Of the Mesocorticolimbic DA System Associated with the DADependent Positive Reinforcing Effects of Nicotine Transcriptional Regulation

Chronic exposure to addictive drugs indirectly stimulates transcription of specific genes by increasing intracellular cyclic adenosine monophosphate (cAMP), which in turn results in activation of multifunctional protein kinases and phosphorylation of several cellular proteins, including transcription factors, in target neurons of the mesocorticolimbic DA system (69,100,101). A limited but growing number of scientific reports indicate that nicotine also produces some adaptive changes in the target neurons of DA terminal fields, namely, the nucleus accumbens, striatum, and prefron-tal cortex, most likely via DA release and D1 receptor mediation. One of the most well studied effects of D1 receptor activation in target neurons is the transcriptional regulation of the immediate early gene (IEG) c-fos, and medium-late genes of the Fos-related antigen family (100,102). Expression of c-fos has been used largely to provide tran-

scriptional activation brain maps induced by various stimuli, ranging from natural to pharmacological (102). Acute nicotine passive administration induces expression of c-fos that is not restricted to the mesocorticolimbic DA system terminal fields, but includes several other rat brain regions, in particular the superior colliculus, the interpeduncular nucleus, the raphe, but not the midbrain DA neurons (101,103,104,105). This activation is inhibited by mecamylamine. In the striatum, a dose-responsiveness was observed, with the lower dose (0.4 mg/kg) inducing c-fos in the medial and central portions. In the nucleus accumbens, the response was smaller than in the striatum, with prevalence in the shell. The acute administration of epibatidine, an nAChR agonist with preferential affinity for the D4D2 form, induced c-fos IR in the prefrontal cortex, medial striatum, nucleus accumbens, amygdala, and superior colliculus (106). In the nucleus accumbens and striatum, but not the prefrontal cortex, this effect was inhibited by pretreatment with D1-D4 receptor antagonists, but not D2 antagonist (107). This result is in agreement with the nicotine-induced DA overflow measured in the terminal fields of the mesocorticolimbic DA system, and the D1 mediation of reinforcing properties of DA released in the nucleus accumbens by addictive drugs (13). Finally, repeated nicotine administration in rats results in preferential Fos increases in visuomotor and limbic structures, including basal ganglia and prefrontal cortex (105,108).

3.6.1. Activation of c-fos and Fos-Related Antigen (FRA) Transcription Factor Expression in the Rat Brain by Nicotine Self-Administration

Nicotine self-administration increases Fos immunoreactivity in most of the brain regions that are activated by passive nicotine treatment (109), in particular the stria-tum, the shell and core of the nucleus accumbens, the lateral septum, the prefrontal cortex, and the cingulate cortex. Also, the retrosplenial cortex, the piriform cortex, the periventricular thalamic nucleus, and the superior colliculus showed increase of Fos immunoreactivity. In contrast to acute nicotine, nicotine self-administration did not increase Fos immunoreactivity in the hypothalamus, locus coeruleus, amygdala, and dentate gyrus (109). Passive chronic nicotine administration produced patterns similar to those obtained with acute nicotine, showing little tolerance to the challenging dose of nicotine, and a more general activation of the core of the nucleus accumbens when compared to the selective increase in the ventral shell observed in nicotine self-administration (105,109). These differences are important, since brain maps of immediate early gene expression simultaneously represent the direct pharmacological effects of nicotine on neural networks and the activation associated with the internal dispositional state that controls nicotine taking, which includes the reinforcing effects of nicotine (110).

A more extensive study of the transcriptional regulation of the c-fos-related antigen family (FRA) expressed in the terminal fields of the mesocorticolimbic DA pathway was performed in rats trained for nicotine and cocaine self-administration. FRA heterodimers constitute the API complex that transcriptionally regulate a large number of plasticity-related genes (111). FRAs, and in particular the 35-kDa component recently identified as D-Fos, are medium-late onset genes and their products persist for several days in the nucleus of the target neurons (100). Therefore, once induced by nicotine or cocaine, FRA-containing API complexes may regulate plasticity-related genes for a long period. In one experiment, rats were trained to self-administering cocaine or nicotine (110). Increased expression levels of FRA immunoreactivity were found in the anterior cingulate cortex, prefrontal cortex, nucleus accumbens, medial striatum, but not amygdala, of rats from both groups. In a second experiment the binding of API complex to neuronal DNA was measured in brain tissues dissected out immediately before the last self-administration session. The results showed a persistent increase of API complexes in the nucleus accumbens in cocaine and nicotine self-administering rats, but not in the prefrontal cortex or striatum. This differential effect points to a primary involvement of target neurons in the nucleus accumbens as cellular substrate for the maintenance of drug self-administration and/or the stimulus-reward learning produced by these two drugs. Interestingly, the lack of increase of API complex in the prefrontal cortex suggests that long-term changes in target neurons of this DA terminal field are of modest relevance in drug self-administration maintenance.

3.6.2. cDNA Microarrays Measurement of Transcriptional Profiles in the Terminal Fields of DA Systems After Chronic Nicotine Treatment cDNA microarray is one among several large-scale biology techniques proposed during the last few years to address the possibility of parallel massive measurement of changes in gene expression products of living cells or tissue following pharmacological manipulations (112).

Only preliminary reports and one full article on nicotine treatments in rodents have been published so far (113). In the latter study, Konu et al. (113) used cDNA microarrays containing 1117 genes and expressed sequence tags (ESTs) to assess the transcriptional response to chronic nicotine treatment in rats, by comparing 4 brain regions, the prefrontal cortex, nucleus accumbens, VTA, and amygdala. The results indicate 94 genes whose expression was altered above threshold. Results from principal component analysis and pairwise correlations suggested that brain regions studied were similar in terms of their absolute expression levels (with amygdala showing the lowest activity), but differed in the composition of transcriptional profiles in response to chronic nicotine (113). Accordingly, prefrontal cortex and nucleus accumbens were significantly more similar to each other than to either VTA or amygdala, supporting the value of the concept of terminal field of the mesocorticolimbic DA system as relatively homogeneous neurobiological substrates for nicotine pharmacological properties.

Several genes involved in cellular signaling, structure/cytoskelectal maintenance, metabolism, cell cycle, and transcriptional regulation were found to be affected by chronic nicotine. In particular, the MAP-kinase, the phosphatidylinositol, and EGFR signaling pathways showed quite consistent changes and were proposed as possible targets in response to nicotine administration.

These results, as exciting as they appear, need a cautionary note for an appropriate interpretation. In fact, technical limitations can importantly question the reproducibil-ity of obtaining the same up- or downregulation for the same gene using different large-scale biological approaches. In addition, given the relatively small percentage of the genome featured into the cDNA microarray (approx 2%), these conclusions can be considered preliminary in all senses. When quasi-exhaustive cDNA microarrays containing >90% of the genome are used, it may well be that other pathways will be most affected, shifting the overall interpretation of comparative relevance.

Independent of any criticisms, these types of experiments are opening the way to a novel "global" approach to understanding the neurobiological substrate of nicotine dependence, where all the cellular biochemical pathways are assessed in parallel, and large-scale computational models will be used to make inferences about results.

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