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Abstract Background The histone demethylase GASC1 (JMJD2C) is an epigenetic factor suspected of involvement in development of different cancers, including breast cancer. It is thought to be overexpressed in the more aggressive breast cancer types based on mRNA expression studies on cell lines and meta analysis of human breast cancer sets. This study aimed to evaluate the prognostic and predictive value of GASC1 for women with invasive breast cancer. Methods All the 355 cases were selected from a cohort enrolled in the Kuopio Breast Cancer Project between April 1990 and December 1995. The expression of GASC1 was studied by immunohistochemistry (IHC) on tissue microarrays. Additionally relative GASC1 mRNA expression was measured from available 57 cases. Results In our material, 56% of the cases were GASC1 negative and 44% positive in IHC staining. Women with GASC1 negative tumors had two years shorter breast cancer specific survival and time to relapse than the women with GASC1 positive tumors (p=0.017 and p=0.034 respectively). The majority of GASC1 negative tumors were ductal cases (72%) of higher histological grade (84% of grade II and III altogether). When we evaluated estrogen receptor negative and progesterone receptor negative cases separately, there was 2 times more GASC1 negative than GASC1 positive tumors in each group (chi2, p= 0.033 and 0.001 respectively). In the HER2 positive cases, there was 3 times more GASC1 negative cases than GASC1 positives (chi2, p= 0.029). Patients treated with radiotherapy (n=206) and hormonal treatment (n=62) had better breast cancer specific survival, when they were GASC1 positive (Cox regression: HR=0.49, p=0.007 and HR=0.33, p=0.015, respectively). The expression of GASC1 mRNA was in agreement with the protein analysis. Conclusions This study indicates that the GASC1 is both a prognostic and a predictive factor for women with invasive breast cancer. GASC1 negativity is associated with tumors of more aggressive histopathological types (ductal type, grade II and III, ER negative, PR negative). Patients with GASC1 positive tumors have better breast cancer specific survival and respond better to radiotherapy and hormonal treatment.

Background: Surgeon volume has been identified as a possible factor that influences outcomes in mitral valve (MV) surgery. The aim of this study was to systematically review all published studies on the association between individual surgeon volume and outcome in MV surgery., Methods: PubMed was searched last on 19 November 2020. The reporting of this systematic review was done in accordance with PRISMA guidelines. Manuscripts were eligible when these studied individual surgeon volumes and its association with repair rate, mortality or reoperation. The methodological quality of the studies was assessed with the Newcastle-Ottawa Scale (NOS). Absolute numbers and percentages of the outcome measures, odds ratios (ORs), P values and threshold values regarding surgeon volume were collected., Results: A total of 7 retrospective cohort studies were included in the qualitative analysis with total of 158488 patients. Definitions of surgeon volumes were found to be heterogenic and therefore pooling of data was not possible. Surgeon volume was significantly associated with repair rate (OR =1.25-5.5) and mortality (OR =0.46-0.84 and OR =1.50-2.27 depending on the reference group). Regarding reoperation, results were not consistent and did not always show a significant lower reoperation rate when surgeon volume increased. A mean threshold of minimally 30 MV surgeries per year was found., Discussion: Higher surgeon volume is significantly associated with improved outcomes of repair rate and mortality. MV should preferentially be performed by high-volume surgeons and centralization of MV surgery might be necessary., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/jtd-21-578). The authors have no conflicts of interest to declare., (2021 Journal of Thoracic Disease. All rights reserved.)

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Aryl hydrocarbon receptor (AHR) activation by tryptophan (Trp) catabolites enhances tumor malignancy and suppresses anti-tumor immunity. The context specificity of AHR target genes has so far impeded systematic investigation of AHR activity and its upstream enzymes across human cancers. A pan-tissue AHR signature, derived by natural language processing, revealed that across 32 tumor entities, interleukin-4-induced-1 (IL4I1) associates more frequently with AHR activity than IDO1 or TDO2, hitherto recognized as the main Trp-catabolic enzymes. IL4I1 activates the AHR through the generation of indole metabolites and kynurenic acid. It associates with reduced survival in glioma patients, promotes cancer cell motility, and suppresses adaptive immunity, thereby enhancing the progression of chronic lymphocytic leukemia (CLL) in mice. Immune checkpoint blockade (ICB) induces IDO1 and IL4I1. As IDO1 inhibitors do not block IL4I1, IL4I1 may explain the failure of clinical studies combining ICB with IDO1 inhibition. Taken together, IL4I1 blockade opens new avenues for cancer therapy., Competing Interests: Declaration of Interests Three patent application families (PCT/US2020/025560, PCT/EP2020/060259, and EP19214890.6) have been filed on aspects of the described work. M. Seiffert. and C.A.O. receive and/or are planning to receive research funding from commercial organizations, like Bayer. C.A.O. is listed as inventor on PCT/EP2012/067504., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: As process measures can be means to change practices, this article presents process measures that impact on outcome measures for surgical aortic valve replacement (SAVR) and transcatheter aortic valve replacement (TAVR) within value-based healthcare., Methods: Desk research and observations of patient trajectories were performed to map the processes involved in TAVR and SAVR. Semistructured interviews were conducted with healthcare professionals (n=8) and patients (n=2) to explore which processes were most important in relation to a standard set of outcome measures that was already monitored. Additionally, open interviews (n=2) were held to prioritise results. A focus group was performed for validation of the formulated process measures. Numerical data for these measures was not collected., Results: Process maps of the full cycle of care of TAVR and SAVR treatments in theory and in practice were developed. 28 processes were found important by interview participants due to their expected impact on patient-relevant outcomes. Seven processes were prioritised to be most important and were formulated into 12 process measures for both TAVR and SAVR: 'Number of times that deficient information provision to SAVR patients causes negative outcomes', 'Type of TAVR/SAVR prosthesis', 'Brand of TAVR prosthesis', 'Number of times the frailty score of a TAVR/SAVR patient >75 years is measured', 'Time between TAVR/SAVR surgery indication and surgery', 'Number of times that anticoagulants are stopped within 3 days before surgery', 'Time in hours between TAVR/SAVR surgery and permanent pacemaker implantation' and 'Percentage of standardised pain measurements'., Conclusion: This study proposes an addition of select process measures to standard sets of outcome measures to improve healthcare quality. It illustrates a clear method for identifying process measures with impact on health outcomes in the future., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Evidence from preclinical studies and trials in healthy volunteers suggests that exercise may modulate the levels of tryptophan (TRP) metabolites along the kynurenine (KYN) pathway. As KYN and downstream KYN metabolites are known to promote cancer progression by inhibiting anti-tumor immune responses and by promoting the motility of cancer cells, we investigated if resistance exercise can also control the levels of KYN pathway metabolites in breast cancer patients undergoing radiotherapy (NCT01468766). Patients and Methods: Chemotherapy-naïve breast cancer patients ( n = 96) were either randomized to an exercise/intervention group (IG) or a control group (CG). The IG participated in a 12-week supervised progressive resistance exercise program twice a week, whereas the CG received a supervised relaxation program. Serum levels of TRP and KYN as well as urine levels of kynurenic acid (KYNA) and neurotoxic quinolinic acid (QUINA) were assessed before (t0), after radiotherapy, and mid-term of the exercise intervention (t1) and after the exercise intervention (t2). Additionally, 24 healthy women (HIG) participated in the exercise program to investigate potential differences in its effects on KYN metabolites in comparison to the breast cancer patients. Results: At baseline (t0) the breast cancer patients showed a significantly elevated serum KYN/TRP ratio and urine QUINA/KYNA ratio, as well as increased urine QUINA levels in comparison to the healthy women. In response to exercise the healthy women and the breast cancer patients differed significantly in the levels of urine QUINA and the QUINA/KYNA ratio. Most importantly, serum KYN levels and the KYN/TRP ratio were significantly reduced in exercising patients (IG) compared to non-exercising patients (CG) both at t1 and t2. Conclusion: Resistance exercise may represent a potent non-pharmacological avenue to counteract an activation of the KYN pathway in breast cancer patients undergoing radiotherapy., (Copyright © 2019 Zimmer, Schmidt, Prentzell, Berdel, Wiskemann, Kellner, Debus, Ulrich, Opitz and Steindorf.)

Tryptophan (Trp) metabolism is an important target in immuno-oncology as it represents a powerful immunosuppressive mechanism hijacked by tumors for protection against immune destruction. However, it remains unclear how tumor cells can proliferate while degrading the essential amino acid Trp. Trp is incorporated into proteins after it is attached to its tRNA by tryptophanyl-tRNA synthestases. As the tryptophanyl-tRNA synthestases compete for Trp with the Trp-catabolizing enzymes, the balance between these enzymes will determine whether Trp is used for protein synthesis or is degraded. In human cancers expression of the Trp-degrading enzymes indoleamine-2,3-dioxygenase-1 (IDO1) and tryptophan-2,3-dioxygenase (TDO2) was positively associated with the expression of the tryptophanyl-tRNA synthestase WARS. One mechanism underlying the association between IDO1 and WARS identified in this study is their joint induction by IFNγ released from tumor-infiltrating T cells. Moreover, we show here that IDO1- and TDO2-mediated Trp deprivation upregulates WARS expression by activating the general control non-derepressible-2 (GCN2) kinase, leading to phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) and induction of activating transcription factor 4 (ATF4). Trp deprivation induced cytoplasmic WARS expression but did not increase nuclear or extracellular WARS levels. GCN2 protected the cells against the effects of Trp starvation and enabled them to quickly make use of Trp for proliferation once it was replenished. Computational modeling of Trp metabolism revealed that Trp deficiency shifted Trp flux towards WARS and protein synthesis. Our data therefore suggest that the upregulation of WARS via IFNγ and/or GCN2-peIF2α-ATF4 signaling protects Trp-degrading cancer cells from excessive intracellular Trp depletion.

Kynurenine formation by tryptophan-catabolic indoleamine-2,3-dioxygenase 1 (IDO1) plays a key role in tumor immune evasion and inhibition of IDO1 is efficacious in preclinical models of breast cancer. As the response of breast cancer to immune checkpoint inhibitors may be limited, a better understanding of the expression of additional targetable immunomodulatory pathways is of importance. We therefore investigated the regulation of IDO1 expression in different breast cancer subtypes. We identified estrogen receptor α (ER) as a negative regulator of IDO1 expression. Serum kynurenine levels as well as tumoral IDO1 expression were lower in patients with ER-positive than ER-negative tumors and an inverse relationship between IDO1 and estrogen receptor mRNA was observed across 14 breast cancer data sets. Analysis of whole genome bisulfite sequencing, 450k, MassARRAY and pyrosequencing data revealed that the IDO1 promoter is hypermethylated in ER-positive compared with ER-negative breast cancer. Reduced induction of IDO1 was also observed in human ER-positive breast cancer cell lines. IDO1 induction was enhanced upon DNA demethylation in ER-positive but not in ER-negative cells and methylation of an IDO1 promoter construct reduced IDO1 expression, suggesting that enhanced methylation of the IDO1 promoter suppresses IDO1 in ER-positive breast cancer. The association of ER overexpression with epigenetic downregulation of IDO1 appears to be a particular feature of breast cancer as IDO1 was not suppressed by IDO1 promoter hypermethylation in the presence of high ER expression in cervical or endometrial cancer.

Background: Hepsin, (also called TMPRSS1) and TMPRSS3 are type II transmembrane serine proteases (TTSPs) that are involved in cancer progression. TTSPs can remodel extracellular matrix (ECM) and, when dysregulated, promote tumor progression and metastasis by inducing defects in basement membrane and ECM molecules. This study investigated whether the gene and protein expression levels of these TTSPs were associated with breast cancer characteristics or survival., Methods: Immunohistochemical staining was used to evaluate hepsin levels in 372 breast cancer samples and TMPRSS3 levels in 373 samples. TMPRSS1 mRNA expression was determined in 125 invasive and 16 benign breast tumor samples, and TMPRSS3 mRNA expression was determined in 167 invasive and 23 benign breast tumor samples. The gene and protein expression levels were analyzed for associations with breast cancer-specific survival and clinicopathological parameters., Results: Low TMPRSS1 and TMPRSS3 mRNA expression levels were independent prognostic factors for poor breast cancer survival during the 20-year follow-up (TMPRSS1, P = 0.023; HR, 2.065; 95 % CI, 1.106-3.856; TMPRSS3, P = 0.013; HR, 2.106; 95 % CI, 1.167-3.800). Low expression of the two genes at the mRNA and protein levels associated with poorer survival compared to high levels (log rank P-values 0.015-0.042). Low TMPRSS1 mRNA expression was also an independent marker of poor breast cancer prognosis in patients treated with radiotherapy (P = 0.034; HR, 2.344; 95 % CI, 1.065-5.160). Grade III tumors, large tumor size, and metastasis were associated with low mRNA and protein expression levels., Conclusions: The results suggest that the TTSPs hepsin and TMPRSS3 may have similar biological functions in the molecular pathology of breast cancer. Low mRNA and protein expression levels of the studied TTSPs were prognostic markers of poor survival in breast cancer.

Recently the increasing prevalence of gastrointestinal diseases, including neoplasm, has resulted in the necessity of characterising not only the tumours, but also healthy mucosa. Research into the morphological changes of healthy mucosa under different experimental conditions, including drugs, special diets and the use of probiotic bacteria, is greatly facilitated by the availability of animal models. In spite of the widespread use of mice in gastrointestinal research, there is a lack of information on the qualitative and quantitative histological characteristics of the intestinal mucosa of the mouse. The aim of this study was to assess the morphological characteristics and the postnatal development of the small intestine of wild type mice -- C57BL/6J. The mice were aged either 5 weeks or 12 weeks. The 12-week-old mice had been weaned at the age of 5 weeks. After dissection the small intestine was divided into 5 equal portions and randomly chosen microscopical sections from each were stained with haematoxylin and eosin. The parameters describing the morphology of the small intestine (villus height, depth of the crypt, villus width near the crypt, width of the villus connective tissue near the crypt, thickness of the muscular layer and the height of the enterocytes and their nuclei) were evaluated under a light microscope. In both age groups the height and width of the villi decreased, while the thickness of the muscular layer increased in the distal direction. The height of the enterocytes decreased and the height of the enterocyte nucleus increased towards the colon in both age groups. The depth of the crypts was greater in the younger animals than in the older ones. Our data provides the baseline morphological description of the small intestinal mucosa in wild type mice, strain C57BL/6J, which can be used as a reference for testing the influence of drugs, toxins, nutrients and inborn mutations on the mouse intestine.

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species including humans and is a potential contaminant in MLV vector preparations for human gene transfer studies. Because MLV replication proceeds through an RNA genome that is generated under the control of viral enhancer and promoter elements, vectors were developed that delete such elements during transduction to reduce the generation of replication-competent virus. It was shown recently that replication of amphotropic MLV in certain human cells is possible without the 75 bp transcription enhancers. It is now demonstrated that enhancer-independent replication requires functional elements within U3 and is repressed by an extended deletion in the U3 region comprising enhancers, promoter and flanking sequences. It is concluded that the transcriptional inactivation of amphotropic MLV in human cells requires the combined deletion of enhancers and of additional elements in U3.

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species including humans and is a potential contaminant in MLV vector preparations for human gene transfer studies. In general, MLV replication depends on the expression of viral genes under the control of 75 bp enhancer elements in the long terminal repeat. However, in specific human fibrosarcoma and lymphoma lines replication of amphotropic MLV is possible without these enhancers. Fibrosarcomas are malignant tumors of fibroblast origin. To test the replication potential of intact and enhancerless amphotropic MLV in untransformed cells, infection studies with these viruses were carried out in three types of primary human fibroblasts. Replication of amphotropic MLV is observed in two of three tested fibroblast strains. None of these primary human fibroblasts is permissive for enhancer-deficient MLV, suggesting that replication of this virus may be limited to transformed cells., (Copyright 2002 Wiley-Liss, Inc.)

Amphotropic murine leukemia virus (MLV) can replicate in human cells and is a potential contaminant in vector preparations for human gene transfer studies. We have recently shown that replication of amphotropic MLV in specific human sarcoma and lymphoma lines is possible in the absence of the viral 75-bp transcription enhancer elements. Here, we have tested the replication of an amphotropic MLV, MLV-(MOA), and an enhancer-deficient mutant of this virus in human breast carcinoma-derived cell lines. The proviral expression plasmids use a cytomegalovirus (CMV) promoter for the initial transcription of virus RNA. We found that all cells analyzed are permissive for replication of MLV-(MOA). Enhancer-deficient virus is unable to replicate. However, in two lines the replication defect can be rescued by the spontaneous insertion of a CMV promoter and enhancer into the U3 region. This recombinant virus MLV-(RCMV) replicates with kinetics similar to that of MLV-(MOA) but is restricted to specific cell lines. The potential formation of RCMV recombinants during MLV vector preparation must be considered., (Copyright 2001 Elsevier Science.)

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes are required for viral transmission and pathogenesis. Due to problems with detection and quantification of the superantigen protein, most reports about the mechanism of superantigen expression from the viral sag gene rely on the quantitative analysis of putative sag mRNAs. The description of multiple promoters as a source of putative sag mRNA has complicated the situation even further. All conclusions about the level of superantigen protein expression based on these data remain circumstantial. To test the effect of the glucocorticoid hormone dexamethasone on the total superantigen expression from an infectious MMTV provirus we used a quantitative assay that is based on a superantigen-luciferase fusion protein. MMTV gene expression from the major promoter in the 5' long terminal repeat (LTR) is strongly induced in the presence of glucocorticoid hormones. We now demonstrate that, in the presence of dexamethasone, sag gene expression is reduced despite increased transcription from the MMTV 5' LTR and increased amounts of putative sag mRNA initiated at the LTR promoter. These data show that the expression of the MMTV sag gene does not correlate with the activity of the major LTR promoter and thus differs from all other MMTV genes., (Copyright 2000 Academic Press.)

Microtubules are present in high concentration in the nervous system and are a prominent component of the neuronal cytoskeleton. Microtubules are composed of tubulin and variety of microtubule-associated proteins (MAPs), which have been implicated in the regulation of microtubule assembly and function. MAP2 is the most abundant of these proteins, and it has been extensively characterized in various functional and pathological conditions. In the present study the distribution of MAP2 was examined in each layer of the CA1 and CA3 regions of the hippocampus proper and dentate gyrus in rat development. A total of 40 brains at various ages starting from postnatal day (P) 0 to P90 were examined. After perfusional fixation the brains were frozen and cut on the coronal plane and stained with either cresyl violet or standard immunohistochemical methods using the anti-MAP2 antibody. MAP2 exhibited a somatodendritic pattern of localization in cells of the hippocampus. Staining was most prominent in dendrites and perikarya as well as granules surrounding cell bodies. In a newborn rat's brain immunostaining was intense in granules and faint in perikarya. Between P4 and P21 immunostaining density for MAP2 was stronger and appeared in perikarya, granules, and dendritic trees. After P21 the perikarya and dendrites of the pyramidal layer and stratum radiatum of the hippocampus proper, as well as the molecular and granular layer of dentate gyrus, showed reduced immunoreactivity. In the stratum oriens of the hippocampus and polymorphic layer of the dentate gyrus immunoreactivity was still strong until P90.

A distinct group of small cells lying in the ventral part of the external capsule in the rat brain is clearly visible at birth. On the basis of its location (medially to the prepiriform claustrum) and probably its function (as a source of neurons for adjacent structures), we define this nucleus as the "paraclaustral reservoir". The present study reveals the cellular changes of the paraclaustral reservoir during postnatal development of the rat brain using unbiased morphometry and in situ DNA end labeling. During the first 4 days after birth the density and total number of cells in the paraclaustral reservoir were stable; after this period a decrease of these parameters was observed until the complete disappearance of this structure at the end of first postnatal week. The rather low number of TUNEL (TdT mediated dUTP nick end labeling of fragmented DNA) positive nuclei in the paraclaustral reservoir suggests that apoptosis is not a crucial mechanism leading to decay of this structure.

Two-laser and two-color approaches were used to observe the colocalization of the calcium-binding proteins, calbindin D28k and parvalbumin, and the retrograde tracer, Fluoro-Gold (FG) in the basolateral amygdala of the rat. The study was performed on five adult rats into which FG was injected to the frontal association cortex. Then, the localization of the retrogradely labeled neurons in the basolateral amygdala was compared with the localization of the neurons labeled by calcium-binding proteins. The present study showed that most of the retrogradely labeled neurons in the posterior part of the basolateral amygdala are also calbindin-positive. Even though a lot of parvalbumin-positive endings were present at the surface of the retrogradely labeled cells, we did not observe the colocalization of the parvalbumin and projective neurons.

For many years the amygdaloid body has been an object of numerous investigations on different species, because the basolateral complex, being the main part of the amygdaloid body, is regarded as "sensory input" to this structure. It plays a very important role in so called emotional memory and learning, what is particularly important in early developmental stages. Impairment at this time may cause psychiatric problems in later life, like neurosis, phobia, unconscious fear or panic attacks. Complicated functions of the basolateral complex require precise control and modulation especially in early development. In this review the morphological changes during the development and maturation will be discussed and compared with neurotransmitter as well as with the expression of the calcium binding proteins at various stages of the development.

The amygdaloid body is a telencephalic structure belonging to the limbic system. The amygdaloid body consists of the two main nuclear groups: corticomedial and basolateral. The former-phylogenetically older group is composed of the central, medial, and cortical nuclei, while the latter, phylogenetically younger one, of the lateral, basolateral and basomedial ones. The results presented in our paper indicate differences in the structure and topography of the specific amygdaloid nuclei. Their subdivisions in the rabbit are not as evident as in the rat. Apart from structural differences, the cellular composition of specific nuclei does not differ distinctly. It can suggest that their intrinsic and extrinsic connections might be similar and the role and function of them is maintained (with few exceptions) through the phylogeny.

The corticoclaustral connections in the rat were investigated by means of the method of the retrograde axonal transport of the fluorescent tracer (Fluoro-Gold; FG). The material consisted of 20 adult Wistar rats. The fluorescent tracer was injected into the anterior, middle or posterior parts of the claustrum. The retrogradely labeled neurons were detected in the layer VI of the neocortex. Injections of the tracer into the anterior part of the claustrum resulted in labeling of the neurons in the motor cortex. After administration of the tracer into the middle part of the insular claustrum, labeled neurons were present both in the motor and somatosensory cortices, while the injections of the tracer into its posterior part resulted in labeling of neurons in the visual cortex. Administration of the fluorescent tracer into the insular claustrum of the rat resulted in labeling of the cortical neurons of the corresponding areas of both hemispheres, however, the contralateral projections seem to be less numerous than the ipsilateral. Our results confirm the existence of reciprocal connections of the claustrum with the neocortex and suggest its role in integration and modification of information reaching the neocortex.

The thalamic reticular nucleus (Rt) is a thin lamina of cells, through which thalamocortical and corticothalamic fibers pass. It is interposed between the thalamic nuclei and the internal capsule and it is composed of GABA-ergic cells with synapses that receive impulses from both kinds of fibers. Rt takes part in the negative feed-back system of controlling the information transfer from the thalamus to the cerebral cortex and it is focused in the sleep-waking cycle. The pattern of parvalbumin reactivity during maturation of Rt becomes the main aim of our study. The study was performed on 36 rats on various postnatal days (P0, P1, P2, P4, P5, P7, P10, P14, P17, P21, P30 and P90). The animals were anesthetized, transcardially perfused, cut on cryostat into 30-microns-thick frontal sections, stained immunocytochemically using standard ABC method and a mouse monoclonal antibody against parvalbumin. A small amount of round and oval, parvalbumin immunopositive cells was detected at stage PO, predominantly in the intermediate part of Rt, whereas the cells in ventral and lateral part at the same time were only slightly immunopositive. At P10 the cells in the intermediate part became more fusiform or oval because of the appearance of dendrites. At P14 we were able to observe separate, punctuated structures interpreted as the axonal endings. There were plenty of them at the time of full maturation of the intermediate portion of reticular nucleus (stage P21). At this time, the dorsal and ventral parts had their first synapses, too, but their maturation ended a week later. At P30 multipolar neurons, with round and fusiform somata were distributed relatively homogeneously throughout Rt. We compared the stage with the parvalbumin reactivity of the adult rat and found no difference in the morphological pattern of PV neurons.

Synaptophysin is an integral membrane protein associated with small, electron-lucent synaptic vesicles. Immunohistochemistry for this protein is a sensitive method to study subtle changes in synaptic density and distribution in various brain regions. In the present study, the synaptogenesis was examined in the rat basolateral amygdala in comparison with the hippocampal hilus, from the day of birth to adulthood. A total of 41 brains at various ages starting from P0 to P90 (P--postnatal day) were examined. After perfusional fixation the brains were frozen and cut in the coronal plane and stained either with cresyl violet or standard immunohistochemical methods using the anti-synaptophysin antibody. Synaptophysin positive granules appeared just after birth in both structures, but their number was very low (about 0.28 x 10(6) and 0.13 x 10(6) per mm3 in the amygdala and hippocampus, respectively). In the basolateral amygdala the number of synapses increased rapidly reaching the maximum at P14 (1.6 x 10(6) per mm3) followed by about 45% decrease in number up to P30 and later being stabile. In the hippocampus two increases of the synaptogenesis were observed. The first at P7 (about 1.7 x 10(6) of synapses per 1 mm3) which was followed by dramatic decrease up to 0.7 x 10(6) per mm3 at P14. The second increase appeared later (about P90) and reached 1.7 x 10(6) per mm3. After that time the density of synapses was stabile. It may be supposed that the first characteristic wave of synaptogenesis observed in the hippocampus and amygdaloid body is due to the overproduction of synapses observed at that time in other cortical regions. The late wave of synaptogenesis found in the hippocampus is related to the great plasticity of the interneuronal connections in this period of development.

Volume and topography of basolateral complex during development and maturation in fetal and postnatal rat brains were studied using morphometrical methods. 39 rat brains of various ages were fixed in formalin, frozen and cut into 25-micron-thick sections and stained with cresyl violet. In cresyl violet preparation the basolateral complex appeared first on 17th prenatal day and it was composed of two homogenous parts--lateral and basolateral nuclei. On about 7th postnatal day each of these nuclei divided into two parts--the first one into the dorsolateral and ventromedial part, while the second one--into the anterior and posterior parts. Morphometric investigations showed that volume of the basolateral complex and its parts underwent the biggest changes up to 14th postnatal day, and we suspected that during this time it was more sensitive to pathological changes. After that day the volume of the basolateral complex had changed only a little.

The study was performed on 19 brains of nondemented patients with age ranging from 36 to 89 years. After embedding in paraffin, coronal 8-microns-thick serial sections were cut and stained either with cresyl violet or with immunocytochemical methods for amyloid and tangles. Morphometrical studies were performed in all parts of the claustrum along its whole extension. Changes related to aging (neuronal loss and decrease in volume) were found in all parts of the claustrum, but the time of origin of these changes is different in various parts of this structure. We did not observe neurofibrillary pathology in any parts of the claustrum. In the oldest subjects a small number of amyloid plaques was found in the paraamygdalar part of the claustrum. We suggest that the neurons of the claustroneocortical loop are affected severely and earlier than those in the claustroentorhinal loop.

The topography and cytoarchitectonics of the claustrum as well as morphometric parameters of its neurons were studied in 10 human brains obtained from patients without any detectable neuropathological changes. We distinguished four parts of the claustrum: dorsal, orbital, temporal and paraamygdalar. The dorsal and orbital parts contain larger cells, than those of the temporal and paraamygdalar parts, although these differences were statistically non significant. The highest neuronal density was observed in the paraamygdalar part. The nucleus and nucleus@cell body area ratio was significantly smaller in the dorsal part than in other parts of the claustrum. We described three types of neurons in the claustrum: (1) medium-sized either fusiform or triangular cells with darkly stained cytoplasm; they predominate in the dorsal and temporal parts, (2) medium-sized as well as large cells, either multipolar or pyramidal-like with lightly stained cytoplasm; they are most numerous in the orbital and paraamygdalar parts, (3) small, multipolar or oval neurons with darkly stained ring of cytoplasm; these types of neurons are uniformly distributed throughout all parts of the claustrum. The subdivision of the human claustrum is in accordance with our observations that each of these parts possesses connections with different cortical regions.

The influence of fixation and histological procedure on morphometric parameters of human and rat brains were analyzed. We found negative correlation of brain weight with the fixation time both in human and rat material. Human brains fixed for shorter period of time than one year showed large differences in weight. The group of brains fixed for longer than one year was characterized by much smaller dispersion. Moreover, we found that during histological preparation the shrinkage of brain tissue of older human subjects is statistically significantly smaller than younger ones (below 65 years). In order to correct these artifacts easily we propose three simple formulas.

Investigation of the dog's insular claustrum was performed using morphometric methods. The brains of 14 dogs (7-14 year-old) were studied. The size, shape and density of neurons are different in various parts of the insular claustrum. The posterior part, related to the visual system, is heterogenous and possesses medium-sized and small densely packed neurons, the average cross-sectional area of cell body being 175.8 +/- 45.5 micron2. The similar average cross-sectional area of cell body (174.1 +/- 55.3 micron2) was found in the central part of the insular claustrum. However, neuronal architecture is here less heterogenous and neurons are less densely arranged. The anterior part, connected mainly with the frontal and motor cortex, is more homogenous and contains loosely arranged neurons of a rather large size (average size--198.7 +/- 53.46 micron2).

Methylazoxymethanol acetate (MAM) is a substance that inhibits migration of neurons in the embryonic brain. After intraperitoneal injection of two different doses of MAM to pregnant rats, microcephaly with or without complete development of the cerebral cortex was observed in every litter. High MAM doses (30 mg/kg) resulted in the lack of superficial layers (II-IV) of the cerebral cortex when the deep layers (V, VI) were seen. The claustrum was present but composed of loosely packed, medium-size, triangular or fusiform neurons with anarchic oriented long axes. After administration of low MAM doses (14 mg/kg) two different parts (medial and lateral) of the insular claustrum were observed. Our results suggest that neurons of the insular claustrum create two different subpopulations of cells, which were similar to that observed in primitive insectivore (e.g., hedgehog), but fuse in development.

The parahippocampal (ventral) surface of the lateral part of the transverse fissure (LTF), formed by the parahippocampal gyrus, was examined in 53 human brain hemispheres without pathological changes. Cytoarchitectonics of this region was studied on the frontal histological sections stained either with cresyl violet or with Weil method. Four types of the shape of the parahippocampal surface of LTF were distinguished. In type 1 the surface is either flat or elevated in its medial part (medial eminence; 34.0%). In type 2 the surface is elevated in its lateral part (lateral eminence, 24.5%). In type 3 there are two eminences--the lateral and the medial; both of similar height (20.8%). In type 4 the lateral eminence is distinctly higher than the medial one (20.7%). The eminences contain cytoarchitectonically different structures. The lateral eminence is formed mainly by the subiculum and CA1 area, the medial by the presubicular region. The shape of the parahippocampal surface of LTF may be of importance in assessment of the Alzheimer's disease pathology.