Carina Helmeke

Juvenile emotional experience alters synaptic composition in the rodent cortex, hippocampus, and lateral amygdala

A quantitative anatomical study in the rodent anterior cingulate and somatosensory cortex, hippocampus, and lateral amygdala revealed region-, cell-, and dendrite-specific changes of spine densities in 3-week-old Octodon degus after repeated parental separation. In parentally separated animals significantly higher spine densities were found on the apical and basal dendrites of the cingulate cortex (up to 143% on apical and 138% on basal dendrite). Branching order analysis revealed that this effect is seen on all segments of the apical dendrite, whereas on the basal dendrites significantly higher spine densities were seen only on the outer branches (third to fifth dendritic segments). Increased spine densities were also observed on the hippocampal CA1 pyramidal neurons (up to 109% on the distal apical segments and up to 106% on the basal segment) compared with the control group. In contrast, significantly reduced spine densities were observed on the granule cell dendrites in the dentate gyrus (down to 92%) and on the apical dendrites in the medial nucleus of the amygdala (down to 95%). No significant changes of spine densities were seen in the somatosensory cortex (except for an increase in the proximal apical segments) and in the lateral nucleus of the dorsal amygdala (except for an increase in the proximal basal dendritic segments). These results demonstrate that repeated stressful emotional experience alters the balance of presumably excitatory synaptic inputs of pyramidal neurons in the limbic system. Such experience-induced modulations of limbic circuits may determine psychosocial and cognitive capacities during later life.

Lack of paternal care affects synaptic development in the anterior cingulate cortex.

Exposure to enriched or impoverished environmental conditions, experience and learning are factors which influence brain development, and it has been shown that neonatal emotional experience significantly interferes with the synaptic development of higher associative forebrain areas. Here, we analyzed the impact of paternal care, i.e. the fathers emotional contribution towards his offspring, on the synaptic development of the anterior cingulate cortex. Our light and electron microscopic comparison of biparentally raised control animals and animals which were raised in single-mother families revealed no significant differences in spine densities on the apical dendrites of layer II/III pyramidal neurons and of asymmetric and symmetric spine synapses. However, significantly reduced densities (-33%) of symmetric shaft synapses were found in layer II of the fatherless animals compared to controls. This finding indicates an imbalance between excitatory and inhibitory synapses in the anterior cingulate cortex of father-deprived animals. Our results query the general assumption that a father has less impact on the synaptic maturation of his offsprings brain than the mother.

Imbalance of immunohistochemically characterized interneuron populations in the adolescent and adult rodent medial prefrontal cortex after repeated exposure to neonatal separation stress

Experimental studies in various animal models have revealed convincing evidence that stressful experience during early developmental periods produces a variety of behavioral, neuroanatomical and endocrine alterations, which are reminiscent of human mental disorders such as depression and various types of anxiety disorders. Since these mental disorders are assumed to be associated with altered GABAergic inhibition in cortical and subcortical brain regions, the current study tested the hypothesis that early postnatal adverse emotional experience (separation stress) interferes with the establishment and functional maturation of distinct inhibitory interneuron populations in different subregions of the medial prefrontal cortex (mPFC) of the precocious rodent degu (Octodon degus). At the age around puberty early stressed animals displayed significantly lower densities of calbindin-D28k-immunoreactive interneurons in the anterior cingulate (down to 79%) and in the precentral medial (down to 64%) subregions of the mPFC compared with age-matched unstressed controls. At this age the densities of two other interneuron types characterized by their expression of the calcium-binding proteins parvalbumin or calretinin remained at control levels. In adulthood, i.e. after an extended period without stress exposure, the density of calbindin-D28k-immunoreactive interneurons in the stressed animals was back to control numbers, whereas parvalbumin-immunoreactive interneurons displayed significantly elevated density in the anterior cingulate (up to 138%) and in the precentral medial cortex (up to 137%) of the stressed animals. In both age groups the density of calretinin- and corticotropin releasing hormone-immunoreactive interneurons did not differ between stressed and control animals, and the prelimbic and infralimbic subregions of the medial prefrontal cortex remained unaffected by stress experience. These results confirm that early adverse emotional experience induces long lasting age-, region- and neuron-specific imbalance of inhibitory systems in some, but not all subregions of the medial prefrontal cortex of the degu.

Neonatal separation stress reduces glial fibrillary acidic protein- and S100β-immunoreactive astrocytes in the rat medial precentral cortex

The interactions between the mother/parents and their offspring provides socioemotional input, which is essential for the establishment and maintenance of synaptic networks in prefrontal and limbic brain regions. Since glial cells are known to play an important role in developmental and experience‐driven synaptic plasticity, the effect of an early adverse emotional experience induced by maternal separation for 1 or 6 h on the expression of the glia specific proteins S100β and glial fibrillary acidic protein (GFAP) was quantitatively analyzed in anterior cingulate cortex, hippocampus, and precentral medial cortex. Three animal groups were analyzed at postnatal day 14: (i) separated for 1 h; (ii) separated for 6 h; (iii) undisturbed (control). Twenty‐four hours after stress exposure, the stressed brains showed significantly reduced numbers of S100β‐immunoreactive (ir) cells in the anterior cingulate cortex (6‐h stress) and in the precentral medial cortex (1‐ and 6‐h stress). Significantly reduced numbers of GFAP‐ir cells were observed only in the medial precentral cortex (1‐ and 6‐h stress); no significant changes were observed in the anterior cingulate cortex. No significant changes of the two glial markers were observed in the hippocampus. Double‐labeling experiments with GFAP and pCREB revealed pCREB labeling only in the hippocampus, where the stressed brains (1 and 6 h) displayed significantly reduced numbers of GFAP/pCREB‐ir glial cells. The observed downregulation of glia‐specific marker proteins is in line with our hypothesis that emotional experience can alter glia cell activation in the juvenile limbic system.

Repeated neonatal separation stress alters the composition of neurochemically characterized interneuron subpopulations in the rodent dentate gyrus and basolateral amygdala.

Emotional experience during early life has been shown to interfere with the development of excitatory synaptic networks in the prefrontal cortex, hippocampus, and the amygdala of rodents and primates. The aim of the present study was to investigate a developmental “homoeostatic synaptic plasticity” hypothesis and to test whether stress‐induced changes of excitatory synaptic composition are counterbalanced by parallel changes of inhibitory synaptic networks. The impact of repeated early separation stress on the development of two GABAergic neuronal subpopulations was quantitatively analyzed in the brain of the semiprecocial rodent Octodon degus. Assuming that PARV‐ and CaBP‐D28k‐expression are negatively correlated to the level of inhibitory activity, the previously described reduced density of excitatory spine synapses in the dentate gyrus of stressed animals appears to be “amplified” by elevated GABAergic inhibition, reflected by reduced PARV‐ (down to 85%) and CaBP‐D28k‐immunoreactivity (down to 74%). In opposite direction, the previously observed elevated excitatory spine density in the CA1 region of stressed animals appears to be amplified by reduced inhibition, reflected by elevated CaPB‐D28k‐immunoreactivity (up to 149%). In the (baso)lateral amygdala, the previously described reduction of excitatory spine synapses appears to be “compensated” by reduced inhibitory activity, reflected by dramatically elevated PARV‐ (up to 395%) and CaPB‐D28k‐immunoreactivity (up to 327%). No significant differences were found in the central nucleus of the amygdala, the piriform, and somatosensory cortices and in the hypothalamic paraventricular nucleus. Thus during stress‐evoked neuronal and synaptic reorganization, a homeostatic balance between excitation and inhibition is not maintained in all regions of the juvenile brain.

Paternal Deprivation Affects the Development of Corticotrophin-Releasing Factor-Expressing Neurones in Prefrontal Cortex, Amygdala and Hippocampus of the Biparental Octodon degus

Although the critical role of maternal care on the development of brain and behaviour of the offspring has been extensively studied, knowledge about the importance of paternal care is comparatively scarce. In biparental species, paternal care significantly contributes to a stimulating socio‐emotional family environment, which most likely also includes protection from stressful events. In the biparental caviomorph rodent Octodon degus, we analysed the impact of paternal care on the development of neurones in prefrontal‐limbic brain regions, which express corticotrophin‐releasing factor (CRF). CRF is a polypeptidergic hormone that is expressed and released by a neuronal subpopulation in the brain, and which not only is essential for regulating stress and emotionality, but also is critically involved in cognitive functions. At weaning age [postnatal day (P)21], paternal deprivation resulted in an elevated density of CRF‐containing neurones in the orbitofrontal cortex and in the basolateral amygdala of male degus, whereas a reduced density of CRF‐expressing neurones was measured in the dentate gyrus and stratum pyramidale of the hippocampal CA1 region at this age. With the exception of the CA1 region, the deprivation‐induced changes were no longer evident in adulthood (P90), which suggests a transient change that, in later life, might be normalised by other socio‐emotional experience. The central amygdala, characterised by dense clusters of CRF‐immunopositive neuropil, and the precentral medial, anterior cingulate, infralimbic and prelimbic cortices, were not affected by paternal deprivation. Taken together, this is the first evidence that paternal care interferes with the developmental expression pattern of CRF‐expressing interneurones in an age‐ and region‐specific manner.

Electron microscopic 3D-reconstruction of dendritic spines in cultured hippocampal neurons undergoing synaptic plasticity

Dendritic spines are assumed to constitute the locus of neuronal plasticity, and considerable effort has been focused on attempts to demonstrate that new memories are associated with the formation of new spines. However, few studies that have documented the appearance of spines after exposure to plasticity‐producing paradigms could demonstrate that a new spine is touched by a bona fida presynaptic terminal. Thus, the functional significance of plastic dendritic spine changes is not clearly understood. We have used quantitative time lapse confocal imaging of cultured hippocampal neurons before and after their exposure to a conditioning medium which activates synaptic NMDA receptors. Following the experiment the cultures were prepared for 3D electron microscopic reconstruction of visually identified dendritic spines. We found that a majority of new, 1‐ to 2‐h‐old spines was touched by presynaptic terminals. Furthermore, when spines disappeared, the parent dendrites were sometime touched by a presynaptic bouton at the site where the previously identified spine had been located. We conclude that new spines are most likely to be functional and that pruned spines can be transformed into shaft synapses and thus maintain their functionality within the neuronal network.

Frühkindliche emotionale Erfahrungen beeinflussen die funktionelle Entwicklung des Gehirns

Zusammenfassung Frühkindliche emotional gesteuerte Lernprozesse, wie die Entstehung der Kind-Eltern Beziehung, sind von grundlegender Bedeutung für die Ausbildung normaler sozioemotionaler und intellektueller Fähigkeiten. Tierexperimentelle Untersuchungen haben gezeigt, dass Störungen dieser emotionalen Bindung zu Veränderungen der Gehirnorganisation und zu Defiziten nicht nur im emotionalen, sondern auch im kognitiven Bereich führen. Das Ziel unserer Forschungsarbeiten ist es, die zellulären und molekularen Mechanismen zu untersuchen, die der erfahrungsabhängigen Gehirnreifung zu Grunde liegen, und herauszufinden, in welcher Weise frühkindliche emotionale Erfahrungen diese Prozesse beeinflussen. In unseren Untersuchungen am Modell der Filialprägung des Haushuhnkükens und der Elterndeprivation bei der Strauchratte (Octodon degus) konnten wir zeigen, dass die Unterbrechung des Kind-Eltern Kontaktes (Elternseparation) zu langfristigen spezifischen synaptischen Veränderungen in limbischen kortikalen Regionen führt, die bei emotionalem Verhalten, Lernen und Gedächtnisbildung eine grundlegende Rolle spielen. Zudem stellte sich heraus, dass es im Verlauf von frühkindlichen Erfahrungen zu erheblichen Veränderungen des Gehirnstoffwechsels kommt, die möglicherweise einen Ausgangspunkt für die später auftretenden synaptischen Veränderungen bilden. Weiterführende, interdisziplinäre Untersuchungen sollen zeigen, inwieweit die aus solchen tierexperimentellen Ansätzen gewonnenen Erkenntnisse einerseits in die Entwicklung neuer pädagogischer Konzepte im Vorschulbereich einfließen können, und ob sie andererseits auch dazu beitragen könnten, die Entstehung von entwicklungs- und umweltinduzierten Verhaltens- und Lernstörungen besser zu verstehen.

Geometrical Modeling and Visualization of Pre- and Post-Synaptic Structures in Double-Labeled Confocal Images

3-D volume samples of selected dendritic segments are scanned by a confocal laser-scanning microscope to investigate the impact of alterations in the shape of dendritic spines for their synaptic functioning. Double-labeling techniques using green fluorescent protein-labeled neurons and synaptophysin-labeled presynaptic terminals allow us to capture images of dendrites and spines in one image channel and the presynaptic boutons in a second image channel. To detect spines and to reconstruct spine shape a parametric model is fitted in the noisy confocal image. Starting with the geometrical model of the spine head, voxels in the second image channel are labeled by a distance map to detect possible associated presynaptic terminals. This method enables a flexible visualization of geometrical models of spines and dendrites together with selected presynapses and an adjustable, distance-dependent quantification of boutons. These functional features are combined with morphological features from the geometrical model for every spine and establish a basis for advanced statistical analysis of the underlying learning processes in brain. The cellular events of changes in spine morphology can be studied in relation to functional properties.

NMDA-induced stimulation of glycolysis in developing hippocampal cell cultures

Developmental changes in energy metabolism of primary hippocampal cell cultures from newborn rats were investigated during the first 3 weeks. These changes were measured by intensity of and number of cells exhibiting NAD(P)H fluorescence in response to NMDA-induced activation of neuronal activity. We observed gradual changes of stimulation-evoked NAD(P)H signaling over the first 3 weeks, such that at day 7 and 16, this stimulation is minimal, while at 5 and 12 days, it is maximal. These results describe a biphasic pattern that was similar to earlier findings from experiments investigating developmental changes in population spike amplitudes or glutamate release in young rats. Inhibition of mitochondrial respiration by KCN revealed that the NMDA-evoked stimulation of energy metabolism is mainly due to increased glycolytic activity.