The role of adenylyl cyclase in the medial prefrontal cortex in cocaine-induced behavioral sensitization in rats

Accepted Manuscript The role of adenylyl cyclase in the medial prefrontal cortex in cocaine-induced behavioral sensitization in rats Kun Liu, Jeffery D. Steketee PII:

S0028-3908(16)30112-5

DOI:

10.1016/j.neuropharm.2016.03.040

Reference:

NP 6237

To appear in:

Neuropharmacology

Received Date: 8 October 2015 Revised Date:

16 February 2016

Accepted Date: 23 March 2016

Please cite this article as: Liu, K., Steketee, J.D., The role of adenylyl cyclase in the medial prefrontal cortex in cocaine-induced behavioral sensitization in rats, Neuropharmacology (2016), doi: 10.1016/ j.neuropharm.2016.03.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title:

The role of adenylyl cyclase in the medial prefrontal cortex in cocaine-induced behavioral sensitization in rats

Kun Liu and Jeffery D. Steketee*

RI PT

Authors:

Department Pharmacology, University of Tennessee Health Science Center,

*Corresponding author: Jeff Steketee

M AN U

SC

Memphis, TN 38163

Department of Pharmacology UTHSC

TE D

874 Union Avenue/Room 115 Crowe Memphis, TN 38163

EP

Tel: 1-901-448-4585 Fax: 1-901-448-7206

AC C

E-mail: [email protected].

Acknowledgments

All research efforts were financially supported by a grant from the National Institute on Drug Abuse (DA023215). Conflicts of interest The authors state no conflict of interest.

Liu and Steketee/ Page

ACCEPTED MANUSCRIPT

2

Abstract Repeated exposure to cocaine progressively increases drug-induced locomotor activity, which is termed behavioral sensitization. Previous research has demonstrated that in the medial

RI PT

prefrontal cortex (mPFC) modulation of cocaine-induced motor activity by agonists of Gi-coupled receptors, such as dopamine D2, GABAB and Group II metabotropic glutamate

SC

receptors, is reduced in sensitized animals, suggesting a loss in receptor function.

Stimulation of each of these receptors acts in part to inhibit adenylyl cyclase activity, and thus,

M AN U

the formation the cAMP. The present studies tested the hypothesis that intra-mPFC inhibition of adenylyl cyclase by infusion of an inhibitor, SQ22536, could bypass the loss of inhibitory receptor function seen in this region, and thereby inhibit the expression of cocaine sensitization.

Additional studies determined whether activation of mPFC adenylyl cyclase

TE D

with NKH 477 could enhance the motor-stimulant response to cocaine. Initial studies demonstrated that cocaine-induced (15 mg/kg, i.p.) motor activity was dose-dependently

EP

reduced by injection of SQ22536 (5-75 nmol/side) into the mPFC, whereas NKH 477 (1.25-40

AC C

nmol/side) produced no significant effects. Additional studies showed that intra-mPFC injection of SQ22536 (50 nmol/side) attenuated the initiation of cocaine-induced behavioral sensitization and blocked the expression of sensitization following 1, 7 or 30 days of abstinence from cocaine. Also, intra-mPFC injection of NKH 477 enhanced cocaine-induced behavioral sensitization following 21 days of abstinence from cocaine. The results of the present study suggest modulation of adenylyl cyclase in the medial prefrontal cortex plays a key role in the expression of cocaine sensitization.

Liu and Steketee/ Page

ACCEPTED MANUSCRIPT

Keywords: Adenylyl cyclase, cocaine, G-protein coupled receptors, locomotor activity,

AC C

EP

TE D

M AN U

SC

RI PT

sensitization

3

Liu and Steketee/ Page

ACCEPTED MANUSCRIPT

4

1. Introduction Repeated administration of psychostimulants such as cocaine can induce a progressive enhancement in locomotor activity, which is termed behavioral sensitization (Downs and Eddy

RI PT

1932; Steketee 2005). Numerous studies have demonstrated that sensitization, which is a long-lasting phenomenon, shares similar neuroadaptations that underlie relapse to

SC

compulsive drug-seeking and drug-taking behavior (Steketee and Kalivas 2011). Thus, much research has been undertaken to elucidate the neural mechanism underlying cocaine-induced

M AN U

sensitization. It has been shown that the mesocorticolimbic dopamine system plays an important role in development of cocaine-induced sensitization (Steketee 2005). The mesolimbic and mesocortical divisions of this system are each comprised of dopaminergic cell bodies within the ventral tegmental area (VTA) that project to several limbic and cortical

TE D

regions, including the nucleus accumbens and medial prefrontal cortex (mPFC), respectively (Oades and Halliday 1987). It is suggested that the VTA is the site of early and transient

EP

neuroadaptations associated with the initiation of behavioral sensitization while the nucleus

AC C

accumbens is the site of persistent drug-induced changes that may underlie the expression of sensitization (Kalivas et al. 1993; Kalivas and Stewart 1991). In addition to the VTA and nucleus accumbens, the role of the mPFC in sensitization has also received attention because of its intricate involvement in the oversight of the mesolimbic system (Steketee 2003; 2005). The mPFC receives dopaminergic projections from the VTA (Lindvall et al. 1978) and glutamatergic projections from the VTA and other principal components of the mesocorticolimbic system including the amygdala and hippocampus (Bacon et al. 1996; Jay et

Liu and Steketee/ Page

ACCEPTED MANUSCRIPT

5

al. 1996; Yamaguchi et al. 2011). In addition, the mPFC provides reciprocal excitatory glutamatergic output to the regions discussed above along with and nucleus accumbens (Carr and Sesack 2000; Omelchenko and Sesack 2007; Sesack et al. 1989). In addition to afferent

RI PT

and efferent connections of the mPFC this region also contains GABAergic local circuit neurons (Retaux et al. 1993; Retaux et al. 1992). Previous studies demonstrated that

SC

dopamine D2, GABAB and Group II metabotropic glutamate (mGluR) receptors within the mPFC are capable of modulating cocaine-induced locomotion (Beyer and Steketee 2000;

M AN U

2002; Steketee and Beyer 2005; Xie and Steketee 2009). However, in animals with a history of repeated cocaine exposure the ability of agonists for these receptors to inhibit cocaine-induced motor activity is reduced (Beyer and Steketee 2002; Steketee and Beyer 2005; Xie and Steketee 2009). Taken with data demonstrating a reduction in receptor

TE D

coupling to G proteins, these data suggest that cocaine sensitization is associated with an attenuation of inhibitory receptor function in the mPFC (Bowers et al. 2004; Febo et al. 2003).

EP

Dopamine D2, GABAB and Group II mGluR receptors all couple to adenylyl cyclase via G

AC C

proteins to inhibit the formation of cAMP (Bettler and Tiao 2006; Jackson and Westlind-Danielsson 1994; Kew and Kemp 2005). As mentioned above, repeated cocaine is known to produce a decrease in GABAB, D2 and Group II mGluR receptor coupling in the mPFC. Thus, while receptor-G protein coupling is reduced the remainder of the second messenger signaling pathway may remain intact in cocaine sensitized animals. The present report tested this hypothesis by examining the effects of intra-mPFC injections of SQ22536, an adenylyl cyclase inhibitor, on the acute locomotor response to cocaine, as well as on the

Liu and Steketee/ Page

ACCEPTED MANUSCRIPT

6

initiation and expression of cocaine-induced sensitization. Since it is hypothesized that sensitization is associated with increased excitability of pyramidal output neurons in the mPFC, follow-up studies were conducted to determine whether stimulation of adenylyl cyclase activity

RI PT

in this region could enhance the locomotor response to cocaine using NKH 477.

SC

2. Materials and methods 2.1 Animals and surgery

M AN U

All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by The University of Tennessee Health Science Center Animal Resources Advisory Committee. Male Sprague-Dawley rats (Harlan, Indianapolis, IN) that weighed 275-300 g at the time of surgery

TE D

were housed under 12-h light/dark cycle and had free access to food and water. Rats were housed in groups of four before surgery and were individually housed after surgery. All

EP

experimental procedures were conducted during the light phase of the light/dark cycle. Rats

AC C

were anesthetized with ketamine hydrochloride and xylazine (80 and 6.0 mg/kg, respectively, i.p.) and their heads were mounted in a stereotaxic frame (Kopf Instruments) with bregma and lambda aligned in the same horizontal plane. Cannulae for microinjections (25 gauge, 14 mm) were bilaterally implanted 1.0 mm above the ventral mPFC (+3.2 mm posterior to bregma, ±0.6 lateral to the midline, and -3.5 mm ventral from dura, (Paxinos and Watson 1986). Cannulae were anchored with three stainless steel screws and dental acrylic. Obturators (32 gauge, 14 mm) were inserted into the cannulae in order to prevent their occlusion. Animals

Liu and Steketee/ Page

ACCEPTED MANUSCRIPT

7

were allowed at least 7 days to recover from surgery.

2.2 Behavior and microinjections

RI PT

Locomotor activity was monitored using a Digiscan system (Accuscan, Columbus, OH, USA) as previously described (Xie and Steketee 2009). Following a 60-min adaption to activity

SC

boxes, animals received intra-mPFC injections 5 min before systemic injections. Intracranial injections (0.5 µl/min, 0.5 µl/side) were made using stainless steel injectors (15 mm, 32 gauge)

M AN U

attached to 1 µl syringes via PE 20 tubing mounted in a Sage syringe pump as described previously (Beyer and Steketee 2002). Injectors were left in place for 20 s to allow for diffusion of the infused solution and obturators were reinserted into guide cannulae after

2.3 Experimental design

TE D

injections. Motor activity was monitored for 2 hr in 15-min intervals following injection.

EP

2.3.1 Acute cocaine studies

AC C

The effects of intra-mPFC injections of the adenylyl cyclase inhibitor, SQ22536 (5, 15, 50 and 75 nmol/side) or the adenylyl cyclase activator, NKH 477 (1.25 and 40 nmol/side), on cocaine (15 mg/kg, ip)-induced motor activity were studied using separate groups of animals for each drug and dose tested. For each group of rats tested each animal received each of the four possible treatment combinations (saline/saline, saline/cocaine, SQ22536 or NKH 477/saline, SQ22536 or NKH 477/cocaine) using a Latin Square design with a minimum 3-day inter-trial interval.

Liu and Steketee/ Page

ACCEPTED MANUSCRIPT

8

2.3.2 Sensitization studies The basic design of the sensitization experiments included an initiation phase of once daily

RI PT

injections of saline (1.0 ml/kg, ip) or cocaine (15 mg/kg, ip) over four consecutive days, followed by an expression phase that involved a challenge injection of cocaine in all animals.

SC

A drug free period of 1, 7 or 30 days separated the initiation and expression phases. Each animal received the same treatment on each injection day during the initiation phase. Motor

M AN U

activity was monitored following injections on test day, but not during the pretreatment (i.e initiation phase) days. The impact of inhibiting cortical adenylyl cyclase on the initiation of sensitization was determined by injecting saline or SQ22536 (50 nmol/side) into the mPFC before saline or cocaine injections on each of the four pretreatment days. Intracranial

TE D

injections were not administered on test day, which occurred 7 days after the last of the daily injections. This drug free period was based on previous experiments in our laboratory in

EP

which sensitization was clearly and reliably expressed at this time point (Beyer and Steketee

AC C

2002; Xie and Steketee 2009). The effects of inhibiting cortical adenylyl cyclase on the expression of sensitization were determined by injecting saline or SQ22536 into the mPFC before the test day cocaine injections that occurred 1, 7 or 30 days after the daily injections, which did not include intracranial injections during the initiation phase. The effects of stimulating cortical adenylyl cyclase on the expression of sensitization was determined by injecting saline or NKH 477 (40 nmol/side) into the mPFC before the test day cocaine injections that occurred 21 days after the initiation phase. As a control for potential

Liu and Steketee/ Page

ACCEPTED MANUSCRIPT

9

nonspecific drug effects animals received saline injections with or without intracranial injections the day before the onset of initiation treatment regimen and the test for sensitization. Test day time points for sensitization studies were based on previous studies (Xie and

RI PT

Steketee 2009).

SC

2.4 Histology

After completion of studies, animals were deeply anesthetized with sodium pentothal (333

M AN U

mg/kg, ip) and were perfused by intracardiac infusion of phosphate-buffered saline (50 ml) and 10% formaldehyde (50 ml). Brains were sectioned (100 µm) and sections were mounted onto gelatin-coated slides and stained with cresyl violet. Injection sites were visualized by light

2.5 Statistics

TE D

microscopy.

EP

Dose-response data were initially analyzed by a two-way analysis of variance (ANOVA; dose

AC C

and treatment) with one repeated measure (treatment) followed by one-way ANOVA for each dose of SQ22536 and multiple comparisons were conducted with a student Newman-Keuls test. A two-way analysis of variance with one repeated measure (time) was used to analyze time courses of behavioral data from sensitization studies. Multiple comparisons were made with a modified least significant differences test (Milliken and Johnson 1984). For some sensitization studies, the first hour of data was totaled and analyzed by a one-way ANOVA followed by a student Newman-Keuls test.

Liu and Steketee/ Page 10

ACCEPTED MANUSCRIPT

2.6 Drugs Cocaine hydrochloride was purchased from Sigma Chemical Company (St. Louis, MO, USA).

RI PT

SQ22536 and NKH 477, a water-soluble form of forskolin, were purchased from Tocris Biosciences (Ellisville, MO). Both compounds have been successfully used in vivo, via

SC

microinjections, to inhibit or stimulate, respectively, adenylyl cyclase in discreet brain locations (Bobeck et al., 2014). All drugs were diluted with sterile isotonic saline (0.9 % sodium

M AN U

chloride).

3. Results

For sensitization studies, animals received multiple treatments that included systemic and/or

TE D

intracranial injections during the pretreatment phase and/or on test day. Thus, the figure legends will indicate sensitization expression test day treatment (daily initiation pretreatments).

EP

Each of these periods could be further subdivided into intracranial pretreatment/systemic

AC C

treatment. Thus, cocaine (saline/saline) would indicate animals that received cocaine on expression test day after receiving intracranial saline/systemic saline during the initiation phase.

3.1 Acute cocaine studies The effects of intra-mPFC SQ22536 (5, 15, 50 and 75 nmol/side) on the acute motor stimulant

Liu and Steketee/ Page 11

ACCEPTED MANUSCRIPT

response to cocaine are illustrated in Fig. 1a. An initial two-way ANOVA with one repeated measure (treatment) revealed a treatment effect (F(3,81)= 78.06, p< 0.0001) and an interaction between treatment and dose (F(9,81)= 2.642, p= 0.0098) suggesting that SQ22536

AC C

EP

TE D

M AN U

SC

RI PT

dose-dependently reduced cocaine-induced motor activity. Subsequent one-way repeated

Fig. 1 The effects of intra-mPFC injection of (a) SQ22536 and (b) NKH 477 on the acute motor response to cocaine (15 mg/kg i.p.). The figure legend indicates intra-mPFC injection/systemic injection and data are expressed as mean photocell counts ± SEM. *p< 0.05 compared to saline/saline and +p< 0.05 compared to saline/cocaine.

measures ANOVAs confirmed this by showing that only 50 nmol/side (F(3, 31)= 12.03, p<

Liu and Steketee/ Page 12

ACCEPTED MANUSCRIPT

0.0001) and 75 nmol/side (F(3, 31)= 13.96, p< 0.0001) reduced the motor-stimulant response to acute cocaine exposure. Additionally, it was also demonstrated that intra-mPFC SQ22536 administration did not significantly alter, at any dose tested, saline-induced motor activity

RI PT

levels, suggesting that the effects of SQ22536 were not the result of a nonspecific suppression of motor activity.

SC

The effects of intra-mPFC injection of NKH 477 (1.25 and 40 nmol/side) on the

motor-stimulant response to cocaine are shown in Fig. 1b. A two-way ANOVA with one

M AN U

repeated measure (treatment) revealed a significant treatment effect (F(3,42)= 24.04, p< 0.0001) but not a significant dose or interaction effect suggesting that cocaine-induced motor activity was not altered by NKH 477 pretreatment in the mPFC. This finding was confirmed by subsequent 1 way ANOVAs (1.25 nmol/side F(3,31)= 29.65, p< 0.0001; 40 nmol/side F(3,31)=

TE D

7.997, p< 0.0001). Similar to SQ22536, NKH 477 was not observed to significantly alter

EP

basal (saline-induced) motor activity.

AC C

3.2 Sensitization studies

The effects on intra-mPFC SQ22536 on the initiation of behavioral sensitization are shown in Fig. 2. The F scores were as follows: treatment F(3,30)= 5.908, p= 0.0007; time F(7, 30)= 49.75, p< 0.0001; and interaction F(21, 224)= 1.340, p= 0.1515. The results showed that the cocaine challenge injection produced a sensitized locomotor response during the first 45 min after injection in animals previously exposed to cocaine [cocaine (saline/cocaine)], as compared to animals receiving cocaine for the first time [cocaine (saline/saline)]. Animals that received

Liu and Steketee/ Page 13

ACCEPTED MANUSCRIPT

repeated intra-mPFC SQ22536 injections before each of their daily cocaine injections [cocaine (SQ22536/cocaine)] also showed an enhanced behavioral response to a challenge injection of cocaine, but only during the first 15 min after cocaine injection compared to

RI PT

cocaine (saline/saline). These data suggest that SQ22536 may have attenuated the initiation of cocaine sensitization. In addition a history of repeated exposure to SQ22536 alone

SC

[cocaine (SQ22536/saline)] did not impact the response to a subsequent exposure to cocaine

EP

TE D

M AN U

for the first time.

Fig. 2 The effects of intra-mPFC SQ22536 (50 nmol/side) injection on the initiation of

AC C

behavioral sensitization to cocaine (15 mg/kg i.p.). The figure legend indicates test day treatment (pretreatment) with pretreatment being shown as intra-mPFC injection/systemic injection. Data are expressed as a mean ± SEM photocell counts for motor activity. *p< 0.05 compared to cocaine (saline/saline) and +p< 0.05 compared to cocaine (saline/cocaine).

Figure 3 illustrates the effects of SQ22536 on the expression of cocaine-induced behavioral sensitization following 1, 7 and 30 days of abstinence from repeated cocaine

Liu and Steketee/ Page 14

ACCEPTED MANUSCRIPT

pretreatment. Following initial time course analysis, the first hour of motor activity was totaled and analyzed in order to more clearly demonstrate the effects of SQ22536 on the expression of sensitization. The F scores for total activity were as follows: day 1 F(3,28)= 3.561, p= 0.0267;

RI PT

day 7 F(3,28)= 7.666, p= 0.0007 and day 30 F(3,27)= 4.842, p= 0.0080. The F scores for time course data were as follows: day 1 treatment F(3,28)= 2.328, p= 0.096; time F(7,28)= 47.50, p<

SC

0.0001; and interaction F(21, 196)=4.408, p= 0.0001; day 7 treatment F(3,28)= 5.692, p= 0.0036; time F(3,28)= 74.48, p< 0.0001; and interaction F(21,196)= 4.439, p< 0.0001; and 30 days

M AN U

treatment F(3,27)= 4.256, p= 0.0138; time F(3,27)= 55.17, p< 0.0001; and interaction F(21,189)= 4.987, p< 0.0001. A cocaine challenge injection, preceded by an intra-cortical saline injection, evoked a sensitized locomotor response in animals previously exposed to cocaine [saline/cocaine (cocaine)] compared to control animals receiving their first cocaine injection

TE D

[saline/cocaine (saline)] 1, 7 and 30 days after the pretreatment regimen. Following one day of abstinence (Fig. 3a and 3b) from repeated cocaine, intra-mPFC SQ22536 injection before a

EP

cocaine challenge injection [SQ22536/cocaine (cocaine)] attenuated the sensitized locomotor

AC C

activity in animals previously exposed to cocaine. Thus, cocaine induced motor activity in sensitized animals pretreated with SQ22536 [SQ22536/cocaine (cocaine)] was not significantly different compared to animals previously exposed to cocaine [saline/cocaine (cocaine)] or saline [saline/cocaine (saline)] when examining total activity for the first hour following cocaine. Analysis of the time course

Liu and Steketee/ Page 15

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

Fig. 3 The effects of intra-mPFC SQ22536 (50 nmol/side) injection on the expression of behavioral sensitization to cocaine (15 mg/kg i.p.) after (a, b) 1 day, (c, d) 7 days and (e, f) 30 days withdrawal from repeated cocaine exposure. The figures legend indicates test day treatment (pretreatment) with test treatment being shown as intra-mPFC injection/systemic injection. Data are expressed as a mean ± SEM photocell counts for motor activity. *p<0.05 compared to saline/cocaine (saline), +p<0.05 compared to saline/cocaine (cocaine) (a, c and + e) and p<0.05 compared to SQ22536/cocaine (cocaine) (b, d and f).

Liu and Steketee/ Page 16

ACCEPTED MANUSCRIPT

data revealed that the animals pretreated with SQ22536 before cocaine were significantly different from both the saline control group and the sensitized group, especially at the 15 min

RI PT

time point. Following 7 and 30 days of abstinence from repeated cocaine, intracortical SQ22536 injection before cocaine challenge injection blocked the expression of the sensitized

SC

response (Fig. 3c-f). Thus, when comparing cocaine pretreated animals, animals receiving intra-mPFC SQ22536 [SQ22536/cocaine (cocaine)] showed a reduced locomotor response

M AN U

compared to animals receiving intra-mPFC saline [saline/cocaine (cocaine)]. Also, animals with a history of repeated cocaine exposure that were pretreated with saline [saline/cocaine (cocaine)] but not SQ22536 [SQ22536/cocaine (cocaine)] in the mPFC before cocaine challenge showed a significantly enhanced response to cocaine compared to animals

TE D

previously exposed to repeated saline [saline/cocaine (saline) or SQ22536/cocaine (saline)]. These effects were most prevalent the first 45 min after injection.

EP

One caveat to the expression studies is that intra-mPFC SQ22536 injection showed no

AC C

significant effects in animals receiving cocaine for the first time [SQ22536/cocaine (saline)] as compared to control animals receiving cocaine for the first time [saline/cocaine (saline)], which differed from the initial dose response studies (Figure 1). It is unclear why intra-mPFC SQ22536 was ineffective against acute cocaine in the expression experiments, although it likely was a result of using a threshold dose of this compound. Nevertheless, the effectiveness of SQ22536 in these studies was confirmed by its ability to reduce the expression of sensitization.

Liu and Steketee/ Page 17

ACCEPTED MANUSCRIPT

Figure 4 shows the effects of NKH 477 (40 nmol/side) on the expression of cocaine-induced behavioral sensitization following 21 days of abstinence from repeated cocaine pretreatment. The F scores were as follows: treatment F(3,28)= 7.229, p=0.001; time

RI PT

F(7,28)= 21.62, p<0.0001; and interaction F(21,196)= 3.603, p<0.0001. Preceded by an intra-cortical saline injection, a cocaine challenge injection evoked a sensitized locomotor

SC

response in animals previously exposed to cocaine [saline/cocaine (cocaine)] compared to

AC C

EP

TE D

M AN U

control animals receiving their first cocaine injection [saline/cocaine (saline)] 21 days after the

Fig. 4 The effects of intra-mPFC NKH 477 (40 nmol/side) injection on the expression of behavioral sensitization to cocaine (15 mg/kg i.p.) after 21 days withdrawal from repeated cocaine exposure. The figures legend indicates test day treatment (pretreatment) with test treatment being shown as intra-mPFC injection/systemic injection. Data are expressed as a mean ± SEM photocell counts for motor activity. *p<0.05 compared to saline/cocaine (saline) + and p<0.05 compared to saline/cocaine (cocaine).

Liu and Steketee/ Page 18

ACCEPTED MANUSCRIPT

pretreatment regimen. Intra-mPFC NKH 477 injection before a cocaine challenge injection [NKH 477/cocaine (cocaine)] enhanced the sensitized locomotor activity in animals previously

RI PT

exposed to cocaine during the first 15 min after injection. Thus, when comparing cocaine pretreated animals, animals receiving intra-mPFC NKH 477 [NKH 477/cocaine (cocaine)]

M AN U

intra-mPFC saline [saline/cocaine (cocaine)].

SC

showed a significantly increased locomotor response compared to animals receiving

3.3 Saline studies

As a control, cocaine treated animals received systemic saline injections before the onset of daily cocaine injections and before the test for behavioral sensitization. Intra-cranial drug

TE D

injections were included before the onset of daily cocaine for initiation experiments or before the test for behavioral sensitization for the expression experiments. In all cases no significant

EP

effects were seen (data not shown) indicating no long-term effects of repeated intra-cranial

AC C

injections of SQ22536 or repeated systemic cocaine on baseline motor activity.

3.4 Histology

Figure 5 shows typical injection sites in the mPFC. Generally, injections sites were located 3.2 mm forward of bregma (Paxinos and Watson 1986) in the ventral portions of the mPFC, specifically in the infralimbic subregion. Animals with incorrect injection sites were removed from the study.

Liu and Steketee/ Page 19

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 5 Injection sites placements in the mPFC. The figure shows a representative illustration

TE D

of injector placement from animals included in the analysis. Illustrations were derived from The Rat Brain in Stereotaxic Coordinates (Paxinos and Watson 1997). fmi forceps minor of the

AC C

4. Discussion

EP

corpus callosum, Cg3 cingulate cortex, area 3, IL infralimbic cortex

The present study demonstrated that intra-mPFC microinjection of SQ22536, an adenylyl cyclase inhibitor, attenuated the acute motor-stimulant response to cocaine, as well as the initiation of behavioral sensitization, while injection of SQ22536 into the mPFC before a cocaine challenge injection reduced the expression of behavioral sensitization at all time points tested. In contrast, intra-mPFC injection of NKH 477, an adenylyl cyclase activator, was not found to alter acute cocaine-induced motor activity, but did enhance the

Liu and Steketee/ Page 20

ACCEPTED MANUSCRIPT

motor-stimulant response to cocaine in sensitized animals. Therefore, the present study suggests that inhibition or activation of adenylyl cyclase in the mPFC can modulate the expression of cocaine sensitization.

RI PT

Previous studies have identified the mPFC as one of the critical regions capable of modulating the stimulant response to cocaine. Thus, the level of motor activity in response to

SC

cocaine is significantly correlated with the level of cocaine in the mPFC (Carey et al. 1994). Repeated electrical stimulation of the mPFC that produces kindling enhances the locomotor

M AN U

response to cocaine (Schenk and Snow 1994). Depletion of dopamine in the mPFC by 6-hydroxydopamine lesions also enhances the stimulant response to cocaine (Beyer and Steketee 1999). Finally, injection of agonists for dopamine D2, GABAB and Group II mGluR receptors into the mPFC reduced cocaine-induced motor activity (Beyer and Steketee 2001;

TE D

Steketee and Beyer 2005; Xie and Steketee 2009). These receptors are coupled to adenylyl cyclase and upon activation inhibit the formation of the second messenger cAMP (Bettler and

EP

Tiao 2006; Jackson and Westlind-Danielsson 1994; Kew and Kemp 2005). Thus, it was

AC C

hypothesized that bypassing these receptors and directly inhibiting adenylyl cyclase activity in the mPFC would also reduced cocaine-induced motor activity. Results of the present study confirmed this hypothesis by demonstrating that intra-mPFC SQ22536 dose-dependently reduced motor activity induced by systemic cocaine. While intra-mPFC SQ22536 was shown to reduce the stimulant response to cocaine, it should be noted that it was only able to attenuate rather than completely block the behavior. It is possible that a lack of complete suppression of cocaine-induced motor activity was due to

Liu and Steketee/ Page 21

ACCEPTED MANUSCRIPT

an insufficient dose. However, when examining the data, it is apparent that the two highest doses produced similar inhibitory effects. Attempts to test higher doses of drug were not possible, as the limits of solubility had been reached. Another potential explanation is that

RI PT

the mPFC is not solely responsible for the modulation of cocaine-mediated behaviors. This is supported by past studies demonstrating that neither APDC, a Group II mGluR agonist nor

SC

quinpirole, a dopamine D2-like agonist, when injected into the mPFC, completely reduced the stimulant response to cocaine (Beyer and Steketee 2000; Xie and Steketee 2009).

M AN U

Furthermore, manipulation of other brain regions, including the nucleus accumbens and ventral tegmental area, has been shown to alter cocaine-induced motor activity (Delfs et al. 1990; Kelly and Iversen 1976; Steketee 1993; Steketee and Braswell 1997). In addition to reducing the acute motor-stimulant response to cocaine, repeatedly pairing

TE D

intra-mPFC quinpirole, APDC or baclofen with systemic cocaine over 4 consecutive days prevented the initiation of behavioral sensitization to cocaine (Beyer and Steketee 2002;

EP

Steketee and Beyer 2005; Xie and Steketee 2009). Since, the receptors these agonists bind

AC C

to are Gi coupled and thus inhibit adenylyl cyclase activity (see above), it was anticipated that direct inhibition of this enzyme in the mPFC would likewise inhibit the initiation of sensitization. However, at best, intra-mPFC SQ22536 attenuated the initiation of sensitization. Several factors could account for the discrepancy between receptor agonists and inhibitors of these receptors’ second messenger systems. First, as discussed earlier, a lack of a complete block of the acute response to cocaine by intra-mPFC SQ22536 would allow for the sensitized response to develop when animals received daily SQ22536 plus systemic cocaine. However

Liu and Steketee/ Page 22

ACCEPTED MANUSCRIPT

similar effects were seen with quinpirole and APDC during acute exposure yet co-treatment with these drugs in the mPFC during daily cocaine injections prevented the initiation of sensitization (Beyer and Steketee 2000; 2002; Xie and Steketee 2009). Another possibility is

RI PT

that onset of action of SQ22536 is delayed such that animals experience the initial surge in the motor stimulant response which could be adequate to induce sensitization. However, when

SC

examining the time course of effects of intra-mPFC SQ22536 on the expression of

sensitization, it is apparent the drug is effective during the early time points following cocaine

M AN U

injection. An additional possibility to consider is that Gi coupled receptors are also known to couple other signaling systems, in particular ion channels (Bettler and Tiao 2006; Jackson and Westlind-Danielsson 1994; Kew and Kemp 2005). Thus, it is possible that inhibition of

initiation of sensitization.

TE D

adenylyl cyclase only bypasses a subset of receptors necessary for the inhibition of the

Similar to the data discussed above, when animals were challenged with cocaine 1 day

EP

after the daily treatment regimen, intra-mPFC SQ22536, also only attenuated the expression

AC C

of sensitization. However, when tested 1 week or 1 month following the daily treatment regimen, injection of SQ22536 into the mPFC was able to completely block the expression of sensitization. These data parallel studies testing the effects of intra-mPFC APDC on the expression of sensitization. Thus, this Group II mGluR agonist was only effective in blocking the expression of sensitization when injected into the mPFC 1, but not 7 or 30 days after the last of the daily cocaine injections (Xie and Steketee 2009). Taken together, these data suggest that receptor-G protein coupling remains intact following short-term, but not long-term

Liu and Steketee/ Page 23

ACCEPTED MANUSCRIPT

abstinence from repeated cocaine exposure. This hypothesis is supported by a report that demonstrates that AGS3 is elevated following long-term, but not short-term withdrawal from cocaine administration (Bowers et al. 2004). Elevated AGS3 is capable of uncoupling

RI PT

receptors from G proteins (Bernard et al. 2001; Natochin et al. 2000). Overall, the data suggest that pharmacological inhibition of adenylyl cyclase activity is only able to completely

SC

modulate cocaine-induced motor activity when receptor modulation of this enzyme is reduced as seen during long-term sensitization.

M AN U

Since the studies discussed above demonstrated that intra-mPFC inhibition of adenylyl cyclase activity reduced the motor-stimulant response to cocaine, subsequent studies examined the impact of stimulation of this enzyme in the mPFC on cocaine-induced locomotion. Initial studies demonstrated that injection of NKH 477, a water soluble form of

TE D

forskolin, into the mPFC did not alter the locomotor response to an acute injection of cocaine. As was the case for SQ22536, it is possible the inability of NKH 477 to enhance

EP

cocaine-induced motor activity was a result of an insufficient concentration being injected into

AC C

the mPFC. However, higher concentrations of NKH 477 were not completely soluble in vehicle. Furthermore, in subsequent studies it was found that NKH 477 enhanced the expression of cocaine sensitization when tested 3 weeks after the daily injections. This time point was chosen based on studies above that showed that SQ22536 was more effective in blocking late expression of sensitization compared to early expression. Thus, as was the case for inhibition of adenylyl cyclase, it appears that stimulation of this enzyme only enhances the motor-stimulant response to cocaine when inhibitory receptor coupling is

Liu and Steketee/ Page 24

ACCEPTED MANUSCRIPT

reduced as seen in late sensitization (Bowers et al. 2004; Xie and Steketee 2009). Similar to our previous work, the present studies focused on the ventral (v) portion of the mPFC. The vmPFC sends dense projections to the VTA, which is a region demonstrated to

RI PT

play a key role in the initiation of sensitization (Sesack and Pickel 1992). Thus, excitotoxic lesions of the vmPFC have been reported to prevent the induction of sensitization (Tzschentke

SC

and Schmidt 1998). Furthermore, as described earlier, inhibitory receptor modulation of excitatory output from the vmPFC is reduced which we have shown leads to enhanced

M AN U

glutamate transmission in the VTA (Jayaram and Steketee 2004; Liu and Steketee 2011; Xie and Steketee 2008). This enhanced glutamate drive in the VTA could be, in part, responsible for the initiation of sensitization that can be reduced by inhibition of adenylyl cyclase activity in the vmPFC. Also, the augmented drive of the VTA could lead to increased dopamine release

TE D

in the nucleus accumbens associated with the expression of sensitization. Thus, inhibiting adenylyl cyclase activity in the vmPFC could reduce the expression of sensitization by

EP

reducing excitation of the VTA.

AC C

In conclusion, the present studies demonstrated that at a time point when agonists of inhibitory receptors in the mPFC have reduced function following a cocaine sensitization treatment regimen (Beyer and Steketee 2002; Steketee and Beyer 2005; Xie and Steketee 2009), this loss of function can be bypassed by treating with an inhibitor of adenylyl cyclase activity. These data argue that signaling systems downstream of receptor-G protein coupling remains intact in animals with a history of repeated cocaine exposure. Thus, the development of treatment strategies that bypass receptors and modulates second messenger

Liu and Steketee/ Page 25

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

systems might prove to be an alternative approach for substance abuse.

Liu and Steketee/ Page 26

ACCEPTED MANUSCRIPT Figure legends

Fig. 1 The effects of intra-mPFC injection of (a) SQ22536 and (b) NKH 477 on the acute motor response to cocaine (15 mg/kg i.p.). The figure legend indicates intra-mPFC

RI PT

injection/systemic injection and data are expressed as mean photocell counts ± SEM. *p<

SC

0.05 compared to saline/saline and +p< 0.05 compared to saline/cocaine.

Fig. 2 The effects of intra-mPFC SQ22536 (50 nmol/side) injection on the initiation of

M AN U

behavioral sensitization to cocaine (15 mg/kg i.p.). The figure legend indicates test day treatment (pretreatment) with pretreatment being shown as intra-mPFC injection/systemic injection. Data are expressed as a mean ± SEM photocell counts for motor activity. *p< 0.05

TE D

+ compared to cocaine (saline/saline) and p< 0.05 compared to cocaine (saline/cocaine).

Fig. 3 The effects of intra-mPFC SQ22536 (50 nmol/side) injection on the expression of

EP

behavioral sensitization to cocaine (15 mg/kg i.p.) after (a, b) 1 day, (c, d) 7 days and (e, f) 30

AC C

days withdrawal from repeated cocaine exposure. The figures legend indicates test day treatment (pretreatment) with test treatment being shown as intra-mPFC injection/systemic injection. Data are expressed as a mean ± SEM photocell counts for motor activity. *p<0.05 compared to saline/cocaine (saline), +p<0.05 compared to saline/cocaine (cocaine) (a, c and e) and +p<0.05 compared to SQ22536/cocaine (cocaine) (b, d and f).

Fig. 4 The effects of intra-mPFC NKH 477 (40 nmol/side) injection on the expression of

Liu and Steketee/ Page 27

ACCEPTED MANUSCRIPT

behavioral sensitization to cocaine (15 mg/kg i.p.) after 21 days withdrawal from repeated cocaine exposure. The figures legend indicates test day treatment (pretreatment) with test treatment being shown as intra-mPFC injection/systemic injection. Data are expressed as a

SC

and +p<0.05 compared to saline/cocaine (cocaine).

RI PT

mean ± SEM photocell counts for motor activity. *p<0.05 compared to saline/cocaine (saline)

Fig. 5 Injection sites placements in the mPFC. The figure shows a representative illustration

M AN U

of injector placement from animals included in the analysis. Illustrations were derived from The Rat Brain in Stereotaxic Coordinates (Paxinos and Watson 1997). fmi forceps minor of the

AC C

EP

TE D

corpus callosum, Cg3 cingulate cortex, area 3, IL infralimbic cortex

Liu and Steketee/ Page 28

ACCEPTED MANUSCRIPT References

Bacon SJ, Headlam AJ, Gabbott PL, Smith AD (1996) Amygdala input to medial prefrontal

RI PT

cortex (mPFC) in the rat: a light and electron microscope study. Brain Res 720: 211-219. Bernard ML, Peterson YK, Chung P, Jourdan J, Lanier SM (2001) Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins. J

SC

Biol Chem 276: 1585-1593.

M AN U

Bettler B, Tiao JY (2006) Molecular diversity, trafficking and subcellular localization of GABAB receptors. Pharmacol Therapeut 110: 533-543.

Beyer CE, Steketee JD (1999) Dopamine depletion in the medial prefrontal cortex induces sensitized-like behavioral and neurochemical responses to cocaine. Brain Res 833:

TE D

133-141.

Beyer CE, Steketee JD (2000) Intra-medial prefrontal cortex injection of quinpirole, but not

EP

SKF 38393, blocks the acute motor-stimulant response to cocaine in the rat. Psychopharmacology 151: 211-218.

AC C

Beyer CE, Steketee JD (2001) Characterization of the role of medial prefrontal cortex dopamine receptors in cocaine-induced locomotor activity. Behav Neurosci 115: 1093-1100.

Beyer CE, Steketee JD (2002) Cocaine sensitization: modulation by dopamine D2 receptors in the rat medial prefrontal cortex. Cereb Cortex 12: 526-535. Bobeck, EN, Chen QL, Morgan MM, Ingram SL (2014) Contribution of adenylyl cyclase modulation of pre- and postsynaptic GABA neurotransmission to morphine antinociception

Liu and Steketee/ Page 29

ACCEPTED MANUSCRIPT and tolerance. Neuropsychopharmacology 39: 2142-2152

Bowers MS, McFarland K, Lake RW, Peterson YK, Lapish CC, Gregory ML, Lanier SM, Kalivas PW (2004) Activator of g protein signaling 3; a gatekeeper of cocaine sensitization

RI PT

and drug seeking. Neuron 42: 269-281. Carey RJ, Dai H, Damianopoulos EN, De Palma G (1994) Cocaine stimulant effects vary with

SC

cocaine levels in medial prefrontal cortex. Neuroreport 5: 2345-2348.

Carr DB, Sesack SR (2000) Projections from the rat prefrontal cortex to the ventral tegmental

M AN U

area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J Neurosci 20: 3864-3873.

Delfs JM, Schreiber L, Kelley AE (1990) Microinjection of cocaine into the nucleus accumbens elicits locomotor activation in the rat. J Neurosci 10: 303-310.

Ther 46: 199-200.

TE D

Downs A, Eddy N (1932) The effect of repeated doses of cocaine on the rat. J Pharmacol Exp

EP

Febo M, Gonzalez-Rodriguez LA, Capo-Ramos DE, Gonzalez-Segarra NY, Segarra AC (2003)

AC C

Estrogen-dependent alterations in D2/D3-induced G protein activation in cocaine-sensitized female rats. J Neurochem 86: 405-412. Jackson DM, Westlind-Danielsson A (1994) Dopamine receptors: molecular biology, biochemistry and behavioural aspects. Pharmacol Therapeut 64: 291-369. Jay TM, Burette F, Laroche S (1996) Plasticity of the hippocampal-prefrontal cortex synapses. Journal of Physiology (Paris) 90: 361-366. Jayaram P and Steketee JD (2004) Effects of repeated cocaine on medial prefrontal cortical

Liu and Steketee/ Page 30

ACCEPTED MANUSCRIPT

GABAB receptor modulation of neurotransmission in the mesocorticolimbic dopamine system. J. Neurochem. 90: 839-847. Kalivas PW, Sorg BA, Hooks MS (1993) The pharmacology and neural circuitry of

RI PT

sensitization to psychostimulants. Behav Pharmacol 4: 315-334. Kalivas PW, Stewart J (1991) Dopamine transmission in the initiation and expression of drug-

SC

and stress-induced sensitization of motor activity. Brain Res Rev 16: 223-244.

Kelly P, Iversen S (1976) Selective 60HDA-induced destruction of mesolimbic dopamine

M AN U

neurons: abolition of psychostimulant-induced locomotor activity in rats. Eur J Pharmacol 40: 45-56.

Kew JN, Kemp JA (2005) Ionotropic and metabotropic glutamate receptor structure and pharmacology. Psychopharmacology (Berl) 179: 4-29.

TE D

Lindvall O, Bjorklund A, Divac I (1978) Organization of catecholamine neurons projecting to the frontal cortex in the rat. Brain Res 142: 1-24.

EP

Liu K and Steketee JD (2011) Repeated exposure to cocaine alters medial prefrontal cortex

AC C

dopamine D2-like receptor modulation of glutamate and dopamine neurotransmission in the mesocorticolimbic system. J. Neurochem. 119: 332-341. Milliken GA, Johnson DE (1984) Analysis of Messy Data; Vol 1, Designed Experiments. Lifetime Learning, Toronto, Ontario, pp 326-337 Natochin M, Lester B, Peterson YK, Bernard ML, Lanier SM, Artemyev NO (2000) AGS3 inhibits GDP dissociation from galpha subunits of the Gi family and rhodopsin-dependent activation of transducin. J Biol Chem 275: 40981-40985.

Liu and Steketee/ Page 31

ACCEPTED MANUSCRIPT

Oades RD, Halliday GM (1987) Ventral tegmental (A10) system: neurobiology. 1. Anatomy and connectivity. Brain Res Rev 12: 117-165. Omelchenko N, Sesack SR (2007) Glutamate synaptic inputs to ventral tegmental area

RI PT

neurons in the rat derive primarily from subcortical sources. Neuroscience 146: 1259-1274.

SC

Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic Coordinates. 2nd Edition. Academic Press, New York

M AN U

Retaux S, Caboche J, Rogard M, Julien JF, Penit-Soria J, Besson MJ (1993) GABA interneurons in the rat medial frontal cortex: characterization by quantitative in situ hybridization of the glutamic acid decarboxylase (GAD67) mRNA. Brain Res 611: 187-196. Retaux S, Julien JF, Besson MJ, Penit-Soria J (1992) Expression of GAD mRNA in GABA

TE D

interneurons of the rat medial frontal cortex. Neurosci Lett 136: 67-71. Schenk S, Snow S (1994) Sensitization to cocaine's motor activating properties produced by

AC C

659: 17-22.

EP

electrical kindling of the medial prefrontal cortex but not of the hippocampus. Brain Res

Sesack SR, Deutch AY, Roth RH, Bunney BS (1989) Topographical organization of the efferent projections of the medial prefrontal cortex in the rat: an anterograde tract-tracing study with Phaseolus vulgaris leucoagglutinin. J Comp Neurol 290: 213-242. Sesack SR, Pickel VM (1992) Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area. J. Comp. Neurol 320: 145-160.

Liu and Steketee/ Page 32

ACCEPTED MANUSCRIPT

Steketee JD (1993) Injection of the protein kinase inhibitor H7 into the A10 dopamine region blocks the acute responses to cocaine: behavioral and in vivo microdialysis studies. Neuropharmacology 32: 1289-1297.

RI PT

Steketee JD (2003) Neurotransmitter systems in the medial prefrontal cortex: potential role in sensitization to psychostimulants. Brain Res Rev 41: 203-228.

SC

Steketee JD (2005) Cortical mechanisms of cocaine sensitization. Crit Rev Neurobiol 17: 69-86.

M AN U

Steketee JD, Beyer CE (2005) Intra-medial prefrontal cortex injections of baclofen blocks the initiation, but not the expression, of cocaine sensitization. Psychoparmacology 180: 352-358.

Steketee JD, Braswell BC (1997) Injection of Sch 23390, but not 7-OH-DPAT, into the ventral

1-7.

TE D

tegmental area blocks the acute motor stimulant response to cocaine. Behav Pharmacol 8:

EP

Steketee JD, Kalivas PW (2011) Drug wanting: behavioral sensitization and relapse to

AC C

drug-seeking behavior. Pharmacol Rev 63: 348-365. Tzschentke TM and Schmidt WJ (1998) The development of cocaine-induced behavioral sensitization is affected by disrete quinlinic acid lesions of the prelimbic medial prefrontal cortex. Brain Res. 795: 71-76. Xie X, Steketee JD (2008) Repeated exposure to cocaine reduces the ability of medial prefrontal cortical Group II metabotropic glutamate receptors to modulate behavioral and neurochemical responses to cocaine in rats. J. Neurochem. 107: 186-196

Liu and Steketee/ Page 33

ACCEPTED MANUSCRIPT

Xie X, Steketee JD (2009) Effects of repeated exposure to cocaine on group II metabotropic glutamate receptor function in the rat medial prefrontal cortex: behavioral and neurochemical studies. Psychopharmacology 203: 501-510.

AC C

EP

TE D

M AN U

SC

pathway. J Neurosci 31: 8476-8490.

RI PT

Yamaguchi T, Wang HL, Li X, Ng TH, Morales M (2011) Mesocorticolimbic glutamatergic

ACCEPTED MANUSCRIPT

1. Repeated exposure to cocaine reduces inhibitory receptor function in the medial prefrontal cortex. 2. Inhibition of adenylyl cyclase activity in the ventral medial prefrontal cortex reduces the initiation and expression of cocaine-induced behavioral sensitization.

RI PT

3. Stimulation of adenylyl cyclase activity in the ventral medial prefrontal cortex enhances the expression of cocaine sensitization.

AC C

EP

TE D

M AN U

SC

4. Thus, the loss of inhibitory receptor function can be reversed by direct inhibition of second messenger systems coupled to these receptors.