alcohol admin induce alteration of parvalbumin and gfap in rat hippocampus and cingulate cortex acta...

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Alcoholadministrationduringadulthoodinducesalterationsofparvalbuminandglialfibrillaryacidicproteinimmunoreactivityinrathippocampusandcingulatecortex.

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Acta histochemica 112 (2010) 392401

0065-1281/$ - sdoi:10.1016/j.

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Alcohol administration during adulthood inducesalterations of parvalbumin and glial fibrillary acidicprotein immunoreactivity in rat hippocampus andcingulate cortex

Uraporn Vongvatcharanona,, Sirirak Mukema, Wandee Udomuksornb,Ekkasit Kumarsitc, Surapong Vongvatcharanond

aDepartment of Anatomy, Faculty of Science, Prince of Songkla University, Hat-Yai 90112, ThailandbDepartment of Pharmacology, Faculty of Science, Prince of Songkla University, Hat-Yai, ThailandcDepartment of Physiology, Faculty of Science, Prince of Songkla University, Hat-Yai, ThailanddDepartment of Oral Surgery (Anesthesiology section), Faculty of Dentistry, Prince of Songkla University, Hat-Yai,Thailand

Received 26 February 2009; received in revised form 18 March 2009; accepted 1 April 2009

KEYWORDSAlcohol;Parvalbumin;Glial fibrillary acidicprotein;Cingulate cortex;Hippocampus;Rats

ee front matter & 2009acthis.2009.04.001

ing author.ess: [email protected].

SummaryAlcohol induces impairment of cognition, learning and memory. Neurotoxic effects ofalcohol on the pathology of the hippocampus and the cingulate cortex wereinvestigated in experimental rats. Parvalbumin (PV), a calcium-binding protein, is acrucial component of GABAergic neurons and glial fibrillary acidic proteinimmunoreactive (GFAP-ir) astrocytes have been used as markers. We investigatedthe effects of ethanol exposure during adulthood on the PV-ir neurons and GFAP-irastrocytes in the hippocampus and the cingulate cortex of 3-month-old male Wistarrats. The rats were divided into 2 groups: control (C) and alcohol-exposed groups.The control group received distilled water whereas the alcohol-exposed groupsreceived either a low dose (20%w/v, LD) or high dose (40%w/v, HD) of ethanol forperiods of 21 days, 3 or 6 months. The brains of the animals were processed forimmunohistochemistry using anti-parvalbumin and anti-GFAP antibodies and thenumbers of PV immunoreactive (PV-ir) neurons and GFAP-ir astrocytes werecounted/unit area. For each period of administration, the number of PV-ir neuronswas significantly reduced for groups exposed to both the low and the high doses ofethanol compared to those of control groups in both the hippocampus and thecingulate cortex (po0.01). In addition, the number of PV-ir neurons wasprogressively reduced after prolonged ethanol exposure. In contrast, there was a

Elsevier GmbH. All rights reserved.

th (U. Vongvatcharanon).
www.elsevier.de/acthisdx.doi.org/10.1016/j.acthis.2009.04.001mailto:[email protected]

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Alcohol alters parvalbumin and glial fibrillary acidic protein levels 393

significantly increased number of GFAP-ir astrocytes observed in the hippocampusand the cingulate cortex in all groups exposed to ethanol and this was a function ofboth the duration and the dose of ethanol exposure, indicating that PV-ir neurons areas sensitive as the GFAP-ir astrocytes to ethanol exposure. Our data indicate thatalcohol exposure induced a reduction of PV-ir neurons and an increase of GFAP-irastrocytes in the hippocampus and the cingulate cortex and this may be associatedwith the impairment of cognition, learning and memory after chronic alcoholadministration.& 2009 Elsevier GmbH. All rights reserved.

Introduction

Ethanol exposure has been shown to haveneurotoxic effects on the morphology and functionof the central nervous system in both humans andexperimental animals (Diamond and Messing,1994). Several studies have demonstrated thatethanol exposure can result in impairment ofcognition, learning and memory (Ollat et al.,1988; Samson and Harris, 1992). The hippocampusplays a critical role in the memory and learningprocesses and is known as one of the sensitivetarget sites for neurotoxic substances (Frankeet al., 1997). Memory impairment of chronicalcoholics is often related to alcohol-mediatedbrain damage, particularly to the hippocampus(Walker et al., 1981; Bowden and McCarter, 1993).Ethanol exposure in utero has been shown to inducea permanent reduction of CA1 pyramidal neurons(Barnes and Walker, 1981), alterations in hippo-campal mossy fiber organization (West et al., 1981)and dendritic arborization (Smith and Davies,1990), delays in synaptogenesis (Hoff, 1988) andchanges in neurochemistry such as choline acetyl-transferase activity (Swanson et al., 1995) in therat septohippocampal system. In addition, pro-longed ethanol treatment has been demonstratedto induce neuronal damage in the hippocampus(Franke et al., 1997). Apart from the hippocampus,the cingulate cortex is a major relay center ofthe limbic lobe and plays an important role inmotor control, attention, emotion and memory(Kupferman, 1991; Paus et al., 1993; Murr et al.,1996; Picard and Strick, 1996). Thus the hippocam-pus and the cingulate cortex have been consistentlychosen for investigation of the adverse effects ofethanol.

Parvalbumin (PV), a calcium-binding protein, ispresent in fast-firing GABAergic neurons, where it isinvolved in the activity of Ca2+-dependent K+

channels and sequesters intracellular calcium(McPhalen et al., 1994; Plogmann and Celio,1993). PV is an important component of theGABAergic neurotransmission system, therefore

any alterations to PV could severely interfere withinhibitory neurotransmission (Moore et al., 1998).An abundance of PV neurons has been identified inthe hippocampus (Sloviter, 1989). Alterations to thePV immunoreactive neurons (PV-ir) due to ethanolexposure was previously studied mostly in thecingulate cortex of prenatal (Moore et al., 1998)and neonatal (Mitchell et al., 2000) rat brains and itwas found that ethanol exposure decreased thePV-ir neurons. Until now, the effect of alcoholadministration during adulthood on PV-ir neurons inthe hippocampus and the cingulate cortex has notbeen investigated.

The central nervous system not only consists ofneurons but also contains a large population of glialcells. Astrocytes are one type of glial cell that havesupportive roles in brain functions including neuro-transmitter uptake (Kimelberg and Katz, 1985) andsynthesis and secretion of neurotrophic factors(Furukawa et al., 1986). It has been demonstratedthat astroglial cells are important targets ofethanol toxicity (Guerri and Renau-Piqueras,1997). Many studies have demonstrated thatfollowing injury, astrocytes are capable of increas-ing the expression of the glial fibrillary acidicprotein (GFAP), the glial-specific cell marker (Enget al., 1992; Norton et al., 1992). Pathologicalchanges in the brain in alcoholism may be relatedto neuronal alterations and may also involve themore abundant glial cells (Miguel-Hidalgo et al.,2002). In addition, both neuronal and glial cellshave been used as markers to indicate braindamage (Gonzalez et al., 2006). As a consequenceof this previous study, we chose the PV-ir neuronsand GFAP-ir astrocytes as appropriate markers toinvestigate brain damage induced by ethanolexposure.

The effects of alcohol on alterations to PV-irneurons or GFAP-ir astrocytes have been studiedseparately using different doses and durations ofethanol exposure. The toxicity of alcohol dependson the amount and duration of ethanol exposure.Thus, it is important to know the dose and durationof exposure that can affect the numbers of PV-ir

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U. Vongvatcharanon et al.394

neurons and GFAP-ir astrocytes. Although alcohol isknown to induce brain damage in both the devel-oping brain (Bonthius and West, 1990) and themature brain (Zou et al., 1996), alcohol is mostlyconsumed by adult humans. Therefore, the aim ofthis study was to investigate the effects of the doseand duration of ethanol exposure on the PV-irneurons and GFAP-ir astrocytes in the hippocampusand cingulate cortex of adult rat brains. Thefindings from this study may help to explain thepathology of the hippocampus and cingulate cortexfollowing chronic alcohol consumption in humanalcoholics.

Materials and methods

Three-month-old male Wistar rats each weighing200250 g were obtained from the SouthernLaboratory Animal Facility, Prince of SongklaUniversity, Thailand. The rats were maintained at22 1C with a 12/12 dark/light cycle (light on at06:00 am). Standard commercial food pellets andfiltered tap water were available ad libitum. Theexperimental protocols described in this study wereapproved and guided by the Animal Ethics Commit-tee of the Prince of Songkla University. The ethanolexposure protocol used in this study is the same asrecently described by Vongvatcharanon et al.,(2009). Briefly, the rats were randomly dividedinto 2 groups: control (C) and alcohol-exposedgroups. They received intragastric administrationof either distilled water (control group) or alcohol(alcohol group) by using a ball-tipped gavage. Thealcohol-exposed group was divided into 6 furthergroups to receive either a low dose (LD, 2 g/kg, 20%w/v) or a high dose (HD, 5 g/kg, 40% w/v) ofethanol once daily for 21 days (21 d, subacute) or3 months (3mo, subchronic) or 6 months (6mo,chronic). There were 10 rats in each treatmentgroup.

At the end of their treatment, rats were deeplyanesthetized by intraperitoneal injection of 75mg/kg pentobarbital sodium (Sigma-Aldrich ChemicalCompany, St. Louis, MO). Blood samples were takenfrom the orbital plexus to measure alcohol levels.Analysis of blood alcohol level was performed usingheadspace gas chromatography (Shimadzu, Japan).The rats were perfused transcardially with 4%paraformaldehyde in phosphate buffered saline(PBS), pH 7.25. The brains were removed andpost-fixed with 4% paraformaldehyde in PBS over-night at room temperature. Subsequently, thetissue was cryoprotected by equilibration with30% sucrose in PBS and rapidly frozen. Serial

40-mm-thick frozen sections were cut coronallyand processed for immunohistochemistry.

The sections were incubated in the followingsolutions and between each of the steps thesections were rinsed with PBS: (1) 10% normalhorse serum (Vector Lab, Burlingame, CA) with 0.2%Triton-X 100 (J.T Baker Inc, Phillipsburg, NJ) for30min; (2) anti-parvalbumin mouse monoclonalantibody (1:200 dilution, Sigma-Aldrich ChemicalCompany, St. Louis, MO) overnight at 4 1C; (3)Texas red conjugated anti-mouse IgG (1:200 dilu-tion, Vector Lab, Burlingame, CA) for 1 h. Allsolutions were prepared in PBS and incubationswere at room temperature unless otherwisespecified. The sections were finally rinsed againwith PBS, mounted with Vectashield (Vector Lab,Burlingame, CA), coverslipped and sealed with nailpolish.

Adjacent series of sections from each control andalcohol-treated animal were immunolabelled inparallel for binding of an anti-GFAP mouse antibody(1:200 dilution, Chemicon, Temecula, CA) using thesame immunohistochemistry protocol as describedabove.

Control sections, omitting the primary antibody,were routinely processed to ensure that anyobserved labelling was due to parvalbumin or GFAP.The morphologies of PV-ir neurons and GFAP-irastrocytes were studied with a BX 50 fluorescentmicroscope (Olympus, Japan).

Every 15th section was selected and coded suchthat all subsequent analyses were carried out in ablinded manner. The number of PV-ir neurons andGFAP-ir astrocytes were counted. For example, the15th, 30th and 45th sections, etc. were selected forcounting PV-ir and the 16th, 31th and 46th, etc. werechosen for counting GFAP-ir, respectively. About 10sections from each animal in each study group wereanalysed for immunolabelling of PV and GFAP.Images from selected sections were captured by adigital camera (DP50, Olympus, Japan). Images ofindividual cells were used in order to avoidinterference from overlapping images. Numbers ofPV-ir neurons and GFAP-ir astrocytes in eachphotomicrograph were counted and the area ofsections examined was measured using imageanalysis software (Olympus, Japan). The resultswere expressed as number of PV-ir neurons andGFAP-ir astrocytes per mm2.

Results are expressed as mean7standard error ofthe mean. The statistical evaluation of the datawas performed using one-way ANOVA and the leastsignificant difference test for post hoc analyses todetermine the significance between means. Differ-ences among means were considered significantwhen Po0.05.

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Results

Blood ethanol levels

The mean blood ethanol level of low-doseethanol exposure was 32717.81mg/dl and follow-ing the high dose of ethanol treatment was 148734.65mg/dl.

Parvalbumin immunoreactive (PV-ir) neuronsin the hippocampus

PV-ir neurons were identified in the hippocampus(Figure 1A, B). A qualitative reduction of PV-irneurons was observed in the hippocampus of theanimals exposed to both low and high dosages ofethanol for 6 months when compared with thecontrols (Figure 2AC).

Parvalbumin immunoreactive neurons in thecingulate cortex

PV-ir neurons were identified in the cingulatecortex (Figure 3A, B). A qualitative reduction of PV-ir neurons was observed in the cingulate cortex ofthe animals exposed to low dose and high doses

Figure 1. (A) PV-ir in the hippocampus of the control animalsshowing the distribution of PV-ir (arrows). Bars 40 and 100

Figure 2. Representative photomicrographs of PV-ir neurons6-month exposure to ethanol in both low dose (B) and high d

after 6 months (Figure 4AC) ethanol administra-tion when compared with the controls.

Quantitative PV-ir neurons in thehippocampus

In the group exposed to ethanol for 21 days,there was a significant reduction of PV-ir neuronsin the hippocampus in both the LD (141.40711.01 cell/mm2) and the HD (136.05730.47 cell/mm2) groups (po0.01) compared to that of thecontrol group (293.71740.18 cell/mm2). In con-trast there was no significant difference of PV-irneuron numbers identified in the LD and the HDethanol-exposed groups (Figure 5).

In the group exposed to ethanol for 3 months, thenumber of PV-ir neurons in the hippocampus wassignificantly reduced in the LD (93.6276.97 cell/mm2) and HD (85.74712.53 cell/mm2) groups(po0.01) compared to that of the control group(274.64754.24 cell/mm2). However, the number ofPV-ir neurons was not significantly different be-tween the LD and the HD ethanol-exposed groups(Figure 5).

In the group exposed to ethanol for 6 months,there was a significant decrease of the numbers ofPV-ir neurons in the hippocampus in the LD (70.24715.52 cell/mm2) and HD (48.2775.50 cell/mm2)

. The framed area of the hippocampus is magnified in (B),mm, respectively.

(arrows) in the hippocampus of control (A) and group withose (C). Bar 200 mm.

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Figure 3. (A) Photomicrographs of PV-ir in the cingulate cortex of the control animal. The framed area of the cingulatecortex is magnified in (B), showing the distribution of PV-ir (arrows). Bars 40 and 100 mm, respectively.

Figure 4. Representative photomicrographs of PV-ir neurons (arrows) in the cingulate cortex of control (A) and groupwith 6-month exposure to ethanol in both low dose (B) and high dose (C). Bar 200 mm.

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Figure 5. Histogram showing the mean numbers of PV-irneurons per mm2 in the hippocampus of the control (C),low dose (LD) and high dose (HD) ethanol exposure afterdifferent durations: 21 days (21 d), 3 months (3mo) and 6months (6mo). ** po0.01, n 6.


groups (po0.01) compared to that of the controlgroup (171.76711.42 cell/mm2). In contrast, therewas no significant difference of PV-ir neuron

numbers between the LD and the HD ethanol-exposed groups (Figure 5).

Quantitative PV-ir neurons in the cingulatecortex

In the group exposed to ethanol for 21 days,there was a significant reduction of PV-ir neurons inthe cingulate cortex in both the LD (150.88712.66 cell/mm2) and the HD (166.35747.48cell/mm2) groups (po0.01) compared to that ofthe control group (333.31746.21 cell/mm2). Incontrast there was no significant difference ofqPV-ir neuron numbers identified in the LD and theHD ethanol-exposed groups (Figure 6).

In the group exposed to ethanol for 3 months, thenumber of PV-ir neurons in the cingulate cortex wassignificantly reduced in the LD (95.21710.64 cell/mm2) and HD (90.40714.42 cell/mm2) groups

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(po0.01) compared to that of the control group(285.40755.52 cell/mm2). However, the number ofPV-ir neurons was not significantly different be-tween the LD and the HD ethanol-exposed groups(Figure 6).

In the group exposed to ethanol for 6 months,there was a significant decrease in numbers of PV-irneurons in the cingulate cortex in the LD (72.48716.63 cell/mm2) and HD (64.84713.33 cell/mm2)groups (po0.01) compared to that of the controlgroup (163.62711.21 cell/mm2). In contrast, therewas no significant difference of PV-ir neuronnumbers between the LD and the HD ethanol-exposed groups (Figure 6).

GFAP-ir astrocytes in the hippocampus andthe cingulate cortex

GFAP-ir astrocytes were found in the hippo-campus (Figure 7AC) and the cingulate cortex(Figure 8AC). A qualitative increase of GFAP-irastrocytes was identified in the hippocampus andthe cingulate cortex of the animals exposed toboth low- and high-dose ethanol after 6 months(Figure 7AC and 8AC) when compared with thecontrols.

Figure 7. Representative photomicrographs of GFAP-ir (arroalcohol-treated animals in both low dose (B) and high dose (

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Figure 6. Histogram showing the mean numbers of PV-irneurons per mm2 in the cingulate cortex of the control(C), low dose (LD) and high dose (HD) ethanol exposureafter different durations: 21 days (21 d), 3 months (3mo)and 6 months (6mo). ** po0.01, n 6.

Quantitative GFAP-ir astrocytes in thehippocampus

In the group exposed to ethanol for 21 days, anincrease of GFAP-ir astrocyte numbers was ob-served in the LD (272.14749.26 cell/mm2) groupsand a significant increase of GFAP-ir astrocytenumbers was found in the HD (384.52738.63 cell/mm2) groups (po0.05) compared to that of thecontrol group (228.19763.88 cell/mm2). In con-trast there was no significant difference of GFAP-irastrocyte numbers in the LD and the HD ethanol-exposed groups (Figure 9).

In the group exposed to ethanol for 3 months, thenumber of GFAP-ir astrocytes was significantlyincreased in the LD (391.01731.75 cell/mm2)(po0.05) groups and HD (458.84738.96 cell/mm2)groups (po0.01) compared to that of the controlgroup (250.29748.92 cell/mm2). However, thenumbers of GFAP-ir astrocytes was not significantlydifferent between the LD and the HD ethanol-exposed groups (Figure 9).

In the group exposed to ethanol for 6 months, asignificant increase of GFAP-ir astrocytes numberwas found in the LD (507.26758.03 cell/mm2)(po0.05) groups and HD (604.15789.77 cell/mm2)groups (po0.01) compared to that of the controlgroup (293.93722.17 cell/mm2). In contrast, therewere no significant difference of GFAP-ir astrocytenumbers between the LD and the HD ethanol-exposed groups (Figure 9).

Quantitative GFAP-ir astrocytes in thecingulate cortex

In the group exposed to ethanol for 21 days, anincrease of GFAP-ir astrocyte numbers was ob-served in the LD (245.55739.98 cell/mm2) and asignificant increase of GFAP-ir astrocyte numberswas found in the HD (365.95737.80 cell/mm2)groups (po0.05) compared to that of the controlgroup (199.95768.09 cell/mm2). In contrast there

ws) in the hippocampus of the control (A) and 6-monthC). Bars 400 mm.

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Figure 8. Representative photomicrographs of GFAP-ir (arrow) in the cingulate cortex of the control (A) and 6-monthalcohol-treated animals in both low dose (B) and high dose (C). Bars 400 mm.

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astro

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Figure 9. Histogram showing the mean numbers of GFAP-ir astrocytes per mm2 in the hippocampus of the control(C), low dose (LD) and high dose (HD) alcohol treatmentat different durations: 21 days (21 d), 3 months (3mo)and 6 months (6mo). * po0.05, ** po0.01, n 6.

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Figure 10. Histogram showing the mean numbers ofGFAP-ir astrocytes per mm2 in the cingulate cortex of thecontrol (C), low dose (LD) and high dose (HD) alcoholtreatment at different durations: 21 days (21 d), 3months (3mo) and 6 months (6mo). * po0.05,** po0.01, n 6.


was no significant difference of GFAP-ir astrocytenumbers in the LD and the HD ethanol-exposedgroups (Figure 10).

In the group exposed to ethanol for 3 months, thenumbers of GFAP-ir astrocytes was increased inthe LD (332.96752.27 cell/mm2) groups and asignificant increase of GFAP-ir astrocyte numberswas found in the HD (411.82766.26 cell/mm2)groups compared to that of the control group(251.55754.28 cell/mm2). However, the numbersof GFAP-ir astrocytes was not significantly differentbetween the LD and the HD ethanol-exposed groups(Figure 10).

In the group exposed to ethanol for 6 months, asignificant increase of GFAP-ir astrocytes numberwas found in the LD (489.55761.53 cell/mm2)(po0.05) and HD (548.32771.52 cell/mm2) groups(po0.01) compared to that of the control group(261.53724.33 cell/mm2). In contrast, there wereno significant differences in GFAP-ir astrocytenumbers between the LD and the HD ethanol-exposed groups (Figure 10).

Discussion

This study demonstrated a significant decrease ofPV-ir neurons in the hippocampus and the cingulatecortex of all groups (21 days, 3 months and6 months) exposed to both low and high doses ofethanol. This indicates that exposure to both thelow dose and the high dose of alcohol, even after21 days, induces a reduction of PV-ir neurons in thehippocampus and cingulate cortex. Furthermore,the reduction of PV-ir neurons induced by ethanolwas augmented by increasing the duration ofethanol exposure. In previous studies, ethanolexposure in both prenatal and neonatal animalsresulted in a decrease of PV-ir neurons in the cingu-late cortex (Moore et al., 1998; Mitchell et al.,2000). In the present study, the ethanol exposure inadulthood reduced PV-ir neurons in both thehippocampus and the cingulate cortex. This in-dicates that exposure to alcohol during adulthoodalso induces a reduction of PV-ir neurons as well as

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in neonatal and prenatal animals. A reduction ofneurons after ethanol exposure was also identifiedin the work of Franke et al. (1997), whichdemonstrated that Wistar rats exposed to 10% v/valcohol during adulthood (10 weeks old) for 4, 12and 36 weeks induced neuronal cell loss in thehippocampus and in the prolonged ethanol expo-sure groups, the neuronal cell loss was furtherincreased. In addition, Satriotomo et al. (2000)have shown that administration of 6% (v/v) ethanolduring adulthood for a short time period (1 week)induced neuronal death and a decrease in the totalnumber of calbindin D28k-ir neurons in the mousehippocampus. Calbindin D28k is another calcium-binding protein (Pfeiffer et al., 1989).

Although many studies have demonstrated thatethanol intoxication can trigger neuronal deathdirectly (Olney et al., 2002; Ikonomidou et al.,2000), diminished PV was observed in adult animalsfollowing prenatal ethanol exposure (Mitchellet al., 2000). Thus, a decrease of PV-ir neurons afterexposure to ethanol may be due to down-regulationof PV protein expression or neuronal loss. Alcoholhas been demonstrated to mediate neuronal deathvia oxidative stress or activation of caspase-3proteases (Goodlett et al., 2005). However, thedecrease of PV-ir neurons has also been suggestedto reflect the loss of neurons. As PV is implicated inbuffering excess calcium (Ca2+) at presynapticnerve terminals after a rapid train of actionpotentials (Heizmann, 1984) and excessively highlevels of intracellular Ca2+ are known to mediatecell death, thus it is possible that an ethanol-induced reduction of PV expression decreases theneurons ability to buffer Ca2+, leading to theinitiation of the process of cell death (Moore et al.,1998).

Apart from the response of the PV-ir neurons, ourdata also demonstrated the effects of the sameethanol exposure on GFAP-ir astrocytes in thehippocampus and the cingulate cortex. An increaseof GFAP-ir astrocytes was observed in the hippo-campus and the cingulate cortex of all ethanol-exposed groups (LD and HD, after 21 days, 3 monthsand 6 months). However, the progressive increaseof GFAP-ir astrocytes was more obvious in pro-longed (3 and 6 months) and HD ethanol-exposedgroups. In contrast to the data on the PV-ir neurons,the reduction of PV-ir neurons was much greaterafter 21 days (about 50%) than the increase of theGFAP-ir astrocytes, although the effect of the HDcompared to the LD and the changes with time wassignificantly higher for the GFAP-ir astrocytes. Thisindicates that PV-ir neurons are more sensitive toethanol exposure and likely to disappear before theincrease of GFAP-ir astrocytes. It is possible that

the loss of neurons induces the proliferation of glialcells to fill in the space. These findings illustratethe importance of the time and doseresponse ofPV-ir neurons and GFAP-ir astrocytes. In addition,quantitative estimation of the changes in PV-irneurons and GFAP-ir astrocytes provides valuableindices for monitoring the neuronal degenerationand reactive astrocytes.

It has been shown that ethanol exposure in brainsof pups from alcohol-fed mothers induced asignificantly reduced increase in the level of GFAPand its mRNA (Valles et al., 1997). However, chronicalcohol intake increased GFAP immunoreactivity inadult rats (Franke et al., 1997). Our data areconsistent with these findings.

Alcohol has been shown to cause an impairmentin cognition, learning and memory (Ollat et al.,1988; Samson and Harris, 1992). The hippocampusand the cingulate cortex have an important rolein cognition, learning and memory and PV is animportant component of the GABAergic system(Miettinen et al., 1993). Thus the reduction of PVlevels in the hippocampus and the cingulate cortexcould interfere with normal inhibitory neurotrans-mission in the hippocampus. In addition, astrocyteshave been shown to be involved in learning andmemory consolidation (Gibbs et al., 2008). Astro-cytes take up most of the synaptically releasedglutamate, terminating transmitter activity andreturning glutamate to neurons in a glutamateglutamine cycle. Thus, an increase of GFAP-irastrocytes may interfere with normal excitatoryneurotransmission (Gibbs et al., 2008). Interfer-ence with normal inhibitory and excitatory neuro-transmissions may result in an impairment ofcognition, learning and memory processes in thehippocampus and the cingulate cortex. This couldexplain the impairment of cognitive, learning andmemory in alcoholics or after chronic alcoholconsumption.

Our study has demonstrated that the same doseand duration of ethanol exposure produced distinctalterations in PV-ir neurons and GFAP-ir astrocytes.A loss of PV-ir neurons was found in both thehippocampus and the cingulate cortex, whereas anincrease of GFAP-ir astrocytes was observed in boththe hippocampus and the cingulate cortex. Thereduction of neurons and the increase of astrocytesindicated pathological changes in the hippocampusand the cingulate cortex after ethanol exposure.This implies that exposure to ethanol duringadulthood also induced mature brain damage andespecially so in the long term and high dose ofexposure to ethanol. Therefore, alcohol consump-tion in adult humans may result in damage to thehippocampus and the cingulate cortex, leading to

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learning and memory impairment in alcoholics orafter chronic alcohol consumption. A further studyis being conducted to investigate the effects ofalcohol exposure during adulthood in other brainregions.

Acknowledgements

This study was financially supported by theGraduate research grant of Prince of SongklaUniversity. We would like to thank Dr. BrianHodgson for assistance with English writing andMiss Wilairat Kankoun, Miss Kanchana Kornchatriand Mr. Pornping Lojanapaiboon for assistance withthe alcohol treatment. Finally, we also thank theDepartment of Anatomy, Faculty of Science, fortheir continuing support.

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