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Both zimelidine and clorgyline decrease preprotachykinin mRNA in adult medullary raphe nuclei
MOLECULAR
AND CELLULAR
NEUROSCIENCES
2,139-144
(1991)
Both Zimelidine and Clorgyline Decrease Preprotachykinin mRNA in Adult Medullary Raphe Nuclei LEIGH Department
A. RILEY, of Biological
RONALD Sciences,
Received
P. HART,
AND
Rutgers
University,
for publication
January
G. MILLER Newark,
New
JONAKAIT Jersey
07102
29, 1991
(13-15) studies. These data show that SP and 5-HT are found together in terminals within the ventral spinal cord-specifically, in the intermediolateral cell column and in the ventral horn. In the spinal cord 5-HT potentiates the potassium-evoked release of SP (16). Moreover, SP appears also to regulate 5-HT release, possibly by an action on presynaptic serotonin receptors (17,X3). These data suggest that colocalized neurotransmitters coordinately regulate release. In recent studies from our laboratory (19), we have begun to ask whether they also mutually regulate biosynthesis. We have found that depletion of 5-HT, using the tryptophan hydroxylase inhibitor p-chlorophenylalanine, increases the levels of mRNA coding for preprotachykinin (PPT), the prohormone precursor of SP (20, 21). The current study was designed to determine quantitatively whether the corollary is true as well: Will augmentation of 5-HT neurotransmission prompt a predictable decline in PPT message levels? A limited study using in situ hybridization suggested this to be the case (22). To increase levels of serotonin, we have used a selective 5-HT reuptake blocker, zimelidine (23), as well as the monoamine oxidase (MAO) inhibitor clorgyline (24). These drugs increase synaptic (zimelidine (23)) or whole brain serotonin (clorgyline; reviewed by Waldmeier (25)) when administered for both acute and chronic time periods. We have examined PPT mRNA levels using Northern blot analysis after both acute and subchronic treatment to discover possible long-term consequences to PPT message levels. Since changes in PPT message might be expected to dictate changes in peptide level, we also determined substance P-like immunoreactivity (SP-LI) in the thoracic ventral spinal cord following treatment.
Neurons of the medullary raphe nuclei contain multiple neurotransmitters including the monoamine neurotransmitter serotonin (5-HT) and the tachykinin substance P (SP). Previously, we showed that inhibition of serotonin synthesis increased levels of mRNA coding for preprotachykinin (PPT), the prohormone precursor of SP (P. D. Walker et al., 1990, Mol. Bruin Res. 8: 113119). To determine whether augmented serotonin transmission would have the predictably opposite effect, we have examined the effects of a 5-HT uptake blocker (zimelidine) and a monoamine oxidase inhibitor (clorgyline) on levels of PPT mRNA. We looked also at the effect of zimelidine and clorgyline on levels of ventral spinal cord substance P-like immunoreactivity (SP-LI) of the same animals. Both zimelidine and clorgyline decreased levels of PPT mRNA in medullary raphe neurons and SP-LI in the ventral spinal cord. The effect on message was transient, with levels returning to control by 14 days of drug treatment. These results show that increases in synaptic 5-HT cause transient decreases in PPT mRNA and SPLI. 0 1991 Academic Press, Inc.
INTRODUCTION
Finding several neurotransmitters colocalized within single neurons raises some new issues of neurotransmitter pharmacology (see (1) for review): Do neurons maintain stable, steady-state levels of coexisting neurotransmitters or do the proportions of neurotransmitters change in response to pharmacological changes in the neuron? More specifically, does the alteration of one neurotransmitter have predictable and obligatory consequences for its colocalized partner(s)? We have begun to address these issues in the medullary raphe nuclei, located in the midline of the medulla. These neurons, long defined by their synthesis and use of the classical monoamine neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) (2,3) also contain the tachykinin neurotransmitter substance P (SP) (4-9). Coexistence of these neurotransmitters in descending terminal projections has been confirmed by both pharmacological (5, 10-12) and immunohistochemical
MATERIALS Animals
and Drug
AND
METHODS
Treatments
Adult Sprague-Dawley rats (8-10 group) were treated with the 5-HT melidine (Astra Lakemadel AB) for mg/kg twice daily) or the MAO 139
weeks, five rats per uptake blocker zi1, 5, or 14 days (10 inhibitor clorgyline
1044-7431/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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AND
(Sigma) for 1,514, or 21 days (1 mg/kg/day). Drugs were administered by ip injection. In some instances, Alzet minipumps (Alza Corp., Palo Alto, CA) were used for chronic treatment. Tissue Dissection After drug treatment, medullary raphe were dissected as previously described (19). For assay of terminal tachykinin, 3-mm cross sections of thoracic (T3) spinal cord were dissected and the dorsal third was discarded to remove SP fibers in the dorsal horn. All tissues were frozen in liquid Nz and stored at -80°C until assayed. Measurement
of PPT RNA Levels
Total RNA from medullary raphe of individual animals was prepared using a modified version of the acid guanidinium thiocyanate-phenol-chloroform extraction technique of Chomczynski and Sacchi (26). After isopropanol precipitation, the RNA pellet was resuspended in buffer containing SDS and ethanol-precipitated in the presence of ammonium acetate. RNA was quantified using a Beckman DU-70 spectrophotometer with a 5 Carat Microcell that can read a 50-~1 volume. All other RNA procedures were as previously described (19). The RNA was subjected to Northern blot hybridization with a 32P-labeled 660-nt RNA probe complementary to rat PPT mRNA. The probe was synthesized from a cDNA template corresponding to a full-length, llOO-nt y PPT mRNA that codes for substance P and substance K (subclone pGEM2-31-1 (20) kindly provided by Dr. James Krause, Washington University School of Medicine, St. Louis, MO). This probe detects three alternately spliced species of PPT mRNA, all of which code for SP (20). Since no change in one species over another has been reported to occur following various drug treatments (27,28), and the three cannot be resolved by agarose gel electrophoresis (27), we refer to them collectively as PPT mRNA. Sizes of mRNAs detected on Northern blots were determined by comparison with synthetic RNA markers (BRL) run in an adjacent lane (not shown). Some filters were stripped of the PPT probe and rehybridized with a probe for actin (29), the 3’ untranslated region of neuron-specific enolase (NSE) (30) or 28 SrRNA (31). RNA from individual animals was run in parallel so that statistical evaluation could be done within a single autoradiogram. Quantitation of mRNA was performed by densitometric scanning with an LKB Ultrascan XL Laser Densitometer and LKB 2400 Gelscan XL software. Varying amounts of pooled RNA from control animals were run alongside experimental samples to test for linearity of the autoradiography. When analyzing RNA from individual animals on the same gel, the use of any of the control probes did not reduce standard errors for those
MILLER
JONAKAIT
blots where the error was already less than 20% of the mean. Measurement
of Substance P-like Immunoreactivity
Tachykinin immunoreactivity in spinal cord was assayed by the radioimmunoassay procedure of Powell et al. (32) as modified by Kessler et al. (33) using an antibody obtained from the laboratory of Dr. Ira Black, Robert Wood Johnson Medical School (Piscataway, NJ). Since our C-terminally directed antibody might recognize other members of the tachykinin family, we use the term “substance P-like immunoreactivity” (SP-LI) throughout. Assay of Monoamine
Oxidase
MAO activity was determined in hippocampal homogenates as previously described (34). The substrate was [ 14C]serotonin (50 Ci/mol; New England Nuclear, Boston MA; 100 &f 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 All data are expressed as the mean & SEM and were compared by a two-tailed Student’s t test. RESULTS Zimelidine Transiently Medullury Raphe
Decreases PPT mRNA Levels in
To determine whether the inhibition of 5-HT uptake altered levels of PPT mRNA, animals were treated for 1, 5, or 14 days with the selective uptake blocker zimelidine (see Materials and Methods). Steady-state levels of PPT mRNA were measured in total RNA isolated from the midline of the ventral medulla of individual animals and subjected to Northern blot hybridization. Following hybridization a single band of approximately 1.2 kb was detected on Northern blots of RNA from ventral medulla but not from rat liver (Fig. 1A). Animals treated with zimelidine for 1 or 5 days showed significant decreases in levels of PPT mRNA (58 + 8% control and 54 +- 8% control, respectively; Fig. 1B). The filter shown in Fig. 1A was stripped of PPT probe and rehybridized with a probe for NSE, a neuronal housekeeping enzyme. No differences in the level of NSE mRNA between saline- and zimelidine-treated animals were found (83 * 3% of control, ns.), suggesting that the effect on PPT mRNA was specific. After 14 days of zimelidine treatment, PPT mRNA had recovered to control levels. Since levels of peptide may have been expected to mirror mRNA levels, we measured SP-LI in ventral spinal cords taken from the animals in which we measured PPT
DRUG-INDUCED
'1
saline 234
CHANGES
zimelidine 5678 \/
91OLl
IN
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mRNA
kg RNA / 23 \
,
5
Days
10
15
FIG. 1.
(A) Northern blot analysis of PPT mRNA isolated from medullary raphe taken from individual animals (l-10) treated with and without zimelidine for 5 days. For each lane total RNA was prepared from the ventral medulla of one animal, and 3 mg was loaded. To assess the linearity of the exposure, varying amounts (1, 2, and 3 pg) of pooled RNA from medullary raphe dissections of control animals were loaded on the same gel. L, 3 ag rat liver total RNA. (B) Northern blots were prepared from individual medullary raphe dissections of animals treated for 1, 5, or 14 days with zimelidine, and the resulting autoradiograms were densitometrically scanned. Results from the scans were expressed as absorbance values summarized over the area of the band and are shown here as percentage control (2 SEM). For the ll-day treatment, some animals received ip injections of zimelidine while others received a constant infusion of drug from Alzet minipumps. From the same animals, SP-LI was determined in ventral spinal cord by radioimmunoassay (see Materials and Methods), and results are expressed as percentage control. SP-LI, solid line; PPT mRNA. dashed line.
message. In these animals peptide levels in ventral spinal cord were unchanged at 1 and 5 days. However, by Day 14 a small but significant decrease in peptide was detected (85 + 4% control; Fig. 1B). Monoamine Oxidase Inhibition Transiently PPT mRNA in Medullury Raphe
Decreases
To determine whether inhibition of MAO would similarly alter PPT mRNA and/or peptide, rats were treated with clorygline for 1, 5, 14, or 21 days. The dose of clorgyline used was sufficient to reduce the oxidation of serotonin by more than 98% in all animals (data not shown). Clorgyline decreased PPT mRNA in the medulla, but only after 5 days of treatment (63 + 10% control; Figs. 2A and 2B). As before, no differences in the level of NSE mRNA between saline- and clorgyline-treated animals were seen when the filter was reprobed (97 +- 17% of control, n.s.). In a profile similar to the one seen with zimelidine, PPT mRNA levels recovered and spinal cord SP-LI was sig-
cloravltne
nificantly decreased at 14 days (48 f 7% control). By 21 days, SP-LI had recovered to control levels. A decrease in spinal cord SP-LI unaccompanied by a change in message occurred after 1 day of clorgyline administration. This may have been the result of increased release of SP (16). DISCUSSION These data suggest that acute augmentation of synaptic serotonin produced by inhibition of either its uptake or its oxidation results in decreased levels of mRNA coding for the prohormone of a colocalized neuropeptide. Previously, Walker et al. (19) demonstrated that levels of this same message are increased by the depletion of serotonin. Together these studies show that predictable changes in preprotachykinin message levels occur in response to pharmacologically evoked changes in serotonin: depletion of serotonin results in increases in PPT mRNA and augmentation of serotonin results in decreases in
uaRNA
05 0 FIG. clorgyline described
2.
(A) Northern for 5 days. RNA in Fig. 1. Animals
5
10
15
20
25
blot analysis of PPT mRNA isolated from medullary raphe of individual animals (l-10) treated with and without was treated as described under Materials and Methods and in Fig. 1. (B) PPT mRNA and SP-LI were determined as were treated for 1, 5, 14, or 21 days with clorgyline. SP-LI, solid line; PPT mRNA, dashed line.
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message. While changes in PPT message levels can result from alterations in transcription rate, RNA processing, or stability, our results do not distinguish among these alternatives. While inhibition of serotonin uptake and oxidation decrease medullary PPT message, this change is transient. By 14 days of treatment with either drug, levels of PPT message returned to control levels. Results with clorgyline suggest that the decrease in SP-LI is transient as well. After 21 days of clorgyline administration, levels of SPLI returned to normal. These observations imply that PPT synthesis is not permanently altered during longterm serotonin uptake blockade or MAO inhibition. Interestingly, while both drugs cause a decrease in PPT mRNA after 5 days of treatment, a decrease after 1 day is seen only with zimelidine. In addition, a dramatic decrease in spinal cord SP-LI is seen after 1 day of clorgyline, but not zimelidine, treatment. These differences may be ascribed to differences in drug specificity. While acute treatment with zimelidine would increase synaptic serotonin specifically, acute clogyline would also increase synaptic catecholamines. Since noradrenergic input activates the raphe (35), the simultaneous activation of raphe by catecholamines together with the increased amount of 5-HT release effected by clorgyline may enhance the serotonergic induction of SP release (16), thereby accounting for the dramatic decrease in SP-LI seen acutely with clorgyline. Similarly an acute change in noradrenergic input might offset any decrease in PPT message that may be induced by increased 5-HT. With either drug, a 14-day exposure resulted in decreased spinal cord SP-LI. It was not surprising to us that a decline in peptide would result from a decline in message. However, our data are not in agreement with the findings of others who have reported increased SP-LI in ventral spinal cord following zimelidine treatment (36-38). This discrepancy may be due to differences in drug dose or route of administration. Compared to Brodin et al. (36, 37), e.g., we administered three times as much drug, and we gave intraperitoneal injections, not oral administration. We used the same dose and route of administration as that used by Schalling et al. (22). While their results are based only on one time point, our data agree with respect to changes in PPT mRNA, but they did not measure spinal cord SP-LI. Alternatively, differences between our data and those of others might result from differences in the antibody used for radioimmunoassay (39) or differing methods of peptide extraction (40). Resolving this discrepancy will be an important line for future investigation. Our results suggest that acutely altered synaptic levels of 5-HT change tachykinin message levels, but the cellular mechanisms through which this occurs are not known. It is possible, however, to envision such a mechanism. Dorsal raphe serotonergic neurons possess autoreceptors which mediate changes in firing rate (41), serotonin release (for
MILLER
JONAKAIT
review see Moret (42)), and biosynthesis (43-45). Similar autoreceptors have been found on serotonergic neurons of the medullary raphe (46, 47). Tachykinin gene transcription might be another process regulated by autoreceptor activation. Receptor binding with associated changes in second messenger systems is capable of changing transcription rates of many neuropeptide-encoding genes (for reviews see Black et al. (48) or Comb et al. (49)). Autoreceptors are, then, one candidate for mediating these changes. The involvement of autoreceptors in the expression of PPT is further suggested by the transient nature of the change. In the dorsal raphe, chronic administration of either uptake blockers (50, 51) or MAO inhibitors (51, 52) results in a desensitization of autoreceptors with an accompanying recovery of firing rate. Similar changes in autoreceptor sensitivity may occur in the medullary raphe as well. The time course of the recovery of PPT mRNA levels is strikingly similar to the time course of the desensitization of serotonin autoreceptors (50-52), suggesting a possible link between autoreceptors and PPT message levels. In conclusion, we have shown that increases in synaptic 5-HT decrease the levels of medullary raphe PPT mRNA and SP-LI. These effects are transient and parallel possible changes in autoreceptor sensitivity and firing rate that accompany chronic treatment with MAO inhibitors and 5-HT uptake blockers. These data in combination with previous work (19,22) suggest that 5-HT can regulate the biosynthesis of colocalized tachykinins. ACKNOWLEDGMENTS This work was supported by grants from the NIMH (MH 43365, MH 06855) and the NSF (BNS 8909551). L.A.R. was supported by fellowships from both the Center for Molecular and Behavioral Neuroscience, Rutgers University, Newark, New Jersey, and the Tourette Syndrome Association.
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2,139-144
(1991)
Both Zimelidine and Clorgyline Decrease Preprotachykinin mRNA in Adult Medullary Raphe Nuclei LEIGH Department
A. RILEY, of Biological
RONALD Sciences,
Received
P. HART,
AND
Rutgers
University,
for publication
January
G. MILLER Newark,
New
JONAKAIT Jersey
07102
29, 1991
(13-15) studies. These data show that SP and 5-HT are found together in terminals within the ventral spinal cord-specifically, in the intermediolateral cell column and in the ventral horn. In the spinal cord 5-HT potentiates the potassium-evoked release of SP (16). Moreover, SP appears also to regulate 5-HT release, possibly by an action on presynaptic serotonin receptors (17,X3). These data suggest that colocalized neurotransmitters coordinately regulate release. In recent studies from our laboratory (19), we have begun to ask whether they also mutually regulate biosynthesis. We have found that depletion of 5-HT, using the tryptophan hydroxylase inhibitor p-chlorophenylalanine, increases the levels of mRNA coding for preprotachykinin (PPT), the prohormone precursor of SP (20, 21). The current study was designed to determine quantitatively whether the corollary is true as well: Will augmentation of 5-HT neurotransmission prompt a predictable decline in PPT message levels? A limited study using in situ hybridization suggested this to be the case (22). To increase levels of serotonin, we have used a selective 5-HT reuptake blocker, zimelidine (23), as well as the monoamine oxidase (MAO) inhibitor clorgyline (24). These drugs increase synaptic (zimelidine (23)) or whole brain serotonin (clorgyline; reviewed by Waldmeier (25)) when administered for both acute and chronic time periods. We have examined PPT mRNA levels using Northern blot analysis after both acute and subchronic treatment to discover possible long-term consequences to PPT message levels. Since changes in PPT message might be expected to dictate changes in peptide level, we also determined substance P-like immunoreactivity (SP-LI) in the thoracic ventral spinal cord following treatment.
Neurons of the medullary raphe nuclei contain multiple neurotransmitters including the monoamine neurotransmitter serotonin (5-HT) and the tachykinin substance P (SP). Previously, we showed that inhibition of serotonin synthesis increased levels of mRNA coding for preprotachykinin (PPT), the prohormone precursor of SP (P. D. Walker et al., 1990, Mol. Bruin Res. 8: 113119). To determine whether augmented serotonin transmission would have the predictably opposite effect, we have examined the effects of a 5-HT uptake blocker (zimelidine) and a monoamine oxidase inhibitor (clorgyline) on levels of PPT mRNA. We looked also at the effect of zimelidine and clorgyline on levels of ventral spinal cord substance P-like immunoreactivity (SP-LI) of the same animals. Both zimelidine and clorgyline decreased levels of PPT mRNA in medullary raphe neurons and SP-LI in the ventral spinal cord. The effect on message was transient, with levels returning to control by 14 days of drug treatment. These results show that increases in synaptic 5-HT cause transient decreases in PPT mRNA and SPLI. 0 1991 Academic Press, Inc.
INTRODUCTION
Finding several neurotransmitters colocalized within single neurons raises some new issues of neurotransmitter pharmacology (see (1) for review): Do neurons maintain stable, steady-state levels of coexisting neurotransmitters or do the proportions of neurotransmitters change in response to pharmacological changes in the neuron? More specifically, does the alteration of one neurotransmitter have predictable and obligatory consequences for its colocalized partner(s)? We have begun to address these issues in the medullary raphe nuclei, located in the midline of the medulla. These neurons, long defined by their synthesis and use of the classical monoamine neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) (2,3) also contain the tachykinin neurotransmitter substance P (SP) (4-9). Coexistence of these neurotransmitters in descending terminal projections has been confirmed by both pharmacological (5, 10-12) and immunohistochemical
MATERIALS Animals
and Drug
AND
METHODS
Treatments
Adult Sprague-Dawley rats (8-10 group) were treated with the 5-HT melidine (Astra Lakemadel AB) for mg/kg twice daily) or the MAO 139
weeks, five rats per uptake blocker zi1, 5, or 14 days (10 inhibitor clorgyline
1044-7431/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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AND
(Sigma) for 1,514, or 21 days (1 mg/kg/day). Drugs were administered by ip injection. In some instances, Alzet minipumps (Alza Corp., Palo Alto, CA) were used for chronic treatment. Tissue Dissection After drug treatment, medullary raphe were dissected as previously described (19). For assay of terminal tachykinin, 3-mm cross sections of thoracic (T3) spinal cord were dissected and the dorsal third was discarded to remove SP fibers in the dorsal horn. All tissues were frozen in liquid Nz and stored at -80°C until assayed. Measurement
of PPT RNA Levels
Total RNA from medullary raphe of individual animals was prepared using a modified version of the acid guanidinium thiocyanate-phenol-chloroform extraction technique of Chomczynski and Sacchi (26). After isopropanol precipitation, the RNA pellet was resuspended in buffer containing SDS and ethanol-precipitated in the presence of ammonium acetate. RNA was quantified using a Beckman DU-70 spectrophotometer with a 5 Carat Microcell that can read a 50-~1 volume. All other RNA procedures were as previously described (19). The RNA was subjected to Northern blot hybridization with a 32P-labeled 660-nt RNA probe complementary to rat PPT mRNA. The probe was synthesized from a cDNA template corresponding to a full-length, llOO-nt y PPT mRNA that codes for substance P and substance K (subclone pGEM2-31-1 (20) kindly provided by Dr. James Krause, Washington University School of Medicine, St. Louis, MO). This probe detects three alternately spliced species of PPT mRNA, all of which code for SP (20). Since no change in one species over another has been reported to occur following various drug treatments (27,28), and the three cannot be resolved by agarose gel electrophoresis (27), we refer to them collectively as PPT mRNA. Sizes of mRNAs detected on Northern blots were determined by comparison with synthetic RNA markers (BRL) run in an adjacent lane (not shown). Some filters were stripped of the PPT probe and rehybridized with a probe for actin (29), the 3’ untranslated region of neuron-specific enolase (NSE) (30) or 28 SrRNA (31). RNA from individual animals was run in parallel so that statistical evaluation could be done within a single autoradiogram. Quantitation of mRNA was performed by densitometric scanning with an LKB Ultrascan XL Laser Densitometer and LKB 2400 Gelscan XL software. Varying amounts of pooled RNA from control animals were run alongside experimental samples to test for linearity of the autoradiography. When analyzing RNA from individual animals on the same gel, the use of any of the control probes did not reduce standard errors for those
MILLER
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blots where the error was already less than 20% of the mean. Measurement
of Substance P-like Immunoreactivity
Tachykinin immunoreactivity in spinal cord was assayed by the radioimmunoassay procedure of Powell et al. (32) as modified by Kessler et al. (33) using an antibody obtained from the laboratory of Dr. Ira Black, Robert Wood Johnson Medical School (Piscataway, NJ). Since our C-terminally directed antibody might recognize other members of the tachykinin family, we use the term “substance P-like immunoreactivity” (SP-LI) throughout. Assay of Monoamine
Oxidase
MAO activity was determined in hippocampal homogenates as previously described (34). The substrate was [ 14C]serotonin (50 Ci/mol; New England Nuclear, Boston MA; 100 &f 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 All data are expressed as the mean & SEM and were compared by a two-tailed Student’s t test. RESULTS Zimelidine Transiently Medullury Raphe
Decreases PPT mRNA Levels in
To determine whether the inhibition of 5-HT uptake altered levels of PPT mRNA, animals were treated for 1, 5, or 14 days with the selective uptake blocker zimelidine (see Materials and Methods). Steady-state levels of PPT mRNA were measured in total RNA isolated from the midline of the ventral medulla of individual animals and subjected to Northern blot hybridization. Following hybridization a single band of approximately 1.2 kb was detected on Northern blots of RNA from ventral medulla but not from rat liver (Fig. 1A). Animals treated with zimelidine for 1 or 5 days showed significant decreases in levels of PPT mRNA (58 + 8% control and 54 +- 8% control, respectively; Fig. 1B). The filter shown in Fig. 1A was stripped of PPT probe and rehybridized with a probe for NSE, a neuronal housekeeping enzyme. No differences in the level of NSE mRNA between saline- and zimelidine-treated animals were found (83 * 3% of control, ns.), suggesting that the effect on PPT mRNA was specific. After 14 days of zimelidine treatment, PPT mRNA had recovered to control levels. Since levels of peptide may have been expected to mirror mRNA levels, we measured SP-LI in ventral spinal cords taken from the animals in which we measured PPT
DRUG-INDUCED
'1
saline 234
CHANGES
zimelidine 5678 \/
91OLl
IN
MEDULLARY
RAPHE
PREPROTACHYKININ
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mRNA
kg RNA / 23 \
,
5
Days
10
15
FIG. 1.
(A) Northern blot analysis of PPT mRNA isolated from medullary raphe taken from individual animals (l-10) treated with and without zimelidine for 5 days. For each lane total RNA was prepared from the ventral medulla of one animal, and 3 mg was loaded. To assess the linearity of the exposure, varying amounts (1, 2, and 3 pg) of pooled RNA from medullary raphe dissections of control animals were loaded on the same gel. L, 3 ag rat liver total RNA. (B) Northern blots were prepared from individual medullary raphe dissections of animals treated for 1, 5, or 14 days with zimelidine, and the resulting autoradiograms were densitometrically scanned. Results from the scans were expressed as absorbance values summarized over the area of the band and are shown here as percentage control (2 SEM). For the ll-day treatment, some animals received ip injections of zimelidine while others received a constant infusion of drug from Alzet minipumps. From the same animals, SP-LI was determined in ventral spinal cord by radioimmunoassay (see Materials and Methods), and results are expressed as percentage control. SP-LI, solid line; PPT mRNA. dashed line.
message. In these animals peptide levels in ventral spinal cord were unchanged at 1 and 5 days. However, by Day 14 a small but significant decrease in peptide was detected (85 + 4% control; Fig. 1B). Monoamine Oxidase Inhibition Transiently PPT mRNA in Medullury Raphe
Decreases
To determine whether inhibition of MAO would similarly alter PPT mRNA and/or peptide, rats were treated with clorygline for 1, 5, 14, or 21 days. The dose of clorgyline used was sufficient to reduce the oxidation of serotonin by more than 98% in all animals (data not shown). Clorgyline decreased PPT mRNA in the medulla, but only after 5 days of treatment (63 + 10% control; Figs. 2A and 2B). As before, no differences in the level of NSE mRNA between saline- and clorgyline-treated animals were seen when the filter was reprobed (97 +- 17% of control, n.s.). In a profile similar to the one seen with zimelidine, PPT mRNA levels recovered and spinal cord SP-LI was sig-
cloravltne
nificantly decreased at 14 days (48 f 7% control). By 21 days, SP-LI had recovered to control levels. A decrease in spinal cord SP-LI unaccompanied by a change in message occurred after 1 day of clorgyline administration. This may have been the result of increased release of SP (16). DISCUSSION These data suggest that acute augmentation of synaptic serotonin produced by inhibition of either its uptake or its oxidation results in decreased levels of mRNA coding for the prohormone of a colocalized neuropeptide. Previously, Walker et al. (19) demonstrated that levels of this same message are increased by the depletion of serotonin. Together these studies show that predictable changes in preprotachykinin message levels occur in response to pharmacologically evoked changes in serotonin: depletion of serotonin results in increases in PPT mRNA and augmentation of serotonin results in decreases in
uaRNA
05 0 FIG. clorgyline described
2.
(A) Northern for 5 days. RNA in Fig. 1. Animals
5
10
15
20
25
blot analysis of PPT mRNA isolated from medullary raphe of individual animals (l-10) treated with and without was treated as described under Materials and Methods and in Fig. 1. (B) PPT mRNA and SP-LI were determined as were treated for 1, 5, 14, or 21 days with clorgyline. SP-LI, solid line; PPT mRNA, dashed line.
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message. While changes in PPT message levels can result from alterations in transcription rate, RNA processing, or stability, our results do not distinguish among these alternatives. While inhibition of serotonin uptake and oxidation decrease medullary PPT message, this change is transient. By 14 days of treatment with either drug, levels of PPT message returned to control levels. Results with clorgyline suggest that the decrease in SP-LI is transient as well. After 21 days of clorgyline administration, levels of SPLI returned to normal. These observations imply that PPT synthesis is not permanently altered during longterm serotonin uptake blockade or MAO inhibition. Interestingly, while both drugs cause a decrease in PPT mRNA after 5 days of treatment, a decrease after 1 day is seen only with zimelidine. In addition, a dramatic decrease in spinal cord SP-LI is seen after 1 day of clorgyline, but not zimelidine, treatment. These differences may be ascribed to differences in drug specificity. While acute treatment with zimelidine would increase synaptic serotonin specifically, acute clogyline would also increase synaptic catecholamines. Since noradrenergic input activates the raphe (35), the simultaneous activation of raphe by catecholamines together with the increased amount of 5-HT release effected by clorgyline may enhance the serotonergic induction of SP release (16), thereby accounting for the dramatic decrease in SP-LI seen acutely with clorgyline. Similarly an acute change in noradrenergic input might offset any decrease in PPT message that may be induced by increased 5-HT. With either drug, a 14-day exposure resulted in decreased spinal cord SP-LI. It was not surprising to us that a decline in peptide would result from a decline in message. However, our data are not in agreement with the findings of others who have reported increased SP-LI in ventral spinal cord following zimelidine treatment (36-38). This discrepancy may be due to differences in drug dose or route of administration. Compared to Brodin et al. (36, 37), e.g., we administered three times as much drug, and we gave intraperitoneal injections, not oral administration. We used the same dose and route of administration as that used by Schalling et al. (22). While their results are based only on one time point, our data agree with respect to changes in PPT mRNA, but they did not measure spinal cord SP-LI. Alternatively, differences between our data and those of others might result from differences in the antibody used for radioimmunoassay (39) or differing methods of peptide extraction (40). Resolving this discrepancy will be an important line for future investigation. Our results suggest that acutely altered synaptic levels of 5-HT change tachykinin message levels, but the cellular mechanisms through which this occurs are not known. It is possible, however, to envision such a mechanism. Dorsal raphe serotonergic neurons possess autoreceptors which mediate changes in firing rate (41), serotonin release (for
MILLER
JONAKAIT
review see Moret (42)), and biosynthesis (43-45). Similar autoreceptors have been found on serotonergic neurons of the medullary raphe (46, 47). Tachykinin gene transcription might be another process regulated by autoreceptor activation. Receptor binding with associated changes in second messenger systems is capable of changing transcription rates of many neuropeptide-encoding genes (for reviews see Black et al. (48) or Comb et al. (49)). Autoreceptors are, then, one candidate for mediating these changes. The involvement of autoreceptors in the expression of PPT is further suggested by the transient nature of the change. In the dorsal raphe, chronic administration of either uptake blockers (50, 51) or MAO inhibitors (51, 52) results in a desensitization of autoreceptors with an accompanying recovery of firing rate. Similar changes in autoreceptor sensitivity may occur in the medullary raphe as well. The time course of the recovery of PPT mRNA levels is strikingly similar to the time course of the desensitization of serotonin autoreceptors (50-52), suggesting a possible link between autoreceptors and PPT message levels. In conclusion, we have shown that increases in synaptic 5-HT decrease the levels of medullary raphe PPT mRNA and SP-LI. These effects are transient and parallel possible changes in autoreceptor sensitivity and firing rate that accompany chronic treatment with MAO inhibitors and 5-HT uptake blockers. These data in combination with previous work (19,22) suggest that 5-HT can regulate the biosynthesis of colocalized tachykinins. ACKNOWLEDGMENTS This work was supported by grants from the NIMH (MH 43365, MH 06855) and the NSF (BNS 8909551). L.A.R. was supported by fellowships from both the Center for Molecular and Behavioral Neuroscience, Rutgers University, Newark, New Jersey, and the Tourette Syndrome Association.
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