Alterations of norepinephrine metabolism in rat locus coeruleus neurons in response to axonal injury

Brain Research, 289 (1983) 205-214 Elsevier

205

Alterations of Norepinephrine Metabolism in Rat Locus Coeruleus Neurons in Response to Axonal Injury BARRY E. LEVIN*

Neurology Service (127), VeteransAdministration Medical Center, East Orange, NJ 07019and Department of Neurosciences, Universityof Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ07103, U.S.A. (Accepted May 3rd, 1983)

Key words: regeneration - - axonal transport - - norepinephrine - - dopamine-fl-hydroxylase- - tyrosine hydroxylase - - 6-hydroxydopamine - - locus coeruleus - - norepinephrine uptake

Adult male Sprague-Dawley rats were injected in the right cerebral hemisphere with the neurotoxin 6-hydroxydopamine (6OHDA) at a site which interrupted the noradrenergic axons ascending from the locus eoeruleus (LC). Distal to the injection site ('posterior corteX'), levels of norepinephrine (NE), dopamine-fl-hydroxylase(DflH) and tyrosine hydroxylase (TH) fell to 39--42% of control levels ipsilateral to the lesion over the first 25 days, while contralateral levels fell to 32-73% of control during this time. These changes were paralleled by a 63% decrease in the high affinity uptake of [3H]NE in the ipsilateral posterior cortex at 12 days after the lesion. Both ipsilateral and contralateral levels of NE and DflH fell in the LC during this time, while LC TH showed variable increases and decreases in activity. At 3 months after right cortical 6-OHDA injections, posterior cortical levels of NE, DflH and TH, as well as the high affinity uptake of [3H]NE, had returned to control levels suggesting that some type of regeneration or axonal sprouting had occurred. Axonal transport of DflH and TH was assessed by measuring the accumulation of enzyme activity proximal to a 6-OHDA lesion made in the more caudal portion of these same LC axons. Transport of DflH fell to 7-40% of control from 2 to 24 days and rose to 160% of control by 3 months after the lesion. TH transport was decreased to only 61% of control only at 24 days and returned to control levels by 3 months. These studies document that there is independent regulation of the metabolism of the NE synthetic enzymes, Dill-/and TH, during the degeneration and subsequent regeneration or collateral sprouting of injured distal axons of LC noradrenergic neurons. INTRODUCTION

dal to these lesions23. Thus this m o d e l system in the rat offers a unique o p p o r t u n i t y to study the changes which occur in the cell b o d y m e t a b o l i s m and subsequent axonal t r a n s p o r t of identified n e u r o t r a n s m i t t e r enzymes during this reaction to axonal injury and repair.

The n o r a d r e n e r g i c neurons of the locus coeruleus (LC) innervate diverse areas of the central nervous system15,29,33,43,44. D e g e n e r a t i o n after destruction of L C axons can be followed by various degrees of restoration of the resulting biochemical defects depending on the type and site of the lesion p r o d u c e d . Small, focal lesions of the distal LC axons can be produced in the cerebral cortex by injection of the neurotoxin 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) 39 since these axons travel in a relatively well defined, circumscribed pathwaylS,29,33,43,44.. F u r t h e r m o r e , axonal

Animals A d u l t male S p r a g u e - D a w l e y rats (200-300 g) were fed s t a n d a r d lab chow and housed 4--6 p e r cage in a r o o m at 22-23 °C with a 12 h light-dark cycle.

transport can be assessed in this p a t h w a y by injecting 6 - O H D A into the m o r e proximal p o r t i o n of these axons and measuring the accumulation of the norepinephrine ( N E ) synthetic enzymes dopamine-fl-hydroxylase (DflH) and tyrosine hydroxylase ( T H ) cau-

Stereotaxic injections, brain dissections and sympathectomy Rats were anesthetized with C h l o r o p e n t (Fort D o d g e Labs) and injected stereotaxicaUy with 6-

* Address all correspondence to the first address. 0006-8993/83/$03.00 (~) 1983 Elsevier Science Publishers B.V.

METHODS

206

A7

Fig. 1. Schematic representation of 6-hydroxydopamine (6OHDA) injection sites and dissection planes shown as crosshatched areas outlined by dotted lines. A and B: lateral and top views of rat brain showing sites of needle tracks for right cortical injections, dissection planes for cortical sections, and right middle cerebral artery (MCA). C: coronal section at 9 mm anterior to intra-aural line (A9) showing sites and approximate sizes of 6-OHDA injections in right cortex and right rostral forebrain as verified by injections of methylene blue. D: coronal section at A7 showing dissection planes for right posterior cortical and anterior hypothalamic sections. E: composite sagittal section showing 6-OHDA injection sites, anterior hypothalamic and locus coeruleus (LC) dissection planes in relationship to coerulocortical noradrenergic projections.

O H D A using the coordinates of KOnig and KlippeW. They were first injected in right lateral fronto-parietal cortex (Fig. 1), with 1 ~1 6 - O H D A (4/~g/pl free base containing 0.1/~g//~l ascorbic acid) or the ascorbic acid carrier solution at approximately the level of the middle cerebral artery (A 9.0, L 5.5, 4.0 mm down from skull surface). At various time intervals (2 days to 3 months) after cortical injections of carrier solution or 6-OHDA, animals also received injections (Fig. 1) of 2/,d 6 - O H D A (4/~g//A) in the right rostral forebrain (A 9.0, L 2.0, 8.0 mm down from skull surface) 1 day before decapitation. This second lesion was used to assess the axonal transport of DflH and TH by measuring the accumulation of these enzymes proximal to the injection site (see below and ref. 23). Biochemical parameters in animals decapitated at 2, 5 and 12 days after cortical lesions were compared to sham injected controls; animals decapitated at 24 days and 3 months after cortical lesions were compared to sham injected or uninjected controls of the same age. At the time of sacrifice, brains were rapidly removed, placed on a chilled glass plate and rapidly dis-

sected (Fig. 1) into left and right anterior hypothalamus 21,24, LC 21.24 and posterior cortex (cerebral cortex distal to the cortical injection site). Enzyme activity was measured in left and right hypothalamic hemisections and transport was calculated by subtracting the enzyme activity in the comparable, uninjected left, from the injected right hypothalamic hemisection 2 mm proximal to the forebrain 6 - O H D A injection site2o,21,23.24. Previous studies 23 have shown that enzyme activity increases linearly proximal to such lesions for 48 h; 24 h was therefore used here to assess axonal transport. In those rats injected with 6 - O H D A 3 months before sacrifice, a subgroup of 6 animals had both superior cervical ganglia removed, using a dissecting microscope, 6 days prior to decapitation, to evaluate the effect on cortical NE and enzyme levels. These rats were compared to groups of uninjected and cortically injected rats sacrificed at the same time.

Tissue preparation and enzyme assays Tissues from LC (5-10 mg), anterior hypothalamic (10-15 mg) and posterior cortical (15-20 rng) brain areas were rapidly dissected, weighed and homogenized in 5 mM Tris buffer (pH 7.4) containing 0,1% Triton X-100. Aliquots were taken for radioenzymatic assay of NE 25, DflH 35, and TI--I7,13.

Immunotitration of axonally transported D~H To confirm that the increased DflH activity which accumulated proximal to 6 - O H D A forebrain injections placed 1 day before represented enzyme protein, pooled hypothalamic hemisections (8-10 rats) from injected (right) and uninjected (left) sites were titrated with progressively increasing amounts of antiserum to DflH, according to the method of Ross et al. 4°. Bovine adrenal DflH was purified and antibodies raised in rabbits by modification of the methods of Hartman et al. 11. There was a 60-75% crossover of antibody to bovine DflH and rat hypothalamic DflH. DflH activity was assayed in the supernatant fluid after precipitation of the antigen-antibody complex.

High affinity posterior cortical uptake of [3H]norepinephrine Separate groups of rats were injected in the right cortex with 6 - O H D A and compared to sham injected controls of comparable age, at 12 days and 3 months

207

after injection, for high affinity uptake of [3H]NE by modification of the methods of Gilad and Reis9. Briefly, posterior cortical tissues were homogenized in 9 vols. of 0.32 M sucrose using a glass-Teflon homogenizer. The supernatant of the initial centrifugation at 1000 g for 10 min was spun at 17,000 g for 10 rain. The pellet of this centrifugation was resuspended in Krebs-Ringer phosphate buffer (pH 7.4) containing 1.7 mM ascorbate and 0.08 mM pargyline. Samples were equilibrated at 37 °C for 15 min in the presence or absence of desmethylimipramine and then incubated with [3H]NE (1-[ring-2, 5, 6-3H]norepinephrine, New England Nuclear; 44.7 Ci/mmol) for 5 min. Incubation was terminated by addition of ice cold saline followed by centrifugation at 17,000 g for 10 min with two washes. Pellets were resuspended in absolute ethanol and counted by liquid scintillation spectroscopy.

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Statistics Results of axonal transport experiments were evaluated by comparing activity in left and right anterior hypothalamic hemisections in the same rat by a twotailed, paired t-test. Intergroup results were compared by two-tailed unpaired Student's t-test.

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2. Sequential changes in regional brain norepinephrine levels after right cortical 6-hydroxydopamine (6-OHDA) injections. Groups of 8-20 rats were sacrificed at various times after right cortical injections of 6 - O H D A . Locus coeruleus, hypothalamic areas and cortex distal to the injection site (posterior cortex) were assayed bilaterally for norepinephrine. Results are means +_ S.E. (vertical bars) NE levels as percent of shamlesioned controls. (See Table I for representative control values.) Separate sets of control rats were used for the 24 day and 3 month groups. * P < 0.05 or less by unpaired Student's t-test when rats with lesions were compared to controls. Fig.

RESULTS

Alterations in NE levels, DflH and TH activities following right cortical 6-OHDA injections Ipsilateral posterior cortical levels of NE (distal to the 6-OHDA lesion site) fell progressively over time; they were 50% of control by day 12, reached their nadir at 39% of control by 24 days and had returned to

control levels by 3 months post-lesion (Fig. 2; Table I). Both LC and hypothalamic NE levels ipsilateral to

TABLE I Levels of NE and activities and axonal transport of D3H and TH in control rats (200-300 g) Units are mean + S.E. ng per locus coeruleus or per g of wet weight tissue for norepinephrine (NE); nmol octopamine/h/LC or g of tissue for dopamine-fl-hydroxylase (DflH); and nmol dihydroxyphenylalanine/h/LC or g of tissue for tyrosine hydroxylase (TH); numbers in parentheses --- n. L, left; R, right brain halves; AT, axonal transport as calculated as the difference in D 3 H and TH activity between the left and right hypothalamic hemisections 1 day after 6 - O H D A injection into the right rostral forebrain (see Methods). Locus coeruleus

NE DflH TH

Hypothalamus

Posterior cortex

L

R

L

R

AT

L

9.4 + 1.0 (15) 22.2 + 3.4 (10) 1.35 + 0.11 (10)

9.3 + 0.8 (16) 23.2 + 3.2 (9) 1.33 + 0.09 (10)

1062 + 117 (20) 140 + 10 (12) 217 + 8 (25)

1169 + 123 (20) 209 + 12 (12) 405 + 25 (25)

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69.0 + 13.2 (12) 188 + 25 (25)

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208 the lesion fell transiently at 12 days but otherwise remained at control levels. Contralateral NE levels also fell in the posterior cortex, LC and hypothalamus with a similar time course to that seen for ipsilateral levels but always to a lesser degree. These contralateral changes were not due to non-specific loss of catecholamines secondary to leakage of 6-OHDA into the subarachnoid space since striatal dopamine and brainstem NE levels (caudal to the LC) were not significantly different from controls at 12 days postlesion (Table II). DflH activity in the posterior cortex ipsilateral to the cortical lesions changed in a pattern similar to that seen for NE; levels fell to 41-54% of control from 5 to 24 days and returned to control levels by 3 months (Fig. 3; Table I). Ipsilateral LC DflH activity fell transiently to 38% of control over the first 12 post-lesion days, returned to control levels by 24 days and was 160% of control by 3 months post-lesion. Contralateral DflH activity fell transiently and to a lesser degree in the posterior cortex and LC over the first 5 post-lesion days. Posterior cortical TH activity ipsilateral to the lesion site (Fig. 4; Table I) fell transiently to 50% of control on day 2, returned to control on day 5, then fell progressively to 50% of control by day 24 (TH levels below 50% of control were below the sensitivity of the assay), and returned to control levels by 3 months. LC TH activity increased transiently to 131% T A B L E II

Levels of brain catecholamines 12 days after 6-OHDA injection in the right cortex Brainstem represents pons and medulla caudal to locus coeruleus; striatum represents rostral pole of striatum.

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** P < 0.001 when norepinephrine levels in brain areas from animals with lesions were compared to comparable brain areas from control animals.

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Fig. 3. Sequential changes in brain dopamine-fl-hydroxylase (DflH) activity and axonal transport after right cortical 6O H D A injections. Groups of 8-20 rats had in vitro DflH activity assayed in their locus coeruleus, posterior cortical and hypothalamic areas bilaterally at various times after right cortical injections of 6-OHDA. Results are mean percent of control + S.E. (vertical bars). Axonal transport represents DflH activity accumulating proximal to a right, rostral forebrain 6-OHDA injection made 1 day before sacrifice. * P < 0:05 or less by unpaired Student's t-test when rats with lesions were compared to controls.

of control on day 2, fell to 71% of control by 12 days and then rose to 147% of control by 24 days where it remained at 3 months. TH activity in contralateral posterior cortex more closely mirrored those seen ipsilateral to the lesion than levels of NE or DflH activity over the 3 months post-lesion period, while contralateral LC TH activity was similar to ipsilateral levels only during the first 12 post-lesion days. The superior cervical ganglia were removed bilaterally 6 days before sacrifice, in a group of rats which had received right cortical 6-OHDA lesions 3 months before, to rule out the possibility that the observed return of cortical NE and enzyme levels was due to ingrowth of peripheral sympathetic fibers (Ta-

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Rats which received right cortical injections of 6O H D A 12 days prior to decapitation showed a significant, 63% decrease in the high affinity cortical uptake of [3H]NE distal to the ipsilateral lesions (Table IV). There was no significant change in the contralateral posterior cortex. By 3 months after the right cortical lesions, high affinity [3H]NE uptake had returned to control levels. There were no significant differences at either time period between desmethylimipramine resistant binding in lesion or control animals. Therefore, the high affinity uptake of [3H]NE in the ipsilateral posterior cortex roughly paralleled the changes seen in levels of N E and activities of T H and DflH during the response to axonal injury.

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Fig. 4. Sequential changes in TH activity and axonal transport after right cortical 6-OHDA injections. Number of animals, methods and statistics for TH activity are the same as for DflH in Fig. 2. ble III). Bilateral ganglionectomy did not alter cortical levels of NE, DflH or T H at this time, showing that the return to control values was due to restoration of neuronal stores within intrinsic axon terminals derived from LC neurons.

As in a previous study 23, the interruption of anterograde transport of DflH and T H with 6 - O H D A lesions of the ascending LC axons was followed by an accumulation of enzyme activities proximal to the lesion site (Table II). For DflH, this increase in activity (49%) was associated with a comparable increase (45%) in the amount of immunotitratable enzyme protein as calculated by extrapolation of the immunotitration curves to baseline for lesioned and unlesioned hypothalamic sections (Fig. 5). When lesions were made in the more distal portions of these same axons in the cerebral cortex, the patterns of accumulation of DflH (Fig. 3) and T H (Fig. 4) activities behind the more proximal hypothalamic lesions varied quite independently of each

TABLE III

Posteriorcorticallevelsof NE, DflH and TH, 3 months after right corticalinjectionsof 6-OHDA, beforeand aftersympathectomy Animals received injections of carrier solution (controls) or 6-OHDA (lesion) in the right cortex 3 months prior to decapitation. Additional animals injected with 6-OHDA in the right cortex also had bilateral sympathectomies performed 6 days prior to decapitation (lesion plus sympathectomy). Units are mean + S.E. ng/g of wet weight tissue for norepinephrine (NE); nmoi octopamine or dihydroxyphenylalanine/h/g wet weight tissue for dopamine-fl-hydroxylase (D/3H) or tyrosine hydroxylase (TH). Numbers in parentheses refer to the number of animals per group.

NE Control Lesion Lesionplus sympathectomy

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TH

L

R

L

R

L

R

133 + 13 (10) 132 + 6 (6) 115 + 16 (6)

139 + 14 (10) 138 + 16 (5) 103 + 27 (6)

60.4 + 5.4 (8) 57.2 _+ 5.6 (19) 68.6 _+ 6.2 (5)

48.9 + 3.1 (9) 55.9 + 5.3 (19) 67.2 _+ 6.2 (6)

8.70 + 1.57 (14) 7.55 + 1.37 (12) 6.90 ___ 0.63 (5)

8.59 + 1.68 (14) 7.00 + 1.18 (11) 6.40 __ 1.15 (5)

210 TABLE IV

Posterior cortical [3H]norepinephrine uptake 12 days and 3 months after right cortex 6-OHDA injection Groups of 8-12 rats were injected with 1/~l 6-OHDA or saline in the right cortex and decapitated after 12 days or 3 months. Uptake of [3H]norepinephrine was assayed in posterior cortical sections ipsilateral (right) and contralateral (left) to the injection sites. Values represent mean cpm/g wet weight tissue _+ S.E. × 102 of desmethylimipramine sensitive uptake.

Lesion

12days 3 months

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L

R

L

R

2228 + 449 (74%) 3450 + 900 (105%)

1150 + 365 (37%)* 2838 + 427 (87%)

3022 + 375 3287 + 373

3123 + 388 3264 + 432

* P < 0.025 when cortical [3H]NE uptake in rats with 6-OHDA lesions were compared to comparable cortical sections from control rats. Numbers in parentheses represent percent of control.

other. This measure of axonal transport of DflH paralleled similar changes in LC and posterior cortical DflH levels, i.e. transport declined steadily over the

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first few days after cortical lesions. By 3 months, both LC DflH activity and the axonal transport of DflH were 160% of control while cortical levels remained at control levels. In contrast to the marked suppression of DflH transport, cortical 6-OHDA lesions had only a mild and transient effect upon T H transport (Fig. 4). While there was a tendency toward decreased transport from 5 days to 3 months following the lesions, this reached statistical significance only at 24 days at 60% of control. Therefore, unlike DflH, the axonal transport of TH mirrored neither cell body nor distal terminal levels of TH activity.

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Fig. 5. Immunoreactive DflH in the ipsilateral (Q) anterior hypothalamic hemisection proximal to a 6-OHDA injection site in the rostral forebrain and in the comparable contralateral (O) hypothalamic sections. Increasing amounts of antiserum to DflH were added to a constant amount of hypothalamic tissue pooled from 6--8 rats and DflH activity (clam) was assayed in the supernatant after the immunoprecipitate was removed. The equivalence point was determined by extrapolation to the baseline of the linear portion of each curve. Each point represents the average of triplicate determinations.

The axons of noradrenergic neurons of the LC are highly ramified and project bilaterally to multiple areas of the central nervous system 15-29-33.43.44, Although these projections are primarily ipsilateral, there are a small number of contralateral projections as well 15,33,43. In the current study, unilateral lesions of these ascending noradrenergic axons with 6O H D A produced contralateral changes in NE levels and the activities of its synthesizing enzymes, DflH and TH which were generally less marked than on the contralateral side. There is currently no good explanation as to why such widespread, bilateral alterations in neurotransmitter metabolism occurred following unilateral lesions, although similar results have been reported in both the nigro-striatat30, 31 and LC systems 36-39. Additionally, Hoff et al.lZ have shown that unilateral ablation of the entorhinai cortex produces a long-lasting increase in synapse turnover without loss of terminals in both ipsilateral and

211 contralateral areas of the hippocampus not innervated by the ablated area. A similar phenomenon may have occurred in the present studies where changes in neurotransmitter metabolism occurred in the posterior cortex contralateral to 6-OHDA lesons without significant loss of nerve terminals (high affinity [3H]NE uptake). This suggests that the contralateral changes and perhaps even those seen in the hypothalamus could have occurred as a remote effect of the primary neurotoxin lesion which produced a functional defect in transmitter synthesis without a structural lesion being present in these non-injured areas. Robinson and Stitt 39 previously reported similar bilateral changes in NE levels following unilateral, focal cortical injections of 6-OHDA in the rat. Furthermore, such unilateral lesions were associated with behavioral hyperactivity, especially when placed in the right hemisphere. The noradrenergic neurons of the LC have also been implicated in the processes of arousal 14, cerebral oxidative metabolismlOA 9 and cerebral vascular permeability 34 among other functions. Unilateral damage to the LC neurons might, therefore, potentially be capable of altering any of these functional properties of the brain because of the bilateral effects of such lesions. There are several ways by which a biochemical balance in the brain might be restored following axonal injury. Either regenerative sprouting of severed axons might occur or, instead, there might be collateral sprouting of surviving axons. Additionally, the synthetic capacity of surviving terminals might be increased by packing them with more synthetic enzyme 1. The site and type of lesion appear to be important determinants of the type of restorative process which will result. Intracerebroventricular injection of 6-OHDA causes extensive damage to noradrenergic neurons and neither complete biochemical nor histological recoveries occur 1,6,45. Surviving terminals do increase their concentrations of TH yielding an increased capacity to synthesize NE per terminal 1. Less extensive damage to noradrenergic neurons occurs after intracerebroventricular injections of 5, 7-dihydroxytryptamine and such lesions are followed by regenerative sprouting 3:. Mechanical lesions of the axon vary in their effectiveness in inducing sprouting of LC neurons. Electrolytic lesions in the ascending noradrenergic pathway from the LC, made close to the cell body in the

posterior hypothalamus, are followed by histologic evidence of a limited degree of regenerative sprouting 16 but little evidence of biochemical recovery36. This may be due to the adverse effect such proximally placed lesions have on the cell body and also to the formation of a glial scar which inhibits effective regenerative sprouting. Partial knife-cut lesions of the coerulocerebellar pathway induce extensive collateral sprouting of surviving noradrenergic fibers32 suggesting that partial lesions made close to the cell body inhibit regenerative sprouting of severed axons but enhance collateral sprouting of remaining ones. As in the present study, lesions of the distal axons of noradrenergic neurons seem to result in better restoration of pre-lesion conditions. Both focal injections of 6-OHDA and ischemic lesions of the distal axons produced by middle cerebral artery occlusion lead to bilateral descreases in brain NE levels, followed by an eventual return to pre-lesion levels 37-39. Ross and Reis 41 found that such ischemic lesions led to a persistent decrease in cortical DflH activity over 60 days, but longer time intervals were not examined. Finally, lesions of the distal noradrenergic axons by cortical freeze lesions have been shown to result in regenerative sprouting in rabbit cortex 5. Collateral sprouting of noradrenergic fibers seems to occur most often after partial lesions of the pathway or following denervation of a target area by lesions of other, non-adrenergic systems 27.28,32. In this circumstance, ingrowth of peripheral sympathetic fibers can also occur8,26 and for this reason sympathectomy controls were used in the present studies to rule out this possibility. Also, the return of cortical NE, DflH and T H to pre-lesion levels was accompanied by a return to normal numbers of cortical noradrenergic nerve terminals as defined by the high affinity uptake of [3H]NE18. This suggests that sprouting did, in fact, occur although the type of sprouting could not be further defined on the basis of biochemical determinations alone. However, the long time period required for recovery suggests that regenerative rather than collateral sprouting occurred. Axonal injury leads to a reordering of metabolic priorities within the neuron. Reis and co-workers36.40 have shown that lesions of the proximal portion of the coerulocortical pathway in the posterior hypothalamus lead to a characteristic triphasic change in both TH and DflH activities in the LC. A similar triphasic

212 pattern for LC TH activity was seen in the present studies after more distal lesions of these same axons, while DflH activity followed only a monophasic decline during the first few days after the lesion. Also, unlike more proximal lesions, both TH and DflH activities in the LC rose to 150-160% of control ipsilateral to the lesion during the recovery period from distal axonal injury, suggesting that a permanent change in the cell body metabolism of these enzymes had occurred. Differences in cortical TH and DflH activities distal to the lesion were also found during the early period following axonal injury. Cortical TH activity followed a triphasic pattern both ipsilateral and contralateral to the lesion, while DflH activity fell early and remained depressed, primarily ipsilateral to the lesion. By 3 months, both cortical TH and DflH activities had returned tO control levels. These data suggest that TH and DflH metabolism were regulated independently of each other during various periods of the reaction to axonal injury. Although the mechanism for this independent regulation remains unclear, differences in either retrograde transport or turn-around of anterogradely transported substances from the lesion site (including the enzymes themselves) might act as the signal for such differential regulation of metabolic changes in the cell body 42. Anterograde axonal transport of enzymes plays an important role in determining the ultimate cell body and terminal levels of that enzyme. TH, DflH and other proteins do not leave the cell body immediately after being synthesized 20.21,23. The ability to control the timing and amount of release and possibly even the selection of which axonal branches will receive newly synthesized proteins 2, gives the neuron another mechanism for modulating the turnover of those proteins under physiologic and pathologic conditions. Normally, DflH and TH have different rates and, presumably, different mechanisms of transport within LC neurons. DflH, a glycoprotein, is transported at a rate similar to NE (24-48 mm/day) and travels with the particulate fraction of the axoplasm20.22,24. TH is transported as a soluble protein at a slower rate of 13--20 mm/day and various neurotoxins differentially affect the transport of the two enzymes 22. In the present study, the transport of TH and DflH in injured LC noradrenergic neurons varied considerably from each other in response to axonal injury. There was only a moderate (39%) de-

crease in the transport of TH, despite marked fluctuations in cell body and terminal activities during the response to injury. Transport of DflH, on the other hand, was markedly depressed to as low as 7-9% of control throughout the first 24 day post-lesion period and this generally followed a pattern similar to that seen for cell body and terminal enzyme activities. Even so, the transport of DflH was much more depressed during the first 24 days and was much higher (160%) at 3 months after the lesion than were cortical levels. Apparently, turnover of cortical DflH varied inversely with anterograde transport while TH transport had a somewhat more parallel relationship to cortical levels from 24 days to 3 months following axonal injury. Regardless of changes in local turnover of these enzymes, differences in the axonal transport of TH and DflH under both physiologic and pathologic conditions must be considered to be an important factor in the differential regulation of their metabolism during the reaction to axonal injury. It should be pointed out that the term 'axonal transport' has been used to describe the accumulation of enzyme activity proximal to a lesion. For DflH, at least, this was also shown to be associated with a comparable accumulation of immunotitratable enzyme protein. Whereas this accumulation represents the algebraic sum of anterograde minus retrograde transport and local turnover of enzyme at the lesion site, the use of a 24 h time period tends to minimize the contribution of the latter two factors since enzyme accumulation is still increasing linearly during this time period23. Even though no clear distinction between changes in the transport rate and the amount of enzyme transported can be made, a general picture of changes in the transport of TH and DflH that result from axonal injury can be gained from the present studies. These data give us an overall picture of the metabolic responses that occur in noradrenergic neurons of the LC in response to axonal injury, and clearly show that these axons retain a significant degree of plasticity in the adult rat. They further demonstrate that different regulatory mechanisms control the metabolism of two closely related synthetic enzymes for NE in the brain during the response of noradrenergic neurons to axonal injury.

213 ACKNOWLEDGEMENTS

p a r a t i o n . This w o r k was s u p p o r t e d by the M e d i c a l R e s e a r c h S e r v i c e of t h e V e t e r a n s A d m i n i s t r a t i o n .

I wish to t h a n k M a r y b e t h F i n n e g a n , E d w a r d A g y k u m and D e b o r a h K i n g e r y for t h e i r e x p e r t t e c h n i c a l

D e s m e t h y l i m i p r a m i n e was k i n d l y s u p p l i e d by M e r rell D o w P h a r m a c e u t i c a l s .

assistance and A n t o i n e t t e Colitti for m a n u s c r i p t pre-

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