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Cellular mechanisms underlying an effect of “early handling” on pCREB and BDNF in the neonatal rat hippocampus
Brain Research 1052 (2005) 187 – 195 www.elsevier.com/locate/brainres
Research Report
Cellular mechanisms underlying an effect of ‘‘early handling’’ on pCREB and BDNF in the neonatal rat hippocampus Efstathios Garoflos, Antonios Stamatakis, Athanasios Mantelas, Helen Philippidis, Fotini Stylianopoulou* Laboratory of Biology-Biochemistry, School of Health Sciences, University of Athens, Papadiamantopoulou 123, 11527 Athens, Greece Accepted 10 June 2005 Available online 15 July 2005
Abstract Early experiences have long-term effects on brain function and behavior. However, the precise mechanisms involved still remain elusive. In an effort to address this issue, we employed the model of ‘‘early handling’’, which is known to affect the ability of the adult organism to respond to stressful stimuli, and determined its effects on hippocampal pCREB and BDNF 2, 4, and 8 h later. 8 h following ‘‘handling’’ on postnatal day 1, there was an increase in pCREB and BDNF positive cells in the hippocampus, a brain area which is a specific target of ‘‘handling’’. On the other hand, vehicle injection resulted in decreased pCREB and BDNF in both handled and non-handled animals 2 and 4 h later. The ‘‘handling’’-induced increase of pCREB and BDNF was cancelled by inhibition of NMDA, AMPA/kainate, GABA-A, 5-HT1A or 5-HT2A/C receptors, as well as L-type voltage-gated Ca2+ channels. It thus appears that ‘‘early handling’’ activates these neurotransmitter receptors, leading to increased intracellular Ca2+, phosphorylation of the transcription factor CREB, and increased BDNF expression. BDNF can then exert its morphogenetic effects and thus ‘‘imprint’’ the effects of ‘‘handling’’ on the brain. D 2005 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Neural plasticity Keywords: Neonatal handling; pCREB; BDNF; Neurotransmitter receptor; L-type voltage-gated Ca2+ channel; Hippocampus
1. Introduction
Abbreviations: 5-HT, serotonin; AMPA, a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid; ANOVA, analysis of variance; BDNF, brainderived neurotrophic factor; CA1 area, field 1 of Ammon’s horn; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt; CPP, [T]-3-(2-carboxypiperazin-4yl)-propyl-1-phosphonic acid; GABA, g-amino-butyric acid; HPA axis, hypothalamic – pituitary – adrenal axis; LVGCCs, L-type voltage-gated Ca2+ channels; NGS, normal goat serum; NMDA, N-methyl-daspartate; NRS, normal rabbit serum; PBS, phosphate-buffered saline; pCREB, phosphorylated cAMP response element binding protein; PKA, protein kinase A; p-MPPI, 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)benzamide hydrochloride; PND, postnatal day; SEM, standard error of mean * Corresponding author. Fax: +30 210 746 1489. E-mail address: [email protected] (F. Stylianopoulou). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.06.032
It is generally acknowledged that early experiences, particularly mother – offspring interactions, play an important role in determining adult behavioral patterns. However, the underlying cellular and molecular mechanisms remain largely elusive. In an effort to address this issue, we have employed the well-described experimental paradigm of ‘‘early handling’’, which has been shown to alter the programming of hypothalamic – pituitary – adrenal axis function, in such a way that the ability of the adult organism to respond, cope, and adapt to stressful stimuli is increased [8,23]. As a consequence, handled rats show less fear in novel environments, more exploratory behavior, and lower emotionality [23,25,51]. Although according to the originally developed model of ‘‘early handling’’ [17] animals are
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exposed to brief periods of ‘‘handling’’ during the first 3 weeks of life, even 1 day of ‘‘early handling’’ has been shown to affect HPA function [21]. Since the effects of ‘‘early handling’’ are long term, the experience must alter the levels of molecules which play a determinant role in brain development and plasticity, such as the neurotrophins. Indeed, it has been shown that ‘‘handling’’ increases NGF in the hippocampus [36]. However, among the neurotrophins, BDNF appears to be the molecule which plays the most important role in brain plasticity. BDNF is involved in neuronal survival and differentiation [11], synaptic plasticity, as well as behavioral interactions between the organism and its environment [50]. BDNF is crucial for regulating cell death, the development of patterned connections, the growth and complexity of dendrites in the cerebral cortex, as well as synaptic strength [11]. The BDNF gene has a CRE element in its promoter, and it has been well documented that BDNF gene expression is controlled by the transcription factor pCREB [43,44]. Activation of this transcription factor by phosphorylation on Ser 133 is effected by PKA, following activation of the cyclic AMP signal transduction pathway [13]. It is noteworthy that ‘‘early handling’’ has been shown to increase hippocampal PKA activity and cyclic AMP levels [24]. CREB has also been shown to be phosphorylated by other kinases as well, such as CaMKII [42], CaMKIV [43], or MAPK [31], depending on the system examined. Following neural activity, phosphorylation of CREB has been shown to be mediated by either CaMKII [42] or CaMKIV [43], which are activated by Ca2+ ions, entering the cell through L-type voltage-gated Ca2+ channels (LVGCCs) or NMDA receptors, following depolarization of the neuronal membrane. Glutamate acting through AMPA/kainate and NMDA receptors is well known to be the major excitatory neurotransmitter in the rat cortex and hippocampus. However, during brain development, including the first postnatal week, GABA-A receptors also act as depolarizing and have been shown to play an important morphogenetic role [3]. Furthermore, ‘‘handling’’ has been shown to increase GABA-A/benzodiazepine receptor levels [4]. On the other hand, serotonin receptors have been implicated in the cellular mechanisms through which ‘‘early handling’’ affects brain function: 5-HT 2A receptors have been shown to mediate the ‘‘handling’’-induced increase in hippocampal GR levels [26], while ‘‘handling’’ increases 5-HT2 receptor binding [48] and type 1A receptor sensitivity [34]. Since the sensory stimulation altered by handling is quite complex (tactile, olfactory, auditory), it is expected that multiple neurotransmitter systems are involved in mediating the effects of handling. Based on the above, we determined BDNF and pCREB levels in the hippocampus—a major brain region controlling the HPA axis, and a target tissue of ‘‘early handling’’ [22 –24,26,36] 2, 4, and 8 h following ‘‘handling’’ on the first postnatal day, in the absence and presence of NMDA, AMPA, GABA-A, serotonin type 1A or 2A/C receptor, or L-type voltage-gated Ca2+ channel blockers.
2. Materials and methods 2.1. Animals Wistar rats of both genders reared in our laboratory were kept under standard conditions (24 -C; 12:12 h light/ dark cycle, lights on at 8:00 a.m.) and received food and water ad libitum. Virgin females were exposed to stud males and pregnancy was determined by the presence of sperm in the vaginal smear (day 0 of pregnancy). Prior to birth, litters from each dam were randomly assigned to either the handled or non-handled category. The average litter size was 8 T 1 pups (mean T SEM, range: 5– 13). Litters were not culled, since it has been shown that litter size within this range (5 –18) does not affect maternal behavior [5]. The gender ratio did not differ among the litters employed in the different animal groups. A total number of 137 male and female Wistar pups were used for all studies. The day of birth was defined as postnatal day 0 (PND0). Two different cohorts of animals were employed: (A) Non-injected (control), handled (n = 19), and nonhandled (n = 20); (B) Injected with either vehicle or one of the drugs, handled, and non-handled (see Drug administration section). All animal experimentations were carried out in agreement with ethical recommendation of the European Communities Council Directive of 24 November 1986 (86/609/EEC). 2.2. ‘‘Early handling’’ (‘‘Neonatal handling’’) The ‘‘early handling’’ protocol employed was as originally described by Levine [17], which involves removal of the pups from the nest for 15 min daily, during the neonatal period and placing them in a separate container, under a lamp in order to keep them warm. In the present experiments, ‘‘handling’’ was performed only on the first postnatal day (PND1). Specifically, in the first cohort of animals (non-injected) on PND1, between 9:00 and 10:00 a.m. (22 – 26 h after birth), mothers of the pups to be subjected to ‘‘handling’’ were removed from their home cages and temporarily placed separately into clean cages. Their pups were then removed and placed into plastic containers, lined with paper towels. After 15 min, the pups, and then their mothers, were returned to their home cages. ‘‘Nonhandled’’ pups were left undisturbed with their mothers in their home cage until sacrifice. Pups in the second cohort (injected either with vehicle or one of the drugs), immediately following the injections on PND1 between 9:00 and 10:00 a.m., were either subjected to ‘‘handling’’ (as described above) or returned to their home cage (‘‘nonhandled’’), as were their mothers. It should be noted that the ‘‘non-handled’’ animals of the second cohort were subjected to the manipulation of the injection, but were not exposed to the 15-min removal from the nest, thus being coined ‘‘non-handled’’.
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2.3. Drug administration For the second cohort of animals (injected), on PND1, the mothers were removed from their cages and pups were injected intraperitoneally (50 Al) with: (A) 7 mg/kg b.w. CPP (n = 13), a competitive NMDA receptor antagonist ([T]-3-(2-carboxypiperazin-4yl)-propyl-1-phosphonic acid, Sigma-Aldrich, USA), (B) 60 mg/kg b.w. CNQX (n = 9), a competitive AMPA/kainate receptor antagonist (6-cyano7-nitroquinoxaline-2,3-dione disodium salt, Sigma-Aldrich, USA), (C) 0.5 mg/kg b.w. bicuculline (n = 12), a GABA-A receptor antagonist (Sigma-Aldrich, USA), (D) 3 mg/kg b.w. nimodipine (n = 11), a potent L-type calcium channel antagonist (Bayer, Germany), (E) 4.75 mg/kg b.w. p-MPPI (n = 10), a 5-HT1A receptor antagonist (4-iodo-N-[2-[4(methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)benzamide hydrochloride, Sigma-Aldrich, USA), (F) 5 mg/kg b.w. ketanserin (n = 13), a 5-HT2A/C receptor antagonist (Sigma-Aldrich, USA), (G) vehicle (n = 32, vehicle control animals). Each animal within a litter was randomly assigned to receive either one of the different drugs or vehicle. 2.4. Tissue preparation 2, 4, or 8 h after the end of the ‘‘early handling’’ procedure, pups were anesthetized by placing on ice and were transcardially perfused with 30 ml of ice-cold 4% paraformaldehyde in phosphate buffer (0.1 M, pH = 7.4). Brains were isolated, post-fixed overnight in the perfusion medium at 4 -C, further processed and embedded in paraffin. Serial sagittal sections (7 Am) were cut and mounted on silane-coated slides. 2.5. Immunocytochemistry Immunocytochemistry was performed as previously described [19]. Sections were deparaffinized, rehydrated, and subsequently subjected to antigen retrieval in a microwave oven for 15 min in citrate buffer (10 mM, pH = 6.0). Following washing with phosphate-buffered saline (PBS) containing 0.4% Triton X-100 (SERVA, Germany), slides were incubated in PBS containing 0.4% Triton X-100 and 10% normal goat serum (NGS) (Dako, Denmark) or 10% normal rabbit serum (NRS) (Dako, Denmark), depending on the primary antibody used, for 1 h at room temperature. Afterwards, sections were incubated overnight at 4 -C with either the rabbit polyclonal anti-BDNF antibody (Santa Cruz, USA) at a concentration of 2 Ag/ml or the goat polyclonal anti-pCREB antibody (Santa Cruz, USA) at a concentration of 1 Ag/ml, diluted in PBS containing 0.4% Triton X-100 and either 4% NGS or 4% NRS, respectively. Following primary antibody incubation, slides were washed in PBS and incubated for 1 h at room temperature with either a biotinylated goat anti-rabbit or a rabbit anti-goat antibody (Dako, Denmark) at a concentration of 5 Ag/ml diluted in PBS containing either 2% NGS or 2% NRS.
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Following several rinses in PBS, slides were exposed to the ABC reagent (Dako, Denmark) for 30 min at room temperature. Slides were then washed in PBS and stained with 3,3V-diaminobenzidine (DAB) (1.7mM, SigmaAldrich, USA) diluted in Tris –HCl buffer (10 mM, pH = 7.6) and 0.03% H2O2 for 2– 5 min at room temperature. Finally, they were washed, dehydrated, and coverslipped with DePex (SERVA, Germany) and analyzed microscopically under a brightfield microscope. 2.6. Quantification of results The image analysis program ‘‘Image Pro Plus’’ (Media Cybernetics, USA) was used for cell counting in (A) handled and non-handled, control (non-injected) or vehicle-injected animals, at 2, 4, or 8 h following drug injection and ‘‘handling’’ (time course); (B) at 8 h in handled and non-handled, vehicle- or drug-injected animals. In all animal groups, the number of pCREB and BDNF immunopositive cells was determined in the same area of the CA1 hippocampal region, blindly by two independent investigators. A ‘‘threshold’’ was set in the image analysis system, in order to include in the counting only cells stained above a certain, pre-set degree. Three to six sections from each brain were evaluated, and in each brain section, cells were counted in 2 different optical fields of the CA1 area. The number of positive cells per optical field of each animal was the average value calculated from the data of the two optical fields in all brain sections evaluated. 2.7. Statistical analysis Data from the time course were analyzed by three-way analysis of variance (ANOVA) with ‘‘handling’’, injection (no injection vs. vehicle injection), and time as independent factors. Two-way analysis of variance (ANOVA) with ‘‘handling’’ and type of drug injected as independent factors was employed for the analysis of data from the 8 h drug or vehicle-injected (handled, non-handled) animals. When interactions between the independent factors were detected, separate one-way ANOVAs followed by Bonferroni post hoc tests were performed in order to identify specific differences between groups. The level of statistical significance was set at 0.05. All tests were performed with the SPSS software (Release 10.0.1, SPSS, USA).
3. Results 3.1. Time course 3.1.1. pCREB immunoreactivity The number of pCREB immunopositive cells in the rat CA1 area, on PND1, was significantly affected by ‘‘handling’’ ( F 1,58 = 37.794, P < 0.001), vehicle injection ( F 1,58 = 62.119, P < 0.001), and time ( F 1,58 = 9.462, P < 0.001), as
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revealed by a three-way ANOVA with ‘‘handling’’, injection, and time as independent factors. Moreover, the statistical analysis indicated the existence of a significant ‘‘handling’’ time interaction ( F 2,58 = 4.188, P = 0.02) and a significant injection time interaction ( F 2,58 = 6.643, P = 0.003). Post hoc analysis of the ‘‘handling’’ time interaction revealed that handled animals had more pCREB immunopositive cells than non-handled animals, 8 h after ‘‘handling’’ (Figs. 1 and 2). No such difference was detected at 2 h. It should be noted that at 4 h, an increase close to statistical significance was found in the number of pCREB immunopositive cells in the handled animals ( P = 0.059). Post hoc analysis of the injection time interaction showed that vehicle-injected animals had less immunopositive cells than non-injected animals both at 2 and 4 h following injection (Fig. 2). No injection effect was evident at 8 h. 3.1.2. BDNF immunoreactivity Likewise, a similar statistical analysis (three-way ANOVA) showed that the number of BDNF immunopositive cells in the CA1 area was significantly affected by ‘‘handling’’ ( F 1,57 = 32.253, P < 0.001), vehicle injection ( F 1,57 = 111.895, P < 0.001), and time ( F 1,57 = 129.915, P < 0.001). Moreover, statistical analysis identified a significant ‘‘handling’’ time interaction ( F 2,57 = 15.459, P < 0.001) and a significant injection time interaction ( F 2,57 =
37.047, P < 0.001). Post hoc analysis of the ‘‘handling’’ time interaction indicated that handled animals had more BDNF immunopositive cells than the non-handled, only at the 8 h after ‘‘handling’’ time point (Figs. 1 and 2). No such difference was detected 2 or 4 h following ‘‘handling’’. Post hoc analysis of the injection time interaction revealed that vehicle-injected animals had less BDNF immunopositive cells than non-injected animals both at 2 and 4 h following ‘‘handling’’ (Fig. 2). No injection effect was evident at 8 h. It should be mentioned that no ‘‘handling’’ injection interaction was observed for either pCREB or BDNF immunopositive cell numbers, at any of the time points studied. 3.2. Drug injection 3.2.1. pCREB immunoreactivity The data regarding the number of pCREB immunopositive cells in the CA1 area 8 h following ‘‘handling’’ were analyzed by two-way ANOVA, with ‘‘handling’’ and type of drug injected as independent factors. This analysis revealed a statistically significant effect of drug ( F 6,65 = 2.814, P = 0.017) and ‘‘handling’’ type of drug injected interaction ( F 6,65 = 7.102, P < 0.001). Post hoc analysis of this interaction showed that there was an effect of ‘‘handling’’ in the vehicle-injected group, with handled animals having more pCREB immunolabeled cells than the
Fig. 1. Representative immunocytochemistry of pCREB and BDNF in the hippocampus of control animals on PND1, 8 h following ‘‘handling’’. The scale bar corresponds to 100 Am. The rectangles denote the area in which the pCREB and BDNF positive cells were counted.
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Fig. 2. (A) High-magnification photomicrographs of pCREB and BDNF positive cells in the hippocampal CA1 area of control animals on PND1, 8 h following ‘‘handling’’. The arrows point to cells counted as pCREB or BDNF positive, respectively. The scale bar corresponds to 20 Am. (B) Time course of the effects of vehicle injection and ‘‘early handling’’ on the numbers of pCREB and BDNF positive cells in the CA1 area of the hippocampus on PND1. Bars represent means T SEM. Numbers within the bars denote the number of animals in each group (n). #Statistically significant injection-induced decrease, in both handled and non-handled animals ( P 0.01, Bonferroni post hoc tests, following 3-way ANOVA). *Statistically significant ‘‘handling’’-induced increase ( P 0.001, Bonferroni post hoc tests, following 3-way ANOVA).
non-handled. This ‘‘handling’’ effect was abolished when animals were injected prior to ‘‘handling’’ with CPP, CNQX, bicuculline, nimodipine, p-MPPI, or ketanserin, since the drugs inhibited the increase in the number of pCREB immunopositive cells observed in the vehicle-injected handled animals. Moreover, in the handled animals, administration of CPP, bicuculline, or nimodipine resulted in a statistically significant reduced number of pCREB positive cells, compared to the administration of vehicle. Interestingly, when handled animals were injected with CPP, the
decrease in the number of pCREB positive cells was so pronounced that an inverse ‘‘handling’’ effect was observed, with handled animals having less pCREB immunolabeled cells than the non-handled (Fig. 3). 3.2.2. BDNF immunoreactivity A similar statistical analysis was performed for the data on the number of BDNF immunolabeled cells in the CA1 area 8 h after ‘‘handling’’. This analysis indicated a significant effect of drug ( F 6,63 = 5.343, P < 0.001) and
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Fig. 3. Effect of CPP, CNQX, bicuculline, p-MPPI, ketanserin (NMDA, AMPA, GABA-A, 5-HT1A, and 5-HT2A/2C receptor antagonists, respectively), as well as nimodipine (L-type VGCC blocker), on the increase in pCREB positive cells observed in the hippocampus 8 h following ‘‘early handling’’. Bars represent means T SEM. Numbers within the bars denote the number of animals in each group (n). *Statistically significant difference between handled and non-handled animals ( P = 0.028 for the vehicle-injected group and P = 0.033 for the CPP-injected group, Bonferroni post hoc tests, following 2-way ANOVA). .Statistically significant difference between drug- and vehicle-injected handled animals ( P < 0.001 for CPP, P = 0.013 for bicuculline, P = 0.003 for nimodipine, Bonferroni post hoc tests, following 2-way ANOVA).
‘‘handling’’ type of drug injected interaction ( F 6,63 = 5.811, P < 0.001). Post hoc analysis of this interaction showed an effect of ‘‘handling’’ in the vehicle-injected group, with handled animals having more BDNF immunolabeled cells than the non-handled. This ‘‘handling’’ effect was abolished when animals were injected prior to ‘‘handling’’ with CPP, CNQX, bicuculline, nimodipine, pMPPI, or ketanserin, since the drugs significantly inhibited the increase in the number of BDNF immunopositive cells observed in the vehicle-injected handled animals. Interestingly, when animals were injected with CPP, an inverse ‘‘handling’’ effect was evident, with handled animals having less BDNF immunolabeled cells than the nonhandled (Fig. 4).
4. Discussion Our results revealed two independent experimentally induced effects on the number of pCREB and BDNF positive cells in the rat CA1 hippocampal area, on PND1. One involved the effect of vehicle injection and the other the effect of ‘‘handling’’. The effect of vehicle injection resulted in decreased numbers of pCREB and BDNF cells, in both handled and non-handled animals. This effect was fairly immediate and transient, being detectable at 2 and 4 h and having ceased at 8 h after ‘‘handling’’. Furthermore, this effect did not appear to be mediated through NMDA, AMPA, GABA-A, 5-HT1A or 2A receptors, or LVGCCs, since their blockade did not
Fig. 4. Effect of CPP, CNQX, bicuculline, p-MPPI, ketanserin (NMDA, AMPA, GABA-A, 5-HT1A, and 5-HT2A/2C receptor antagonists, respectively), as well as nimodipine (L-type VGCC blocker), on the increase in BDNF positive cells observed in the hippocampus 8 h following ‘‘early handling’’. Bars represent means T SEM. Numbers within the bars denote the number of animals in each group (n). *Statistically significant difference between handled and non-handled animals ( P = 0.031 for the vehicle-injected group and P = 0.049 for the CPP-injected group, Bonferroni post hoc tests, following 2-way ANOVA). .Statistically significant difference between drug- and vehicle-injected handled animals ( P < 0.001 for CPP, CNQX, nimodipine, or ketanserin, P = 0.007 for pMPPI, Bonferroni post hoc tests, following 2-way ANOVA).
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inhibit the injection-induced effect (data not shown). The decrease in pCREB and BDNF can be viewed as a response of the brain to the ‘‘stress’’ of vehicle injection. It has also been reported previously that injection of vehicle to neonatal rats is a stressful stimulus, and elicits cellular responses in the brain [47], in spite of the fact that it occurs within the stress-hyporesponsive period [40]. It should be noted, however, that the ‘‘hyporesponsiveness’’ refers to the function of the HPA axis [47]. The other component mediating the stress response, the locus coeruleus/noradrenergic system, is functional at this developmental stage [6] and can probably mediate the observed effects, either by increasing the activity of a phosphatase – which can dephosphorylate pCREB – or decrease that of a respective kinase. Relevant to this point is the report that noradrenaline induces mitogen-activated protein kinase phosphatase-1 expression [37]. The decreased levels of pCREB can in turn result in decreased expression of BDNF [44]. ‘‘Handling’’ resulted in increased numbers of BDNF and pCREB cells 8 h later, in both vehicle-injected and noninjected animals, while at 2 or 4 h, no ‘‘handling’’-induced effect was evident. The ‘‘handling’’-induced increase in BDNF and pCREB was not observed, when CPP, CNQX, bicuculline, p-MPPI, ketanserin, or nimodipine (NMDA, AMPA/kainate, GABA-A, 5HT-1A, 5HT-2A/C receptor, or LVGCC antagonist, respectively) were injected prior to the ‘‘handling’’ procedure. These results indicate that these types of receptors and channels are involved in mediating the effects of ‘‘early handling’’ on the developing rat brain. Regarding the role of NMDA, AMPA/kainate, GABA-A receptors, and the L-type voltage-gated Ca2+ channels, we can propose the following possible mechanism: ‘‘Handling’’, which alters tactile and possibly olfactory and auditory stimulation of the pups, initiates neuronal activity. This activates AMPA and GABA-A receptors and results in membrane depolarization, which in turn leads to activation of NMDA receptors and opening of LVGCCs. It has been well documented that GABA-A receptors act as depolarizing during brain development and along with AMPA receptors mediate NMDA receptor activation and opening of LVGCCs [2,3]. Calcium ions can then enter the cell and activate Ca2+-dependent kinases, which can phosphorylate and activate the transcription factor CREB. Among Ca2+dependent kinases, CaMK II and IV have been shown to be particularly important in mediating neuronal activitydependent CREB phosphorylation [42 – 44]. Furthermore, CREB can be phosphorylated by PKA following activation of Ca2+-sensitive adenylate cyclase [13]. Interestingly, ‘‘handling’’ has been shown to increase PKA activity [24]. The activated, by phosphorylation, transcription factor CREB is a key molecule in the cellular mechanisms underlying brain plasticity [28,46,49]. In the final step of the mechanism mediating the effects of ‘‘early handling’’, pCREB can bind to the BDNF gene control region and
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induce its expression [43,44]. The functional relationship between pCREB and BDNF is also supported by the finding that partial deletion of the CREB gene results in decreased BDNF mRNA expression and promotes alcohol-drinking behaviors [33]. It is worth noting that ‘‘early handling’’ decreases alcohol consumption [10]. Induction of BDNF by pCREB has been shown to occur as a result of activitydependent neuronal plasticity [43,44]. Furthermore, chronic stress results in decreased protein levels of both CREB and BDNF [15,38], while physical exercise increases the mRNA of both molecules [27]. In our study, both BDNF and pCREB are significantly increased at 8 h, while at 4 h, no change in BDNF is observed, whereas pCREB shows a trend to increase. It thus appears that the BDNF gene is the target of the transcription factor pCREB. On the other hand, it has been shown that BDNF signaling can lead to increased pCREB [50]. Hence, the pCREB-induced BDNF can lead to further increases in pCREB, establishing a positive feedback loop between these two plasticity-related molecules following ‘‘handling’’. The increased BDNF induced by ‘‘handling’’ can exert its morphogenetic roles and ‘‘imprint’’ the effects of the neonatal experience on the developing rat brain. Indeed, it has been shown that BDNF has an important morphogenetic role [20] in the development of the serotonergic system, which is also influenced by ‘‘early handling’’ [32,34,48]. BDNF can increase dendritic spine density [45] and mediate LTP (Long-Term Potentiation) [14], effects which have also been described to be induced by ‘‘handling’’ [30,52]. Interestingly, rearing in an enriched environment, which has been shown to have similar effects as ‘‘early handling’’ [8], also results in increased BDNF protein [39]. On the other hand, maternal deprivation, which has opposite effects from those of ‘‘early handling’’ on brain function and plasticity [16], results in decreased protein levels of pCREB [12] and BDNF [38]. Most interestingly, the increase in BDNF induced by 1 day of ‘‘handling’’ has long-term consequences, since it is maintained in the brain of the adult handled animals [9]. The ‘‘handling’’-induced increased BDNF could be related to the effects of ‘‘early handling’’ in improving active avoidance acquisition [7] and preventing the spatial learning deficits, which accompany aging [22,23], since it is well documented that BDNF plays an important role in learning and memory [53]. Moreover, the increased BDNF could be related to the decreased vulnerability of male handled animals in chronic stress-induced animal models of depression [35], since current data implicate BDNF in depression [29]. Furthermore, the neuroprotective effect of ‘‘early handling’’, which has been shown to increase neuronal survival in the aging hippocampus [22,23], can also be attributed to the neurotrophic effects [11] of the ‘‘handling’’-induced increased BDNF. It is noteworthy that after administration of the competitive NMDA receptor antagonist CPP, not only was the ‘‘handling’’-induced increase in pCREB and BDNF abol-
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ished, but a ‘‘reverse’’ effect was observed: handled animals had lower numbers of pCREB and BDNF positive cells than the non-handled. This result could be attributed to a parallel ‘‘handling’’-induced activation of NMDA receptors, both following AMPA and GABA-A receptor-mediated depolarization. Indeed, it has been shown that these two mechanisms operate in hippocampal cells [2,3]. Our findings point to the NMDA receptor as a key molecule in the cellular mechanisms underlying the effects of ‘‘early handling’’ on the developing brain. The involvement of serotonin receptors both type 1A and 2A/C in the mechanism of the ‘‘handling’’-induced increase in pCREB and BDNF may be indirect, i.e., the cells possessing these receptors are probably different from those in which the levels of pCREB and BDNF are increased. On one hand, 5HT-1A receptors are located in the hippocampus, but they are inhibitory [1] and on the other, 5HT2A/C receptors are located mainly in the cortex and their levels are very low in the hippocampus at this developmental stage [18]. 5HT-1A receptors are localized on inhibitory hippocampal interneurons [1] which normally exert modulatory control on hippocampal neurotransmission. Thus, 5HT1A receptors physiologically mediate disinhibition [41] and activation of hippocampal neuronal activity, which – as mentioned previously [42,46] – is accompanied by an increase in pCREB. Based on our results, we can infer that neuronal circuits involving the abovementioned 5HT1A receptor-containing interneurons are activated by ‘‘handling’’, since their blockade cancels the increase in pCREB and BDNF induced by ‘‘handling’’. The cortical cells having the 5HT-2A/C receptors could be activated by the ‘‘handling’’-induced sensory stimulation, and in turn excite trans-synaptically the hippocampal cells, resulting in increased pCREB and BDNF, in the latter. Other previously reported effects of handling, such as an increase in hippocampal GR [26] and cAMP [24] levels, have also been shown to be mediated via 5-HT2A/C receptors. Based on our results, we propose that the altered sensory stimulation of ‘‘early handling’’ leads to depolarization of hippocampal cells via AMPA and GABA-A receptors, which then results in activation of NMDA receptors and opening of LVGCCs, through which calcium ions enter the cell. The increased intracellular calcium in turn activates calciumdependent kinases and leads to phosphorylation of the transcription factor pCREB, which induces the expression of BDNF. ‘‘Handling’’ also stimulates 5HT-1A or 5-HT2A/ 2C receptor-containing cells, which trans-synaptically activate the abovementioned calcium-dependent mechanism in hippocampal cells having NMDA, AMPA, and GABA-A receptors, resulting in increased pCREB and BDNF. These molecules participate in neuronal plasticity processes, and mediate the effects of ‘‘early handling’’ leading to improved adaptability and ability to cope with novel environmental stimuli.
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Research Report
Cellular mechanisms underlying an effect of ‘‘early handling’’ on pCREB and BDNF in the neonatal rat hippocampus Efstathios Garoflos, Antonios Stamatakis, Athanasios Mantelas, Helen Philippidis, Fotini Stylianopoulou* Laboratory of Biology-Biochemistry, School of Health Sciences, University of Athens, Papadiamantopoulou 123, 11527 Athens, Greece Accepted 10 June 2005 Available online 15 July 2005
Abstract Early experiences have long-term effects on brain function and behavior. However, the precise mechanisms involved still remain elusive. In an effort to address this issue, we employed the model of ‘‘early handling’’, which is known to affect the ability of the adult organism to respond to stressful stimuli, and determined its effects on hippocampal pCREB and BDNF 2, 4, and 8 h later. 8 h following ‘‘handling’’ on postnatal day 1, there was an increase in pCREB and BDNF positive cells in the hippocampus, a brain area which is a specific target of ‘‘handling’’. On the other hand, vehicle injection resulted in decreased pCREB and BDNF in both handled and non-handled animals 2 and 4 h later. The ‘‘handling’’-induced increase of pCREB and BDNF was cancelled by inhibition of NMDA, AMPA/kainate, GABA-A, 5-HT1A or 5-HT2A/C receptors, as well as L-type voltage-gated Ca2+ channels. It thus appears that ‘‘early handling’’ activates these neurotransmitter receptors, leading to increased intracellular Ca2+, phosphorylation of the transcription factor CREB, and increased BDNF expression. BDNF can then exert its morphogenetic effects and thus ‘‘imprint’’ the effects of ‘‘handling’’ on the brain. D 2005 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Neural plasticity Keywords: Neonatal handling; pCREB; BDNF; Neurotransmitter receptor; L-type voltage-gated Ca2+ channel; Hippocampus
1. Introduction
Abbreviations: 5-HT, serotonin; AMPA, a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid; ANOVA, analysis of variance; BDNF, brainderived neurotrophic factor; CA1 area, field 1 of Ammon’s horn; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt; CPP, [T]-3-(2-carboxypiperazin-4yl)-propyl-1-phosphonic acid; GABA, g-amino-butyric acid; HPA axis, hypothalamic – pituitary – adrenal axis; LVGCCs, L-type voltage-gated Ca2+ channels; NGS, normal goat serum; NMDA, N-methyl-daspartate; NRS, normal rabbit serum; PBS, phosphate-buffered saline; pCREB, phosphorylated cAMP response element binding protein; PKA, protein kinase A; p-MPPI, 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)benzamide hydrochloride; PND, postnatal day; SEM, standard error of mean * Corresponding author. Fax: +30 210 746 1489. E-mail address: [email protected] (F. Stylianopoulou). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.06.032
It is generally acknowledged that early experiences, particularly mother – offspring interactions, play an important role in determining adult behavioral patterns. However, the underlying cellular and molecular mechanisms remain largely elusive. In an effort to address this issue, we have employed the well-described experimental paradigm of ‘‘early handling’’, which has been shown to alter the programming of hypothalamic – pituitary – adrenal axis function, in such a way that the ability of the adult organism to respond, cope, and adapt to stressful stimuli is increased [8,23]. As a consequence, handled rats show less fear in novel environments, more exploratory behavior, and lower emotionality [23,25,51]. Although according to the originally developed model of ‘‘early handling’’ [17] animals are
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exposed to brief periods of ‘‘handling’’ during the first 3 weeks of life, even 1 day of ‘‘early handling’’ has been shown to affect HPA function [21]. Since the effects of ‘‘early handling’’ are long term, the experience must alter the levels of molecules which play a determinant role in brain development and plasticity, such as the neurotrophins. Indeed, it has been shown that ‘‘handling’’ increases NGF in the hippocampus [36]. However, among the neurotrophins, BDNF appears to be the molecule which plays the most important role in brain plasticity. BDNF is involved in neuronal survival and differentiation [11], synaptic plasticity, as well as behavioral interactions between the organism and its environment [50]. BDNF is crucial for regulating cell death, the development of patterned connections, the growth and complexity of dendrites in the cerebral cortex, as well as synaptic strength [11]. The BDNF gene has a CRE element in its promoter, and it has been well documented that BDNF gene expression is controlled by the transcription factor pCREB [43,44]. Activation of this transcription factor by phosphorylation on Ser 133 is effected by PKA, following activation of the cyclic AMP signal transduction pathway [13]. It is noteworthy that ‘‘early handling’’ has been shown to increase hippocampal PKA activity and cyclic AMP levels [24]. CREB has also been shown to be phosphorylated by other kinases as well, such as CaMKII [42], CaMKIV [43], or MAPK [31], depending on the system examined. Following neural activity, phosphorylation of CREB has been shown to be mediated by either CaMKII [42] or CaMKIV [43], which are activated by Ca2+ ions, entering the cell through L-type voltage-gated Ca2+ channels (LVGCCs) or NMDA receptors, following depolarization of the neuronal membrane. Glutamate acting through AMPA/kainate and NMDA receptors is well known to be the major excitatory neurotransmitter in the rat cortex and hippocampus. However, during brain development, including the first postnatal week, GABA-A receptors also act as depolarizing and have been shown to play an important morphogenetic role [3]. Furthermore, ‘‘handling’’ has been shown to increase GABA-A/benzodiazepine receptor levels [4]. On the other hand, serotonin receptors have been implicated in the cellular mechanisms through which ‘‘early handling’’ affects brain function: 5-HT 2A receptors have been shown to mediate the ‘‘handling’’-induced increase in hippocampal GR levels [26], while ‘‘handling’’ increases 5-HT2 receptor binding [48] and type 1A receptor sensitivity [34]. Since the sensory stimulation altered by handling is quite complex (tactile, olfactory, auditory), it is expected that multiple neurotransmitter systems are involved in mediating the effects of handling. Based on the above, we determined BDNF and pCREB levels in the hippocampus—a major brain region controlling the HPA axis, and a target tissue of ‘‘early handling’’ [22 –24,26,36] 2, 4, and 8 h following ‘‘handling’’ on the first postnatal day, in the absence and presence of NMDA, AMPA, GABA-A, serotonin type 1A or 2A/C receptor, or L-type voltage-gated Ca2+ channel blockers.
2. Materials and methods 2.1. Animals Wistar rats of both genders reared in our laboratory were kept under standard conditions (24 -C; 12:12 h light/ dark cycle, lights on at 8:00 a.m.) and received food and water ad libitum. Virgin females were exposed to stud males and pregnancy was determined by the presence of sperm in the vaginal smear (day 0 of pregnancy). Prior to birth, litters from each dam were randomly assigned to either the handled or non-handled category. The average litter size was 8 T 1 pups (mean T SEM, range: 5– 13). Litters were not culled, since it has been shown that litter size within this range (5 –18) does not affect maternal behavior [5]. The gender ratio did not differ among the litters employed in the different animal groups. A total number of 137 male and female Wistar pups were used for all studies. The day of birth was defined as postnatal day 0 (PND0). Two different cohorts of animals were employed: (A) Non-injected (control), handled (n = 19), and nonhandled (n = 20); (B) Injected with either vehicle or one of the drugs, handled, and non-handled (see Drug administration section). All animal experimentations were carried out in agreement with ethical recommendation of the European Communities Council Directive of 24 November 1986 (86/609/EEC). 2.2. ‘‘Early handling’’ (‘‘Neonatal handling’’) The ‘‘early handling’’ protocol employed was as originally described by Levine [17], which involves removal of the pups from the nest for 15 min daily, during the neonatal period and placing them in a separate container, under a lamp in order to keep them warm. In the present experiments, ‘‘handling’’ was performed only on the first postnatal day (PND1). Specifically, in the first cohort of animals (non-injected) on PND1, between 9:00 and 10:00 a.m. (22 – 26 h after birth), mothers of the pups to be subjected to ‘‘handling’’ were removed from their home cages and temporarily placed separately into clean cages. Their pups were then removed and placed into plastic containers, lined with paper towels. After 15 min, the pups, and then their mothers, were returned to their home cages. ‘‘Nonhandled’’ pups were left undisturbed with their mothers in their home cage until sacrifice. Pups in the second cohort (injected either with vehicle or one of the drugs), immediately following the injections on PND1 between 9:00 and 10:00 a.m., were either subjected to ‘‘handling’’ (as described above) or returned to their home cage (‘‘nonhandled’’), as were their mothers. It should be noted that the ‘‘non-handled’’ animals of the second cohort were subjected to the manipulation of the injection, but were not exposed to the 15-min removal from the nest, thus being coined ‘‘non-handled’’.
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2.3. Drug administration For the second cohort of animals (injected), on PND1, the mothers were removed from their cages and pups were injected intraperitoneally (50 Al) with: (A) 7 mg/kg b.w. CPP (n = 13), a competitive NMDA receptor antagonist ([T]-3-(2-carboxypiperazin-4yl)-propyl-1-phosphonic acid, Sigma-Aldrich, USA), (B) 60 mg/kg b.w. CNQX (n = 9), a competitive AMPA/kainate receptor antagonist (6-cyano7-nitroquinoxaline-2,3-dione disodium salt, Sigma-Aldrich, USA), (C) 0.5 mg/kg b.w. bicuculline (n = 12), a GABA-A receptor antagonist (Sigma-Aldrich, USA), (D) 3 mg/kg b.w. nimodipine (n = 11), a potent L-type calcium channel antagonist (Bayer, Germany), (E) 4.75 mg/kg b.w. p-MPPI (n = 10), a 5-HT1A receptor antagonist (4-iodo-N-[2-[4(methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)benzamide hydrochloride, Sigma-Aldrich, USA), (F) 5 mg/kg b.w. ketanserin (n = 13), a 5-HT2A/C receptor antagonist (Sigma-Aldrich, USA), (G) vehicle (n = 32, vehicle control animals). Each animal within a litter was randomly assigned to receive either one of the different drugs or vehicle. 2.4. Tissue preparation 2, 4, or 8 h after the end of the ‘‘early handling’’ procedure, pups were anesthetized by placing on ice and were transcardially perfused with 30 ml of ice-cold 4% paraformaldehyde in phosphate buffer (0.1 M, pH = 7.4). Brains were isolated, post-fixed overnight in the perfusion medium at 4 -C, further processed and embedded in paraffin. Serial sagittal sections (7 Am) were cut and mounted on silane-coated slides. 2.5. Immunocytochemistry Immunocytochemistry was performed as previously described [19]. Sections were deparaffinized, rehydrated, and subsequently subjected to antigen retrieval in a microwave oven for 15 min in citrate buffer (10 mM, pH = 6.0). Following washing with phosphate-buffered saline (PBS) containing 0.4% Triton X-100 (SERVA, Germany), slides were incubated in PBS containing 0.4% Triton X-100 and 10% normal goat serum (NGS) (Dako, Denmark) or 10% normal rabbit serum (NRS) (Dako, Denmark), depending on the primary antibody used, for 1 h at room temperature. Afterwards, sections were incubated overnight at 4 -C with either the rabbit polyclonal anti-BDNF antibody (Santa Cruz, USA) at a concentration of 2 Ag/ml or the goat polyclonal anti-pCREB antibody (Santa Cruz, USA) at a concentration of 1 Ag/ml, diluted in PBS containing 0.4% Triton X-100 and either 4% NGS or 4% NRS, respectively. Following primary antibody incubation, slides were washed in PBS and incubated for 1 h at room temperature with either a biotinylated goat anti-rabbit or a rabbit anti-goat antibody (Dako, Denmark) at a concentration of 5 Ag/ml diluted in PBS containing either 2% NGS or 2% NRS.
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Following several rinses in PBS, slides were exposed to the ABC reagent (Dako, Denmark) for 30 min at room temperature. Slides were then washed in PBS and stained with 3,3V-diaminobenzidine (DAB) (1.7mM, SigmaAldrich, USA) diluted in Tris –HCl buffer (10 mM, pH = 7.6) and 0.03% H2O2 for 2– 5 min at room temperature. Finally, they were washed, dehydrated, and coverslipped with DePex (SERVA, Germany) and analyzed microscopically under a brightfield microscope. 2.6. Quantification of results The image analysis program ‘‘Image Pro Plus’’ (Media Cybernetics, USA) was used for cell counting in (A) handled and non-handled, control (non-injected) or vehicle-injected animals, at 2, 4, or 8 h following drug injection and ‘‘handling’’ (time course); (B) at 8 h in handled and non-handled, vehicle- or drug-injected animals. In all animal groups, the number of pCREB and BDNF immunopositive cells was determined in the same area of the CA1 hippocampal region, blindly by two independent investigators. A ‘‘threshold’’ was set in the image analysis system, in order to include in the counting only cells stained above a certain, pre-set degree. Three to six sections from each brain were evaluated, and in each brain section, cells were counted in 2 different optical fields of the CA1 area. The number of positive cells per optical field of each animal was the average value calculated from the data of the two optical fields in all brain sections evaluated. 2.7. Statistical analysis Data from the time course were analyzed by three-way analysis of variance (ANOVA) with ‘‘handling’’, injection (no injection vs. vehicle injection), and time as independent factors. Two-way analysis of variance (ANOVA) with ‘‘handling’’ and type of drug injected as independent factors was employed for the analysis of data from the 8 h drug or vehicle-injected (handled, non-handled) animals. When interactions between the independent factors were detected, separate one-way ANOVAs followed by Bonferroni post hoc tests were performed in order to identify specific differences between groups. The level of statistical significance was set at 0.05. All tests were performed with the SPSS software (Release 10.0.1, SPSS, USA).
3. Results 3.1. Time course 3.1.1. pCREB immunoreactivity The number of pCREB immunopositive cells in the rat CA1 area, on PND1, was significantly affected by ‘‘handling’’ ( F 1,58 = 37.794, P < 0.001), vehicle injection ( F 1,58 = 62.119, P < 0.001), and time ( F 1,58 = 9.462, P < 0.001), as
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revealed by a three-way ANOVA with ‘‘handling’’, injection, and time as independent factors. Moreover, the statistical analysis indicated the existence of a significant ‘‘handling’’ time interaction ( F 2,58 = 4.188, P = 0.02) and a significant injection time interaction ( F 2,58 = 6.643, P = 0.003). Post hoc analysis of the ‘‘handling’’ time interaction revealed that handled animals had more pCREB immunopositive cells than non-handled animals, 8 h after ‘‘handling’’ (Figs. 1 and 2). No such difference was detected at 2 h. It should be noted that at 4 h, an increase close to statistical significance was found in the number of pCREB immunopositive cells in the handled animals ( P = 0.059). Post hoc analysis of the injection time interaction showed that vehicle-injected animals had less immunopositive cells than non-injected animals both at 2 and 4 h following injection (Fig. 2). No injection effect was evident at 8 h. 3.1.2. BDNF immunoreactivity Likewise, a similar statistical analysis (three-way ANOVA) showed that the number of BDNF immunopositive cells in the CA1 area was significantly affected by ‘‘handling’’ ( F 1,57 = 32.253, P < 0.001), vehicle injection ( F 1,57 = 111.895, P < 0.001), and time ( F 1,57 = 129.915, P < 0.001). Moreover, statistical analysis identified a significant ‘‘handling’’ time interaction ( F 2,57 = 15.459, P < 0.001) and a significant injection time interaction ( F 2,57 =
37.047, P < 0.001). Post hoc analysis of the ‘‘handling’’ time interaction indicated that handled animals had more BDNF immunopositive cells than the non-handled, only at the 8 h after ‘‘handling’’ time point (Figs. 1 and 2). No such difference was detected 2 or 4 h following ‘‘handling’’. Post hoc analysis of the injection time interaction revealed that vehicle-injected animals had less BDNF immunopositive cells than non-injected animals both at 2 and 4 h following ‘‘handling’’ (Fig. 2). No injection effect was evident at 8 h. It should be mentioned that no ‘‘handling’’ injection interaction was observed for either pCREB or BDNF immunopositive cell numbers, at any of the time points studied. 3.2. Drug injection 3.2.1. pCREB immunoreactivity The data regarding the number of pCREB immunopositive cells in the CA1 area 8 h following ‘‘handling’’ were analyzed by two-way ANOVA, with ‘‘handling’’ and type of drug injected as independent factors. This analysis revealed a statistically significant effect of drug ( F 6,65 = 2.814, P = 0.017) and ‘‘handling’’ type of drug injected interaction ( F 6,65 = 7.102, P < 0.001). Post hoc analysis of this interaction showed that there was an effect of ‘‘handling’’ in the vehicle-injected group, with handled animals having more pCREB immunolabeled cells than the
Fig. 1. Representative immunocytochemistry of pCREB and BDNF in the hippocampus of control animals on PND1, 8 h following ‘‘handling’’. The scale bar corresponds to 100 Am. The rectangles denote the area in which the pCREB and BDNF positive cells were counted.
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Fig. 2. (A) High-magnification photomicrographs of pCREB and BDNF positive cells in the hippocampal CA1 area of control animals on PND1, 8 h following ‘‘handling’’. The arrows point to cells counted as pCREB or BDNF positive, respectively. The scale bar corresponds to 20 Am. (B) Time course of the effects of vehicle injection and ‘‘early handling’’ on the numbers of pCREB and BDNF positive cells in the CA1 area of the hippocampus on PND1. Bars represent means T SEM. Numbers within the bars denote the number of animals in each group (n). #Statistically significant injection-induced decrease, in both handled and non-handled animals ( P 0.01, Bonferroni post hoc tests, following 3-way ANOVA). *Statistically significant ‘‘handling’’-induced increase ( P 0.001, Bonferroni post hoc tests, following 3-way ANOVA).
non-handled. This ‘‘handling’’ effect was abolished when animals were injected prior to ‘‘handling’’ with CPP, CNQX, bicuculline, nimodipine, p-MPPI, or ketanserin, since the drugs inhibited the increase in the number of pCREB immunopositive cells observed in the vehicle-injected handled animals. Moreover, in the handled animals, administration of CPP, bicuculline, or nimodipine resulted in a statistically significant reduced number of pCREB positive cells, compared to the administration of vehicle. Interestingly, when handled animals were injected with CPP, the
decrease in the number of pCREB positive cells was so pronounced that an inverse ‘‘handling’’ effect was observed, with handled animals having less pCREB immunolabeled cells than the non-handled (Fig. 3). 3.2.2. BDNF immunoreactivity A similar statistical analysis was performed for the data on the number of BDNF immunolabeled cells in the CA1 area 8 h after ‘‘handling’’. This analysis indicated a significant effect of drug ( F 6,63 = 5.343, P < 0.001) and
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Fig. 3. Effect of CPP, CNQX, bicuculline, p-MPPI, ketanserin (NMDA, AMPA, GABA-A, 5-HT1A, and 5-HT2A/2C receptor antagonists, respectively), as well as nimodipine (L-type VGCC blocker), on the increase in pCREB positive cells observed in the hippocampus 8 h following ‘‘early handling’’. Bars represent means T SEM. Numbers within the bars denote the number of animals in each group (n). *Statistically significant difference between handled and non-handled animals ( P = 0.028 for the vehicle-injected group and P = 0.033 for the CPP-injected group, Bonferroni post hoc tests, following 2-way ANOVA). .Statistically significant difference between drug- and vehicle-injected handled animals ( P < 0.001 for CPP, P = 0.013 for bicuculline, P = 0.003 for nimodipine, Bonferroni post hoc tests, following 2-way ANOVA).
‘‘handling’’ type of drug injected interaction ( F 6,63 = 5.811, P < 0.001). Post hoc analysis of this interaction showed an effect of ‘‘handling’’ in the vehicle-injected group, with handled animals having more BDNF immunolabeled cells than the non-handled. This ‘‘handling’’ effect was abolished when animals were injected prior to ‘‘handling’’ with CPP, CNQX, bicuculline, nimodipine, pMPPI, or ketanserin, since the drugs significantly inhibited the increase in the number of BDNF immunopositive cells observed in the vehicle-injected handled animals. Interestingly, when animals were injected with CPP, an inverse ‘‘handling’’ effect was evident, with handled animals having less BDNF immunolabeled cells than the nonhandled (Fig. 4).
4. Discussion Our results revealed two independent experimentally induced effects on the number of pCREB and BDNF positive cells in the rat CA1 hippocampal area, on PND1. One involved the effect of vehicle injection and the other the effect of ‘‘handling’’. The effect of vehicle injection resulted in decreased numbers of pCREB and BDNF cells, in both handled and non-handled animals. This effect was fairly immediate and transient, being detectable at 2 and 4 h and having ceased at 8 h after ‘‘handling’’. Furthermore, this effect did not appear to be mediated through NMDA, AMPA, GABA-A, 5-HT1A or 2A receptors, or LVGCCs, since their blockade did not
Fig. 4. Effect of CPP, CNQX, bicuculline, p-MPPI, ketanserin (NMDA, AMPA, GABA-A, 5-HT1A, and 5-HT2A/2C receptor antagonists, respectively), as well as nimodipine (L-type VGCC blocker), on the increase in BDNF positive cells observed in the hippocampus 8 h following ‘‘early handling’’. Bars represent means T SEM. Numbers within the bars denote the number of animals in each group (n). *Statistically significant difference between handled and non-handled animals ( P = 0.031 for the vehicle-injected group and P = 0.049 for the CPP-injected group, Bonferroni post hoc tests, following 2-way ANOVA). .Statistically significant difference between drug- and vehicle-injected handled animals ( P < 0.001 for CPP, CNQX, nimodipine, or ketanserin, P = 0.007 for pMPPI, Bonferroni post hoc tests, following 2-way ANOVA).
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inhibit the injection-induced effect (data not shown). The decrease in pCREB and BDNF can be viewed as a response of the brain to the ‘‘stress’’ of vehicle injection. It has also been reported previously that injection of vehicle to neonatal rats is a stressful stimulus, and elicits cellular responses in the brain [47], in spite of the fact that it occurs within the stress-hyporesponsive period [40]. It should be noted, however, that the ‘‘hyporesponsiveness’’ refers to the function of the HPA axis [47]. The other component mediating the stress response, the locus coeruleus/noradrenergic system, is functional at this developmental stage [6] and can probably mediate the observed effects, either by increasing the activity of a phosphatase – which can dephosphorylate pCREB – or decrease that of a respective kinase. Relevant to this point is the report that noradrenaline induces mitogen-activated protein kinase phosphatase-1 expression [37]. The decreased levels of pCREB can in turn result in decreased expression of BDNF [44]. ‘‘Handling’’ resulted in increased numbers of BDNF and pCREB cells 8 h later, in both vehicle-injected and noninjected animals, while at 2 or 4 h, no ‘‘handling’’-induced effect was evident. The ‘‘handling’’-induced increase in BDNF and pCREB was not observed, when CPP, CNQX, bicuculline, p-MPPI, ketanserin, or nimodipine (NMDA, AMPA/kainate, GABA-A, 5HT-1A, 5HT-2A/C receptor, or LVGCC antagonist, respectively) were injected prior to the ‘‘handling’’ procedure. These results indicate that these types of receptors and channels are involved in mediating the effects of ‘‘early handling’’ on the developing rat brain. Regarding the role of NMDA, AMPA/kainate, GABA-A receptors, and the L-type voltage-gated Ca2+ channels, we can propose the following possible mechanism: ‘‘Handling’’, which alters tactile and possibly olfactory and auditory stimulation of the pups, initiates neuronal activity. This activates AMPA and GABA-A receptors and results in membrane depolarization, which in turn leads to activation of NMDA receptors and opening of LVGCCs. It has been well documented that GABA-A receptors act as depolarizing during brain development and along with AMPA receptors mediate NMDA receptor activation and opening of LVGCCs [2,3]. Calcium ions can then enter the cell and activate Ca2+-dependent kinases, which can phosphorylate and activate the transcription factor CREB. Among Ca2+dependent kinases, CaMK II and IV have been shown to be particularly important in mediating neuronal activitydependent CREB phosphorylation [42 – 44]. Furthermore, CREB can be phosphorylated by PKA following activation of Ca2+-sensitive adenylate cyclase [13]. Interestingly, ‘‘handling’’ has been shown to increase PKA activity [24]. The activated, by phosphorylation, transcription factor CREB is a key molecule in the cellular mechanisms underlying brain plasticity [28,46,49]. In the final step of the mechanism mediating the effects of ‘‘early handling’’, pCREB can bind to the BDNF gene control region and
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induce its expression [43,44]. The functional relationship between pCREB and BDNF is also supported by the finding that partial deletion of the CREB gene results in decreased BDNF mRNA expression and promotes alcohol-drinking behaviors [33]. It is worth noting that ‘‘early handling’’ decreases alcohol consumption [10]. Induction of BDNF by pCREB has been shown to occur as a result of activitydependent neuronal plasticity [43,44]. Furthermore, chronic stress results in decreased protein levels of both CREB and BDNF [15,38], while physical exercise increases the mRNA of both molecules [27]. In our study, both BDNF and pCREB are significantly increased at 8 h, while at 4 h, no change in BDNF is observed, whereas pCREB shows a trend to increase. It thus appears that the BDNF gene is the target of the transcription factor pCREB. On the other hand, it has been shown that BDNF signaling can lead to increased pCREB [50]. Hence, the pCREB-induced BDNF can lead to further increases in pCREB, establishing a positive feedback loop between these two plasticity-related molecules following ‘‘handling’’. The increased BDNF induced by ‘‘handling’’ can exert its morphogenetic roles and ‘‘imprint’’ the effects of the neonatal experience on the developing rat brain. Indeed, it has been shown that BDNF has an important morphogenetic role [20] in the development of the serotonergic system, which is also influenced by ‘‘early handling’’ [32,34,48]. BDNF can increase dendritic spine density [45] and mediate LTP (Long-Term Potentiation) [14], effects which have also been described to be induced by ‘‘handling’’ [30,52]. Interestingly, rearing in an enriched environment, which has been shown to have similar effects as ‘‘early handling’’ [8], also results in increased BDNF protein [39]. On the other hand, maternal deprivation, which has opposite effects from those of ‘‘early handling’’ on brain function and plasticity [16], results in decreased protein levels of pCREB [12] and BDNF [38]. Most interestingly, the increase in BDNF induced by 1 day of ‘‘handling’’ has long-term consequences, since it is maintained in the brain of the adult handled animals [9]. The ‘‘handling’’-induced increased BDNF could be related to the effects of ‘‘early handling’’ in improving active avoidance acquisition [7] and preventing the spatial learning deficits, which accompany aging [22,23], since it is well documented that BDNF plays an important role in learning and memory [53]. Moreover, the increased BDNF could be related to the decreased vulnerability of male handled animals in chronic stress-induced animal models of depression [35], since current data implicate BDNF in depression [29]. Furthermore, the neuroprotective effect of ‘‘early handling’’, which has been shown to increase neuronal survival in the aging hippocampus [22,23], can also be attributed to the neurotrophic effects [11] of the ‘‘handling’’-induced increased BDNF. It is noteworthy that after administration of the competitive NMDA receptor antagonist CPP, not only was the ‘‘handling’’-induced increase in pCREB and BDNF abol-
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ished, but a ‘‘reverse’’ effect was observed: handled animals had lower numbers of pCREB and BDNF positive cells than the non-handled. This result could be attributed to a parallel ‘‘handling’’-induced activation of NMDA receptors, both following AMPA and GABA-A receptor-mediated depolarization. Indeed, it has been shown that these two mechanisms operate in hippocampal cells [2,3]. Our findings point to the NMDA receptor as a key molecule in the cellular mechanisms underlying the effects of ‘‘early handling’’ on the developing brain. The involvement of serotonin receptors both type 1A and 2A/C in the mechanism of the ‘‘handling’’-induced increase in pCREB and BDNF may be indirect, i.e., the cells possessing these receptors are probably different from those in which the levels of pCREB and BDNF are increased. On one hand, 5HT-1A receptors are located in the hippocampus, but they are inhibitory [1] and on the other, 5HT2A/C receptors are located mainly in the cortex and their levels are very low in the hippocampus at this developmental stage [18]. 5HT-1A receptors are localized on inhibitory hippocampal interneurons [1] which normally exert modulatory control on hippocampal neurotransmission. Thus, 5HT1A receptors physiologically mediate disinhibition [41] and activation of hippocampal neuronal activity, which – as mentioned previously [42,46] – is accompanied by an increase in pCREB. Based on our results, we can infer that neuronal circuits involving the abovementioned 5HT1A receptor-containing interneurons are activated by ‘‘handling’’, since their blockade cancels the increase in pCREB and BDNF induced by ‘‘handling’’. The cortical cells having the 5HT-2A/C receptors could be activated by the ‘‘handling’’-induced sensory stimulation, and in turn excite trans-synaptically the hippocampal cells, resulting in increased pCREB and BDNF, in the latter. Other previously reported effects of handling, such as an increase in hippocampal GR [26] and cAMP [24] levels, have also been shown to be mediated via 5-HT2A/C receptors. Based on our results, we propose that the altered sensory stimulation of ‘‘early handling’’ leads to depolarization of hippocampal cells via AMPA and GABA-A receptors, which then results in activation of NMDA receptors and opening of LVGCCs, through which calcium ions enter the cell. The increased intracellular calcium in turn activates calciumdependent kinases and leads to phosphorylation of the transcription factor pCREB, which induces the expression of BDNF. ‘‘Handling’’ also stimulates 5HT-1A or 5-HT2A/ 2C receptor-containing cells, which trans-synaptically activate the abovementioned calcium-dependent mechanism in hippocampal cells having NMDA, AMPA, and GABA-A receptors, resulting in increased pCREB and BDNF. These molecules participate in neuronal plasticity processes, and mediate the effects of ‘‘early handling’’ leading to improved adaptability and ability to cope with novel environmental stimuli.
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