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

Abstract

Little is known about the morphological effects of alcoholism on the developing adolescent brain and its consequences into adulthood. We studied here the relationship between two neurotransmitter systems (the serotoninergic and nitrergic) and the astrocytic and neuronal cytoskeleton immediately and long after drinking cessation of a chronic, but low, ethanol administration. Adolescent male Wistar rats were exposed to ethanol 6.6% (v/v) in drinking water for 6 weeks and studied after ending exposure or after a 10-week recovery period drinking water. Control animals received water. Brain sections were processed by immunohistochemistry using antibodies to serotonin (5-HT); glial fibrillary acidic protein (GFAP); astroglial S-100b protein; microtubule associated protein-2 (MAP-2); 200 kDa neurofilaments (Nf-200); and neuronal nitric oxide synthase (nNOS). The mesencephalic dorsal and median raphe nucleus (DRN; MRN) and three prosencephalic areas closely related to cognitive abilities (CA1 hippocampal area, striatum and frontal cortex) were studied by digital image analysis. 5-HT immunoreactivity (-ir) decreased in the DRN and recovered after abstinence and was not changed in the MRN. In the three prosencephalic areas, astrocytes' cell area (GFAP-ir cells) increased after EtOH exposure and tended to return to normality after abstinence, while cytoplasmic astroglial S100b protein-ir, relative area of MAP-2-ir and Nf-200-ir fibers decreased, and later partially recovered. In the striatum and frontal cortex, nNOS-ir decreased only after abstinence. In conclusion, in the adolescent brain, drinking cessation can partially ameliorate the ethanol-induced morphological changes on neurons and astrocytes but cannot fully return it to the basal state.

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-HT_1A 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).