Microinjection procedure

Two days before testing, females were gently handled once daily to habituate them to the experimenter handling associated with the intracranial drug administration. On PPD8, just before behavioral testing, females were gently hand-held while their stylets were removed and replaced by 28-gauge stainless steel injectors that extended 0.75 mm for Cg1, and 1.5 mm for PrL, and IL past the tip of the guide cannulae. Injectors were connected by PE-10 tubing to 10-μl Hamilton syringes, and bilateral infusions were driven simultaneously by a two-syringe infusion pump (Harvard 22 syringe pump; Harvard Apparatus).

Inactivation of mPFC subregions was achieved by infusion of bupivacaine hydrochloride (2% w/v solution; Sigma) as previously published (Pereira and Morrell, 2009, 2010). Bupivacaine reversibly blocks voltage-gated sodium channels and hence prevents initiation and propagation of action potentials both in neuronal cell bodies and axons (Hille, 1966). We chose bupivacaine over other pharmacological agents due to its fast onset of action (within minutes), shorter duration of action (up to 1 h), and more discrete functional spread (∼radius of 500–620 μm from the tip of the injector; Tehovnik and Sommer, 1997; Boehnke and Rasmusson, 2001; Edeline et al., 2002; Pereira de Vasconcelos et al., 2006).

Infusions of bupivacaine or saline were administered bilaterally into one of the mPFC subregions over 120 s at a rate of 0.5 μl/min. The injectors were left in place for an additional 60 s to allow for diffusion of the drug. Immediately after, stylets were replaced. Each rat remained in its home cage for a 5-min period before behavioral testing. An infusion volume of 1.0 μl rather than a smaller 0.5-μl infusion volume was selected based on our prior results and those of others demonstrating a more stable inactivation over time while producing a similar functional spread (Tehovnik and Sommer, 1997; Pereira and Morrell, 2009). Additional groups of postpartum females that did not undergo surgery but were similarly handled, trained and tested as the surgical groups were used to provide behavioral baseline. All females remained healthy throughout the experiment, fully exhibiting typical maternal behaviors, and their pups gained weight and developed normally (Figs. 1 and ​and22).

Effect of transient mPFC subregion-specific inactivation on preference decision making in a concurrent pup/cocaine choice CPP. A, Schematic representation of cross-sections of the rat brain showing the location of injection sites in mother rats receiving infusions of saline (white circles) or bupivacaine (inactivation, black circles) into each of three mPFC subregions. Numbers beside each plate indicate the distance caudal to bregma in millimeters. B, Experimental timeline. C, Locomotor scores during 1.0 mg/kg intraperitoneal cocaine conditioning in the CPP apparatus across the four conditioning days. D, CPP scores (time spent in the preferred compartment during the postconditioning test minus the time spent in the same compartment during the preconditioning session), (E) conditioned chamber preferences, and (F) mean time spent in each chamber of the CPP apparatus during the test session by behavioral control (n = 12), mPFC vehicle treated (n = 20), and mPFC Cg1 (n = 7), PrL (n = 7), and IL (n = 9) bupivacaine-treated postpartum females. Immediately before the start of the 60-min postconditioning CPP test, mPFC cannulated females randomly received bilateral intracranial infusions of either 2% bupivacaine or saline vehicle. Statistical analysis revealed that there was no significant difference in distribution of preference on concurrent pup/cocaine choice between postpartum females that received vehicle into Cg1, PrL, or IL subregions. As such, these groups were combined into a single vehicle-treated group for graphing purposes. Data are expressed as the mean ± SEM; *significant difference at p < 0.05.

Effect of transient mPFC subregion-specific inactivation on maternal behavior. A, Number of active maternal responses over the 30-min maternal behavior test following infusion of either 2% bupivacaine or saline vehicle into the Cg1, PrL, or IL. B, C, Latency to first retrieval and reunion of the litter, and (D) duration of total time with pups over the maternal behavior test. Data are expressed as the mean ± SEM; *significant difference at p < 0.05.