[Ethical Considerations of Including Minors in Clinical Trials Using the Example of the Indicated Prevention of Psychotic Disorders]. / Minderjährige in klinischen Prüfungen: Ethische Abwägungen ihres Einbezugs am Beispiel der indizierten Prävention psychotischer Erkrankungen.

Ethical Considerations of Including Minors in Clinical Trials Using the Example of the Indicated Prevention of Psychotic Disorders Abstract: As a vulnerable group, minors require special protection in studies. For this reason, researchers are often reluctant to initiate studies, and ethics committees are reluctant to authorize such studies. This often excludes minors from participating in clinical studies. This exclusion can lead to researchers and clinicians receiving only incomplete data or having to rely on adult-based findings in the treatment of minors. Using the example of the study "Computer-Assisted Risk Evaluation in the Early Detection of Psychotic Disorders" (CARE), which was conducted as an 'other clinical investigation' according to the Medical Device Regulation, we present a line of argumentation for the inclusion of minors which weighs the ethical principles of nonmaleficence (especially regarding possible stigmatization), beneficence, autonomy, and fairness. We show the necessity of including minors based on the development-specific differences in diagnostics and early intervention. Further, we present specific protective measures. This argumentation can also be transferred to other disorders with the onset in childhood and adolescence and thus help to avoid excluding minors from appropriate evidence-based care because of insufficient studies.

Impaired active avoidance learning in infant rats appears to be related to insufficient metabolic recruitment of the lateral septum.

The temporal dissociation between early information acquisition and output of complex behaviors is a common principle during development. Thus, although infant rats are not able to generate sufficient avoidance behavior during two-way active avoidance (TWA) training they obviously deposit a certain "memory trace" (Schäble, Poeggel, Braun, & Gruss, 2007). The ontogeny of learning is probably mirrored by the maturing functionality of different basal forebrain regions. Two of the basal forebrain regions involved in TWA learning are the medial septum/diagonal band of Broca (MS/DB), which is essential for the encoding and retrieval of memory and the lateral septum (LS) that plays a role in the generation of behavior. Mapping 2-fluoro-deoxy-glucose utilization in freely behaving animals, the aim of this study was to assess the functional recruitment of the MS/DB and LS in infant (P17-P21) and adolescent (P38-P42) rats during the first (acquisition) and fifth (retrieval) TWA training. Metabolic activity in the MS/DB was similar in both age groups during acquisition and retrieval indicating that this region is already mature in the infant rat. In contrast, metabolic activity in the LS was generally lower in the infant rats suggesting that this region is not yet fully functional during P17 and P21. This insufficient recruitment may be one reason for the poor TWA performance of infant rats. Finally, the LS displayed significantly higher activity during acquisition than during retrieval indicating that the highest amount of energy is consumed during the initial learning phase.

Dopaminergic modulation of auditory cortex-dependent memory consolidation through mTOR.

Previous studies in the auditory cortex of Mongolian gerbils on discrimination learning of the direction of frequency-modulated tones (FMs) revealed that long-term memory formation involves activation of the dopaminergic system, activity of the protein kinase mammalian target of rapamycin (mTOR), and protein synthesis. This led to the hypothesis that the dopaminergic system might modulate memory formation via regulation of mTOR, which is implicated in translational control. Here, we report that the D1/D5 dopamine receptor agonist SKF-38393 substantially improved gerbils' FM discrimination learning when administered systemically or locally into the auditory cortex shortly before, shortly after, or 1 day before conditioning. Although acquisition performance during initial training was normal, the discrimination of FMs was enhanced during retraining performed hours or days after agonist injection compared with vehicle-injected controls. The D1/D5 receptor antagonist SCH-23390, the mTOR inhibitor rapamycin, and the protein synthesis blocker anisomycin suppressed this effect. By immunohistochemistry, D1 dopamine receptors were identified in the gerbil auditory cortex predominantly in the infragranular layers. Together, these findings suggest that in the gerbil auditory cortex dopaminergic inputs regulate mTOR-mediated, protein synthesis-dependent mechanisms, thus controlling for hours or days the consolidation of memory required for the discrimination of complex auditory stimuli.

Vesicular glutamate transporter 3-immunoreactive pericellular baskets ensheath a distinct population of neurons in the lateral septum.

The lateral septum (LS) plays a role in the adjustment of behavioral responses according to environmental demands. This is a complex integrative process wherein a variety of modulatory systems, i.e. cholinergic, dopaminergic and serotonergic projections forming pericellular baskets around LS neurons, are involved. Recently, vesicular glutamate transporter 3 (VGLUT3)-immunoreactive (-ir) structures outlining unlabeled somata and their proximal dendrites were described in the LS. However, the vesicular transporters for acetylcholine and GABA were not or only rarely co-expressed with VGLUT3. In this study, the morphology and distribution of these VGLUT3-ir structures were systematically analyzed revealing that (1) they form distinct pericellular baskets (PBs) displaying variable shapes, (2) they are arranged in a layer-like pattern similar to the terminals of other modulatory systems, (3) beside a few exceptions (e.g., choline acetyltransferase), they are generally not or very sparsely co-localized with other neurochemical markers characterizing major neuron populations or afferent systems of the LS, i.e. calcium-binding proteins, tyrosine hydroxylase, tryptophan hydroxylase, vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2) and the vesicular GABA transporter. Thus, in the LS, a separate population of neurons is covered by VGLUT3-ir PBs. The distribution pattern and the lack of co-localization indicate that the VGLUT3-expressing cells of origin are located in the brainstem and that they could be pure glutamatergic projection neurons-different from the well-defined canonical VGLUT1- and VGLUT2-expressing neurons. Alternatively, they could simultaneously express VGLUT3 and second transmitter, but use different release sites inside the LS for both.

Increased sensitivity to agonist-induced seizures, straub tail, and hippocampal theta rhythm in knock-in mice carrying hypersensitive alpha 4 nicotinic receptors.

We studied a strain of exon replacement mice ("L9'S knock-in") whose alpha4 nicotinic receptor subunits have a leucine to serine mutation in the M2 region, 9' position (Labarca et al., 2001); this mutation renders alpha4-containing receptors hypersensitive to agonists. Nicotine induced seizures at concentrations (1 mg/kg) approximately eight times lower in L9'S than in wild-type (WT) littermates. At these concentrations, L9'S but not WT showed increases in EEG amplitude and theta rhythm. L9'S mice also showed higher seizure sensitivity to the nicotinic agonist epibatidine, but not to the GABA(A) receptor blocker and proconvulsant bicuculline. Dorsiflexion of the tail (Straub tail) was the most sensitive nicotine effect found in L9'S mice (0.1 mg/kg). The L9'S mice were hypersensitive to galanthamine- and tacrine-induced seizures and Straub tails. There were no apparent neuroanatomical differences between L9'S and WT mice in several brain regions. [(125)I]Epibatidine binding to brain membranes showed that the mutant allele was expressed at approximately 25% of WT levels, presumably because of the presence of a neomycin selection cassette in a nearby intron. (86)Rb efflux experiments on brain synaptosomes showed an increased fraction of function at low agonist concentrations in L9'S mice. These data support the possible involvement of gain-of-function alpha4 receptors in autosomal dominant nocturnal frontal-lobe epilepsy.

Reelin-immunoreactivity in the hippocampal formation of 9-month-old wildtype mouse: effects of APP/PS1 genotype and ovariectomy.

Reelin, an extracellular matrix protein has an important role in the migration, correct positioning and maturation of neurons during development. Though it is generally down-regulated in the postnatal period, expression of this large glycoprotein continues in the adult brain in some cell populations. In the present study, we examined the distribution of reelin-immunoreactivity (-ir) in the hippocampal formation of 9-month-old wildtype mice (WT). Then, reelin-ir in normal mice was compared to that of transgenic mice (APP/PS1) carrying mutated human APP and PS1 genes, which are linked to the familial form of Alzheimer's disease (AD). The APP/PS1 mice were additionally burdened with a second risk factor for AD, namely depletion of circulating gonadal hormones by ovariectomy (APP/PS1 + OVX). The analyses revealed that in adult WT reelin-ir is expressed by Cajal-Retzius cells and a subgroup of interneurons throughout the hippocampal formation. In addition, layer II projection neurons in the lateral entorhinal subfields are reelin-ir. Interestingly, ovariectomy decreases the number of reelin-ir cells in the hilus in WT mice, whereas AD-related genotype alone induces only a non-significant reduction. Unexpectedly, additional stress, e.g., depletion of gonadal hormones, does not aggravate the slight reduction in the reelin cell number in the APP/PS1 mice. We propose that the changes in normal reelin-ir are linked to disturbances in repair mechanisms in which APP/PS1 and gonadal hormones are involved and which are perturbed in neurodegenerative conditions, namely AD.

Complementary distribution of vesicular glutamate transporters 1 and 2 in the nucleus accumbens of rat: Relationship to calretinin-containing extrinsic innervation and calbindin-immunoreactive neurons.

The caudomedial shell of the rat nucleus accumbens exhibits inhomogeneous distribution patterns of the vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2). This paper focuses on the question of whether patterns of VGLUT1 and VGLUT2 correspond to cytoarchitectonically and cytochemically defined subterritories of the caudomedial shell region. VGLUT2 was shown to be coexpressed with calretinin in the dense axonal plexus known to emanate from the paraventricular thalamic nucleus. In regions termed corridors, which are spared by this paraventricular thalamic innervation, axonal terminals were found to be clustered and VGLUT1-immunoreactive. It is assumed that these fibers originate in the prelimbic cortex and/or in the parvicellular basal amygdaloid nucleus known to project to accumbal shell components. Our findings confirm the existence of two well-separated neuronal circuits in the caudomedial shell that are dominated by two different excitatory input systems originating from either thalamic, cortical, or cortex-like amygdaloid sources. The large lateral corridors-which resemble the accumbal core not only in respect to their VGLUT1 immunolabeling but also concerning their content of calbindin-positive cells-may represent a component of the anatomically weakly defined accumbal shore region.

Immunocytochemical evidence for the striatal nature of the rat lateral part of interstitial nucleus of the posterior limb of the anterior commissure (IPAC).

The present study focuses on the basal forebrain region originally designated as fundus striati, but currently known as 'interstitial nucleus of the posterior limb of the anterior commissure' (IPAC). Using multiple immunofluorescence of the calcium-binding proteins parvalbumin and calbindin, the GABA(A) receptor alpha1-subunit, vasoactive intestinal polypeptide (VIP), met(5)-enkephalin (MENK) and glutamic acid decarboxylase (GAD), it was shown that VIP-immunostained axons, which are typical for major parts of the extended amygdala, densely innervate only the medial part of IPAC, while they are absent in the lateral part. On the other hand, large-sized GABAergic, parvalbumin- and GABA(A) receptor alpha1-subunit-immunoreactive neurons, which are densely covered by separate GAD- and MENK-immuno reactive terminals and a type of medium-sized alpha1-subunit-monolabelled cells, occur in the dorsal striatum and in the adjacent lateral part of IPAC as well. Large-sized neurons double labelled for parvalbumin and the GABA(A) receptor alpha1-subunit are also widely distributed in the neighbouring ventral pallidum. Neurons of this type are absent, however, in the medial part of IPAC and other extended amygdala subunits. Our findings confirm the recent suggestion of a morphofunctional dichotomy of IPAC (Comp. Neurol. 439 (2001) 104), as only the medial part reveals characteristics as typical for extended amygdala, while its lateral part exhibits cytochemical peculiarities of striatal tissue. Therefore, the term 'lateral part of IPAC' should be replaced by the term 'putaminal fundus (fundus putaminis)' according to recently published designations of corresponding striatal constituents (Atlas of the Human Brain, 2002, Academic Press, San Diego, CA; J. Chem. Neuroanat. 23 (2002) 75).

VGLUT3-immunoreactive afferents of the lateral septum: ultrastructural evidence for a modulatory role of glutamate.

Through its extensive connections with various brain regions, the lateral septum (LS) participates in the processing of cognitive, emotional and autonomic information. It is decisively involved in the generation of behavioral responses according to environmental demands. Modulatory afferents reaching the LS from the brain stem (e.g. dopaminergic, serotonergic) play a role in the adjustment of these behavioral responses. Recently, a population of vesicular glutamate transporter 3-immunoreactive (VGLUT3-ir) fibers forming prominent pericellular basket-like structures (PBLS) was described in the rat LS. These VGLUT3-ir PBLS are distributed in a layer-like pattern, which is very typical for modulatory afferents of the LS. There is meanwhile broad evidence that glutamate can act as a modulatory or co-transmitter and that those neurons, which make use of this transmission mode, primarily express VGLUT3. Thus, the VGLUT3-ir fibers within the LS could also display features typical for non-canonical glutamatergic transmission. Employing pre-embedding electron microscopy for VGLUT3 in rats, we show now that the VGLUT3-ir fibers outlining LS neurons represent axonal terminals, which primarily form symmetric synapses with somata and proximal dendrites of their target neurons. Occasionally, we also found VGLUT3-ir terminals that make canonical asymmetric synapses on distal dendrites and spines. Thus, VGLUT3-ir boutons in the LS form two different, disproportionate, populations of synaptic contacts with their target neurons. The larger one of them is indicative of employing glutamate as a modulatory transmitter.

Differential changes of metabolic brain activity and interregional functional coupling in prefronto-limbic pathways during different stress conditions: functional imaging in freely behaving rodent pups.

The trumpet-tailed rat or degu (Octodon degus) is an established model to investigate the consequences of early stress on the development of emotional brain circuits and behavior. The aim of this study was to identify brain circuits, that respond to different stress conditions and to test if acute stress alters functional coupling of brain activity among prefrontal and limbic regions. Using functional imaging (2-Fluoro-deoxyglucose method) in 8-day-old male degu pups the following stress conditions were compared: (A) pups together with parents and siblings (control), (B) separation of the litter from the parents, (C) individual separation from parents and siblings, and (D) individual separation and presentation of maternal calls. Condition (B) significantly downregulated brain activity in the prefrontal cortex, hippocampus, nucleus accumbens (NAcc), and sensory areas compared to controls. Activity decrease was even more pronounced during condition (C), where, in contrast to all other regions, activity in the PAG was increased. Interestingly, brain activity in stress-associated brain regions such as the amygdala and habenula was not affected. In condition (D) maternal vocalizations "reactivated" brain activity in the cingulate and precentral medial cortex, NAcc, and striatum and in sensory areas. In contrast, reduced activity was measured in the prelimbic and infralimbic cortex (IL) and in the hippocampus and amygdala. Correlation analysis revealed complex, region- and situation-specific changes of interregional functional coupling among prefrontal and limbic brain regions during stress exposure. We show here for the first time that early life stress results in a widespread reduction of brain activity in the infant brain and changes interregional functional coupling. Moreover, maternal vocalizations can partly buffer stress-induced decrease in brain activity in some regions and evoked very different functional coupling patterns compared to the three other conditions.

Estimation of the total number of hippocampal CA1 pyramidal neurons: new methodology applied to helpless rats.

We have recently reported that in the learned helplessness model of depression, the less hippocampal spine synapses rats have, the more helpless they become. It remains unclear, however, whether the observed synaptic changes are associated with the loss of CA1 pyramidal cells. Cell bodies in the CA1 pyramidal layer are very densely packed, making cell counting difficult in this hippocampal subregion. To address this issue, we developed a new approach that (1) yields excellent preservation of the three-dimensional tissue structure; (2) utilizes osmium tetroxide to unambiguously label nucleoli; and (3) facilitates and accelerates unbiased, reliable counting of densely packed cell bodies. Our method provides an improved tool for studies aiming to evaluate hippocampal atrophy and cell loss, the most characteristic features in many neurodegenerative diseases, such as Alzheimer's disease, temporal lobe epilepsy and ischemia, as well as in several psychiatric disorders. Using this new method, we demonstrated no significant changes in the number of CA1 pyramidal cells in the rat learned helplessness paradigm. In addition, volumes of the CA1 pyramidal cell layer and the entire CA1 subfield remained unchanged among treatment groups. We conclude that previously observed synaptic alterations in helpless rats are not associated with CA1 pyramidal cell loss. This finding suggests that behavioral outcome in the learned helplessness paradigm is related to plastic events at the synaptic level, rather than at the level of principal cells.

Reelin-immunoreactive Cajal-Retzius cells: the entorhinal cortex in normal aging and Alzheimer's disease.

Alzheimer's disease (AD) is a disorder of brain self organization associated with morphodysregulation at the synaptic level. Disturbances follow a hierarchical spatio-temporal pattern throughout the cortex and involve the re-activation of developmental molecular programs. The large glycoprotein reelin, synthesized by Cajal-Retzius (CR) cells, is an important component of a signaling pathway involved in embryonic development and modulation of synaptic circuitry, but is also implicated in the pathogenetic cascade in AD. Although the majority of CR cells sequentially disappears from the postnatal cortical layer I, a few of them persist in the normal adult brain. They continue to produce reelin, express a variety of other proteins, and are characterized by a typical morphology. Recently, CR cells have been reported to be altered in number and morphology in a variety of neurological and psychiatric diseases linked to maldevelopment. In the present study we show that reelin-positive CR cells persist in the layer I of the entorhinal cortex in normal senescent brains and are also preserved in AD. The majority of CR cells in AD is morphologically and cytochemically-as revealed by double labeling with calcium-binding proteins-indistinguishable from normal cases, suggesting that they are not dramatically altered in the entorhinal cortex of AD patients. Nevertheless, CR cells seem to be partially affected by the formation of paired helical filaments, indicating subtle changes that are suggested to be a result rather than a cause of the pathogenetic cascade of AD.