- Email: [email protected]
The effect of aging on the response of striatal preproenkephalin and preprotachykinin mRNA contents to chronic haloperidol treatment in rats: measurement by solution-hybridization RNase protection assay
Neuroscience Letters 246 (1998) 33–36
The effect of aging on the response of striatal preproenkephalin and preprotachykinin mRNA contents to chronic haloperidol treatment in rats: measurement by solution-hybridization RNase protection assay S.M. Lau, F. Tang* Department of Physiology, Faculty of Medicine, The University of Hong Kong, Hong Kong, China Received 11 December 1997; received in revised form 5 March 1998; accepted 6 March 1998
Abstract The preproenkephalin (PPeK) and preprotachykinin (PPT) mRNA contents in 3-, 10- and 23-month-old rats in the striatum were measured by solution hybridization-RNase protection assay after 3 weeks of haloperidol injection. Haloperidol increased striatal PPek mRNA. There was no age-related difference in the response of striatal PPeK mRNA to chronic haloperidol treatment. The PPT mRNA decreased by 21% after the haloperidol treatment in young rats only. Meanwhile, age decreased the PPT mRNA by 27 and 24% in 10- and 23-month-old rats, respectively. It is concluded that there is a difference in the effects of aging on the response of PPek and PPT mRNA contents to haloperidol and that the loss of PPT mRNA response in 10- and 23-month-old rats might be due to the change of dopamine system of the striatum in these rats. 1998 Elsevier Science Ireland Ltd.
Keywords: Haloperidol; Preproenkephalin mRNA; Preprotachykinin mRNA; Striatum; Solution hybridization-RNase; Protection assay; Aging; Dopamine receptors
The regulation of the enkephalin system in the striatum by dopamine activity is well established. Haloperidol, a dopamine receptor antagonist, has been reported to elevate striatal met-enkephalin (ME) content [8,20] through the increase of gene expression. Both pro-enkephalin mRNA [19,21] and pre-proenkephalin (PPeK) mRNA [1,3] have been measured in the striatum and found to increase after haloperidol treatment. In contrast to the enkephalin system, dopamine exerts a stimulatory effect on the tachykinin system in the striatum. Dopamine (DA) receptor agonists increased substance P (SP) content of substantia nigra (SN) [18] and reduced SP of the striatum [6,12]. The increase of SP in SN was shown to be due to an increase of SP biosynthesis in striatum [7]. The inhibitory effect of haloperidol was later proved to be due to a decrease of SP biosynthesis by measuring the preprotachykinin mRNA * Corresponding author. Tel.: +853 281 99269; fax: +852 2 8559730.
content in the striatum [2,13]. However, DA receptor antagonists seemed to have no effect on SP level in the striatum. In the central nervous system, dopamine activity is decreased with age in different brain areas of several animal species including man [16]. The decline of DA found in the striatum, substantia nigra and limbic system is because the age-related decrease in the activity and concentration of the rate-limiting enzyme, tyrosine hydroxylase (TH) [17]. The decrease was particularly obvious in the striatum and the brainstem in senescent rats [15]. Meanwhile, the D1 and D2 dopamine receptors decrease in the striatum of old rats [10]. The age-related decrease in DA content and D1 and D2 receptors may modify the response of preproenkephalin and preprotachykinin systems to chronic haloperidol treatment in the striatum. This possibility is studied by measuring the mRNA content of PPT and PPeK with a sensitive solution hybridization-RNase protection assay. Specific pathogen free male Sprague–Dawley rats
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00202- X
34
S.M. Lau, F. Tang / Neuroscience Letters 246 (1998) 33–36
(Charles River Laboratories, Japan) aged 3 months (young), 10 months (adult) and 20–23 months (old) received daily haloperidol (Sigma) injections for 3 weeks at 2 mg/kg body weight. The control groups received vehicle consisting of 0.9% saline at the same pH. Rats were maintained on a 12:12 h cycle (light on at 0600 h) at 22°C and 45–55% humidity. Rat pellets (PMI Laboratory Autoclavable Rodent Diet 5010, from PMI feeds Inc., Richmond, IN, USA) and drinking water (UV-treated) were provided ad libitum. The experiments were performed twice. In the first experiment, only PPeK mRNA was measured while in the second experiment, both PPeK and PPT mRNAs were measured. Rats were sacrificed by decapitation. The RNAs of the striatum was extracted by guanidinium thiocynate solution [5]. The striatum was homogenized in 1 ml cold solution D (4 M guanidinium thiocynate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, 10 mM 2-mercarptoethanol) by a polytron for three bursts of 10 s each. One hundred microliters sodium acetate (2 M, pH 4.0), 1 ml water-saturated phenol (pH 7.0) and 200 ml chloroform were added. The mixture was vigorously vortexed for 10 s and then cooled down on ice. The two phases were partitioned by centrifugation for 20 min. An equal volume of isopropanol was added to the upper aqueous phase. The precipitation proceeded at −20°C overnight. After centrifugation, the RNA pellet was re-dissolved in 0.3 ml solution D. The RNAs were again precipitated by isopropanol and washed by 1 ml 70% ethanol. Finally, RNA samples were reconstituted in 30 ml Tris EDTA buffer. The mRNAs of PPT or PPeK in the striatal samples were quantified by a solution hybridization-RNase protection assay technique [22]. The plasmid containing cDNA of bPPT were obtained from Prof. J.E. Krause (School of Medicine, Washington University, St. Louis, MO, USA) and the pYSEC 1 and pYSEA 1 plasmids were provided by Dr. S. Sabol (NIH, Bethesda, MD, USA) and Dr. J. Hong (NIEHS, Research Traingle Park, NC, USA). The b-actin plasmid was the gift of Dr. D. Autelitano (Baker Medical Institute, Melbourne, Australia). A kit (Rioboprobe Tanscription System, Promega, Madison, WI, USA) was used for in vitro transcription. For the synthesis of the antisense RNA, 10 mM dithiothreitol, 40 units RNasin, 0.5 mM ATP, CTP, GTP, 15 mM UTP, 1 mg linearized RNA (cut by Eco RI), 25 mCi 32P-UTP, 7.5U SP6 RNA polymerase, and 1× transcription buffer in a total volume of 20 ml were incubated at 37°C for 1 h. The average specific activity of antisense PPeK (930 nt) b−PPT (567 nt) and actin (387 nt) were 210 c.p.m/pg, 250 c.p.m/pg and 225 c.p.m/pg, respectively. The sense RNAs (standard) were also synthesized using T7 RNA polymerase, and 10 mg of linearized template DNA (cut by Hind III). RNA samples/standard and 32P-riboprobe (100 000 c.p.m) were denatured at 85°C for 5 min. Approximately 1.5 mg, 2 mg and 10 mg total RNA were used for actin mRNA, PPT mRNA and PPeK mRNA assays, respectively. For the standard points, yeast total RNA (similar amount as in sample used) was added. The mixture was allowed to
hybridize at 45°C overnight in hybridization buffer (80% formamide, 40 mM PIPE, 400 mM NaCl and 1 mM EDTA (pH 8.0)). The unhybridized RNAs were digested at 37°C for 45 min by RNase A and RNase T3. After adding 20 ml 10% sodium dodecyl sulfate, the enzymes were digested by proteinase K (5 ml) and 1 mg of yeast RNA carrier was added. The hybrids were separated by 4% polyacrylamide gel (PAGE) with 1× Tris–Borate EDTA buffer as a running buffer at 160 V and 34 mA for 1 h. The hybrid bands on the gel were cut out using the X-ray film exposed to the blot as template and counted. A standard curve could be drawn by plotting the radioactivity versus the pg of standard points. The result was presented as pg mRNA per pg b-actin mRNA. The values of the mRNAs were not corrected to their native lengths. Statistical analysis was performed by 2way analysis of variance (ANOVA) and Student’s t-test. Statistical significance was set at P , 0.05. The separation of the hybrids for PPek was shown in Fig. 1, in which different amounts of total striatal RNA were used to demonstrate a linear relationship between this and radioactivity in the hybrids. The X-ray film of polyacrylamide gel electrolysis of RNA hybrids of standard and striatal PPT mRNA is shown in Fig. 2. In both experiments, haloperidol treatment increased PPeK mRNA in the striatum of rats of all three ages. In the first experiment, the values (mean ± SE) for the control rats were 51.0 ± 6.6 (n = 10), 46.8 ± 5.3 (n = 8) and 38.4 ± 6.9 (n = 9) for the young, adult and old rats, respectively. The values for the haloperidol rats were 87.5 ± 7.3 (n = 8), 67.3 ± 8.4 (n = 6) and 77.6 ± 8.5 (n = 6). The data obtained from experiment 2 is shown in Fig. 3. By two-way ANOVA, a treatment effect was noted. After haloperidol treatment, the striatal PPT mRNA decreased significantly by 21% in young rat group (Fig. 4). Meanwhile, aging decreased the striatal PPT mRNA by 27 and 24% in the middle-aged and old rat group. No haloperidol effect was noted in adult or old rats. The PPeK mRNA was elevated after haloperidol injection in all age groups (Fig. 3). A corresponding increment of met-enkephalin (ME) was found in previous studies [9,11]. Haloperidol increases striatal ME by acting on D2 receptors; the finding of an increase of PPeK mRNA after haloperidol
Fig. 1. The X-ray film of polyacryamide gel electrophoresis of RNA hybrid of striatal PPeK mRNA with 32P-labeled PPeK probe. There is a linear relationship between the radioactivity of the hybrid and the amount of striatal RNA used. The numbers on the top of the lanes are mg total RNA used. The probe was shown on the left (arrow). The hybrids of the PPeK standards were not shown here but could be found in Tang and Lau [22].
S.M. Lau, F. Tang / Neuroscience Letters 246 (1998) 33–36
35
Fig. 2. The X-ray film of polyacryamide gel electrophoresis of RNA hybrids of standards and striatal PPT mRNA with 32P-labeled b-PPT probe. The three arrows marked the positions (from above) of b−PPT hybrid (508 bp), gPPT hybrid (295 bp) and gPPT hybrid (168 bp). The details could be found in Carter and Krause [4]. Lanes 1–6, standard; lane 1, 0 pg; lane 2, 10 pg; lane 3, 50 pg; lane 4, 100 pg; lane 5, 500 pg; lane 6, 1000 pg.
was in agreement with earlier reports [1,3,19,21]. Dopamine concentration declines with age [16] while the D1 and D2 dopamine receptors decrease in the striatum of old rats [10]. We have previously observed a blunted response in the ME content in the striatum in old rats upon haloperidol treatment [11]. This blunting in response, however, is not found for striatal PPeK mRNA levels in the present study. Aging therefore does not modify the response of striatal PPek mRNA to haloperidol treatment in spite of a decrease in dopamine system in the old rats. As much as both aging and haloperidol lead to a decrease in dopamine activity but only haloperidol results in an increase in PPeK mRNA content, aging must have its own effect in decreasing PPeK mRNA content. This is supported by the finding of a lack of increase of PPeK mRNA (the present study) and ME [11,24] with age. It is perhaps pertinent to note that in one of our experiments, we found a borderline decrease in PPeK mRNA content in the striatum of the old rats. Meanwhile, haloperidol caused a decrease in total PPT mRNA in the striatum. The result was supported by previous findings [2,6,7]. However, there was no significant decrease of substance P (SP) in our previous study [11] and in other studies [8,14]. The explanation of this result might be that the striato-nigral SP containing neurons project to SN with their cell bodies situated in the striatum.
Fig. 3. The PPeK mRNA levels in the striatum in different age groups after 3 weeks of haloperidol injection. RNA samples were extracted by GnSCN solution. Data are the mean ± SE with n value in parentheses. By a Student’s t-test, *P , 0.05 when compared to the control group. Two-way ANOVA: P = 0.093 (d.f. = 2/53, F = 2.5) for the aging effect, P , 0.001 (d.f. = 1/53, F = 28) for the haloperidol effect, P = 0.82 (d.f. = 2/53, F = 0.2) for the interaction between aging and haloperidol treatment.
Fig. 4. The PPT mRNA levels in the striatum in different age groups after 3 weeks of haloperidol injection. RNA samples were extracted by GnSCN solution. Data are the mean ± SE with n value in parentheses. Student’s T-test: *P , 0.05 when compared to the control group, **P , 0.05 when compared to young control group. Two-way ANOVA: P , 0.001 (d.f. = 2/50, F = 9.8) for the aging effect, P = 0.006 (d.f. = 1/50, F = 8.3) for haloperidol effect and P = 0.262 (d.f. = 2/50, F = 1.4) for the interaction between aging and haloperidol treatment.
Therefore, it is expected that DA receptor antagonism decreases the SP level in SN and reduces the PPT mRNA in the striatum. The decrease of SP caused by DA antagonist in SN had been extensively documented [6,8,12]. In the adult and old rats, the significant decrease of PPT mRNA contents compared with young rats, which is a developmental change before maturity, also agrees with the previous findings of an age-related decline in dopamine activity [10,16]. Here, both aging (as defined by change with time) and haloperidol decrease dopamine activity, and decrease PPT mRNA content so that the effect of aging seems to be mediated through the dopamine system. However, the effect of haloperidol is already abolished in 10month-old rats, a finding which correlates with the decrease of D1 and D2 receptors at 7 and 12 months of age [10]. It is possible that aging and haloperidol work via a common pathway to decrease dopamine activity and as a result, no haloperidol effect can be observed in the older rats. Alternatively, aging might have modified the dopamine system (e.g. by decreasing its receptors) such that it will not respond to haloperidol any more. Perhaps it should be noted that rats live to a maximum life span of 36 months and if the last quarter is taken as ‘senescence’ (Finch, as quoted by Pradhan [16]), the rat has to be 27 months old before it can be ‘old’. However, lots of publications have used 24-, 20-, or even 18-month-old rats as ‘old rats’ and even at 18 months of age, there are already lots of physiological changes [23]. In summary, this study provides strong evidence that the dopaminergic system stimulates the tachykininergic system but inhibits the enkephaline system in the striatum through the change of gene expression of their precursors. Because of the differences in the effects of aging and haloperidol on the two systems, there is a difference in the effect of aging on the response to haloperidol. The abolition of the tachykinin response to haloperidol may mean that in the older rats some peptidergic systems may have lost the ability to
36
S.M. Lau, F. Tang / Neuroscience Letters 246 (1998) 33–36
respond or there may be a change in the threshold of either dopaminergic or the peptidergic systems in the response to haloperidol. This may have important repercussions in the physiology of old animals as these systems play important roles in neurotransmission and neuromodulation. The study of the interaction of DA with peptidergic systems is particularly important as additive losses of neurotransmitters and receptors could have pathophysiological consequences and pharmacological implications in the elderly.
[10]
[11]
[12]
We thank Professor J.E. Krause (School of Medicine, Washington University, St. Louis, MO, USA) for the gift of cDNA of b−PPT, Dr. S. Sabol (NIH, Bethesda, MD, USA) and Dr. J. Hong (NIEHS, Research Triangle Park, NC, USA) for the gift of cDNA of pYSEC1 and pYSEA1. We are also indebted to Dr. D. Autelitano (Baker Medical Research Institution, Melbourne, Australia) for the gift of b-actin plasmid and his help in setting up the solution hybridization-RNase protection assay. [1] Abood, M.E., Eberwine, J.H., Erdelyi, E. and Evans, C.J., Regulation of both preproenkephalin mRNA and its derived opioids by haloperidol – a method for measurement of peptides and mRNA in the same tissue extract, Mol. Brain Res., 8 (1990) 243–248. [2] Bannon, M.J., Lee, J.M., Giraud, P., Young, A., Affolter, H.U. and Bonner, T.I., Dopamine antagonist haloperidol decrease substance P, substance K and preprotachykinin mRNAs in rat striatonigral neurons, J. Biol. Chem., 261 (1986) 6640–6642. [3] Bernard, V., Le Moine, C. and Bloch, B., Striatal neurons express increased level of dopamine D2 receptor mRNA in response to haloperidal treatment: a quantitative in situ hybridization study, Neuroscience, 45 (1991) 117–126. [4] Carter, M.S. and Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat pre-protachykinin gene encoding substance P, neurokinin A, neuropeptide K and neuropeptide, J. Neurosci., 10 (1990) 2203–2214. [5] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156–159. [6] Hanson, G.R., Alphs, L., Wolf, W., Pradhan, S. and Lovenberg, W., Response of striatonigral substance P systems to a dopamine receptor agonist and antagonist, Neuropharmacology, 20 (1981) 541–548. [7] Haverstick, D.M., Rubenstein, A. and Bannon, M.J., Striatal tachykinin gene expression regulated by interaction of D1 and D2 dopamine receptors, J. Pharmacol. Exp. Ther., 248 (1989) 858–862. [8] Hong, J.S., Yang, H.Y. and Costa, E., Substance P content of substantia nigra after chronic treatment with antischizophrenic drugs, Neuropharmacology, 17 (1978) 83–85. [9] Hong, J.S., Yang, H.Y.T., Gillin, J.C., Di Giulio, A.M., Fratta, W.
[13]
[14]
[15]
[16] [17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
and Costa, E., Chronic treatment with haloperidol accelerates the biosynthesis of enkephalin in rat striatum, Brain Res., 160 (1979) 192–195. Hyttel, J., Age related decrease in the density of dopamine D1 and D2 receptors in corpus striatum of rats, Pharmacol. Toxicol., 61 (1987) 126–129. Lau, S.M. and Tang, F., The effect of haloperidol on met-enkephalin, beta-endorphin, cholecytokinin and substance P in the pituitary, the hypothalamus and the striatum of rats during aging, Prog. Neuro-Psychopharmacol. Biol. Psychiatr., 19 (1995) 1163–1175. Li, S.J., Sivam, P., McGinty, J.F., Huang, Y.S. and Hong, J.S., Dopaminergic regulation of tachykinin metabolism in the striatonigral pathway, J. Pharmacol. Exp. Ther., 243 (1987) 792– 798. Lindefors, N., Amphetamine and haloperidol modulate preprotachykinin A mRNA expression in rat nucleus accumbens and caudate-putamen, Mol. Brain Res., 13 (1992) 151–154. Nylander, I. and Terenius, L., Chronic haloperidol and clozapine differentially affect dynorphin peptides and substance P in basal ganglia of the rat, Brain Res., 380 (1986) 34–41. Ponzio, F., Calderini, G., Lomuscio, G., Vantini, G., Toffano, G. and Algeri, S., Changes in monoamines and their metabolite levels in some brain regions of aged rats, Neurobiol. Aging, 3 (1982) 23–29. Pradhan, S.N., Central neurotransmitters and aging, Life Sci., 26 (1980) 1643–1656. Reis, D.J., Ross, R.A. and Tong, H.J., Changes in the activity and amounts of enzymes synthesizing catecholamines and acetylcholine in brain, adrenal medulla and sympathetic ganglia of aged rat and mouse, Brain Res., 136 (1977) 465–474. Ritter, J.K., Schmidt, C.J., Gibb, J.W. and Hanson, G.R., Increases of substance P-like immunoreactivity within striatalnigral structures after subacute methamphetamine treatment, J. Pharmacol. Exp. Ther., 229 (1984) 487–492. Sabol, S.L., Yoshikawa, K. and Hong, J.S., Regulation of methionine-enkephalin precursor messenger RNA in rat striatum by haloperidol and lithium, Biochem. Biophys. Res. Commun., 113 (1983) 391–399. Sivam, S.P., Strunk, C., Smith, D.R. and Hong, J.S., Proenkephalin-A gene regulation in the rat striatum: influence of lithium and haloperidol, Mol. Pharmacol., 30 (1986) 186–191. Tang, F., Costa, E. and Schwarz, J.P., Increase of proenkephalin mRNA and enkephalin content of rat striatum after daily injection of haloperidol for 2 to 3 weeks, Proc. Natl. Acad. Sci. USA, 80 (1983) 3841–3844. Tang, F. and Lau, S.M., Chronic haloperidol treatment decreases preproenkephalin mRNA in the anterior pituitary: a study by solution-hybridization-RNase protection assay, Neurosci. Lett., 221 (1996) 66–68. Tang, F. and Man, S.Y., Hypothyroidism and pituitary contents of immunoactive met-enkephalin and b-endorphin in male rats of different ages, Proc. Soc. Exp. Biol. Med., 188 (1988) 82–86. Wang, Z.P., Man, S.Y. and Tang, F., Age related changes in the content of neuropeptides in the rat brain and pituitary, Neurobiol. Aging, 14 (1993) 529–534.
The effect of aging on the response of striatal preproenkephalin and preprotachykinin mRNA contents to chronic haloperidol treatment in rats: measurement by solution-hybridization RNase protection assay S.M. Lau, F. Tang* Department of Physiology, Faculty of Medicine, The University of Hong Kong, Hong Kong, China Received 11 December 1997; received in revised form 5 March 1998; accepted 6 March 1998
Abstract The preproenkephalin (PPeK) and preprotachykinin (PPT) mRNA contents in 3-, 10- and 23-month-old rats in the striatum were measured by solution hybridization-RNase protection assay after 3 weeks of haloperidol injection. Haloperidol increased striatal PPek mRNA. There was no age-related difference in the response of striatal PPeK mRNA to chronic haloperidol treatment. The PPT mRNA decreased by 21% after the haloperidol treatment in young rats only. Meanwhile, age decreased the PPT mRNA by 27 and 24% in 10- and 23-month-old rats, respectively. It is concluded that there is a difference in the effects of aging on the response of PPek and PPT mRNA contents to haloperidol and that the loss of PPT mRNA response in 10- and 23-month-old rats might be due to the change of dopamine system of the striatum in these rats. 1998 Elsevier Science Ireland Ltd.
Keywords: Haloperidol; Preproenkephalin mRNA; Preprotachykinin mRNA; Striatum; Solution hybridization-RNase; Protection assay; Aging; Dopamine receptors
The regulation of the enkephalin system in the striatum by dopamine activity is well established. Haloperidol, a dopamine receptor antagonist, has been reported to elevate striatal met-enkephalin (ME) content [8,20] through the increase of gene expression. Both pro-enkephalin mRNA [19,21] and pre-proenkephalin (PPeK) mRNA [1,3] have been measured in the striatum and found to increase after haloperidol treatment. In contrast to the enkephalin system, dopamine exerts a stimulatory effect on the tachykinin system in the striatum. Dopamine (DA) receptor agonists increased substance P (SP) content of substantia nigra (SN) [18] and reduced SP of the striatum [6,12]. The increase of SP in SN was shown to be due to an increase of SP biosynthesis in striatum [7]. The inhibitory effect of haloperidol was later proved to be due to a decrease of SP biosynthesis by measuring the preprotachykinin mRNA * Corresponding author. Tel.: +853 281 99269; fax: +852 2 8559730.
content in the striatum [2,13]. However, DA receptor antagonists seemed to have no effect on SP level in the striatum. In the central nervous system, dopamine activity is decreased with age in different brain areas of several animal species including man [16]. The decline of DA found in the striatum, substantia nigra and limbic system is because the age-related decrease in the activity and concentration of the rate-limiting enzyme, tyrosine hydroxylase (TH) [17]. The decrease was particularly obvious in the striatum and the brainstem in senescent rats [15]. Meanwhile, the D1 and D2 dopamine receptors decrease in the striatum of old rats [10]. The age-related decrease in DA content and D1 and D2 receptors may modify the response of preproenkephalin and preprotachykinin systems to chronic haloperidol treatment in the striatum. This possibility is studied by measuring the mRNA content of PPT and PPeK with a sensitive solution hybridization-RNase protection assay. Specific pathogen free male Sprague–Dawley rats
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00202- X
34
S.M. Lau, F. Tang / Neuroscience Letters 246 (1998) 33–36
(Charles River Laboratories, Japan) aged 3 months (young), 10 months (adult) and 20–23 months (old) received daily haloperidol (Sigma) injections for 3 weeks at 2 mg/kg body weight. The control groups received vehicle consisting of 0.9% saline at the same pH. Rats were maintained on a 12:12 h cycle (light on at 0600 h) at 22°C and 45–55% humidity. Rat pellets (PMI Laboratory Autoclavable Rodent Diet 5010, from PMI feeds Inc., Richmond, IN, USA) and drinking water (UV-treated) were provided ad libitum. The experiments were performed twice. In the first experiment, only PPeK mRNA was measured while in the second experiment, both PPeK and PPT mRNAs were measured. Rats were sacrificed by decapitation. The RNAs of the striatum was extracted by guanidinium thiocynate solution [5]. The striatum was homogenized in 1 ml cold solution D (4 M guanidinium thiocynate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, 10 mM 2-mercarptoethanol) by a polytron for three bursts of 10 s each. One hundred microliters sodium acetate (2 M, pH 4.0), 1 ml water-saturated phenol (pH 7.0) and 200 ml chloroform were added. The mixture was vigorously vortexed for 10 s and then cooled down on ice. The two phases were partitioned by centrifugation for 20 min. An equal volume of isopropanol was added to the upper aqueous phase. The precipitation proceeded at −20°C overnight. After centrifugation, the RNA pellet was re-dissolved in 0.3 ml solution D. The RNAs were again precipitated by isopropanol and washed by 1 ml 70% ethanol. Finally, RNA samples were reconstituted in 30 ml Tris EDTA buffer. The mRNAs of PPT or PPeK in the striatal samples were quantified by a solution hybridization-RNase protection assay technique [22]. The plasmid containing cDNA of bPPT were obtained from Prof. J.E. Krause (School of Medicine, Washington University, St. Louis, MO, USA) and the pYSEC 1 and pYSEA 1 plasmids were provided by Dr. S. Sabol (NIH, Bethesda, MD, USA) and Dr. J. Hong (NIEHS, Research Traingle Park, NC, USA). The b-actin plasmid was the gift of Dr. D. Autelitano (Baker Medical Institute, Melbourne, Australia). A kit (Rioboprobe Tanscription System, Promega, Madison, WI, USA) was used for in vitro transcription. For the synthesis of the antisense RNA, 10 mM dithiothreitol, 40 units RNasin, 0.5 mM ATP, CTP, GTP, 15 mM UTP, 1 mg linearized RNA (cut by Eco RI), 25 mCi 32P-UTP, 7.5U SP6 RNA polymerase, and 1× transcription buffer in a total volume of 20 ml were incubated at 37°C for 1 h. The average specific activity of antisense PPeK (930 nt) b−PPT (567 nt) and actin (387 nt) were 210 c.p.m/pg, 250 c.p.m/pg and 225 c.p.m/pg, respectively. The sense RNAs (standard) were also synthesized using T7 RNA polymerase, and 10 mg of linearized template DNA (cut by Hind III). RNA samples/standard and 32P-riboprobe (100 000 c.p.m) were denatured at 85°C for 5 min. Approximately 1.5 mg, 2 mg and 10 mg total RNA were used for actin mRNA, PPT mRNA and PPeK mRNA assays, respectively. For the standard points, yeast total RNA (similar amount as in sample used) was added. The mixture was allowed to
hybridize at 45°C overnight in hybridization buffer (80% formamide, 40 mM PIPE, 400 mM NaCl and 1 mM EDTA (pH 8.0)). The unhybridized RNAs were digested at 37°C for 45 min by RNase A and RNase T3. After adding 20 ml 10% sodium dodecyl sulfate, the enzymes were digested by proteinase K (5 ml) and 1 mg of yeast RNA carrier was added. The hybrids were separated by 4% polyacrylamide gel (PAGE) with 1× Tris–Borate EDTA buffer as a running buffer at 160 V and 34 mA for 1 h. The hybrid bands on the gel were cut out using the X-ray film exposed to the blot as template and counted. A standard curve could be drawn by plotting the radioactivity versus the pg of standard points. The result was presented as pg mRNA per pg b-actin mRNA. The values of the mRNAs were not corrected to their native lengths. Statistical analysis was performed by 2way analysis of variance (ANOVA) and Student’s t-test. Statistical significance was set at P , 0.05. The separation of the hybrids for PPek was shown in Fig. 1, in which different amounts of total striatal RNA were used to demonstrate a linear relationship between this and radioactivity in the hybrids. The X-ray film of polyacrylamide gel electrolysis of RNA hybrids of standard and striatal PPT mRNA is shown in Fig. 2. In both experiments, haloperidol treatment increased PPeK mRNA in the striatum of rats of all three ages. In the first experiment, the values (mean ± SE) for the control rats were 51.0 ± 6.6 (n = 10), 46.8 ± 5.3 (n = 8) and 38.4 ± 6.9 (n = 9) for the young, adult and old rats, respectively. The values for the haloperidol rats were 87.5 ± 7.3 (n = 8), 67.3 ± 8.4 (n = 6) and 77.6 ± 8.5 (n = 6). The data obtained from experiment 2 is shown in Fig. 3. By two-way ANOVA, a treatment effect was noted. After haloperidol treatment, the striatal PPT mRNA decreased significantly by 21% in young rat group (Fig. 4). Meanwhile, aging decreased the striatal PPT mRNA by 27 and 24% in the middle-aged and old rat group. No haloperidol effect was noted in adult or old rats. The PPeK mRNA was elevated after haloperidol injection in all age groups (Fig. 3). A corresponding increment of met-enkephalin (ME) was found in previous studies [9,11]. Haloperidol increases striatal ME by acting on D2 receptors; the finding of an increase of PPeK mRNA after haloperidol
Fig. 1. The X-ray film of polyacryamide gel electrophoresis of RNA hybrid of striatal PPeK mRNA with 32P-labeled PPeK probe. There is a linear relationship between the radioactivity of the hybrid and the amount of striatal RNA used. The numbers on the top of the lanes are mg total RNA used. The probe was shown on the left (arrow). The hybrids of the PPeK standards were not shown here but could be found in Tang and Lau [22].
S.M. Lau, F. Tang / Neuroscience Letters 246 (1998) 33–36
35
Fig. 2. The X-ray film of polyacryamide gel electrophoresis of RNA hybrids of standards and striatal PPT mRNA with 32P-labeled b-PPT probe. The three arrows marked the positions (from above) of b−PPT hybrid (508 bp), gPPT hybrid (295 bp) and gPPT hybrid (168 bp). The details could be found in Carter and Krause [4]. Lanes 1–6, standard; lane 1, 0 pg; lane 2, 10 pg; lane 3, 50 pg; lane 4, 100 pg; lane 5, 500 pg; lane 6, 1000 pg.
was in agreement with earlier reports [1,3,19,21]. Dopamine concentration declines with age [16] while the D1 and D2 dopamine receptors decrease in the striatum of old rats [10]. We have previously observed a blunted response in the ME content in the striatum in old rats upon haloperidol treatment [11]. This blunting in response, however, is not found for striatal PPeK mRNA levels in the present study. Aging therefore does not modify the response of striatal PPek mRNA to haloperidol treatment in spite of a decrease in dopamine system in the old rats. As much as both aging and haloperidol lead to a decrease in dopamine activity but only haloperidol results in an increase in PPeK mRNA content, aging must have its own effect in decreasing PPeK mRNA content. This is supported by the finding of a lack of increase of PPeK mRNA (the present study) and ME [11,24] with age. It is perhaps pertinent to note that in one of our experiments, we found a borderline decrease in PPeK mRNA content in the striatum of the old rats. Meanwhile, haloperidol caused a decrease in total PPT mRNA in the striatum. The result was supported by previous findings [2,6,7]. However, there was no significant decrease of substance P (SP) in our previous study [11] and in other studies [8,14]. The explanation of this result might be that the striato-nigral SP containing neurons project to SN with their cell bodies situated in the striatum.
Fig. 3. The PPeK mRNA levels in the striatum in different age groups after 3 weeks of haloperidol injection. RNA samples were extracted by GnSCN solution. Data are the mean ± SE with n value in parentheses. By a Student’s t-test, *P , 0.05 when compared to the control group. Two-way ANOVA: P = 0.093 (d.f. = 2/53, F = 2.5) for the aging effect, P , 0.001 (d.f. = 1/53, F = 28) for the haloperidol effect, P = 0.82 (d.f. = 2/53, F = 0.2) for the interaction between aging and haloperidol treatment.
Fig. 4. The PPT mRNA levels in the striatum in different age groups after 3 weeks of haloperidol injection. RNA samples were extracted by GnSCN solution. Data are the mean ± SE with n value in parentheses. Student’s T-test: *P , 0.05 when compared to the control group, **P , 0.05 when compared to young control group. Two-way ANOVA: P , 0.001 (d.f. = 2/50, F = 9.8) for the aging effect, P = 0.006 (d.f. = 1/50, F = 8.3) for haloperidol effect and P = 0.262 (d.f. = 2/50, F = 1.4) for the interaction between aging and haloperidol treatment.
Therefore, it is expected that DA receptor antagonism decreases the SP level in SN and reduces the PPT mRNA in the striatum. The decrease of SP caused by DA antagonist in SN had been extensively documented [6,8,12]. In the adult and old rats, the significant decrease of PPT mRNA contents compared with young rats, which is a developmental change before maturity, also agrees with the previous findings of an age-related decline in dopamine activity [10,16]. Here, both aging (as defined by change with time) and haloperidol decrease dopamine activity, and decrease PPT mRNA content so that the effect of aging seems to be mediated through the dopamine system. However, the effect of haloperidol is already abolished in 10month-old rats, a finding which correlates with the decrease of D1 and D2 receptors at 7 and 12 months of age [10]. It is possible that aging and haloperidol work via a common pathway to decrease dopamine activity and as a result, no haloperidol effect can be observed in the older rats. Alternatively, aging might have modified the dopamine system (e.g. by decreasing its receptors) such that it will not respond to haloperidol any more. Perhaps it should be noted that rats live to a maximum life span of 36 months and if the last quarter is taken as ‘senescence’ (Finch, as quoted by Pradhan [16]), the rat has to be 27 months old before it can be ‘old’. However, lots of publications have used 24-, 20-, or even 18-month-old rats as ‘old rats’ and even at 18 months of age, there are already lots of physiological changes [23]. In summary, this study provides strong evidence that the dopaminergic system stimulates the tachykininergic system but inhibits the enkephaline system in the striatum through the change of gene expression of their precursors. Because of the differences in the effects of aging and haloperidol on the two systems, there is a difference in the effect of aging on the response to haloperidol. The abolition of the tachykinin response to haloperidol may mean that in the older rats some peptidergic systems may have lost the ability to
36
S.M. Lau, F. Tang / Neuroscience Letters 246 (1998) 33–36
respond or there may be a change in the threshold of either dopaminergic or the peptidergic systems in the response to haloperidol. This may have important repercussions in the physiology of old animals as these systems play important roles in neurotransmission and neuromodulation. The study of the interaction of DA with peptidergic systems is particularly important as additive losses of neurotransmitters and receptors could have pathophysiological consequences and pharmacological implications in the elderly.
[10]
[11]
[12]
We thank Professor J.E. Krause (School of Medicine, Washington University, St. Louis, MO, USA) for the gift of cDNA of b−PPT, Dr. S. Sabol (NIH, Bethesda, MD, USA) and Dr. J. Hong (NIEHS, Research Triangle Park, NC, USA) for the gift of cDNA of pYSEC1 and pYSEA1. We are also indebted to Dr. D. Autelitano (Baker Medical Research Institution, Melbourne, Australia) for the gift of b-actin plasmid and his help in setting up the solution hybridization-RNase protection assay. [1] Abood, M.E., Eberwine, J.H., Erdelyi, E. and Evans, C.J., Regulation of both preproenkephalin mRNA and its derived opioids by haloperidol – a method for measurement of peptides and mRNA in the same tissue extract, Mol. Brain Res., 8 (1990) 243–248. [2] Bannon, M.J., Lee, J.M., Giraud, P., Young, A., Affolter, H.U. and Bonner, T.I., Dopamine antagonist haloperidol decrease substance P, substance K and preprotachykinin mRNAs in rat striatonigral neurons, J. Biol. Chem., 261 (1986) 6640–6642. [3] Bernard, V., Le Moine, C. and Bloch, B., Striatal neurons express increased level of dopamine D2 receptor mRNA in response to haloperidal treatment: a quantitative in situ hybridization study, Neuroscience, 45 (1991) 117–126. [4] Carter, M.S. and Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat pre-protachykinin gene encoding substance P, neurokinin A, neuropeptide K and neuropeptide, J. Neurosci., 10 (1990) 2203–2214. [5] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156–159. [6] Hanson, G.R., Alphs, L., Wolf, W., Pradhan, S. and Lovenberg, W., Response of striatonigral substance P systems to a dopamine receptor agonist and antagonist, Neuropharmacology, 20 (1981) 541–548. [7] Haverstick, D.M., Rubenstein, A. and Bannon, M.J., Striatal tachykinin gene expression regulated by interaction of D1 and D2 dopamine receptors, J. Pharmacol. Exp. Ther., 248 (1989) 858–862. [8] Hong, J.S., Yang, H.Y. and Costa, E., Substance P content of substantia nigra after chronic treatment with antischizophrenic drugs, Neuropharmacology, 17 (1978) 83–85. [9] Hong, J.S., Yang, H.Y.T., Gillin, J.C., Di Giulio, A.M., Fratta, W.
[13]
[14]
[15]
[16] [17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
and Costa, E., Chronic treatment with haloperidol accelerates the biosynthesis of enkephalin in rat striatum, Brain Res., 160 (1979) 192–195. Hyttel, J., Age related decrease in the density of dopamine D1 and D2 receptors in corpus striatum of rats, Pharmacol. Toxicol., 61 (1987) 126–129. Lau, S.M. and Tang, F., The effect of haloperidol on met-enkephalin, beta-endorphin, cholecytokinin and substance P in the pituitary, the hypothalamus and the striatum of rats during aging, Prog. Neuro-Psychopharmacol. Biol. Psychiatr., 19 (1995) 1163–1175. Li, S.J., Sivam, P., McGinty, J.F., Huang, Y.S. and Hong, J.S., Dopaminergic regulation of tachykinin metabolism in the striatonigral pathway, J. Pharmacol. Exp. Ther., 243 (1987) 792– 798. Lindefors, N., Amphetamine and haloperidol modulate preprotachykinin A mRNA expression in rat nucleus accumbens and caudate-putamen, Mol. Brain Res., 13 (1992) 151–154. Nylander, I. and Terenius, L., Chronic haloperidol and clozapine differentially affect dynorphin peptides and substance P in basal ganglia of the rat, Brain Res., 380 (1986) 34–41. Ponzio, F., Calderini, G., Lomuscio, G., Vantini, G., Toffano, G. and Algeri, S., Changes in monoamines and their metabolite levels in some brain regions of aged rats, Neurobiol. Aging, 3 (1982) 23–29. Pradhan, S.N., Central neurotransmitters and aging, Life Sci., 26 (1980) 1643–1656. Reis, D.J., Ross, R.A. and Tong, H.J., Changes in the activity and amounts of enzymes synthesizing catecholamines and acetylcholine in brain, adrenal medulla and sympathetic ganglia of aged rat and mouse, Brain Res., 136 (1977) 465–474. Ritter, J.K., Schmidt, C.J., Gibb, J.W. and Hanson, G.R., Increases of substance P-like immunoreactivity within striatalnigral structures after subacute methamphetamine treatment, J. Pharmacol. Exp. Ther., 229 (1984) 487–492. Sabol, S.L., Yoshikawa, K. and Hong, J.S., Regulation of methionine-enkephalin precursor messenger RNA in rat striatum by haloperidol and lithium, Biochem. Biophys. Res. Commun., 113 (1983) 391–399. Sivam, S.P., Strunk, C., Smith, D.R. and Hong, J.S., Proenkephalin-A gene regulation in the rat striatum: influence of lithium and haloperidol, Mol. Pharmacol., 30 (1986) 186–191. Tang, F., Costa, E. and Schwarz, J.P., Increase of proenkephalin mRNA and enkephalin content of rat striatum after daily injection of haloperidol for 2 to 3 weeks, Proc. Natl. Acad. Sci. USA, 80 (1983) 3841–3844. Tang, F. and Lau, S.M., Chronic haloperidol treatment decreases preproenkephalin mRNA in the anterior pituitary: a study by solution-hybridization-RNase protection assay, Neurosci. Lett., 221 (1996) 66–68. Tang, F. and Man, S.Y., Hypothyroidism and pituitary contents of immunoactive met-enkephalin and b-endorphin in male rats of different ages, Proc. Soc. Exp. Biol. Med., 188 (1988) 82–86. Wang, Z.P., Man, S.Y. and Tang, F., Age related changes in the content of neuropeptides in the rat brain and pituitary, Neurobiol. Aging, 14 (1993) 529–534.