NAC-1, a rat brain mRNA, is increased in the nucleus accumbens three weeks after chronic cocaine self-administration

Abstract

Chronic cocaine use leads to biochemical and behavioral changes that can persist for weeks to months after drug administration is discontinued. Alterations in gene expression in the mammalian CNS may contribute to these long-term neural consequences of cocaine abuse. A combined in situ transcription-PCR amplification strategy was used to isolate a novel mRNA, NAC-1, from the nucleus accumbens of rats 3 weeks after discontinuing 3 weeks of intravenous cocaine self-administration. In rats that self-administered cocaine, levels of NAC-1 were increased approximately 50% in the nucleus accumbens but not in the dorsal striatum or hippocampus, when compared with levels from yoked-saline controls. In situ hybridization analysis demonstrated increased numbers of NAC-1-expressing cells in the nucleus accumbens of rats who had self-administered cocaine. NAC-1 mRNA exists as one form, approximately 4400 nucleotides (nt) in size, and also is present at much lower amounts in non-neural tissues. A full-length cDNA clone was isolated from a whole brain library. The predicted polypeptide sequence contains a POZ domain in the first 120 amino acids; the same POZ domain sequence mediates protein-protein interactions among some transcriptional regulators. NAC-1 mRNA levels were also increased in the nucleus accumbens 1 week after 6 d of noncontingent cocaine treatments. Regulation of NAC-1 mRNA in the nucleus accumbens demonstrates a long-term effect of cocaine use on cellular function that may be relevant in behavioral sensitization or cocaine self-administration.

Fig. 1.

NAC-1 mRNA is detected in multiple CNS regions. Northern blots were performed using poly(A⁺) RNA isolated from discrete CNS regions of individual rats. Partial-length cDNAs for NAC-1 and cyclophilin (IB15) were used as probes. Digitized images are shown after 4 d (NAC-1) or 8 hr (IB15) phosphorscreen exposures. These representative Northern blots demonstrate that NAC-1 mRNA occurs as a single size, ∼4400 nt. The amount of NAC-1 mRNA relative to cyclophilin mRNA was greater in nucleus accumbens samples from rats that had self-administered cocaine (C) than from yoked-saline controls (S). The relative amounts of NAC-1 mRNA did not differ between the two treatment groups in the hippocampus and the caudate–putamen. Thearrowhead indicates the position of 28S rRNA.

Fig. 2.

NAC-1 mRNA is present at increased levels in the nucleus accumbens after withdrawal from cocaine self-administration. The levels of NAC-1 mRNA as a percentage of cyclophilin mRNA are shown as values normalized to the values from saline-exposed rats. Cyclophilin mRNA, a constitutively expressed gene, was used as a measure of RNA transfer in each blot. The level of NAC-1 mRNA in the nucleus accumbens of cocaine-exposed rats (C) is ∼50% higher when compared with that of saline-treated rats (S). No significant changes were observed in levels of NAC-1 mRNA in the hippocampus or caudate–putamen. A minimum of four rats was used for each group (*p < 0.05; Student’s t test).

Fig. 3.

DNA and the predicted amino acid sequence for NAC-1. The open reading frame and surrounding 5′ and 3′ regions are shown. The first underlined region represents a consensus sequence for the initiation of protein synthesis. The two other underlined regions indicate the locations where the PCR primers SSTR₄ and TM7 hybridized in the PCR amplification of a partial-length cDNA. Potential N-glycosylation sites are marked in italics (amino acids 60–62, 199–201, and 475–477). The predicted termination codon is noted (*); GenBank accession number AF015911.

Fig. 4.

NAC-1 contains a POZ domain consensus sequence. The first 120 amino acids of NAC-1 are aligned above the consensus sequence for the POZ domain from 28 different proteins (Bardwell and Treisman, 1994). The N terminal of NAC-1 matches the POZ conserved sequence in 34 of 37 residues (boxed letters); the nonmatching amino acids are indicated by asterisks. There is a conservative substitution in position 71 (Ala instead of other neutral, hydrophobic residues). The predicted structure of this N terminal of NAC-1 consists of α-helices and β-pleated sheets, a uniform characteristic observed in other POZ proteins.

Fig. 5.

Tissue distribution of NAC-1 mRNA in the rat. A single mRNA species is detected in total RNA from multiple tissues. The hybridization signal is approximately three- to fourfold more intense for the four CNS regions, as compared with peripheral organs. The relative levels of NAC-1 mRNA in the nucleus accumbens and caudate–putamen are similar to those in the CNS regions shown in this figure. NAC-1 mRNA is also present in the brain from a day 18 embryo (E18). A signal is not obvious in liver mRNA. The distribution of an RNA molecular ladder is listed on theleft.

Fig. 6.

In situ hybridization studies of NAC-1 expression. A, Hybridization of a NAC-1 cRNA probe in the olfactory tubercle. Intense staining is seen throughout the pyramidal cell layer. B, Hybridization of a sense cRNA probe to a tissue section adjacent to the section shown inA. There is not any obvious staining of cells, demonstrating the specificity of the cRNA probe. C, NAC-1 mRNA expression in the nucleus accumbens of a yoked-saline control rat. An antisense probe detected NAC-1-positive cells scattered throughout the core and shell regions. D, NAC-1 mRNA expression in a similar region of the nucleus accumbens of a rat after withdrawal from daily cocaine self-administration. Note the increased numbers of stained cells, as compared with those in C. The appearance of individual NAC-1-expressing cells is indicated by thearrowheads. The inset is a high-power view of the outlined region in D; the cytoplasmic location of the stain is visible. The insetalso shows the presence of cells in which NAC-1 was not detected. Medial is to the left inA–C and to theright in D. Ventral is to thebottom in all pictures. The anterior portion of the anterior commissure is indicated by an asterisk inC and D. Scale bars:A–D, 120 μm; inset inD, 60 μm.