BACKGROUND
iodine is an essential trace element and critical for the synthesis of triiodothyronine  and thyroxine .
thereby, iodine deficiency  is the most common cause of hypothyroidism worldwide particularly in the developing countries  <cit> .
in the process of early growth and development of most organs, hypothyroidism results in impaired brain development, cognitive deficits, impaired memory, impaired cognitive function and inattentiveness, and mental retardation in offspring  <cit> .
during these developmental periods, the mother is the only source of iodine for the fetus  <cit> .
not until the day of birth is the offspring able to make sufficient levels of its own thyroid hormone.
in line with this conclusion, recent researches show that children born to mothers experiencing subclinical perturbations of the thyroid axis during pregnancy have lower iq scores and subtle deficits in cognition and memory  <cit> .
importantly, on one hand, id induces irreversible developmental alterations in the fetuses and children central nervous system   <cit> .
on the other hand, id is one of the greatest preventable cause of mental retardation and an important public-health problem  <cit> .
this has been proven true in china where, after complying with universal salt iodization  in  <dig>  the id disorders have been significantly reduced  <cit> .
however, we recently reported that  <dig> of all  <dig> chinese provinces or  <dig> counties with a population of  <dig>  billion suffered from id  <cit> .
the hippocampus, a major component of the brain of humans and other mammals, belongs to the limbic system and plays important roles in long-term memory, learning processes and spatial navigation.
the structural integrity of the hippocampus is dependent on sufficient thyroid hormone during development  <cit> .
the classical genomic molecular mechanism of thyroid hormone is well understood  <cit> .
after the uptake of t <dig> or t <dig> by target cells, t <dig> gains access to the cell nucleus and binds to nuclear thyroid hormone receptor   <cit> .
in the nucleus, the facilitate binding of heterodimeric of tr and retinoic acid x receptor to thyroid hormone response elements regulates the consequent gene transcription through the action of co-repressors and co-activators  <cit> .
several lines of evidence imply that mitogen-activated protein kinases  mediate multiple cellular processes during normal brain development including gene expression, migration or trafficking, metabolism, differentiation, proliferation and apoptosis  <cit> .
the mapks are also called extracellular signal-regulated kinases   <cit> , which convey signals from cell surface receptors to the nucleus.
this process is important in triggering the genomic response in neurons, and integrates signals from other transduction pathways  <cit> .
it has been reported that erk inhibition in the hippocampus led to disruption of spatial memory  <cit> .
this is further supported by a recent study from alzoubi and colleagues  <cit> , showing that late long-term potentiation  depends on new protein synthesis through kinases-induced activation of camp-mapk-creb signaling pathway, leading to alteration of synaptic structure.
ltp is a well-accepted synaptic model of learning and memory  <cit>  and thyroid hormone may play an indirect role in ltp by affecting mapks independent of nuclear thyroid receptors  <cit> .
firstly, thyroid hormone activates g-protein-coupled receptors, which activates erk1/ <dig>  leading to creb phosphorylation and camp response element  transcription  <cit> .
it has been reported that mapk/erk activation is part of the non-genomic action of thyroid hormone  <cit> .
mapk signal transduction cascade is activated by t <dig> and a plasma membrane receptor on integrin αvβ <dig> via phospholipase c and protein kinase cα.
the activated mapk can translocate to the nucleus to phosphorylate nuclear thyroid hormone receptor trβ <dig>  step de-repress tr and modulate intracellular protein trafficking of tr from cytoplasm to nucleus  <cit> .
furthermore, thyroid hormone has also been shown to regulate the expression and phosphorylation of erk1/ <dig> and creb.
phosphorylation of erk1/ <dig> and creb, in turn, causes important downstream effects and regulates the expression of a variety of proteins, such as immediate early genes, which are important in memory  <cit> .
therefore, it is not surprising that erk1/ <dig> and creb play a critical role in ltp impairment following hypothyroidism  <cit> .
however, little is know about how id resulting in hypothyroidism regulates developmental hippocampus during lactational and adolescent period.
it is widely accepted that neocorticogenesis begins at about embryonic day  <dig>  and the postnatal development and maturation of the cns persist for the lactation  and adolescence  in rat  <cit> .
so, transition from gestation to adolescent period is critical for cns development and maturation.
in adult rats, it has been shown that, thyroid hormones reduction by perchlorate irreversibly impairs synaptic transmission  <cit> , where the restored thyroid hormone can not recover the developmental cns impairments.
in line with this study, our group has also previously shown in adult rats, developmental id and hypothyroidism impairs ltp in ca <dig> region  <cit> .
in contrast to many researches on adult animals exposed to developmental thyroid hormone insufficiency, there are very few experimental studies available to evaluate the alterations in early developmental period, following developmental id and hypothyroidism.
so far, it is still unclear whether id and hypothyroidism have similar effects on erk1/ <dig>  creb and their phosphorylated fraction in hippocampal subregions in lactational and adolescent animals.
in the present study, we shown that, in lactational and adolescent rats, hippocampal erk1/ <dig> and creb are implicated in the brain impairment by developmental exposure to id and  <dig> or  <dig> propylthiouracil .
methods
animals
wistar rats  were obtained from the center for experimental animals at china medical university  with a national animal use license number of scxk-ln 2003- <dig>  all experiments and surgical procedures were approved by the animal care and use committee at china medical university, which complies with the national institutes of health guide for the care and use of laboratory animals.
all efforts were made to minimize the number of animals used and their suffering.
rats were housed under the following standard conditions temperature  <dig> ± 1°c and 12/ <dig> h light/dark cycles food and water was provided ad libitum.
animals were kept for  <dig> week before mating .
the day of the vaginal plug was taken as gestational day   <dig>  the pregnant rats were randomly assigned into four groups : control group,  <dig> ppm ptu-treatment group,  <dig> ppm ptu-treatment group and iodine-deficient group.
control group received tap water and normal diet  during the experiment.
iodine-deficient group was administered with iodine-deficient diet  and tap water from gd <dig> till pn <dig>  ptu-treated groups were administered  <dig> ppm and  <dig> ppm ptu  in the drinking water and fed with normal diet  from gd <dig> to pn <dig>  the animal diet is made up of corn , rice , soybean , calcium carbonate , and sodium chloride .
for iodine-deficient diet, corn, rice, and soybean were obtained from the severe iodine-deficient area.
on gd <dig>  the control group gave birth to 12- <dig> pups per litter.
in contrast, 8- <dig> pups were born per litter in the treated groups.
each litter was culled to nine-ten pups on pn <dig> .
pups were weaned and each cage housed only two pups on pn <dig>  pup weights were recorded from pn <dig> to pn <dig>  eye opening was examined by daily observation between pn <dig> and pn <dig>  the percent of pups per litter with both eyes open was calculated accordingly.
prior to the sacrifice on pn <dig>  pn <dig> , pn <dig> and pn <dig> , the same number of pups were randomly taken from different litters with a similar sex ratio in each group and weighed.
thyroid hormones
thyroid hormone concentrations were determined via blood sampling collected from heart puncture of 8- <dig> pups in each group on pn <dig>  pn <dig>  pn <dig> and pn <dig>  briefly, after the pup was anesthetized using 99% ether and laid on its back, a 25-gauge needle attached to a  <dig> ml syringe was introduced at 10-30° from the horizontal axis of the sternum into the thoracic cavity to collect heart blood.
blood samples were centrifuged at  <dig>  g for  <dig> min.
serum was then separated and stored at -70°c.
all the serum was assayed by super-sensitive chemiluminescence immunoassay  to measure thyroid stimulating hormone , free triiodothyronine  and free thyroxine .
hormones were measured in  <dig> μl aliquots.
all samples were run in duplicate and the intra- and inter-assay variations were below 10%.
the sensitivity of detection for tsh was  <dig> miu/l, ft <dig> was  <dig> - <dig> pmol/l, and ft <dig> was  <dig> - <dig>  pmol/l.
results below these limits of quantification were recorded as  <dig> miu/l ,  <dig>  pmol/l  and  <dig>  pmol/l , respectively for statistical purposes.
nissl staining
on pn <dig>  pn <dig>  pn <dig>  and pn <dig>   <dig> pups in each group under deep anesthesia were intracardiac perfused with 50- <dig> ml normal saline containing  <dig> % heparin followed by 200- <dig> ml 4% paraformaldehyde in  <dig>  m potassium phosphate buffer .
the fixed brains were embedded in paraffin and sectioned into 6-μm-thick coronal sections on a microtome.
every third serial section was collected on gelatin-coated microscope slides.
after deparaffinization in xylene for  <dig> min followed by 100% ethanol, the slides were washed in deionized water.
then, the slides were performed with routine nissl staining based on the thionine technique and then analyzed under a microscope.
the hippocampal subregions of interest were selected: ca <dig>  ca <dig>  and dentate gyrus .
all images were obtained under the same conditions of light illumination, at a magnification of 400×, with the microscope light source stabilized.
for each group, quantitative data were acquired from the hippocampus on both sides of the brain.
cells with round and palely stained nuclei were considered to be surviving cells, whereas shrunken neurons with pyknotic nuclei were considered to be non-surviving cells.
every fifth brain section was selected from each animal and processed for cell counting in order to obtain an overall mean value for subsequent statistical analysis.
data were expressed as the number of surviving cells per field.
the experimenter was blind to the experimental treatment of the individual animals during all data measurements.
western blot
on pn <dig>  pn <dig>  pn <dig> and pn <dig>   <dig> pups in each group, including males and females, were deeply anesthetized and euthanized by ether.
brains were removed and kept in an ice-cold artificial cerebrospinal fluid  composed in milimolar :  <dig> mm nacl,  <dig> mm kcl,  <dig> mm cacl,  <dig> mm mgso <dig>   <dig>  mm nah2po <dig>   <dig> mm nahco <dig>  and  <dig> mm glucose.
according to the paxions and wastson atlas of the rat brain  <cit> , the ca <dig>  ca <dig> and dg regions of the hippocampus were immediately dissected out on ice and frozen at -70°c.
tissue samples were homogenized in  <dig> μl of buffered isotonic cocktail containing protease and phosphatase inhibitors .
the samples were sonicated and incubated on ice for  <dig> min, and centrifuged at  <dig>  g for  <dig> min at 4°c.
the supernatants were centrifuged again and then removed.
the total protein was estimated using coomassie brilliant blue assay.
the samples were stored at -70°c until use.
tissue lysates were diluted in sample buffer , 50% glycerol, 10% sds, 25% β-mercaptoethanol, and  <dig> % bromophenol blue) to contain the same concentration of protein  and were then boiled at 100°c for  <dig> min.
<dig> μl aliquots of each sample  were loaded onto 10% sds-acrylamide gels.
proteins were separated by the application of a constant voltage of  <dig> v for  <dig>  h and then transferred onto nitrocellulose membranes at a constant voltage of  <dig> v for  <dig> min.
after blocking the aspecific sites with pbs containing  <dig> % tween  <dig>  and 5% defatted dried milk, membranes were washed and incubated with rabbit anti-phospho-creb monoclonal antibody  for  <dig> h at room temperature.
rabbit polyclonal antibody for glyceraldehyde phosphodehydrogenase  was used as a loading control.
the ratio of protein bands intensity to gapdh band intensity was compared among the different groups.
membranes were washed and incubated in a horseradish peroxidase-conjugated anti-rabbit igg  for  <dig> h at room temperature before reaction with enhanced chemiluminescence  solution .
initial control experiments determined the optimal time of exposure to film, which was maintained throughout the experimental procedure.
membranes were exposed to film optimal time, and developed by hand.
after developed and fixed, film was scanned.
protein bands were quantified with an image-analysis program  and recorded with net optical density  corrected for background chemiluminescence.
for all western blots, on each gel gapdh lanes were reserved for a quality control sample.
the signals from target bands on a gel were normalized to the average signal for the quality control sample bands to simplify comparison across gels and reduce inter-gel variability.
membranes were washed in restore western blotting stripping buffer  for  <dig> min at room temperature after all the steps, and then washed in pbst.
following the operating instructions, we incubated the membrane with new supersignal west working solution and exposed it to film.
if no signal was detected using a 5-min exposure, the hrp conjugate was successfully removed from the antigen or primary antibody.
the membranes were stripped and re-incubated in rabbit anti-phospho-erk1/ <dig> monoclonal antibody, rabbit anti-creb monoclonal antibody, and rabbit anti-erk1/ <dig> monoclonal antibody  for  <dig> h at room temperature in turn.
after incubation with primary antibodies, the membranes were washed and incubated in the horseradish peroxidase-conjugated anti-rabbit igg  for  <dig> h at room temperature before reaction with ecl solution respectively.
following the films were developed, fixed, and scanned, the protein band intensities were quantified with an image-analysis program.
statistics
results were expressed as mean ± sem.
all analyses were carried out using the spss  <dig>  software.
for western blots, all of the values were normalized with gapdh, which served as the internal control.
the target band intensities in the treatment groups were normalized to those of the control group.
the data differences among the multiple groups were analyzed using one-way analysis of variance .
if f was significant, tukey test was also used.
p-values of less than  <dig>  were considered statistically significant.
RESULTS
animal models
numerous studies have shown that ptu treatment reduces offspring body weight  <cit> .
our data shown that the offspring's body weights in iodine-deficient,  <dig> ppm and  <dig> ppm ptu-treatment groups were statistically significant lower than the controls from pn <dig> to pn <dig> .
eye opening is a parameter that reflects the early physiological development of rats.
pups open their eyes normally between pn <dig> and pn <dig>  <cit> .
id and  <dig> ppm ptu-treatment delayed the pup eye opening to pn <dig>  further, comparing with the controls, the iodine-deficient and  <dig> ppm ptu-treated pups had a significantly lower eye opening percentage with both eye open per litter on pn <dig> and pn <dig> .
dose-dependent reductions in thyroid hormones with concomitant elevations in tsh were observed in hypothyroid offspring by many researchers  <cit> .
interestingly and in line with this statement, our data showed that the offspring displayed hypothyroxinemia in iodine-deficient group  and hypothyroidism in  <dig> ppm and  <dig> ppm ptu-treatment groups.
tsh levels were increased significantly in  <dig> ppm and  <dig> ppm ptu-treatment offspring vs controls .
the iodine-deficient and  <dig> ppm ptu-treatment groups had significantly lower serum ft <dig> and ft <dig> than the controls on pn <dig>  pn <dig> and pn <dig> .
on pn <dig>  the concentrations of serum ft <dig>  ft <dig> and tsh in iodine-deficient and ptu-treatment groups were restored.
taken together, our results demonstrate that the iodine-deficient and  <dig> ppm ptu-treatment caused pup id and hypothyroidism with developmental delay.
the pup weights of iodine-deficient,  <dig> ppm and  <dig> ppm ptu-treatment groups were significantly lower than controls from pn <dig> to pn <dig>  each values represents mean ± sem.
*p <  <dig> , as compared to control group.
#p <  <dig> , as compared to  <dig> ppm ptu-treatment group.
Δp <  <dig> , as compared to  <dig> ppm ptu-treatment group.
the circulating levels of tsh were increased in ptu-induced hypothyroidism pups, while there were no significant differences in iodine-deficient pups on pn <dig>  pn <dig> and pn <dig>  id and ptu-induced hypothyroidism decreased the serum ft <dig> and ft <dig> levels on pn <dig>  pn <dig> and pn <dig>  on pn <dig>  the concentrations of serum ft <dig>  ft <dig> and tsh in iodine-deficient and ptu-treatment groups were restored gradually.
each values represents mean ± sem .
*p <  <dig> , as compared to control group.
#p <  <dig> , as compared to  <dig> ppm ptu-treatment group.
Δp <  <dig> , as compared to  <dig> ppm ptu-treatment group.
id and hypothyroidism increase hippocampal neuronal loss
to investigate whether or not hippocampal neuronal survival is impaired by id and hypothyroidism, histological examination of hippocampal neurons was performed on nissl-stained sections.
the results reveal greater nuclear breakdown in the hippocampal neurons of offspring with low circulating thyroid hormone levels, in the ca <dig> , ca <dig> , and dg  regions on pn <dig>  pn <dig>  pn <dig>  and pn <dig>  the mean number of surviving cells in the hippocampus of the iodine-deficient and  <dig> ppm ptu-treated rats was significantly reduced compared to controls .
these findings suggest that id and hypothyroidism led to morphological damage in the hippocampus.
assessment of the simple effects of group showed that neuronal loss was increased at each time point in the hippocampus of rats exposed to the iodine-deficient or ptu-adulterated diet.
id and hypothyroidism reduce t-erk1/ <dig> and p-erk1/2
regulated by thyroid hormone and the role that they play in the hippocampus, erk1/ <dig> are important in the generation of learning and memory  <cit> .
in the present study, we detected t-erk1/ <dig> and p-erk1/ <dig> changes in the pups following developmental id and hypothyroidism using western blot approach.
both t-erk1/ <dig> and p-erk1/ <dig> were measured in ca <dig>  ca <dig> and dg regions on pn <dig>  pn <dig>  pn <dig> and pn <dig> .
in ca <dig> and ca <dig> regions of the hippocampus, id and hypothyroidism significantly reduced t-erk <dig>  or t-erk <dig>  .
p-erk <dig> and p-erk <dig> were significantly lower on pn <dig>  pn <dig> and pn <dig> .
on the other hand, p-erk1/ <dig> was hardly detected on pn <dig>  this might be due to lower t-erk in early postnatal period in pups, and thus p-erk1/ <dig> signal becomes too weak to capture.
in the dg region, however, id and hypothyroidism did not change t-erk1/ <dig> or p-erk1/ <dig> expression.
id and hypothyroidism reduce t-creb and p-creb
as a downstream target molecule of erk1/ <dig>  creb plays a critical role in the generation of protein synthesis-dependent long-term changes in the brain  <cit>  and is necessary for the fear-associated memory  <cit> .
in order to investigate the effects of id and hypothyroidism on creb, t-creb and p-creb were detected via western blot.
in the present study, t-creb and p-creb were clearly expressed in ca <dig>  ca <dig> and dg regions on pn <dig>  pn <dig>  pn <dig> and pn <dig> .
however, the signals of p-creb were very weak on pn <dig>  id and hypothyroidism significantly reduced both t-creb  and p-creb  in ca <dig>  ca <dig> and dg regions .
discussion
the major findings of this study are that, in lactation and adolescent stage of development rats, developmental id and hypothyroidism  significantly reduced the mean number of surviving cells in hippocampus and decreased erk1/ <dig> and creb expression in hippocampal ca <dig> and ca3; even after the thyroid hormones back to normal, surviving cells, erk1/ <dig> and creb were still lower than the controls.
the present study demonstrates that developmental id and hypothyroidism down-regulate hippocampal erk1/ <dig> and creb in lactational and adolescent rats.
our previous study has shown that id was still a serious public health problem in china  <cit> .
given so many chinese children exposed to developmental id, this study sought to make three lactational and adolescent animal models to mimic the developmental exposure to id and hypothyroidism.
many lines of literature using adult animal models have demonstrated that developmental hypothyroidism alters synaptic function in the hippocampus  <cit> .
in agreement with these studies, the lactational and adolescent rats in this study have an irreversible impairment in hippocampal erk1/ <dig> and creb following developmental exposure to id and hypothyroidism.
this is because that, during these developmental periods, the mother is the only source of iodine for the fetus and neonate  <cit> .
therefore, maternal id leads to maternal and filial hypothyroxinemia.
taking together, developmental periods during pregnancy and lactation are critical in the brain development.
thyroid hormone is well known to regulate morphological and biochemical alterations in brain during critical periods of brain development.
this regulation can be done by genomic, posttranslational, and direct actions on neural substrates.
therefore, thyroid hormone is a fundamental factor that regulates normal development of the cns.
logically, thyroid hormone insufficiency during early brain development is detrimental to synaptic transmission in hippocampus and to a cellular and behavioral model of learning and memory  <cit> .
in need, our previous study has clearly demonstrated that id-exposure lowers the children iqs in china  <cit> .
experimentally, we have also shown the id can lead to an inhibition of ltp in the rat ca <dig> area  <cit> .
it is well known that mapk mediates neuronal metabolism, differentiation and proliferation  <cit> .
also, the persisting alteration in developmental brain involves changes in myelination and migration patterns of neurons  <cit> .
the present study further confirmed the hypothesis that developmental id and hypothyroidism induce irreversible impairment of two key proteins expression in the ca <dig> and ca <dig> regions, where both erk1/ <dig> and creb expression was significantly reduced in either total protein level or phorsphorylated fraction.
in agreement with our findings, gilbert and colleagues observed the irreversibly cns impairments in rats induced by developmental hypothyroidism from gd <dig> to pn <dig>  <cit> .
this study provides us a clue to explore the mechanism by which thyroid hormone regulates brain development during lactational and adolescent periods.
as a member of mapk, erk1/ <dig> is a critical memory-related protein in the generation of learning and memory.
thyroid hormone activates g-protein-coupled receptors, leads to erk1/ <dig> phosphorylation, and in turn enhances the transcription of some proteins that are important in memory  <cit> .
it has been shown that hypothyroidism reduces the basal p-erk1/ <dig> in hippocampal ca <dig> region without affecting t-erk1/ <dig>  <cit> .
in the dg region, however, hypothyroidism had no effect on t-erk1/ <dig> and p-erk1/ <dig>  <cit> .
in the lactational and adolescent rats, here we show that t-erk1/ <dig> and p-erk1/ <dig> in iodine-deficient,  <dig> ppm ptu-treatment and  <dig> ppm ptu-treatment groups in ca <dig> and ca <dig> regions were significantly lower than controls; while there was no difference in dg region.
this difference might be due to the different cell types of neurons in the different regions.
in the dg region, the granule cells are more resistant to conditioned stimulus than the pyramidal cells  <cit> .
this is in line with the importance of the ca <dig> and ca <dig> in the learning and memory generation.
recently, it is reported that mapk genes are the direct targets of thyroid hormone  <cit>  and mapk activation is part of the non-genomic action of thyroid hormone  <cit> .
unlike the t3-dependent genomic mechanism, the non-genomic pathway involves t <dig> not t <dig>  in present study, id and hypothyroidism caused low t <dig> concentration in the lactational and adolescent rats.
it is reported that in ptu treated pups, serum t <dig> concentration is negatively correlated with cerebrocortical type ii 5'-deiodinase  activity, a sensitive marker of tissue thyroid status and an indicator of brain's compensatory response to maintain cerebrocortical t <dig>  <cit> .
since t <dig> is correlated with synaptic response, in the present study, we speculate that the reduced serum ft <dig> may increase d <dig> activity, decrease cerebrocortical t <dig> concentration, and lead to bound-tr reduction.
taken together, erk1/ <dig> might be down-regulated by developmental id and hypothyroidism via non-genomic pathway.
different from the present study, calloni et al.
reported that hypothyroidism promoted an increase in p-erk1/ <dig>  <cit> .
it might be attributed to difference of the method on establishing hypothyroid animal model or the concentration of medicine.
calloni et al.
administered pregnant rats with  <dig> % methimazole  in drinking water from gd <dig> to the birth  <cit> .
since the neocorticogenesis and maturation of the cns persist for the adolescence in rat  <cit> , the early developmental impairments may be minor and reversible.
therefore, the increased p-erk1/ <dig> might be ascribed to the recovered morphological and physiological changes because of the termination of administration at birth.
another key protein for the long-term memory processes is creb.
the activation of creb and creb-dependent transcriptional pathway is essential for memory consolidation  <cit> .
different from increased p-creb in thyroid hormone treated neural cells  <cit> , in the present study, developmental id and hypothyroidism significantly lowered both t-creb and p-creb in ca <dig>  ca <dig> and dg regions.
it has been proposed that p-creb decrease might attribute to protein reduction of t-creb, ca2+/camodulin dependent protein kinase iv , and adenylyl cyclase type i , or elevation of calcineurin  <cit> .
in addition, tr activation can antagonize creb-mediated transcription and inhibit the phosphorylation of creb  <cit> .
so far, the detailed mechanism by which developmental id and hypothyroidism regulate creb is still unclear.
since creb is a downstream target molecule of erk1/ <dig>  our present data imply that developmental id and hypothyroidism may induce p-erk1/ <dig> reduction, which in turn lead to p-creb reduction.
however, we can not exclude the involvement of genomic pathway, where thyroid hormone modulates the creb by regulating the transcriptions of camkiv.
moreover, the number and morphology of neuron in hippocampus are affected by neonatal and adult hypothyroidism  <cit> .
as a marker of neuronal development, nissl body diminishes when neuronal impairment.
we found here that the number of surviving neuronal cells in hippocampus in iodine-deficient and  <dig> ppm ptu-treatment groups were significantly lower than the controls.
importantly, the reduction of nissle bodies in the developmental id and hypothyroidism fits very well with the decreased protein production of erk1/ <dig> and creb.
this might be due to the decrease in cell number, and to a lower expression of the investigated genes in each neuron.
therefore, our observation on nissl bodies further confirmed the irreversible cns impairments following developmental id and hypothyroidism.
in summary, this study has shed some light on one aspect of the id/hypothyroidism-induced learning and memory impairment.
the following questions are still unanswered: how does thyroid hormone regulate erk and creb?
why is there more reduction of p-creb in dg region than p-erk1/2?
how do erk1/ <dig> and creb regulate ltp production?
is erk protein regulation due to a specific gene-expression modulation or are related to a general decrease in gene expression and/or protein synthesis following hypothyroidism?
further investigations are needed to answer these questions.
CONCLUSIONS
our previous study has confirmed the impairment of ltp induction following developmental id and hypothyroidism  <cit> .
the present study has further shown that, in lactational and adolescent rats, developmental id and hypothyroidism induce irreversible reduction of erk1/ <dig> and creb in hippocampal ca <dig> and ca <dig> regions.
in conclusion, erk1/ <dig> and creb may play an important role in id and hypothyroidism-induced brain impairment in lactational and adolescent rats.
abbreviations
aci: adenylyl cyclase type i; acsf: artificial cerebrospinal fluid; anova: analysis of variance; camkiv: ca2+/camodulin dependent protein kinase iv; cns: central nervous system; cre: camp response element; creb: camp response element-binding protein; d2: type ii 5'-deiodinase; dab:  <dig>  30-diaminobenzidine; ecl: chemiluminescence; erk: extracellular signal-regulated kinase;ft3: free triiodothyronine; ft4: free thyroxine; gapdh: glyceraldehyde phosphodehydrogenase; gd: gestational day; he: hematoxylin; id: iodine deficiency; iegs: immediate early genes; l-ltp: late long-term potentiation; ltp: long-term potentiation; mapks: mitogen-activated protein kinases; mek: mitogen extracellular regulating kinase; mmi: methimazole; nmda: n-methyl-d-asparticacid; nod: optical density; pbs: phosphate-buffered saline; pn: postnatal day; ptu: propylthiouracil; snk: student-newman-keuls; t3: triiodothyronine; t4: thyroxine; tr: thyroid hormone receptor; tsh: thyroid stimulating hormone.
competing interests
the authors declare that they have no competing interests.
authors' contributions
jd, wl and jc conceived of the study, and participated in its design and coordination.
jd drafted the manuscript.
jd, yw and yh carried out the experiments, collected and analyzed the data.
qx revised the manuscript.
all authors participated in writing, and read and approved the final manuscript.
supplementary material
additional file 1
id and hypothyroidism induced neuronal loss in ca <dig> region .
nissl staining was used to assess the numbers of surviving cells  and dead cells  in the hippocampus.
more dead pyramidal neurons were found in the ca <dig> region of the iodine-deficient and ptu-treated rats at pn <dig>  pn <dig> and pn <dig> compared to controls.
representative photomicrographs are shown for each time point.
scale bar represents  <dig> μm, shown on lower left in , and applies to all panels.
the nissl-positive survival cell number of iodine-deficient and  <dig> ppm ptu-treatment groups was significantly lower than controls .
each value represents mean ± sem.
significant differences from control group: *, p <  <dig> .
click here for file
additional file 2
id and hypothyroidism induced neuronal loss in ca <dig> region .
more dead pyramidal neurons were observed in the ca <dig> region of the iodine-deficient and  <dig> ppm ptu-treated rats on pn <dig> and pn <dig> compared to controls.
representative photomicrographs are shown for each time point.
scale bar represents  <dig> μm, shown on lower left in , and applies to all panels.
the nissl-positive survival cell number of iodine-deficient and  <dig> ppm ptu-treatment groups was significantly lower than controls .
each value represents mean ± sem.
significant differences from control group: *, p <  <dig> .
click here for file
additional file 3
id and hypothyroidism induced neuronal loss in dg region .
more dead granular cells were found in the dg region of the iodine-deficient and  <dig> ppm ptu-treated rats on pn <dig>  pn <dig>  and pn <dig> compared to controls.
representative photomicrographs are shown for each time point.
scale bar represents  <dig> μm, shown on lower left in , and applies to all panels.
the nissl-positive survival cell number of iodine-deficient and  <dig> ppm ptu-treatment groups was significantly lower than controls .
each value represents mean ± sem.
significant differences from control group: *, p <  <dig> .
click here for file
acknowledgements
this work was supported by the national natural science foundation committee of china .
the authors are very grateful for excellent theoretical and technical support from prof. mingqi gao in the college of clinical medical sciences, prof. hong lai in the college of basic medical sciences and prof. zhongyan shan in the endocrine research institute at china medical university, pedro san-cristobal md department of physiology, radboud university nijmegen medical centre, for critical reading the manuscript.

 iodine is an essential trace element and critical for the synthesis of triiodothyronine and thyroxine. thereby, iodine deficiency is the most common cause of hypothyroidism worldwide particularly in the developing countries. in the process of early growth and development of most organs, hypothyroidism results in impaired brain development, cognitive deficits, impaired memory, impaired cognitive function and inattentiveness, and mental retardation in offspring. during these developmental periods, the mother is the only source of iodine for the fetus. not until the day of birth is the offspring able to make sufficient levels of its own thyroid hormone. in line with this conclusion, recent researches show that children born to mothers experiencing subclinical perturbations of the thyroid axis during pregnancy have lower iq scores and subtle deficits in cognition and memory. importantly, on one hand, id induces irreversible developmental alterations in the fetuses and children central nervous system. on the other hand, id is one of the greatest preventable cause of mental retardation and an important public-health problem. this has been proven true in china where, after complying with universal salt iodization in the id disorders have been significantly reduced. however, we recently reported that of all chinese provinces or counties with a population of billion suffered from id. the hippocampus, a major component of the brain of humans and other mammals, belongs to the limbic system and plays important roles in long-term memory, learning processes and spatial navigation. the structural integrity of the hippocampus is dependent on sufficient thyroid hormone during development. the classical genomic molecular mechanism of thyroid hormone is well understood. after the uptake of t or t by target cells, t gains access to the cell nucleus and binds to nuclear thyroid hormone receptor. in the nucleus, the facilitate binding of heterodimeric of tr and retinoic acid x receptor to thyroid hormone response elements regulates the consequent gene transcription through the action of co-repressors and co-activators. several lines of evidence imply that mitogen-activated protein kinases mediate multiple cellular processes during normal brain development including gene expression, migration or trafficking, metabolism, differentiation, proliferation and apoptosis. the mapks are also called extracellular signal-regulated kinases, which convey signals from cell surface receptors to the nucleus. this process is important in triggering the genomic response in neurons, and integrates signals from other transduction pathways. it has been reported that erk inhibition in the hippocampus led to disruption of spatial memory. this is further supported by a recent study from alzoubi and colleagues, showing that late long-term potentiation depends on new protein synthesis through kinases-induced activation of camp-mapk-creb signaling pathway, leading to alteration of synaptic structure. ltp is a well-accepted synaptic model of learning and memory and thyroid hormone may play an indirect role in ltp by affecting mapks independent of nuclear thyroid receptors. firstly, thyroid hormone activates g-protein-coupled receptors, which activates erk1/ leading to creb phosphorylation and camp response element transcription. it has been reported that mapk/erk activation is part of the non-genomic action of thyroid hormone. mapk signal transduction cascade is activated by t and a plasma membrane receptor on integrin αvβ via phospholipase c and protein kinase cα. the activated mapk can translocate to the nucleus to phosphorylate nuclear thyroid hormone receptor trβ step de-repress tr and modulate intracellular protein trafficking of tr from cytoplasm to nucleus. furthermore, thyroid hormone has also been shown to regulate the expression and phosphorylation of erk1/ and creb. phosphorylation of erk1/ and creb, in turn, causes important downstream effects and regulates the expression of a variety of proteins, such as immediate early genes, which are important in memory. therefore, it is not surprising that erk1/ and creb play a critical role in ltp impairment following hypothyroidism. however, little is know about how id resulting in hypothyroidism regulates developmental hippocampus during lactational and adolescent period. it is widely accepted that neocorticogenesis begins at about embryonic day and the postnatal development and maturation of the cns persist for the lactation and adolescence in rat. so, transition from gestation to adolescent period is critical for cns development and maturation. in adult rats, it has been shown that, thyroid hormones reduction by perchlorate irreversibly impairs synaptic transmission, where the restored thyroid hormone can not recover the developmental cns impairments. in line with this study, our group has also previously shown in adult rats, developmental id and hypothyroidism impairs ltp in ca region. in contrast to many researches on adult animals exposed to developmental thyroid hormone insufficiency, there are very few experimental studies available to evaluate the alterations in early developmental period, following developmental id and hypothyroidism. so far, it is still unclear whether id and hypothyroidism have similar effects on erk1/ creb and their phosphorylated fraction in hippocampal subregions in lactational and adolescent animals. in the present study, we shown that, in lactational and adolescent rats, hippocampal erk1/ and creb are implicated in the brain impairment by developmental exposure to id and or propylthiouracil. methods animals wistar rats were obtained from the center for experimental animals at china medical university with a national animal use license number of scxk-ln 2003- all experiments and surgical procedures were approved by the animal care and use committee at china medical university, which complies with the national institutes of health guide for the care and use of laboratory animals. all efforts were made to minimize the number of animals used and their suffering. rats were housed under the following standard conditions temperature ± 1°c and 12/ h light/dark cycles food and water was provided ad libitum. animals were kept for week before mating. the day of the vaginal plug was taken as gestational day the pregnant rats were randomly assigned into four groups: control group, ppm ptu-treatment group, ppm ptu-treatment group and iodine-deficient group. control group received tap water and normal diet during the experiment. iodine-deficient group was administered with iodine-deficient diet and tap water from gd till pn ptu-treated groups were administered ppm and ppm ptu in the drinking water and fed with normal diet from gd to pn the animal diet is made up of corn, rice, soybean, calcium carbonate, and sodium chloride. for iodine-deficient diet, corn, rice, and soybean were obtained from the severe iodine-deficient area. on gd the control group gave birth to 12- pups per litter. in contrast, 8- pups were born per litter in the treated groups. each litter was culled to nine-ten pups on pn. pups were weaned and each cage housed only two pups on pn pup weights were recorded from pn to pn eye opening was examined by daily observation between pn and pn the percent of pups per litter with both eyes open was calculated accordingly. prior to the sacrifice on pn pn, pn and pn, the same number of pups were randomly taken from different litters with a similar sex ratio in each group and weighed. thyroid hormones thyroid hormone concentrations were determined via blood sampling collected from heart puncture of 8- pups in each group on pn pn pn and pn briefly, after the pup was anesthetized using 99% ether and laid on its back, a 25-gauge needle attached to a ml syringe was introduced at 10-30° from the horizontal axis of the sternum into the thoracic cavity to collect heart blood. blood samples were centrifuged at g for min. serum was then separated and stored at -70°c. all the serum was assayed by super-sensitive chemiluminescence immunoassay to measure thyroid stimulating hormone, free triiodothyronine and free thyroxine. hormones were measured in μl aliquots. all samples were run in duplicate and the intra- and inter-assay variations were below 10%. the sensitivity of detection for tsh was miu/l, ft was - pmol/l, and ft was - pmol/l. results below these limits of quantification were recorded as miu/l, pmol/l and pmol/l, respectively for statistical purposes. nissl staining on pn pn pn and pn pups in each group under deep anesthesia were intracardiac perfused with 50- ml normal saline containing % heparin followed by 200- ml 4% paraformaldehyde in m potassium phosphate buffer. the fixed brains were embedded in paraffin and sectioned into 6-μm-thick coronal sections on a microtome. every third serial section was collected on gelatin-coated microscope slides. after deparaffinization in xylene for min followed by 100% ethanol, the slides were washed in deionized water. then, the slides were performed with routine nissl staining based on the thionine technique and then analyzed under a microscope. the hippocampal subregions of interest were selected: ca ca and dentate gyrus. all images were obtained under the same conditions of light illumination, at a magnification of 400×, with the microscope light source stabilized. for each group, quantitative data were acquired from the hippocampus on both sides of the brain. cells with round and palely stained nuclei were considered to be surviving cells, whereas shrunken neurons with pyknotic nuclei were considered to be non-surviving cells. every fifth brain section was selected from each animal and processed for cell counting in order to obtain an overall mean value for subsequent statistical analysis. data were expressed as the number of surviving cells per field. the experimenter was blind to the experimental treatment of the individual animals during all data measurements. western blot on pn pn pn and pn pups in each group, including males and females, were deeply anesthetized and euthanized by ether. brains were removed and kept in an ice-cold artificial cerebrospinal fluid composed in milimolar: mm nacl, mm kcl, mm cacl, mm mgso mm nah2po mm nahco and mm glucose. according to the paxions and wastson atlas of the rat brain, the ca ca and dg regions of the hippocampus were immediately dissected out on ice and frozen at -70°c. tissue samples were homogenized in μl of buffered isotonic cocktail containing protease and phosphatase inhibitors. the samples were sonicated and incubated on ice for min, and centrifuged at g for min at 4°c. the supernatants were centrifuged again and then removed. the total protein was estimated using coomassie brilliant blue assay. the samples were stored at -70°c until use. tissue lysates were diluted in sample buffer, 50% glycerol, 10% sds, 25% β-mercaptoethanol, and % bromophenol blue) to contain the same concentration of protein and were then boiled at 100°c for min. μl aliquots of each sample were loaded onto 10% sds-acrylamide gels. proteins were separated by the application of a constant voltage of v for h and then transferred onto nitrocellulose membranes at a constant voltage of v for min. after blocking the aspecific sites with pbs containing % tween and 5% defatted dried milk, membranes were washed and incubated with rabbit anti-phospho-creb monoclonal antibody for h at room temperature. rabbit polyclonal antibody for glyceraldehyde phosphodehydrogenase was used as a loading control. the ratio of protein bands intensity to gapdh band intensity was compared among the different groups. membranes were washed and incubated in a horseradish peroxidase-conjugated anti-rabbit igg for h at room temperature before reaction with enhanced chemiluminescence solution. initial control experiments determined the optimal time of exposure to film, which was maintained throughout the experimental procedure. membranes were exposed to film optimal time, and developed by hand. after developed and fixed, film was scanned. protein bands were quantified with an image-analysis program and recorded with net optical density corrected for background chemiluminescence. for all western blots, on each gel gapdh lanes were reserved for a quality control sample. the signals from target bands on a gel were normalized to the average signal for the quality control sample bands to simplify comparison across gels and reduce inter-gel variability. membranes were washed in restore western blotting stripping buffer for min at room temperature after all the steps, and then washed in pbst. following the operating instructions, we incubated the membrane with new supersignal west working solution and exposed it to film. if no signal was detected using a 5-min exposure, the hrp conjugate was successfully removed from the antigen or primary antibody. the membranes were stripped and re-incubated in rabbit anti-phospho-erk1/ monoclonal antibody, rabbit anti-creb monoclonal antibody, and rabbit anti-erk1/ monoclonal antibody for h at room temperature in turn. after incubation with primary antibodies, the membranes were washed and incubated in the horseradish peroxidase-conjugated anti-rabbit igg for h at room temperature before reaction with ecl solution respectively. following the films were developed, fixed, and scanned, the protein band intensities were quantified with an image-analysis program. statistics results were expressed as mean ± sem. all analyses were carried out using the spss software. for western blots, all of the values were normalized with gapdh, which served as the internal control. the target band intensities in the treatment groups were normalized to those of the control group. the data differences among the multiple groups were analyzed using one-way analysis of variance. if f was significant, tukey test was also used. p-values of less than were considered statistically significant.  animal models numerous studies have shown that ptu treatment reduces offspring body weight. our data shown that the offspring's body weights in iodine-deficient, ppm and ppm ptu-treatment groups were statistically significant lower than the controls from pn to pn. eye opening is a parameter that reflects the early physiological development of rats. pups open their eyes normally between pn and pn. id and ppm ptu-treatment delayed the pup eye opening to pn further, comparing with the controls, the iodine-deficient and ppm ptu-treated pups had a significantly lower eye opening percentage with both eye open per litter on pn and pn. dose-dependent reductions in thyroid hormones with concomitant elevations in tsh were observed in hypothyroid offspring by many researchers. interestingly and in line with this statement, our data showed that the offspring displayed hypothyroxinemia in iodine-deficient group and hypothyroidism in ppm and ppm ptu-treatment groups. tsh levels were increased significantly in ppm and ppm ptu-treatment offspring vs controls. the iodine-deficient and ppm ptu-treatment groups had significantly lower serum ft and ft than the controls on pn pn and pn. on pn the concentrations of serum ft ft and tsh in iodine-deficient and ptu-treatment groups were restored. taken together, our results demonstrate that the iodine-deficient and ppm ptu-treatment caused pup id and hypothyroidism with developmental delay. the pup weights of iodine-deficient, ppm and ppm ptu-treatment groups were significantly lower than controls from pn to pn each values represents mean ± sem. *p <, as compared to control group. #p <, as compared to ppm ptu-treatment group. Δp <, as compared to ppm ptu-treatment group. the circulating levels of tsh were increased in ptu-induced hypothyroidism pups, while there were no significant differences in iodine-deficient pups on pn pn and pn id and ptu-induced hypothyroidism decreased the serum ft and ft levels on pn pn and pn on pn the concentrations of serum ft ft and tsh in iodine-deficient and ptu-treatment groups were restored gradually. each values represents mean ± sem. *p <, as compared to control group. #p <, as compared to ppm ptu-treatment group. Δp <, as compared to ppm ptu-treatment group. id and hypothyroidism increase hippocampal neuronal loss to investigate whether or not hippocampal neuronal survival is impaired by id and hypothyroidism, histological examination of hippocampal neurons was performed on nissl-stained sections. the results reveal greater nuclear breakdown in the hippocampal neurons of offspring with low circulating thyroid hormone levels, in the ca, ca, and dg regions on pn pn pn and pn the mean number of surviving cells in the hippocampus of the iodine-deficient and ppm ptu-treated rats was significantly reduced compared to controls. these findings suggest that id and hypothyroidism led to morphological damage in the hippocampus. assessment of the simple effects of group showed that neuronal loss was increased at each time point in the hippocampus of rats exposed to the iodine-deficient or ptu-adulterated diet. id and hypothyroidism reduce t-erk1/ and p-erk1/2 regulated by thyroid hormone and the role that they play in the hippocampus, erk1/ are important in the generation of learning and memory. in the present study, we detected t-erk1/ and p-erk1/ changes in the pups following developmental id and hypothyroidism using western blot approach. both t-erk1/ and p-erk1/ were measured in ca ca and dg regions on pn pn pn and pn. in ca and ca regions of the hippocampus, id and hypothyroidism significantly reduced t-erk or t-erk. p-erk and p-erk were significantly lower on pn pn and pn. on the other hand, p-erk1/ was hardly detected on pn this might be due to lower t-erk in early postnatal period in pups, and thus p-erk1/ signal becomes too weak to capture. in the dg region, however, id and hypothyroidism did not change t-erk1/ or p-erk1/ expression. id and hypothyroidism reduce t-creb and p-creb as a downstream target molecule of erk1/ creb plays a critical role in the generation of protein synthesis-dependent long-term changes in the brain and is necessary for the fear-associated memory. in order to investigate the effects of id and hypothyroidism on creb, t-creb and p-creb were detected via western blot. in the present study, t-creb and p-creb were clearly expressed in ca ca and dg regions on pn pn pn and pn. however, the signals of p-creb were very weak on pn id and hypothyroidism significantly reduced both t-creb and p-creb in ca ca and dg regions. discussion the major findings of this study are that, in lactation and adolescent stage of development rats, developmental id and hypothyroidism significantly reduced the mean number of surviving cells in hippocampus and decreased erk1/ and creb expression in hippocampal ca and ca3; even after the thyroid hormones back to normal, surviving cells, erk1/ and creb were still lower than the controls. the present study demonstrates that developmental id and hypothyroidism down-regulate hippocampal erk1/ and creb in lactational and adolescent rats. our previous study has shown that id was still a serious public health problem in china. given so many chinese children exposed to developmental id, this study sought to make three lactational and adolescent animal models to mimic the developmental exposure to id and hypothyroidism. many lines of literature using adult animal models have demonstrated that developmental hypothyroidism alters synaptic function in the hippocampus. in agreement with these studies, the lactational and adolescent rats in this study have an irreversible impairment in hippocampal erk1/ and creb following developmental exposure to id and hypothyroidism. this is because that, during these developmental periods, the mother is the only source of iodine for the fetus and neonate. therefore, maternal id leads to maternal and filial hypothyroxinemia. taking together, developmental periods during pregnancy and lactation are critical in the brain development. thyroid hormone is well known to regulate morphological and biochemical alterations in brain during critical periods of brain development. this regulation can be done by genomic, posttranslational, and direct actions on neural substrates. therefore, thyroid hormone is a fundamental factor that regulates normal development of the cns. logically, thyroid hormone insufficiency during early brain development is detrimental to synaptic transmission in hippocampus and to a cellular and behavioral model of learning and memory. in need, our previous study has clearly demonstrated that id-exposure lowers the children iqs in china. experimentally, we have also shown the id can lead to an inhibition of ltp in the rat ca area. it is well known that mapk mediates neuronal metabolism, differentiation and proliferation. also, the persisting alteration in developmental brain involves changes in myelination and migration patterns of neurons. the present study further confirmed the hypothesis that developmental id and hypothyroidism induce irreversible impairment of two key proteins expression in the ca and ca regions, where both erk1/ and creb expression was significantly reduced in either total protein level or phorsphorylated fraction. in agreement with our findings, gilbert and colleagues observed the irreversibly cns impairments in rats induced by developmental hypothyroidism from gd to pn. this study provides us a clue to explore the mechanism by which thyroid hormone regulates brain development during lactational and adolescent periods. as a member of mapk, erk1/ is a critical memory-related protein in the generation of learning and memory. thyroid hormone activates g-protein-coupled receptors, leads to erk1/ phosphorylation, and in turn enhances the transcription of some proteins that are important in memory. it has been shown that hypothyroidism reduces the basal p-erk1/ in hippocampal ca region without affecting t-erk1/. in the dg region, however, hypothyroidism had no effect on t-erk1/ and p-erk1/. in the lactational and adolescent rats, here we show that t-erk1/ and p-erk1/ in iodine-deficient, ppm ptu-treatment and ppm ptu-treatment groups in ca and ca regions were significantly lower than controls; while there was no difference in dg region. this difference might be due to the different cell types of neurons in the different regions. in the dg region, the granule cells are more resistant to conditioned stimulus than the pyramidal cells. this is in line with the importance of the ca and ca in the learning and memory generation. recently, it is reported that mapk genes are the direct targets of thyroid hormone and mapk activation is part of the non-genomic action of thyroid hormone. unlike the t3-dependent genomic mechanism, the non-genomic pathway involves t not t in present study, id and hypothyroidism caused low t concentration in the lactational and adolescent rats. it is reported that in ptu treated pups, serum t concentration is negatively correlated with cerebrocortical type ii 5'-deiodinase activity, a sensitive marker of tissue thyroid status and an indicator of brain's compensatory response to maintain cerebrocortical t. since t is correlated with synaptic response, in the present study, we speculate that the reduced serum ft may increase d activity, decrease cerebrocortical t concentration, and lead to bound-tr reduction. taken together, erk1/ might be down-regulated by developmental id and hypothyroidism via non-genomic pathway. different from the present study, calloni et al. reported that hypothyroidism promoted an increase in p-erk1/. it might be attributed to difference of the method on establishing hypothyroid animal model or the concentration of medicine. calloni et al. administered pregnant rats with % methimazole in drinking water from gd to the birth. since the neocorticogenesis and maturation of the cns persist for the adolescence in rat, the early developmental impairments may be minor and reversible. therefore, the increased p-erk1/ might be ascribed to the recovered morphological and physiological changes because of the termination of administration at birth. another key protein for the long-term memory processes is creb. the activation of creb and creb-dependent transcriptional pathway is essential for memory consolidation. different from increased p-creb in thyroid hormone treated neural cells, in the present study, developmental id and hypothyroidism significantly lowered both t-creb and p-creb in ca ca and dg regions. it has been proposed that p-creb decrease might attribute to protein reduction of t-creb, ca2+/camodulin dependent protein kinase iv, and adenylyl cyclase type i, or elevation of calcineurin. in addition, tr activation can antagonize creb-mediated transcription and inhibit the phosphorylation of creb. so far, the detailed mechanism by which developmental id and hypothyroidism regulate creb is still unclear. since creb is a downstream target molecule of erk1/ our present data imply that developmental id and hypothyroidism may induce p-erk1/ reduction, which in turn lead to p-creb reduction. however, we can not exclude the involvement of genomic pathway, where thyroid hormone modulates the creb by regulating the transcriptions of camkiv. moreover, the number and morphology of neuron in hippocampus are affected by neonatal and adult hypothyroidism. as a marker of neuronal development, nissl body diminishes when neuronal impairment. we found here that the number of surviving neuronal cells in hippocampus in iodine-deficient and ppm ptu-treatment groups were significantly lower than the controls. importantly, the reduction of nissle bodies in the developmental id and hypothyroidism fits very well with the decreased protein production of erk1/ and creb. this might be due to the decrease in cell number, and to a lower expression of the investigated genes in each neuron. therefore, our observation on nissl bodies further confirmed the irreversible cns impairments following developmental id and hypothyroidism. in summary, this study has shed some light on one aspect of the id/hypothyroidism-induced learning and memory impairment. the following questions are still unanswered: how does thyroid hormone regulate erk and creb? why is there more reduction of p-creb in dg region than p-erk1/2? how do erk1/ and creb regulate ltp production? is erk protein regulation due to a specific gene-expression modulation or are related to a general decrease in gene expression and/or protein synthesis following hypothyroidism? further investigations are needed to answer these questions.  our previous study has confirmed the impairment of ltp induction following developmental id and hypothyroidism. the present study has further shown that, in lactational and adolescent rats, developmental id and hypothyroidism induce irreversible reduction of erk1/ and creb in hippocampal ca and ca regions. in conclusion, erk1/ and creb may play an important role in id and hypothyroidism-induced brain impairment in lactational and adolescent rats. abbreviations aci: adenylyl cyclase type i; acsf: artificial cerebrospinal fluid; anova: analysis of variance; camkiv: ca2+/camodulin dependent protein kinase iv; cns: central nervous system; cre: camp response element; creb: camp response element-binding protein; d2: type ii 5'-deiodinase; dab: 30-diaminobenzidine; ecl: chemiluminescence; erk: extracellular signal-regulated kinase;ft3: free triiodothyronine; ft4: free thyroxine; gapdh: glyceraldehyde phosphodehydrogenase; gd: gestational day; he: hematoxylin; id: iodine deficiency; iegs: immediate early genes; l-ltp: late long-term potentiation; ltp: long-term potentiation; mapks: mitogen-activated protein kinases; mek: mitogen extracellular regulating kinase; mmi: methimazole; nmda: n-methyl-d-asparticacid; nod: optical density; pbs: phosphate-buffered saline; pn: postnatal day; ptu: propylthiouracil; snk: student-newman-keuls; t3: triiodothyronine; t4: thyroxine; tr: thyroid hormone receptor; tsh: thyroid stimulating hormone. competing interests the authors declare that they have no competing interests. authors' contributions jd, wl and jc conceived of the study, and participated in its design and coordination. jd drafted the manuscript. jd, yw and yh carried out the experiments, collected and analyzed the data. qx revised the manuscript. all authors participated in writing, and read and approved the final manuscript. supplementary material additional file 1 id and hypothyroidism induced neuronal loss in ca region. nissl staining was used to assess the numbers of surviving cells and dead cells in the hippocampus. more dead pyramidal neurons were found in the ca region of the iodine-deficient and ptu-treated rats at pn pn and pn compared to controls. representative photomicrographs are shown for each time point. scale bar represents μm, shown on lower left in, and applies to all panels. the nissl-positive survival cell number of iodine-deficient and ppm ptu-treatment groups was significantly lower than controls. each value represents mean ± sem. significant differences from control group: *, p <. click here for file additional file 2 id and hypothyroidism induced neuronal loss in ca region. more dead pyramidal neurons were observed in the ca region of the iodine-deficient and ppm ptu-treated rats on pn and pn compared to controls. representative photomicrographs are shown for each time point. scale bar represents μm, shown on lower left in, and applies to all panels. the nissl-positive survival cell number of iodine-deficient and ppm ptu-treatment groups was significantly lower than controls. each value represents mean ± sem. significant differences from control group: *, p <. click here for file additional file 3 id and hypothyroidism induced neuronal loss in dg region. more dead granular cells were found in the dg region of the iodine-deficient and ppm ptu-treated rats on pn pn and pn compared to controls. representative photomicrographs are shown for each time point. scale bar represents μm, shown on lower left in, and applies to all panels. the nissl-positive survival cell number of iodine-deficient and ppm ptu-treatment groups was significantly lower than controls. each value represents mean ± sem. significant differences from control group: *, p <. click here for file acknowledgements this work was supported by the national natural science foundation committee of china. the authors are very grateful for excellent theoretical and technical support from prof. mingqi gao in the college of clinical medical sciences, prof. hong lai in the college of basic medical sciences and prof. zhongyan shan in the endocrine research institute at china medical university, pedro san-cristobal md department of physiology, radboud university nijmegen medical centre, for critical reading the manuscript.
three developmental rat models were created by administrating dam rats with either iodine-deficient diet or propylthiouracil -added drinking water from gestational day till postnatal day then, the total and phorsporylated erk1/ and total and phorsporylated creb in the hippocampus were detected with western blot on pn pn pn and pn here, we show that hippocampal extracellular signal-regulated kinase and camp response element-binding protein are implicated in the impaired learning and memory in lactational and adolescent rat hippocampus following developmental id and hypothyroidism. developmental id and hypothyroidism down-regulate hippocampal erk1/ and creb in lactational and adolescent rats.
 developmental iodine deficiency leads to inadequate thyroid hormone that impairs learning and memory with an unclear mechanism. here, we show that hippocampal extracellular signal-regulated kinase and camp response element-binding protein are implicated in the impaired learning and memory in lactational and adolescent rat hippocampus following developmental id and hypothyroidism. methods three developmental rat models were created by administrating dam rats with either iodine-deficient diet or propylthiouracil -added drinking water from gestational day till postnatal day then, the total and phorsporylated erk1/ and total and phorsporylated creb in the hippocampus were detected with western blot on pn pn pn and pn  the iodine-deficient and hypothyroid pups showed lower serum ft and ft levels, smaller body size, and delayed eyes opening. the mean number of surviving cells in the hippocampus of the iodine-deficient and ppm ptu-treated rats was significantly reduced compared to controls. iodine-deficient and ppm ptu-treatment groups demonstrated significantly lower level of total and phosphorylated erk1/ and creb than the controls on pn pn and pn. the reduction of erk1/ and creb was not reversible with the restoration of serum thyroid hormone concentrations on pn  developmental id and hypothyroidism down-regulate hippocampal erk1/ and creb in lactational and adolescent rats. 