Cocaine self-administration differentially alters mRNA expression of striatal peptides

Molecular Brain Research, 13 (1992) 165-170 Elsevier Science Publishers B.V.

165

BRESM 80117

Cocaine self-administration differentially alters mRNA expression of striatal peptides Y.L. Hurd 1'2, E.E. Brown 3, J.M. Finlay 3'* H.C. F i b i g e r 3 a n d C . R . G e r f e n 1 1Laboratory of Cell Biology and 2Clinical Neuroscience Branch, National Institute of Mental Health, Bethesda, MD (U.S.A.) and 3Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C. (Canada) (Accepted 19 November 1991) Key words: Cocaine; Dopamine; Peptide; Striatum

The influence of cocaine self-administration on the expression of messenger RNAs for dynorphin, enkephalin and substance P was analyzed in the rat striatum with in situ hybridization histochemistry. Cocaine, an indirect dopamine agonist, was found to differentially affect the levels of mRNA encoding these neuropeptides in different subregions of the striatum. Following a 7 day period of variable free access to cocaine, dynorphin and substance P mRNA levels were elevated throughout the striatum, but the increases were substantially greater in the dorsal striatum than in the nucleus accumbens. Enkephalin mRNA was not significantly altered in the dorsal striatum but was slightly elevated in the nucleus accumbens. These results suggest that cocaine self-administration has differential effects on striatonigral and striatopallidal projection neurons, and that these effects vary in subregions of the striatum. The psychomotor stimulant effects of cocaine (e.g. euphoria, locomotion, stereotopy) are thought to be mediated primarily by dopaminergic mechanisms in brain regions such as the striatum, including the nucleus accumbens 8'26'28. Dopaminergic afferents to the striatum are directed principally to the medium spiny neurons 1°, which constitute 90-95% of the striatal neurons and give rise to the major striatal efferent pathways 3°. On the basis of their axonal projections, striatal outputs can be divided into two types, those which project to the globus pallidus and those which project to the entopeduncular nucleus/substantia nigra complex 19. Neurons contributing to both pathways contain glutamic acid decarboxylase ( G A D ) 21 and utilize y-aminobutyric acid ( G A B A ) as a transmitter 5 but each contains different neuropeptides. For example, the majority of striatonigral neurons contain dynorphin and substance p3,13,32 whereas the majority of striatopallidal neurons contain enkephalin 14. Pharmacological and lesion studies have demonstrated that dopamine differentially modulates the expression of these peptides as determined by changes in immunoreactivity 16"17'22'23'33or m R N A levels 2'12'25'36. The contrasting effects of dopamine on peptide m R N A regulation appears to be dependent on the differential expression of the D~ and D2 dopamine receptors on striatonigral and striatopallidal neurons, respectively 11. Thus, dopamine-induced alterations in striatal peptide m R N A levels have provided a useful paradigm for studies of receptor-

mediated regulation of specific subtypes of striatal output neurons. Because cocaine increases dopaminergic transmission in the striatum 18'24 the present study examined the effects of this indirect agonist on the m R N A levels of the striatal peptides dynorphin, substance P and enkephalin using in situ hybridization histochemistry (ISHH). Additionally, a treatment schedule was used in which rats were given unlimited access to intravenous cocaine, in order to examine effects that may be related to the abuse of this drug. Subjects were naive, male L o n g - E v a n s rats (Charles River, Quebec), weighing 300-350 g at the start of the experiment. Initially, the rats were trained to bar press for food in standard operant boxes (BRS-LVE). Following the acquisition of responding, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and a chronic indwelling silastic cannula was surgically implanted into the right jugular vein of each animal. For the remainder of the experiment, rats were individually housed in standard operant boxes equipped with a houselight, a removable response lever and a cue light located directly above the lever (BRS-LVE). Each operant box was located in a sound-attenuating chamber with a continuously operating fan. Food and water were available ad libitum throughout the experiment unless noted otherwise. All experimental contingencies were programmed and data were recorded by a N O V A IV/X minicomputer (Data General) equipped with a M A N X interface and software

Current address: Department of Behavioral Neuroscience, University of Pittsburgh, Pittsburgh, PA, U.S.A. Correspondence: C.R. Gerfen, Laboratory of Cell Biology, NIMH, Bldg. 36, Rm. 2D-10, Bethesda, MD 20892, U.S.A.

166

..

contro|

A

;~m,

cocai~:

~.,...:~

DYN

J~

DYN

JJJ•l C

~..

" sP

b

r

sP

....

ENK

Fig. 1. Autoradiographic film images of dynorphin (DYN; A,B), substance P (SP; C,D) and enkephalin (ENK; E,F) ISHH labeling in the striatum in a control (A,B,C) and cocaine self-administration animal (B,D,F).

167 TABLE I Mean numbers of responses for animals in the cocaine self-administration group given for each day of the experiment

On day 1 the actual time of access varied, with some animals being stopped prior to 3 h to prevent overdosing and some given more time if they did not initiate responding within 3 h. On days 2 and 3, most rats were given 7 h of access time, except for those which had not initiated a significant level of responding, which received additional access. Each infusion was 0.18 ml (1.25 mg cocaine/ml or 0.225 mg/infusion) over 4.5 s and the average amount of cocaine delivered was determined.

Duration of access Responses (mean + S.E.M.) Range of responses Total cocaine per animal (average in mg)

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

3h

7h

7h

24 h

24 h

24 h

24 h

36.1 + 9.0 7-83

73.4 + 15.5 7-121

74.4 + 15.7 11-120

275.4 + 29.8 128-400

230.6 + 20.5 127-307

265.5 + 37.6 42-367

274.3 + 21.5 180-347

8.1

16.5

16.7

62.0

51.9

59.7

61.7

(GC controls). This procedure has been described in detail previously 28. The day following surgery, each rat in the self-administration group was given access to a lever, responses on which delivered a 4.5 s infusion of 0.225 mg of cocaine HC1 in 0.18 ml of sterile saline and a 30 s illumination of the light cue. Responses during illumination of the light cue were recorded but had no programmed consequences. On the first day of the experiment, drug access was limited to 3 h or 60 responses, whichever occurred first. Subjects that failed to initiate responding within 3 h were given 6 h access. The second and third days of the experiment were the same as the first, except that access was increased to 7 h and there was no limit on the number of infusions that could be taken. Again, low responders were given extended access. The rats were given continuous access (23.75 h/day) to cocaine on days 4 through 7. A reversed 12 h light-dark cycle was maintained by the houselight. All self-administration sessions began at the start of the dark cycle (10.00 h). Following the completion of the final self-administration session, rats were sacrificed by decapitation, their brains were removed and frozen in isopentane (-70°C for 15 s) and stored frozen at -70°C until processed for ISHH. The training and surgical procedures for the control rats were identical to the self-administration group. The day following surgery, control rats received 31 infusions of sterile saline (0.18 ml over 4.5 s) over a 3 h period. On days 2 and 3, control animals received 71 infusions over a 7 h period, and on the final four days of the experiment these animals received 71 infusions over a 23.75 h period. The number of saline infusions (1 infusion/6 min) was based on the average rate of responding in the cocaine self-administration group. Saline infusions were accompanied by the illumination of the cue light (30 s). Control subjects were further subdivided into two groups, one of which received food ad libitum and another which had restricted access to food. Weight matched controls

were given a limited amount of food in order to approximate the weight loss observed in the rats that self-administered cocaine. All subjects received water ad libitum. Following the final session, control animals were sacrificed and their brains processed in the same manner as the self-administration group. The brains were cut in a cryostat into coronal sections (12 pm), thaw-mounted onto slides (twice coated with gelatin), dried for 1 min at 37°C and then stored at -20°C. Sections were taken through the rostral-caudal extent of the nucleus accumbens, which also included more dorsal parts of the striatum. In situ hybridization histochemistry was performed according to standard methods 1a'36. To prepare sections for I S H H they were warmed to room temperature, fixed in 4% formaldehyde (in 0.9% saline) for 10 min, placed in 0.25% acetic anhydride (in 0.1 M triethanolamine/0.9% saline, p H 8) for 10 min, dehydrated in ascending concentrations of ethanol, defatted in chloroform (2 x 5 min), rehydrated, dried and stored frozen. Synthetic c D N A oligonucleotide probes of 48 bases in length directed against the m R N A s encoding dynorphin, substance P and enkephalin, which have been used in previous studies 13'36, were labeled at the 3" terminal end using terminal deoxynucleotidyl transferase (TdT, Boehringer-Mannheim) and [35S]dATP (New England Nuclear). The reaction parameters add a 'tail' of approximately 20-30 d A M P s to each oligonucleotide. Labeled probes were then added to hybrid.ization buffer containing 50% formamide, 10% dextran sulfate, 600 mM NaC1, 80 mM Tris (pH 7.5), 0.1% sodium pyrophosphate, 0.2% SDS, 0.02% heparin sulfate and 100 mM dithiothreitol. A volume of 25/A of hybridization buffer containing 500-1000 kcpm of labeled probe was added to each section, these were covered and incubated overnight at 37°C. Following hybridization sections were rinsed four times in 1 x SSC, in 50% formamide and 2 x SSC 4 x 15 min at 41°C, rinsed in l x SSC at room temperature for 60 min, and dried. Sections

168 TABLE II Average values of dynorphin, substance P and enkephalin 1SHH labeling in the dorsal striatum and nucleus accumbens from animals in control, food-restricted and cocaine self-administration groups

Values are given as average optical density measurements plus or minus the standard error of the means (S.E.M.). There was no statistical difference between the control and food restricted groups. The average increases in ISHH labeling in cocaine versus control groups are listed and those which are statistically significant (P > 0.05) are denoted with an asterisk. Dynorphin

Control + S.E.M. Food-restricted _+ S.E.M. Cocaine + S.E.M. Cocaine vs control

Substance P

Enkephalin

Dorsal caudate

Nucleus accumbens

Dorsal caudate

Nucleus accumbens

Dorsal caudate

Nucleus accumbens

154.0 + 1.9 154.9 + 2.3 183.0 + 2.1 +29.0*

176.2 + 2.5 177.6 _+ 2.3 192.6 _+ 1.0 +16.4"

150.4 + 1.4 150.2 + 2.1 173.6 _+ 2.3 +23.2*

152.4 + 1.0 151.0 _+ 1.l 160.9 + 0.8 +8.6*

161.9 + 1.1 164.6 _+ 1.4 162.4 + 1.4 +0.5

159.9 + 0.9 161.3 _+ 0.9 164.3 + 1.1 +4.4*

were then apposed to film ( K o d a k X - O m a t ) for 7-28 days and developed. In situ hybridization labeling was measured from the d e v e l o p e d film using a Macintosh based image analysis system (Image, W a y n e Rasband, N I M H ) . D a t a were obtained from 8 animals in each group (6 in the food restricted group). F o r each animal the average optical density of I S H H labeling in a circular field of constant size was measured in the right and left striatum of two coronal sections, matched across animals for their rostral-caudal level, in the dorsal striatum and nucleus accumbens (4 measurements p e r area per animal). Statistical significance between optical density measurements in control and drug treated groups was d e t e r m i n e d with a Student's t-test. Cocaine self-administration values are given in Table I. The average amount of cocaine self-administered by these animals when given 24 h access to the drug was 58.8 mg/day (approximately 200 mg/kg/day). Rates of self-administration were relatively stable throughout the light-dark cycle. Fig. 1 shows photomicrographs of I S H H labeling for dynorphin, substance P and enkephalin in coronal sections through the striatum of a control animal and a cocaine self-administration animal. Table II provides a comparison of the average labeling (measured in optical density units) in the dorsal striatum and nucleus accumhens of dynorphin, substance P and enkephalin in control, food restricted, and cocaine self-administering groups. These data show increased dynorphin m R N A I S H H labeling in both the dorsal striatum and nucleus accumbens in the cocaine self-administration group. The increase in the dorsal striatum is substantially greater than that in the nucleus accumbens, although it should be noted that the basal level of dynorphin m R N A I S H H labeling in the nucleus accumbens is higher than that in the dorsal striatum. Substance P m R N A I S H H labeling also shows a significant cocaine-induced elevation in both the dorsal striatum and the nucleus accumbens, and

again the increase in the dorsal striatum is substantially greater than in the nucleus accumbens. E n k e p h a l i n m R N A I S H H labeling is not significantly altered in the dorsal striatum, but shows a slight, significant increase in the nucleus accumbens. None of the peptide m R N A I S H H levels is significantly different between the control and the food restricted groups. The present data indicate that cocaine self-administration differentially alters the levels of m R N A s that encode dynorphin, substance P and enkephalin in both the dorsal and ventral striatum. H o w e v e r , there are regional differences in these cocaine-induced changes. Both dynorphin and substance P m R N A levels show substantial cocaine-induced increases in the dorsal striatum, whereas in the nucleus accumbens these increases are relatively modest, particularly in the case of substance P. Enkephalin m R N A is elevated only in the nucleus accumbens. As dynorphin and substance P m R N A are expressed by most striatonigral neurons and enkephalin m R N A is expressed p r e d o m i n a n t l y by striatopallidal neurons 13, the present data suggest that cocaine self-administration differentially alters gene regulation in these two striatal output systems. The most p r o n o u n c e d effect of cocaine is an elevation of both dynorphin and substance P m R N A levels in the dorsal striatum, whereas enkephalin m R N A levels are not significantly altered in this region. Thus in the dorsal striatum there appears to be a difference in gene regulation in striatonigral and striatopallidal neurons. In the ventral striatum, which includes the nucleus accumbens, the cocaine-induced affects are m o r e subtle and complex. H e r e the dynorphin m R N A levels show the largest increase, c o m p a r e d to the other peptide m R N A s , but this increase is considerably less p r o n o u n c e d than in the dorsal striatum. Both substance P and enkephalin m R N A levels show a slight significant cocaine-induced elevation in the nucleus accumhens. Ventral striatal neurons that express substance P project to the ventral pallidum TM, however, it has not

169 been established whether these neurons belong to a set that co-expresses either enkephalin or dynorphin and that provides axon collaterals to both the ventral pallidum and substantia nigra. Nonetheless, the elevation in dynorphin, substance P and enkephalin m R N A levels suggests that gene regulation is increased, albeit only modestly, in most output neurons in the nucleus accumbens during cocaine self-administration. This is in contrast to the dorsal striatum, where cocaine self-administration results in a rather selective and substantial increase in gene regulation in striatonigral neurons thus generating a relative imbalance between striatonigral and striatopallidal neurons compared to the control condition. Although it has been proposed that the differential effects of dopamine on striatonigral and striatopallidal neurons are mediated by the specific expression of D1 and D 2 dopamine receptors by these two types of output neurons, respectively n, the cocaine-induced affects reported here do not necessarily indicate a predominant D 1 receptor mediated response. In a previous study peptide m R N A levels in striatonigral and striatopallidal neurons were shown to be specifically altered by D 1- o r D 2s e l e c t i v e agonists, respectively 1~. However, those drug treatments were given to animals with lesions of the nigrostriatal dopamine pathway, and thus examined the consequence of activation mediated by one receptor type in the absence of the other. In animals self-administering cocaine both types of dopamine receptors would presumably be activated simultaneously. In this circumstance there is considerable evidence that there are complex synergistic interactions between D1 and D 2 dopamine receptors 4'6'34'35. The cellular mechanisms of D 1 and D 2 dopamine receptor synergism have not been elucidated but may involve: (1) co-activation of both receptors on individual neurons that express both receptors, (2) interactions between striatal neurons that express only one of the dopamine receptor subtypes, and/or (3) interconnections between striatal interneurons, such as cholinergic neurons, and striatal output neurons. Further study is needed to delineate the details of interactions of D1 and D 2 dopamine receptor activation that may occur in animal models of cocaine self-administration. The present data suggest that cocaine-induced gene regulation differs in the dorsal and ventral striatum. Several mechanisms may be considered. For example, D 1 and D 2 receptor synergism may differ in dorsal and ventral striatal subregions. Alternatively, as acetylcholine affects dopaminergic mediated regulation of dynorphin gene expression 7, dorsal-ventral regional differences in D2 dopamine receptor mediated inhibition of acetylcho-

line release 31 may be involved. Additionally, as the action of cocaine on the dopamine transporter has been suggested to be causally related to its self-administration 27, regional differences in the distribution of this transporter may be of interest. The present data are somewhat consistent with those of Sivam 29 who demonstrated that experimenter administered injections of cocaine elevate dynorphin in the striatum. However, that study did not find a significant cocaine-induced alteration in substance P. This disparity may reflect differences in the route and dose of drug administration. For example, the dose utilized by Sivam z9 was considerably less than the amount self-administered in the present study. A model for dopamine's functional effects in the dorsal striatum has been proposed that suggests that this neurotransmitter modulates the balance of activity in the striatopallidal and striatonigral output systems l'n. The consequences of altering the balance in these output pathways are related to the ultimate effects on the activity of GABAergic neurons in the entopeduncular nucleus and substantia nigra. Increased striatopallidal activity results in an increase in the activity of nigral GABAergic neurons by way of the intervening subthalamic nucleus 2°, which is thought to inhibit voluntary motor behavior. Conversely, increased striatonigral activity directly inhibits nigral GABAergic neurons 5, which is thought to facilitate motor activity 15. Following cocaine self-administration there appears to be an increase in the relative function of striatonigral versus striatopallidal output systems in the dorsal striatum, which, according to this model, would result in a facilitation of movement related behavior. Whether such alterations are directly or indirectly related to the effects of cocaine are unclear. A model that relates alterations in the relative activity of ventral striatal output systems to specific behavioral responses is yet to be developed. However, the present results suggest that although cocaine self-administration may not produce as great an imbalance in the output systems of the nucleus accumbens, the fact that both systems show elevated gene regulation indicates that normal activity in this striatal subregion is also altered by cocaine. Although the functional consequences of these changes are not known, it is interesting to speculate that they may be related to the well-known psychotogenic effects of chronically administered stimulant drugs 9. Supported by a grant to HCF from the Medical Research Council of Canada (PG-23). E.E.B. is a Medical Research Council of Canada Student. Y.L.H. is a Pharmacology Research Associate of the National Institute of General Medical Sciences. We thank Miles Herkenham for his help.

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