Effects of neonatal stress on markers of synaptic plasticity in the hippocampus: Implications for spatial memory

Early stressful adverse situations may increase the vulnerability to cognitive deficits and psychiatric disorders, such as depression. Maternal separation (MS) has been used as an animal model to study changes in neurochemistry and behavior associated with exposure to early‐life stress. This study investigated the effects of neonatal stress (MS) on the expression of synaptic plasticity markers in the hippocampus and a purported relationship to cognitive processes. Spatial learning (Morris water maze) significantly increased the expression of total levels of the neural cell adhesion molecule (NCAM), as well as its three major isoforms (NCAM‐120, ‐140, and ‐180) both in the control and MS groups. Interestingly, these increases in NCAM expression after learning were lower in MS animals when compared with control rats. MS induced a significant decrease in total levels of NCAM, and specifically, in the NCAM‐140 isoform expression. In the hippocampus of MS rats there was a significant decrease in brain‐derived neurotrophic factor and synaptophysin mRNA densities. Cell proliferation, measured as BrdU‐positive cells, was also decreased in the dentate gyrus of MS rats. Altogether these results suggest that MS can alter normal brain development, providing a potential mechanism by which early environmental stressors may influence vulnerability to show cognitive impairments later in life. © 2009 Wiley‐Liss, Inc.     

Pyramidal cells of the rat basolateral amygdala: synaptology and innervation by parvalbumin-immunoreactive interneurons

The generation of emotional responses by the basolateral amygdala is determined largely by the balance of excitatory and inhibitory inputs to its principal neurons, the pyramidal cells. The activity of these neurons is tightly controlled by gamma-aminobutyric acid (GABA)-ergic interneurons, especially a parvalbumin-positive (PV(+)) subpopulation that constitutes almost half of all interneurons in the basolateral amygdala. In the present semiquantitative investigation, we studied the incidence of synaptic inputs of PV(+) axon terminals onto pyramidal neurons in the rat basolateral nucleus (BLa). Pyramidal cells were identified by using calcium/calmodulin-dependent protein kinase II (CaMK) immunoreactivity as a marker. To appreciate the relative abundance of PV(+) inputs compared with excitatory inputs and other non-PV(+) inhibitory inputs, we also analyzed the proportions of asymmetrical (presumed excitatory) synapses and symmetrical (presumed inhibitory) synapses formed by unlabeled axon terminals targeting pyramidal neurons. The results indicate that the perisomatic region of pyramidal cells is innervated almost entirely by symmetrical synapses, whereas the density of asymmetrical synapses increases as one proceeds from thicker proximal dendritic shafts to thinner distal dendritic shafts. The great majority of synapses with dendritic spines are asymmetrical. PV(+) axon terminals form mainly symmetrical synapses. These PV(+) synapses constitute slightly more than half of the symmetrical synapses formed with each postsynaptic compartment of BLa pyramidal cells. These data indicate that the synaptology of basolateral amygdalar pyramidal cells is remarkably similar to that of cortical pyramidal cells and that PV(+) interneurons provide a robust inhibition of both the perisomatic and the distal dendritic domains of these principal neurons.     

Early‐life stress affects the structural and functional plasticity of the medial prefrontal cortex in adolescent rats

Early life experiences are crucial factors that shape brain development and function due to their ability to induce structural and functional plasticity. Among these experiences, early‐life stress (ELS) is known to interfere with brain development and maturation, increasing the risk of future psychopathologies, including depression, anxiety, and personality disorders. Moreover, ELS may contribute to the emergence of these psychopathologies during adolescence. In this present study, we investigated the effects of ELS, in the form of maternal separation (MS), on the structural and functional plasticity of the medial prefrontal cortex (mPFC) and anxiety‐like behavior in adolescent male rats. We found that the MS procedure resulted in disturbances in mother–pup interactions that lasted until weaning and were most strongly demonstrated by increases in nursing behavior. Moreover, MS caused atrophy of the basal dendritic tree and reduced spine density on both the apical and basal dendrites in layer II/III pyramidal neurons of the mPFC. The structural changes were accompanied by an impairment of long‐term potentiation processes and increased expression of key proteins, specifically glutamate receptor 1, glutamate receptor 2, postsynaptic density protein 95, αCa²⁺/calmodulin‐dependent protein kinase II and αCa²⁺/calmodulin‐dependent protein kinase II phosphorylated at residue Thr305, that are engaged in long‐term potentiation induction and maintenance in the mPFC. We also found that the MS animals were more anxious in the light/dark exploration test. The results of this study indicate that ELS has a significant impact on the structural and functional plasticity of the mPFC in adolescents. ELS‐induced adaptive plasticity may underlie the pathomechanisms of some early‐onset psychopathologies observed in adolescents.     

Serotonin-immunoreactive axon terminals innervate pyramidal cells and interneurons in the rat basolateral amygdala

The basolateral nuclear complex of the amygdala (BLC) receives a dense serotonergic innervation that appears to play a critical role in the regulation of mood and anxiety. However, little is known about how serotonergic inputs interface with different neuronal subpopulations in this region. To address this question, dual-labeling immunohistochemical techniques were used at the light and electron microscopic levels to examine inputs from serotonin-immunoreactive (5-HT+) terminals to different neuronal subpopulations in the rat BLC. Pyramidal cells were labeled by using antibodies to calcium/calmodulin-dependent protein kinase II, whereas different interneuronal subpopulations were labeled by using antibodies to a variety of interneuronal markers including parvalbumin (PV), vasoactive intestinal peptide (VIP), calretinin, calbindin, cholecystokinin, and somatostatin. The BLC exhibited a dense innervation by thin 5-HT+ axons. Electron microscopic examination of the anterior basolateral nucleus (BLa) revealed that 5-HT+ axon terminals contained clusters of small synaptic vesicles and a smaller number of larger dense-core vesicles. Serial section reconstruction of 5-HT+ terminals demonstrated that 76% of these terminals formed synaptic junctions. The great majority of these synapses were symmetrical. The main targets of 5-HT+ terminals were spines and distal dendrites of pyramidal cells. However, in light microscopic preparations it was common to observe apparent contacts between 5-HT+ terminals and all subpopulations of BLC interneurons. Electron microscopic analysis of the BLa in sections dual-labeled for 5-HT/PV and 5-HT/VIP revealed that many of these contacts were synapses. These findings suggest that serotonergic axon terminals differentially innervate several neuronal subpopulations in the BLC.     

Cholinergic innervation of pyramidal cells and parvalbumin-immunoreactive interneurons in the rat basolateral amygdala

The basolateral nucleus of the amygdala receives an extremely dense cholinergic innervation from the basal forebrain that is critical for memory consolidation. Although previous electron microscopic studies determined some of the postsynaptic targets of cholinergic afferents, the majority of postsynaptic structures were dendritic shafts whose neurons of origin were not identified. To make this determination, the present study analyzed the cholinergic innervation of the anterior subdivision of the basolateral amygdalar nucleus (BLa) of the rat using electron microscopic dual-labeling immunocytochemistry. The vesicular acetylcholine transporter (VAChT) was used as a marker for cholinergic terminals; calcium/calmodulin-dependent protein kinase II (CaMK) was used as a marker for pyramidal cells, the principal neurons of the BLa; and parvalbumin (PV) was used as a marker for the predominant interneuronal subpopulation in this nucleus. VAChT(+) terminals were visualized by using diaminobenzidine as a chromogen, whereas CAMK(+) or PV(+) neurons were visualized with Vector very intense purple (VIP) as a chromogen. Quantitative analyses revealed that the great majority of dendritic shafts receiving cholinergic inputs were CAMK(+) , indicating that they were of pyramidal cell origin. In fact, 89% of the postsynaptic targets of cholinergic terminals in the BLa were pyramidal cells, including perikarya (3%), dendritic shafts (47%), and dendritic spines (39%). PV(+) structures, including perikarya and dendrites, constituted 7% of the postsynaptic targets of cholinergic axon terminals. The cholinergic innervation of both pyramidal cells and PV(+) interneurons may constitute an anatomical substrate for the generation of oscillatory activity involved in memory consolidation by the BLa.     

Minimal residues in linker domain of syntaxin 1A required for binding affinity to Ca2+/calmodulin-dependent protein kinase II

The linker domain is important for the conformational change syntaxin 1A, which enables it to act as a SNARE for exocytosis. We found that when applied exogenously, the linker domain is a potent inhibitor of exocytosis through inhibiting interaction between autophosphorylated CaMKII and endogenous syntaxin 1A (Ohyama et al. [2002] J. Neurosci. 22:3342-3351). To identify the simplest and the most potent inhibitor for exocytosis, we further characterized the linker domain and determined the minimal number of residues required for CaMKII binding. The minimal length of the CaMKII-binding site was 145-172 residues and a loss of G172 considerably weakened affinity for CaMKII. The basic amino acid clusters, R151 and K146, were indispensable for binding, whereas R148 was not. A comparison of the CaMKII-binding in several syntaxin isoforms revealed that the substitution of S162 attenuated CaMKII-binding activity. These results suggest that S162 is an important residue as well as the basic amino acid cluster within region 145-172 of the linker domain.     

Transforming growth factor-beta1 enhances expression of brain-derived neurotrophic factor and its receptor, TrkB, in neurons cultured from rat cerebral cortex.

The effects of transforming growth factor (TGF)-beta1 on expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, TrkB, in neurons cultured from the cerebral cortex of 18-day-old embryonic rats were examined. BDNF mRNA was significantly increased from 24-48 hr after the TGF-beta1 treatment over 20 ng/ml. Accumulation of BDNF protein in the culture medium was also potentiated by TGF-beta1, although the intracellular content of BDNF was nearly unchanged. The enhancement of BDNF mRNA expression was suppressed by the co-presence of decorin, a small TGF-beta-binding proteoglycan that inhibits the biological activities of TGF-betas. mRNA expression of full-length TrkB, the bioactive high-affinity receptor for BDNF, was also upregulated after treatment with TGF-beta1. These observations suggest that: 1) TGF-beta1 potentiates BDNF/TrkB autocrine or local paracrine system; and 2) the neurotrophic activity of TGF-beta1 is partly responsible for the BDNF induced by TGF-beta1 itself. To test this latter possibility, we examined the neuronal survival activity of TGF-beta1 with or without K252a, a selective inhibitor of Trk family tyrosine kinases. TGF-beta1 significantly enhanced neuronal survival, but the co-presence of K252a completely suppressed the activity, demonstrating the involvement of Trk receptor signaling in TGF-beta1-mediated neuronal survival in cultured rat cortical neurons. These results seem to be in line with recent findings by other investigators that some neurotrophic factors including BDNF require TGF-betas as a cofactor to exert their neurotrophic activities.     

CaMKII is differentially localized in synaptic regions of Kenyon cells within the mushroom bodies of the honeybee brain

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been linked to neuronal plasticity associated with long-term potentiation as well as structural synaptic plasticity. Previous work in adult honeybees has shown that a single CaMKII gene is strongly expressed in the mushroom bodies (MBs), brain centers associated with sensory integration, and learning and memory formation. To study a potential role of CaMKII in synaptic plasticity, the cellular and subcellular distribution of activated (phosphorylated) pCaMKII protein was investigated at various life stages of the honeybee using immunocytochemistry, confocal microscopy, and western blot analyses. Whereas at pupal stages 3-4 most parts of the brain showed high levels of pCaMKII immunoreactivity, the protein was predominantly concentrated in the MBs in the adult brain. The results show that pCaMKII is present in a specific subpopulation of Kenyon cells, the noncompact cells. Within the olfactory (lip) and visual (collar) subregion of the MB calyx neuropil pCaMKII was colocalized with f-actin in postsynaptic compartments of microglomeruli, indicating that it is enriched in Kenyon cell dendritic spines. This suggests a potential role of CaMKII in Kenyon cell dendritic plasticity. Interestingly, pCaMKII protein was absent in two other types of Kenyon cells, the inner compact cells associated with the multimodal basal ring and the outer compact cells. During adult behavioral maturation from nurse bees to foragers, pCaMKII distribution remained essentially similar at the qualitative level, suggesting a potential role in dendritic plasticity of Kenyon cells throughout the entire life span of a worker bee.     

Activation of phenotypically distinct neuronal subpopulations in the anterior subdivision of the rat basolateral amygdala following acute and repeated stress

The effects of acute and repeated stress on expression of the early immediate gene c-fos in the basolateral amygdala have previously been reported; however, characterization of which neuronal subpopulations are activated by these stimuli has not been investigated. This question is of considerable relevance, insofar as the basolateral amygdala houses a heterogeneous population of neurons, including those of gamma-aminobutyric acid (GABA)-ergic and glutamatergic phenotypes that may be subcategorized based on their expression of various calcium-binding proteins, including parvalbumin, calbindin, calretinin, and the calcium-sensitive enzyme calcium/calmodulin-dependent kinase II. Characterization of these subpopulations has revealed unique differences in their physiology, synaptology, and morphology, suggesting that each distinct phenotype may have profound effects on the local circuitry of the amygdala. Therefore, we examined the effects of acute and repeated restraint stress on expression of the immediate early gene c-fos in neurons containing parvalbumin, calbindin, calretinin, or calcium/calmodulin-dependent kinase II in the basolateral amygdala. Double-label immunohistochemistry revealed that acute restraint stress activated a proportion of parvalbumin-, calbindin-, or calcium/calmodulin-dependent kinase II-positive neurons. Prior exposure to repeated restraint stress markedly attenuated acute-stress mediated activation of these neuronal populations, although not equally. Expression of c-Fos protein was not detected in calretinin-positive neurons in any experimental group. These results demonstrate that distinct neuronal phenotypes in the basolateral amygdala are activated by acute restraint stress and that prior repeated restraint stress differentially affects this response.     

Prenatal exposure to valproic acid enhances synaptic plasticity in the medial prefrontal cortex and fear memories

The prefrontal cortex has been extensively implicated in autism to explain deficits in executive and other higher brain functions related to cognition, language, sociability and emotion. Hyper-connectivity and hyper-plasticity at the level of the neuronal microcircuit in the medial prefrontal cortex (mPFC) in the valproic acid (VPA) animal model of autism has been suggested. However, the possible alterations at the system levels are not well understood. The present study investigated the basal synaptic transmission and synaptic plasticity in the mPFC in vivo in the VPA rat model of autism. Furthermore, short-term and long-term retention of fear memories were also examined. The findings displayed that paired-pulse facilitation (PPF) and long-term potentiation (LTP), representing short- and long-term synaptic plasticity, were enhanced by the prenatal exposure to VPA. In addition, the short- and long-term fear memories were enhanced. These results suggest that enhanced synaptic plasticity in the mPFC and fear memories might be one of the mechanisms underlying some symptoms of autism.     

Structural and functional asymmetry in the normal and epileptic rat dentate gyrus

The rat dentate gyrus is usually described as relatively homogeneous. Here, we present anatomic and physiological data which demonstrate that there are striking differences between the supra- and infrapyramidal blades after status epilepticus and recurrent seizures. These differences appear to be an accentuation of a subtle asymmetry present in normal rats. In both pilocarpine and kainic acid models, there was greater mossy fiber sprouting in the infrapyramidal blade. This occurred primarily in the middle third of the hippocampus. Asymmetric sprouting was evident both with Timm stain as well as antisera to brain-derived neurotrophic factor (BDNF) or neuropeptide Y (NPY). In addition, surviving NPY-immunoreactive hilar neurons were distributed preferentially in the suprapyramidal region of the hilus. Extracellular recordings from infrapyramidal sites in hippocampal slices of pilocarpine-treated rats showed larger population spikes and weaker paired-pulse inhibition in response to perforant path stimulation relative to suprapyramidal recordings. A single stimulus could evoke burst discharges in infrapyramidal granule cells but not suprapyramidal blade neurons. BDNF exposure led to spontaneous epileptiform discharges that were larger in amplitude and longer lasting in the infrapyramidal blade. Stimulation of the infrapyramidal molecular layer evoked larger responses in area CA3 than suprapyramidal stimulation. In slices from the temporal pole, in which anatomic evidence of asymmetry waned, there was little evidence of physiological asymmetry either. Of interest, some normal rats also showed signs of greater evoked responses in the infrapyramidal blade, and this could be detected with both microelectrode recording and optical imaging techniques. Although there were no signs of hyperexcitability in normal rats, the data suggest that there is some asymmetry in the normal dentate gyrus and this asymmetry is enhanced by seizures. Taken together, the results suggest that supra- and infrapyramidal blades of the dentate gyrus could have different circuit functions and that the infrapyramidal blade may play a greater role in activating the hippocampus.     

ERK1/2 and ERK5 have distinct roles in the regulation of brain-derived neurotrophic factor expression

Neurotrophins play essential roles in the development, differentiation, and survival of neuronal and nonneuronal cells. Alterations in neurotrophin expression have been implicated in a variety of neurodegenerative disorders. Dysregulation of brain-derived neurotrophic factor (BDNF) has been implicated in deficits of long-term potentiation and cognition and may contribute to the development of Alzheimer's disease (AD). In this study, we used complementary pharmacological and molecular approaches to evaluate the role of ERK1/2 and ERK5, two members of the MAPK pathway associated with neuroprotection, in regulating BDNF expression in C6 glial cells and primary astrocytes. Our data revealed that U0126, an inhibitor of both ERK5 and ERK1/2, increased the levels of BDNF mRNA, whereas the MEK1/2-specific inhibitor PD184352 did not, suggesting that ERK5 exerts negative control over BDNF expression. This was supported by experiments in which RNAi-mediated depletion of ERK5 led to an increase in BDNF. In contrast, transfection with constitutively active MEK5 resulted in an inhibition of BDNF expression, confirming the inhibitory role of ERK5 in the regulation of BDNF. Interestingly, transfection with the dominant active mutant of MEK1 (MEKR4F), the upstream activator of ERK1/2, resulted in a modest increase in BDNF levels. Collectively, our data suggest that ERK5 and ERK1/2 exert opposite effects on BDNF expression and support the hypothesis that an imbalance of these two signaling pathways may contribute to the pathology of diseases in which neurotrophin dysregulation is noted.     

Effects of intrathecal injection of nimodipine, ω-conotoxin GVIA, calmidazolium, and KN-62 on the antinociception induced by cold water swimming stress in the mouse

The present study was designed to determine if spinal calcium channels, calmodulin, and calcium/calmodulin-dependent protein kinase II were involved in the production of antinociception induced by cold water swimming stress (CWSS). The effects of intrathecal (i.t.) injection of nimodipine, ω-conotoxin GVIA, calmidazolium, or (S)-5-isoquinolinesulfonic acid, 4-[2-[(5-isoquinolinyl-sulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)-propyl]phenyl ester (KN-62) on CWSS-induced antinociception were studied in ICR mice. The antinociception was assessed by the tail-flick test. CWSS produced inhibition of the tail-flick response. Various doses of nimodipine (10–40 ng), ω-conotoxin GVIA (5–40 ng), calmidazolium (10–40 ng), or KN-62 (5–40 ng) injected i.t. alone did not show any antinociceptive effect in the tail-flick test. I.t. pretreatment with ω-conotoxin GVIA, calmidazolium, or KN-62 dose dependently attenuated the CWSS-induced inhibition of the tail-flick response. However, i.t. pretreatment with nimodipine did not affect the inhibition of the tail-flick response induced by CWSS. Our results suggest that spinal N-type calcium channel, calmodulin and calcium/calmodulin-dependent protein kinase II may be involved in the production of antinociception induced by CWSS. On the other hand, CWSS-induced antinociception appears not to be mediated via the spinal L-type calcium channel.     

Effects of exercise and muscle type on BDNF,NT‐4/5, and TrKB expression in skeletal muscle

Muscle‐derived neurotrophins are thought to contribute to the adaptation of skeletal muscle to exercise, but the effects of brief exercise interventions on BDNF, NT‐4/5, and trkB are not understood. RNA was extracted for RT‐PCR from soleus and medial gastrocnemius of Sprague‐Dawley rats exercised on a treadmill at speeds up to 20 m/min at 5% incline for 5 or 10 days. BDNF expression was elevated in soleus following 5 days (184%, P < 0.001) but not 10 days of exercise. NT‐4/5 and trkB were not affected at either time‐point. BDNF mRNA was significantly higher in soleus at rest when compared with medial gastrocnemius (193%, P < 0.05). No significant effects of muscle type were detected for NT‐4/5 and trkB. Our results indicate differential control of BDNF expression between soleus and medial gastrocnemius following 5 days of exercise. BDNF may be a protein with an uncharacterized contribution to the acute adaptation of skeletal muscle to exercise, whereas NT‐4/5 shows no response. Muscle Nerve, 2009     

Knockdown of tropomyosin‐related kinase B receptor expression in the nucleus accumbens shell prevents intermittent social defeat stress‐induced cross‐sensitization to amphetamine in rats

The nucleus accumbens (NAc) is a critical brain region for the rewarding effects of drugs of abuse. Brain‐derived neurotrophic factor (BDNF) can facilitate stress‐ and drug‐induced neuroadaptation in the mesocorticolimbic system. BDNF‐containing projections to the NAc originate from the ventral tegmental area (VTA) and the prefrontal cortex, and BDNF release activates tropomyosin‐related kinase B (TrkB). In this study, we examined the necessity for BDNF‐TrkB signaling in the NAc shell during social defeat stress‐induced cross‐sensitization to amphetamine. Adeno‐associated virus expressing short hairpin RNA directed against TrkB (AAV‐shTrkB) was infused bilaterally into the NAc shell to knock down TrkB, whereas AAV‐GFP (green fluorescent protein) was used as the control virus. Rats were exposed to intermittent social defeat stress or handling procedures; amphetamine challenge was given at 10 days after the last defeat and locomotor activity was measured. Stressed rats that received the control virus showed cross‐sensitization to amphetamine compared with the handled rats. In contrast, NAc TrkB knockdown prevented social defeat stress‐induced cross‐sensitization. TrkB knockdown in the NAc was found to reduce the level of phospho‐extracellular signal‐regulated kinase 1 in this region. NAc TrkB knockdown also prevented stress‐induced elevation of BDNF and the glutamate receptor type 1 (GluA1) subunit of AMPA receptor in the VTA, as well as ΔFosB expression in the NAc. These findings indicated that BDNF‐TrkB signaling in the NAc shell was required for social defeat stress‐induced cross‐sensitization. NAc TrkB‐BDNF signaling also appeared to be involved in the regulation of GluA1 in the VTA, as well as in the NAc ΔFosB accumulation that could trigger cross‐sensitization after social defeat stress.     

Increased expression of calcium/calmodulin-dependent protein kinase IIbeta in frontal cortex in schizophrenia and depression

In searching for genes dysregulated in schizophrenia, we measured the expression of the two splice variants of calcium/calmodulin-dependent protein kinase II (CaMKIIalpha and CaMKIIbeta) in postmortem frontal cerebral cortex tissues from patients who had died with schizophrenia, bipolar disorder, or severe depression. The mRNA levels of expression of these two splice variants were measured by real-time Quantitative PCR, using an Mx4000 instrument. The values for the expression of CaMKIIalpha and CaMKIIbeta were normalized by the expression of beta-glucuronidase in the tissues. The expression of CaMKIIalpha was significantly elevated in the depression tissues by 29%. The expression of CaMKIIbeta was significantly elevated in the schizophrenia tissues by 27%, and in the depression tissues by 36%. Because CaMKIIbeta influences the expression of many neuroreceptors and influences neural outgrowth and pruning, its altered expression in the cerebral cortex in schizophrenia or depression may contribute to these diseases.     

Differential expression of human placental neurotrophic factors in preterm and term deliveries

Neurotrophic factors such as brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are involved in development of the placenta and fetal brain. A series of human and animal studies in our department have shown that micronutrients (folic acid, vitamin B₁₂) and omega 3 fatty acids like DHA are all interlinked in the one carbon cycle. Any alterations in one carbon components will lead to changes in methylation patterns that further affect the gene expression at critical periods of development resulting in complications during pregnancy. This may further contribute to risk for neurodevelopmental disorders in children born preterm. Therefore this study for the first time examines the mRNA levels from preterm and term placentae. A total number of 38 women delivering preterm (<37 weeks gestation) and 37 women delivering at term (=>37 weeks gestation) were recruited. The mRNA levels of BDNF and NGF were analyzed by real time quantitative polymerase chain reaction. Our results indicate that BDNF and NGF mRNA levels were lower in preterm group as compared to term group. There was a positive association of placental BDNF and NGF mRNA levels with cord plasma BDNF and NGF levels. The differential expression of BDNF and NGF gene in preterm placentae may also alter the vascular development in preterm deliveries. Our data suggests that the reduced mRNA levels of BDNF and NGF may possibly be a result of altered epigenetic mechanisms and may have an implication for altered fetal programming in children born preterm.