease in the uptake site affinity for NE may exist, it could not, by itself, explain the entire age-related decline in specific NE uptake by these terminals. The finding of a K m in young animals (0.17 pM), which is significantly higher than the estimated mean intrasynaptic NE concentration 25, suggests that any possible age-related changes in K m could have important functional consequences. However, the rela-

tively similar impairment in pineal function in both young and aged animals during the first night after surgery implies that, at least under these conditions, the uptake process is functionally relatively intact in aged animals. Reuss et al. 4z have recently demonstrated a major decrease in the glyoxylic acid fluorescence in pineal glands from aged rats. While not quantitated, their findings suggest the possibility, also raised by our NE uptake data, of an age-related decline in the numbers of sympathetic nerve terminals. However, the age-associated decrease in catecholamine fluorescence in the pineal gland 42, and also in other sympathetically innervated tissues such as the heart 2, could be explained on the basis of a decrease in NE content per nerve terminal since such an effect would lead to a decrease in catecholamine histofluorescence even in the absence of a decrease in the number of nerve terminals z9. Our results showing a decrease in the pineal content of NE are consistent with either decreased numbers of nerve terminals or depletion in NE content per nerve terminal.

A biochemical index of collateral sprouting in aging The finding that specific neuronal NE uptake in 10-, 16- and 25-month-old animals was 50% lower in animals lesioned 1.5 days earlier as compared to animals shamoperated 10 days earlier suggested that this lesion resulted in the removal of one-half the innervation at all of these ages. Lesion studies in 10-month-old animals suggest an increase in NE uptake in the pineal gland between 1.5 and 10 days, which, though not statistically significant, was similar to that seen in the 4-month-old animals discussed above. However, studies in 16- and 25-month-old animals showed that specific neuronal uptake was nearly identical in the animals lesioned 10 days earlier, as compared to those of the same age lesioned 1.5 days earlier, suggesting that collateral sprouting did not occur in these groups. Aprevious study has also established a decrease in sprouting by pexipheral sympathetic fibers into the hippocampus .following a septal lesion in aged animals 45. Studies of reactive synaptogenesis by Cholinergic septo-hippocampal fibers in the hippocampus following a unilateral entorhinal lesion have established a delay of several days in the initiation of new nerve terminal formation in aged as compared to young animals 19'46.We have not examined collateral sprouting later than 10 days after a unilateral denervation and the highly different nature of the two systems make any extrapolation difficult. Interestingly, though the age-related delay in synapse formation in the hippocampal model was significant in the middle, it was not significant in the outer molecular layer of the hippocampus 19. The nerve terminal formation in the former region would require, much

202 greater axonal ingrowth than in the latter. This raises the possibility that the delay in nerve terminal formation in the d e n e r v a t e d , aged h i p p o c a m p u s was the result of decreased axonal growth rather than nerve terminal formation, which is the p r e d o m i n a n t process likely to occur in the pineal system. While the specific mechanisms involved in the regulation of either axonal or nerve terminal formation in collateral sprouting are not known, a potential explanation for o u r observations in aged animals could be d e c r e a s e d expression of a target tissue-derived signal for the initiation of sprouting or a failure in the responsiveness of nerve cells to this signal. The former mechanism has been suggested to explain the age-related decline in neuronal plasticity by cholinergic 36 and sympathetic fibers 11 in the h i p p o c a m p a l system. Nerve growth factor ( N G F ) is a leading candidate as the tissue-derived factor involved in trophic support of sympathetic fibers 39 and a p p e a r s to be involved in the collateral sprouting response of sensory nociceptive fibers xS. It remains to be established w h e t h e r N G F is involved in collateral sprouting by the pineal sympathetic fibers and whether agerelated declines in N G F expression or responsiveness to N G F could be responsible for our observations. The precise role for collateral sprouting, if any,

remains to be established in the functional recovery taking place after unilateral denervation of the pineal gland. While sprouting m a y not be essential for recovery of pineal function in the p a r a d i g m we have examined, sprouting may play a role following nerve lesions resulting in greater than 50% losses in innervation. As suggested by lesion studies in the nigrostriatal system 4°'5° and by p o s t m o r t e m analysis on subjects afflicted with Parkinson's disease 4, in certain systems nerve cell losses greater than 80% must take place prior to evidence of a functional deficit. In the absence of collateral sprouting, function may be m a i n t a i n e d by the presence of redundancy in the system's function and/or structure or by c o m p e n s a t o r y mechanisms o t h e r than sprouting. These c o m p e n s a t o r y mechanisms may include losses of neurotransmitter uptake sites, increased nerve firing and increased n e u r o t r a n s m i t t e r release by the intact nerve cellsS0,52. Acknowledgements. This work was supported by the MacArthur Foundation Program on Successful Aging and by a U.S. Public Health Service Research Grant NS17512 from the National Institutes of Health. G.A.K. is a Brookdale National Fellow of The Brookdale Foundation. R.E.Z. was supported by a Research Scientist Award (MH 00162). We thank Dr John W. Rowe for his advice and support, and Dr. Tina Williams McKeon, Hilary Hyatt Sachs and Claire Baldwin for careful reading of the manuscript.

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