Deniz Yilmazer-Hanke

Anxiety-related behavior and densities of glutamate, GABAA, acetylcholine and serotonin receptors in the amygdala of seven inbred mouse strains

The amygdala is a brain region involved in the regulation of anxiety-related behavior. The purpose of this study was to correlate anxiety-related behavior of inbred mouse strains (BA//c, BALB/cJ, C3H/HeJ, C57BL/6J, CPB-K, DBA/2J, NMRI) to receptor binding in the amygdala. Binding site densities of receptors (NMDA, AMPA, kainate, GABA(A), serotonin, muscarinergic M(1)-M(2)) were measured with quantitative receptor autoradiography using tritiated ligands. Measurements of fear-sensitized acoustic startle response (ASR; induced by footshocks), elevated plus maze (EPM) behavior and receptor binding studies showed differences between the strains except for AMPA and muscarinergic M(2) receptors. Factor analysis revealed a Startle Factor with positive loadings of the density of serotonin and kainate receptors, and the amplitudes of the baseline and fear-sensitized ASRs. A second Anxiety-related Factor only correlated with the fear-sensitized ASR and anxiety parameters on the EPM but not receptor densities. There were also two General Activity Factors defined by (negative) correlations with entries to closed arms of the EPM. Because the density of NMDA and muscarinergic M(1) receptors also correlated negatively with the two factors, these receptors had a positive effect on general activity. In contrast, correlations of GABA(A), serotonin, and kainate receptors had the opposite sign as compared to closed arm entries. It is concluded that hereditary variations in the amygdala, particularly in kainate and serotonin receptors, play a role for the baseline and fear-sensitized ASR, whereas the general activity is influenced by many neurotransmitter receptor systems.

THE CORTICOTROPIN-RELEASING FACTOR (CRF)-SYSTEM AND MONOAMINERGIC AFFERENTS IN THE CENTRAL AMYGDALA: INVESTIGATIONS IN DIFFERENT MOUSE STRAINS AND COMPARISON WITH THE RAT

Corticotropin-releasing-factor (CRF) containing systems and monoaminergic afferents of the central amygdaloid nucleus (Ce) are crucial players in central nervous stress responses. For functional analyses of specific roles of these systems, numerous mouse models have been generated which lack or overexpress individual signal transduction components. Since data concerning system morphologies in murine brain are rarely available, mouse studies are usually designed and interpreted based on previous findings in rats, although interspecies differences are frequent. In the present study, in situ hybridization for CRF mRNA and correlative immunocytochemistry for CRF and monoaminergic afferents revealed numerous CRF mRNA-reactive neurons in the lateral Ce subnucleus (CeL) codistributed with dense dopaminergic fiber plexus in mice as has been demonstrated in rats. However, while in rats the lateral capsular Ce (CeLc) displays only scarce CRF immunoreactive (CRF-ir) innervation, particularly dense CRF-ir fiber plexus were observed in the CeLc in mice, with differences in labeling densities between different strains. CRF-ir terminal fibers overlap with the moderate serotonergic innervation of this subnucleus in mice. Additionally, CRF mRNA-reactive neurons were found immediately dorsal to the amygdala in the region of the interstitial nucleus of the posterior limb of the anterior commissure/amygdalostriatal transition area in both species. In mice, this region displayed dense CRF-ir fiber plexus, with variations between the strains. The results indicate that in mice and rats dopaminergic afferents represent the primary monoaminergic input to the CRF neurons in the CeL. In mice only, CRF-ir afferents provide dense innervation of CeLc neurons. Since the CeLc lacks dopaminergic input in both species but possesses moderate serotonergic afferents, CRF/serotonin interactions may occur selectively in mouse CeLc. The observed interspecies and interstrain differences in CRF input and CRF/monoaminergic interactions may influence the interpretation of findings concerning Ce functions in stress and fear in mouse models.

Contribution of amygdala neurons containing peptides and calcium-binding proteins to fear-potentiated startle and exploration-related anxiety in inbred Roman high- and low-avoidance rats

The purpose of this study was to investigate amygdala‐related fear and anxiety in two inbred rat lines differing in emotionality (RHA/Verh and RLA/Verh), and to relate the behaviour of the animals to neuronal types in different nuclei of the amygdala. The behavioural tests used were the motility test, elevated plus‐maze and fear‐potentiated startle response. The neurons investigated were immunoreactive for the anxiogenic peptide corticotropin‐releasing factor (CRF‐ir), the anxiolytic peptide neuropeptide Y (NPY‐ir), and the calcium‐binding proteins parvalbumin (PARV‐ir) and calbindin (CALB‐ir). The NPY‐ir, PARV‐ir and CALB‐ir neurons studied were subpopulations of GABAergic neurons. RLA/Verh rats, which showed a significant fear‐potentiation of the acoustic startle response, had more CRF‐ir projection neurons in the central nucleus of the amygdala. The same RLA/Verh rats were either less or equally anxious in the motility test (similar to open field) and elevated plus‐maze as compared with RHA/Verh rats. In accordance with this behaviour, the RLA/Verh rats had more NPY‐ir neurons in the lateral, and more PARV‐ir neurons in basal nuclei of the amygdala than RHA/Verh rats, but no differences were detected in the number of CRF‐ir and CALB‐ir neurons of the basolateral complex. In conclusion, the RLA/Verh rats displayed an opposite behaviour in the fear‐potentiated startle model and the exploratory tests measuring anxiety based on choice behaviour. Thus, the anxiogenic systems in the central nucleus and anxiolytic systems in the basolateral complex of the amygdala might be differentially involved in the fear‐potentiated startle paradigm and exploratory tests in the Roman rat lines.

Impaired Pavlovian fear extinction is a common phenotype across genetic lineages of the 129 inbred mouse strain.

Fear extinction is impaired in psychiatric disorders such as post‐traumatic stress disorder and schizophrenia, which have a major genetic component. However, the genetic factors underlying individual variability in fear extinction remain to be determined. By comparing a panel of inbred mouse strains, we recently identified a strain, 129S1/SvImJ (129S1), that exhibits a profound and selective deficit in Pavlovian fear extinction, and associated abnormalities in functional activation of a key prefrontal‐amygdala circuit, as compared with C57BL/6J. The first aim of the present study was to assess fear extinction across multiple 129 substrains representing the strains four different genetic lineages (parental, steel, teratoma and contaminated). Results showed that 129P1/ReJ, 129P3/J, 129T2/SvEmsJ and 129X1/SvJ exhibited poor fear extinction, relative to C57BL/6J, while 129S1 showed evidence of fear incubation. On the basis of these results, the second aim was to further characterize the nature and specificity of the extinction phenotype in 129S1, as an exemplar of the 129 substrains. Results showed that the extinction deficit in 129S1 was neither the result of a failure to habituate to a sensitized fear response nor an artifact of a fear response to (unconditioned) tone per se. A stronger conditioning protocol (i.e. five × higher intensity shocks) produced an increase in fear expression in 129S1, relative to C57BL/6J, due to rapid rise in freezing during tone presentation. Taken together, these data show that impaired fear extinction is a phenotypic feature common across 129 substrains, and provide preliminary evidence that impaired fear extinction in 129S1 may reflect a pro‐fear incubation‐like process.

Morphological correlates of emotional and cognitive behaviour: insights from studies on inbred and outbred rodent strains and their crosses.

Every study in rodents is also a behavioural genetic study even if only a single strain is used. Outbred strains are genetically heterogeneous populations with a high intrastrain variation, whereas inbred strains are based on the multiplication of a unique individual. The aim of the present review is to summarize findings on brain regions involved in three major components of rodent behaviour, locomotion, anxiety-related behaviour and cognition, by paying particular attention to the genetic context, genetic models used and interstrain comparisons. Recent trends correlating gene expression in inbred strains with behavioural data in databases, morpho-behavioural-haplotype analyses and problems arising from large-scale multivariate analyses are discussed. Morpho-behavioural correlations in multiple strains are presented, including correlations with projection neurons, interneurons and fibre systems in the striatum, midbrain, amygdala, medial septum and hippocampus, by relating them to relevant transmitter systems. In addition, brain areas differentially activated in different strains are described (hippocampus, prefrontal cortex, nucleus accumbens, locus ceruleus). Direct interstrain comparisons indicate that strain differences in behavioural variables and neuronal markers are much more common than usually thought. The choice of the appropriate genetic model can therefore contribute to an interpretation of positive results in a wider context, and help to avoid misleading interpretations of negative results.

Neonatal thyroxine treatment: changes in the number of corticotropin-releasing-factor (CRF) and neuropeptide Y (NPY) containing neurons and density of tyrosine hydroxylase positive fibers (TH) in the amygdala correlate with anxiety-related behavior of wistar rats.

Neonatal hyperthyroidism induces persisting alterations in the adult brain, e.g. in spatial learning and hippocampal morphology. In the present study, the relationship between anxiety-related behavior and amygdala morphology was investigated in the adult rat after transient neonatal hyperthyroidism (daily s.c. injections of 7.5 microg L-thyroxine in 0.5 ml 0.9% NaCl solution from postnatal day p1 to p12). The behavioral tests used to study anxiety-related behavior were the motility test, elevated plus-maze and fear-sensitized acoustic startle response. In the amygdala, the number of neurons containing the anxiogenic peptide corticotropin releasing factor (CRF-ir and CRF mRNA) and anxiolytic neuropeptide Y (NPY-ir), the total number of neurons and the density of tyrosine hydroxylase immunoreactive (TH-ir) fibers were quantified. Thyroxine-treated pups presented an accelerated development including opening of eyes and snout elongation as typical signs of hyperthyroidism. Thyroxine-treated adult animals displayed a reduced anxiety in the motility box and elevated plus maze, a reduction in the number of CRF-ir neurons in the central nucleus of the amygdala, as well as an increase in the number of NPY-ir neurons and density of TH-ir fibers in nuclei of the basolateral complex of the amygdala. Moreover, there was a reduction in the total number of neurons in all nuclei of the basolateral complex (despite the higher number of NPY-ir neurons), but not central nucleus of the amygdala. The number of CRF-ir neurons in the central nucleus correlated positively with anxiety-related behavior, and the number of NPY-ir neurons and the density of TH-ir fibers in the basolateral complex correlated inversely with anxiety-related behavior. The findings suggested a shift toward an anxiolytic rather than anxiogenic distribution of peptidergic neurons and fibers in the amygdala at adult age following transient neonatal hyperthyroidism.

Interrelations between monoaminergic afferents and corticotropin-releasing factor-immunoreactive neurons in the rat central amygdaloid nucleus: ultrastructural evidence for dopaminergic control of amygdaloid stress systems.

Ample evidence implicates corticotropin-releasing factor (CRF)-producing neurons of the central amygdaloid nucleus (CeA) in vegetative, endocrine, and behavioral responses to stress and anxiety in laboratory rats. Monoaminergic systems are involved in modulating these responses. In the present paper, interrelations between CRF-immunoreactive (ir) neurons, and noradrenergic, serotonergic, and dopaminergic afferents were studied using single and double immunolabeling for light and electron microscopy in the rat CeA. Dopaminergic axons formed dense plexus in the CeA overlapping with the localization of CRF-ir neurons, and their terminals formed frequent associations with CRF-ir somata. Contacts of serotonergic axons on CRF-ir neurons were few, and contacts of noradrenergic axons were the exception. Ultrastructurally, symmetric synapses of dopaminergic terminals on CRF-ir somata and dendrites were found. More than 83% of CRF-ir somata were contacted in single ultrathin sections. About half of these possessed two or more contacts. Of non-ir somata, 37% were contacted by dopaminergic terminals, and only 13% of these had two or more contacts. Correlative in situ hybridization indicated that CeA CRF-ir neurons may express receptor subtype dopamine receptor subtype 2. In conclusion, dopaminergic afferents appear to specifically target CeA CRF neurons. They are thus in a position to exert significant influence on the rat amygdaloid CRF stress system.

Topography of thalamic and parabrachial calcitonin gene‐related peptide (CGRP) immunoreactive neurons projecting to subnuclei of the amygdala and extended amygdala

Injections of calcitonin gene‐related peptide (CGRP) into the amygdala evoke fear‐related behaviors and antinociceptive effects. In the present study we therefore characterized CGRP‐containing amygdaloid afferents by injecting the retrograde tracer FluoroGold (FG) into subnuclei of the amygdala and adjacent divisions of the extended amygdala, namely, the lateral (LA) and central (CE) amygdaloid nuclei, interstitial nucleus of the posterior limb of the anterior commissure (IPAC), and the amygdalostriatal area (AStr). The distribution of retrogradely FG‐labeled neurons and colocalization of CGRP‐immunoreactivity with FG‐labeling were mapped in the posterior paralaminar thalamic complex and parabrachial nuclei. The analysis of the posterior thalamus revealed that about 50% of CGRP‐containing neurons projected to the AStr, the projections originating in the medial part of the medial geniculate body, posterior intralaminar nucleus, parvicellular subparafascicular nucleus, and peripeduncular nucleus. However, the percentage of CGRP‐containing thalamic neurons projecting to the adjacent LA, medial part of the CE, and ventrocaudal part of the caudatoputamen rapidly dropped to 3–9%. There were no double‐labeled cells after injections into the lateral and capsular parts of the CE and the IPAC. Thus, the AStr received the heaviest CGRP‐containing projection from the posterior thalamus. CGRP‐containing parabrachial neurons projected to the AStr and lateral, capsular, and medial parts of the CE, the projections originating in the external, crescent, and central parts of the lateral parabrachial nucleus and external part of the medial parabrachial nucleus. The results demonstrate a distinct projection pattern of CGRP‐containing thalamic and parabrachial neurons to subnuclei of the amygdala and extended amygdala. J. Comp. Neurol. 505:268–291, 2007.

Two Wistar rat lines selectively bred for anxiety-related behavior show opposite reactions in elevated plus maze and fear-sensitized acoustic startle tests.

Two Wistar rat lines selectively bred for high (HAB), and low anxiety-related behavior (LAB) on the elevated plus maze were tested for the fear-sensitized acoustic startle response. The study of male rats from the F9 generation revealed a higher anxiety level of HAB rats on the elevated plus maze. However, the LAB rats displayed a higher baseline and fear-sensitized acoustic startle response compared to HAB rats, although the two rat lines did not differ in freezing duration during the interstimulus intervals in the startle experiment (neither before, nor after, footshocks). Counts of neurons immunoreactive for corticotropin-releasing factor and neuropeptide Y in amygdaloid nuclei did not reveal any differences between the two lines, which is in marked contrast to findings in the Roman rat lines (Yilmazer-Hanke, D. M. et al. [2002]). The data indicate that opposite types of anxiety/fear responses are elicited in HAB/LAB rats in the elevated plus maze and fear-sensitized startle tests. Moreover, the animals displayed a differential fear response in the startle experiment, as assessed by measuring the fear-sensitized startle response and freezing.

Progression of Alzheimer-Related Neuritic Plaque Pathology in the Entorhinal Region, Perirhinal Cortex and Hippocampal Formation

Extracellular deposits of the β-amyloid protein and intraneuronal neurofibrillary changes are hallmarks of Alzheimer’s disease. Neurofibrillary changes in the cell body of neurons are the neurofibrillary tangles, while β-amyloid deposits containing dystrophic neurites with neurofibrillary changes are called neuritic plaques. β-Amyloid deposits and neurofibrillary tangles display a sequential accumulation in the cerebral cortex. In the present study, the topographical distribution of β-amyloid deposits and neuritic plaques in the entorhinal region, perirhinal cortex and hippocampal formation was investigated in relationship to the amyloid and neurofibrillary staging proposed by Braak. The number of subregions displaying β-amyloid deposits and neuritic plaques continuously increases in correlation with the amyloid stage (for β-amyloid deposits r = 0.90, p < 0.0001, for neuritic plaques r = 0.74, p < 0.0001) and neurofibrillary stage (for β-amyloid deposits r = 0.53, p < 0.0001, for neuritic plaques r = 0.68, p < 0.0001). Parallel to the advancement in the neurofibrillary stage, early and late predilection sites of β-amyloid deposits and neuritic plaques can be distinguished. The early predilection sites correspond to projection areas of regions which exhibit incipient neurofibrillary tangles. Furthermore, neuritic plaques only occur in the presence of neurofibrillary tangles in the areas investigated. The findings indicate that neuritic plaques gradually develop in the projection areas of tangle-bearing neurons.