Histological and ultrastructural evidence thatd -amphetamine causes degeneration in neostriatum and frontal cortex of rats

Brain Research, 518 (1990) 67-77 Elsevier

67

BRES 15516

Histological and ultrastructural evidence that D-amphetamine causes degeneration in neostriatum and frontal cortex of rats Lawrence J. Ryan 1, Jean C. Linder 2, Maryann E. Martone 3 and Philip M.

G r o v e s 2'3

1Department of Psychology, Oregon State University, Corvallis, OR 97331 (U.S.A.) and Departments of ePsychiatry and 3Neuroscience, University of California San Diego, La Jolla, CA 92093 (U.S.A.) (Accepted 7 November 1989)

Key words: Amphetamine; Degeneration; Neostriatum; Dopamine; Frontal cortex; Motor cortex; Neurotoxicity; Ultrastructure o-Amphetamine sulfate, continuously administered for 3 days subcutaneously via an implanted minipump, induced neural degeneration in Long-Evans and Sprague-Dawley rats at doses between 20 and 60 mg/kg/day. Using Fink-Heimer silver staining, axonal degeneration was detected in the neostriatum and the dorsal agranular insular cortex and degenerating pyramidal cells were observed in portions of the somatosensory neocortex in both strains. In contrast, dense axonal degeneration largely confined to layers 2 and 3 of frontal motor areas (Frl, Fr2 and Fr3 of Zilles 36) with occasional degenerating cells was seen reliably in Long-Evans rats but rarely in Sprague-Dawley rats. In the electron microscope, cortical degeneration consisted mainly of disrupted cell bodies and dark processes, including axons making asymmetric synapses. Damage in all cortical areas represents damage to non-monoamine neurons and processes since tyrosine hydroxylase and serotonin immunolabeling were normal. In contrast, the damage in neostriatum probably includes damage to dopamine axonal terminals since tyrosine hydroxylase immunolabeling was patchy with many swollen and distorted labeled axons. Serotonin and Leu-enkephalin labeling were normal. Electron microscopy confirmed that the neostriatum contained many tyrosine hydroxylase-labeled axons that were swollen and disrupted, although other labeled processes made normal symmetric synapses onto spines and dendrites. Additional degeneration found only in amphetamine-treated rats included many dark, shrunken profiles, Some of these appeared to be astrocytic processes and a few were myelinated axons, suggesting that some non-monoamine, possibly cortical afferents, are also degenerating in the neostriatum. Since similar degrees of behavioral activation, weight loss and lethality were seen in both strains, a genetic predisposition constrain amphetamine-induced motor cortex damage but not neostriatal damage. INTRODUCTION Repeated administration of o-amphetamine, methamphetamine and other amphetamine analogues produces persisting reductions in many indices of dopamine neurotransmission including an enduring depletion of dopamine 7'9'24'26, a reduction in dopamine uptake sites 32'33, reduced dopamine histofluorescence 17,19, and decreased tyrosine hydroxylase activity 15. These changes occur primarily in axonal terminals of the nigrostriatal dopamine system; extensive degeneration of these projections is suggested by the appearance of degenerating axonal terminals within the neostriatum as identified by F i n k Heimer and related silver stains 13'17'24'25. Dopamine cell bodies in the substantia nigra and the other dopamine systems of the brain appear to be unaffected. Similar changes in serotonergic projections have also been reported, especially following administration of methylated and halogenated amphetamines 2,334,22,23,31. Two types of degeneration have also been reported in the neocortex following the repeated administration of methamphetamine: local degeneration of cortical pyramidal cells in circumscribed regions of somatosensory

cortex in albino rats 2'3 and dense terminal degeneration in circumscribed layers of medial frontal neocortex in immature gerbils 34. In this report we demonstrate that amphetamine can cause extensive degeneration in several regions of agranular frontal cortex. This damage is dose- and straindependent, whereas damage to other regions and behavioral activation are only dose-dependent. Furthermore, we extend earlier findings by (1) demonstrating that neostriatal and somatosensory pyramidal cell degeneration occur in L o n g - E v a n s as well as Sprague-Dawley albino rats, (2) characterizing this degeneration in the electron microscope and (3) showing that the presence of axonal degeneration detected with F i n k - H e i m e r silver stains in neostriatum but not in cortex is associated with altered tyrosine hydroxylase immunolabeling. Neither serotonin nor Leu-enkephalin immunostaining is altered anywhere. MATERIALS AND METHODS

Continuous drug administration Adult male Sprague-Dawley albino (n = 19) and Long-Evans

Correspondence: P. M. Groves, Department of Psychiatry, M-003, University of California at San Diego, La Jolla, CA 92093, U.S.A. 0006-8993/90/$03.50 t~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

68 black-hooded (n = 39) rats weighing between 25(1 and 45(I g were anesthetized with 60 mg/kg sodium pentobarbital supplemented with 1.0 mg/kg atropine methyl nitrate, i.p. An Alzet model 2MLI minipump was inserted through a small incision in the skin between the shoulder blades. Pumps filled with D-amphetamine sulfate (Sigma) dissolved in 0.9% saline vehicle discharged their contents subcutaneously. Sham-operated control rats (n = 6) were implanted with a silastic plug the size of the minipump. Naive controls (n 14) were untreated. Animals to be examined for electron microscopy received drug for 24, 48, or 72 h and most were then immediately sacrificed and perfused. All other animals received continuous drug infusions for 3 days, at which point the pumps were removed under light pentobarbital (45 mg/kg, i.p.) anesthesia. These animals were given one day to recover prior to sacrifice.

Light microscopy procedures Animals were deeply anesthetized (100 mg/kg pentobarbital, i.p.) and perfused intracardially using one of three fixation protocols: (1) 70 ml 0.9% saline flush followed by 250 ml 10% formalin, (2) for serotonin immunoreactivity, 70 ml 0.9% saline flush followed by 250 ml 4% paraformaldehyde plus 0.1% glutaraldehyde or (3) for best tyrosine hydroxylase immunoreactivity, 50 ml 0.9% saline followed by 200 ml of 4% paraformaldehyde plus 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer at pH 6.5 which was then followed by 200 ml of fixative in 0.1 M phosphate buffer at pH 8.5; the brains were postfixed for 24 h in 4% paraformaldehyde in 0.1 M phosphate buffer at pH 8.5 and then stored in 20% sucrose in 0.1 M buffer until cut. Pairs of adjacent frozen coronal sections (60 ktm) were collected throughout the extent of the neostriatum. In some cases, one section of each pair was processed for Leu-enkephalin-like (antibody obtained from Immunonuclear) and the other for tyrosine hydroxylase-like (Eugene Tech) immunoreactivity using the avidin-biotin (Vector) technique of Hsu 16 as previously described by us 12. In other cases, pairs were processed for tyrosine hydroxylase and serotonin-like (Immunonuclear) immunoreactivity. Sections to be labeled for tyrosine hydroxylase and serotonin were also collected from frontal cortex, the substantia nigra, dorsal raphe and the locus coeruleus. Frozen sections (30/~m) were also collected throughout the anterior 5 mm of the rat brain, including frontal neocortex and the extent of the neostriatum. These were processed for axonal and terminal degeneration using the protocol of Fink and Heimer 8. Tissue processed using the Fink-Heimer technique was examined for the presence of silver grains above the level of staining seen in control tissue processed in the same tray. The presence of degeneration was scored only when the staining was unequivocally denser than in control tissue. Serotonin and dopamine immunolabeled tissue were scored as indicating degeneration if at least 2 abnormal, swollen axonal profiles were observed within a structure.

Behavioral analysis While implanted with the minipumps, all animals were videotaped at least once per day, between 10.00 h and 13.00 h for at least 20 rain in their home cages. Between minutes 5 and 20, the animal's behavior was scored using an arbitrary system based upon the dose-related emergence of various behaviors. Briefly, the following behaviors were scored: (A) sleep (score = 0), (B) grooming (= 0), (C) quiet waking (= 1), (D) walking and rearing (= 2), (E) sniffing associated with side-to-side head movements occurring in concert with locomotion (= 3), (F) in-place sniffing, often directed downward, with associated side-to-side head movements at one or more fixed locations in the cage (= 4), (G) focussed, stereotyped sniffing and chewing directed towards the animal's body, at one location in the cage for extended periods, with the animal standing upright on his hind paws (= 5), (H) as in G but with animal lying on his back, no longer supported by the hind legs (= 6). The occurrence of eating, drinking and scratching were also noted, but not scored.

RESULTS

Behavioral analysis A m p h e t a m i n e c a u s e d a d o s e - d e p e n d e n t i n c r e a s e in the daily m e a n b e h a v i o r a l rating (Fig. 1). E q u i v a l e n t d e g r e e s of b e h a v i o r a l activation w e r e e v i d e n t in b o t h strains. A t m o s t doses, a m p h e t a m i n e p r o d u c e d less b e h a v i o r a l act i v a t i o n on day 3 t h a n on day 1. H o w e v e r , this r e d u c t i o n m a y not reflect the d e v e l o p m e n t of t o l e r a n c e since the s h a m - o p e r a t e d c o n t r o l rats also s h o w e d a day-3 versus day-1 r e d u c t i o n . T h e r e was no e v i d e n c e for t h e develo p m e n t of b e h a v i o r a l sensitization across the 3 days.

Weight loss and lethality A m p h e t a m i n e p r o d u c e d substantial w e i g h t loss in b o t h strains after 3 days of a d m i n i s t r a t i o n (Fig. 2). A t the 4 0 - 5 0 m g / k g / d a y r a n g e , 2 5 % of t h e S p r a g u e - D a w l e y rats d i e d c o m p a r e d to 4 4 % of the L o n g - E v a n s rats.

Neostriatum: Fink-Heimer terminal degeneration In b o t h L o n g - E v a n s and S p r a g u e - D a w l e y rats, silver grain d e p o s i t i o n , indicative of the p r e s e n c e of d e g e n e r -

Electron microscopic procedure

ating a x o n t e r m i n a l s , was s e e n in t h e n e o s t r i a t u m at

Vibratome sections (100 ktm) from brains of 16 rats were prepared for electron microscopy by methods described previously TM. Tissue from the 12 rats (8 experimental, 4 control) with the best tissue preservation was coded and analyzed blind for the presence of abnormal or degenerating profiles (dark, disrupted or swollen) in neostriatum and agranular frontal cortex. Separate portions of tissue from one additional animal, given a suprathreshold dose of amphetamine (35-40 mg/kg/day), was labeled for either tyrosine hydroxylase (using the immunocytochemical procedure described above but without Triton X-100 in the solutions) or for FinkHeimer degeneration. Prior to making ultrastructural observations, the presence of typical degeneration was confirmed in the light microscope with both Fink-Heimer and tyrosine hydroxylase labeling. The immunolabeled sections were embedded for electron microscopy between plastic coverslips. Some of that tissue was cut into ribbons of 10-35 serial sections and both degenerating and tyrosine hydroxylase-labeled profiles were evaluated with a computerized three-dimensional reconstruction program 35.

doses a b o v e 20 mg/kg/day.

N o e v i d e n c e of r e g i o n a l

differences within t h e n e o s t r i a t u m was s e e n (Table I).

Neostriatum: tyrosine hydroxylase, serotonin and Leuenkephalin irnrnunohistochemistry In L o n g - E v a n s and S p r a g u e - D a w l e y rats r e c e i v i n g at least 20 m g / k g / d a y a m p h e t a m i n e , t y r o s i n e h y d r o x y l a s e i m m u n o s t a i n i n g typically was p a t c h y 27 in c o n t r a s t to the m o r e u n i f o r m p a t t e r n s e e n in controls. A t h i g h e r m a g nification m a n y i m m u n o r e a c t i v e p r o f i l e s w e r e s e e n to be swollen and e n l a r g e d (Fig. 3). T h e a b n o r m a l profiles p r o b a b l y reflect t y r o s i n e h y d r o x y l a s e a c c u m u l a t e d proximal to a x o n a l d a m a g e . T h e s e profiles w e r e o b s e r v e d throughout

the n e o s t r i a t u m

differences (Table I).

with no o b v i o u s r e g i o n a l

69 No abnormal serotonin-immunolabeled profiles (Table I, Fig. 3) nor enkephalin immunolabeled profiles (data not shown) were observed after any treatment.

Neostriatum: electron microscopy Ultrastructural evidence of neostriatal degeneration was assessed in coded samples from rats treated with 28-60 mg/kg/day amphetamine for 0 (control, n = 4), 1 (n = 2), 2 (n = 3) or 3 (n = 3) days. Large areas of tissue were scanned for each animal and the presence or absence of particular types of abnormalities noted in a qualitative fashion (e.g. absent, sparse, frequent, dense). An effort was made to perform an equally comprehensive survey of each animal. Even before the code was broken, it was easy to classify the animals into two distinct groups based upon the amount and type of degeneration noted. All of the control and 1-day animals fell into one group which showed only a few potential abnormalities. These included a few dark, but undisrupted, cell bodies as well as a small number of very dense profiles which could have Behavioral Ratlnge -6

.

/I ?

3

t

2

.

.

.

.

Long Evans .

I

IH]

!',o " I

Control

0-10 10-~0 ~10-30 $0-40 40-60 )60

DOSE RANGE (mg/kg/day)

Behavioral Ratlngl

-- 8prague-Dawley

,.liHi*''" JiJ]l 5

[i

Day1 /

I

reflected either degeneration or pigment deposition. Glial processes occasionally contained dark or membranous debris within multivesicular bodies. When the code was broken, all of the 2- and 3-day animals belonged to a second group, which contained much greater amounts of the specific types of degeneration described below, in addition to those few abnormalities seen in the first group. Tissue from Long-Evans and Sprague-Dawley rats appeared similar. The specific degeneration in this second group was typically not distributed homogeneously, which precluded making a detailed quantitative analysis. We did, however, compare the degeneration found by normal electron microscopic techniques with electron microscopic observations made on tissue immunolabeled for tyrosine hydroxylase from an animal with extensive degeneration initially detected in the light microscope. Two main types of degeneration were found in neostriatal tissue treated with amphetamine for at least two days. Some processes were swollen and either empty of organelles or filled with membranous debris. This type of degenerating process was sometimes labeled for tyrosine hydroxylase in the immunolabeled animal (Fig. 4A,B). Many apparently normal immunolabeled axons were seen in this same tissue and seemingly normal symmetric tyrosine hydroxylase-positive synapses onto spines and dendrites could be found (Fig. 4C,D). However, most cytological details that might have revealed early stages of degeneration of the dopaminergic terminals were obscured by the diaminobenzidine reaction product used in this tissue. The second category of degenerating processes were very dark profiles which often had irregular outlines. The distinctive tyrosine hydroxylase reaction product was never observed in these profiles. They frequently appeared to have shrunk away from the surrounding neuropil and were sometimes engulfed by glia. The

•

I I

dl

t

,° I

?

Sprague-Dawley

k' ijjil I

0 W

E I

Control

0-10 10-10 Ira-00 go-4o 4040

DOSE RANGE (nql/Iql/day)

Fig. 1. Mean behavioral ratings for Long-Evans and SpragueDawley rats for each day of amphetamine infusion. The same sham-operated control group (Long-Evans (n = 6)) is shown for both strains. Long-Evans: 0-10 mg/kg/day, n = 2; 10-20, n = 4; 20-30, n = 3; 30-40, n = 4; 40-50, n = 4; > 50, n = 4. Sprague-Dawley: 10-20, n = 1; 20-30, n = 1; 30-40, n = 3; 40-50, n=5.

.

.

.

.

i

-6

li

HGT -10 f C -t6 H ^ N -20 EG -25

L Cont. O-IO -20 -30 -40 -50 ,60

10-20 -30 -40 -60

DOSE RANGE (mg/kg/day)

Fig. 2. Mean weight loss after 3 days of amphetamine administration. Same rats as in Fig. 1.

70 TABLE 1

Degenerative changes observed in basal ganglia using Fink-Heimer, tyrosine hydroxylase and serotonin histochemistry Numbers shown in table are: number of animals in which degeneration was detected/number of animals in which tissue was examined. Stains used: FH: Fink-Heimer silver stain; TOH: tyrosine hydroxylase immunohistochemistry; 5-HT: serotonin immunohistochemistry. D (dorsal) and V (ventral) were defined as the upper and lower halves of the neostriatum. Anterior striatum was defined as the region anterior to the decussation of the anterior commissure. Middle was between this landmark and the appearance of the globus pallidus. Posterior was caudal to this landmark.

Stain

n

Nucleus accumbens

Neostriatum

Globus pallidus

Anterior D Long-Evans <20 mg/kg/day FH TOH 5-HT 20-60 mg/kg/day FH TOH 5- HT Sprague-Dawley <20 mg/kg/day FH TOH 5-HT 20-50 mg/kg/day FH TOH 5-HT Controls Both strains FH TOH 5-HT

Middle V

D

Posterior V

D

V

6 0/6 0/6 0/1

0/6 0/6 0/1

0/6 0/6 0/1

0/6 0/6 0/1

0/6 0/6 0/1

0/6 0/6 0/1

0/6 0/6 0/1

0/6 0/6 0/1

0/14 5/13 0/4

10/14 11/13 0/4

11/14 13/13 0/4

12/14 12/13 0/4

12/14 11/13 0/4

12/14 12/13 0/4

12/14 12/13 0/4

0/14 4/13 (1/4

0/1 0/1 .

0/1 0/1

0/1 0/1

0/1 0/1

0/1 0/1

0/1 0/1

0/1 0/1

0/1 0/1

14

1

.

.

.

.

.

.

9 0/8 1/7 0/2

8/8 5/7 0/2

7/8 5/7 0/2

8/8 7/7 0/2

8/8 6/7 0/2

8/8 6/7 0/2

8/8 5/7 0/2

0/8 1/7 0/2

0/16 0/12 0/2

0/16 0/12 0/2

0/16 0/12 0/2

0/16 0/12 0/2

0/16 0/12 0/2

0/16 0/12 0/2

0/16 0/12 0/2

0/15 0/12 0/1

16

identity of these profiles was often not determined because most were too dark to reveal vesicles or synapses, even in the 2-day tissue prepared by normal ultrastructural techniques. However, the few synaptic terminals that could be identified included both symmetric and asymmetric types. Some of this dark degeneration resembled astrocytic processes (Fig. 4E). When reconstructed from serial sections, these narrow black profiles branched extensively and encircled normal axon terminals. Other ultrastructural alterations were more rarely observed in amphetamine-treated tissue, but were never seen in controls. A few of these were disrupted myelinated axons (Fig. 4F). Some profiles could not be easily categorized. They often had denser than normal cyto-

Fig. 3. Neostriatal tyrosine hydroxylase, but not serotonin, immunostaining was abnormal in rats treated with over 20 mg/kg/day amphetamine. A: neostriatal tyrosine hydroxylase labeling in a control rat as compared to B: an amphetamine-treated rat in which many large, swollen densely labeled processes (arrows) are seen. No swollen, distorted or otherwise abnormal processes were seen labeled for serotonin in these same rats: C: control, D: amphetamine treated.

71 plasm and a proliferation or disorganization of various types of membranous organdies and usually lacked synaptic vesicles.

Frontal neocortex: Fink-Heimer terminal degeneration In 11 of 14 L o n g - E v a n s rats receiving greater than 20

mg/kg/day amphetamine, dense silver grain deposition was seen in one or more regions o f m o t o r neocortex (Table II), including areas F r l , Fr2 and Fr3 of Zilles 36 (Fig. 5). D a m a g e within Fr2 and Fr3 was always confined to regions anterior to the nucleus accumbens. Damage to Frl could be seen in several cases posteriorly to approx-

Fig. 4. Ultrastructural changes reflecting degeneration in the neostriatum include many large, swollen and abnormal processes labeled for tyrosine hydroxylase (A,B) which may be compared to more normal appearing axon terminals making symmetric synapses (arrows) onto spines (C) and onto dendritic shafts (D). Some dark degeneration is not reactive for tyrosine hydroxylase but instead resembles astrocytic processes in its complex, branching form and its tendency to encircle axon terminals (E). Degenerating myelinated fibers (F) were occasionally observed. All bars = 0.5/~m.

72 imately the crossing of the anterior commissure. In contrast, only 2 of 7 S p r a g u e - D a w l e y rats (above 30 mg/kg/day) showed any (sparse) degeneration in F r l and never in F R 2 or FR3. This difference between strains is significant (P < 0.025 for all animals receiving doses above 20 mg/kg/day (n = 14 L o n g - E v a n s , n = 8 S p r a g u e - D a w l e y ) ; P < 0.05 for animals matched by dose across strains for doses above 30 mg/kg (n = 7 and 7); Fisher exact probability test29). D e g e n e r a t i o n was much denser in layers 2 and 3 than elsewhere (Fig. 5). D e g e n e r a t i n g neurons were sometimes observed scattered among the debris of axonal degeneration. D e g e n e r a t i o n was also seen in region A I D (dorsal agranular insular cortex) of ZiUes 36 in 4 of 13 L o n g Evans and 4 of 8 S p r a g u e - D a w l e y rats receiving 20 mg/kg/day or m o r e of a m p h e t a m i n e . Frontal cortex: tyrosine hydroxylase and serotonin immunohistochemistry N e i t h e r tyrosine hydroxylase nor serotonin immunola-

beling a p p e a r e d to be altered in the frontal cortex of L o n g - E v a n s and S p r a g u e - D a w l e y rats, even in rats showing dense F i n k - H e i m e r labeling on adjacent sections (Table II). M a n y fine processes with regularly

spaced varicosities were seen; no swollen or otherwise distorted i m m u n o l a b e l e d profiles were o b s e r v e d in areas F r l , Fr2, Fr3, and A I D . Frontal cortex: electron microscopy Blind, coded electron microscopic analysis was con-

ducted on tissue from the frontal cortex of 6 L o n g - E v a n s rats receiving over 30 mg/kg/day a m p h e t a m i n e for 1 (n -2), 2 (n = 3) or 3 (n = 1) days, of one S p r a g u e - D a w l e y rat receiving a m p h e t a m i n e for 3 days and of two L o n g - E v a n s naive control rats. The evaluation procedures were the same as those described for the neostriatum, except that all tissue was p r e p a r e d by normal electron microscopy without i m m u n o c y t o c h e m i s t r y since no alterations of tyrosine hydroxylase were detected in frontal cortex in the light microscope. Significant degeneration was o b s e r v e d in some areas of frontal cortex of all L o n g - E v a n s rats that received a m p h e t a m i n e for at least 2 days, but only rare profiles a p p e a r e d abnormally d a r k in S p r a g u e - D a w l e y , control o r 1-day treated tissue. Most cortical d e g e n e r a t i o n consisted of disrupted cell bodies (Fig. 6A) and d a r k , shrunken processes (Fig. 6B). Some of these dark processes were axons that m a d e clear asymmetric synapses (Fig. 6 C , D ) whereas o t h e r dark processes were postsynaptic dendrites and dendritic spines. A f t e r only 2 days of a m p h e t a m i n e t r e a t m e n t ,

TABLE II Degenerative changes observed in frontal cortex using Fink-Heimer, tyrosine hydroxylase and serotonin histochemistry

Numbers shown in table are: number of animals in which degeneration was detected/number of animals in which tissue was examined. Stains used: FH: Fink-Heimer silver stain; TOH: tyrosine hydroxylase immunohistochemistry; 5-HT: serotonin immunohistochemistry. Smin

Long-Evans <20 mg/kg/day FH TOH 5-HT 20-60 mg/kg/day FH TOH 5-HT Sprague-Dawley <20 mg/kg/day FH TOH 5-HT 20-50 mg/kg/day FH TOH 5-HT Controls Both strains FH TOH 5-HT

n

FR1

FR2

FR3

AID

SS

0/6 0/6 0/1

0/6 0/6 0/1

0/6 0/6 0/1

0/6 0/6 0/1

0/6 0/6 0/1

8/13 0/11 0/4

5/8 0/8 0/4

5/13 0/11 0/4

4 / 1 3 9/11 0/8 0/11 0/4 0/4

6

14

1 0/1 0/1 0/1 0/1 . . .

0/1 0/1 0/1 0/1 . .

1/I 0/1

2/8 0/6 0/2

0/8 0/6 0/2

0/7 0/6 0/2

4/6 0/7 0/2

0/16 0/12 0/2

0 / 1 6 0 / 1 6 0 / 1 6 0/16 0 / 1 2 0 / 1 2 0 / 1 2 0/12 0/2 0/2 0/2 0/2

9 4/8 0/6 0/2

16

Fig. 5. Dense axonal degeneration was seen reliably (especially in layers 2 and 3) in the frontal motor cortical areas in Long-Evans but not Sprague-Dawley rats that received over 20 mg/kg/day of D-amphetamine sulfate. A: Fink-Heimer-labeled section showing dense silver grain deposition in layers 2 and 3. Much less is seen in layer 5 (near bottom). B: four unilateral sections through frontal cortex (anterior-most at top) in dark-field illumination. Dense silver grain deposition (at arrows) may be seen in circumscribed areas of frontal cortex (Areas Frl and Fr3 of Zilles36).

73

Fig. 6. Ultrastructural changes indicative of degeneration seen in the neocortex include the following. A: somatic changes such as this dark and vacuolated cell body. B: more common were scattered processes (arrows) that appeared dark and shrunken. C,D: some of these made asymmetric synapses onto dendritic spines. Bars = 1.0/~m in A and B; 0.5/zm in C and D.

many degenerating profiles were already too dark to identify. Additional abnormalities seen only in treated rats included disrupted myelinated fibers, very large empty profiles, and processes with a proliferation of organelles, perhaps indicative of reactive astrocytes. Dense areas of degeneration were often found adjacent to normal tissue.

Somatosensory neocortex: Fink-Heimer, tyrosine hydroxylase and serotonin labeling At doses above 10 mg/kg/day amphetamine in Sprag u e - D a w l e y and above 20 mg/kg/day in L o n g - E v a n s rats, circumscribed clusters of degenerating pyramidal neurons and debris associated with degenerating dendrites and axons were seen in layers 2 and 3 of somatosensory cortex of most rats (Table 1I, Fig. 7) in the region previously described as sensitive to degeneration by Commins et al. z'3. In immunolabeled tissue, no small circumscribed irregularities of staining that might correspond to these regions of degeneration could be found.

Fig. 7. Amphetamine-induced pyramidal cell degeneration in local regions of somatosensory neocortex. A,B: two degenerating neurons (arrows) are shown in sections stained with Fink-Heimer silver stain. Note the shrunken cell body and the densely labeled disintegrating dendrites. Other Fink-Heimer-labeled debris may be seen surrounding the cells.

74 TABLE III

Degenerativechanges observedin otherbrain regions using Fink-Heimer, tyrosinehydroxylase and serotonin histochernistry Numbers shown in table are: number of animals in which degeneration was detected/number of animals in which tissue was examined. Stains used: FH: Fink-Heimer silver stain; TOH: tyrosine hydroxylase immunohistochemistry; 5-HT: serotonin immunohistochemistry.

Stain Long-Evans <20mg/kg/day FH TOH 5-HT 20-60mg/kg/day FH TOH 5-HT Sprague-Dawley <20 mg/kg/day FH TOH 5-HT 20-50mg/kg/day FH TOH 5-HT Controls Both strains FH TOH 5-HT

n

SN

VTA

LC

D. Raphe

Cerebellum L. Septurn

Hippocampus

0/3 0/4 0/1

0/3 0/4 0/1

0/3 0/4 0/1

0/1 0/1

0/3 0/2 -

0/5 0/6 0/1

0/4 0/1 -

0/6 0/6 0/4

0/6 0/6 0/4

0/5 0/5 0/4

0/4 0/1 [)/2

0/5 0/5 0/2

0/10 0/13 014

0/10 0/4 0/4

0/1 0/1

0/1 0/1

-

0/1 0/1 -

0/1 0/1

6

14

1 . 0/1 0/1

.

. 0/1 0/1

.

8 0/4 0/3 0/3

0/4 0/4 0/3

0/2 0/4 0/3

0/2 0/3 0/3

0/2 -

0/6 0/7 0/2

0/6 0/4 0/2

0/7 0/8 0/3

0/7 0/8 0/3

0/6 0/8 0/3

0/2 0/2 0/1

0/5 0/5 -

0/10 0/6 0/1

0/15 0/13 0/3

16

Substantia nigra, locus coeruleus and dorsal raphe nuclei: Fink-Heimer and tyrosine hydroxylase and serotonin immunohistochemistry In no case, in neither L o n g - E v a n s nor S p r a g u e Dawley rats, was silver grain deposition observed in F i n k - H e i m e r - l a b e l e d sections containing the nuclei of origin of ascending d o p a m i n e , norepinephrine and serotonin projections. In cases where adjacent sections were labeled for tyrosine hydroxylase or serotonin immunoreactivity, dense somatic and dendritic labeling was observed. No a b n o r m a l profiles or labeling were seen (Table III). Other neural targets of monoamine neurons: FinkHeimer and immunolabeling F i n k - H e i m e r degeneration was never observed in other structures, including the globus pallidus, nucleus accumbens, h i p p o c a m p u s , septum and cerebellum, that receive a d o p a m i n e , n o r a d r e n e r g i c or serotonergic input (Tables I and III). A few a b n o r m a l tyrosine hydroxylase profiles were occasionally seen in the nucleus accumbens and globus pallidus in animals receiving 30 mg/kg/day or m o r e of a m p h e t a m i n e . N o a b n o r m a l serotonin labeling was o b s e r v e d anywhere. DISCUSSION The continuous administration of a m p h e t a m i n e caused

neural d e g e n e r a t i o n in several regions of the rat brain including the neostriatum and neocortex. The precise pattern was strain- and d o s e - d e p e n d e n t . A x o n a l degeneration was usually seen in the neostriatum in both S p r a g u e - D a w l e y albino rats and L o n g - E v a n s blackh o o d e d rats above a dose of a m p h e t a m i n e of approximately 20 mg/kg/day. In contrast, d e g e n e r a t i o n was seen in frontal m o t o r cortex significantly m o r e frequently in L o n g - E v a n s than in S p r a g u e - D a w l e y rats. This strain difference is likely to reflect a genetic predisposition for the induction of frontal m o t o r cortical d a m a g e rather than t r e a t m e n t differences. First, an extensive d o s e response curve, extending up to lethal doses, was examined for both strains. Second, the doses were matched across strains in the sense that at one or more doses in which d e g e n e r a t i o n was seen in L o n g - E v a n s but not S p r a g u e - D a w l e y rats, equivalent degrees of behavioral activation, weight loss, and lethality were observed. Third, damage was observed in all other regions at similar doses in both strains. Thus it seems unlikely that difference in sensitivity of the frontal m o t o r cortex to a m p h e t a m i n e - i n d u c e d d a m a g e is due to drug-dispositional or dosage factors. H o w e v e r , the nature of this predisposition is unknown. Much of the damage revealed in the n e o s t r i a t u m of both strains using F i n k - H e i m e r silver staining p r o b a b l y reflects degeneration of d o p a m i n e axons and terminals. Many tyrosine hydroxylase-immunoreactive processes in

75 the neostriatum show evidence of damage in both the light and electron microscopes. Many profiles are swollen and distended and the even feltwork appearance of tyrosine hydroxylase-positive fibers typically seen with the light microscope in control tissue 11 is replaced by a sparse network of readily distinguishable fibers in amphetamine-treated rats. Since there are few noradrenergic fibers in the neostriatum, it seems likely that much of the axonal degeneration seen in the neostriatum reflects degeneration of dopamine axons and terminals. In contrast, neither serotonin-immunoreactive fibers nor Leu-enkephalin-immunoreactive processes showed any obvious alterations in size or shape in amphetaminetreated rats. This attempt to identify the electron microscopic characteristics of degeneration faced the problem of distinguishing amphetamine-induced degeneration from the inherent background abnormalities typically found in control tissue. Although we were unable to perform a detailed quantitative comparison of treated and control tissue due to the inhomogeneous distribution of the degeneration, it was still possible for a blind observer to correctly divide the coded tissue into two groups. Because all of the animals receiving amphetamine for at least two days (and none of the controls) comprised the high degeneration group, we feel confident that by analyzing the abnormalities present only or especially in that group we have correctly identified a set of ultrastructural alterations that continuously administered amphetamine can produce. The swollen and disrupted tyrosine hydroxylase-labeled profiles seen in the electron microscope probably correspond to the swollen tyrosine hydroxylase-positive processes seen in the light microscope. However, the lack of tyrosine hydroxylase-immunolabel in the dark degenerating profiles seen in the electron microscope may not indicate that these are non-dopaminergic processes. It might reflect a different stage of dopaminergic axonal degeneration in which tyrosine hydroxylase has been digested or deactivated or reflect a different region of the dopamine axon. Two additional types of degeneration occurring in the neostriatum were observed. First, some degenerating profiles, reconstructed in 3 dimensions from serial electron micrographs 35, resemble the darkened glial processes seen after MPTP treatment TM. Second, a few degenerating myelinated fibers were reliably seen in tissue from amphetamine-treated rats. The dopamine axons in the neostriatum are unmyelinated, as were our tyrosine hydroxylase-labeled processes. Thus it is likely that these myelinated axons arise from non-monoamine neurons. These may be cortical afferents since some of the degenerating myelinated axons were seen in the fascicles of the internal capsule and may arise from the

degenerating cortical neurons seen in frontal cortex or somatosensory cortex. These histological and ultrastructural results confirm and extend earlier reports demonstrating that chronic amphetamine administration produces enduring alterations of dopamine neurotransmission in the neostriaturn. However, not all dopamine systems are equally sensitive to amphetamine's neurodegenerative effects. Examination of dopamine terminal fields in the septum, hippocampus, nucleus accumbens and globus pallidus using Fink-Heimer silver staining revealed no evidence of degeneration. In some rats, mostly at the higher doses, a few swollen and distorted tyrosine hydroxylase-immunoreactive profiles were seen in the globus pallidus and nucleus accumbens. The labeling in globus pallidus may represent dopaminergic fibers of passage, the distal ends of which were damaged in the neostriatum. In nucleus accumbens and possibly in globus pallidus, amphetamine may indeed be causing slight damage, below the threshold for detection using the Fink-Heimer technique. In previous experiments it was shown that in both Sprague-Dawley and Long-Evans rats, tyrosine hydroxylase staining in the neostriatum was made patchy by amphetamine treatment at doses that also produced degeneration seen on adjacent sections with FinkHeimer staining27. These patches were in register with Leu-enkephalin patches seen on adjacent sections, indicating that the tyrosine hydroxylase patches corresponded to striosomes. Thus, even within the neostriatum, different dopamine systems may have different sensitivities for amphetamine-induced axonal degeneration. The dopamine neurons of the substantia nigra and the ventral tegmental area, the axons of which form the dopamine innervation of the neostriatum, do not appear to be damaged by amphetamine. This result confirms previous studies that also demonstrate axonal damage in the absence of somatic damage (e.g. refs. 17,24,26). In addition, the noradrenergic nucleus, the locus coeruleus, the serotonergic nucleus and the dorsal raphe, which provide much of the ascending monoaminergic innervation of the forebrain, also do not appear to be damaged by amphetamine. Three types of neocortical degeneration were observed. First, in both Sprague-Dawley and Long-Evans rats, isolated pockets of degenerating cortical pyramidal cells were seen in somatosensory neocortex. This pattern is the same as previously reported for analogues of D-amphetamine, including methamphetamine 1, parachloroamphetamine 3, and MDMA 2. The second site of neocortical degeneration was the frontal AID cortex. Although the AID receives a dopamine projection 5, the degeneration detected with

76 F i n k - H e i m e r probably is that of non-monoaminergic neurons. Neither tyrosine hydroxylase nor serotonin immunolabeling was abnormal. This damage may occur independently from the frontal motor cortex damage since it occurs equally often in Sprague-Dawley and L o n g - E v a n s rats. The third pattern of cortical degeneration was seen predominantly in L o n g - E v a n s rats. Dense F i n k - H e i m e r silver staining was seen localized to layers 2 and 3 of frontal motor cortex extending from near the orbital pole posteriorly to the level of the anterior commissure in some cases. This pattern has not been reported previously for any of the amphetamines and is different from the pattern of degeneration reported to occur in medial frontal cortex of immature gerbils 34. It is likely that the degenerating axons are not monoaminergic. First, the layering of the degeneration is not consistent with the pattern of innervation of motor cortex by monoamine fibers 5'2~. Second, no abnormal tyrosine hydroxylase or serotonergic immunolabeled processes were seen in these regions. Instead, it seems likely that these degenerating axons are cortico-cortical processes. The degenerating axons occupy the layers of termination of some intracortical connections and, in the electron microscope, many asymmetric synapses, typical of cortico-corticai connections, were observed to be degenerating. Indeed, some degenerating neurons, seen both in the light and electron microscopes, were scattered throughout motor cortex, suggesting that the degenerating axons may be callosal or recurrent collateral connections. Although there were far fewer degenerating cells than might be expected to account for the density of the terminal degeneration, the time course of somatic degeneration may be different (and was not explored), and, indeed, somatic degeneration may not occur in most cases. Degenerating neurons in the somatosensory cortex may also contribute degenerating axons to motor cortex; however, these degenerating neurons were seen in both rat strains, whereas the degenerating motor cortex axons were usually seen in just L o n g - E v a n s rats. A curious feature of the frontal motor cortex damage is that the precise pattern is unpredictable from one animal to the next. For example, in one animal receiving

REFERENCES 1 Commins, D.L. and Seiden, L.S., Alpha-methyltyrosine blocks methylamphetamine-induced degeneration in the rat somatosensory cortex, Brain Research, 365 (1986) 15-20. 2 Commins, D.B., Axt, K.J., Vosmer, G. and Seiden L.S., Endogenously produced 5,6-dihydroxytryptamine may mediate the neurotoxic effects of para-chloroamphetamine, Brain Research, 419 (1987) 253-261. 3 Commins, D.L., Vosmer, G., Virus, R.M., Wollverton, W.L., Schuster, C.R. and Seiden, L.S., Biochemical and histochemical

between 30 and 40 mg/kg/day of amphetamine, damage was very dense in Fr2, while Frl was intact. Another L o n g - E v a n s rat, receiving this same dose, showed dense degeneration in Frl and Fr3, but not in Fr2. Although the precise location was variable, the density of the silver stain was always, in L o n g - E v a n s rats, many times greater in these frontal cortical areas than in the neostriatum. This variable pattern, plus the lack of evidence for degenerating monoamine axons in the cortex, suggests that some of the mechanisms inducing degeneration in these areas may be different from the mechanisms in the neostriatum. Part of the apparent variability in the location of damage could reflect different areal time courses of degeneration, but this is unlikely since all animals were treated on the same schedule. Instead, this variability might reflect dynamic variables. For instance, degeneration in one area might decrease the likelihood of damage in adjacent areas. This could occur, for example, if the axonal damage were due to a type of excitotoxicity, where damage in one area might decrease the excitatory influence on connected areas. Recently, Sonsalla and co-workers 3° demonstrated that antagonists of N M D A receptors protect against methamphetamine-induced depletions of neostriatal dopamine, suggesting that excitotoxic actions may contribute to amphetamine-induced neurodegeneration. Similar excitotoxic action might occur in the neocortex. The origin of the genetic predisposition of frontal cortex sensitivity is unknown. The most obvious difference between L o n g - E v a n s black-hooded and SpragueDawley albino rats is pigmentation. It is known that melanin can sequester and prolong the action of amphetamine 4 and it has been suggested that neuromelanin (produced via a different synthetic pathway than melanin and thus not necessarily affected by albinism) may participate in the sequestration and targeting of another dopamine neuron toxin, MPTP, and its important degradative product, MPP ÷ (ref. 6). In this regard L o n g - E v a n s rats are also more sensitive to the degenerative effects of MPTP than are Sprague-Dawley rats TM. Acknowledgements. This research was supported by Grants DA 02854 and RSA 00079 from the National Institute on Drug Abuse to P.M.G.

evidence that methylenedioxymethylamphetamine (MDMA) is toxic to neurons in the rat brain, J. Pharmacol. Exp. Ther., 24l (1987) 338-345. 4 Creel, D., Inappropriate use of albino animals as models in research, Pharmacol. Biochem. Behav., 12 (1980) 969-977. 5 Descarries, L., Lemay, B., Doucet, G. and Berger, B., Regional and laminar density of the dopamine innervation in adult rat cerebral cortex, Neuroscience, 21 (1987) 807-824. 6 D'Amato, R.J., Alexander, R.J., Schwartzman, R.J., Kitt, C.A., Price, D.L. and Snyder, S.H., Evidence for neuromelanin involvement in MPTP-induced neurotoxicity, Nature (Lond.),

77 327 (1987) 324-326. 7 Ellison, G., Eison, M.S., Huberman, H.S. and Daniel, E, Long-term changes in dopaminergic innervation of caudate nucleus after continuous amphetamine administration, Science, 201 (1978) 276-278. 8 Fink, R.P. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 9 Fukui, K., Kariyama, H., Kashiba, A., Kato, N. and Kimura, H., Further confirmation of heterogeneity of the rat striatum: different mosaic patterns of dopamine fibers after administration of methamphetamine or reserpine, Brain Research, 382 (1986) 81-86. 10 Fuller, R.W. and Hemrick-Leucke, S., Long-lasting depletion of striatal dopamine by a single injection of amphetamine in iprindole-treated rats, Science, 209 (1980) 305-307. 11 Gerfen, C.R., Herkenham, M. and Thibault, J., The neostriatal mosaic: II. Patch- and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems, J. Neurosci., 7 (1987) 3915-3934. 12 Groves, P.M., Martone, M., Young, S.J. and Armstrong, D.M., Three-dimensional pattern of enkephalin-like immunoreactivity in the neostriatum of the cat, J. Neurosci., 8 (1988) 892-900. 13 Groves, P.M., Ryan, L.J. and Linder, J.C., Amphetamine changes neostriatal morphology. In D.P. Friedman and D.H. Clouet (Eds.), The Role of Neuroplasticity in the Response to Drugs, N.I.D.A. Res. Monogr. No. 78, Washington, 1987, pp. 132-142. 14 Harvey, J.A., McMaster, S.E. and Fuller, R.W., Comparison between the neurotoxic and serotonin-depleting effects of various halogenated derivatives of amphetamine in the rat, J. Pharmacol. Exp. Ther., 202 (1977) 581-589. 15 Hotchkiss, A.J. and Gibb, J.W., Long-term effects of multiple doses of methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase activity in rat brain, J. Pharmacol. Exp. Ther., 214 (1980) 257-262. 16 Hsu, S.M., Raine, L. and Fanger, H., The use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 17 Jonsson, G. and Nwanze, E., Selective amphetamine neurotoxicity on striatal dopamine nerve terminals in the mouse, Br. J. Pharmacol., 77 (1982) 335-345. 18 Linder, J.C., Klemfuss, H. and Groves, P.M., Acute ultrastructural and behavioral effects of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) in mice, Neurosci. Lett., 82 (1987) 221-226. 19 Lorez,,H., Fluorescence histochemistry indicates damage of striatal dopamine nerve terminals in rats after multiple doses of methamphetamine, Life Sci., 28 (1981) 911-916. 20 Markey, S.P. and Schmuff, N.R., The pharmacology of the parkinsonian syndrome producing neurotoxin MPTP (1-methyl4-phenyl-l,2,3,6-tetrahydropyridine) and structurally related compounds, Med. Res. Rev., 6 (1986) 389-429. 21 Morrison, J.H., Grzanna, R., Molliver, M.E. and Coyle, J.T., The distribution and orientation of noradrenergic fibers in the neocortex of the rat: an immunofluorescence study, J. Comp. Neurol., 181 (1978) 17-40.

22 O'Hearn, E., Battaglia, G., De Souza, E.B., Kuhar, M.J. and Molliver, M.E., Methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA) cause selective ablation of serotonergic axon terminals in the forebrain: immunocytochemical evidence for neurotoxicity, J. Neurosci., 8 (1988) 2788-2803. 23 Peat, M.A., Warren, EE, Bakhit, C. and Gibb, J.W., The acute effects of methamphetamine and p-chloroamphetamine on the cortical serotonergic system of the rat brain: evidence for differences in the effects of methamphetamine and amphetamine, Eur. J. Pharmacol., 116 (1985) 11-16. 24 Ricaurte, G.A., Guillery, R.W., Seiden, L.S., Schuster, C.R. and Moore, R.Y., Dopamine nerve terminal degeneration produced by high doses of methylamphetamine in the rat brain, Brain Research, 235 (1982) 93-103. 25 Ricaurte, G.A., Guillery, R.W., Selden, L.S. and Schuster, C.R., Nerve terminal degeneration after a single injection of D-amphetamine in iprindol-treated rats: relation to selective long-lasting dopamine depletion, Brain Research, 291 (1984) 378-382. 26 Ricaurte, G.A., Seiden, L.S. and Schuster, C.R., Further evidence that amphetamines produce long-lasting dopamine neurochemical deficits by destroying dopamine nerve fibers, Brain Research, 303 (1984) 359-364. 27 Ryan, L.J., Martone, M.E., Linder, J.C. and Groves, P.M., Continuous amphetamine induces tyrosine hydroxylase immunoreactive patches in the adult rat neostriatum, Brain Res. Bull., 21 (1988) 133-137. 28 Seiden, L.S., Fischman, M.W. and Schuster, C.R., Long-term methamphetamine-induced changes in brain catecholamines in tolerant rhesus monkeys, Drug Alcohol Depend., 1 (1975/76) 215-219. 29 Siegel, S., Non-parametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956, 312 pp. 30 Sonsalla P.K., Nicklas, W.J. and Heikkila, R.E., Role for excitatory amino acids in methamphetamine-induced nigrostriatal dopaminergic toxicity, Science, 243 (1989) 398-400. 31 Stone, D.M., Stahl, D.C., Hanson, G.R. and Gibb, J.W., The effects of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) on monoaminergic systems in the rat brain, Eur. J. Pharmacol., 128 (1986) 41-48. 32 Wagner, G.C., Ricaurte, G.A., Johanson, C.E., Schuster, C.R. and Seiden, L.S., Amphetamine induces depletion of dopamine and loss of dopamine uptake sites in the caudate, Neurology, 30 (1980) 547-549. 33 Wagner, G.C., Ricaurte, G.A., Seiden, L.S., Schuster, C.R., Miller, R.J. and Westley, L., Long-lasting depletion of striatal dopamine and loss of dopamine uptake sites following repeated administration of methamphetamine, Brain Research, 181 (1980) 151-160. 34 Wahnschaffee, U. and Esslen, J., Structural evidence for the neurotoxicity of methylamphetamine in the frontal cortex of gerbils (Meriones unguiculatus): a light and electron microscopical study, Brain Research, 337 (1985) 299-310. 35 Young S.J., Royer, S.M., Groves, P.M. and Kinnamon, J.C., Three-dimensional reconstructions from serial micrographs using the IBM PC, J, Electron. Microsc. Tech., 6 (1987) 207-217. 36 Zilles, K., The Cortex of the Rat, Springer, Berlin, 1985, 113 pp.