Differences in dopamine responsiveness to drugs of abuse in the nucleus accumbens shell and core of Lewis and Fischer 344 rats

Authors

  • Cristina Cadoni,

    1. CNR Institute of Neuroscience, Section of Cagliari, Cagliari, Italy
    2. Centre of Excellence for Studies on the Neurobiology of Addiction, University of Cagliari, Cagliari, Italy
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  • Gaetano Di Chiara

    1. CNR Institute of Neuroscience, Section of Cagliari, Cagliari, Italy
    2. Department of Toxicology, University of Cagliari, Cagliari, Italy
    3. Centre of Excellence for Studies on the Neurobiology of Addiction, University of Cagliari, Cagliari, Italy
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Address correspondence and reprint requests to Prof. Gaetano Di Chiara, Department of Toxicology, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy. E-mail: gadichia@tiscali.it

Abstract

The use of inbred rat strains provides a tool to investigate the role of genetic factors in drug abuse. Two such strains are Lewis and Fischer 344 rats. Although several biochemical and hormonal differences have been observed between Lewis and Fischer 344 strains, a systematic comparison of the effect of different drugs of abuse on dopamine (DA) transmission in the shell and core of the nucleus accumbens of these strains is lacking. We therefore investigated, by means of dual probe microdialysis, the effect of different doses of morphine (1.0, 2.5, and 5.0 mg/kg), amphetamine (0.25, 0.5, and 1.0 mg/kg) and cocaine (5, 10, and 20 mg/kg) on DA transmission in the shell and in the core of nucleus accumbens. Behavior was monitored during microdialysis. In general, Lewis rats showed greater DA responsiveness in the NAc core compared to F344 rats except after 2.5 mg/kg of morphine and 20 mg/kg of cocaine. In the NAc shell, different effects were obtained depending on drug and dose: after 1.0 mg/kg of morphine no strain differences were observed, at 2.5 and 5.0 mg/kg Lewis rats showed greater increase in DA in the NAc shell. Following amphetamine and cocaine challenge, Lewis rats showed greater DA increase in the shell after 0.25 mg/kg of amphetamine and 20 mg/kg of cocaine. Behavioral activation was greater in Lewis rats in response to the lowest dose of morphine (1.0 mg/kg), to the highest dose of amphetamine (1.0 mg/kg) and to all doses of cocaine. These differences might be the basis for the different behavioral responses of these strains to drugs of abuse.

Abbreviations used
DA

dopamine

DAT

dopamine transporter

F344

Fischer 344

LEW

Lewis

NAc

nucleus accumbens

TH

tyrosine hydroxylase

VTA

ventral tegmental area

Increasing evidence suggests that genetic factors play a fundamental role in the vulnerability to drug addiction (Kendler et al. 2000; Tsuang et al. 2001). One way to study the role of genetic factors in the reinforcing and dependence producing properties of drugs is to look for biochemical differences in the brain of genetically inbred strains and to relate these differences to behavioral effects of drugs (Crabbe et al. 1994). Two inbred rat strains that are suitable for such approach are the Lewis (LEW) and Fischer 344 (F344) rats. LEW and F344 show marked differences in their response to stress and to drugs of abuse (see for review, Kosten and Ambrosio 2002). LEW rats more readily acquire self-administration of alcohol, opiates and cocaine, and support drug self-administration at higher breaking points in progressive ratio schedules (Suzuki et al. 1988a, 1992; George and Goldberg 1989; Ambrosio et al. 1995; Kosten et al. 1997; Martin et al. 1999). Recently, it has been reported that LEW rats readily acquire nicotine self-administration whereas F344 rats do not (Brower et al. 2002). However, once self-administration behavior has been acquired, F344 rats show higher response rates towards cocaine (Kosten et al. 2007). LEW rats also show a stronger conditioned place preference to morphine, cocaine, and nicotine compared to F344 rats (Guitart et al. 1992; Kosten et al. 1994; Horan et al. 1997; but see also Davis et al. 2007) although the reverse applies to amphetamine (Stöhr et al. 1998). Moreover, LEW but not F344 rats were reported to show facilitation of brain self-stimulation reward by Δ9-tetrahydrocannabinol (Lepore et al. 1996).

Given the role of mesolimbic dopamine (DA) in the locomotor activating and rewarding properties of drugs of abuse the research on the neurochemical basis of this different sensitivity to drugs of abuse of LEW and F344 rats has been focused on differences in mesolimbic DA function and responsiveness to drugs. Previous studies have shown that LEW rats have higher levels of tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) and lower levels in the nucleus accumbens (NAc) compared to F344 rats (Beitner-Johnson et al. 1991; Guitart et al. 1992; Ortiz et al. 1995; Haile et al. 2001). F344 rats have higher numbers of spontaneously active neurons in VTA and substantia nigra pars compacta (SNC) but LEW rats have a greater percentage of substantia nigra pars compacta and VTA DA neurons exhibiting a burst firing pattern (Minabe et al. 1995). In the NAc, LEW rats show lower levels of Giα1/2 (Guitart et al. 1993; Brodkin et al. 1998; Haile et al. 2001) and higher levels of adenylate cyclase and cyclic AMP-dependent protein kinase compared to F344 rats (Guitart et al. 1993). Although there are no significant differences in the levels of D1-like DA receptors between strains, at least in the striatum and NAc, the levels of D2 like DA receptors are lower in the striatum and NAc core, and D3 DA receptors are lower in the NAc shell and olfactory tubercle of LEW rats compared to F344 rats (Flores et al. 1998). Other differences between strains include differences in the levels of DA transporter (DAT), that are lower in the striatum, NAc shell and core and in the olfactory tubercle of LEW rats (Flores et al. 1998).

Studies aimed at detecting differences in the response of extracellular DA induced by different drugs of abuse reported contrasting results. Thus, when Camp et al. (1994) observed a greater increase in DA in the ventral striatum of LEW rats after methamphetamine and cocaine, Strecker et al. (1995) and Fernandez et al. (2003) did not find significant differences between LEW and F344 rats in the increase in extracellular DA induced by cocaine or by 3,4-methylendioxymethamphetamine in the NAc and Mocsary and Bradberry (1996) found a greater increase in extracellular DA after ethanol in the NAc of F344 rats. Apart from several experimental differences, an important role in these discrepancies might be played by the specific subdivision of the NAc selected for microdialysis studies. In fact, an increasing body of evidences shows that the NAc is not homogenous and that two anatomical and functional distinct compartments can be distinguished: a core and a shell region, whose DA innervation shows differential responses to drug and non-drug rewards (see for review, Zahm 1999; Di Chiara and Bassareo 2007). To better understand the neurochemical basis of the differences between LEW and F344 strains in the vulnerability to drugs of abuse, we performed dual probe microdialysis experiments to monitor the effects of acute challenge with morphine, amphetamine, and cocaine on extracellular DA in the NAc shell and core and on behavior.

Materials and methods

Animals

Male Lewis and Fischer 344 rats (Charles River, Calco, Italy) weighing 270–300 g were housed in group of six per cage with food and water ad libitum under an artificial 12 h light: 12 h dark cycle (lights on at 8:00 am) and standard conditions of temperature (23°C) and humidity (60%). Animals were allowed at least 1 week to habituate to the novel housing conditions before testing. All animal experimentations have been conducted in accordance with the guidelines for care and use of experimental animals of the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Surgery

The rats were anesthetized with chloral hydrate (400 mg/kg i.p.) and implanted with vertical dialysis probes prepared essentially according to the method of Di Chiara et al. (1993) modified by Tanda et al. (1996). The length of the dialysing area was of 1.5 mm. Each rat was implanted with two probes, one at the level of the NAc shell (A 2.2, L 1.1 from bregma and V 7.8 from dura) and the other at the level of the NAc core of the opposite emisphere (A 1.6, L 1.8 from bregma and V 7.5 from dura) according to the atlas of Paxinos and Watson (1998). After surgery, the rats were individually housed in plexiglass hemispherical bowls (diameter 50 cm) which also served as the experimental environment.

Analytical procedure

On the day following surgery, the rats were connected to an infusion pump and perfused with Ringer’s solution (147 mmol/L NaCl, 2.2 mmol/L CaCl2, 4 mmol/L KCl) at a constant rate of 1 μL/min. Dialysate samples were collected every 20 min and injected into an HPLC apparatus equipped with a reverse phase ODS column (Symmetry C-18, 3.9 × 150 mm, 5 μm particle size, Waters, Milan, Italy) and a coulometric detector (ESA Coulochem II, Alfatech, Genova, Italy) in order to quantitate DA. The first electrode was set at +100 mV (oxidation) and the second at –125 mV (reduction). The composition of the mobile phase was 50 mmol/L NaH2PO4, 0.1 mmol/L Na2-EDTA, 0.5 mmol/L n-octyl sodium sulfate and 15% methanol; pH was adjusted to 5.5. The sensitivity of the assay was 2 fmol/sample. The experiments were performed between 8 am and 7 pm. Baseline samples were collected for at least 3 h before drug challenge.

Behavior

In order to estimate the behavioral response to drug treatments during the dialysis experiments the animals were videotaped and two behavioral categories were distinguished: non-stereotyped activity, consisting of normal exploratory behavior including forward locomotion, sniffing around, and rearing and stereotyped activity, consisting of activity confined to a restricted area of the cage not directed to any specific goal and involving confined gnawing, downward sniffing, and licking. In the case of morphine a third behavioral category was evaluated that is opiate catalepsy consisting of frozen postures and trunk rigidity. The behavior was evaluated by an observer unaware of the rat strain. The percentage of time spent by the rat performing each behavior was recorded, throughout the test session.

Histology

At the end of the experiment, rats were anesthetized and transcardially perfused with 50 mL saline and 50 mL of a 4% formaldehyde/1% calcium acetate/100 mmol/L NaCl solution. The probes were removed and brains were cut with a Vibratome in serial coronal slices oriented according to the atlas of Paxinos and Watson (1998). The location of the probes was reconstructed and referred to the atlas of Paxinos and Watson (1998). Fig. 1 shows the location of the probes in the NAc shell and core of F344 and LEW rats.

Figure 1.

 Location of the dialysis probes (dialysing portion) within the NAc shell and core of F344 and LEW rats. Although the side of implant of the probes was randomly assigned to the shell and core probes, in this drawing all the probes in one area are represented on one side and the others in the opposite side. The numbers in each forebrain section (redrawn from Paxinos and Watson 1998) indicate millimeters anterior from bregma. Stippled lines refer to probes not correctly placed and therefore animals not included in the statistical analysis. Sh, Co shell and core of the NAc.

Drugs

Cocaine hydrochloride (5, 10, and 20 mg/kg of salt, Mc Farlan Smith, Edimburgh, Scotland, UK) was dissolved in saline and injected intraperitoneally (i.p.) in a volume of 3 mL/kg of body weight. Amphetamine sulfate (0.25, 0.5, and 1.0 mg/kg of salt, Sigma, Milan, Italy), morphine hydrochloride (1.0, 2.5, and 5.0 mg/kg of salt, Franchini Prodotti Chimici S.r.l., Mozzate, Como, Italy) were dissolved in saline and injected subcutaneously (s.c.) in a volume of 1 mL/kg of body weight. These drug doses were selected on the basis of previous studies on Sprague–Dawley rats (Cadoni and Di Chiara 1999; Cadoni et al. 2000).

Statistics

Statistical analysis was carried out by Statistica for Windows. Differences in behavioral scores and in the levels of dialysate DA between groups were assessed by two-way analysis of variance (anova) for repeated measures with strain and drug dose as independent variables. Results showing significant overall changes were subjected to post hoc Tukey’s test. Basal values of extracellular DA were the means of three consecutive samples differing by no more than 10%. The data were expressed as percent of basal values (100%). The data from animals with incorrect placement of the dialysis probes were excluded.

Results

The mean of DA basal levels in dialysates from shell and core of NAc of the two strains were the following: F344 shell 95 ± 6 (n = 67) core 128 ± 9 (n = 60); LEW shell 88 ± 7 (n = 61) core 106 ± 7 (n = 71). Although there is a trend toward lower DA levels in the core of LEW rats statistical analysis did not show any significant difference between strains (shell F1,126 = 1.79, p > 0.05; core F1,129 = 3.11, p > 0.05).

Morphine

Figure 2 shows the effect of 1.0, 2.5, and 5.0 mg/kg of morphine in NAc shell and core of F344 and LEW rats. Basal DA output in the shell and core of F344 and LEW rats were the following: F344 shell 96 ± 11 core 125 ± 14; LEW shell 88 ± 7 core 103 ± 11 with no significantly differences in each area between the two strains (shell: F1,42 = 0.37, p > 0.05; core F1,44 = 1.74, p > 0.05). While 1.0 mg/kg of morphine induced an equivalent increase in extracellular DA in the shell of the two strains (Fstrain (1,13) = 0.05, p > 0.05, Fstrain × time (9,117) =  0.83, p > 0.05), higher doses of morphine elicited a greater DA increase in the shell of LEW compared to the shell of F344 rats (2.5 mg/kg: Fstrain (1,11) = 7.28, p < 0.05, post hoc p < 0.05, Fstrain × time (9,99) = 4.34, p < 0.0001; 5 mg/kg: Fstrain (1,14) = 1.62, p > 0.05, Fstrain × time (9,126) = 3.82, p < 0.001). DA responsiveness to morphine in the NAc core was greater in LEW as compared to F344 rats after 1.0 and 5.0 mg/kg (1.0 mg/kg: Fstrain (1,13) = 6.70, p < 0.05, post hoc p < 0.05, Fstrain × time (9,117) = 2.13, p < 0.05; 5.0 mg/kg: Fstrain (1,13) =  3.43, p > 0.05, Fstrain × time (9,117) = 2.73, p < 0.01) but equal after the intermediate dose (Fstrain (1,15) = 0.95, p > 0.05, Fstrain × time (9,135) = 1.08, p > 0.05). Two-way anova applied to the data from the shell and core of each strain (Table 1) reveals that after 1.0 mg/kg of morphine, F344 rats show a preferential stimulation of DA transmission in the shell compared to the core, after 2.5 and 5.0 mg/kg of morphine, only LEW rats show a preferential stimulation of DA transmission in the shell compared to the core.

Figure 2.

 Effect of different doses of morphine on basal extracellular DA in dialysates from the NAc shell and core of Fischer 344 (circles) and Lewis (squares) rats. Results are expressed as means ± SEM of the percent of basal values. Filled symbols represent points significantly different from respective basal values by two-way anova for repeated measures followed by Tukey’s test. *p < 0.05 and **p < 0.01 significantly different from the correspondent value of the other strain by two-way anova followed by Tukey’s test.

Table 1.   Results of statistical analysis (two-way anova for repeated measures followed by Tukey’s test) applied to the data (percent of basal values) from the shell and core within each strain
 LewisFischer 344
Morphine (mg/kg)
 1.0Sh = Co F(1,12) = 0.00, p > 0.05Sh > Co F(1,14) = 12.16, p < 0.01
 2.5Sh > Co F(1,16) = 17.43, p < 0.01Sh = Co F(1,10) = 0.81, p > 0.05
 5.0Sh > Co F(1,13) = 6.53, p < 0.05Sh = Co F(1,13) = 3.80, p > 0.05
Amphetamine (mg/kg)
 0.25Sh = Co F(1,10) = 0.74, p > 0.05Sh = Co F(1,9) = 2.71, p > 0.05
 0.5Sh = Co F(1,12) = 0.02, p > 0.05Sh > Co F(1,15) = 4.85, p < 0.05
 1.0Sh = Co F(1,9) = 0.71, p > 0.05Sh > Co F(1,13) = 25.58, p < 0.01
Cocaine (mg/kg)
 5.0Sh = Co F(1,14) = 2.63, p > 0.05Sh > Co F(1,11) = 4.94, p < 0.05
 10Sh = Co F(1,14) = 0.08, p > 0.05Sh = Co F(1,14) = 3.15, p > 0.05
 20Sh > Co F(1,13) = 6.59, p < 0.01Sh = Co F(1,11) = 2.62, p > 0.05

Following challenge with all the doses of morphine LEW rats showed opiate catalepsy absent in F344 rats (1.0 mg/kg: F1,15 = 86.44, p < 0.0001, post hoc p < 0.001; 2.5 mg/kg: F1,15 = 64.24, p < 0.001, post hoc p < 0.001; 5.0 mg/kg: F1,14 = 37.04, p < 0.0001, post hoc p < 0.001), Fig. 3. While no significant difference in motor activation was observed after 5.0 mg/kg of morphine between strains (Fig. 4) neither in non-stereotyped (Fstrain (1,14) = 0.30, p > 0.05, Fstrain × time (9,126) = 1.15, p > 0.05) nor in stereotyped activity (Fstrain (1,14) = 0.01, p > 0.05, Fstrain × time (9,126) = 0.28, p > 0.05), a significant effect of strain and strain × time interaction was obtained after 1.0 mg/kg of morphine (non-stereotyped activity: Fstrain (1,15) = 5.91, p < 0.05, Fstrain × time (9,135) = 3.09, p < 0.01; stereotyped activity: Fstrain (1,15) = 3.75, p < 0.05, Fstrain × time (9,135) = 1.75, p > 0.05). Post hoc analysis showed that LEW rats had a greater motor activation (p < 0.05). After the intermediate dose of morphine no significant difference was obtained in the non-stereotyped as well as in the stereotyped activity elicited in the two strains (non-stereotyped activity: Fstrain (1,15) = 0.23, p > 0.05; stereotyped activity: Fstrain (1,15) =  0.00, p > 0.05, Fstrain × time (9,135) = 1.87, p > 0.05) but only a difference in the time course of appearance of non-stereotyped activity (Fstrain × time (9,135) = 2.1, p < 0.05) that indeed was marginal compared to the time spent in stereotyped movements (mainly gnawing), Fig. 4.

Figure 3.

 Time spent by each strain (Fischer 344 open bars, Lewis filled bars) in frozen postures and trunk rigidity (opiate catalepsy) during the 3 h of observation after morphine challenge. The number of animals for each dose group is the same as reported in Fig. 4. These effects are observed immediately after morphine challenge. Results are expressed as means ± SEM of the time in min spent by the rat in this status. **p < 0.001 by one way anova followed by Tukey’s test.

Figure 4.

 Behavioral effects induced in Fischer 344 (open bars) and Lewis (filled bars) rats by challenge with different doses of morphine. The results are expressed as means ± SEM of the percentage of time spent performing each behavioral item. The time points at which the sum of the percentage of time spent in the different behavioral items is not 100, the difference has to be referred to sedation or no activity. *p < 0.05 significantly different from the correspondent value of the other strain by two-way anova for repeated measures followed by Tukey’s test.

Amphetamine

Basal DA output in the shell and core of F344 and LEW rats were the following: F344 shell 93 ± 8 core 134 ± 12, LEW shell 90 ± 10 core 112 ± 10 with no significantly differences in each area between the two strains (shell:F1,38 =  0.05, p > 0.05; core F1,37 =  1.70, p > 0.05). While all the doses of amphetamine tested elicited a greater increase in extracellular DA in the core of LEW compared to the core of F344 rats (0.25 mg/kg: Fstrain (1,10) = 5.71, p < 0.05, post hoc p < 0.05, Fstrain × time (9,90) = 2.17, p < 0.05; 0.5 mg/kg: Fstrain (1,14) = 10.86, p < 0.01, post hoc p < 0.01, Fstrain × time (9,126) = 4.12, p < 0.001; 1.0 mg/kg: Fstrain (1,9) = 5.67, p < 0.05, post hoc p < 0.05, Fstrain × time (9,81) = 3.72, p < 0.001), Fig. 5, the effect in the shell of the two strains was variable depending on the dose. While at the dose of 0.25 mg/kg the increase in extracellular DA was greater in LEW than in F344 animals (Fstrain (1,9) = 6.98, p < 0.05, post hoc p < 0.05, Fstrain × time (9,81) = 1.79, p > 0.05), after 1.0 mg/kg of amphetamine F344 rats showed a greater DA responsiveness (Fstrain (1,12) = 3.78, p > 0.05, Fstrain × time (9,108) = 3.17, p < 0.01) and no differences between strains were observed after the intermediate dose of 0.5 mg/kg (Fstrain (1,13) = 1.6, p > 0.05, Fstrain × time (9,117) = 1.43, p > 0.05), Fig. 5. While LEW rats did not show preferential stimulation of DA transmission in the shell compared to the core at all doses of amphetamine tested, F344 showed a preferential stimulation in the shell compared to the core at 0.5 and 1.0 mg/kg (Table 1).

Figure 5.

 Effect of different doses of amphetamine on basal extracellular DA in dialysates from the NAc shell and core of Fischer 344 (circles) and Lewis (squares) rats. Results are expressed as means ± SEM of the percent of basal values. Filled symbols represent points significantly different from respective basal values by two-way anova for repeated measures followed by Tukey’s test. *p < 0.05 and **p < 0.01 significantly different from the correspondent value of the other strain by two-way anova followed by Tukey’s test.

Administration of 0.25 mg/kg of amphetamine elicited a greater stereotyped activation in F344 compared to LEW rats (Fstrain (1,10) = 6.98, p < 0.05, post hoc p < 0.05, Fstrain × time (6,60) = 1.49, p > 0.05) but no significant differences were observed between strains in the non-stereotyped activity (Fstrain (1,10) = 0.38, p > 0.05) except for the time course of the activation that was prolonged in LEW rats (Fstrain × time (6,60) = 2.4, p < 0.05), Fig. 6. While no significant difference between strains was observed in the activation elicited by 0.5 mg/kg of amphetamine (non-stereotyped activity: Fstrain (1,15) = 0.11, p > 0.05, Fstrain × time (9,135) = 1.46, p > 0.05; stereotyped activity: Fstrain (1,15) = 0.46, p > 0.05, Fstrain × time (9,135) = 1.0, p > 0.05), a significant greater locomotor activation was observed in LEW compared to F344 rats after the dose of 1.0 mg/kg (non-stereotyped activity: Fstrain (1,12) = 4.62, p < 0.05, post hoc p < 0.05, Fstrain × time (9,108) = 1.84, p < 0.05) without differences between strains in the stereotyped activity (Fstrain (1,12) = 0.07, p > 0.05, Fstrain × time (9,108) = 0.10, p > 0.05), Fig. 6.

Figure 6.

 Behavioral effects induced in Fischer 344 (open bars) and Lewis (filled bars) rats by challenge with different doses of amphetamine. The results are expressed as means ± SEM of the percentage of time spent performing each behavioral item. The time points at which the sum of the percentage of time spent in the different behavioral items is not 100, the difference has to be referred to sedation or no activity. *p < 0.05 and **p < 0.01 significantly different from the correspondent value of the other strain by two-way anova for repeated measures followed by Tukey’s test.

Cocaine

Basal DA output in the shell and core of F344 and LEW rats were the following: F344 shell 96 ± 5 core 126 ± 17 LEW shell 85 ± 7 core 102 ± 6 with no significant differences in each area between the two strains (shell:F1,42 =  0.93, p > 0.05; core F1,43 =  3.37, p > 0.05). Fig. 7 shows the effect of 5, 10 and 20 mg/kg i.p. of cocaine to F344 and LEW rats. While 5 and 10 mg/kg of cocaine elicited an increase in extracellular DA in the shell of the two strains that was superimposable (5 mg/kg: Fstrain (1,11) = 0.51, p > 0.05, Fstrain × time (9,99) = 0.92, p > 0.05; 10 mg/kg: Fstrain (1,16) = 0.10, p > 0.05, Fstrain × time (9,144) = 0.75, p > 0.05) the effect observed in the core was significantly different in the two strains (5 mg/kg: Fstrain (1,14) = 6.97, p < 0.01, Fstrain × time (9,126) = 4.03, p < 0.001; 10 mg/kg: Fstrain (1,12) = 0.89, p > 0.05, Fstrain × time (9,108) = 5.29, p < 0.0001) with LEW rats showing greater DA increases compared to F344 (post hoc p < 0.01). After the higher dose of cocaine while no significant effect of strain and strain × time interaction was observed in the stimulation of DA transmission in the NAc core (F1,13 = 1.37, p > 0.05, F9,117 = 0.79, p > 0.05) a significant strain x time interaction was observed in the stimulation of DA transmission in the NAc shell (Fstrain (1,11) = 0.19, p > 0.05, Fstrain × time (9,99) = 8.88, p < 0.0001) with a significantly greater increase in extracellular DA in the shell of LEW rats at 20, 40 and 60 min (p < 0.01). Two-way anova applied to the data from the shell and core of each strain (tab. 1) revealed that F344 rats show a preferential stimulation of DA transmission in the shell compared to the core after 5 mg/kg of cocaine and LEW after 20 mg/kg of cocaine while no preferential stimulation was observed in either strain after 10 mg/kg.

Figure 7.

 Effect of different doses of cocaine on basal extracellular DA in dialysates from the NAc shell and core of Fischer 344 (circles) and Lewis (squares) rats. Results are expressed as means ± SEM of the percent of basal values. Filled symbols represent points significantly different from respective basal values by two-way anova for repeated measures followed by Tukey’s test. *p < 0.05 and **p < 0.001 significantly different from the correspondent value of the other strain by two-way anova followed by Tukey’s test.

As regards the behavioral stimulation induced by cocaine, LEW rats appeared to be more sensitive than F344 rat to the locomotor activating properties of cocaine (Fig. 8) both in terms of non-stereotyped (cocaine 5 mg/kg Fstrain (1,15) = 7.45, p < 0.01, post hoc p < 0.01, Fstrain × time (6,90) = 3.43, p < 0.01; cocaine 10 mg/kg Fstrain (1,18) = 5.97, p < 0.05, post hoc p < 0.05, Fstrain × time (6,108) = 1.23, p > 0.05; cocaine 20 mg/kg Fstrain (1,16) = 7.2, p < 0.01, post hoc p < 0.01, Fstrain × time (9,144) = 3.3, p < 0.001) and stereotyped activity (cocaine 5 mg/kg Fstrain (1,15) = 6.21, p < 0.05, post hoc p < 0.05, Fstrain × time (6,90) = 2.88, p < 0.01; cocaine 10 mg/kg Fstrain (1,18) = 5.99, p < 0.05, post hoc p < 0.05, Fstrain × time (6,108) = 2.10, p < 0.05) with the only exception of the higher dose of cocaine after which the stereotyped activation was not different in the two strains (Fstrain (1,16) = 0.12, p > 0.05, Fstrain × time (9,144) = 0.30, p > 0.05).

Figure 8.

 Behavioral effects induced in Fischer 344 (open bars) and Lewis (filled bars) rats by challenge with different doses of cocaine. The results are expressed as means ± SEM of the percentage of time spent performing each behavioral item. The time points at which the sum of the percentage of time spent in the different behavioral items is not 100, the difference has to be referred to sedation or no activity. *p < 0.05 significantly different from the correspondent value of the other strain by two-way anova for repeated measures followed by Tukey’s test.

In Tables 2 and 3 are summarized all the biochemical and behavioral effects of drugs in the two strains.

Table 2.   Summary of the effects of the different drugs and drug doses in raising extracellular DA in the shell and core of nucleus accumbens of the two strains
 ShellCore
  1. See results and figures for statistical details.

Morphine (mg/kg)
 1.0LEW = F344LEW > F344
 2.5LEW > F344LEW = F344
 5.0LEW > F344LEW > F344
Amphetamine (mg/kg)
 0.25LEW > F344LEW > F344
 0.5LEW = F344LEW > F344
 1.0LEW < F344LEW > F344
Cocaine (mg/kg)
 5.0LEW = F344LEW > F344
 10LEW = F344LEW > F344
 20LEW > F344LEW = F344
Table 3.   Summary of the behavioral effects of the different drugs and drug doses in the two strains
 Non-stereotyped activityStereotyped activity
  1. See results and figures for statistical details.

Morphine (mg/kg)
 1.0LEW > F344LEW > F344
 2.5LEW = F344LEW = F344
 5.0LEW = F344LEW = F344
Amphetamine (mg/kg)
 0.25LEW = F344LEW < F344
 0.5LEW = F344LEW = F344
 1.0LEW > F344LEW = F344
Cocaine (mg/kg)
 5.0LEW > F344LEW > F344
 10LEW > F344LEW > F344
 20LEW > F344LEW = F344

Discussion

The results of this study show that F344 and LEW rats are differentially sensitive to the DA releasing and motor stimulant effects of morphine, amphetamine, and cocaine. In general, LEW rats show a greater DA responsiveness in the NAc core compared to F344 rats with the only exception of the intermediate dose of morphine (2.5 mg/kg) and of the highest dose of cocaine (20 mg/kg). In the NAc shell different effects are obtained depending on the drug and the drug dose: while at the dose of 1.0 mg/kg of morphine no differences between strains were obtained, at the dose of 2.5 and 5.0 mg/kg LEW rats show a greater increase in extracellular DA in the NAc shell. These differences might be the basis for the greater place-preference to morphine of LEW rats compared to F344 rats (Guitart et al. 1992). As suggested by several studies (see for review, Di Chiara et al. 2004) DA transmission in the NAc shell is involved in the rewarding effects of drugs of abuse. Guitart et al. (1992) utilized a dose of 4 mg/kg of morphine that should correspond to that of 5.0 mg/kg utilized here. Although at 5.0 mg/kg there is no effect of strain in the NAc shell, but only a significant strain × time interaction, the analysis for each strain (Table 1) shows that only LEW rats have a preferential stimulation of the DA transmission in the NAc shell compared to the core, a feature observed in male Sprague–Dawley rats (Di Chiara 2002). Indeed, the shell/core ratio of DA responsiveness to the drug, rather than the absolute amount of DA released in the NAc shell of the two strains, might be important for drug reward. If this was the case, F344 rats should show place preference after a dose of 1.0 mg/kg of morphine that induces a preferential increase in dialysate DA in the shell over the core. Indeed, according to a recent study (Davis et al. 2007), F344 but not LEW rats acquire conditioned place preference at 1.0 mg/kg of morphine. However these authors did not find any preference at 4 and 10 mg/kg of morphine in either strains, in contrast with previous reports (Guitart et al. 1992; Grakalic et al. 2006). The reason of these discrepancies might be due to differences in the nature, biased (Davis et al. 2007) versus unbiased (Guitart et al. 1992; Grakalic et al. 2006) of the conditioned place preference paradigm utilized.

Dopamine transmission in the NAc core has been suggested to play a role in acquiring (Smith-Roe and Kelley 2000), directing (Corbit et al. 2001) and sustaining (Salamone et al. 1997) instrumental action. Therefore, the greater increase in extracellular DA in the NAc core of LEW compared to F344 rats after 1.0 and 5.0 mg/kg of morphine would be in agreement with the higher rate of acquisition (Suzuki et al. 1988b; Ambrosio et al. 1995; Martin et al. 1999), maintenance (Martin et al. 2003) and higher breaking points in progressive ratio schedules of morphine self-administration (Martin et al. 1999).

The strain differences in the ability of morphine to stimulate DA transmission in the NAc shell and core are not accounted for by pharmacokinetic differences (Guitart et al. 1992; Gosnell and Krahn 1993; Lancellotti et al. 2001; Davis et al. 2007). It has been hypothesized that morphine and heroin stimulate DA transmission in the NAc shell through an action on μ1 opioid receptors located in the VTA (Tanda et al. 1997; Tanda and Di Chiara 1998). Although no differences between strains were observed in the number of μ receptors in the brain cortex and spinal cord (Herradon et al. 2003), a recent paper shows that LEW rats have lower levels of μ opioid receptors in several brain areas included VTA and NAc shell and core and caudate putamen (Sanchez-Cardoso et al. 2007). The relationship of these differences to the differences in the responsiveness of NAc shell and core DA to the various drugs of abuse is at the moment obscure.

Major strain differences were observed in the behavioral effects of morphine. A first difference is that opiate catalepsy was observed only in LEW rats. This, however, is likely to be a non-dopaminergic effect. Differences in motor activation are observed at 1.0 mg/kg morphine, with LEW rats showing greater activation both in terms of locomotion and stereotyped activity. This result is consistent with a greater DA response to morphine in the NAc core of LEW compared to F344 rats. With higher doses of morphine the prevalent expression of motor activation is stereotyped activity with no significant difference between strains.

Administration of different doses of amphetamine stimulated to a greater extent DA transmission in the NAc core of LEW compared to F344 rats, while DA response in the NAc shell was variable depending on the dose. Thus, after 0.25 mg/kg LEW rats showed a greater increase in extracellular DA compared to F344, after 0.5 mg/kg no difference between strains was observed and 1.0 mg/kg of amphetamine induced a significant greater increase in extracellular DA at 40 and 60 min in F344 strain. Moreover, F344 rats show a preferential stimulation of DA transmission in the shell compared to the core at 0.5 and 1.0 mg/kg (Table 1), while LEW rats did not show any difference in DA responsiveness in the shell and in the core at all doses of amphetamine tested. Since only the F344 strain shows place preference for amphetamine (Stöhr et al. 1998), these results are consistent with the idea that amphetamine-conditioned place preference is related to a preferential stimulation of DA transmission in the shell rather than to the absolute response of DA in the NAc shell.

A previous study reported that methamphetamine enhances to a greater extent extracellular DA in the ventral striatum of LEW compared to F344 rats (Camp et al. 1994). These results are difficult to compare with the present ones given the failure to distinguish between NAc shell and core placement of the probes but might reflect their prevalent core location. Camp et al. (1994) attributed these differences to pharmacokinetics differences. This, however, is not consistent with the present observation that depending on the dose amphetamine is more effective in raising dialysate DA in the F344 than LEW strain. Given the mechanism of action of amphetamine (Chiueh and Moore 1975; Raiteri et al. 1979; Liang and Rutledge 1982) differences in TH (Beitner-Johnson et al. 1991; Guitart et al. 1992; Ortiz et al. 1995; Haile et al. 2001) and DAT levels (Flores et al. 1998) in the mesolimbic system fully explain the differences between LEW and F344 rats in the DA stimulant properties of amphetamine.

As regards the motor effects of amphetamine in LEW and F344 strains, we observed a greater stereotyped activity in F344 rats at 0.25 mg/kg and a greater non-stereotyped activity in LEW rats at 1.0 mg/kg. Stöhr et al. (1998) observed more locomotor activation in response to amphetamine in F344 than LEW strain but results were not analyzed by a post hoc test. According to George et al. (1991) LEW rats are less sensitive to the locomotor stimulant effect of amphetamine. However, in this study behavior was monitored for a time interval too short (1 h instead of 3 h, present study) compared to the total duration of the drug-induced behavioral stimulation.

Following challenge with cocaine LEW rats displayed higher increases in extracellular DA in the NAc core after 5.0 and 10 mg/kg but no significant difference between strains was obtained after 20 mg/kg. In the NAc shell we observed a larger increase in DA after 20 mg/kg of cocaine in LEW as compared to F344 rats. As in the case of amphetamine, failure to distinguish between shell and core might account for inconsistencies between previous studies. Thus, while Camp et al. (1994) found a significant effect of strain after the dose of 10 mg/kg, with LEW rats showing greater DA increase, Strecker et al. (1995) found no difference between strains after 3, 10, and 30 mg/kg of cocaine. As regards, cocaine pharmacokinetics data in literature are contrasting. While Guitart et al. (1992) and Kosten et al. (1997) did not find any difference between strains after a cocaine dose of 15 mg/kg i.p. and an intravenous cocaine infusion (1.0 mg/kg) respectively, Camp et al. (1994) reported higher blood and brain levels of cocaine in LEW as compared to F344 rats after 10 mg/kg.

In the present study, 10 mg/kg of cocaine failed to preferentially stimulate DA transmission in the shell of NAc of any strain while 20 mg/kg did it only in the LEW strain. In relation to this it is notable that Kosten et al. (1994) did not obtained place preference after 7.5 mg/kg of cocaine in both strains and obtained place preference at 15 and 30 mg/kg of cocaine only in LEW rats. Faster rate of acquisition of cocaine self-administration (Kosten et al. 1997) might be related to greater absolute increases in extracellular DA in the NAc core of LEW rats compared to F344 ones.

As to the behavioral stimulant properties of cocaine, our results are in agreement with George et al. (1991) and Camp et al. (1994) who reported that LEW rats are more sensitive to the locomotor stimulating effect of cocaine.

In summary, although the differences observed in DA responsiveness in NAc shell and core between strains might be at the basis of the different vulnerability observed in place conditioning and self-administration studies this conclusion should be considered with caution. In fact, it should be taken into account that here we analyzed the effect of an acute challenge while place conditioning and self-administration studies involve more than one injection that might be non-contingent or contingent. These repeated drug injections might change the DA responsiveness of the two strains due to sensitization phenomenon.

In conclusion, LEW rats seem more responsive than F344 rats to the increase in DA transmission in the NAc shell and core induced by morphine, cocaine, and amphetamine. The increase in dialysate DA was preferential in the NAc shell except for amphetamine in LEW rats. Although it is difficult to correlate the neurochemical effects of acute drug exposure (present study) to the behavioral effects of repeated drug exposure, the strain differences in the effect of amphetamine (preferential increase in DA in the NAc shell of F344 but not LEW rats) could explain why amphetamine is the only drug of abuse reported to induce conditioned place preference in F344 but not LEW rats (Stöhr et al. 1998). In turn, the observation that LEW rats show, compared to F344 rats, greater increases in extracellular DA in the NAc core in response to all drug tested is reminiscent of the effect observed in outbred rats after pharmacological or stress induced sensitization (Cadoni and Di Chiara 1999, 2000; Cadoni et al. 2000, 2003). This property might result in facilitation of the acquisition and expression of instrumental responding for drug self-administration. This is in turn consistent with the proposal that NAc core DA is involved in acquiring (Smith-Roe and Kelley 2000), directing (Corbit et al. 2001) and sustaining (Salamone et al. 1997) instrumental action according to the incentive value of the predicted outcome.

If LEW strain is as a rule more responsive than F344 strain in operant paradigms of addictive drug effetcs, this is not the case in a drug-conditioned place preference paradigm, where, depending on the drug and dose, F344 strain is more responsive than LEW one. Strain differences, in this case, might be related to preferential stimulation of DA transmission in the NAc shell versus core. This in turn agrees with the role assigned to NAc shell DA in the acquisition of drug conditioned place preference (Fenu et al. 2006; Spina et al. 2006). This, while consistent with the importance assigned to DA in the rewarding and reinforcing properties of drugs of abuse, raises some doubts as to the drug specificity of the differences between LEW and F344 strains in terms of drug reinforcement in operant paradigms. Thus, LEW rats show higher sensitivity to temporal discounting (Anderson and Woolverton 2005) and acquire more efficiently than F344 strain in non-resetting delay paradigms of instrumental responding for food (Anderson and Elcoro 2007). This suggests that differences between LEW and F344 strains in the reinforcing effects of drugs of abuse in operant paradigms is related to basic differences in the impact of reinforcers on instrumental response emission rather than to specific differences in the rewarding properties of drugs of abuse. Finally, an additional factor of confound is the observation that although LEW rats acquire faster than F344 rats cocaine self-administration, F344 rats show higher rates of responding to cocaine self-administration in low-ratio schedules (FR3) and higher premature responses on active lever (Kosten et al. 2007). These properties might indicate a higher impulsivity of F344 as compared to LEW rats and a higher vulnerability to compulsive drug self-administration. Therefore, paradoxically, it might be the F344 rather than LEW strain to provide a model of human addiction (Kosten et al. 2007).

Acknowledgements

This study was supported by funds from Ministero dell’Università e Ricerca Scientifica (MIUR) (ex 40%) to GDC and by funds from Presidenza del Consiglio dei Ministri (DNPA) to GDC.

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