Antonios Stamatakis

Effects of an Early Experience Involving Training in a T-Maze Under either Denial or Receipt of Expected Reward through Maternal Contact

The mother is the most salient stimulus for the developing pups and a number of early experience models employ manipulation of the mother-infant interaction. We have developed a new model which in addition to changes in maternal behavior includes a learning component on the part of the pups. More specifically, pups were trained in a T-maze and either received (RER rats) or were denied (DER) the reward of maternal contact, during postnatal days 10–13. Pups of both experimental groups learn the T-maze, but the RER do so more efficiently utilizing a procedural-type of learning and memory with activation of the dorsal basal ganglia. On the other hand, the DER experience leads to activation of the hippocampus, prefrontal cortex, and amygdala in the pups. In adulthood, male DER animals exhibit better mnemonic abilities in the Morris water maze and higher activation of the hippocampus, while they have decreased brain serotonergic activity, exhibit a depressive-like phenotype and proactive aggressive behavior in the resident-intruder test. While male RER animals assume a reactive coping style in this test, and showed increased freezing during both contextual and cued memory recall following fear conditioning.

Enhanced neuronal plasticity and elevated endogenous sAPPα levels in mice over-expressing MMP9

J. Neurochem. (2012) 121, 239–251.

IGF-I ameliorates hippocampal neurodegeneration and protects against cognitive deficits in an animal model of temporal lobe epilepsy

Epilepsy is a major neurological disease, and patients often show spatial memory deficits. Thus, there is a need of effective new therapeutic approaches. IGF-I has been shown to be neuroprotective following a number of experimental insults to the nervous system, and in a variety of animal models of neurodegenerative diseases. In the present work, we investigated the possible neuroprotective effects of IGF-I following unilateral intrahippocampal administration of kainic acid (KA), an animal model of temporal lobe epilepsy (TLE). KA induced cell death, as shown by FluoroJade B, and extensive cell loss in both the ipsilateral and contralateral CA3 and CA4 areas, as well as granule cell dispersal in the DG, as revealed by Cresyl violet staining. KA also resulted in intense astrogliosis and microgliosis, as assessed by the number of GFAP and CD11b immunopositive cells, respectively, and increased hippocampal neurogenesis. Exposure to the Morris Water Maze task revealed that mice injected with KA were deficient in spatial learning and both short- and long-term memories, when tested in a larger diameter pool, which requires the use of allocentric strategies. When tested in a smaller pool, only long-term memory was impaired. Administration of IGF-I decreased seizure severity, hippocampal neurogenesis, and protected against neurodegeneration at the cellular level as assessed by FluoroJade B and Cresyl violet staining, as well as the number of GFAP and CD11b immunopositive cells. Furthermore, IGF-I abolished the cognitive deficits. Our results support that IGF-I could have a possible therapeutic potential in TLE.

Rat dams exposed repeatedly to a daily brief separation from the pups exhibit increased maternal behavior, decreased anxiety and altered levels of receptors for estrogens (ERα, ERβ), oxytocin and serotonin (5-HT1A) in their brain

In the present study we investigated the neurobiological mechanisms underlying expression of maternal behavior. Increased maternal behavior was experimentally induced by a brief 15-min separation between the mother and the pups during postnatal days 1 to 22. On postnatal days (PND) 12 and 22, we determined in experimental and control dams levels of anxiety in the elevated plus maze (EPM) as well as the levels of receptors for estrogens (ERα, ERβ), oxytocin (OTR) and serotonin (5-HT1AR) in areas of the limbic system (prefrontal cortex-PFC, hippocampus, lateral septum-SL, medial preoptic area-MPOA, shell of nucleus accumbens-nAc-Sh, central-CeA and basolateral-BLA amygdala), involved in the regulation of maternal behavior. Experimental dams, which showed increased maternal behavior towards their offspring, displayed reduced anxiety in the EPM on both PND12 and PND22. These behavioral differences could be attributed to neurochemical alterations in their brain: On both PND12 and PND22, experimental mothers had higher levels of ERα and OTRs in the PFC, hippocampus, CeA, SL, MPOA and nAc-Sh. The experimental manipulation-induced increase in ERβ levels was less widespread, being localized in PFC, the hippocampal CA2 area, MPOA and nAc-Sh. In addition, 5-HT1ARs were reduced in the PFC, hippocampus, CeA, MPOA and nAc-Sh of the experimental mothers. Our results show that the experience of the daily repeated brief separation from the pups results in increased brain ERs and OTRs, as well as decreased 5-HT1ARs in the dams brain; these neurochemical changes could underlie the observed increase in maternal behavior and the reduction of anxiety.

Learning of a T-maze by rat pups when contact with the mother is either permitted or denied.

Mother-pup interactions constitute an important component of environmental stimulation of the offspring during the neonatal period. Employing maternal contact as either a positive reinforcer or, its denial, as a frustrative, non-rewarding stimulus, we developed a novel experimental paradigm involving learning by rat neonates of a T-maze. When trained under the reward of maternal contact during postnatal days 10-13 Wistar rat pups learned the choice leading to the mother in a T-maze. When tested 2h later, in the absence of the mother, pups showed a clear preference for the arm of the T-maze leading to the position of the mother during training. Furthermore, pups receiving the expected reward of maternal contact had higher numbers of c-Fos immunopositive cells in the dorsal striatum compared to either naïve or pups denied the expected reward. The above behavioral and cellular results indicate that pups receiving the expected reward developed a procedural-like memory. When trained under frustrative non-reward pups learned to make the correct choice in the T-maze, albeit less efficiently than pups receiving the expected reward. Following this training condition c-Fos immunohistochemistry revealed increased activation of the CA1 area of the hippocampus and the orbitofrontal cortex. Expression of the information learned by the pups denied the expected reward was contingent upon the presence of the mother in the experimental setup in exactly the same configuration as during the training.

Denial of reward in the neonate shapes sociability and serotonergic activity in the adult rat.

Background Manipulations of the early environment are linked to long-lasting alterations of emotionality and social capabilities. Denial of rewarding mother-pup interactions in early life of rats could serve as model for child neglect. Negative consequences for social competence in later life, accompanied by changes in the serotonergic system would be expected. In contrast, rewarding mother-pup contact should promote adequate social abilities. Methodology/Principal Findings Male Wistar rats trained in a T-maze during postnatal days 10–13 under denial (DER) or permission (RER) of maternal contact were tested for play behavior in adolescence and for coping with defeat in adulthood. We estimated serotonin (5-HT) levels in the brain under basal conditions and following defeat, as well as serotonin receptor 1A (5-HT1A) and serotonin transporter (SERT) expression. DER rats exhibited increased aggressive-like play behavior in adolescence (i.e. increased nape attacks, p<0.0001) and selected a proactive coping style during defeat in adulthood (higher sum of proactive behaviors: number of attacks, flights, rearings and defensive upright posture; p = 0.011, p<0.05 vs RER, non-handled-NH). In adulthood, they had lower 5-HT levels in both the prefrontal cortex (p<0.05 vs RER) and the amygdala (p<0.05 vs NH), increased 5-HT levels following defeat (PFC p<0.0001) and decreased serotonin turnover (amygdala p = 0.008). The number of 5-HT1A immunopositive cells in the CA1 hippocampal area was increased (p<0.05 DER, vs RER, NH); SERT levels in the amygdala were elevated (p<0.05 vs RER, NH), but were lower in the prefrontal cortex (p<0.05 vs NH). Conclusions/Significance Denial of expected maternal reward early in life negatively affects sociability and the serotonergic system in a complex manner. We propose that our animal model could contribute to the identification of the neurobiological correlates of early neglect effects on social behavior and coping with challenges, but also in parallel with the effects of a rewarding early-life environment.

Selective effects of neonatal handling on rat brain N-methyl-D-aspartate receptors.

Neonatal handling, an experimental model of early life experiences, is known to affect the hypothalamic-pituitary-adrenal axis function thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related processes. A molecule that plays a most critical role in such processes is the N-methyl-D-aspartate (NMDA) receptor, a tetramer consisting of two obligatory, channel forming NR1 subunits and two regulatory subunits, usually a combination of NR2A and NR2B. Since the subunit composition of the NMDA receptor affects brain plasticity, in the present study we investigated the effect of neonatal handling on NR1, NR2A and NR2B mRNA levels using in situ hybridization, and on NR2B binding sites, using autoradiography of in vitro binding of [(3)H]-ifenprodil, in adult rat limbic brain areas. We found that neonatal handling specifically increased NR2B mRNA and binding sites, while it had no effect on the NR1 and NR2A subunits. More specifically, neonatally handled animals, both males and females, had higher NR2B mRNA and binding sites in the dorsal CA1 hippocampal area, as well as the prelimbic, the anterior cingulate and the somatosensory cortex, compared to the non-handled. Moreover NR2B binding sites were increased in the dorsal CA3 area of handled animals of both sexes. Furthermore, neonatal handling had a sexually dimorphic effect, increasing NR2B mRNA and binding sites in the central and medial amygdaloid nuclei only of the females. The neonatal handling-induced increase in the NR2B subunit of the NMDA receptor could underlie the higher brain plasticity, which neonatally handled animals exhibit.

Long‐term effects of neonatal handling on mu‐opioid receptor levels in the brain of the offspring

Neonatal handling is an experimental paradigm of an early experience which permanently alters hypothalamic-pituitary-adrenal axis function resulting in increased ability to cope with stress, and decreased emotionality. In the present work we investigated the effect of neonatal handling on adult rat brain mu-opioid receptor levels, since the opioid system is known to play an important role in emotional processing, anxiety and stress responses. Neonatal handling resulted in increased levels of mu-opioid receptors in the basolateral and central amygdaloid nuclei, in the CA3 and CA4 hippocampal areas, in the ventral tegmental area, the nucleus accumbens and the prefrontal cortex. Handled animals of both sexes had lower anxiety as measured in the elevated plus maze. The increased mu receptor levels could participate in the molecular mechanisms underlying the well-documented decreased stress and anxiety responses of handled animals.

Neural stem/progenitor cells differentiate into oligodendrocytes, reduce inflammation, and ameliorate learning deficits after transplantation in a mouse model of traumatic brain injury

The central nervous system has limited capacity for regeneration after traumatic injury. Transplantation of neural stem/progenitor cells (NPCs) has been proposed as a potential therapeutic approach while insulin‐like growth factor I (IGF‐I) has neuroprotective properties following various experimental insults to the nervous system. We have previously shown that NPCs transduced with a lentiviral vector for IGF‐I overexpression have an enhanced ability to give rise to neurons in vitro but also in vivo, upon transplantation in a mouse model of temporal lobe epilepsy. Here we studied the regenerative potential of NPCs, IGF‐I‐transduced or not, in a mouse model of hippocampal mechanical injury. NPC transplantation, with or without IGF‐I transduction, rescued the injury‐induced spatial learning deficits as revealed in the Morris Water Maze. Moreover, it had beneficial effects on the host tissue by reducing astroglial activation and microglial/macrophage accumulation while enhancing generation of endogenous oligodendrocyte precursor cells. One or two months after transplantation the grafted NPCs had migrated towards the lesion site and in the neighboring myelin‐rich regions. Transplanted cells differentiated toward the oligodendroglial, but not the neuronal or astrocytic lineages, expressing the early and late oligodendrocyte markers NG2, Olig2, and CNPase. The newly generated oligodendrocytes reached maturity and formed myelin internodes. Our current and previous observations illustrate the high plasticity of transplanted NPCs which can acquire injury‐dependent phenotypes within the host CNS, supporting the fact that reciprocal interactions between transplanted cells and the host tissue are an important factor to be considered when designing prospective cell‐based therapies for CNS degenerative conditions. GLIA 2016;64:763–779

Subventricular Zone-Derived Neural Stem Cell Grafts Protect Against Hippocampal Degeneration and Restore Cognitive Function in the Mouse Following Intrahippocampal Kainic Acid Administration

Temporal lobe epilepsy (TLE) is a major neurological disease, often associated with cognitive decline. Since approximately 30% of patients are resistant to antiepileptic drugs, TLE is being considered as a possible clinical target for alternative stem cell‐based therapies. Given that insulin‐like growth factor I (IGF‐I) is neuroprotective following a number of experimental insults to the nervous system, we investigated the therapeutic potential of neural stem/precursor cells (NSCs) transduced, or not, with a lentiviral vector for overexpression of IGF‐I after transplantation in a mouse model of kainic acid (KA)‐induced hippocampal degeneration, which represents an animal model of TLE. Exposure of mice to the Morris water maze task revealed that unilateral intrahippocampal NSC transplantation significantly prevented the KA‐induced cognitive decline. Moreover, NSC grafting protected against neurodegeneration at the cellular level, reduced astrogliosis, and maintained endogenous granule cell proliferation at normal levels. In some cases, as in the reduction of hippocampal cell loss and the reversal of the characteristic KA‐induced granule cell dispersal, the beneficial effects of transplanted NSCs were manifested earlier and were more pronounced when these were transduced to express IGF‐I. However, differences became less pronounced by 2 months postgrafting, since similar amounts of IGF‐I were detected in the hippocampi of both groups of mice that received cell transplants. Grafted NSCs survived, migrated, and differentiated into neurons—including glutamatergic cells—and not glia, in the host hippocampus. Our results demonstrate that transplantation of IGF‐I producing NSCs is neuroprotective and restores cognitive function following KA‐induced hippocampal degeneration.