Abstract5: The influence of postnatal socio-emotional deprivation on the development of nicotinamide adenine dinucleotide phosphate (NADPH)- diaphorase-reactive neurons in prefrontal cortical areas and in subdivisions of the nucleus accumbens was quantitatively investigated in the precocious rodent Octodon degus. Forty-five-days-old O. degus from two animal groups were compared: (i) degus which were repeatedly separated from their mothers during the first three postnatal weeks and after weaning reared in complete isolation; and (ii) degus which were reared under normal undisturbed social conditions. Socially-deprived animals displayed a significant decrease of NADPH-diaphorase-containing neurons in anterior cingulate cortex (85.5%), the same tendency was observed in the infralimbic, precentral medial and prelimbic prefrontal areas. Similarly, the core region of nucleus accumbens expressed reduced NADPH-diaphorase-reactive neuron numbers in deprived animals (70%), whereas the shell region remained unchanged. Since during normal postnatal development the number of NADPH-diaphorase-reactive neurons gradually decreases in all prefrontal cortical and accumbal regions, the observed deprivation-induced changes may reflect either an excessive reduction of NADPH-diaphorase-positive neurons or a down- regulation of the enzyme in neurons that normally express it. Since some NADPH-diaphorase-containing neurons in the prefrontal cortex have been shown to be GABAergic, it is tempting to speculate that a reduction of these inhibitory neurons in the anterior cingulate cortex may result in an enhanced excitatory output activity of disinhibited projection neurons in this cortical region, including those that project to the core region of the nucleus accumbens. Our results indicate a link between early adverse socio-emotional experience and the maturation of NADPH-reactive neurons and further studies are required to analyse the functional implication for this experience- induced brain pathology

Abstract6: The stimulation of adenylyl cyclase (AC) by dopamine was investigated in membrane fractions of the forebrain areas mediorostral neostriatum/hyperstriatum ventrale (MNH) and lobus parolfactorius (LPO) of 8-day-old domestic chicks that had been raised under different social conditions: group A, socially isolated; group B, imprinted on an acoustic stimulus; group C, trained but nonimprinted; and group D, reared in small groups. Only in the brain of the socially experienced groups could cyclic AMP (cAMP) synthesis be stimulated by dopamine, but not in the socially isolated animals (group A). Ligand binding studies of dopamine D1- and D2-type receptors in membrane fractions did not reveal differences between socially experienced and isolated animals. Forskolin stimulation of total AC in MNH and LPO membrane fractions revealed a significantly enhanced AC stimulation in the socially reared but not in the imprinted group compared with isolated controls. Stimulation of AC by the G protein activator guanylylimidodiphosphate was significantly increased in the MNH and the LPO of socially reared chicks compared with isolated control animals. These results suggest that early postnatal social experience modulates the rate of cAMP synthesis and that these lasting changes are not due to changes of dopamine receptors but are related to increased AC activities and to increased sensitivity of Gs protein Leibniz Institute for Neurobiology, Magdeburg, GermanyPMID- 0010461924

Abstract7: Degu mothers (Octodon degus) utter specific maternal calls during nursing which presumably stimulate and reinforce suckling. Pups from surgically muted mothers show a reduced gain of body weight during postnatal development compared to pups from normally vocalizing mothers. Our behavioral studies suggest that the pups have to learn the meaning of the maternal calls during the first two weeks of life. Two-week-old pups from normally vocalizing mothers expressed a preference for the maternal call in a behavioral discrimination test, in contrast to pups from surgically muted mothers. Investigation of brain activities using the 2-[ ¹⁴ C]fluoro -deoxyglucose (2-FDG) method revealed that pups from normal mothers display a significantly higher 2-FDG uptake in precentral medial, anterior cingulate cortex and a slight, non-significant increase in the prelimbic cortex and orbital PFC upon presentation of the maternal call, compared to pups from muted mothers, for which the maternal call was unfamiliar and meaningless. These prefrontal cortical areas are known to be involved in associative learning processes and our data suggest that they are involved in the association between the maternal call and the positive emotional situation during nursing

Abstract8: We studied the influence of single intracerebroventricular (ICV) and intraperitoneal (IP) injections of the neurotoxin aluminum on the retrieval of a passive avoidance task in rats and on the vasopressin- evoked improvement of the recall of the task. It was found that ICV administration of the metal alone strongly decreases the retention time of a passive avoidance task, whereas IP application of aluminum prolongs it. Vasopressin given ICV and IP leads to an enhancement of retrieval (prolongation of the retention time). Vasopressin in combination with aluminum does not improve the recall of the task when both substances are given ICV. Intraperitoneal injection of the neuropeptide together with the metal improves the recall of the task. Our data point to the crucial importance of the route of application of aluminum for behavioral studies Institute for Anatomy, Medical Academy Magdeburg, GermanyPMID- 8208778

Abstract9: L-Ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis and thus a unique marker of the proliferation and maturation processes, has been localized by means of immunohistochemistry in the human central nervous system. The enzyme protein has not only been found in the developing nervous system but also in the adult brain. The first immunopositive neuroblasts occurred in the 15th week of gravidity, whereas an increasing number of structures in the medulla oblongata and cerebellum showed ODC immunoreactivity in weeks 18- 24 of gravidity. ODC immunoreactivity was revealed in multiple neurons and in the ependymal lining of ventricles. In the adult human brain ODC immunoreactivity was found in the hippocampus and in the spinal cord. In no case were immunoreactive glial cells observed Federal Institute for Neurobiology, Magdeburg, FRGPMID- 8168440

Abstract10: The distribution of guanylate cyclase, phosphodiesterase, and NADPH- diaphorase [nitric oxide (NO) synthase] was studied in rat brain both at the light and electron microscopic level with special emphasis on the vascular system. We showed that the cGMP-generating enzyme is located in cells (glial cells and pericytes) surrounding cerebral vessels, but not in the endothelium. For NO synthase, a dual localization was observed. The enzyme is present in parts of the endothelium and in nerve endings apparently innervating larger brain vessels. We propose, therefore, that NO acts on guanylate cyclase both from a "synaptic" and endothelial source

Abstract11: Knowledge about second messenger metabolizing enzymes in neuroglia is still rather fragmentary. Therefore, the aim of the present investigation was to localize adenylate cyclase, guanylate cyclase, cyclic nucleotide phosphodiesterase and protein kinase A in glial cells of the rat hippocampus and cerebellum. Enzyme histochemical and immunohistochemical methods were used to detect the enzymes at the light and electron microscopic level. Astroglial cells were found to contain all 4 enzymes. Especially the microvascular glial cell processes were reactive. Oligodendroglial cells were only stained for adenylate cyclase acticity. Intracellularly, microtubules and intracellular membranes were frequently stained. The results point to the regulation of glial cell metabolism and of transport processes by cyclic nucleotides Institut for Neurobiology and Brain Research, Division of Neuromorphology Magdeburg, GermanyPMID- 1687099

Abstract12: Guanylate cyclase activity was demonstrated in the rat hippocampus at the electron microscopy level by a cerium precipitation technique. Enzymatic activity is localized in the granular cells of the dentate gyrus and in other, medium-sized neurons. The reaction product was found on the cytoplasmic membrane, in the cytoplasm, on the surface of endoplasmic reticulum and in postsynaptic densities. Glial cell processes and small dendritic processes were also stained. Besides the neurons, glial cells contained strong enzymatic activity, especially the perivascular glia and protoplasmatic glial cells neighbouring to neurons. Only little activity was detected in pyramidal cells and no activity was found in the endothelium cells of capillaries Institute for Neurobiology and Brain Research, Academy of Sciences, Magdeburg, GDRPMID- 2569303

Abstract13: A procedure for the separation of cyclic AMP phosphodiesterase from a commercially available preparation and for raising antibodies against this enzyme in rabbits is described. An antiserum thus obtained was used for the immunocytochemical detection of cyclic nucleotide phosphodiesterase in rat cerebellum. The molecular layer, the granular layer and the cerebellar white matter exhibited different degrees of immunoreactivity. Only a few cell bodies (possibly glial cells) were stained. Most of the antigenic sites were present in the neuropil of the molecular layer and around Purkinje cells. Cerebellar glomeruli, sites of synaptic interactions between mossy fibres, Golgi cells and granule cells, were also stained by this antiserum. Control reactions using preimmune serum were consistently negative Acadamy of Sciences, Institute for Neurobiology and Brain Research, Madgeburg, German Democratic RepublicPMID- 2835339

Abstract14: In target cells with receptors of the V2 type, vasopressin activates the adenylate cyclase and induces, finally, a clustering of intramembranous particles in the plasma membrane. Both characteristic effects, however, could not be observed at hippocampal capillaries of the rat. The presumed vasopressin receptors at cerebral capillaries that represent the blood-brain barrier are, therefore, probably not of the V2 type

Abstract15: Guanylate cyclase in rat cerebellum was investigated on the light microscopical level with guanylyl imidodiphosphate as substrate. Several attempts for activation of enzymatic activity and delimitation to other enzymes were made by sodium azide, aminophylline, sodium fluoride and dithioerythrole. The localization was similar but less strong compared to adenylate cyclase (Poeggel and Luppa 1984) and differs in behaviour to the above mentioned substances. Nucleotide pyrophosphatases seem to play an unimportant role in guanylyl imidodiphosphate conversion, while alkaline phosphatase is possibly of more importance. A light microscopical demonstration of guanylate cyclase by its enzymatic activity must be considered with caution. Main reasons are the low activity and therefore the great importance of interfering enzymes with high activities

Abstract16: A new method for the histochemical demonstration of adenylate cyclase activity, introduced and biochemically tested by Poeggel et al. (1981 a), was employed in nervous tissue. Using this method a multiple pattern of activity was detectable. Activity occurs in nervous as well as glial elements. Biochemical results and physiological conclusions could be confirmed by ultrahistochemical visualization of adenylate cyclase activity in nervous tissue. The specificity of the reaction is controlled by a number of variations of the incubation methods

Abstract17: methodische Untersuchungen zum Verhalten des Enzyms unter histochemischen Bedingungen, The adequate histochemical demonstration of adenylate cyclase still involves serious methodical problems (low basal activity, fixation procedures, heavy metal ions as capture agents etc.). We have tried to revise fixation approach and composition of incubation medium in order to specify the mode of demonstration of the enzyme. The influence of strontium and barium (instead of lead) as well as dithiothreitole and sodium fluoride on the survive of adenylate cyclase activity was studied using a radiochemical model system

Abstract18: Adenylate cyclase from rat hippocampus was separated by electrophoresis in polyacryl amide microgels and stained for enzymatic activity using a new histochemical procedure. This method involves the use of AMP-PNP, aminophylline, dithiotreitole, and Sr2+ as "primary" capture ions, thus fulfilling all the demands for a really specific histochemical incubation medium for the enzyme. The incubation of the gels with this medium resulted in the inhibition of other enzymes, which are capable of splitting AMP-PNP (ATP: pyrophosphatase, alkaline phosphatase), whereas adenylate cyclase remained highly active under these conditions. The enzyme was found to be present in two forms in the gels. Both protein bands were stimulated by the addition of various biogenic amines to the incubation medium. One protein band was fully GMP-PNP dependent in its activity. It is reasonable to suppose that these forms are either differently high aggregated molecules of the enzyme or enzyme molecules bound to their regulatory sites

Abstract19: The fate of ATP exposed to rat hippocampal extracts was investigated after their separation by a microdisc electrophoresis tenhnique. It could be demonstrated that the histochemical adenylate cyclase procedure using ATP as substrate is not suitable for specific localization of the enzyme, since other ATP hydrolysing enzymes were also able to convert ATP unless the concentrations of inhibitors reached 1 mM (ouabain) and 40 mM (NaF). With a prolonged incubation time of 18 h further substrate splitting protein zones could be revealed, possibly reflecting activities of enzymes involved in the hydrolysis of degradation products of ATP

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Abstract20: Analogous to the experience-driven development of sensory systems, the functional maturation of limbic circuits is significantly influenced by early socio-emotional experience. In a combined light and electron microscopic study in the anterior cingulate cortex of Octodon degus, the densities of spine and shaft synapses on apical dendrites of layer III pyramidal neurons were compared in 45 day old (1) undisturbed control animals; (2) handled animals; (3) animals which were repeatedly maternally deprived during the first three postnatal weeks; (4) animals which were treated similarly to group 3 and thereafter kept in chronic social isolation. Animals in groups 2-4 showed significantly higher spine densities (up to 121%, 142% and 151% respectively) compared to control group 1. Group 3 displayed significantly longer apical dendrites compared to control group 1. The electron microscopic analysis in cortical layer II revealed significantly higher spine synapses in group 4 (up to 166%) and fewer shaft synapses in groups 3 and 4 (down to 53% and 65% respectively) compared to group 1. These results demonstrate that early traumatic emotional experience alters synaptic input of pyramidal neurons. Such experience-induced modulation of limbic cortex development may determine psychosocial and cognitive capacities during later life.


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Abstract21:The cellular and spatial distribution of the presumptive retrograde transmitter nitric oxide, detected by NADPH-diaphorase histochemistry and of immunocytochemically labeled monoaminergic fiber systems was compared in regions of the medial prefrontal cortex of the precocious rodent Octodon degus. The staining patterns at two postnatal stages (P0 and P14) were compared to those found in adult animals (P90). At birth, NADPH-diaphorase positive neurons were found in all cortical layers of the anterior cingulate, infralimbic and prelimbic cortex. During postnatal development the number of diaphorase-positive cells gradually decreased in layers Il-VI and remained unchanged in layer I of these cortical regions. All NADPH-diaphorase positive neurons appeared to be spineless and pyramidal cells never contained NADPH-diaphorase. No colocalization of NADPH-diaphorase with either serotonin or TH was detectable. However, at all developmental stages the somata and proximal dendritic shafts of some of the NADPH-diaphorase containing bi-or multipolar neurons in layers V-VI were contacted by serotonin-and tyrosine hydroxylase-immunoreactive fibers. In a subpopulation of GABAergic interneurons in the deeper layers (V-VI) NADPH-diaphorase activity was colocalized with calbindin-D 28 k immunoreactivity. The abundance of nitridergic systems in regions of the medial prefrontal cortex already at birth together with their close spatial relationship with monoaminergic afferent systems as well as local GABAergic units may indicate a yet to determine role of these transmitter systems in early learning

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Abstract22:An increase of nitric oxide synthase (NOS) activity was found in the brain of few day old chicks after exposure to different social stress situations. The cerebellum, which showed the highest NOS activity, displayed the most sensitive response. Increases of NOS activity were found in the chronically separated tall regions) and in the acutely separated animals (cerebellum). In the cerebellum and the midbrain/brainstem/tectum NOS activity was significantly different also between the chronically separated and chronically isolated groups. NOS activity was higher in the cerebellum of the acutely separated group compared to the chronically isolated and social controls, a similar trend was found in the midbrain/brainstem/tectum sample, the values in the forebrain remained unchanged. Chronic isolation did not induce significant changes of NOS activity. These data confirm that the newborn chick is a suitable model for the experimental investigation of stress-induced alterations of brain maturation and behavioural development.

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Abstract23: The repeated separation from the family during very early stages of life is a stressful emotional experience, which induce a variety of neuronal and synaptic changes in limbic cortical areas that may be related to behavioral alterations. In a first series of experiments we investigated in eight day old Octodon degus if repeated parental separation and handling without separation from the family, leads to altered spontaneous exploratory behavior in a novel environment (open field test). In a second series of experiments we tested if the parentally deprived and handled animals display different stimulus-evoked exploratory behaviors in a modified open field version, in which a positive emotional stimulus, the maternal call, was presented. In the open field test a significant influence of previous emotional experience was found for the parameters, running, rearing and vocalization. Parentally deprived degus displayed increased horizontal (running) and vertical (rearing) motoric activities, but decreased vocalization, compared to normal and handled controls. The presentation of maternal vocalizations significantly modified running, vocalization and grooming activities, which in the case of running activity was dependent on previous emotional experience. Deprivation-induced locomotor hyperactivity together with the reduced behavioral response towards an familiar acoustic emotional signal are similar to behavioral disturbances which are observed in human attachment disorders.

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Abstract24: Although the potential vulnerability of the juvenile brain towards adverse environmental influences is generally recognized, relatively little is known about the basic mechanisms involved. The plasticity and adaptability of the juvenile brain in response to adverse emotional experiences was analyzed in  the South American Octodon degus. Our study revealed that repeated brief separation from the parents and exposure to an unfamiliar environment induces an upregulation of D1- and 5HT1A- receptor density in the precentral medial, anterior cingulate, prelimbic and infralimbic cortices in female pups. No significant changes of GABAA receptor density were found in deprived animals of both genders.
The acoustic presence of the mother during parental deprivation suppressed the D1-receptor upregulation as well as the 5-HT1A-receptor upregulation, again only in the female pups. These results demonstrate that that early adverse emotional experience alters aminergic function within the prefrontal cortex in the female but not the male brain. The mother’s voice, a powerful emotional signal, can protect the developing cortex from separation -induced receptor changes.

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Abstract25: The influence of early parental deprivation on the development of tyrosine hydroxylase- and 5-hydroxytryptamine-immunoreactive fiber innervation of subregions of the orbital prefrontal cortex (ventrolateral orbital, lateral orbital and agranular insular cortex) was quantitatively investigated in the precocious lagomorph Octodon degus. 45 day old degus from two groups were compared: 1) degus which were repeatedly separated from their parents during the first three postnatal weeks, after weaning they were reared in social  isolation; and 2) degus which were reared undisturbed in their families. Compared to the normal control animals the ventrolateral orbital prefrontal cortex and agranular insular cortex of the deprived animals displayed significantly increased density of tyrosine hydroxylase-immunoreactive fibers (up to 172% in the ventrolateral orbital prefrontal cortex and up to 143% in the agranular insular cortex). The lateral orbital prefrontal cortex showed increased 5-hydroxytryptamine-positive fiber densities (up to 118%).
This altered balance between the serotonergic and dopaminergic cortical innervation in the orbital prefrontal cortex may reflect an anatomical and functional adaptation, which may be triggered by an altered activity of these transmitter systems during the phases of parental separation and social isolation.



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Abstract26: Relatively little is known about the basic mechanisms, which play a role in the vulnerability of the developing brain towards adverse environmental influences. In  the South American Octodon degus we analyzed if changes of dopaminergic, serotonergic and GABAergic transmitter receptors are mediators of the adaptability of the developing brain in response to adverse emotional experiences. Our study revealed that repeated brief separation from the parents and exposure to an unfamiliar environment induces in the hippocampal formation of male and female pups an upregulation of D1- and 5-HT1A- receptor density in the stratum radiatum and stratum lacunosum moleculare of the CA1region. In the CA3 region, only the 5-HT1A receptors were upregulated, no changes were observed for D1 receptors in this region. GABAA receptor density in the hippocampus and amygdala was downregulated (non-significant trend) after parental separation.
The acoustic presence of the mother during parental separation suppressed the D1- and 5-HT1A-receptor upregulation in some regions of the hippocampus, no such suppressing influence was observed for the GABAA -receptors. In the basomedial amygdala the maternal calls enhanced the separation-induced 5-HT1A -receptor upregulation in the male pups, whereas in the female pups, the separation-induced receptor densities were not only suppressed by the maternal call, but further downregulated, compared to the control group. These results demonstrate that that early adverse emotional experience alters aminergic function within the hippocampus and amygdala, and that the mother’s voice, a powerful emotional signal, can modulate these effects in the developing limbic system.

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Abstract27: 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.


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Abstract28: The influence of early parental deprivation on the development of NADPH-diaphorase-(NO-synthase) reactive neuron numbers in subregions of the orbital prefrontal cortex (ventrolateral orbital, lateral orbital and agranular insular cortex) was quantitatively investigated in the precocious lagomorph Octodon degus. 45 day old degus from three groups were compared: 1) repeated parental separation: degus which were repeatedly separated from their parents during the first three postnatal weeks and thereafter raised in undisturbed social conditions, 2) chronic isolation: degus which were raised under undisturbed social conditions until postnatal day 21, and then were reared in chronic social  isolation; and 3) control: degus which were reared undisturbed in their families. Compared to the control animals the ventrolateral orbital prefrontal cortex and agranular insular cortex of the two deprived groups displayed significantly decreased density of NADPH-diaphorase-reactive neurons (down to 62% in the ventrolateral orbital prefrontal cortex of males, 70% in the agranular insular cortex and in the lateral orbital prefrontal cortex 80% in both genders). These results confirm that early changes of social environment interferes with the development of limbic circuits, which might determine normal or pathological behaviors in later life.


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