Rate of onset of dopamine transporter inhibitors assessed with intracranial self-stimulation and in vivo dopamine photometry in rats

Abstract

Drug self-administration and intracranial self-stimulation (ICSS) are two preclinical behavioral procedures used to predict abuse potential of drugs, and abuse-related drug effects in both procedures are thought to depend on increased mesolimbic dopamine (DA) signaling. Drug self-administration and ICSS yield concordant metrics of abuse potential across a diverse range of drug mechanisms of action. The "rate of onset," defined as the velocity with which a drug produces its effect once administered, has also been implicated as a determinant of abuse-related drug effects in self-administration procedures, but this variable has not been systematically examined in ICSS. Accordingly, this study compared ICSS effects produced in rats by three DA transporter inhibitors that have different rates of onset (fastest to slowest: cocaine, WIN-35428, RTI-31) and that produced progressively weaker metrics of abuse potential in a drug self-administration procedure in rhesus monkeys. Additionally, in vivo photometry using the fluorescent DA sensor dLight1.1 targeted to the nucleus accumbens (NAc) was used to assess the time course of extracellular DA levels as a neurochemical correlate of behavioral effects. All three compounds produced ICSS facilitation and increased DA levels assessed by dLight. In both procedures, the rank order of onset rate was cocaine > WIN-35428 > RTI-31; however, in contrast to monkey drug self-administration results, maximum effects did not differ across compounds. These results provide additional evidence that drug-induced increases in DA drive ICSS facilitation in rats and illustrate the utility of both ICSS and photometry to evaluate the time course and magnitude of abuse-related drug effects in rats.

Keywords: Addiction; Dopamine release; Fluorimetry; Latency; Psychostimulants; Reuptake inhibitors.

Figure 1.. Time course of ICSS difference scores for cocaine, WIN-35428, and RTI-31.

Abscissae: Time in min (log scale) after administration of IP saline or test drug, with numerals indicating the midpoint of each 10-min component (e.g., the number “5” indicates the midpoint of the component conducted from 0–10 min after injection). Ordinates: Number of stimulations per component expressed as Difference Scores (%BL Drug - %BL Vehicle). Each data point represents the mean ± SEM of 6–7 rats, and filled points indicate a significant difference from saline vehicle at that time point using a repeated-measures two-way ANOVA (cocaine, RTI-31) or mixed-effects analysis (WIN-34528) followed by a Dunnett post-hoc test (p < 0.05). Statistics for significant dose x time interactions were as follows: cocaine (F(3.155, 15.78) = 7.244; p = 0.0026), WIN-35428 (F(3.062, 15.15) = 5.600; p = 0.0084), RTI-31 (F(4.123, 24.74) = 9.908; p < 0.0001). A mixed-effects analysis was used for WIN-34528 because there was missing data for one rat at the 1445-min time point for doses of 0.10 and 0.18 mg/kg.

Figure 2.. Potency comparisons for effects produced by cocaine, WIN-35428, and RTI-31.

Panel A shows dose-effect curves determined at the approximate T_max for each drug. Abscissa: Drug dose in mg/kg (log scale). Ordinate: E_max for each drug dose. Dotted line indicates the difference score used for relative potency comparisons (ED₇₅). Each point represents the mean ± SEM of 6–7 rats. Statistics for linear regressions are as follows: cocaine (p = 0.0035; r² = 0.5901), WIN-35428 (p = 0.0001; r² = 0.6118), RTI-31 (p < 0.0001; r² = 0.6012). Panels B-D show frequency-rate curves for cocaine, WIN-35428, and RTI-31 during the component closest to their calculated T_max. Abscissae: Frequency of electrical brain stimulation in Hz (log scale). Ordinates: Number of stimulations per trial expressed as a percentage of daily maximum control rate (MCR). Each data point represents the mean ± SEM from 6–7 rats, and filled points represent frequencies at which ICSS rates were different compared to saline as determined by a repeated-measures two-way ANOVA followed by a Dunnett post-hoc test (p < 0.05). Two-way ANOVA for frequency x dose interactions were as follows: cocaine (F(4.028, 20.14) = 5.873; p = 0.0026), WIN-35428 (F(3.355, 16.77) = 3.922; p = 0.0242), RTI-31 (F(3.888, 23.33) = 2.651; p = 0.0600). Although the frequency x dose interaction was not significant for RTI-31, there was a significant main effect of dose (F(1.553, 9.316) = 40.88; p < 0.0001). RTI-31 only: Asterisks indicate doses at which ICSS rates collapsed across all frequencies were different compared to saline as determined by a repeated-measures one-way ANOVA followed by a Dunnett post-hoc test (p < 0.05). One-way ANOVA for dose F(1.328, 7.968) = 51.98; p < 0.0001).

Figure 3.. Measurement of dopamine signals using ratiometric photometry targeting the NAc in vivo.

Four to six weeks after dLight1.1-AAV injection and optic canula implantation, DA signals were measured in awake and behaving rats. A) representative 30 s recording of the demodulated signal arising from the 470 nm excitation. B) Simultaneous signal demodulated from the isosbestic 405 nm excitation. C) The ratio between channels (F_470/405) serves as a “corrected” signal, revealing transients of ~1s duration characteristic of spontaneous DA release (see insert). D) dLight1.1 signals were recorded beginning immediately after cocaine (or saline) injection 90 min. Each 30s sweep (F_470/405) was averaged to get a single intensity point per minute. The resulting trace was divided by the first point (F_470/405/F₀). E) The “saline” trace was subtracted from the “cocaine” trace (cocaine – vehicle) yielding a new trace showing the mean DA changes along the full experimental time. A logistic fit (red dashed trace) was used to estimate T₅₀ and E_max (see Table 1). F) The experiments were performed for cocaine (black trace), WIN-35428 (blue trace), and RTI-31 (red trace) in 5 rats. The traces represent the mean + SEM. The maximal response (E_max), half time to maximal response (T₅₀), and time for maximal response (T_max) were calculated and are shown in Table 1.

Figure 4.. Thirty second traces of the DA signal immediately after injection (T₀) and at the time of peak response (T_max).

The 470/405 signals were normalized to the mean value of the T₀ sweep for comparison. The T₀ sweep and one T_max sweep are shown. Event analysis for the 8–15 min time band for cocaine, 30–35 min for WIN-35428 (WIN), and 60–65 min for RTI-31 (RTI) are shown in Table 2. The response for the same animal at a matched time after saline injection is shown in the right panels (corresponding control).

Figure 5.. The expression of abuse-related behavior (ICSS facilitation) matches the time course of increased DA levels in the NAc (dLight) in awake rats.

Overlay of ICSS and dLight results shown in Fig 1 and Fig 3F, respectively. The mean values obtained for A) cocaine (IP, 10 mg/kg), B) WIN-35428 (IP, 0.32 mg/kg), and C) RTI-31 (IP, 0.1 mg/kg) were expressed as a percentage of the maximal response for each trace, and ICSS and dLight responses were plotted together as function of time. ICSS and dLight experiments were performed in different groups of rats (ICSS N = 6–7 rats and dLight N = 5 rats).