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The relationship between MRNA levels and the locomotor response to novelty
BRAIN RESEARCH ELSEVIER
Brain Research 663 (1994) 312-316
Research report
The relationship between M R N A levels and the locomotor response to novelty M. Stacy Hooks 1, Barbara A. Sorg, Peter W. Kalivas * Alcohol and Drug Abuse Program and the Department of Veterinary and Comparatit, e Anatomy, Pharmacology and Physiology, Washington State Uniuersity, Pullman, WA 99164-6520, USA Accepted 16 August 1994
Abstract Differences in behavioral and neurochemical responses to drugs of abuse and environmental stress have been observed between rats that have a greater locomotor response in a novel environment (high responders: HR) compared to those that have a low response to novelty (low responders: LR). This study examined nuclei associated with the nigrostriatal and mesolimbic systems for differences in mRNA content between HR and LR using Northern blot analysis. These brain regions were chosen because of their role in both drug abuse and stress responses. The mRNAs examined code for either peptide transmitters that interact with the dopaminergic system or components of the dopaminergic system that have not been previously examined for differences between HR and LR. HR rats had approximately 50% lower levels of mRNA for/3-preprotachykinin (PPT) in the core of the nucleus accumbens (NACC) compared to LR. No differences between HR and LR in mRNA levels for dynorphin (DYN), preproenkephalin (PPE), glutamic acid decarboxylase (GAD) or neurotensin (NT) were observed in the core of the NACC. In the shell region of the NACC, HR exhibited a 25% reduction in the level of mRNA for NT compared to LR. No differences between HR and LR in mRNA levels for PPT, DYN, PPE or GAD were observed in the shell of the NACC. In the medial frontal cortex and the dorsal striatum, no differences between HR and LR in mRNA levels for PPT, DYN, PPE, GAD or NT were found. In the substantia nigra and ventral tegmental area no differences between HR and LR in mRNA levels for tyrosine hydroxylase, GAD, cholecystokinin, or NT were noted. This work indicates that neurotransmitters other than dopamine may contribute to the differences between HR and LR and that these differences may reside to a greater extent in the mesolimbic than the nigrostriatal systems.
Keywords: Nucleus accumbens; mRNA; Neurotensin; Tachykinin; Novelty; Individual difference
I. Introduction The locomotor response expressed by rats in response to a novel environment has been shown to be predictive of an animal's behavioral response to drugs of abuse [5-9,16]. Recent studies have investigated the neurochemical underpinnings of individual variability in response to novelty. Subjects with a high locomotor response to novelty, high responders (HR), have a greater increase in extracellular dopamine in the nucleus accumbens (NACC) than animals showing a low locomotor response to novelty, low responders (LR),
* Corresponding author. Fax: (1) (509) 335-4650. Current address: CytoTherapeutics, Inc., Two Richmond Square, Providence, RI 02906, USA. Elsevier Science B.V.
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 0 0 4 - 8
following administration of either cocaine [5,7] or amp h e t a m i n e [2,8]. It has also been shown that H R have higher basal levels of extracellular dopamine in the N A C C in addition to higher dopamine turnover as m e a s u r e d by the tissue content of dopamine metabolites c o m p a r e d to LR. Recently, several studies were conducted to determine if basal differences existed in other components of the dopamine system [7-9,17]. The number of dopamine D 2 receptors were reduced in H R c o m p a r e d to LR in both the N A C C and striatum (STR) [9]. Moreover, the m R N A content for the D z receptor was also reduced [9]. A similar reduction in m R N A for D 2 receptors was measured in both the shell of the N A C C (Shell-NACC) and the core of the N A C C (Core-NACC). While the role of the dopaminergic system in producing individual differences has been extensively stud-
M.S. Hooks et al. ~Brain Research 663 (1994) 312-316
ied, other neurotransmitter systems have not been examined. Considering the apparent importance of mesoaccumbens dopamine transmission, it seemed most likely that neurotransmitters known to interact with dopamine transmission would most likely reflect differences between HR and LR. Several studies have identified neurotransmitter systems that modulate mesolimbic and nigrostriatal dopamine transmission. Axonal dopamine release was produced following stimulation of GABA A, neurotensin (NT), enkephalin, or tachykinin (PPT) receptors in the ventral tegmental area (VTA) or substantia nigra (SN) [12,13]. In contrast, dynorphin (DYN) reduced the axonal release of dopamine while cholecystokinin (CCK) had no effect [13]. Dendritic dopamine release was increased by GABAA, NT and enkephalin agonists and decreased by tachykinin agonists administered into the VTA or SN. Moreover, GABAA, NT, enkephalin, CCK, DYN and PPT altered dopamine cell firing [13]. Likewise, in dopamine terminal fields such as the NACC and striatum dopamine modulates neurons containing GABA, NT, enkephalin, PPT and DYN [13]. In the present study, we hypothesized that a change in the content of mRNAs coding for the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase (TH), in addition to mRNA coding for other neurotransmitter synthesizing enzymes or neuropeptides would be observed between HR and LR. The mRNAs were chosen because of their regulatory interactions with the mesolimbic and/or nigrostriatal dopamine systems. These brain regions were examined because of their role in mediating the effects of stress and drugs of abuse [12,20]. Northern blot analysis was performed to measure the content of several mRNAs, including /3preprotachykinin (PPT), dynorphin (DYN), preproenkephalin (PPE), glutamic acid decarboxylase (GAD), neurotensin (NT), tyrosine hydroxylase (TH) and cholecystokinin (CCK).
2. Materials and methods
313
have found that a 3-day interval between behavioral tests permits a stable behavioral baseline (see [13] for a review). Nonetheless, it remains possible that an interaction between the novelty test and mRNA levels may be present in this study. The tissue was dissected on a cooled glass plate and rapidly frozen at -80°C until use. 2.2. RNA isolation and northern blot analysis Total RNA was isolated from dissected tissue by the method of Bingham and Zachar [1]. The concentration of total RNA was estimated by optical absorption (A260). Denatured total RNA was subjected to agarose formaldehyde gel electrophoresis, transferred to a nylon membrane and cross-linked to the membrane by UV irradiation. Oligonucleotides were end-labeled with 32p-dATP (New England Nuclear; Boston, MA) and hybridized to RNA. Non-specifically bound probe was washed from the membrane, using a final stringency of 1XSSC/0.1% SDS at 42°C (1XSSC=0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0). The membranes were exposed to preflashed FUJI-RX film at -80°C with an intensifying screen using exposures that were within the linear range. A 32p_ labeled /3-actin was used to control for loading equal amounts of RNA in each lane of the gel, because/3-actin mRNA levels were not altered as a result of response to novelty. Sizes of mRNA bands were determined using an RNA standard (GIBCO BRL, Gaithersburg, MD). Relative optical density for each band was quantified by computerized videodensitometry (Image 1.41, Dr. Wayne Rasband, National Institutes of Health). The system was calibrated using a photographic step tablet # 2 (Eastman Kodak, Rochester, NY)
2.3. Oligonucleotides probes Probes for /3-actin (48-bases) [15], PPT (bases 777-818), DYN (bases 862-909) [18], PPE (bases 388-435) [23], GAD (bases 12071254) [22] and NT (bases 443-487) [14] were used to measure the corresponding mRNA levels in the terminal field regions, including Core-NACC, ShelI-NACC, Dorsal-STR and MFC. Probes for fl-actin, GAD, NT, TH (bases 1266-1315) [4] and CCK (bases 313-362) [3] were used for measurement of these mRNA levels in the cell body regions, including the VTA and SN. For analysis, the value for relative optical density was divided by the value of the control fl-actin mRNA and the resulting ratio was expressed as a percentage of the average of the LR rats.
2.4. Statistical analysis The optical density of the /3-actin bands for HR and LR were analyzed using an unpaired Student's t-test. After it was determined that fl-actin did not differ between the two groups, the other bands
2.1. Animal housing, behavioral screening and tissue preparation
ShelI-NACC
Core-NACC Male Sprague-Dawley rats ( n = 2 4 , Simonsen, Gilroy, CA) weighing 275-320 g were housed 3-4 per cage and maintained on a 12 h light/dark cycle with free access to food and water. Subjects were screened for their locomotor response to novelty in Plexiglas photocell cages between 09:00-13:00 h. Rats were placed in the photocell cages for a 1-h period and were classified as HR if their locomotor response to novelty was above the median for the population screened. LR were rats whose locomotor response to novelty was below the median. Three days following novelty screening, tissue was taken from the Core-NACC, ShelI-NACC, Dorsal-STR, MFC, SN and the VTA. Rats were decapitated 3 days after novelty testing in an effort to minimize the potential alterations in mRNA levels that may have been induced by exposure to a novel environment. Three days was chosen because in many microinjection studies we
LR HR
LR H R NT
PPT ii
fl-act
fl-act
Fig. 1. Northern blots of PPT and fl-actin in the Core-NACC and of NT and fl-actin in the Shell of the NACC.
314
M.S. Hooks et al. / B r a i n Research 663 (1994) 312-316
125
f
A, Core-NACC
~ 125
t B. ShelI-NACC
;i1 / li'O01 E 125
1
1 t°
1
~o. 75
50
25
'PPT
DYN
PPE
GAD
0
NT
PPT
DYN
PPE
GAD
NT
Fig. 2. As seen in Panel A the m R N A level for PPT was lower in high responders compared to low responders. In the shell of the N A C C (Panel B) the m R N A level for neurotensin was lower in high responders compared to low responders. No significant differences between high responders and low responders were observed in the Dorsal-STR (Panel C) or the MFC. Open bars represent high responders (n = 12) and closed bars represent low responders (n = 12). * P < 0.05.
were analyzed as a ratio of m R N A to fl-actin to control for small differences in loading. A n unpaired Student's t-test was used for group comparison of m R N A levels following Northern analysis. In addition, linear regression was performed to determine the relationship between m R N A levels and response to novelty.
NACC and SheI1-NACC. The measured sizes for m R N A were as follows: PPT - 1.1 kb; DYN - 2.6; PPE - 1.5; G A D - 3.7; NT - 1.5; TH - 1.9; CCK 0.8; /3-ACT - 2.3. The two significant changes observed between H R and LR were PPT and NT m R N A in the Core- and Shell-NACC, respectively. The level of PPT m R N A was approximately 40% lower in H R than in LR in the Core-NACC (T(1,22)= 3.56, P << 0.005, Fig. 2). The m R N A concentration for PPT in the Core-NACC and the locomotor response to novelty were inversely correlated ( r = - 0 . 5 3 , P<<0.01). NT m R N A levels were reduced approximately 28% in H R compared to LR in the ShelI-NACC (T(1,22)= 3.46, P < 0.005, Fig. 2B). The m R N A concentration for NT in the Shell-NACC and the locomotor response to novelty were inversely correlated, ( r = - 0 . 6 0 , P<
3. Results High responders (counts = 12,939 _+ 932; n = 12) had approximately a 30% greater locomotor response to novelty than low responders (counts = 9,837 + 756; n = 12; T(1,22) = 4.78, P < 0.0001). The /3-actin concentration did not differ between H R and LR in any of the regions examined (data not included). Fig. 1 illustrates representative Northern Blots from the Core-
P 125 #0 '1o c 100 0 o~ 75 Q3 r~ •
5 N
50
25
e
A. SN
125
1111117/ '1o c 100 e~
75
r~ •
5O
2~
25
Iii1111 o
0
TH GAD NT CCK TH GAD NT CCK Fig. 3. There were no detectable differences between high responders (open bars; n = 12) and low responders (darken bars; n = 12) in either the substantia nigra (Panel A) or the ventral tegmental area (Panel B) in the mRNA examined.
M.S. Hooks et al. / Brain Research 663 (1994) 312-316
0.0025). A typical autoradiograph for PPT and /3-actin and NT and b-actin in the Core-NACC and ShellNACC, respectively, is shown in Fig. 1. The m R N A levels for PPT or N T were not significantly different between H R and LR in the other terminal fields (Fig. 2). The m R N A levels for DYN, PPE or G A D also did not significantly differ between H R and LR in the terminal fields examined (Fig. 2). Fig. 3 shows that there were no differences in m R N A levels between H R and LR in the VTA and SN, including m R N A for TH, CCK, N T and GAD.
4. Discussion The data further demonstrate the importance of the NACC as the site of changes that produce individual differences in the locomotor response to novelty. Variation in P P T m R N A levels in the Core-NACC and in neurotensin m R N A in the ShelI-NACC was observed. Previous studies have shown that the NACC dopamine system is involved in producing the differences between H R and LR rats [2,5,7-9,17]. While the current data further implicate the NACC in production of individual differences, they also demonstrate that T H m R N A levels in the SN and VTA are not different between H R and LR. The differences between H R and LR are often compared with the differences observed between amphetamine or cocaine sensitized subjects and controls, since the behavioral differences of these groups are similar [8,9]. In the present experiment we observed a significant reduction in the concentration of PPT m R N A in the Core-NACC. Previous work with repeated cocaine has observed small increases in PPT m R N A levels [11]. In some cases these were only transient increases [21]. Both of these studies observed increases in m R N A content for PPT, DYN and PPE in the dorsal striatum after repeated cocaine administration. Because of variability in the treatment schedules and withdrawal periods used in the repeated cocaine experiments, it is difficult to determine if underlying differences between H R and LR and sensitized and control subjects are mediated by the same differences in PPT, DYN or PPE. Hurd et al. [10] showed that repeated amphetamine produced an increase in the amount of T H and CCK m R N A in the VTA and SN. Specifically, there was a reduction in the number of ceils containing CCK m R N A with an increase in T H and CCK m R N A levels per cell. In addition, Sorg et al. [19] have shown no differences between cocaine sensitized and control subjects in T H m R N A content in the VTA. These results do not show a consistent pattern of m R N A changes following psychomotor stimulant sensitization. Therefore, it is difficult to make comparisons between stud-
315
ies that use different treatment and withdrawal schedules to determine the role of m R N A changes in drug vulnerability. The results support the importance of the NACC in producing individual differences in response to novelty and indicate that neurotransmitters located postsynaptic to dopamine innervation show variation between H R and LR. Both N T an PPT neurons in the NACC project to the ventral pallidum [13]. This projections critical to the expression motor activity elicited from the NACC and there is evidence that in the ventral pallidum both NT and tachykinins regulate the expression of motor activity [13]. Although the tachykinin projection emanates from both the shell-NACC and the core-NACC, the N T neurons contribute selectively to the projection from the shell-NACC to the ventral pallidum [13]. This latter anatomical observation is consistent with the selective reduction in N T m R N A measured in the shell-NACC of the H R compared to LR. The results from the present study indicate that not all components of the dopaminergic system are different between H R and LR and that the tachykinin and neurotensin systems may contribute to the differences observed between H R and LR. This is further evidence that the differences between H R and LR are not the result of a simple change in one neurotransmitter system, but are a consequence of a more complex pattern of neurochemical changes in a multicomponent circuit involving several transmitters.
Acknowledgements We thank Jenny Baylon for assistance in preparing the manuscript. The research was supported by National Research Service Award DA-05391 (MSH), National Institutes of Health Grants DA-07827 and DA08212 (BAS) and a Research Career Development award DA-OO158 (PWK).
References [1] Bingham, P.M. and Zachar, Z., Evidence that two mutations, W DZL and zl, affect synapsis-dependent genetic behavior of white and transcriptional regulatory mutations, Cell, 40 (1985) 819-823. [2] Bradberry, C.W., Gruen, R.J., Berridge, C.W. and Roth, R.H., Individual differences in behavioral measures: correlations with nucleus accumbens dopamine measured by microdialysis, Pharmacol. Biochem. Behav., 39 (1991) 877-882. [3] Deschenes, R.J., Lorenz, L.J., Haun, R.S., Roos, B.A., Collier, K.J. and Dixon, J.E., Cloning and sequence analysis of a cDNA encoding rat preprocholecystokinin,Proc. Natl. Acad. Sci., 81 (1984) 726-730. [4] Grima, B., Lamouroux,L., Blanot, F., Biguet, N.F. and Mallet, J., Completecoding sequenceof rat tyrosinehydroxylasemRNA, Proc. Natl. Acad. Sci., 82 (1985) 617-621.
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[5] Hooks, M.S., Jones, G.H., Smith, A.D., Neill, D.B. and Justice, J.B., Response to novelty predicts the locomotor and nucleus accumbens dopamine response to cocaine, Synapse, 9 (1991) 121-128. [6] Hooks, M.S., Jones, G.H., Liem, B.J. and Justice, J.B., Sensitization and individual differences to IP amphetamine, cocaine or caffeine following repeated intra-cranial amphetamine infusions, Pharmacol. Biochem. Behav., 43 (1992) 815-823. [7] Hooks, M.S., Colvin, A,, Juncos, J.L. and Justice, J.B. Individual differences in basal and cocaine stimulated dopamine levels in the nucleus accumbens, Brain Res., 587 (1992) 306-312. [8] Hooks, M.S., Individual Differences in Response to Drugs of Abuse: Behavioral and Neurochemical Correlates, Ph.D. Thesis, Emory University, 1992. [9] Hooks, M.S., Juncos, J.L., Justice, J.B., Meiergerd, S.M., Povlock, S.L., Schenk, J.O. and Kalivas, P.W., Individual locomotor response to novelty predicts selective alterations in D I and D 2 receptors and mRNAs, J. Neurosci., in press. [10] Hurd, Y.L., Lindefors, N., Brodin, E., Brene, S., Persson, H., Ungerstedt, U. and Hokfelt, T., Amphetamine regulation of mesolimbic dopamine/cholecystokinin neurotransmission, Brain Res., 578 (1992) 317-326. [11] Hurd, Y.L., Brown, E.E., Finlay, J.M., Fibiger, H.C. and Gerfen, C.R., Cocaine self-administration differentially alters mRNA expression of striatal peptides, Mol. Brain Res., 13 (1992) 165-170. [12] Kalivas, P.W., Sorg, B.A. and Hooks, M.S., The pharmacology and neural circuitry of sensitization to psychostimulants, BehaL'. Pharmacol., 4 (1993) 315-334. [13] Kalivas, P.W., Churchill, L. and Klitenick, M.A. The circuitry mediating the translation of motivational stimuli into adaptive motor response. In P.W. Kalivas and C.D. Barnes, Limbic Motor Systems and Neuropsychiatry, CRC Press, Boca Raton, FL, 1993, pp. 237-288.
[14] Kislauskis, E., Bullock, B., McNeil, S. and Dobner, P.R., The rat gene encoding neurotensin and neuromedin N, J. Biol. Chem., 263 (1988) 4963-4968. [15] Nudel, U., Zakut, R., Shani, M., Neuman, S., Lew, Z. and Yaffe, D., The nucleotide sequence of the rat cytoplasmic-actin gene, Nucleic Acid Res., 11 (1983) 1759-1771. [16] Piazza, P.V., Deminiere, J.M., Le Moal, M. and Simon, H., Factors that predict individual vulnerability to amphetamine self-administration, Science, 29 (1989) 1511-1513. [17] Piazza, P.V., Rouge-Pont, F., Deminiere, P.V., Kharoubi, M., Le Moal, M. and Simon, S., Dopaminergic activity is reduced in the prefrontal cortex and increased in the nucleus accumbens of rats predisposed to develop amphetamine self-administration, Brain Res., 567 (1991) 169-174. [18] Rosen, J.B., Cain, C.J., Weiss, S.R.B. and Post, R.M., Alterations in mRNA of enkephalin, dynorphin and thyrotropin releasing hormone during amygdala kindling: an in situ hybridization study, MoL Brain Res., 15 (1992) 247-255. [19] Sorg, B.A., Chen, S.Y. and Kalivas, P.W., Time course of tyrosine hydroxylase expression following behavioral sensitization to cocaine, J. Pharmac. Exp. Then., 266 (1993) 424-430. [20] Sorg, B.A. and Kalivas, P.W., Behavioral sensitization to stress and psychostimulants: role of dopamine and excitatory amino acids in the mesocorticolimbic systems, Sem. Neurosci., 5 (1993) 343-350. [21] Steiner, H. and Gerfen, C.R., Time course alterations in substance P, dynorphin, enkephalin and c-fos gene expression in striatal neurons during chronic cocaine treatment, Soc. Neurosci. Abstr., 18 (1992) 437.11. [22] Wyborski, R.J., Bond, R.W. and Gottlieb, DT, Characterization of a cDNA coding rat glutamic acid decarboxylase, Mol. Brain Res., 8 (1990) 193-198. [23] Yoshikawa, K., Williams, C. and Sabol, S.L., Rat brain preproenkephalin mRNA, Z Biol. Chem., 259 (1984) 14301-14308.
Brain Research 663 (1994) 312-316
Research report
The relationship between M R N A levels and the locomotor response to novelty M. Stacy Hooks 1, Barbara A. Sorg, Peter W. Kalivas * Alcohol and Drug Abuse Program and the Department of Veterinary and Comparatit, e Anatomy, Pharmacology and Physiology, Washington State Uniuersity, Pullman, WA 99164-6520, USA Accepted 16 August 1994
Abstract Differences in behavioral and neurochemical responses to drugs of abuse and environmental stress have been observed between rats that have a greater locomotor response in a novel environment (high responders: HR) compared to those that have a low response to novelty (low responders: LR). This study examined nuclei associated with the nigrostriatal and mesolimbic systems for differences in mRNA content between HR and LR using Northern blot analysis. These brain regions were chosen because of their role in both drug abuse and stress responses. The mRNAs examined code for either peptide transmitters that interact with the dopaminergic system or components of the dopaminergic system that have not been previously examined for differences between HR and LR. HR rats had approximately 50% lower levels of mRNA for/3-preprotachykinin (PPT) in the core of the nucleus accumbens (NACC) compared to LR. No differences between HR and LR in mRNA levels for dynorphin (DYN), preproenkephalin (PPE), glutamic acid decarboxylase (GAD) or neurotensin (NT) were observed in the core of the NACC. In the shell region of the NACC, HR exhibited a 25% reduction in the level of mRNA for NT compared to LR. No differences between HR and LR in mRNA levels for PPT, DYN, PPE or GAD were observed in the shell of the NACC. In the medial frontal cortex and the dorsal striatum, no differences between HR and LR in mRNA levels for PPT, DYN, PPE, GAD or NT were found. In the substantia nigra and ventral tegmental area no differences between HR and LR in mRNA levels for tyrosine hydroxylase, GAD, cholecystokinin, or NT were noted. This work indicates that neurotransmitters other than dopamine may contribute to the differences between HR and LR and that these differences may reside to a greater extent in the mesolimbic than the nigrostriatal systems.
Keywords: Nucleus accumbens; mRNA; Neurotensin; Tachykinin; Novelty; Individual difference
I. Introduction The locomotor response expressed by rats in response to a novel environment has been shown to be predictive of an animal's behavioral response to drugs of abuse [5-9,16]. Recent studies have investigated the neurochemical underpinnings of individual variability in response to novelty. Subjects with a high locomotor response to novelty, high responders (HR), have a greater increase in extracellular dopamine in the nucleus accumbens (NACC) than animals showing a low locomotor response to novelty, low responders (LR),
* Corresponding author. Fax: (1) (509) 335-4650. Current address: CytoTherapeutics, Inc., Two Richmond Square, Providence, RI 02906, USA. Elsevier Science B.V.
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 0 0 4 - 8
following administration of either cocaine [5,7] or amp h e t a m i n e [2,8]. It has also been shown that H R have higher basal levels of extracellular dopamine in the N A C C in addition to higher dopamine turnover as m e a s u r e d by the tissue content of dopamine metabolites c o m p a r e d to LR. Recently, several studies were conducted to determine if basal differences existed in other components of the dopamine system [7-9,17]. The number of dopamine D 2 receptors were reduced in H R c o m p a r e d to LR in both the N A C C and striatum (STR) [9]. Moreover, the m R N A content for the D z receptor was also reduced [9]. A similar reduction in m R N A for D 2 receptors was measured in both the shell of the N A C C (Shell-NACC) and the core of the N A C C (Core-NACC). While the role of the dopaminergic system in producing individual differences has been extensively stud-
M.S. Hooks et al. ~Brain Research 663 (1994) 312-316
ied, other neurotransmitter systems have not been examined. Considering the apparent importance of mesoaccumbens dopamine transmission, it seemed most likely that neurotransmitters known to interact with dopamine transmission would most likely reflect differences between HR and LR. Several studies have identified neurotransmitter systems that modulate mesolimbic and nigrostriatal dopamine transmission. Axonal dopamine release was produced following stimulation of GABA A, neurotensin (NT), enkephalin, or tachykinin (PPT) receptors in the ventral tegmental area (VTA) or substantia nigra (SN) [12,13]. In contrast, dynorphin (DYN) reduced the axonal release of dopamine while cholecystokinin (CCK) had no effect [13]. Dendritic dopamine release was increased by GABAA, NT and enkephalin agonists and decreased by tachykinin agonists administered into the VTA or SN. Moreover, GABAA, NT, enkephalin, CCK, DYN and PPT altered dopamine cell firing [13]. Likewise, in dopamine terminal fields such as the NACC and striatum dopamine modulates neurons containing GABA, NT, enkephalin, PPT and DYN [13]. In the present study, we hypothesized that a change in the content of mRNAs coding for the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase (TH), in addition to mRNA coding for other neurotransmitter synthesizing enzymes or neuropeptides would be observed between HR and LR. The mRNAs were chosen because of their regulatory interactions with the mesolimbic and/or nigrostriatal dopamine systems. These brain regions were examined because of their role in mediating the effects of stress and drugs of abuse [12,20]. Northern blot analysis was performed to measure the content of several mRNAs, including /3preprotachykinin (PPT), dynorphin (DYN), preproenkephalin (PPE), glutamic acid decarboxylase (GAD), neurotensin (NT), tyrosine hydroxylase (TH) and cholecystokinin (CCK).
2. Materials and methods
313
have found that a 3-day interval between behavioral tests permits a stable behavioral baseline (see [13] for a review). Nonetheless, it remains possible that an interaction between the novelty test and mRNA levels may be present in this study. The tissue was dissected on a cooled glass plate and rapidly frozen at -80°C until use. 2.2. RNA isolation and northern blot analysis Total RNA was isolated from dissected tissue by the method of Bingham and Zachar [1]. The concentration of total RNA was estimated by optical absorption (A260). Denatured total RNA was subjected to agarose formaldehyde gel electrophoresis, transferred to a nylon membrane and cross-linked to the membrane by UV irradiation. Oligonucleotides were end-labeled with 32p-dATP (New England Nuclear; Boston, MA) and hybridized to RNA. Non-specifically bound probe was washed from the membrane, using a final stringency of 1XSSC/0.1% SDS at 42°C (1XSSC=0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0). The membranes were exposed to preflashed FUJI-RX film at -80°C with an intensifying screen using exposures that were within the linear range. A 32p_ labeled /3-actin was used to control for loading equal amounts of RNA in each lane of the gel, because/3-actin mRNA levels were not altered as a result of response to novelty. Sizes of mRNA bands were determined using an RNA standard (GIBCO BRL, Gaithersburg, MD). Relative optical density for each band was quantified by computerized videodensitometry (Image 1.41, Dr. Wayne Rasband, National Institutes of Health). The system was calibrated using a photographic step tablet # 2 (Eastman Kodak, Rochester, NY)
2.3. Oligonucleotides probes Probes for /3-actin (48-bases) [15], PPT (bases 777-818), DYN (bases 862-909) [18], PPE (bases 388-435) [23], GAD (bases 12071254) [22] and NT (bases 443-487) [14] were used to measure the corresponding mRNA levels in the terminal field regions, including Core-NACC, ShelI-NACC, Dorsal-STR and MFC. Probes for fl-actin, GAD, NT, TH (bases 1266-1315) [4] and CCK (bases 313-362) [3] were used for measurement of these mRNA levels in the cell body regions, including the VTA and SN. For analysis, the value for relative optical density was divided by the value of the control fl-actin mRNA and the resulting ratio was expressed as a percentage of the average of the LR rats.
2.4. Statistical analysis The optical density of the /3-actin bands for HR and LR were analyzed using an unpaired Student's t-test. After it was determined that fl-actin did not differ between the two groups, the other bands
2.1. Animal housing, behavioral screening and tissue preparation
ShelI-NACC
Core-NACC Male Sprague-Dawley rats ( n = 2 4 , Simonsen, Gilroy, CA) weighing 275-320 g were housed 3-4 per cage and maintained on a 12 h light/dark cycle with free access to food and water. Subjects were screened for their locomotor response to novelty in Plexiglas photocell cages between 09:00-13:00 h. Rats were placed in the photocell cages for a 1-h period and were classified as HR if their locomotor response to novelty was above the median for the population screened. LR were rats whose locomotor response to novelty was below the median. Three days following novelty screening, tissue was taken from the Core-NACC, ShelI-NACC, Dorsal-STR, MFC, SN and the VTA. Rats were decapitated 3 days after novelty testing in an effort to minimize the potential alterations in mRNA levels that may have been induced by exposure to a novel environment. Three days was chosen because in many microinjection studies we
LR HR
LR H R NT
PPT ii
fl-act
fl-act
Fig. 1. Northern blots of PPT and fl-actin in the Core-NACC and of NT and fl-actin in the Shell of the NACC.
314
M.S. Hooks et al. / B r a i n Research 663 (1994) 312-316
125
f
A, Core-NACC
~ 125
t B. ShelI-NACC
;i1 / li'O01 E 125
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Fig. 2. As seen in Panel A the m R N A level for PPT was lower in high responders compared to low responders. In the shell of the N A C C (Panel B) the m R N A level for neurotensin was lower in high responders compared to low responders. No significant differences between high responders and low responders were observed in the Dorsal-STR (Panel C) or the MFC. Open bars represent high responders (n = 12) and closed bars represent low responders (n = 12). * P < 0.05.
were analyzed as a ratio of m R N A to fl-actin to control for small differences in loading. A n unpaired Student's t-test was used for group comparison of m R N A levels following Northern analysis. In addition, linear regression was performed to determine the relationship between m R N A levels and response to novelty.
NACC and SheI1-NACC. The measured sizes for m R N A were as follows: PPT - 1.1 kb; DYN - 2.6; PPE - 1.5; G A D - 3.7; NT - 1.5; TH - 1.9; CCK 0.8; /3-ACT - 2.3. The two significant changes observed between H R and LR were PPT and NT m R N A in the Core- and Shell-NACC, respectively. The level of PPT m R N A was approximately 40% lower in H R than in LR in the Core-NACC (T(1,22)= 3.56, P << 0.005, Fig. 2). The m R N A concentration for PPT in the Core-NACC and the locomotor response to novelty were inversely correlated ( r = - 0 . 5 3 , P<<0.01). NT m R N A levels were reduced approximately 28% in H R compared to LR in the ShelI-NACC (T(1,22)= 3.46, P < 0.005, Fig. 2B). The m R N A concentration for NT in the Shell-NACC and the locomotor response to novelty were inversely correlated, ( r = - 0 . 6 0 , P<
3. Results High responders (counts = 12,939 _+ 932; n = 12) had approximately a 30% greater locomotor response to novelty than low responders (counts = 9,837 + 756; n = 12; T(1,22) = 4.78, P < 0.0001). The /3-actin concentration did not differ between H R and LR in any of the regions examined (data not included). Fig. 1 illustrates representative Northern Blots from the Core-
P 125 #0 '1o c 100 0 o~ 75 Q3 r~ •
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TH GAD NT CCK TH GAD NT CCK Fig. 3. There were no detectable differences between high responders (open bars; n = 12) and low responders (darken bars; n = 12) in either the substantia nigra (Panel A) or the ventral tegmental area (Panel B) in the mRNA examined.
M.S. Hooks et al. / Brain Research 663 (1994) 312-316
0.0025). A typical autoradiograph for PPT and /3-actin and NT and b-actin in the Core-NACC and ShellNACC, respectively, is shown in Fig. 1. The m R N A levels for PPT or N T were not significantly different between H R and LR in the other terminal fields (Fig. 2). The m R N A levels for DYN, PPE or G A D also did not significantly differ between H R and LR in the terminal fields examined (Fig. 2). Fig. 3 shows that there were no differences in m R N A levels between H R and LR in the VTA and SN, including m R N A for TH, CCK, N T and GAD.
4. Discussion The data further demonstrate the importance of the NACC as the site of changes that produce individual differences in the locomotor response to novelty. Variation in P P T m R N A levels in the Core-NACC and in neurotensin m R N A in the ShelI-NACC was observed. Previous studies have shown that the NACC dopamine system is involved in producing the differences between H R and LR rats [2,5,7-9,17]. While the current data further implicate the NACC in production of individual differences, they also demonstrate that T H m R N A levels in the SN and VTA are not different between H R and LR. The differences between H R and LR are often compared with the differences observed between amphetamine or cocaine sensitized subjects and controls, since the behavioral differences of these groups are similar [8,9]. In the present experiment we observed a significant reduction in the concentration of PPT m R N A in the Core-NACC. Previous work with repeated cocaine has observed small increases in PPT m R N A levels [11]. In some cases these were only transient increases [21]. Both of these studies observed increases in m R N A content for PPT, DYN and PPE in the dorsal striatum after repeated cocaine administration. Because of variability in the treatment schedules and withdrawal periods used in the repeated cocaine experiments, it is difficult to determine if underlying differences between H R and LR and sensitized and control subjects are mediated by the same differences in PPT, DYN or PPE. Hurd et al. [10] showed that repeated amphetamine produced an increase in the amount of T H and CCK m R N A in the VTA and SN. Specifically, there was a reduction in the number of ceils containing CCK m R N A with an increase in T H and CCK m R N A levels per cell. In addition, Sorg et al. [19] have shown no differences between cocaine sensitized and control subjects in T H m R N A content in the VTA. These results do not show a consistent pattern of m R N A changes following psychomotor stimulant sensitization. Therefore, it is difficult to make comparisons between stud-
315
ies that use different treatment and withdrawal schedules to determine the role of m R N A changes in drug vulnerability. The results support the importance of the NACC in producing individual differences in response to novelty and indicate that neurotransmitters located postsynaptic to dopamine innervation show variation between H R and LR. Both N T an PPT neurons in the NACC project to the ventral pallidum [13]. This projections critical to the expression motor activity elicited from the NACC and there is evidence that in the ventral pallidum both NT and tachykinins regulate the expression of motor activity [13]. Although the tachykinin projection emanates from both the shell-NACC and the core-NACC, the N T neurons contribute selectively to the projection from the shell-NACC to the ventral pallidum [13]. This latter anatomical observation is consistent with the selective reduction in N T m R N A measured in the shell-NACC of the H R compared to LR. The results from the present study indicate that not all components of the dopaminergic system are different between H R and LR and that the tachykinin and neurotensin systems may contribute to the differences observed between H R and LR. This is further evidence that the differences between H R and LR are not the result of a simple change in one neurotransmitter system, but are a consequence of a more complex pattern of neurochemical changes in a multicomponent circuit involving several transmitters.
Acknowledgements We thank Jenny Baylon for assistance in preparing the manuscript. The research was supported by National Research Service Award DA-05391 (MSH), National Institutes of Health Grants DA-07827 and DA08212 (BAS) and a Research Career Development award DA-OO158 (PWK).
References [1] Bingham, P.M. and Zachar, Z., Evidence that two mutations, W DZL and zl, affect synapsis-dependent genetic behavior of white and transcriptional regulatory mutations, Cell, 40 (1985) 819-823. [2] Bradberry, C.W., Gruen, R.J., Berridge, C.W. and Roth, R.H., Individual differences in behavioral measures: correlations with nucleus accumbens dopamine measured by microdialysis, Pharmacol. Biochem. Behav., 39 (1991) 877-882. [3] Deschenes, R.J., Lorenz, L.J., Haun, R.S., Roos, B.A., Collier, K.J. and Dixon, J.E., Cloning and sequence analysis of a cDNA encoding rat preprocholecystokinin,Proc. Natl. Acad. Sci., 81 (1984) 726-730. [4] Grima, B., Lamouroux,L., Blanot, F., Biguet, N.F. and Mallet, J., Completecoding sequenceof rat tyrosinehydroxylasemRNA, Proc. Natl. Acad. Sci., 82 (1985) 617-621.
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[5] Hooks, M.S., Jones, G.H., Smith, A.D., Neill, D.B. and Justice, J.B., Response to novelty predicts the locomotor and nucleus accumbens dopamine response to cocaine, Synapse, 9 (1991) 121-128. [6] Hooks, M.S., Jones, G.H., Liem, B.J. and Justice, J.B., Sensitization and individual differences to IP amphetamine, cocaine or caffeine following repeated intra-cranial amphetamine infusions, Pharmacol. Biochem. Behav., 43 (1992) 815-823. [7] Hooks, M.S., Colvin, A,, Juncos, J.L. and Justice, J.B. Individual differences in basal and cocaine stimulated dopamine levels in the nucleus accumbens, Brain Res., 587 (1992) 306-312. [8] Hooks, M.S., Individual Differences in Response to Drugs of Abuse: Behavioral and Neurochemical Correlates, Ph.D. Thesis, Emory University, 1992. [9] Hooks, M.S., Juncos, J.L., Justice, J.B., Meiergerd, S.M., Povlock, S.L., Schenk, J.O. and Kalivas, P.W., Individual locomotor response to novelty predicts selective alterations in D I and D 2 receptors and mRNAs, J. Neurosci., in press. [10] Hurd, Y.L., Lindefors, N., Brodin, E., Brene, S., Persson, H., Ungerstedt, U. and Hokfelt, T., Amphetamine regulation of mesolimbic dopamine/cholecystokinin neurotransmission, Brain Res., 578 (1992) 317-326. [11] Hurd, Y.L., Brown, E.E., Finlay, J.M., Fibiger, H.C. and Gerfen, C.R., Cocaine self-administration differentially alters mRNA expression of striatal peptides, Mol. Brain Res., 13 (1992) 165-170. [12] Kalivas, P.W., Sorg, B.A. and Hooks, M.S., The pharmacology and neural circuitry of sensitization to psychostimulants, BehaL'. Pharmacol., 4 (1993) 315-334. [13] Kalivas, P.W., Churchill, L. and Klitenick, M.A. The circuitry mediating the translation of motivational stimuli into adaptive motor response. In P.W. Kalivas and C.D. Barnes, Limbic Motor Systems and Neuropsychiatry, CRC Press, Boca Raton, FL, 1993, pp. 237-288.
[14] Kislauskis, E., Bullock, B., McNeil, S. and Dobner, P.R., The rat gene encoding neurotensin and neuromedin N, J. Biol. Chem., 263 (1988) 4963-4968. [15] Nudel, U., Zakut, R., Shani, M., Neuman, S., Lew, Z. and Yaffe, D., The nucleotide sequence of the rat cytoplasmic-actin gene, Nucleic Acid Res., 11 (1983) 1759-1771. [16] Piazza, P.V., Deminiere, J.M., Le Moal, M. and Simon, H., Factors that predict individual vulnerability to amphetamine self-administration, Science, 29 (1989) 1511-1513. [17] Piazza, P.V., Rouge-Pont, F., Deminiere, P.V., Kharoubi, M., Le Moal, M. and Simon, S., Dopaminergic activity is reduced in the prefrontal cortex and increased in the nucleus accumbens of rats predisposed to develop amphetamine self-administration, Brain Res., 567 (1991) 169-174. [18] Rosen, J.B., Cain, C.J., Weiss, S.R.B. and Post, R.M., Alterations in mRNA of enkephalin, dynorphin and thyrotropin releasing hormone during amygdala kindling: an in situ hybridization study, MoL Brain Res., 15 (1992) 247-255. [19] Sorg, B.A., Chen, S.Y. and Kalivas, P.W., Time course of tyrosine hydroxylase expression following behavioral sensitization to cocaine, J. Pharmac. Exp. Then., 266 (1993) 424-430. [20] Sorg, B.A. and Kalivas, P.W., Behavioral sensitization to stress and psychostimulants: role of dopamine and excitatory amino acids in the mesocorticolimbic systems, Sem. Neurosci., 5 (1993) 343-350. [21] Steiner, H. and Gerfen, C.R., Time course alterations in substance P, dynorphin, enkephalin and c-fos gene expression in striatal neurons during chronic cocaine treatment, Soc. Neurosci. Abstr., 18 (1992) 437.11. [22] Wyborski, R.J., Bond, R.W. and Gottlieb, DT, Characterization of a cDNA coding rat glutamic acid decarboxylase, Mol. Brain Res., 8 (1990) 193-198. [23] Yoshikawa, K., Williams, C. and Sabol, S.L., Rat brain preproenkephalin mRNA, Z Biol. Chem., 259 (1984) 14301-14308.