www.elsevier.com/locate/ijdevneu

Int. J. Devl Neuroscience 25 (2007) 133–139

Effects of maternal hyperhomocysteinemia induced by high methionine

diet on the learning and memory performance in offspring

Giyasettin Baydas a,*, Sema T. Koz a, Mehmet Tuzcu b,Victor S. Nedzvetsky c, Ebru Etem d

a Department of Physiology, Faculty of Medicine, Firat University, 23119 Elazig, Turkeyb Department of Biology, Faculty of Science, Firat University, 23119 Elazig, Turkey

c Department of Biophysics and Biochemistry, Faculty of Biology, Dnepropetrovsk National University, Dnepropetrovsk, Ukrained Department of Medical Biology, Faculty of Medicine, Firat University, 23119 Elazig, Turkey

Received 14 July 2006; received in revised form 6 February 2007; accepted 2 March 2007

Abstract

In this study, we suggest that chronic maternal hyperhomocysteinemia results in learning deficits in the offspring due to delayed brain

maturation and altered expression pattern of neural cell adhesion molecule. Although the deleterious effects of hyperhomocysteinemia were

extensively investigated in the adults, there is no clear evidence suggesting its action on the developing fetal rat brain and cognitive functions of the

offspring. Therefore, in the present work we aimed to investigate effects of maternal hyperhomocysteinemia on the fetal brain development and on

the behavior of the offspring. A group of pregnant rats received daily methionine (1 g/kg body weight) dissolved in drinking water to induce

maternal hyperhomocysteinemia, starting in the beginning of gestational day 0. The levels of glial fibrillary acidic protein, S100B protein, and

neural cell adhesion molecule were determined in the tissue samples from the pups. Learning and memory performances of the young-adult

offsprings were tested using Morris water maze test. There were significant reductions in the expressions of glial fibrillary acidic protein and S100B

protein in the brains of maternally hyperhomocysteinemic pups on postnatal day 1, suggesting that hyperhomocysteinemia delays brain maturation.

In conclusion, maternal hyperhomocysteinemia changes the expression pattern of neural cell adhesion molecule and therefore leads to an

impairment in the learning performance of the offspring.

# 2007 ISDN. Published by Elsevier Ltd. All rights reserved.

Keywords: Hyperhomocysteinemia; Cognitive functions; Brain development; NCAM; GFAP

1. Introduction

Homocysteine (Hcy) is a sulfur containing amino acid

derived from the metabolism of methionine (Clarke et al., 1991;

Reis et al., 2002). Dietary methionine contributes to a rise in

circulating homocysteine concentration (Hirche et al., 2006).

Thus, many pathogenic effects of hypermethioninemia may

be related to the metabolic link between methionine and

homocysteine. Accumulating evidence suggest that there is an

association between plasma Hcy levels and various vascular

diseases including coronary, cerebral and peripheral arterial

and venous thrombosis (Clarke et al., 1991; Kruman et al.,

* Corresponding author. Tel.: +90 424 237 00 00; fax: +90 424 237 91 38.

E-mail addresses: baydas@hotmail.com, gbaydas@firat.edu.tr

(G. Baydas).

0736-5748/$30.00 # 2007 ISDN. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.ijdevneu.2007.03.001

2000; Ho et al., 2002). More recently, clinical studies implicate

increased levels of Hcy and reduced levels of folate with

neurodegenerative conditions including Alzheimer’s disease

(Shea et al., 2002). Increased levels of Hcy are associated with

structural brain changes like cerebral infarcts, white matter

lesions and cerebral atrophy (Vermeer et al., 2002; den Heijer

et al., 2003).

Hcy is an excitatory amino acid and markedly enhances the

vulnerability of neuronal cells to excitotoxic and oxidative

injury in vitro and in vivo (Hankey and Eikelboom, 1999; Streck

et al., 2003; Baydas et al., 2006). Moreover, Hcy has ability

to inhibit the expression of antioxidant enzymes such as

glutathione peroxidase, superoxide dismutase, which might

potentate the toxic effects of free radicals (Ho et al., 2002;

Baydas et al., 2003a, 2006). Recently, Rosenquist and Finnell

(2001) have reported that an increase in the level of maternal

homocysteine induces abnormal development, inhibiting the

G. Baydas et al. / Int. J. Devl Neuroscience 25 (2007) 133–139134

function of N-methyl-D-aspartate (NMDA) receptors in the

neural epithelium.

Evidence from literature indicates that Hcy is an important

risk factor for cognitive dysfunctions (Miller, 2003). Recently,

our group (Baydas et al., 2005a) and others (Streck et al., 2004)

have suggested that chronic administration of Hcy to rats

impairs both short-term and long-term memory, implying that

hyperhomocysteinemia causes memory impairment through

the enhancement of oxidative stress (Baydas et al., 2005a).

Development of the nervous system requires numerous

factors including neural cell adhesion molecules (NCAM).

NCAM are likely to be involved in synaptogenesis and neuronal

plasticity. Furthermore, there is also evidence suggesting

strongly that they also participate in synaptic changes

underlying memory formation in adult individuals (Schachner,

1997). In addition, NCAM is involved in cellular migration,

axonal growth, and regeneration of peripheral axons (Walsh

and Doherty, 1997; Tzeng et al., 2001). One of the maturational

events in the brain is the alteration in the pattern of expression

of the polysialylated form of the NCAM (PSA-NCAM). PSA-

NCAM is expressed in immature neurons and is important for

morphological plasticity in the hippocampus (Seki and Arai,

1993). PSA-NCAM plays a critical role in axonal growth and

fasciculation during development. The level of PSA-NCAM

decreases dramatically during development in some brain areas

(Doherty et al., 1990).

Intermediate filament proteins comprise a major portion of

the brain’s cytoskeleton with nestin, vimentin, and glial

fibrillary acidic protein (GFAP) differentially expressed with

respect to neurologic development (Hutchins and Casagrande,

1989; Hewicker-Trautwein and Trautwein, 1993). Immature

astrocytes of the CNS usually express vimentin as the major

intermediate filament. Maturation of astrocytes is followed by a

switch between vimentin and GFAP expression, with the latter

being recognized as an astrocyte maturation marker (Gomes

et al., 1999). It is also demonstrated that glial S100B is involved

in neuronal differentiation and maturation (Yang et al., 1996).

The effects of Hcy were extensively investigated in postnatal

life, however, little is known about its deleterious action in

prenatal life. Thus, in the present work we investigated, for the

first time, effects of maternal hyperhomocysteinemia on brain

development by evaluating the expression of NCAM and

intermediate filaments in the brain and behavioral changes of

offsprings.

2. Materials and methods

2.1. Animals and treatments

Female Wistar rats were obtained from the Animal Research Unit, Firat

University, Elazig, and housed in standard plastic cages. Animal room was

maintained on a 12-h light, 12-h dark photoperiod, and all animals were

permitted free access to food and tap water. Daily vaginal smear was performed

on all animals. Pro-estrous animals were left overnight with male rats and the

following day was recorded as gestational day 0 when spermatozoa were

observed in a smear of the vaginal contents.

Pregnant rats were randomly divided into two groups (n = 5 animals/

group) as follows: one group of animals was assigned as control and the second

group was assigned as Hcy group and received daily methionine (1 g/kg body

weight) dissolved in drinking water, starting the first day of gestation through-

out pregnancy. Based upon our (Baydas et al., 2005a) and other published

reports (Bagi et al., 2003) the orally administered methionine dose in this study

increase plasma Hcy levels approximately three to six times compared with

the control values. This is an adequate methionine dose to induce chronic

hyperhomocysteinemia. Due to unattractive option of sulfur group, rats

drinked less water including methionine on the first day, however, from the

second day and onwards they drank adequate water as control rats did. All rats

were housed in individual cages until delivery. All animal procedures were

done according to the Guide for the Care and Use of Laboratory Animals, and

were reviewed by the Institutional Animal Care and Use Committee of the

Firat University.

After delivery, the litter size and the number of live offspring were recorded.

On postnatal day (PND) 1, the body weight and gender of the offsprings of each

litter were recorded. Total Hcy levels in plasma of mothers were determined

with an enzyme immunoassay kit (Axis-Shield AS, Oslo, Norway). Fifteen pups

per group (three from each litter) were dissected and their brains were stored at

�70 8C for the analysis of glial and neuronal markers on PND1. The remaining

pups from each experimental litter were weaned from their dams when they

were 21 days old. After weaning, pups were separated into male and female

cages. Behavioral testing commenced at approximately postnatal day 75.

Twenty-four hours after the behavioral testing, young-adult offsprings from

both groups were decapitated. Brain tissues were removed and hippocampus

were dissected and stored at �70 8C.

2.2. Immunoblotting

Briefly, frozen tissues were thawed; hand-homogenized using glass-glass

homogenisator on ice 1:10 (w/v) in a buffer containing 50 mM Tris (pH 7.4),

0.1 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1.0 mM phenylmethylsulfonyl

fluoride, 10 mg/mL aprotinin and 10 mg/mL leupeptin. Tissue homogenates

were then centrifuged at 40,000 � g at 4 8C for 60 min, and supernatants were

collected into fresh tubes. Protein determinations in the homogenates were

performed according to the Lowry procedure using a protein assay kit (Sigma,

St. Louis, MO, USA). Equal quantities of proteins were separated by SDS-

polyacrylamide gel electrophoresis on 7.5–17.5 polyacrylamide gradient gels as

described previously (Baydas et al., 2002a,b). Separated proteins were trans-

ferred to nitrocellulose membranes (Schleicher and Schuell Inc., Keene, NH,

USA). Nitrocellulose blots were washed twice for 5 min each in phosphate-

buffered saline and blocked with 1% bovine serum albumin in phosphate-

buffered saline for 1 h prior to application of the primary antibody. Blots were

then incubated overnight at 4 8C with primary antibodies including rabbit

polyclonal antibody (Santa Cruz Biotechnology, Inc., USA) against NCAM

(diluted 1:1000), GFAP (diluted 1:2000) and S100B (diluted 1:1500), and

mouse monoclonal antibody (Chemicon Inc., CA, USA) against PSA-NCAM

(diluted at 1:500). The blots were washed and incubated for 1 h with a

secondary antibody, goat anti-rabbit or anti-mouse Ig peroxidase conjugated

(Santa Cruz Biotechnology, Inc., USA) at a dilution of 1:500. Specific binding

was detected using diaminobenzidine and H2O2 as substrates. The relative

amount of immunoreactive bands on Western blots was quantified in arbitrary

units by scanning blots using a computerized software program (LabWorks 4.0;

UVP, Inc., Cambridge, UK).

2.3. Morris water maze

Male (n = 4) and female (n = 4) rat offsprings (8 per group), on approxi-

mately PND75 from the two groups were tested in the Morris water maze test as

we described previously (Baydas et al., 2005b). Briefly, a circular galvanized

steel tank (120 cm diameter, 50 cm height) was filled to a depth of 30 cm with

water at 25 � 1 8C and the tank was divided virtually into four equal quadrants,

labeled N–S–E–W. The water was made opaque by the addition of semi-

skimmed milk. An escape platform (10 cm diameter) was placed in one of the

four maze quadrants (the target quadrant) and submerged 1.5 cm below the

water surface. For the spatial training, the platform was placed in a fixed

quadrant. The rats were required to find the platform using only distal spatial

cues available in the testing room. Cues were maintained constant throughout

testing. The rats were trained to locate and escape onto the platform for four

Fig. 1. Effects of chronic maternal hyperhomocysteinemia on the acquisition of

spatial learning of rats in the Morris water maze test (n = 8 per group). Mean

swimming times of the four trials per day for 5 days for each of the two groups are

shown. Escape latencies required by rats to find the hidden platform along five

consecutive days. Average latency time (days 3 and 5) to find hidden platform was

prolonged by hyperhomocysteinemia (ANOVA; P < 0.05 vs. control).

Fig. 2. Effects of chronic hyperhomocysteinemia on the mean percentage time

spent in the target quadrant in which the platform had previously been located

during acquisition (n = 8 per group). There is no significant difference between

the groups.

G. Baydas et al. / Int. J. Devl Neuroscience 25 (2007) 133–139 135

trials per day for five consecutive days. A different starting position was used on

each trial (in a quadrant not containing the platform). A trial began by placing

the rat into the water facing the wall of the pool at one of the starting points. The

animals were allowed to swim freely to find the hidden platform within 60 s and

after reaching the platform they were allowed to stay on it for 30 s and then

returned to the cage, which was warmed with a heating pad, to await its next

trial. There was a 30 s intertrial interval. If the rat had failed to escape, then the

rat was directed to the platform by the experimenter and allowed to remain there

for the same amount of time. Data collection was automated by a video tracking

system. Tracking was achieved by a system consisting of a video camera

mounted over the center of the pool. The tracker’s digitized coordinate values

were sampled in turn using a computer.

2.3.1. Probe trial

Twenty-four hours after the last training trial, a probe test was performed

wherein the extent of memory consolidation was assessed. The time spent in the

target quadrant indicates the degree of memory consolidation that has taken

place after learning. In the probe trial, the rat was placed into the pool as in the

training trial, except that the hidden platform was removed from the pool. The

time of crossing the former platform quadrant and the total time of crossing all

quadrants were recorded for 1 min.

Following the memory probe trial, a visible target task (cued trial) was

conducted using a raised platform of a contrasting color to confirm the tested

animals were not visually impaired and to test possible deficits in sensorimotor

processes (Baydas et al., 2003b). Latency times to reach the platform were

recorded for each trial.

2.4. Statistics

Data are reported as the mean � S.E. The data from spatial learning were

analyzed by two-way analysis of variance (ANOVA) for repeated measures. If a

main effect was found, post hoc analyses were done with the Tukey test.

Densitometric results of Western blotting and probe test were analyzed by

Mann–Whitney U test.

3. Results

Daily methionine administration via drinking water to the

pregnant rats significantly increased the plasma Hcy level

measured at the end of pregnancy (control: 6 mM/L and

Hcy group: 26 mM/L; P < 0.001). There was no significant

difference in the number of pups per litter between groups, as

follows (mean number per litter � S.E.): control 11.5 � 1.0 and

Hcy 10.2 � 0.6. The brain and body weights of pups from Hcy

group were slightly but not significantly lower than the control

values. During behavioral test, the mean weights of rats were as

follows: control 195 � 10 g and Hcy 188 � 8 g.

3.1. Morris water maze test

No significant differences occurred between male and female

rats, and the data were therefore pooled for subsequent analysis.

Although the latencies to reach the submerged platform

decreased gradually in both groups during the 5 days of training

in the Morris water maze test, the mean latency was significantly

prolonged in Hcy group compared to control group, indicating a

poorer learning performance due to maternal hyperhomocys-

teinemia (Fig. 1). Significant differences between groups were

observed in the time spent to find the platform on acquisition days

3 and 5. Hcy group showed longer swim latencies than those in

the control groups. ANOVA revealed a significant effect of Hcy

on escape latency (P < 0.05). During probe trials, all the rats

spent the most of the times searching in the quadrant where the

platform had been located (Fig. 2). Hcy offsprings showed no

significant impairment in spatial memory compared with the

controls. The control animals spent a slightly greater percentage

of swimming time in the target quadrant in which the platform

had previously been located compared to Hcy group, however

this greater percentage of time was statistically insignificant

(P > 0.05). Furthermore, the performances of control and Hcy

groups were similar in the trial with the visible platform

(latencies, 10.8 � 0.5 s in control and 11.0 � 0.5 s in Hcy

group), indicating that swimming motivation and ability were

similar and that the observed differences in spatial performance

were not due to sensorimotor disturbances. However, the number

of target crossings was significantly reduced in the rats from

hyperhomocysteinemic mother suggesting a spatial navigation

deficit (Fig. 3; P < 0.05).

Fig. 3. The mean number of platform region crossings from the hidden platform

(n = 8 per group). The number of the target crossings was significantly reduced

in the rats with chronic hyperhomocysteinemia (Mann–Whitney U test;

P < 0.05 vs. control group).

Fig. 5. The levels of S100B in total brain of PND1 pups from control and

hyperhomocysteinemic dams. Brain homogenates were electrophoresed, blotted,

and stained with S100B monoclonal antibody. A representative Western blot of

S100B protein is shown in (A). Computerized densitometry was performed and

the averages (�S.E.) for the two groups (n = 15 per group) are shown in (B). A

significant reduction in S100B contents was observed in the Hcy group of PND1

pups compared to the control pups (Mann–Whitney U test P < 0.001).

G. Baydas et al. / Int. J. Devl Neuroscience 25 (2007) 133–139136

3.2. Immunoblots

GFAP, the predominant intermediate filament protein of

mature astrocytes, exhibited decreased abundance in Hcy pups

on PND1 relative to the control pups (Fig. 4). The main band of

GFAP protein (49 kDa) is expressed in very low levels in Hcy

pups on PND1 (Fig. 4; P < 0.001). Similar results were also

found in the expression levels of S100B protein. The amount of

this protein was less in Hcy pups compared to the control values

on PND1 (Fig. 5). Western blotting of total brain homogenates

from pups of control and maternally hyperhomocysteinemic

rats revealed differences in the expression of NCAM. On

PND1, a decreased PSA-NCAM expression was observed in

Fig. 4. Effects of maternally induced hyperhomocysteinemia on the level of

GFAP in total brain of pups (n = 15 per group). Hyperhomocysteinemia was

induced by giving daily methionine (1 g/kg body weight) dissolved in drinking

water. Supplementation with methionine was continued throughout their preg-

nancies. Brain homogenates were electrophoresed, blotted, and stained with

GFAP polyclonal antibody. A representative Western blot of GFAP protein is

shown in (A). Computerized densitometry was performed and the averages

(�S.E.) for the two groups (control and Hcy) are shown in (B). A significant

reduction in GFAP contents was observed in the Hcy group of PND1 pups

compared to the control pups (Mann–Whitney U test; P < 0.001).

whole homogenates from Hcy pups compared to the control

values (Fig. 6; P < 0.001). On PND82, the amounts of whole

brain PSA-NCAM in both control and treated animals were

below the level of immunodetection. Thus, on PND82, the

amount of PSA-NCAM was measured by immunoblotting in

hippocampus. Densitometric comparison of immunoblots from

control and treated rats showed a non-significant decrease in the

level of PSA-NCAM expression in the hippocampal homo-

genates from Hcy rats (Fig. 7; P < 0.05).

Densitometric comparison of NCAM blots from Hcy group

showed a significant reduction in NCAM-140 expression in

whole homogenates of Hcy pups (Fig. 8; P < 0.001). No gender

differences in the expression of PSA-NCAM or other NCAM

isoforms were observed in control or Hcy rat pups at any time-

point. On PND82, while the level of NCAM-180 was markedly

decreased in the hippocampus of Hcy group (P < 0.001),

NCAM-120 and NCAM-140 isoforms appeared unaffected

(Fig. 9).

Fig. 6. Mean amount of PSA-NCAM in total brain of PND1 pups (n = 15 per

group) from the control and hyperhomocysteinemic mothers. Mouse mono-

clonal antibody against PSA-NCAM (diluted at 1:500) was used to stain blots.

PSA-NCAM expression was lower in Hcy group compared to the control values

(Mann–Whitney U test; P < 0.001).

Fig. 7. The expression of the PSA-NCAM in hippocampus from the young-

adult offsprings from control and Hcy groups (n = 8 per group). Representatvive

blot is shown in (A) and densitometric results are shown in (B).

Fig. 8. Western blot results of NCAM polypeptide in homogenates of total

brain (A) and densitometric analysis (B) of these polypeptide bands on Western

blot of pups (n = 15 per group). Rabbit polyclonal NCAM antibodies for NCAM

120, 140 and 180 isoform were used in this study. The expression of NCAM 140

isoform was significantly lower than control values (Mann–Whitney U test;

P < 0.001).

G. Baydas et al. / Int. J. Devl Neuroscience 25 (2007) 133–139 137

4. Discussion

Recently we have suggested that chronic hyperhomocys-

teinemia impaired cognitive performance of adult rats in a

Fig. 9. Representative Western blot (A) and densitometric analysis (B) of

NCAM (120, 180, and 140 kDa) from control and Hcy offsprings (n = 8 per

group). Homogenates were prepared from hippocampus. NCAM 180 levels

were significantly lower in young-adult offsprings from hyperhomocysteinemic

rat dams compared to the control group (Mann–Whitney U test; P < 0.001).

water maze task and the retention of long-term memory in a

passive avoidance task. Furthermore, we have suggested that

Hcy in high levels can impair the ability of spatial navigation

(Baydas et al., 2005a). However, to our best knowledge, there is

no study evaluating effects of maternal hyperhomocysteinemia

on the cognitive behaviors in the offspring.

We postulated that chronic maternal hyperhomocysteinemia

disrupts fetal brain development and therefore cognitive

functions. In order to test this hypothesis, we developed a

hyperhomocysteinemic rat dam model. It is well known that

administration of methionine significantly increase plasma Hcy

levels (McAuley et al., 1999; Labinjoh et al., 2001; Bagi et al.,

2003; Hidiroglou et al., 2004; Baydas et al., 2005a).

Furthermore, it has been reported that increased maternal

plasma homocysteine significantly correlate with their infants

(Bohles et al., 1999; Molloy et al., 2002). Thus, maternal

hyperhomocysteinemia leads an increase in the fetal Hcy

concentration. Hcy can be produced in the brain itself from

methionine metabolism or increased plasma Hcy can be

transferred from blood brain barrier to the brain tissue via a

specific saturable receptor in addition to simple diffusion

(Grieve et al., 1992; Griffiths et al., 1992). Streck et al. (2002)

have suggested that maximal concentration of homocysteine in

cerebrum can be achieved 15 min after the injection of

homocysteine. Therefore, administration of methionine to the

pregnant rat significantly increase plasma Hcy concentration in

feutus leading to an increase of fetal brain Hcy concentration.

Recently, Rees et al. (2006) have reported that excess

methionine in the diet indirectly influence fetal development

through the production of homocysteine.

We demonstrated here for the first time that there is an

association between maternal hyperhomocysteinemia and the

learning and memory retrieval deficits in their young-adult

offsprings. Significant differences between groups were

observed in the time spent to find the platform on acquisition

days 3 and 5. Hcy group showed a longer swim latency than that

in the control group. The learning deficits in the Hcy offsprings

appear to result from impairments in spatial learning, not from

swimming ability or visual acuity because there was no

significant difference between the groups in latency to reach

visible platform. Deficits in spatial learning in the Hcy

offsprings may be due to developmental delays in brain

maturation and neurogenesis during gestation. Therefore, we

determined the levels of glial intermediate filaments and

NCAM expression in the whole brain from PND1 pups.

GFAP is the major intermediate filament protein of mature

astrocytes. One of the key events during astrocytes differentia-

tion is the onset of GFAP expression (McCall et al., 1996).

Immature astrocytes initially express vimentin, switching to

GFAP as they mature (Dahl, 1981; Bovolenta et al., 1984).

Therefore, GFAP is recognized as an astrocyte maturation

marker (Gomes et al., 1999). It is also demonstrated that glial

S100B is involved in neuronal differentiation and maturation

(Yang et al., 1996). Herein we show that the levels of these two

proteins (GFAP and S100B) were markedly low in the Hcy pups

on PND1 compared to the control values. Impaired astrocytic

maturation of the fetal brain seems to be due to maternal

G. Baydas et al. / Int. J. Devl Neuroscience 25 (2007) 133–139138

hyperhomocysteinemia. It is reported that GFAP is involved

neuronal–glial interaction (McCall et al., 1996). Thus,

alterations in the levels of GFAP may result in disturbed

neuron–neuron and neuron–glia connectivity.

Development of the nervous system is a complex process.

Evidence suggests that NCAM participate in several aspects

of neural development (Edelman and Crossin, 1991). NCAM

mediates cell–cell interaction, modulates developmental pro-

cesses including neuronal migration and neurite extension, and

participates in neuronal regeneration and hippocampal neuro-

genesis in adult brain (Seki and Arai, 1993; Walsh and Doherty,

1997). One of the latest maturational events in the brain is the

alteration in the pattern of expression of the PSA-NCAM. This

immature form of NCAM plays a critical role in axonal growth

and fasciculation during brain development (Doherty et al.,

1990). In the present study, although we found that PSA-

NCAM was highly expressed in the whole brain homogenates

from the PND1 pups, it was undetectable in the whole brains of

mature rats. However, on the PND82, PSA-NCAM was

detected in hippocampal homogenates. In the adults, PSA-

NCAM remains expressed in the regions exhibiting a robust

synaptic plasticity such as hippocampus, which is well

known to retain a high degree of plasticity in adult rat

(Hoffman et al., 2001).

In the present study, the expression pattern of NCAM

isoforms in adult rats was different from that in the PND1 pups.

On PND1, the level of NCAM 180 was higher than that of other

isoforms, whereas the level of NCAM 120 was higher in mature

brain. This is in accordance with the previous findings that

NCAM 120 is the latest isoform to appear during development

(Chuong and Edelman, 1984; Nagata and Schachner, 1986).

There seems to be an association between maternal hyperho-

mocysteinemia and the decrease in the expression of PSA-

NCAM both in the whole brain from the PND1 pups and in the

hippocampus from the young-adult offsprings. The addition of

PSA to NCAM polypeptides seems to be a critical functional

feature for NCAM-mediated cell–cell interaction and function.

High expression of PSA-NCAM during early development

appears to play a permissive role, allowing structural remo-

deling by decreasing cell adhesion mediated by NCAM and

thereby facilitating the guidance and targeting of axons and

migration of neuronal and glial precursors (Rutishauser, 1996;

Minana et al., 2000). Decreased PSA-NCAM expression in

the developing brain might create a suitable environment to

reduce the rate of axonal growth at the target, making NCAM

available in its highly adhesive state to modulate glial–neuronal

interaction-dependent processes (Minana et al., 2000). Thus,

maternal hyperhomocysteinemia seems to impair the expected

increase in NCAM polysialylation, possibly leading to

persisting neuroplastic deficits, which may cause impairments

of learning and memory performance.

In the present study, there was an imbalance in the expression

of NCAM isoforms with a significant decrease in the expression

of NCAM 140 in the brains of pups from the hyperhomocys-

teinemic mothers on PND1. However, NCAM 180 isoform was

expressed less in the hippocampus from the offsprings of Hcy

group on PND82 compared to the control ones. NCAM 140 is

expressed on both pre- and post-synaptic membranes and plays a

pivotal role in cell–cell adhesion and neurite outgrowth

(Schachner, 1997). Therefore, we have proposed that the

reduction in NCAM 140 expression might be implicated in

the structural alterations of brain during development. However,

the causal involvement still needs to be established.

The fact that NCAM 180 is the isoform particularly affected in

the hippocampus of offspring from mother with hyperhomo-

cysteinemia supports a key role for this isoform on cognitive

processing-associated synaptic plasticity in this brain region.

NCAM 180 has been suggested to be an essential determinant in

synaptic plasticity and to be critically implicated in the

stabilization of synaptic strength (Schuster et al., 1998; Dityatev

et al., 2000). Given the importance of NCAM 180 in synaptic

function, the decreased level of NCAM 180 expression in the

hippocampus of the offspring from hyperhomocysteinemic dams

could lead to a synaptic destabilization in neural circuits relevant

to the storage of information.

In conclusion, present results suggest that maternal hyperho-

mocysteinemia results in the learning deficits in offspring due to

delayed brain maturation and altered expression pattern of

NCAM.

Acknowledgement

This work was supported by the Firat University Research

Foundation (FUBAP-1148).

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