Understanding the fate of adult-generated neurons and the mechanisms that influence them requires consistent labeling and tracking of large numbers of stem cells. We generated a nestin-CreER T2 /R26R-yellow fluorescent protein (

Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (Experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (Experiment 2). In Experiment 1, mice received sham or morphine pellets (subcutaneous, 0 and 48 hrs) and bromodeoxyuridine (BrdU) 2 hrs prior to sacrifice (24, 72 or 96 hrs). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 hrs of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 hrs. In Experiment 2, nestin-GFP mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 hrs, sacrifice 96 hrs) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells were less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were Type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage.

The reconsolidation of cocaine memories following retrieval is necessary for the sustained ability of a cocaine-paired environmental context to elicit cocaine seeking. Extracellular signal-regulated kinase (ERK) is an intracellular signaling molecule involved in nucleus accumbens core (NACc)-mediated reconsolidation of Pavlovian cocaine memories. Here, we used a rodent model of drug context-elicited relapse to test the hypothesis that ERK would be similarly required for the reconsolidation of context-response-cocaine memories that underlie drug context-induced reinstatement of instrumental cocaine-seeking behavior, with a focus on the NACc and on the basolateral amygdala (BLA), another important locus for the reconsolidation of cocaine memories. We show that the mitogen-activated protein kinase (MEK)/ERK1/2 inhibitor, U0126 (1.0 μg/0.5 μl/hemisphere), microinfused bilaterally into the BLA--but not the NACc--immediately after brief re-exposure to a previously cocaine-paired context (that is, cocaine-memory reactivation), significantly attenuated subsequent drug context-induced cocaine seeking relative to vehicle (VEH). This effect in the BLA was associated with a transient inhibition of ERK1/2 phosphorylation, and it depended on memory reactivation given that U0126 administered following exposure to a novel context did not alter subsequent cocaine seeking. Furthermore, similar to U0126, baclofen+muscimol-induced (B+M; 106.8/5.7 ng/0.5 μl/hemisphere) neural inactivation of the NACc, following cocaine-memory reactivation, failed to alter subsequent cocaine seeking. These findings demonstrate that ERK activation in the BLA, but not the NACc, is required for the reconsolidation of context-response-cocaine associative memories. Together with prior research, these results suggest that contextual drug-memory reconsolidation in Pavlovian and instrumental settings involves distinct neuroanatomical mechanisms.

Environmentally induced relapse to cocaine seeking requires the retrieval of context-response-cocaine associative memories. These memories become labile when retrieved and must undergo reconsolidation into long-term memory storage to be maintained. Identification of the molecular underpinnings of cocaine-memory reconsolidation will likely facilitate the development of treatments that mitigate the impact of cocaine memories on relapse vulnerability. Here, we used the rat extinction-reinstatement procedure to test the hypothesis that the Src family of tyrosine kinases (SFK) in the dorsal hippocampus (DH) critically controls contextual cocaine-memory reconsolidation. To this end, we evaluated the effects of bilateral intra-DH microinfusions of the SFK inhibitor, PP2 (62.5 ng per 0.5 μl per hemisphere), following re-exposure to a cocaine-associated (cocaine-memory reactivation) or an unpaired context (no memory reactivation) on subsequent drug context-induced instrumental cocaine-seeking behavior. We also assessed alterations in the phosphorylation state of SFK targets, including GluN2A and GluN2B N-methyl-D-aspartate (NMDA) and GluA2 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits at the putative time of memory restabilization and following PP2 treatment. Finally, we evaluated the effects of intra-DH PEAQX (2.5 μg per 0.5 μl per hemisphere), a GluN2A-subunit-selective NMDAR antagonist, following, or in the absence of, cocaine-memory reactivation on subsequent drug context-induced cocaine-seeking behavior. GluN2A phosphorylation increased in the DH during putative memory restabilization, and intra-DH PP2 treatment inhibited this effect. Furthermore, PP2-as well as PEAQX-attenuated subsequent drug context-induced cocaine-seeking behavior, in a memory reactivation-dependent manner, relative to VEH. These findings suggest that hippocampal SFKs contribute to the long-term stability of cocaine-related memories that underlie contextual stimulus control over cocaine-seeking behavior.

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Opiates, such as morphine, decrease neurogenesis in the postnatal hippocampal subgranular zone (SGZ) by inhibiting progenitor proliferation and maturation. However, it is not known how morphine influences the growth factors and vasculature that encompass the neurogenic SGZ microenvironment. We examined morphine’s effect on pro- and anti-proliferative factors in the dentate gyrus (DG; Experiment 1) as well as the DG neurovasculature itself (Experiment 2). For Experiment 1, mice were implanted with sham or morphine pellets subcutaneously (s.c.; 0 and 48 hrs) and were decapitated 24 or 96 hrs later. One brain hemisphere was post fixed to examine proliferation by immunohistochemistry, and a DG-enriched sample was dissected from the other hemisphere to examine the neurogenic microenvironment via immunoblotting for known pro- and anti-proliferative factors. Consistent with previous results, morphine decreased the number of proliferating cells in the SGZ, as the number of Ki67-immunoreactive (IR) cells was decreased at 96 hrs. Morphine did not alter DG levels of the pro-proliferative factor BDNF, anti-proliferative factor IL1β, or their receptors TrkB and IL1R1 at either time point. However, morphine increased the pro-proliferative factor VEGF at 96 hrs. Given that VEGF is also a potent angiogenic factor, Experiment 2 examined whether the morphine-induced increase in VEGF correlated with altered DG neurovasculature. Mice were implanted with morphine pellets as in Experiment 1, and two hrs before perfusion (24 or 96 hrs) were administered bromodeoxyuridine intraperitoneally (BrdU, i.p.; 150 mg/kg). Tissue was co-stained for BrdU and the endothelial cell marker endoglin to enable examination of DG vessels and proximity of BrdU-IR cells to endoglin-IR vessels. At 96 hrs, endoglin-IR vessel area and perimeter were increased, but proximity of BrdU-IR cells to endoglin-IR vessels remained unchanged. These data suggest that following chronic morphine exposure, factors within the neurogenic microenvironment are maintained or upregulated to compensate for decreased SGZ proliferation.

Rationale Contextual control over drug relapse depends on the successful reconsolidation and retention of context-response-cocaine associations in long-term memory stores. The basolateral amygdala (BLA) plays a critical role in cocaine memory reconsolidation and subsequent drug context-induced cocaine-seeking behavior; however, less is known about the cellular mechanisms of this phenomenon. Objectives The present study evaluated the hypothesis that protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) activation in the BLA is necessary for the reconsolidation of context-response-cocaine memories that promote subsequent drug context-induced cocaine-seeking behavior. Methods Rats were trained to lever-press for cocaine infusions in a distinct context, followed by extinction training in a different context. Rats were then briefly re-exposed to the previously cocaine-paired context or an unpaired context in order to reactivate cocaine-related contextual memories and initiate their reconsolidation or to provide a similar behavioral experience without explicit cocaine-related memory reactivation, respectively. Immediately after this session, rats received bilateral microinfusions of vehicle, the PKA inhibitor, Rp-Adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS), or the CaMKII inhibitor, KN-93, into the BLA or the posterior caudate putamen (anatomical control region). Rats were then tested for cocaine-seeking behavior (responses on the previously cocaine-paired lever) in the cocaine-paired context and the extinction context. Results Intra-BLA infusion of Rp-cAMPS, but not KN-93, following cocaine memory reconsolidation impaired subsequent cocaine-seeking behavior in a dose-dependent, site-specific, and memory reactivation-dependent fashion. Conclusions PKA, but not CaMKII, activation in the BLA is critical for cocaine memory re-stabilization processes that facilitate subsequent drug context-induced instrumental cocaine-seeking behavior.

Cocaine addiction is a disease characterized by chronic relapse despite long periods of abstinence. The lateral orbitofrontal cortex (lOFC) and basolateral amygdala (BLA) promote cocaine-seeking behavior in response to drug-associated conditioned stimuli (CS) and share dense reciprocal connections. Hence, we hypothesized that monosynaptic projections between these brain regions mediate CS-induced cocaine-seeking behavior. Male Sprague-Dawley rats received bilateral infusions of a Cre-dependent adeno-associated viral (AAV) vector expressing enhanced halorhodopsin 3.0 fused with a reporter protein (NpHR-mCherry) or a control AAV (mCherry) plus optic fiber implants into the lOFC (Experiment 1) or BLA (Experiment 2). The same rats also received bilateral infusions of a retrogradely transported AAV vector expressing Cre recombinase (Retro-Cre-GFP) into the BLA (Experiment 1) or lOFC (Experiment 2). Thus, NpHR-mCherry or mCherry expression was targeted to lOFC neurons that project to the BLA or to BLA neurons that project to the lOFC in different groups. Rats were trained to lever press for cocaine infusions paired with 5-s CS presentations. Responding was then extinguished. At test, response-contingent CS presentation was discretely coupled with optogenetic inhibition (5-s laser activation) or no optogenetic inhibition while lever responding was assessed without cocaine/food reinforcement. Optogenetic inhibition of lOFC to BLA, but not BLA to lOFC, projections in the NpHR-mCherry groups disrupted CS-induced reinstatement of cocaine-seeking behavior relative to (i) no optogenetic inhibition or (ii) manipulations in mCherry control or (iii) NpHR-mCherry food control groups. These findings suggest that the lOFC sends requisite input to the BLA, via monosynaptic connections, to promote CS-induced cocaine-seeking behavior.