Neurogenesis during Abstinence Is Necessary for Context-Driven Methamphetamine-Related Memory

Abstract

Abstinence from methamphetamine addiction enhances proliferation and differentiation of neural progenitors and increases adult neurogenesis in the dentate gyrus (DG). We hypothesized that neurogenesis during abstinence contributes to context-driven drug-seeking behaviors. To test this hypothesis, the pharmacogenetic rat model (GFAP-TK rats) was used to conditionally and specifically ablate neurogenesis in the DG. Male GFAP-TK rats were trained to self-administer methamphetamine or sucrose and were administered the antiviral drug valganciclovir (Valcyte) to produce apoptosis of actively dividing GFAP type 1 stem-like cells to inhibit neurogenesis during abstinence. Hippocampus tissue was stained for Ki-67, NeuroD, and DCX to measure levels of neural progenitors and immature neurons, and was stained for synaptoporin to determine alterations in mossy fiber tracts. DG-enriched tissue punches were probed for CaMKII to measure alterations in plasticity-related proteins. Whole-cell patch-clamp recordings were performed in acute brain slices from methamphetamine naive (controls) and methamphetamine experienced animals (+/-Valcyte). Spontaneous EPSCs and intrinsic excitability were recorded from granule cell neurons (GCNs). Reinstatement of methamphetamine seeking enhanced autophosphorylation of CaMKII, reduced mossy fiber density, and induced hyperexcitability of GCNs. Inhibition of neurogenesis during abstinence prevented context-driven methamphetamine seeking, and these effects correlated with reduced autophosphorylation of CaMKII, increased mossy fiber density, and reduced the excitability of GCNs. Context-driven sucrose seeking was unaffected. Together, the loss-of-neurogenesis data demonstrate that neurogenesis during abstinence assists with methamphetamine context-driven memory in rats, and that neurogenesis during abstinence is essential for the expression of synaptic proteins and plasticity promoting context-driven drug memory.SIGNIFICANCE STATEMENT Our work uncovers a mechanistic relationship between neurogenesis in the dentate gyrus and drug seeking. We report that the suppression of excessive neurogenesis during abstinence from methamphetamine addiction by a confirmed phamacogenetic approach blocked context-driven methamphetamine reinstatement and prevented maladaptive changes in expression and activation of synaptic proteins and basal synaptic function associated with learning and memory in the dentate gyrus. Our study is the first to demonstrate an interesting and dysfunctional role of adult hippocampal neurogenesis during abstinence to drug-seeking behavior in animals self-administering escalating amounts of methamphetamine. Together, these results support a direct role for the importance of adult neurogenesis during abstinence in compulsive-like drug reinstatement.

Keywords: CaMKII; NeuroD; electrophysiology; methamphetamine; self-administration; synaptoporin.

Figure 1.

a, b, Schematic of experimental design indicating the time frame for methamphetamine (Meth; a) and sucrose (b) operant behaviors. Rats either experienced intravenous methamphetamine self-administration (Meth sessions; 6 h/session, 0.05 mg/kg Meth/infusion delivered in 100 μl volume) or oral sucrose self-administration [sucrose sessions; 30 min/session, 10% sucrose solution (w/v), 100 μl solution/infusion]. After 13 sessions rats were initiated on Valcyte diet or vehicle (peanut butter) to ablate or maintain neurogenesis and continued this diet until the end of the experiment. After the last reinstatement session, brain tissue was processed for IHC, Western blotting (WB) and electrophysiology (Ephys). c, d, Escalation of methamphetamine and sucrose taking in GFAP-TK rats: lever responses during 6 h methamphetamine sessions (c) and 30 min sucrose sessions (d). Animals received Valcyte or vehicle on days 13–17, and self-administration behavior did not differ between the two groups in methamphetamine and sucrose animals. n = 19, methamphetamine TK−Valcyte; n = 17 methamphetamine;TK+Valcyte; n = 7 sucrose TK−Valcyte; and n = 9 sucrose TK+Valcyte. Data shown are represented as the mean ± SEM. *p < 0.05, compared with session 1 (c, d) by repeated-measures two-way ANOVA and Student–Newman–Keuls post-tests.

Figure 2.

Extinction and reinstatement of drug seeking in GFAP-TK rats. a–b, Active and inactive lever responses during extinction sessions in TK−Valcyte and TK+Valcyte methamphetamine (a) and sucrose (b) animals. Lever responses from the last day of self-administration are indicated for comparison. c–e, The percentage change in active lever responses on day 1 of extinction compared with the last day of self-administration (c), the percentage change in active lever responses on context-driven reinstatement compared with last day of extinction (d), and the percentage change in active lever responses on cue-driven reinstatement compared with last day of extinction (e). Data shown are represented as the mean ± SEM. ⁺p < 0.05 vs TK+Valcyte in a; $p < 0.05 vs. last methamphetamine session in a; #p < 0.05 vs. active lever responses in a, b; *p < 0.05, compared with extinction session 6 (a, b) by repeated-measures two-way ANOVA and Student–Newman–Keuls post-tests. *p < 0.05 vs TK−Valcyte methamphetamine rats in c, d; +p < 0.05 main effect of treatment in b; #p < 0.05 main effect of treatment in e.

Figure 3.

Valcyte reduces proliferation and inhibits neurogenesis in methamphetamine and sucrose rats. a–e, Photomicrographs of labeled cells from control rats: Ki-67 in the DG (a), NeuroD in the DG (b), NeuroD in the SVZ (c, d), and doublecortin in the DG (e). Scale bars: (in a) a–c, e, 100 μm; (in a) d, 20 μm. Arrowheads in a–e point to immunoreactive cells. Hil, Hilus; Mol, molecular layer. f–i, Quantitative analysis of the total number of cells labeled with Ki-67 (f), NeuroD (g, h), and doublecortin (i) in all rats. Drug-naive age-matched controls, n = 6; methamphetamine TK−Valcyte, n = 16; methamphetamine TK+Valcyte, n = 14; sucrose TK−Valcyte, n = 7; sucrose TK+Valcyte, n = 9. Data shown are represented as mean ± SEM. *p < 0.05 compared with drug-naive controls by one-way ANOVA; ***p < 0.001 vs TK−Valcyte groups and controls by one-way ANOVA.

Figure 4.

Plasticity-related proteins are affected by methamphetamine seeking, and Valcyte treatment normalizes these effects. a, b, Schematic showing the location of tissue punches taken in the dorsal (a) and ventral (b) DG of the hippocampus. c–f, Representative Western blots for protein expression in dorsal (c, d) and ventral (e, f) DG-enriched tissue of total and phosphorylated CaMKII from methamphetamine (c, e) and sucrose (d, f) rats. g, h, Density of protein expression for total and phosphorylated CaMKII in dorsal (g) and ventral (h) DG from methamphetamine and sucrose rats. *p < 0.05 compared with drug- and sucrose-naive age-matched controls by one-way ANOVA followed by unpaired t tests. $p < 0.05 vs +Valcyte group by two-way ANOVA followed by significant effect of Valcyte. #p < 0.05 vs sucrose group by two-way ANOVA followed by significant effect of treatment. Data shown are represented as the mean ± SEM. Drug-naive age-matched controls, n = 6; methamphetamine TK−Valcyte, n = 13–16; methamphetamine TK+Valcyte, n = 14; sucrose TK−Valcyte, n = 7; sucrose TK+Valcyte, n = 9.

Figure 5.

Methamphetamine seeking alters mossy fiber tracts in the DG and Valcyte normalizes these effects. a–c, Photomicrographs of sections through the anterior dorsal hippocampus stained with synaptoporin. Staining revealed mossy fiber tracts and terminal fields in the hilus (b) and the CA3 pyramidal projections (c). The arrow in b points to the area in the hilus used for density measures, and the arrow in c points to the pyramidal projections used for density measures. Scale bar, a, 500 μm. d, e, Quantitative analysis of the density measures in the hilus (d) and CA3 pyramidal projections (e). Data shown are represented as the mean ± SEM. Drug-naive age-matched controls, n = 6; methamphetamine TK−Valcyte, n = 16; methamphetamine TK+Valcyte, n = 12; sucrose TK−Valcyte, n = 7; sucrose TK+Valcyte, n = 9. *p < 0.05 compared with drug- and sucrose-naive age-matched controls. #p < 0.05 vs respective treatment groups.

Figure 6.

(a) Representative image of the granule cell layer with GCNs and indication of the patch pipette on a selected neuron. Scale bar is 25µm applies to a. (b-d) Representative spontaneous excitatory postsynaptic currents (sEPSCs) traces in voltage-clamp recording from control (b), TK-Valcyte (c) and TK+Valcyte (d) rats. (e-g) Representative traces of action potentials elicited by depolarizing current injections. Traces were recorded from GCNs from control (e; drug and behavior naïve n=2 male GFAP-TK rats and n=2 male Wistar rats; the electrophysiology data between the two strains were not significantly different and therefore were combined), TK-Valcyte (f; n=3 rats) and TK+Valcyte (g; n=2 rats) rats. (h) Frequency of sEPSCs in Hz from neurons. (i) Average of all amplitudes of all sEPSCs above -10pA from GCNs. (j) Graphical relationship between the number of spikes elicited by increasing current injections in current-clamp recording. (k) Electrophysiological properties of GCNs from control, TK-Valcyte and TK+Valcyte rats. Data shown are represented as mean +/- SEM. Number of GCNs, n = 8-13 controls, n = 7-8 TK-Valcyte, n = 15 TK+Valcyte. *p < 0.05 vs. controls and #p < 0.05 vs. TK+Valcyte by ANOVA.