0145-6008/94/1803-057 1 $3.00/0 zyxwvutsrqp ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH Vol. 18, No. 3 May/June 1994 Reduced Serotonergic Immunoreactive Fibers in the Forebrain of Alcohol-Preferring Rats zy Feng C. Zhou, Sharon Bledsoe, Larry Lumeng, and Ting-Kai Li Our previous study indicated that 5-hydroxytryptamine (5-HT) im- munoreactive fiber densities were decreased in specific areas of the brain in alcohol-preferring rats (P) when compared with alcohol- nonpreferring rats (NP). The results of our current study show that there are quantitative and qualitative differences in 5-HT innervation in other selected regions of the forebrains of P rats. The 5-HT fiber density in the brains of young adult P and NP rats was measured by immunocytochemistry and quantitative image analysis. A routine error of two-dimensionalquantitation of nerve fiber was addressed and an adjustment was made. The amount of 5-HT fibers was significantly lower in CA4 and fasciola cinereum of the dorsal hip- pocampus, caudate-putamen, and hypothalamus of the P as com- pared with NP rats (unpaired Student’s zyxwvutsr t tests). In examining the fiber types, we found that, in the frontal cortical and hippocampalregions, where normally fine 5-HT fibers with small varicosities and thick 5-HT fibers with large varicosities coexist, fewer fine 5-HT fibers were seen in P rats as comparedwith NP rats. the fine fibers are known to be vulnerable to abusive drugs. These observations indicate that (a) there are quantitative differences in 5- zyxwvu HT innervation or that the 5-HT in some 5-HT fibers is reduced to a level undetectable by immunocytochemistry, and (b) the fine 5-HT fibers are specifically reduced to a greater degree in the selected brain regions of P rats when compared with that of NP rats. The involvement of the 5-HT system in the alcohol abuse is discussed. Key Words: Serotonin, Immunocytochemistry, Animal Model, Al- coholism, Image Analysis. TUDIES IN RECENT years have produced increasing S evidence that serotonin neurons may be involved in alcohol drinking behavior. First, the level of 5-hydroxy- tryptamine (5-HT), as well as its metabolite 5-hydroxyin- dole-acetic acid (5-HIAA), has been consistently found to be lower by 15-20% in the hippocampus, nucleus accum- bens, striatum, cortex, and hypothalamus of different alcohol-preferring(P) animals as compared with nonpre- femng (NP) animals (P/NP,3 N/Nih genetically hetero- geneou~,~ HAD/LAD,’ and inbred mice6).Second, 5-HTi receptor affinity were higher in the rats as compared with NP rats; B,,, of 5-HT1receptors in the cellular membrane was increased 30-50% in the hippocampus and prefrontal zyxwvu From the Departments ofAnatomy (F.C. zyxwvutsrqp Z., S.B.) and Medicine (L.L., T.-K. LJ, Indiana University School @Medicine and VeteransAdminis- tration Medical Cenier (L.L.,T.-K.L.). Indianapolis, Indiana. Received for publication August 18, 1993; accepted October 19, 1993 This research is supported by the National Institutes of Health Grants A0761 1 and ,4408553. Reprint requests: Feng C. Zhou, Ph.D., Department of Anatomy, MS 508, Indiana University School of Medicine. 635 Barnhill Drive, Indi- anapolis, IN 45202. Alcohol Clin Exp Res. Vol 18, No 3, 1994: pp 51 1-519 Copyright zyxwvutsrqpo 0 I994 by The Research Society on Alcoholism. Third, increased 5-HT synthesis or administra- tion of 5-HT agonists markedly reduced alcohol consump- tion by P and HAD Fourth, 5-HT uptake blocker, which increases synaptic serotonin, also reduced alcohol consumption by P and HAD rat^.^.'^ These results suggest that the abnormality in the 5-HT neurons may be associated with high alcohol-seeking char- acteristics. The lower levels of 5-HT and/or 5-HIAA may indicate a lower functioning and/or innervation of the 5- HT system projecting to the previously specified brain regions. Our pilot data indicated that 5-HT-immunostaining (5- HT-IM) was altered in the anterior frontal cortex, cingu- late cortex, nucleus accumbens, and ventral hippocam- pus” in the P rats, as compared with NP rats. In the present study, we demonstrate that 5-HT-IM fibers are altered in a number of structures in the forebrain that are related to rewarding behavior. In addition, a detailed qualitative difference in the 5-HT fiber morphology is reported in two major areas: the frontal cortex and hip- pocampus. MATERIALS AND METHODS P and NP Rats The animal model P and NP rat lines were established in the Alcohol Research Center at Indiana University. The P and NP rats were devel- oped by mass selection for high and low dcohoi preference from a foundation stock of Wistar rats.12-15 The P line of rats meets the criteria for an animal model of alcoholism as outlined by Cicero.16 Immunocytochemistry Young ethanol-naive female P and NP rats (200-250 g body weight) were used. All animals were treated with pargyline (200 mg/kg, an inhibitor of the major 5-HT degradation enzyme monoamine oxidase, MAO, Sigma, St. Louis, MO) 80 min, prior to perfusion, and with L- tryptophan (200 mg/kg, a precursor of 5-HT; Sigma) 60 min prior to perfusion. The pretreatment of animals with 5-HT precursor L-trypto- phan and pargyline, known to increase 5-HT levels, has been used to increase visualization of 5-HT fibers to a level closer to that of the actual density. Without 5-HT enhancement, the known available antibodies can only detect a portion of the entire fiber population. The 5-HT- enhancing treatment, shown to increase immunolabeling in 5-HT neu- rons, increases the number of 5-HT-IM fibers and the number of 5-HT- IM cell bodies in both P and NP rats.” The P rat levels may even increase more, but not to a significant degree as compared with the increase of NP rats.” The animals were perfused intracardially with formaldehyde (reagent grade) freshly made from zyx 4% paraformaldehyde and 0.1 M phosphate-buffered saline (PBS) under deep ketamine cocktail 571