33
Brain Research, 546 (1991) 33-39 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391164848 BRES 16484
Serotonin regulation of tachykinin biosynthesis in the rat neostriatum Paul D. Walker, Leigh A. Riley, Ronald P. Hart and G. Miller Jonakait Department of Biological Sciences, Rutgers University, Newark, NJ 07102 (U.S.A.) (Accepted 30 October 1990) Key words: Preprotachykinin; Substance P; Serotonin; Tachykinin; Neostriatum; mRNA
Serotonin (5-HT) neurotransmission was altered to determine its role in regulating the biosynthesis of tachykinins in the neostriatum (NS). Depletion of 5-HT with subchronic p-chiorophenylalanine (pCPA) treatment decreased preprotachykinin (PPT, the prohormone precursor to SP) mRNA levels in the NS. By contrast, raising extracellular 5-HT levels with zimelidine (a 5-HT uptake inhibitor) or ciorgyline (a monoamine oxidase inhibitor) resulted in increased levels of PPT mRNA. To determine whether 5-HT receptors played a role in mediating the changes in PPT mRNA, animals were treated with the 5-HT2 agonist DOI. This drug significantly increased both PPT mRNA and SP-like immunoreactivity in the NS. These results together indicate that neostriatal tachykinin biosynthesis is sensitive to alterations in 5-HT neurotransmission. INTRODUCTION D u e to the many neurological disorders affecting the biochemistry and function of the neostriatum (NS), and particularly because of the dire consequences resulting from the loss of nigrostriatal dopaminergic innervation, much attention has been focused upon the role of afferent pathways in the regulation of neostriatal neurotransmitter biosynthesis. A well-documented example of such trans-synaptic regulation is the influence that afferent nigrostriatal d o p a m i n e ( D A ) exerts on the biosynthetic rate of tachykinins, n e u r o p e p t i d e s utilized by neostriatal projection neurons 14"37'47'62'63, and shown to be d e p l e t e d in Parkinsonian brains 5°'66. Treatment with D A antagonists 5'6 or 6 - h y d r o x y d o p a m i n e 58'59'73 significantly decreases steady-state levels of the m R N A coding for p r e p r o t a c h y k i n i n (PPT), the p r o h o r m o n e precursor for substance P (SP) and neurokinin A ( N K A ) . In contrast, administration of direct or indirect D A agonists p r o d u c e s the opposite effect 6'33. These results suggest a m o d u l a t o r y role for the nigrostriatal pathway in neostriatal tachykinin biosynthesis. H o w e v e r , in addition to d o p a m i n e innervation, the NS receives afferent input from serotonin (5-HT) cell groups located in midbrain raphe regions 4'19'38'61'64'68"69. Little information exists on the role of this m o n o a m i n e neurotransmitter in neostriatal tachykinin biosynthesis. Therefore, the present studies were designed to determine w h e t h e r alterations in 5-HT neurotransmission would
affect tachykinin biosynthesis in the NS. By measuring steady-state levels of PPT m R N A following 5-HT manipulation, we have found that agents that p r o m o t e 5-HT neurotransmission tend to increase striatal PPT synthesis, while agents that d e p l e t e 5-HT lower synthesis. MATERIALS AND METHODS Animals and drug treatments. Adult Sprague-Dawley rats (8-10 weeks old) were treated subchronically with the following drugs via i.p. injections or subcutaneous Alzet (Alza Corp., Palo Alto, CA) minipump infusion: p-chlorophenylalanine (pCPA; Sigma, 100 mg/kg/day for 7 or 14 days); zimelidine (Astra Lakemedal AB; 10 mg/kg twice daily for 5 or 14 days); clorgyline (Sigma, 1 mg/kg/day for 5, 14, or 21 days); DOI (RBI; 7.5 mg/kg/day for 9 days). Following drug treatment, animals were sacrificed and brain tissue was dissected as previously described71. The tissue was frozen in liquid N 2 and stored at -80 °C until assay. Northern blot analysis. Initial experiments with pCPA utilized total RNA obtained from pooled NS collected from groups of rats. However, in all other experiments total RNA was obtained from individual striata and was subjected to the acid guanidinium thiocyanate-phenol-chloroform extraction procedure of Chomczynski and Sacchi 18. This method enabled individual RNA samples from each experimental and control animal to be analyzed separately for statistical comparisons. All remaining methods for PPT mRNA analysis were as previously described7°'71. Northern blots were hybridized with a [32p]-labeled 660 nt RNA probe complementary to PPT mRNA. This probe was synthesized from a cDNA template corresponding to a full length, 1100 nt gamma PPT mRNA which codes for substance P and substance K (subclone pGEM2-31-1, kindly provided by Dr. James Krause, Washington University School of Medicine, St. Louis, MO) 45. The PPT mRNA probe was then removed and the blot reprobed with a [32p]-labeled DNA probe complementary for mouse 28S ribosomal RNA to correct potential differences in RNA loaded. In experiments using RNA
Correspondence: P.D. Walker. Present address: Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 East Canfield Ave., Detroit, MI 48201, U.S.A.
34 from individual animals, the PPT probe was removed from filters that had a >20% standard error and the blots reprobed with neuron-specific enolase control probe as described previously71. Alternatively, the Northern blot was repeated, RNA was stained, and 28S ribosomal RNA was quantified by densitometry prior to hybridization. Sizes of mRNAs detected on Northern blots were determined by comparison with synthetic RNA markers (BRL) run in an adjacent lane. Quantitation of mRNA was performed by densitometric scanning with an LKB Ultrascan XL Laser Dcnsitometer and LKB 2400 Gelscan XL software. Radioimmunoassay. SP-like immunoreactivity was determined by radioimmunoassays6 as modified by Kessler and Black 42 using an antibody obtained from the laboratory of Dr. Ira Black, Cornell University Medical College. Data from the RIA is expressed as the mean + standard error. Tryptophan hydroxylase (TPH) assay. TPH enzymatic activity was determined as previously described41 using the radioenzymatic microassay of Kizer et al. 43 modified by substituting the homogenization buffer of Cash et a1.15. Standard curves run with each assay revealed linearity up to 20 ng 5-hydroxytryptophan. Sensitivity of the assay is between 1-2 ng 5-hydroxytryptophan formed/h (twice blank). MAO assay. Monoamine oxidase activity was determined in hippocampal homogenates by modification57 of Wurtman and Axelrod72. The substrate was [14C]serotonin (50 Ci/mmol; New England Nuclear, Boston MA; 100/~M final concentration) in 0.1
M potassium phosphate buffer, pH 7.3. Assay time was 10 min and samples were diluted appropriately to assure linearity of the assays with time. Statistics. Data were compared by Student's t-test
RESULTS
Decreased 5-HT decreases striatal P P T m R N A
In order to determine effects of 5-HT depletion on tachykinin biosynthesis, Alzet minipumps designed to dispense pCPA (an inhibitor of tryptophan hydroxylase39"44; TPH) for two weeks were implanted in adult rats. Effectiveness of the drug was monitored by assay of TPH in the midbrain raphe of each animal. In pCPAtreated animals, TPH activity in the raphe was inhibited 70-80% at 1, 2, and 3 weeks following pump implantation. To determine whether specific, steady-state PPT mRNA levels in the NS were affected by pCPA treatment, Northern mRNA analysis was performed for PPT
pCPA l wk
2wk
3wk
A
200
0
100
100
80
i°°
6.-5%
~///////;,
4.a
/ / / / / / / / / / / / / / / / / /
¢1 40
G
/ / ~ / t 1 1 / I / I J / / / / / /
~ 2o
ff~fff~ff
/ / / / / / / / /
(///////~
I week
2 weeks
3 weeks
Fig. 1. A: Northern blot analysis of PPT mRNA taken from animals treated with and without pCPA for varying periods of time. For each lane 3 striata were pooled, and 1/xg of total RNA was loaded. Following probing with [32p]-labeled PPT cRNA, the blot was stripped and reprobed for 28S ribosomal RNA (see Materials and Methods). C, control; E, experimental. B: the autoradiograms in (A) were densitometrically scanned, PPT was expressed as a ratio to 28S RNA, and shown as percent of control.
0
5 days
14 days
Fig. 2. A: representative Northern blot analysis of PPT mRNA taken from individual striata from animals treated with and without zimelidine for 5 days. For each lane total RNA was prepared from one striatum, and 3/tg were loaded. To assess the linearity of the exposure, varying amounts (3, 4.5 and 6/~g) of NS RNA were loaded on the same gel. L: 3/zg rat liver RNA. B: Northern blots were prepared from individual striata from animals treated for 5 or 14 days with and without zimelidine (4-5 per group). These autoradiograms were densitometrically scanned, and PPT mRNA levels were expressed as percent of control. Two experiments are shown for each time point. The hatched bar at 5 days is the scan of the autoradiogram in (AL
35
and 28S ribosomal R N A (see Materials and Methods; Fig. 1A). As described previously 7°'71, a single 1.2 kb band was detected in lanes of total RNA probed for PPT mRNA. A 4.5 kb band was detected for 28S. Levels of PPT m R N A were expressed as a ratio of PPT/28S m R N A to obtain a measure of specific PPT m R N A (Fig. 1B). One week following p C P A pump implantation, specific PPT m R N A was depleted 82% in the NS. PPT m R N A levels were still depressed 64% two weeks following pump implantation. After 3 weeks (one week after depletion of p C P A from the pump), PPT m R N A had recovered substantially (Fig. 1B). Because of the potential vagaries of Northern analysis, the experiment was performed 4 times with consistent results.
saline 123
300
DOI
456
789101112
B p < 0.0005
200
I
o
o o
p < 0.0005 100
0
Serotonin uptake inhibition increases striatal P P T m R N A In order to determine whether increases in 5-HT neurotransmission would produce the opposite effect, adult rats received the uptake inhibitor zimelidine for two weeks. Northern blot analysis was performed on individual (not pooled) striata after 5 or 14 days of treatment (Fig. 2A). In two separate experiments, PPT m R N A was
saline 1
2
3
clorgyline 4
5
6
7
8
~tg R N A
9 10
PP'I" ~ R N A
SP peptlde
Fig. 4. A: representative Northern blot analysis of PPT m R N A taken from individual striata from animals treated with and without D O I for 9 days. For each lane total R N A was prepared from a single striatum and 2/~g were loaded. Lanes shown were from the same blot and exposure. B: in two separate experiments Northern blots were prepared from single striata taken from animals treated with and without D O I for 9 days. A u t o r a d i o g r a m s were scanned densitometrically and PPT m R N A was expressed as a ratio to NSE m R N A , and is shown as percent of control. SP-like immunoreactivity was assessed in contralateral striata.
L 3 4.5 6 significantly increased in the NS after 5 days of zimelidine treatment, but was at control levels after a 14-day treatment (Fig. 2B). A slight, but significant increase in SP-like immunoreactivity was detected in NS after 5 days (123 _+ 2%; P < 0.025).
1~0.0"2.~ ,-,
o I,,.i
200
p
Brain Research, 546 (1991) 33-39 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391164848 BRES 16484
Serotonin regulation of tachykinin biosynthesis in the rat neostriatum Paul D. Walker, Leigh A. Riley, Ronald P. Hart and G. Miller Jonakait Department of Biological Sciences, Rutgers University, Newark, NJ 07102 (U.S.A.) (Accepted 30 October 1990) Key words: Preprotachykinin; Substance P; Serotonin; Tachykinin; Neostriatum; mRNA
Serotonin (5-HT) neurotransmission was altered to determine its role in regulating the biosynthesis of tachykinins in the neostriatum (NS). Depletion of 5-HT with subchronic p-chiorophenylalanine (pCPA) treatment decreased preprotachykinin (PPT, the prohormone precursor to SP) mRNA levels in the NS. By contrast, raising extracellular 5-HT levels with zimelidine (a 5-HT uptake inhibitor) or ciorgyline (a monoamine oxidase inhibitor) resulted in increased levels of PPT mRNA. To determine whether 5-HT receptors played a role in mediating the changes in PPT mRNA, animals were treated with the 5-HT2 agonist DOI. This drug significantly increased both PPT mRNA and SP-like immunoreactivity in the NS. These results together indicate that neostriatal tachykinin biosynthesis is sensitive to alterations in 5-HT neurotransmission. INTRODUCTION D u e to the many neurological disorders affecting the biochemistry and function of the neostriatum (NS), and particularly because of the dire consequences resulting from the loss of nigrostriatal dopaminergic innervation, much attention has been focused upon the role of afferent pathways in the regulation of neostriatal neurotransmitter biosynthesis. A well-documented example of such trans-synaptic regulation is the influence that afferent nigrostriatal d o p a m i n e ( D A ) exerts on the biosynthetic rate of tachykinins, n e u r o p e p t i d e s utilized by neostriatal projection neurons 14"37'47'62'63, and shown to be d e p l e t e d in Parkinsonian brains 5°'66. Treatment with D A antagonists 5'6 or 6 - h y d r o x y d o p a m i n e 58'59'73 significantly decreases steady-state levels of the m R N A coding for p r e p r o t a c h y k i n i n (PPT), the p r o h o r m o n e precursor for substance P (SP) and neurokinin A ( N K A ) . In contrast, administration of direct or indirect D A agonists p r o d u c e s the opposite effect 6'33. These results suggest a m o d u l a t o r y role for the nigrostriatal pathway in neostriatal tachykinin biosynthesis. H o w e v e r , in addition to d o p a m i n e innervation, the NS receives afferent input from serotonin (5-HT) cell groups located in midbrain raphe regions 4'19'38'61'64'68"69. Little information exists on the role of this m o n o a m i n e neurotransmitter in neostriatal tachykinin biosynthesis. Therefore, the present studies were designed to determine w h e t h e r alterations in 5-HT neurotransmission would
affect tachykinin biosynthesis in the NS. By measuring steady-state levels of PPT m R N A following 5-HT manipulation, we have found that agents that p r o m o t e 5-HT neurotransmission tend to increase striatal PPT synthesis, while agents that d e p l e t e 5-HT lower synthesis. MATERIALS AND METHODS Animals and drug treatments. Adult Sprague-Dawley rats (8-10 weeks old) were treated subchronically with the following drugs via i.p. injections or subcutaneous Alzet (Alza Corp., Palo Alto, CA) minipump infusion: p-chlorophenylalanine (pCPA; Sigma, 100 mg/kg/day for 7 or 14 days); zimelidine (Astra Lakemedal AB; 10 mg/kg twice daily for 5 or 14 days); clorgyline (Sigma, 1 mg/kg/day for 5, 14, or 21 days); DOI (RBI; 7.5 mg/kg/day for 9 days). Following drug treatment, animals were sacrificed and brain tissue was dissected as previously described71. The tissue was frozen in liquid N 2 and stored at -80 °C until assay. Northern blot analysis. Initial experiments with pCPA utilized total RNA obtained from pooled NS collected from groups of rats. However, in all other experiments total RNA was obtained from individual striata and was subjected to the acid guanidinium thiocyanate-phenol-chloroform extraction procedure of Chomczynski and Sacchi 18. This method enabled individual RNA samples from each experimental and control animal to be analyzed separately for statistical comparisons. All remaining methods for PPT mRNA analysis were as previously described7°'71. Northern blots were hybridized with a [32p]-labeled 660 nt RNA probe complementary to PPT mRNA. This probe was synthesized from a cDNA template corresponding to a full length, 1100 nt gamma PPT mRNA which codes for substance P and substance K (subclone pGEM2-31-1, kindly provided by Dr. James Krause, Washington University School of Medicine, St. Louis, MO) 45. The PPT mRNA probe was then removed and the blot reprobed with a [32p]-labeled DNA probe complementary for mouse 28S ribosomal RNA to correct potential differences in RNA loaded. In experiments using RNA
Correspondence: P.D. Walker. Present address: Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 East Canfield Ave., Detroit, MI 48201, U.S.A.
34 from individual animals, the PPT probe was removed from filters that had a >20% standard error and the blots reprobed with neuron-specific enolase control probe as described previously71. Alternatively, the Northern blot was repeated, RNA was stained, and 28S ribosomal RNA was quantified by densitometry prior to hybridization. Sizes of mRNAs detected on Northern blots were determined by comparison with synthetic RNA markers (BRL) run in an adjacent lane. Quantitation of mRNA was performed by densitometric scanning with an LKB Ultrascan XL Laser Dcnsitometer and LKB 2400 Gelscan XL software. Radioimmunoassay. SP-like immunoreactivity was determined by radioimmunoassays6 as modified by Kessler and Black 42 using an antibody obtained from the laboratory of Dr. Ira Black, Cornell University Medical College. Data from the RIA is expressed as the mean + standard error. Tryptophan hydroxylase (TPH) assay. TPH enzymatic activity was determined as previously described41 using the radioenzymatic microassay of Kizer et al. 43 modified by substituting the homogenization buffer of Cash et a1.15. Standard curves run with each assay revealed linearity up to 20 ng 5-hydroxytryptophan. Sensitivity of the assay is between 1-2 ng 5-hydroxytryptophan formed/h (twice blank). MAO assay. Monoamine oxidase activity was determined in hippocampal homogenates by modification57 of Wurtman and Axelrod72. The substrate was [14C]serotonin (50 Ci/mmol; New England Nuclear, Boston MA; 100/~M final concentration) in 0.1
M potassium phosphate buffer, pH 7.3. Assay time was 10 min and samples were diluted appropriately to assure linearity of the assays with time. Statistics. Data were compared by Student's t-test
RESULTS
Decreased 5-HT decreases striatal P P T m R N A
In order to determine effects of 5-HT depletion on tachykinin biosynthesis, Alzet minipumps designed to dispense pCPA (an inhibitor of tryptophan hydroxylase39"44; TPH) for two weeks were implanted in adult rats. Effectiveness of the drug was monitored by assay of TPH in the midbrain raphe of each animal. In pCPAtreated animals, TPH activity in the raphe was inhibited 70-80% at 1, 2, and 3 weeks following pump implantation. To determine whether specific, steady-state PPT mRNA levels in the NS were affected by pCPA treatment, Northern mRNA analysis was performed for PPT
pCPA l wk
2wk
3wk
A
200
0
100
100
80
i°°
6.-5%
~///////;,
4.a
/ / / / / / / / / / / / / / / / / /
¢1 40
G
/ / ~ / t 1 1 / I / I J / / / / / /
~ 2o
ff~fff~ff
/ / / / / / / / /
(///////~
I week
2 weeks
3 weeks
Fig. 1. A: Northern blot analysis of PPT mRNA taken from animals treated with and without pCPA for varying periods of time. For each lane 3 striata were pooled, and 1/xg of total RNA was loaded. Following probing with [32p]-labeled PPT cRNA, the blot was stripped and reprobed for 28S ribosomal RNA (see Materials and Methods). C, control; E, experimental. B: the autoradiograms in (A) were densitometrically scanned, PPT was expressed as a ratio to 28S RNA, and shown as percent of control.
0
5 days
14 days
Fig. 2. A: representative Northern blot analysis of PPT mRNA taken from individual striata from animals treated with and without zimelidine for 5 days. For each lane total RNA was prepared from one striatum, and 3/tg were loaded. To assess the linearity of the exposure, varying amounts (3, 4.5 and 6/~g) of NS RNA were loaded on the same gel. L: 3/zg rat liver RNA. B: Northern blots were prepared from individual striata from animals treated for 5 or 14 days with and without zimelidine (4-5 per group). These autoradiograms were densitometrically scanned, and PPT mRNA levels were expressed as percent of control. Two experiments are shown for each time point. The hatched bar at 5 days is the scan of the autoradiogram in (AL
35
and 28S ribosomal R N A (see Materials and Methods; Fig. 1A). As described previously 7°'71, a single 1.2 kb band was detected in lanes of total RNA probed for PPT mRNA. A 4.5 kb band was detected for 28S. Levels of PPT m R N A were expressed as a ratio of PPT/28S m R N A to obtain a measure of specific PPT m R N A (Fig. 1B). One week following p C P A pump implantation, specific PPT m R N A was depleted 82% in the NS. PPT m R N A levels were still depressed 64% two weeks following pump implantation. After 3 weeks (one week after depletion of p C P A from the pump), PPT m R N A had recovered substantially (Fig. 1B). Because of the potential vagaries of Northern analysis, the experiment was performed 4 times with consistent results.
saline 123
300
DOI
456
789101112
B p < 0.0005
200
I
o
o o
p < 0.0005 100
0
Serotonin uptake inhibition increases striatal P P T m R N A In order to determine whether increases in 5-HT neurotransmission would produce the opposite effect, adult rats received the uptake inhibitor zimelidine for two weeks. Northern blot analysis was performed on individual (not pooled) striata after 5 or 14 days of treatment (Fig. 2A). In two separate experiments, PPT m R N A was
saline 1
2
3
clorgyline 4
5
6
7
8
~tg R N A
9 10
PP'I" ~ R N A
SP peptlde
Fig. 4. A: representative Northern blot analysis of PPT m R N A taken from individual striata from animals treated with and without D O I for 9 days. For each lane total R N A was prepared from a single striatum and 2/~g were loaded. Lanes shown were from the same blot and exposure. B: in two separate experiments Northern blots were prepared from single striata taken from animals treated with and without D O I for 9 days. A u t o r a d i o g r a m s were scanned densitometrically and PPT m R N A was expressed as a ratio to NSE m R N A , and is shown as percent of control. SP-like immunoreactivity was assessed in contralateral striata.
L 3 4.5 6 significantly increased in the NS after 5 days of zimelidine treatment, but was at control levels after a 14-day treatment (Fig. 2B). A slight, but significant increase in SP-like immunoreactivity was detected in NS after 5 days (123 _+ 2%; P < 0.025).
1~0.0"2.~ ,-,
o I,,.i
200
p
