Therapeutic concentrations of valproate but not amitriptyline increase neuropeptide Y (NPY) expression in the human SH-SY5Y neuroblastoma cell line.

Neuropeptide Y (NPY) is a peptide found in the brain and autonomic nervous system, which is associated with anxiety, depression, epilepsy, learning and memory, sleep, obesity and circadian rhythms. NPY has recently gained much attention as an endogenous antiepileptic and antidepressant agent, as drugs with antiepileptic and/or mood-stabilizing properties may exert their action by increasing NPY concentrations, which in turn can reduce anxiety and depression levels, dampen seizures or increase seizure threshold. We have used human neuroblastoma SH-SY5Y cells to investigate the effect of valproate (VPA) and amitriptyline (AMI) on NPY expression at therapeutic plasma concentrations of 0.6mM and 630nM, respectively. In addition, 12-O-tetradecanoylphorbol-13-acetate (TPA) known to differentiate SH-SY5Y cells into a neuronal phenotype and to increase NPY expression through activation of protein kinase C (PKC) was applied as a positive control (16nM). Cell viability after drug treatment was tested with a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. NPY expression was measured using immunofluorescence and quantitative RT-PCR (qRT-PCR). Results from immunocytochemistry have shown NPY levels to be significantly increased following a 72h but not 24h VPA treatment. A further increase in expression was observed with simultaneous VPA and TPA treatment, suggesting that the two agents may increase NPY expression through different mechanisms. The increase in NPY mRNA by VPA and TPA was confirmed with qRT-PCR after 72h. In contrast, AMI had no effect on NPY expression in SH-SY5Y cells. Together, the data point to an elevation of human NPY mRNA and peptide levels by therapeutic concentrations of VPA following chronic treatment. Thus, upregulation of NPY may have an impact in anti-cancer treatment of neuroblastomas with VPA, and antagonizing hypothalamic NPY effects may help to ameliorate VPA-induced weight gain and obesity without interfering with the desired central effects of VPA.

Alterations in prefrontal cortical serotonin and antidepressant-like behavior in a novel C3H/HeJxDBA/2J recombinant inbred mouse strain.

In the present study, two genetically related inbred mouse strains selectively bred for high and low fear-sensitized acoustic startle reflex (FSS) were assessed in the forced swim test model of anti-depressant action and central monoamine concentrations in several brain regions were investigated. These mice were generated through backcrossing C3H/HeJ mice on DBA/2J mice, followed by inbreeding for several generations. The high-FSS and low-FSS strains are known to differ in their acquisition and extinction of fear following auditory fear conditioning. Significantly increased concentrations of 5-HT and its metabolite 5-HIAA were observed in the medial prefrontal cortex (mPFC) but not in the hypothalamus, striatum, hippocampus, amygdala, or midbrain of high-FSS mice compared to low-FSS mice. In addition the concentration of DOPAC, the major metabolite of dopamine was also significantly increased in the mPFC. Furthermore, the high-FSS mice displayed significantly higher levels of immobility in the forced swim test but not the tail suspension test in comparison to the low-FSS group. The mPFC is not only important in the regulation of fear extinction, but also a key region of interest in the study of depression and maintenance of depressive-like behaviors. These data implicate serotonergic modulation in the mPFC in the maintenance of antidepressant-like behavior in a highly fearful mouse strain.

Ultrastructural and functional characterization of satellitosis in the human lateral amygdala associated with Ammon's horn sclerosis.

The amygdala displays neuronal cell loss and gliosis in human temporal lobe epilepsy (TLE). Therefore, we investigated a certain type of gliosis, called satellitosis, in the lateral amygdala (LA) of TLE patients with Ammon's horn sclerosis (AHS, n = 15) and non-AHS (n = 12), and in autopsy controls. Satellite cells were quantified using light and electron microscopy at the somata of Nissl-stained and glutamic acid decarboxylase-negative projection neurons, and their functional properties were studied using electrophysiology. Non-AHS cases suffered from ganglioglioma, cortical dysplasia, Sturge-Weber syndrome, astrocytoma WHO III-IV, Rasmussen's encephalitis, cerebral infarction and perinatal brain damage. TLE cases with AHS had a more prominent satellitosis as compared to non-AHS and/or autopsy cases, which correlated with epilepsy duration but not age. At ultrastructural level, the predominant type of satellite cells occurring in both AHS and non-AHS cases displayed a dark cytoplasm and an irregularly shaped dark nucleus, whereas perineuronal glial cells with a light cytoplasm and light oval nucleus were much rarer. Satellite cells expressed time- and voltage-dependent transmembrane currents as revealed by patch-clamp recordings typical for 'complex' glia, although only 44% of satellite cells were immunostained for the chondroitin sulfate proteoglycan NG2. Together, the perineuronal cells described here were a heterogenous cell population regarding their NG2 expression, although they resembled NG2 cells rather than bona fide oligodendrocytes and astrocytes based on their ultrastructural and electrophysiological characteristics. Thus, perineuronal satellitosis as studied in the LA seems to be a hallmark of AHS-associated TLE pathology in patients suffering from intractable epilepsy.

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.

Maternal and genetic effects on the acoustic startle reflex and its sensitization in C3H/HeN, DBA/2JHd and NMRI mice following blastocyst transfer.

In the present study, reciprocal embryo transfers were conducted to examine genetic and maternal effects on the baseline and fear-sensitized acoustic startle response (ASR) in the two inbred strains C3H/HeN and DBA/2JHd and the outbred strain NMRI. The largest differences in the ASR were found in untreated strains (effect size 0.6). The transfer procedure per se had a significant effect on the behavior of NMRI mice resulting in a reduction in the baseline, and an increase in the fear-sensitized ASR. In contrast, there were no significant effects of the transfer procedure in the two inbred strains. Autosomal genetic effects had a stronger impact on the amplitude of the ASR (effect sizes 0.5) than sex (effect sizes 0.06) as revealed by reciprocal embryo transfer. Nevertheless, the genetic effects on the fear-sensitized ASR were somewhat more variable and strain-dependent (effect sizes 0.1-0.2). Global maternal effects were detected after embryo transfer into NMRI mothers resulting in a larger reduction of the ASR in the offspring of DBA and NMRI donors than C3H donors (effect sizes 0.1-0.2). An additional fostering procedure was introduced to dissect uterine and postnatal maternal effects in NMRI offspring. Uterine factors changed the baseline ASR of the offspring in direction of the recipient mother strain. Surprisingly, postnatal maternal effects on the ASR were contrary to the behavior of the rearing mother. In conclusion, both genetic and prenatal/postnatal maternal factors persistently influenced the ASR of the offspring, whereas the fear-sensitized ASR was mainly influenced by genetic factors. Our study shows that uterine and postnatal maternal influences deserve more attention when determining the phenotype of genetically engineered mice at least in the first generation following embryo transfer.

Axo-somatic inhibition of projection neurons in the lateral nucleus of amygdala in human temporal lobe epilepsy: an ultrastructural study.

Here, we report ultrastructural alterations in the synaptic circuitry of the human amygdala related to neuronal cell densities in surgical specimens of patients suffering from temporal lobe epilepsy (TLE). The neuronal cell densities quantified in the basolateral complex of amygdala were significantly reduced only in the lateral nucleus (LA) of TLE patients as compared to autopsy or non-Ammon's horn sclerosis (AHS) controls (Nissl staining, immunostaining against the neuronal marker NeuN). For this reason, we focussed on the LA to perform a more detailed quantitative ultrastructural analysis, which revealed an inverse correlation between the number of axo-somatic inhibitory synaptic profiles at the somata of glutamic acid decarboxylase (GAD)-negative projection neurons and the extent of perisomatic fibrillary gliosis. In contrast, the density of GAD-immunoreactive interneurons positively correlated with the number of axo-somatic inhibitory synaptic profiles. The fibrillary material in perisomatic glial cell processes was preferentially labeled by the astroglial marker S100B. In addition, a qualitative study of the dendrites of GAD- and parvalbumin (PARV)-containing interneurons showed that they were often contacted by asymmetrical excitatory synapses. Our results are in line with anatomical data from rodents and cats, which show that amygdalar interneurons form axo-somatic inhibitory synapses on GAD-negative projection neurons, whereas the interneurons themselves receive excitatory input from recurrent collaterals of projection neurons and from cortico- and thalamo-amygdalar afferents. The structural reorganization patterns observed in the GABAergic circuitry are compatible with a reduced feedback or feed forward inhibition of amygdalar projection neurons in human TLE.

Differential effects of embryo transfer and maternal factors on anxiety-related behavior and numbers of neuropeptide Y (NPY) and parvalbumin (PARV) containing neurons in the amygdala of inbred C3H/HeN and DBA/2J mice.

The effects of reciprocal embryo transfers were studied on anxiety-related behavior of inbred C3H/HeN and DBA/2J mice on the elevated plus maze (EPM), and related to amygdaloid neuropeptide Y (NPY)- and parvalbumin (PARV)-immunoreactive neurons. Embryo transfer significantly reduced closed arm entries in in-stain-transferred C3H mice, and maternal factors influenced open arm entries only in interaction with genetic background and sex. In DBA/2J-mice, embryo transfer resulted in a reduced number of NPY-immunoreactive (NPY-ir) neurons, while PARV-immunoreactive (PARV-ir) cells were not affected. In C3H/HeN mice, however, in-strain embryo transfer only resulted in a reduction of the number of PARV-immunoreactive neurons. Maternal factors mainly induced changes in the number of NPY-ir neurons in the basolateral complex of the amygdala either directly or in interaction with genetic factors. In summary, in-strain embryo transfer had a minor effect on the behavior of C3H/HeN mice, and a differential influence on the numbers of amygdaloid NPY-ir and PARV-ir neurons of inbred C3H/HeN and DBA/2J mice. Maternal factors had a stronger impact on the numbers of NPY-ir neurons than PARV-ir neurons. The present results indicate that alterations in behavior and amygdala morphology induced by embryo transfer or maternal factors depend on the genetic background of the mouse strains used.

Maternal and genetic effects on anxiety-related behavior of C3H/HeN, DBA/2J and NMRI mice in a motility-box following blastocyst transfer.

Reciprocal embryo transfers were conducted to examine genetic and maternal effects on the behavior of inbred C3H/HeN and DBA/2J mice, and outbred NMRI mice using a motility-box. The behavioral variables measured were (i) horizontal locomotor activity assessed as the path and time spent during traveling; (ii) vertical activity assessed as the time spent with and numbers of rearings/leanings; (iii) and the time spent in the more anxiogenic central field. The transfer procedure per se resulted in a minor increase in vertical activity of inbred C3H/HeN mice, but had no effect in inbred DBA/2J mice. In contrast, outbred NMRI mice displayed a lower central field activity following embryo transfer indicating a higher anxiety level. Moreover, genetic differences between the mouse strains studied remained stable following embryo transfer for locomotor and vertical activity, but not central field activity depending on the recipient mother strain. Maternal effects were found for (i) vertical activity in the two inbred mouse strains, (ii) all behavioral variables studied in outbred NMRI mice, and (iii) an interaction with gender for the time spent in the anxiogenic central field. An additional fostering procedure revealed that the vertical activity of NMRI mice was modified towards the behavior of the recipient C3H/HeN strain by uterine factors, whereas the postnatal maternal effect of C3H/HeN mothers was the opposite. In summary, the effects of the embryo transfer procedure per se, stability of genetic characteristics following embryo transfer and maternal effects were related to the mouse strains used as donators and recipients, and the behavioral variables studied.

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.

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.