A low chronic ethanol exposure induces morphological changes in the adolescent rat brain that are not fully recovered even after a long abstinence: An immunohistochemical study
Introduction
Normal adolescent animals have brains that are clearly under development; their neurotransmitter systems are actively and rapidly changing; their axons are still being subjected to extensive myelination (Flechsig, 1896); their cognitive and behavioral abilities are expanding, getting more complex and finer every day. Adolescence is a developmental stage in which a tremendous amount of learning takes place and when the brain is essentially built to be shaped and molded by experience (Dahl, 2004, Juraska and Markham, 2004, Monti et al., 2005).
Ethanol (EtOH) is perhaps the most important and common neurotoxin which humans make use or abuse of. It is a known neurotoxic for adults (for review, see Crews et al., 2004) and it is during adolescence when most humans have their first contact with EtOH and when most alcoholics begin to drink. Once people begin to drink and become alcoholics, the drinking patterns they exhibit are very variable. It has been demonstrated in vitro and in vivo that both in adults and developing animals, EtOH can exert diverse and even contradictory effects depending mainly on: (a) the developmental period when it is administered, (b) the pattern of exposure (continuous or intermittent) and (c) the blood ethanol concentrations (BEC) that are reached (Ahluwalia et al., 2000, Bonthius and West, 1988, Evrard et al., 2003, Ramos et al., 2002a). Therefore, a low and chronic level of EtOH exposure may be very different in its deleterious effects from a situation in which an adolescent consumes EtOH intermittently in a binge-like fashion (thus reaching higher BEC), even if the consumed total amount was the same in both cases. Most animal models of EtOH exposure (both in prenatal, adolescent or adult animals) are commonly focused in producing high BEC by means of acute binge-like or high chronic administration patterns and in generating evident and rough brain damages.
It has been noted that involvement in excessive EtOH drinking is highly prevalent in late human adolescence (Monti et al., 2005, Spear, 2004), but little or nothing is known about the effects of low chronic levels of EtOH use.
Much evidence exists supporting the fact that the serotoninergic system is one of the main targets for the EtOH neurotoxic effects, both in prenatal developing (Evrard et al., 2003, Sari et al., 2001, Tajuddin and Druse, 1999) and adult animals (Baker et al., 1996a, Baker et al., 1996b, Berggren et al., 2002, Halliday et al., 1995, Lovinger, 1999, Mantere et al., 2002, Pistis et al., 1997). Serotonin, acting through 5-HT1A receptors, induces astrocytes to release the S-100b protein (Ramos et al., 2000, Whitaker-Azmitia et al., 1990). Among other functions, S-100b protein directly interacts with different cytoskeletal proteins and inhibits their phosphorylation, thus inducing a cytoskeletal stabilization that promotes neurite outgrowth. Both the glial fibrillary acidic protein (GFAP), the type 2 of microtubule associated proteins (MAP-2) and the 200 kDa neurofilament (Nf-200) are influenced by S-100b protein action (Azmitia, 2001, Donato, 2003). The neuronal nitric oxide synthase (nNOS)-containing neurons are also related to the serotoninergic system (Kaehler et al., 1999, Tagliaferro et al., 2001, Tagliaferro et al., 2003, Wang et al., 1995). Nitric oxide (NO), a diffusible gaseous neurotransmitter, may inhibit tryptophan hydroxylase (TPH), the rate-limiting enzyme of the 5-HT biosynthetic pathway (Kuhn and Geddes, 1999). High concentrations of NO may induce neuronal death (Hu et al., 1997).
In the present work, we aimed at testing two hypotheses: (a) EtOH given to adolescent male rats in a chronic fashion, even at low levels, induces toxic morphological changes in the serotoninergic system, astrocytes, neuronal cytoskeleton and nitrergic system; and (b) given that adolescents still have recovery capacity after an insult, if the brain of adolescent male rats is subjected to the neurotoxic effects of low chronic EtOH levels, their high plastic capacity will completely (or almost completely) repair the damage provoked, if permitted to recover during a time of at least the same duration of that in which the noxious stimulus was applied.
Morphological studies have the advantage to show pathological or experimental changes with a very high anatomical resolution, that is very difficult to attain with other experimental methods. To our knowledge, there are no morphological studies evaluating the effects of an in vivo low chronic EtOH exposure on the serotoninergic system and its relationship with astrocytes, neuronal cytoskeleton and the nitrergic system. Therefore, in the present work, we carried out a series of morphometric studies by digital image analysis in order to contrast the previous hypotheses. We studied two mesencephalic nuclei from the rostral brainstem raphe group of serotoninergic nuclei that are the source of the distal prosencephalic serotoninergic innervation: the dorsal raphe nucleus (DRN) and the median raphe nucleus (MRN). Additionally, we studied three prosencephalic areas that receive a dense serotoninergic innervation and subserve important cognitive abilities, known to be affected after EtOH exposure: the stratum radiatum of the CA1 area of the hippocampus (Hipp), the corpus striatum (Strt) and the frontal cortex (FCx).
Section snippets
Materials
EtOH administered to the animals was of analytical grade (Merck KgaA; Darmstadt, Germany). For the immunohistochemical experiments, we used primary antibodies directed to: (a) 5-HT (rabbit polyclonal, previously developed in our laboratory; see Brusco et al., 1983); (b) GFAP (rabbit polyclonal anti-cow, purchased from Dako Corp.; Glostrup, Denmark; lot 096, edition 05.07.00); (c) S-100b protein (mouse monoclonal, purchased from Sigma Chemical Co., St. Louis, MO, USA; clone SH-B1, lot 099H4812);
Rats weight, food and liquid consumption and blood ethanol concentrations
There were no weight differences among the four treatment groups before and after the EtOH exposure and abstinence periods. At the beginning of the EtOH exposure period, all rats were at age P45 to P50 (late adolescent rats) and weighed 194.5 ± 33.0 g. By the end of this period, animals were at age P87 to P92 (adult rats) and weighed 366.4 ± 18.15 g (Control; n = 10) and 351.8 ± 29.55 (EtOH; n = 10) (P = 0.1997). By the end of the recovery period, rats were at age P157 to P162 and weighed
Discussion
In this study, we found for the first time that when a group of adolescent Wistar male rats were subjected to a low chronic EtOH exposure for 6 weeks their brains showed: (a) in the mesencephalon, a decrease in the 5-HT-ir of the DRN neurons and no change in the MRN, and (b) in three prosencephalic areas (Hipp, Strt and FCx), an increase in GFAP-ir and a consistent and significant decrease in S-100b-ir, MAP-2-ir and Nf-200-ir; in the Strt and FCx, there were no changes in the nNOS-ir neurons.
Acknowledgments
We thank Mrs. Emérita Jorge Vilela for her expertise and technical assistance and Ms. Marcela Di Paolo for her expertise and helpful grammatical correction of the manuscript. This work was supported by grant UBACyT M-072 from the Universidad de Buenos Aires to Prof. Dr Brusco.
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