Gene expression evidence for remodeling of lateral hypothalamic circuitry in cocaine addiction

Abstract

By using high-density oligonucleotide arrays, we profiled gene expression in reward-related brain regions of rats that developed escalated cocaine intake after extended access to cocaine (6 h per day). Rats allowed restricted daily access to cocaine (only 1 h) that displayed a stable level of cocaine intake and cocaine naive rats were used for controls. Four analysis methods were compared: Affymetrix microarray suite 4 and microarray suite 5, which use perfect-match-minus-mismatch models, and dchip and rma, which use perfect-match-only models to generate expression values. Results were validated by RT-PCR in individual animals from an independent replication of the experiment. A small number of genes was associated with escalated cocaine intake (ESC genes). Unexpectedly, of the brain regions examined [prefrontal cortex, nucleus accumbens, septum, lateral hypothalamus (LH), amygdala, and ventral tegmental area], the LH was the most transcriptionally responsive in escalation of cocaine intake. Most of the ESC genes identified are also expressed during synaptogenesis and synaptic plasticity and include genes that code for several presynaptic and postsynaptic proteins involved in neurotransmission. These results suggest that LH intrinsic circuitry undergoes a structural reorganization during escalation of cocaine use. This remodeling of LH circuitry could contribute to the chronic deficit in reward function that has been hypothesized to drive the transition to drug addiction. Results also support the value of using multiple analysis strategies to identify the most robust changes in gene expression and to compensate for the biases that affect each strategy.

Fig. 1.

Effects of access time to cocaine self-administration on drug intake and brain gene expression levels. (A) Escalation of i.v. cocaine consumption in rats. Rats had access to cocaine for either 1 h (ShA rats, n = 8) or 6 h per day (LgA rats, n = 8). Data represent the mean (±SEM) number of cocaine injections obtained during the first hour of each daily self-administration session. *, Different from ShA rats; P < 0.05, tests of simple main effects after appropriate two-way analyses of variance. (B) Total number of probe sets per brain region that significantly changed in LgA and ShA rats compared with drug-naive control rats (CSA genes, white bars) and probe sets that significantly changed in LgA rats from ShA and drug-naive rats (ESC genes, black bars). AMY, amygaloid complex; MPF, medial prefrontal cortex.

Fig. 2.

Correlation between changes in gene expression levels and patterns of cocaine intake. (A) NAc. (B) Amygdala. (C) LH. (D) Medial prefrontal cortex. (E) SEP. (F) VTA. In LgA and ShA groups, the expression levels corresponding to each probe set were normalized to control levels (drug-naive rats), as described in Materials and Methods, to obtain values ranging from 0 to 1, with 0.5 corresponding to no change from the control level. Only probe sets with average expression levels ≥100 in at least one condition were included in the regression analysis. ESC genes were defined as significantly different in LgA vs. control and LgA vs. ShA, regardless of whether they were significant in ShA vs. controls. Regression analysis showed a positive correlation between the change in expression in ShA from controls and the change in expression in LgA from controls. This correlation suggests that, in all of the brain regions considered, the majority of genes whose expression levels were affected by a history of cocaine self-administration were not differentially affected by the pattern of cocaine intake (stable/moderate in ShA rats vs. escalating/excessive in LgA rats). The diagonal lines in each graph represent the 1.4-fold cutoffs used in the analysis of mas4 and mas5 data (see Materials and Methods for details). In this plot, deviations from the center within the area defined by the diagonal lines indicate changes in ShA and LgA rats from controls (CSA genes). Points outside the area defined by the diagonal lines indicate genes that differed in LgA from ShA and control rats (ESC genes).

Fig. 3.

Coordinated presynaptic and postsynaptic gene expression changes in the LH after cocaine intake escalation. The levels of the mRNAs for several synaptic proteins were altered in the LH of cocaine-escalating rats. ESC genes are indicated in bold within yellow boxes. The diagram shows their presynaptic and postsynaptic distributions and some of their possible interactions. In some cases, e.g., Map1a and PKCγ, the proteins could be present presynaptically and postsynaptically. Changes in elements of the release machinery were usually increases and could reflect changes in protein content or the result of increased synaptic contacts. SNAP-25, synaptosome-associated protein-25; ERK, extracellular signal-regulated kinase; ANIA-6, activity and neurotransmitter-induced early gene 6; VAMP, vesicle-associated membrane protein; Nf1, neurofibromin; PSD-95, postsynaptic density-95; CamKII, Ca²⁺/calmodulin protein kinase II; mGlur, metabotropic GluR; NMDAR, NMDA receptor. The differential regulation of several genes related to structural plasticity in the LH of animals with escalated cocaine intake but not in rats with stable cocaine intake suggests that a remodeling of LH circuitry involving synaptogenesis and neuritogenesis contributes to the transition to cocaine addiction.

Fig. 4.

Hierarchical clustering of genes specifically associated with escalated cocaine intake (ESC genes) obtained using mas5-generated expression values. The dendrogram was generated by using genespring 4.2.1. The first node separates genes up-regulated from genes down-regulated in LgA rats relative to controls (C) and ShA rats. Darker shades of gray represent higher expression levels.