D2 vector assessment
Histological examination of the area of injection, as previously described did not reveal any unusual neuropathology or significant signs of inflammation associated with sites infected with AdCMV.DRD2 and those receiving control treatments. In addition, as demonstrated in our previous studies (Ikari et al. 1995, 1999; Umegaki et al. 1997; Ingram et al. 1998; Ogawa et al. 2000) rats receiving the DRD2 vector exhibited an increase in the DRD2 binding only at the injection site (NAc). This binding extended across several serial sections and no [3 H] raclopride binding was detected in contralateral sides. Figure 1 illustrates the NAc microinjection sites in these animals and an example of DRD2 binding in the NAc.
Figure 1. (a) A coronal section of the rat brain illustrating the NAc and the location of injection sites (adopted from Paxinos and Watson 1986). (b) Qualitative assessment of DRD2 binding 4 days after treatment with the DRD2 vector into the left NAc. Autoradiography of a rat coronal brain section treated with AdCMV.DRD2. Intense concentration of DRD2 demonstrates binding [3H]raclopride in the NAc near the injection site (see arrow).
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Autoradiography assessment of DRD2 in the NAc after AdCMV.DRD2 microinjection revealed a localized increase of DRD2 levels in animals examined 4, 6, 8 or 10 days following vector treatment (Fig. 2). Specifically, a one-way anova comparing NAc DRD2 levels in different groups of rats revealed a significant difference [(4, 3.99) p < 0.05]. Subsequently, post hoc t-test comparisons between DRD2 levels on the vector-injected side versus the contralateral control side revealed the following increases in DRD2: day 4, 52.1% (Tobs= 12.28, *p < 0.001); day 6, 37.4% (Tobs= 6.60, *p < 0.001); day 8, 25.2% (Tobs= 3.7, *p < 0.01); day 10, 8.3% (Tobs= 1.28, ns), respectively. Rats treated a second time with a vector injection at day 10 showed an increase in DRD2 levels at day 13 of 25.6% (Tobs= 4.37, *p < 0.01). Furthermore, as a control we examined DRD2 levels in the caudate putamen of the same sections. A one-way anova revealed no significant differences between DRD2 levels on the vector-injected side versus the contralateral control side [(4, 0.36) p > 0.05].
Figure 2. Mean (+ SE) percent increase in DRD2 levels of the NAc over time (*p < 0.05; **p < 0.001). Animals were treated with AdCMV.DRD2 on day 0 and day 10.
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Drinking preference was assessed as the amount of ethanol consumed divided by total fluid consumed × 100. In the initial preference test it was found that all animals showed little ethanol intake and followed water as it alternated positions each day. The mean intakes were expressed as milliliters and grams of ethanol/kg of body weight/day (Table 1).
Table 1. Twenty-four hour ethanol intakes in a two-bottle choice condition measured before and after sucrose-fading technique
| ||Ethanol (mL)||Ethanol (g/kg)||Water (mL)|
|Before training||6.2 (5.1)||0.57 (0.48)||28.1 (6.2)|
| Non-preferring rats||24.0 (8.4)**||2.53 (0.28)**||13.1 (5.9)*|
| Preferring rats||8.11 (1.9)||0.85 (0.13)||32.3 (6.8)|
Assessment of each animal's ethanol drinking preference following the sucrose-fading procedure (previously described) resulted in six rats in the alcohol preferring group [ > 60% preference of ethanol (7% v/v) versus water], and nine rats in the alcohol non-preferring group [ > 60% preference of water versus ethanol (7% v/v)]. The mean weight of the animals increased by the end of the sucrose-fading technique to 522 g (range 442–611). Furthermore, it was found that the non-preferring rats showed little ethanol intake and followed water as it alternated positions each day. In contrast, preferring rats increased their ethanol intake (Table 1). A series of one-way anova tests revealed significant differences in ethanol intake before and after sucrose fading. Specifically, significant differences were found in ethanol intake [(27, 43.09) p < 0.0001], water intake [(27, 17.86) p < 0.0001], and g/kg/day ethanol intake [(27, 50.26) p < 0.0001]. Post hoc analyses using a Duncan test revealed that ethanol intake in the preferring rats, was significantly different from both the non-preferring rats, p < 0.0001; and ethanol intake prior to training; p < 0.0001 (before; see Table 1). In contrast, the same type of post hoc analysis between the non-preferring rats and ethanol intake prior to training, revealed no significant differences.
In addition, given a mean total fluid intake of 37.1 mL and 40.4 mL for preferring and non-preferring rats, this resulted in a mean fluid intake of 7.1 mL and 7.7 mL/100 g of body weight. Calculation of the same intake measures for the initial preference test resulted in 7.9 mL/100 g of body weight, and is not significantly different between the two test periods. Thus on a per weight basis fluid intake had not changed but the amount of fluid ingested daily as ethanol was altered.
Cannula placement was confirmed as in the previous experiment through histological examination as previously described. One animal was not included in the behavioral assessment because the cannula placement was too dorsal. No unusual neuropathology or significant signs of inflammation associated with injection sites was observed regardless of treatment and this was consistent with our previous studies (Ikari et al. 1995, 1999; Umegaki et al. 1997; Ingram et al. 1998; Ogawa et al. 2000).
Furthermore, repeated measures anova comparing drinking preference between the preoperative and postoperative drinking phases revealed no statistical difference (p > 0.05) and this data was pooled together and referred to as baseline (Fig. 3). Subsequently, animals were treated with the AdCMV.Null (vehicle) vector on day 0 and drinking preference was recorded for 7 days (Fig. 3). A repeated measures anova comparing baseline ethanol drinking and ethanol drinking after treatment with the control vector revealed no significant difference (p > 0.05; Fig. 3).
Figure 3. Mean (+ SE) percent ethanol preference over time in (a) preferring and (b) non-preferring rats (*p < 0.05). Both groups of animals were treated at two time points with the DRD2 and vehicle (null) vectors.
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On day 8, all animals were treated with the DRD2 vector and ethanol preference drinking monitored until day 28 (Fig. 3). Again, a repeated measures anova comparing drinking preference in both groups of rats across time after treatment with the DRD2 vector revealed the following significant differences: group effect (12, 45.414), p < 0.0001; Time effect (21, 13.668), p < 0.0001 and the interaction group × time effect (21, 3.761) p < 0.0001.
Post hoc t-test comparisons were then performed between baseline drinking preference and drinking at different times after DRD2 vector treatment and revealed several significant differences illustrated in Fig. 3 by an asterisk (p < 0.05). At day 12, 4 days after DRD2 vector treatment (time when peak DRD2 levels were observed in experiment 1), ethanol preference was decreased in the preferring rats from 70% to 27% and in the non-preferring rats from 20% to 4% (Fig. 3). Ethanol preference in the preferring rats returned to pretreatment levels at day 16, but preference in the non-preferring rats did not return to pretreatment levels until day 25 (Fig. 3). At day 28 half the animals in each group received a second DRD2 vector treatment, which decreased ethanol preference at day 32 to the same extent as it had done during the first treatment (Fig. 3). In contrast, the other half of the animals received a vehicle (null vector) treatment at day 28, and showed no significant effect on their ethanol preference on day 32 (Fig. 3).
Ethanol intake (g/kg/day) was also examined in both groups of animals (Fig. 4). A repeated measures anova comparing ethanol intake in both groups of rats across time after treatment with the DRD2 vector revealed the following significant differences: group effect (12, 263.05), p < 0.0001; time effect (21, 17.77), p < 0.0001 and the interaction group × time effect (21, 4.54) p < 0.0001. Post hoc t-test comparisons were then performed on ethanol intake between baseline and at different times after DRD2 vector treatment and revealed several significant differences illustrated in Fig. 4 by an asterisk (p < 0.05). At day 12, four days after DRD2 vector treatment (time when peak DRD2 levels were observed in experiment i), ethanol intake was decreased in the preferring rats from 2.2 to 0.8 g/kg/day and in the non-preferring rats from 0.8 to 0.2 g/kg/day (Fig. 4). Ethanol intake returned to baseline levels 8 days later and a second challenge with either the vehicle or DRD2 vector at day 28 revealed similar effects on ethanol intake.
Figure 4. Mean (+ SE) ethanol intake (g/kg/day) over time in (a) preferring and (b) non-preferring rats (*p < 0.05). Both groups of animals were treated at two time points with the DRD2 and vehicle (null) vectors.
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Overall, it should be noted, there was no significant decrease in total fluid intake after treatment with the vector, but rather a decrease in ethanol preference (drinking from the ethanol bottle versus the water bottle) and ethanol intake. No visible decrease in locomotor activity was observed in animals following vector treatment. In addition, rats did not show any signs of malaise or weight loss due to treatment with the vector and this was consistent with previous studies (Ikari et al. 1995, 1999; Umegaki et al. 1997; Ingram et al. 1998).