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Serotonin content is elevated in the dopamine deficient striatum of the weaver mutant mouse
267
Brain Research, 606 (1993) 267-272 Elsevier Science Publishers B.V.
BRES 18632
Serotonin content is elevated in the dopamine deficient striatum of the weaver mutant mouse E.H. Stotz d9 L.C. T r i a r h o u b,d 9 B. G h e t t i a,b,d a n d J . R . S i m o n a,c,d Departments of a Psychiatry (Institute of Psychiatric Research), b Pathology (Neuropathology), c Biochemistry and Molecular Biology, and dprogram in Medical Neurobiology, Indiana University School of Medicine, Indianapolis, IN 46202-4887 (USA) (Accepted 20 October 1992)
Key words: Immunocytochemistry; Serotonin; Striatum; Weaver mutant mouse
In the present study, we measured the striatal serotonin content of weaver and control mice at different ages. Overall, weaver mutant mice exhibited 50% more striatal serotonin than controls. Neither a rostrocaudal gradient nor an age effect was found for either genotype. An analysis of serotonin content across the dorsoventral extent of the striatum revealed that in the dorsal striatum of the weaver, serotonin content was increased 200%, and in the ventral striatum, the increase amounted to 50% relative to control mice. Serotonin immunocytochemistry also revealed an increase in the dorsal striata of weaver mice. The major increase in striatal serotonin content seen in the weaver striatum occurs in the same region that exhibits the severest dopamine depletion. This observation is consistent with the notion that the increase in serotonin levels may be secondary to the decrease in dopamine content and may play an adaptive or compensatory role.
INTRODUCTION The weaver mouse has a recessive mutation which results in the loss of the dopaminergic cells in the substantia nigra (SN) 29. By 3 months of age, homozygous weaver mice (wv/wv) have lost approximately 70% of their tyrosine hydroxylase immunoreactive cells in the SN compared to control ( + / + ) mice 29. The nigral loss is accompanied by depletions in DA terminal markers in the striatum (STR). Dopamine content in the STR is decreased by 6 5 - - 7 0 % 16'17'20'21'26, and the activity of tyrosine hydroxylase (TH) is reduced by about 6 0 % 16'21'26. Striatal DA uptake is affected to a greater extent than either DA content or TH activity, exhibiting a decrease of approximately 9 0 % 7'16'20'26. The administration of 6-hydroxydopamine (6OHDA) into the cerebrospinal fluid of neonatal rats (intracisternally or intracerebroventricularly) results in the reduction of striatal DA content 3'4'13'23'28. Furthermore, the serotonergic system appears to hyperinnervate the S T R 1'3'4'13'22'23'28. Depending upon the age of the rat at the time of the lesion and the extent of the
lesion, striatal serotonin (5-HT) content is increased from 25%-100% 3'4'13'23'28 and 5-HT uptake is also elevated 13,23. Immunocytochemical studies have shown substantial increases in the density of 5-HT-like immunoreactive terminals in both the rostral and caudal STR t3. Horseradish peroxidase tracing methods have shown that an increased number of 5-HT cells are labeled in the dorsal raphe nucleus 1'22, suggesting the presence of an increased number of 5-HT cells projecting from the dorsal raphe to the STR. Using the weaver mutant mouse as a model of dopamine loss, we examined the hypothesis that a loss of striatal dopamine terminals will lead to an increase in striatal serotonergic innervation. Serotonin content of weaver and control mice was examined along the rostrocaudal extent of the STR as the rostral portion of the STR has been shown to be the principal site of the 5-HT hyperinnervation in rats with 6-OHDA lesions 13'23. The weaver mutation is known to cause a severer depletion of DA in the dorsal aspect of the STR than in the ventral aspect. Since the severity of the DA depletion has been shown to influence the
Correspondence: J.R. Simon, Institute of Psychiatric Research, Indiana University School of Medicine, 791 Union Drive, Indianapolis, IN 46202-4887, USA. Fax: (1) (317) 274-1365.
268 extent of the 5-HT hyperinnervation l, the dorsal and the ventral portions of the weaver STR were investigated separately. MATERIALS
AND METHODS
Homozygous weaver (we~we) and wild-type ( + / + ) control mice were obtained from a colony maintained at Indiana University Medical Center. The colony originated from heterozygous mice (we~+) purchased from Jackson Laboratory. The weaver mutation is maintained on a B6CBA-AW-J/A hybrid background. For the determination of 5-HT content as a function of age, mice were used at the ages of 60_+5 days (2 months), 2 4 5 + 9 days (8 months) or 430 + 17 days (14 months). Forty-two mice were used for the study of 5-HT content as a function of location (rostrocaudal or dorsoventral). For the immunocytochemistry study, four mice were used. For the determination of 5-HT content, age-matched pairs consisting of a weaver and a control mouse were sacrificed by decapitation. The brains were rapidly removed, m o u n t e d on a chuck and placed in a cryostat/microtome set at - 1 2 ° C . The frozen brains were cut in an alternating pattern of 40 /zm and 480 p,m thick sections which were m o u n t e d on glass slides. Collection of sections began with the appearance of the striatum and ceased with the appearance of the decussation of the superior cerebellar peduncles. The 480 / l m thick sections were numbered such that the coronal section containing the midline crossing of the anterior commissure was assigned '0'. More rostral sections were numbered with negative integers such that the more rostral a section, the larger the absolute value of the negative integer. More caudal sections were numbered with positive integers such that the more caudal a section, the larger the positive integer. The 480 p,m thick sections were stored overnight at - 7 0 ° C while the 40 /~m thick sections were stained with Cresyl violet and were used for orientation. The following day, the slides with the 480 p.m thick sections were placed on a Petri dish with ice and viewed under a dissecting microscope. For the analysis of 5-HT content along the rostrocaudal extent of the STR, bilateral striata from each section were dissected and pooled, whereas for the dorsoventral study, bilateral striata were dissected into dorsal and ventral aspects which were then pooled from all the rostrocaudal sections. In all experiments, care was taken to exclude the globus pallidus and the nucleus accumbens. Dissected samples were placed in microfuge tubes with 150 ~1 of 0.5 M perchloric acid containing dihydroxybenzylamine as an internal standard. The tissue was sonicated until it was homogeneous and then placed on ice for 30 min. Following centrifugation for 5 min in a Beckman Model B microfuge, 100/zl of the supernatant was injected on an H P L C for m o n o a m i n e determination and the pellet was resuspended in 150 p.1 of 0.1 N N a O H for protein determination 12. For monoamine determination, the area under the curve from the H P L C chromatograph was converted to picomoles by using a standard curve and correcting for recovery with the internal standard. The H P L C - E C was conducted at a flow rate of 1.0 m l / m i n and a potential of 0.8 V. The C-18 column had a sphere size of 5 izm with the dimensions 4.6 m m (inner diameter) ×25 cm. The limit of sensitivity of this system was approximately 0.5 pmol for 5-HT. T h e mobile phase contained 60 m M citric acid, 30 m M sodium phosphate, 0.39 m M octylsodium sulfate, 0.11 m M E D T A and 11.6% methanol (v/v). The statistical analyses were performed using a repeated-measures analysis of variance with one grouping factor (age) and two repeated-measures factors (genotype and location) 31. Each 'subject' in an age group was a pair of mice, one + / + and one wc/wu, whose tissues were assayed at the same time. There were six such pairs in each age group and there were three age groups (36 mice). For each mouse, five locations were analyzed, thus resulting in 180 data points. The data were transformed to log scale to remove correlation between m e a n s and variances. Ten missing values were estimated by maximum likelihood using program B M D P 5 V 6. The analysis was then completed using the ' A N O V A ' c o m m a n d in the M I N I T A B package TM. The degrees of freedom for the highest order
error term were reduced by ten (from 60 to 50) to compensate for the estimation of the missing values. For the serotonin immunocytochemistry study, the peroxidase antiperoxidase method of Sternberger et al. 25 was used as modified by Steinbusch and Tilders 24. Briefly, age-matched pairs of weaver and control mice were pretreated with 100 m g / k g pargyline i.p. 2 h before perfusion. Mice were given 600 units of heparin i.p. before being anesthetized with 50 m g / k g pentobarbital sodium i.p. Mice were perfused transcardially with 60 ml of ice-cold, oxygen-enriched, calcium-free Tyrode's buffer followed by 120 ml of ice-cold perfusion medium containing 4% (w/v) formaldehyde, 0.05% (v/v) glutaraldehyde and 0.2% (w/v) picric acid in 0.1 M sodium phosphate buffer (pH 7.4). Brains were removed and were left in perfusion medium without glutaraldehyde overnight. Fifty-~m-thick coronal sections were cut with a vibratome and were collected free-floating in Trisbuffered saline (pH 7.6)(TBS). Sections were rinsed consecutively for 5 min with TBS, TBS containing 0.5% Triton X-100 (TBS-T), and TBS. Sections were then incubated at 4°C for 40 h with rabbit anti-5-HT antiserum diluted 1:1800. After rinsing the sections for 5 min in TBS-T, TBS and TBS-T, goat anti-rabbit IgG at a dilution of 1:40 was applied to the sections for 2 h at room temperature. Sections were rinsed with TBS-T, TBS and TBS-T. Rabbit peroxidase anti-peroxidase complex at a dilution of 1 : 250 was applied to the sections for a 1 h incubation at room temperature. Following three TBS rinses, the sections were reacted with 0.01% 3,3'-diaminobenzidine-HCl, 0.67% nickel ammonium sulfate, 0.05 M imidazole and 0.01% H 2 0 2. After final TBS rinses, the sections were mounted on gelatinized slides and coverslipped. RESULTS
Serotonin content Examination of the rostrocaudal distribution of 5-HT in 14-month-old mice revealed an increase in 5-HT content in the wv/wv at every striatal level examined (Fig. 1). No rostrocaudal gradient for 5-HT was found
c
40
-~ ~?_
3o
~ZIZ "-~
20
C O
~
O E
-2 rostra]
-I
0
section number
i
2 caudal
Fig. 1. The rostrocaudal distribution of 5-HT content in striatum of 14-month-old + / + and wc/wc mice. T h e data are presented in p m o l / m g protein and represent the m e a n s _+S.E.M. for n = 6 for each genotype. Sections are numbered such that section '0' contains the midline crossing of the anterior commissure. Rostral sections are numbered with negative integers such that the more rostral a section, the larger the absolute value of the negative integer. Caudal sections are numbered with positive integers such that the more caudal a section, the larger the positive integer. A repeated measures A N O V A was performed on a log transformation of the data over the three ages (2, 8 and 14 months), the five locations and the two genotypes. T h e analysis showed no effect of a g e (F2,15 = 0.36, P > 0.05) nor of location (F4,60 = 0.81, P > 0.05). There was an effect of genotype ( F l j s = 68.08, P < 0.05), but none of the possible interactions were significant at the P < 0.05 lwel.
269 in either genotype; n o r was such a g r a d i e n t f o u n d in 8or 2 - m o n t h - o l d mice (data n o t shown). Analysis a m o n g the ages (2, 8 a n d 14 m o n t h s ) i n d i c a t e d that t h e r e was n o significant effect of age o n 5 - H T c o n t e n t within
25
each genotype. Thus, with n o significant effect of age or r o s t r o c a u d a l location, 5 - H T c o n t e n t data could be pooled for each genotype. Pooling the d a t a in this m a n n e r r e s u l t e d in a striatal 5 - H T c o n t e n t of 17.3 + 0.8 p m o l / m g p r o t e i n in + / +
mice a n d a striatal 5 - H T
c o n t e n t of 26.6 _+ 1.0 p m o l / m g p r o t e i n in w v / w v mice (Fig. 2). T h e 50% increase in striatal 5 - H T c o n t e n t in the weaver was significant with P < 0.001 ( n = 18 for each genotype). F i n d i n g n o effect of age o n striatal 5 - H T c o n t e n t , a single age, 3 m o n t h s , was used to study the dorsoventral g r a d i e n t ( T a b l e I). I n + / + mice, the v e n t r a l S T R c o n t a i n e d a p p r o x i m a t e l y 65% m o r e 5 - H T t h a n the dorsal STR. I n w v / w v mice, the r a n k o r d e r of striatal
o
,=~ o E
T
10 5
+/+ wv/wv Fig. 2. Striatal 5-HT content in + / + and wv/wv mice. The data are presented in pmol/mg protein and represent the means + S.E.M. for n = 18 for each genotype. 5-HT content was measured along the rostrocaudal extent of the STR in 2-, 8- and 14-month-old mice. A repeated measures ANOVA revealed only a significant genotype effect (F1,15= 68.08, P < 0.05). Therefore the data were pooled for age and location within each genotype.
Fig. 3. Serotonin immunocytochemistryin the dorsal and ventral striatum of + / + and wv / wv mice. (a) The dorsal striatum of the + / + mouse displays few 5-HT immunoreactive fibers while (b) the dorsal striatum of the wv/wv mouse contains many more 5-HT immunoreactive fibers. No clear difference in the density of fiber staining can be seen between (c) the ventral striatum of the + / + mouse and (d) the ventral striatum of the weaver mouse. Mice were 21 months of age at the time of sacrifice. Bar = 50 p.m.
270 TABLE I
Dorsoventral gradient of 5-HT content in + / + and wv / wv mice The data are presented as p m o l / m g protein and represent the means_+S.E.M, for n = 3 for each genotype. 5-HT content was determined in the dorsal and ventral STR by H P L C - E C in 3-monthold mice. An A N O V A was performed on the data revealing an effect of genotype (F1, 4 = 91.391, P < 0.05) but not an effect of region (F1, 4 = 0.326, P > 0.05). The interaction of genotype and region was also significant (F1. 4 = 15.414, P < 0.05). + / + dorsal STR vs + / + ventral STR, t ( 7 ) = - 3 . 3 5 , P < 0.05; w v / w v dorsal STR vs w v / w v ventral STR, t(7)=2.50, P < 0 . 0 5 ; + / + dorsal STR vs w v / w v dorsal STR, t(7)=9.27, P < 0 . 0 5 ; + / + ventral STR vs w v / w v ventral STR, t(7) = 3.44, P < 0.05.
Striatal location Dorsal Ventral
5-HT Content
+/+
wv / wv
15.6 + 1.8 26.1 + 4.3
44.7 + 1.5 36.9 + 2.4
5-HT content was reversed, with the dorsal STR containing 20% more 5-HT than the ventral STR. Comparisons between the two genotypes showed that both the dorsal and the ventral STR of the w v / w v had a greater 5-HT content compared to control. The 5-HT content of the dorsal STR of the weaver was increased 3-fold over the corresponding region in the control, while 5-HT in the ventral STR of the weaver was increased 50% over the corresponding region in the control. Serotonin immunocytochemistry
The 5-HT immunoreactive fibers seen in this study were very fine with small fusiform or granular varicosities, suggesting that they are 'type D' 5-HT axons which originate in the dorsal raphe nuclei 11. Serotonin immunocytochemistry of control S T R revealed a dorsoventral gradient of 5-HT immunoreactive fibers (Fig. 3). Despite the sparse distribution of the stained fibers in the dorsal STR, once located, single fibers were easily traced. In the ventral STR of control mice, 5-HT immunoreactive fibers were more numerous and tracing of individual fibers was more difficult due to extensive overlapping in the 50 /zm thick sections. In the STR of weaver mice, the dorsoventral gradient of 5-HT immunoreactive fibers seen in controls was not found. The distribution of 5-HT immunoreactive fibers appeared to favor the dorsal STR. In the dorsal STR, tracing of individual fibers was more difficult than in the ventral S T R due to crowding, suggesting that the dorsal STR contained more fibers. While comparisons between control ventral STR and weaver ventral STR revealed no clear differences between genotypes, the dorsal STR of weaver mice clearly had a higher density of 5-HT immunoreactive fibers than the same region in
control mice. Increases in both the number of fibers and darkly stained varicosities could be seen in the weaver dorsal STR as compared to control mice. DISCUSSION The results of the present study indicate that the weaver STR contains more 5-HT than the control STR. This is seen both as an expression of 5-HT content and of 5-HT immunoreactivity. The increase in 5-HT occurs predominantly in the dorsal aspect of the STR. Neither a rostrocaudal distribution nor an age effect was found for 5-HT content within either genotype. In rats, striatal 5-HT content has been shown to follow a rostrocaudal gradient in which the caudal S T R contains 3 - 6 times more 5-HT than the rostral STR 23'27. In rats which have received 6 - O H D A lesions as neonates this gradient is diminished as 5-HT content in the rostral STR is increased to a greater extent than in the caudal STR ~3'23. In the present study, no rostrocaudal gradient for 5-HT was found in control mice. Despite an increase in 5-HT content which was found in the weaver mice, still no rostrocaudal gradient was observed. Previous work has shown a dorsoventral gradient for striatal 5-HT in rats in which the ventral aspect of the STR contains more 5-HT than the dorsal aspect 27. A similar pattern was seen in the present study in + / + mice. In control mice, the ventral S T R contains 67% more 5-HT than the dorsal STR. However, in weaver mice, the ventral STR contains 17% less 5-HT than the dorsal STR. Such a loss of the serotonergic dorsoventral gradient is the result of a greater increase in 5-HT content in the dorsal portion of the weaver STR compared to the ventral portion. A comparison of corresponding striatal regions between the two genotypes reveals increases in 5-HT content of 187% and 41% in the weaver dorsal and ventral STR respectively. The histochemical data show an increase in 5-HT immunoreactive fibers in the dorsal STR of weaver mice compared to the same region in the control mice. Immunocytochemistry provides the anatomical resolution to clearly see the increase in 5-HT in the dorsal portion of the weaver STR. While the ventral S T R of the weaver mouse has been shown to have a 41% increase in 5-HT content, no striking increase in immunolabeling of 5-HT fibers was seen. It is possible that immunocytochemistry alone may not reveal the 40% increase in the ventral STR of the weaver. The argument could be made that the increase in striatal 5-HT content in weaver mice might be an artifact associated with striatal atrophy. Thus, if striatal
271 volume and protein content decrease, the content of 5-HT on a p m o l / m g protein basis may be artificially elevated. We have not found substantial evidence for striatal shrinkage in as much as striatal protein content in the weaver decreases by only 10-15% (data not shown). Such a small reduction in protein is not sufficient to account for the 50% increase in 5-HT content. Because striatal shrinkage may be a contributing factor to the elevated 5-HT levels, we analyzed the data as 5-HT per STR to eliminate the possibility that the increase in 5-HT is merely an effect of shrinkage. Such a comparison demonstrated a 35% increase in 5:HT content in weaver mice. Thus, the increase in 5-HT, seen both on a p m o l / m g protein basis and on a p m o l / S T R basis, is unlikely to result solely from striatal shrinkage. Using rats which had received lesions as neonates, Luthman et al. 13 demonstrated that while 6-OHDA led to a decrease in D A content and an increase in 5-HT content in the STR, 6 - O H D A given with the D A uptake blocker amfonelic acid did not alter the levels of D A or 5-HT. These results suggest that 6-OHDA per se is not responsible for the increase in 5-HT content but rather that the loss of D A induced by this toxin is required for the changes in 5-HT levels to occur. As opposed to intracisternal or intracerebroventricular injections of 6-OHDA, injections of 6-OHDA given directly into the SN of adult rats lead to increases in striatal 5-HT levels 32. Since the SN and the STR may be innervated by the same serotonergic raphe neurons 2, Zhou et al. 32 discussed the idea that the increase in striatal 5-HT seen might be due to sprouting of striatal afferents triggered by damage to nigral 5-HT collaterals. However, rats treated with the 5-HT neurotoxin 5,7-dihydroxytryptamine in the SN did not exhibit increased striatal 5-HT levels 32. Thus, these results suggest that the increase in striatal 5-HT seen in rats given 6 - O H D A is not caused by sprouting of raphe afferents triggered by damage to the nigral 5-HT terminals. Serotonin content of the weaver SN is not different from that seen in the control SN (unpublished results), suggesting that in the weaver, there is no damage to nigral 5-HT collaterals. Thus, the weaver model supports the idea that 5-HT hyperinnervation of the STR is not triggered by damage to nigral 5-HT collaterals, but rather by the striatal D A depletion. It has been demonstrated that the dorsal aspect of the weaver STR is the main site of D A terminal lOSS7-10'15'17-19'30. Thus, the increase in 5-HT content in the weaver occurs in the same striatal region which exhibits the major D A terminal loss. This pattern of neurotransmitter changes suggests a causal relationship between the decrease in D A and the increase in 5-HT.
It can be hypothesized that the increase in striatal 5-HT is secondary to the loss of DA, as appears to be the case in rats with 6-OHDA lesions t3'32. Two possible explanations for the increase in 5-HT content are that (1) there are more 5-HT terminals innervating the weaver STR or (2) the number of 5-HT terminals is the same, but each terminal contains more 5-HT. Our immunocytochemical data support the contention that there are more 5-HT terminals innervating the weaver STR. The increased immunoreactivity in the weaver STR, especially the dorsal aspect, indicates that there are more fibers and terminals and thus suggests a hyperinnervation of the weaver STR by serotonergic afferents. The changes in 5-HT levels, possibly in response to the loss of DA, may play a compensatory role in the weaver STR. Electrophysiological studies have shown that the dopaminergic nigrostriatal pathway and the serotonergic raphe striatal pathway converge upon the same striatal neurons 5. Thus it is possible that the increase in 5-HT might functionally replace the lost DA. Studies involving 5-HT uptake and release, which offer more insight into serotonergic function, need to be carried out on the weaver STR in order to determine if the additional 5-HT can functionally replace the lost DA. Acknowledgements. We would like to thank Dr. J. Norton for his help with the statistical analyses of the data and Ms. Constance Alyea and Ms. Lillian Fitzpatrick for their excellent care of the animal colony. We would also like to thank H.W.M. Steinbusch for the gracious gift of serotonin antibodies. This work was supported in part by US PHS Grants R01 NS14426 and P01 NS27613.
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Brain Research, 606 (1993) 267-272 Elsevier Science Publishers B.V.
BRES 18632
Serotonin content is elevated in the dopamine deficient striatum of the weaver mutant mouse E.H. Stotz d9 L.C. T r i a r h o u b,d 9 B. G h e t t i a,b,d a n d J . R . S i m o n a,c,d Departments of a Psychiatry (Institute of Psychiatric Research), b Pathology (Neuropathology), c Biochemistry and Molecular Biology, and dprogram in Medical Neurobiology, Indiana University School of Medicine, Indianapolis, IN 46202-4887 (USA) (Accepted 20 October 1992)
Key words: Immunocytochemistry; Serotonin; Striatum; Weaver mutant mouse
In the present study, we measured the striatal serotonin content of weaver and control mice at different ages. Overall, weaver mutant mice exhibited 50% more striatal serotonin than controls. Neither a rostrocaudal gradient nor an age effect was found for either genotype. An analysis of serotonin content across the dorsoventral extent of the striatum revealed that in the dorsal striatum of the weaver, serotonin content was increased 200%, and in the ventral striatum, the increase amounted to 50% relative to control mice. Serotonin immunocytochemistry also revealed an increase in the dorsal striata of weaver mice. The major increase in striatal serotonin content seen in the weaver striatum occurs in the same region that exhibits the severest dopamine depletion. This observation is consistent with the notion that the increase in serotonin levels may be secondary to the decrease in dopamine content and may play an adaptive or compensatory role.
INTRODUCTION The weaver mouse has a recessive mutation which results in the loss of the dopaminergic cells in the substantia nigra (SN) 29. By 3 months of age, homozygous weaver mice (wv/wv) have lost approximately 70% of their tyrosine hydroxylase immunoreactive cells in the SN compared to control ( + / + ) mice 29. The nigral loss is accompanied by depletions in DA terminal markers in the striatum (STR). Dopamine content in the STR is decreased by 6 5 - - 7 0 % 16'17'20'21'26, and the activity of tyrosine hydroxylase (TH) is reduced by about 6 0 % 16'21'26. Striatal DA uptake is affected to a greater extent than either DA content or TH activity, exhibiting a decrease of approximately 9 0 % 7'16'20'26. The administration of 6-hydroxydopamine (6OHDA) into the cerebrospinal fluid of neonatal rats (intracisternally or intracerebroventricularly) results in the reduction of striatal DA content 3'4'13'23'28. Furthermore, the serotonergic system appears to hyperinnervate the S T R 1'3'4'13'22'23'28. Depending upon the age of the rat at the time of the lesion and the extent of the
lesion, striatal serotonin (5-HT) content is increased from 25%-100% 3'4'13'23'28 and 5-HT uptake is also elevated 13,23. Immunocytochemical studies have shown substantial increases in the density of 5-HT-like immunoreactive terminals in both the rostral and caudal STR t3. Horseradish peroxidase tracing methods have shown that an increased number of 5-HT cells are labeled in the dorsal raphe nucleus 1'22, suggesting the presence of an increased number of 5-HT cells projecting from the dorsal raphe to the STR. Using the weaver mutant mouse as a model of dopamine loss, we examined the hypothesis that a loss of striatal dopamine terminals will lead to an increase in striatal serotonergic innervation. Serotonin content of weaver and control mice was examined along the rostrocaudal extent of the STR as the rostral portion of the STR has been shown to be the principal site of the 5-HT hyperinnervation in rats with 6-OHDA lesions 13'23. The weaver mutation is known to cause a severer depletion of DA in the dorsal aspect of the STR than in the ventral aspect. Since the severity of the DA depletion has been shown to influence the
Correspondence: J.R. Simon, Institute of Psychiatric Research, Indiana University School of Medicine, 791 Union Drive, Indianapolis, IN 46202-4887, USA. Fax: (1) (317) 274-1365.
268 extent of the 5-HT hyperinnervation l, the dorsal and the ventral portions of the weaver STR were investigated separately. MATERIALS
AND METHODS
Homozygous weaver (we~we) and wild-type ( + / + ) control mice were obtained from a colony maintained at Indiana University Medical Center. The colony originated from heterozygous mice (we~+) purchased from Jackson Laboratory. The weaver mutation is maintained on a B6CBA-AW-J/A hybrid background. For the determination of 5-HT content as a function of age, mice were used at the ages of 60_+5 days (2 months), 2 4 5 + 9 days (8 months) or 430 + 17 days (14 months). Forty-two mice were used for the study of 5-HT content as a function of location (rostrocaudal or dorsoventral). For the immunocytochemistry study, four mice were used. For the determination of 5-HT content, age-matched pairs consisting of a weaver and a control mouse were sacrificed by decapitation. The brains were rapidly removed, m o u n t e d on a chuck and placed in a cryostat/microtome set at - 1 2 ° C . The frozen brains were cut in an alternating pattern of 40 /zm and 480 p,m thick sections which were m o u n t e d on glass slides. Collection of sections began with the appearance of the striatum and ceased with the appearance of the decussation of the superior cerebellar peduncles. The 480 / l m thick sections were numbered such that the coronal section containing the midline crossing of the anterior commissure was assigned '0'. More rostral sections were numbered with negative integers such that the more rostral a section, the larger the absolute value of the negative integer. More caudal sections were numbered with positive integers such that the more caudal a section, the larger the positive integer. The 480 p,m thick sections were stored overnight at - 7 0 ° C while the 40 /~m thick sections were stained with Cresyl violet and were used for orientation. The following day, the slides with the 480 p.m thick sections were placed on a Petri dish with ice and viewed under a dissecting microscope. For the analysis of 5-HT content along the rostrocaudal extent of the STR, bilateral striata from each section were dissected and pooled, whereas for the dorsoventral study, bilateral striata were dissected into dorsal and ventral aspects which were then pooled from all the rostrocaudal sections. In all experiments, care was taken to exclude the globus pallidus and the nucleus accumbens. Dissected samples were placed in microfuge tubes with 150 ~1 of 0.5 M perchloric acid containing dihydroxybenzylamine as an internal standard. The tissue was sonicated until it was homogeneous and then placed on ice for 30 min. Following centrifugation for 5 min in a Beckman Model B microfuge, 100/zl of the supernatant was injected on an H P L C for m o n o a m i n e determination and the pellet was resuspended in 150 p.1 of 0.1 N N a O H for protein determination 12. For monoamine determination, the area under the curve from the H P L C chromatograph was converted to picomoles by using a standard curve and correcting for recovery with the internal standard. The H P L C - E C was conducted at a flow rate of 1.0 m l / m i n and a potential of 0.8 V. The C-18 column had a sphere size of 5 izm with the dimensions 4.6 m m (inner diameter) ×25 cm. The limit of sensitivity of this system was approximately 0.5 pmol for 5-HT. T h e mobile phase contained 60 m M citric acid, 30 m M sodium phosphate, 0.39 m M octylsodium sulfate, 0.11 m M E D T A and 11.6% methanol (v/v). The statistical analyses were performed using a repeated-measures analysis of variance with one grouping factor (age) and two repeated-measures factors (genotype and location) 31. Each 'subject' in an age group was a pair of mice, one + / + and one wc/wu, whose tissues were assayed at the same time. There were six such pairs in each age group and there were three age groups (36 mice). For each mouse, five locations were analyzed, thus resulting in 180 data points. The data were transformed to log scale to remove correlation between m e a n s and variances. Ten missing values were estimated by maximum likelihood using program B M D P 5 V 6. The analysis was then completed using the ' A N O V A ' c o m m a n d in the M I N I T A B package TM. The degrees of freedom for the highest order
error term were reduced by ten (from 60 to 50) to compensate for the estimation of the missing values. For the serotonin immunocytochemistry study, the peroxidase antiperoxidase method of Sternberger et al. 25 was used as modified by Steinbusch and Tilders 24. Briefly, age-matched pairs of weaver and control mice were pretreated with 100 m g / k g pargyline i.p. 2 h before perfusion. Mice were given 600 units of heparin i.p. before being anesthetized with 50 m g / k g pentobarbital sodium i.p. Mice were perfused transcardially with 60 ml of ice-cold, oxygen-enriched, calcium-free Tyrode's buffer followed by 120 ml of ice-cold perfusion medium containing 4% (w/v) formaldehyde, 0.05% (v/v) glutaraldehyde and 0.2% (w/v) picric acid in 0.1 M sodium phosphate buffer (pH 7.4). Brains were removed and were left in perfusion medium without glutaraldehyde overnight. Fifty-~m-thick coronal sections were cut with a vibratome and were collected free-floating in Trisbuffered saline (pH 7.6)(TBS). Sections were rinsed consecutively for 5 min with TBS, TBS containing 0.5% Triton X-100 (TBS-T), and TBS. Sections were then incubated at 4°C for 40 h with rabbit anti-5-HT antiserum diluted 1:1800. After rinsing the sections for 5 min in TBS-T, TBS and TBS-T, goat anti-rabbit IgG at a dilution of 1:40 was applied to the sections for 2 h at room temperature. Sections were rinsed with TBS-T, TBS and TBS-T. Rabbit peroxidase anti-peroxidase complex at a dilution of 1 : 250 was applied to the sections for a 1 h incubation at room temperature. Following three TBS rinses, the sections were reacted with 0.01% 3,3'-diaminobenzidine-HCl, 0.67% nickel ammonium sulfate, 0.05 M imidazole and 0.01% H 2 0 2. After final TBS rinses, the sections were mounted on gelatinized slides and coverslipped. RESULTS
Serotonin content Examination of the rostrocaudal distribution of 5-HT in 14-month-old mice revealed an increase in 5-HT content in the wv/wv at every striatal level examined (Fig. 1). No rostrocaudal gradient for 5-HT was found
c
40
-~ ~?_
3o
~ZIZ "-~
20
C O
~
O E
-2 rostra]
-I
0
section number
i
2 caudal
Fig. 1. The rostrocaudal distribution of 5-HT content in striatum of 14-month-old + / + and wc/wc mice. T h e data are presented in p m o l / m g protein and represent the m e a n s _+S.E.M. for n = 6 for each genotype. Sections are numbered such that section '0' contains the midline crossing of the anterior commissure. Rostral sections are numbered with negative integers such that the more rostral a section, the larger the absolute value of the negative integer. Caudal sections are numbered with positive integers such that the more caudal a section, the larger the positive integer. A repeated measures A N O V A was performed on a log transformation of the data over the three ages (2, 8 and 14 months), the five locations and the two genotypes. T h e analysis showed no effect of a g e (F2,15 = 0.36, P > 0.05) nor of location (F4,60 = 0.81, P > 0.05). There was an effect of genotype ( F l j s = 68.08, P < 0.05), but none of the possible interactions were significant at the P < 0.05 lwel.
269 in either genotype; n o r was such a g r a d i e n t f o u n d in 8or 2 - m o n t h - o l d mice (data n o t shown). Analysis a m o n g the ages (2, 8 a n d 14 m o n t h s ) i n d i c a t e d that t h e r e was n o significant effect of age o n 5 - H T c o n t e n t within
25
each genotype. Thus, with n o significant effect of age or r o s t r o c a u d a l location, 5 - H T c o n t e n t data could be pooled for each genotype. Pooling the d a t a in this m a n n e r r e s u l t e d in a striatal 5 - H T c o n t e n t of 17.3 + 0.8 p m o l / m g p r o t e i n in + / +
mice a n d a striatal 5 - H T
c o n t e n t of 26.6 _+ 1.0 p m o l / m g p r o t e i n in w v / w v mice (Fig. 2). T h e 50% increase in striatal 5 - H T c o n t e n t in the weaver was significant with P < 0.001 ( n = 18 for each genotype). F i n d i n g n o effect of age o n striatal 5 - H T c o n t e n t , a single age, 3 m o n t h s , was used to study the dorsoventral g r a d i e n t ( T a b l e I). I n + / + mice, the v e n t r a l S T R c o n t a i n e d a p p r o x i m a t e l y 65% m o r e 5 - H T t h a n the dorsal STR. I n w v / w v mice, the r a n k o r d e r of striatal
o
,=~ o E
T
10 5
+/+ wv/wv Fig. 2. Striatal 5-HT content in + / + and wv/wv mice. The data are presented in pmol/mg protein and represent the means + S.E.M. for n = 18 for each genotype. 5-HT content was measured along the rostrocaudal extent of the STR in 2-, 8- and 14-month-old mice. A repeated measures ANOVA revealed only a significant genotype effect (F1,15= 68.08, P < 0.05). Therefore the data were pooled for age and location within each genotype.
Fig. 3. Serotonin immunocytochemistryin the dorsal and ventral striatum of + / + and wv / wv mice. (a) The dorsal striatum of the + / + mouse displays few 5-HT immunoreactive fibers while (b) the dorsal striatum of the wv/wv mouse contains many more 5-HT immunoreactive fibers. No clear difference in the density of fiber staining can be seen between (c) the ventral striatum of the + / + mouse and (d) the ventral striatum of the weaver mouse. Mice were 21 months of age at the time of sacrifice. Bar = 50 p.m.
270 TABLE I
Dorsoventral gradient of 5-HT content in + / + and wv / wv mice The data are presented as p m o l / m g protein and represent the means_+S.E.M, for n = 3 for each genotype. 5-HT content was determined in the dorsal and ventral STR by H P L C - E C in 3-monthold mice. An A N O V A was performed on the data revealing an effect of genotype (F1, 4 = 91.391, P < 0.05) but not an effect of region (F1, 4 = 0.326, P > 0.05). The interaction of genotype and region was also significant (F1. 4 = 15.414, P < 0.05). + / + dorsal STR vs + / + ventral STR, t ( 7 ) = - 3 . 3 5 , P < 0.05; w v / w v dorsal STR vs w v / w v ventral STR, t(7)=2.50, P < 0 . 0 5 ; + / + dorsal STR vs w v / w v dorsal STR, t(7)=9.27, P < 0 . 0 5 ; + / + ventral STR vs w v / w v ventral STR, t(7) = 3.44, P < 0.05.
Striatal location Dorsal Ventral
5-HT Content
+/+
wv / wv
15.6 + 1.8 26.1 + 4.3
44.7 + 1.5 36.9 + 2.4
5-HT content was reversed, with the dorsal STR containing 20% more 5-HT than the ventral STR. Comparisons between the two genotypes showed that both the dorsal and the ventral STR of the w v / w v had a greater 5-HT content compared to control. The 5-HT content of the dorsal STR of the weaver was increased 3-fold over the corresponding region in the control, while 5-HT in the ventral STR of the weaver was increased 50% over the corresponding region in the control. Serotonin immunocytochemistry
The 5-HT immunoreactive fibers seen in this study were very fine with small fusiform or granular varicosities, suggesting that they are 'type D' 5-HT axons which originate in the dorsal raphe nuclei 11. Serotonin immunocytochemistry of control S T R revealed a dorsoventral gradient of 5-HT immunoreactive fibers (Fig. 3). Despite the sparse distribution of the stained fibers in the dorsal STR, once located, single fibers were easily traced. In the ventral STR of control mice, 5-HT immunoreactive fibers were more numerous and tracing of individual fibers was more difficult due to extensive overlapping in the 50 /zm thick sections. In the STR of weaver mice, the dorsoventral gradient of 5-HT immunoreactive fibers seen in controls was not found. The distribution of 5-HT immunoreactive fibers appeared to favor the dorsal STR. In the dorsal STR, tracing of individual fibers was more difficult than in the ventral S T R due to crowding, suggesting that the dorsal STR contained more fibers. While comparisons between control ventral STR and weaver ventral STR revealed no clear differences between genotypes, the dorsal STR of weaver mice clearly had a higher density of 5-HT immunoreactive fibers than the same region in
control mice. Increases in both the number of fibers and darkly stained varicosities could be seen in the weaver dorsal STR as compared to control mice. DISCUSSION The results of the present study indicate that the weaver STR contains more 5-HT than the control STR. This is seen both as an expression of 5-HT content and of 5-HT immunoreactivity. The increase in 5-HT occurs predominantly in the dorsal aspect of the STR. Neither a rostrocaudal distribution nor an age effect was found for 5-HT content within either genotype. In rats, striatal 5-HT content has been shown to follow a rostrocaudal gradient in which the caudal S T R contains 3 - 6 times more 5-HT than the rostral STR 23'27. In rats which have received 6 - O H D A lesions as neonates this gradient is diminished as 5-HT content in the rostral STR is increased to a greater extent than in the caudal STR ~3'23. In the present study, no rostrocaudal gradient for 5-HT was found in control mice. Despite an increase in 5-HT content which was found in the weaver mice, still no rostrocaudal gradient was observed. Previous work has shown a dorsoventral gradient for striatal 5-HT in rats in which the ventral aspect of the STR contains more 5-HT than the dorsal aspect 27. A similar pattern was seen in the present study in + / + mice. In control mice, the ventral S T R contains 67% more 5-HT than the dorsal STR. However, in weaver mice, the ventral STR contains 17% less 5-HT than the dorsal STR. Such a loss of the serotonergic dorsoventral gradient is the result of a greater increase in 5-HT content in the dorsal portion of the weaver STR compared to the ventral portion. A comparison of corresponding striatal regions between the two genotypes reveals increases in 5-HT content of 187% and 41% in the weaver dorsal and ventral STR respectively. The histochemical data show an increase in 5-HT immunoreactive fibers in the dorsal STR of weaver mice compared to the same region in the control mice. Immunocytochemistry provides the anatomical resolution to clearly see the increase in 5-HT in the dorsal portion of the weaver STR. While the ventral S T R of the weaver mouse has been shown to have a 41% increase in 5-HT content, no striking increase in immunolabeling of 5-HT fibers was seen. It is possible that immunocytochemistry alone may not reveal the 40% increase in the ventral STR of the weaver. The argument could be made that the increase in striatal 5-HT content in weaver mice might be an artifact associated with striatal atrophy. Thus, if striatal
271 volume and protein content decrease, the content of 5-HT on a p m o l / m g protein basis may be artificially elevated. We have not found substantial evidence for striatal shrinkage in as much as striatal protein content in the weaver decreases by only 10-15% (data not shown). Such a small reduction in protein is not sufficient to account for the 50% increase in 5-HT content. Because striatal shrinkage may be a contributing factor to the elevated 5-HT levels, we analyzed the data as 5-HT per STR to eliminate the possibility that the increase in 5-HT is merely an effect of shrinkage. Such a comparison demonstrated a 35% increase in 5:HT content in weaver mice. Thus, the increase in 5-HT, seen both on a p m o l / m g protein basis and on a p m o l / S T R basis, is unlikely to result solely from striatal shrinkage. Using rats which had received lesions as neonates, Luthman et al. 13 demonstrated that while 6-OHDA led to a decrease in D A content and an increase in 5-HT content in the STR, 6 - O H D A given with the D A uptake blocker amfonelic acid did not alter the levels of D A or 5-HT. These results suggest that 6-OHDA per se is not responsible for the increase in 5-HT content but rather that the loss of D A induced by this toxin is required for the changes in 5-HT levels to occur. As opposed to intracisternal or intracerebroventricular injections of 6-OHDA, injections of 6-OHDA given directly into the SN of adult rats lead to increases in striatal 5-HT levels 32. Since the SN and the STR may be innervated by the same serotonergic raphe neurons 2, Zhou et al. 32 discussed the idea that the increase in striatal 5-HT seen might be due to sprouting of striatal afferents triggered by damage to nigral 5-HT collaterals. However, rats treated with the 5-HT neurotoxin 5,7-dihydroxytryptamine in the SN did not exhibit increased striatal 5-HT levels 32. Thus, these results suggest that the increase in striatal 5-HT seen in rats given 6 - O H D A is not caused by sprouting of raphe afferents triggered by damage to the nigral 5-HT terminals. Serotonin content of the weaver SN is not different from that seen in the control SN (unpublished results), suggesting that in the weaver, there is no damage to nigral 5-HT collaterals. Thus, the weaver model supports the idea that 5-HT hyperinnervation of the STR is not triggered by damage to nigral 5-HT collaterals, but rather by the striatal D A depletion. It has been demonstrated that the dorsal aspect of the weaver STR is the main site of D A terminal lOSS7-10'15'17-19'30. Thus, the increase in 5-HT content in the weaver occurs in the same striatal region which exhibits the major D A terminal loss. This pattern of neurotransmitter changes suggests a causal relationship between the decrease in D A and the increase in 5-HT.
It can be hypothesized that the increase in striatal 5-HT is secondary to the loss of DA, as appears to be the case in rats with 6-OHDA lesions t3'32. Two possible explanations for the increase in 5-HT content are that (1) there are more 5-HT terminals innervating the weaver STR or (2) the number of 5-HT terminals is the same, but each terminal contains more 5-HT. Our immunocytochemical data support the contention that there are more 5-HT terminals innervating the weaver STR. The increased immunoreactivity in the weaver STR, especially the dorsal aspect, indicates that there are more fibers and terminals and thus suggests a hyperinnervation of the weaver STR by serotonergic afferents. The changes in 5-HT levels, possibly in response to the loss of DA, may play a compensatory role in the weaver STR. Electrophysiological studies have shown that the dopaminergic nigrostriatal pathway and the serotonergic raphe striatal pathway converge upon the same striatal neurons 5. Thus it is possible that the increase in 5-HT might functionally replace the lost DA. Studies involving 5-HT uptake and release, which offer more insight into serotonergic function, need to be carried out on the weaver STR in order to determine if the additional 5-HT can functionally replace the lost DA. Acknowledgements. We would like to thank Dr. J. Norton for his help with the statistical analyses of the data and Ms. Constance Alyea and Ms. Lillian Fitzpatrick for their excellent care of the animal colony. We would also like to thank H.W.M. Steinbusch for the gracious gift of serotonin antibodies. This work was supported in part by US PHS Grants R01 NS14426 and P01 NS27613.
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