Preventative treatment in an animal model of ADHD: Behavioral and biochemical effects of methylphenidate and its interactions with ovarian hormones in female rats

Abstract

Clinical and preclinical studies on attention deficit hyperactivity disorder (ADHD) show that juvenile males that are exposed to methylphenidate (MPH) show reduced risk for substance use later in life. In contrast, little is known about whether females have the same enduring treatment response to stimulants and how gonadal hormones influence their behavior later in life. Females received either a sham or 6-hydroxydopamine (6-OHDA) microinjection in the prefrontal cortex (PFC) at postnatal day (P)10. Subjects were then treated with Vehicle or MPH (2 mg/kg, p.o.) between P20–35 and tested during late adolescence/young adulthood (P60); half of these subjects underwent ovariectomy at P55 to determine hormonal influences. Females with 6-OHDA were depleted of PFC dopamine by 61% and demonstrated increased impulsive choice (delayed discounting) and preferences for cocaine-associated environments relative to control females. Both MPH and ovariectomy reduced impulsive choice and cocaine preferences in 6-OHDA females, but had no enduring effect in Sham females. Ovariectomy itself did not significantly affect impulsivity. Juvenile MPH interacted strongly with 6-OHDA to increase D4, D5, Alpha-1A, Alpha-2A, and 5-HT-1A mRNA receptor expression in the PFC. MPH alone effected D1 mRNA, while 6-OHDA increased BDNF; all markers were decreased by ovariectomy. Together, these data suggest that 6-OHDA changes in dopamine are not only relevant for ADHD-like behaviors, but their long-term modulation by treatment and the influence of cyclical differences in menstrual cycle.

The use of laser capture microdissection to identify specific pathways and mechanisms involved in impulsive choice in rats

“…4Correlation analyses of qPCR values for D1 receptors in the projection of the A) orbital frontal cortex (OFC) to the Nucleus accumbens core (OFC→NAc) and B) prelimbic PFC (plPFC) projections to the basolateral amygdala (plPFC→BLA) as a function of each subject's impulsivity score. The delay discounting 50 (DDT 50 ) refers to the delay period when the choice between large and small reinforcers is 50% (Lukkes et al., 2016). Lower DDT 50 values reflect greater impulsivity.…”

“…5Correlation analyses of qPCR values for 5-HT1a receptors in the orbital frontal cortex (OFC; A) and the prelimbic PFC (plPFC; B) as a function of each subject's impulsivity score. The delay discounting 50 (DDT 50 ) refers to the delay period when the choice between large and small reinforcers is 50% (Lukkes et al., 2016). Four different pathways are represented: innervation from the OFC→basolateral amygdala (BLA; OFC→BLA; open circles) or the nucleus accumbens core: OFC→NAc (closed circles); innervation from the plPFC→BLA (open circles) or plPFC→NAc (closed circles).…”

“…Subjects were food deprived to a target goal of 90% of free-feeding weight, consistent with other studies (Winstanley et al., 2004; Zeeb et al., 2010). The original paradigm was based on Evenden and Ryan (1996) and modified by (Lukkes et al., 2016) and is shown in Fig. 1A.…”

“…Next, 1 ng RNA was reverse transcribed to cDNA using Superscript® IV First-Strand Synthesis System for RT-PCR Kit (Life Technologies, Beverly, MA) by random hexamers, or cDNA was synthesized from 250 pgs RNA using the SMARTer Ultra Low Input RNA Kit - v3 to yield more product (Clontech, Mountain View, CA). Fifty to one hundred pgs cDNA per reaction was then PCR amplified using the iTaq SYBR Green Supermix (BioRad, Hercules, CA) and 50 nM gene-specific primers (Table 2), according to our published protocols (Lukkes et al., 2016). Replicates whose C T value was more than one away from the average were excluded; the average raw C T values for the receptors were normalized to the housekeeping gene of GAPDH and quantified based on the 2 − ΔCt method by Livak-Schmittgarten method (Livak and Schmittgen, 2001).…”

“…The delay when 50% of the large reinforcers (referred to as DDT 50 for delay discounting at 50% of the large rewards received) was then determined from the best fit of the line between delay and the number of large reinforcers received for each individual subject. This line was often non-linear in nature and we required that R 2 was greater than 0.8 to assure goodness of fit (Lukkes et al., 2016). The calculated fit of the line to estimate the DDT 50 using a non-linear curve fitting approach is more appropriate than estimating the delay at which 50% of large reward responding occurred based on a linear relationship (Doremus-Fitzwater et al., 2012).…”