Localization of latexin-immunoreactive neurons in the adult cat cerebral cortex and claustrum/endopiriform formation.

The distribution of neurons that are immunoreactive to latexin, which is an endogenous inhibitor of the A/B subfamily of metallocarboxypeptidases, was investigated in the adult cat telencephalon. Latexin-immunoreactive neurons were distributed in the lower layers of the neocortex and adjacent ventral mesocortex, as well as in the claustrum/endopiriform formation. There were marked regional and laminar differences in density and distribution of latexin-immunoreactive neurons in the cerebral cortex. The density followed a roughly lateral-to-medial decreasing gradient: it was high in lateral cortical regions, which included the insular, second somatosensory, and anterior sylvian areas, and in the temporal auditory field; moderate in laterodorsal cortical regions, which included the primary and second auditory fields; and low in dorsal cortical regions, which included visual areas 18 and 19. Latexin-immunoreactive neurons were absent in medial cortical regions, which included the motor, premotor, prefrontal, prelimbic, cingulate, and retrosplenial areas. The lateral-to-medial gradient was apparent even within a single cytoarchitectonic area in certain cortical regions. The allocortex was devoid of latexin-immunoreactive neurons, with the exception of the anteroventral part of the dentate gyrus. The majority of cortical latexin-immunoreactive neurons were localized in layers V and VI and appeared to correspond to the "modified pyramidal cells in the infragranular layers." The remaining latexin-immunoreactive neurons were localized in layer IV, as well as in lower layer III and in the white matter. There were no latexin-immunoreactive neurons from layer I through upper layer III. Latexin-immunoreactive neurons were present in telencephalic structures outside the cerebral cortex, with particularly high density in the claustrum/endopiriform formation. All these features, with the exception of that detected in the archicortex, are compatible with the features observed previously in the rat telencephalon. The similar pattern of distribution of latexin-immunoreactive neurons in several mammalian species from different orders suggests that latexin plays an important role in a specific cortical network.

Chemically defined feedback connections from infragranular layers of sensory association cortices in the rat.

The primary visual (V1), auditory (AI), and somatosensory (SI) cortices are reciprocally connected with their respective sensory association cortices. In the rat, we have previously demonstrated that some of the connections arising from the secondary somatosensory (SII) and parietal insular (PA) cortices and terminating in the SI, are characterized by the expression of latexin, a candidate protein of carboxypeptidase A inhibitor. Here, by using retrograde tracing and latexin-immunohistochemistry, we show that latexin-expressing neurons in other association cortices of different sensory modalities also contribute to the feedback projections to the corresponding primary sensory cortices. These are the lateral part of the secondary visual cortex (V2L), temporal association cortex, and the dorsal and ventral (AIIv) parts of the secondary auditory belt cortex. Within sublayer VIa of the V2L, AIIv and SII, the majority of the V1-, AI- and SI-projecting neurons respectively, are latexin-immunopositive. In contrast to feedback connections, far fewer latexin-expressing neurons participate in callosal or intrahemispheric feedforward connections. The latexin-expressing neurons constitute a virtually completely different population from corticothalamic neurons within the infragranular layers. Given that latexin might participate in the modulation of neuronal activity by controlling the protease activity, latexin-expressing feedback pathways would play a unique role in the modulation of sensory perception.

Distinct neuronal populations specified to form corticocortical and corticothalamic projections from layer VI of developing cerebral cortex.

Layer VI of the cerebral cortex contains heterogeneous populations of pyramidal neurons whose axons project either cortically or subcortically. It has been shown that a subset of layer VI neurons expressing latexin projects ipsilaterally to other cortical areas but does not contribute to the corticothalamic projections. Taking advantage of the connectional specificity of latexin-expressing neurons, we here determine whether corticocortical and corticothalamic neurons are generated at different times, and at which stage the connectional distinction develops in corticogenesis. Our experimental findings indicate that: (1) thalamic-projecting neurons in layer VI of the rat secondary somatosensory cortex (SII) are born at embryonic day 14 or before while latexin-expressing neurons in the same layer are generated at embryonic day 15 or later; (2) axonal invasion by SII neurons into ipsilateral cortical areas and into the posterior dorsal thalamus mainly takes place early in the postnatal period; (3) latexin-expressing neurons never project toward the dorsal thalamus in normal development; (4) presumptive latexin-expressing neurons in the neonatal SII are able to grow into a cortical slice in vitro, but do not invade a thalamic slice even transiently; (5) thalamic-projecting neurons, on the other hand, fail to simultaneously establish connections with a cortical slice. Taken together, our findings suggest that the time frame in which presumptive corticocortical and corticothalamic neurons are generated differs, and that the two populations are restricted in connectional fate potential by the perinatal period prior to target innervation.

Latexin, a carboxypeptidase A inhibitor, is expressed in rat peritoneal mast cells and is associated with granular structures distinct from secretory granules and lysosomes.

Latexin, a protein possessing inhibitory activity against rat carboxypeptidase A1 (CPA1) and CPA2, is expressed in a neuronal subset in the cerebral cortex and cells in other neural and non-neural tissues of rat. Although latexin also inhibits mast-cell CPA (MCCPA), the expression of latexin in rat mast cells has not previously been confirmed. In the present study we examined the expression and subcellular localization of latexin in rat peritoneal mast cells. Western blot and reverse-transcriptase-mediated PCR analyses showed that latexin was contained and expressed in the rat peritoneal mast cells. Immunocytochemically, latexin immunofluorescence was localized on granular structures distinct from MCCPA-, histamine- or cathepsin D-immunopositive granules. Immunoelectron microscopy revealed that latexin was associated with a minority population of granules. The latexin-associated granules were separated from MCCPA- or histamine-containing granules on a self-generating density gradient of polyvinylpyrrolidone-coated silica-gel particles (Percoll). Treatments with high ionic strength and heparinase released latexin from the granules, suggesting that latexin is non-covalently associated with a heparin-like component of the granules. MCCPA and histamine were released from the mast cells after non-immunological and immunological stimulation with compound 48/80, A23187 and anti-IgE antibody, whereas latexin was not released. These results show that latexin is synthesized in rat peritoneal mast cells and suggest that it is associated with a unique type of intracellular granules distinct from MCCPA- and histamine-containing secretory granules and lysosomes.

Corticocortical associative neurons expressing latexin: specific cortical connectivity formed in vivo and in vitro.

Latexin, a carboxypeptidase A inhibitor, is expressed in a subset of neurons in the infragranular layers of the lateral cortex in the rat. We here show that latexin-expressing neurons exhibit ultrastructural features common to cortical pyramidal neurons. We show in combined retrograde tracing and immunofluorescent experiments that latexin-expressing neurons contribute to specific corticocortical pathways. Thus, injections of the retrograde tracer fluorogold into either the primary somatosensory (SI) or the primary motor (MI) cortical area labeled many latexin-expressing neurons in the infragranular layers of the secondary somatosensory (SII) and visceral sensory (Vi) areas. In contrast, tracer injections involving the thalamus, striatum, or contralateral SII and Vi exclusively labeled latexin-nonexpressing neurons in both the SII and Vi. Finally, we show that the correct corticocortical projections can be formed in organotypic slice cultures in vitro from latexin-expressing neurons: when slices of developing SII were cocultured with those from the SI and the thalamus, latexin-immunoreactive neurons in the SII projected preferentially to their normal SI target. The specific connectivity formed in vivo and in vitro by this molecularly distinct neuronal population reveals its characteristic manner of cortical organization and provides a unique model system to analyze mechanisms underlying the formation of precise corticocortical pathways.

Foreign gene expression in an organotypic culture of cortical anlage after in vivo electroporation.

A high level of foreign gene expression in organotypic cultures of the cerebral cortical anlage was achieved by electroporation-mediated gene transfer in vivo. A mammalian expression plasmid for green fluorescent protein (GFP) gene was injected into the lateral ventricle of rat embryos. Immediately after the plasmid DNA injection, the head of the embryo was electroporated between a pair of tweezer-type electrodes. The cortical anlage was isolated and maintained organotypically up to 21 days in vitro (DIV). The GFP-transgene was expressed intensely in neural progenitor cells at 1 DIV. GFP-expressing cells were still detectable and were demonstrated to differentiate into neurons and glia at 21 DIV. This system is expected to be useful for molecular analysis of cerebral cortical development and function.

Genomic organization and regulatory elements of the rat latexin gene, which is expressed in a cell type-specific manner in both central and peripheral nervous systems.

Latexin, a carboxypeptidase A inhibitor, is expressed in a cell type-specific manner in both central and peripheral nervous systems in the rat. In the neocortex, a specific subpopulation of neurons in layers V and VI expresses latexin. In the primary sensory ganglia, the expression is restricted to smaller diameter neurons. As a first step to clarify regulatory mechanisms underlying cell type-specific expression of latexin, we have determined the organization of the rat latexin gene and analyzed its regulatory elements. The latexin gene spans approximately 5.8 kb, and consists of six exons and five introns. Three transcription initiation sites were mapped. The upstream region lacks typical TATA or CAAT boxes but has several GC-rich sites. To assess promoter activity, the luciferase reporter gene fused to the 5'-flanking region (6.4 kb) of the latexin gene was transiently transfected into several cell lines. Luciferase activity was 2-8 times higher in latexin-expressing cells (PC12) than non-expressing cells (NS20 and L6). Deletion analysis with PC12 cells revealed that a core promoter is located between nucleotide positions -261 and -201 relative to the A of the initiation codon. Nerve growth factor (NGF)-responsive element(s) is located between positions -518 and -262, in which AP-1, AP-2 and NF-kappaB binding sites are found. Furthermore, we demonstrate that a 1.3 kb genomic fragment containing the first intron has transcriptional enhancing activity in PC12 cells. These results suggest that up and downstream regulatory elements are involved in the control of cell type-specific expression of latexin.

Area- and lamina-specific organization of a neuronal subpopulation defined by expression of latexin in the rat cerebral cortex.

The aim of the present study was to investigate the density, laminar distribution, size, morphology, and neurotransmitter phenotype of rat cortical neurons expressing latexin, an inhibitor of carboxypeptidase A. Immunohistochemical analyses established that latexin-immunoreactive neurons are restricted essentially to the infragranular layers of lateral cortical areas in the rat. The overall density, laminar or sublaminar localization, and cell size distribution of latexin-positive neurons differed substantially across cytoarchitectonic areas within lateral cortex. Numerous latexin-positive neurons had the morphology of modified pyramidal cells especially of layer VI. The vast majority of latexin-positive neurons were glutamate-immunoreactive in the six lateral neocortical areas examined, while neurons immunoreactive for both latexin and GABA were virtually absent. Thus the majority of latexin-positive neurons are likely to be excitatory projection neurons. The area- and lamina-specific distribution of the latexin-expressing subpopulation of glutamate-immunoreactive neurons is a distinctive feature that may contribute to the functional specialization of the lateral cortical areas.

Cerebral cortical specification by early potential restriction of progenitor cells and later phenotype control of postmitotic neurons.

Neurons expressing latexin, a carboxypeptidase A inhibitor, are restricted to lateral areas in the cerebral cortex of adult and early postnatal rats. To address the precise timing of cortical regional specification at the cellular level, we monitored latexin expression in developing cortical cells under specific conditions in vitro. Individual cortical cells were labeled with 5-bromo-2'-deoxyuridine in vivo, dissociated and exposed to a defined new environment in a monolayer or a reaggregated-cell culture system. While a substantial fraction of early progenitor cells derived from the lateral cerebral wall became latexin-expressing neurons in both systems, far fewer progenitors from dorsal cortex did so under the same environmental conditions, indicating early establishment of cortical regional specification at the progenitor cell level. Furthermore, it was shown that the probability for postmitotic cells within lateral cortex to become latexin-expressing neurons was influenced by temporally regulated regional environmental signals. These findings suggest that developing cortical cells are progressively specified for a regional molecular phenotype during both their proliferative and postmitotic periods.

Latexin expression in smaller diameter primary sensory neurons in the rat.

Most of the smaller diameter neurons of dorsal root and trigeminal ganglia in adult rats expressed latexin, which has the inhibitor activity of carboxypeptidase A. Most of the dorsal root ganglion (DRG) neurons containing either calcitonin gene-related peptide (CGRP), substance P (SP) or somatostatin (SST) coexpressed latexin. Latexin was widely distributed in the cytoplasm of the cell body and in axonal fibers of cultured DRG neurons which were sensitive to capsaicin. In addition, latexin-immunoreactivity was observed throughout lamina II of the spinal cord in normal animals, but was lost following sciatic nerve-axotomy, suggesting the presence of latexin-immunoreactive axonal fibers and/or terminals from DRG neurons. Immunoelectron microscopy indeed revealed latexin-immunoreactive axonal terminals and thinly myelinated and unmyelinated axonal fibers within the dorsal horn. These observations suggest that latexin may be involved in nociceptive information transmission or its modulation.

Early patterning of the rat cerebral wall for regional organization of a neuronal population expressing latexin.

The exact timing of regional patterning in the developing cerebral cortex and other telencephalic structures remains to be elucidated. In the present study, we addressed this issue by comparing the distribution and density of neuronal population expressing latexin in the adult rat telencephalon, with the regional pattern in the fetal cerebral wall as to the potential to generate latexin-expressing neurons. Immunohistochemical analyses on adult animals have shown that latexin-expressing neurons are restricted to a lateral cortical field, within which they are most abundant at the middle level, decreasing in number rostrally and caudally. Substantial numbers of latexin-immunopositive neurons were recorded in the claustrum and endopiriform nuclei, both of which are located from rostral to middle level in the lateral telencephalon. By examining the number and density of latexin-immunopositive neurons in organotypic slice cultures from various portions of the developing rat cerebral wall, it has been shown that the regional pattern within the early cerebral wall as to the potential to generate latexin-expressing neurons matches well the distribution and density of latexin-expressing neurons in the adult telencephalon. Thus, in cultures derived from either embryonic day 13 or 16 fetuses, latexin-immunopositive neurons appeared most prominently in those from rostral-to-middle portions of the lateral cerebral wall, decreasing in number rostrally and caudally. In cultures from the dorsal cerebral wall, the number was generally very low. In light of our previous finding that most prospective latexin-expressing neurons are still dividing at embryonic day 13, it can be concluded that some kind of pattern formation event occurs within the early cerebral wall even prior to the genesis of the postmitotic neurons that would be later allocated in a region-specific manner.

Restricted expression of latexin in dorsal midline cells of developing rat forebrain.

Latexin is a novel 29 kDa protein which is expressed in a subpopulation of neurones in the lateral cerebral cortex of adult rats. Here, we report the distribution of immunohistochemically detectable latexin in the rat brain during early phases of development. Latexin was first detected at embryonic day 11 (E11) along the dorsal midline of the diencephalon. At E12, the expression domain of latexin corresponded to the dorsal midline regions of the diencephalon and also of the mesencephalon. At E14, the expression domain was more restricted than that at E12. Strong latexin expression was restricted within the pineal anlage, and dorsal midline cells anterior to the pineal anlage did not express latexin. These findings suggest that latexin plays a role in regional specification and/or morphogenesis of the forebrain, especially of its dorsal midline structures including the pineal gland.

Latexin: a molecular marker for regional specification in the neocortex.

It largely remains to be elucidated how the mammalian neocortex is regionally specified during development. In an attempt to obtain molecular markers in the neocortex, we have generated a monoclonal antibody PC3.1 which recognizes a subset of neurons located in lateral, but not dorsal, neocortical areas. The antigen is a novel class of protein, named latexin, having a molecular weight of 29,000. Our in vitro studies have revealed that the neocortical regional specification for the production of latexin-positive neurons occurs very early prior to thalamocortical interactions and the completion of neurogenesis, indicating that elements intrinsic to the neocortex play important roles in the neocortical specification. Furthermore, our recent analyses have suggested that this regional specification is attributable, at least in part, to an early restriction of developmental potential in neocortical progenitor cells to become latexin-positive neurons.

Intracortical regionality represented by specific transcription for a novel protein, latexin.

The monoclonal antibody (mAb) PC3.1 recognizes a subset of neurons distributed in the infragranular layers of the lateral neocortex of the rat. Immunoaffinity chromatography with mAb PC3.1 showed that this antibody specifically binds a peptide epitope on a 29 kDa protein named latexin. To study the molecular details of the protein, we isolated four independent cDNA clones for latexin from cDNA libraries of the rat cerebral cortex and whole brain using the amino acid sequences of latexin fragments. Analysis of these cDNA clones showed that the predicted primary structure of latexin consists of 223 amino acids, and has no strict homology to any sequences so far known. Western and Northern blots demonstrated that the latexin and its mRNA were expressed predominantly in neural tissues with some expression in non-neural tissues. The gene that encodes latexin in the rat appeared to have homologues in other mammalian species and in the chick. In situ hybridization showed that latexin mRNA is synthesized in a subset of neurons in the lateral but not the dorsal neocortex, and that the distribution profile of these neurons is quite similar to that of neurons expressing latexin. These results indicate that latexin is a novel class of neuronal protein which represents intracortical regionality, and suggest that the regional specification of the neocortex involves selective parcellation of neurons which express a particular gene.

Cogeneration of neurons with a unique molecular phenotype in layers V and VI of widespread lateral neocortical areas in the rat.

Monoclonal antibody PC3.1 detects a unique subpopulation of neurons located mainly in layer VI and, to a lesser extent, in layer V within the lateral neocortical areas in the rat. In an attempt to characterize these neurons, we determined the time of their generation in selected neocortical areas by a double-labeling experiment combining quantitative long-survival 3H-thymidine autoradiography and immunohistochemistry for the PC3.1 antigen. We found that the vast majority of PC3.1-positive neurons in both layers V and VI were generated concurrently at embryonic day 15 in all areas examined, demonstrating a strict correlation between the molecular identity of neurons and the time of their generation, irrespective of their final positions along the radial and tangential axes. In contrast, PC3.1-negative neurons, which should represent more diverse phenotypic identities, were generated during a more extended period of cortical development and tended to exhibit radial (inside-to-outside) and tangential (ventral-to-dorsal and rostral-to-caudal) neurogenetic gradients. Our findings indicate that laminar and tangential locations of cortical neurons are not established solely by a combination of mechanisms for the inside-out movement of newly generated neurons in each cortical area and for the broad tangential neurogenetic gradients. The results of this study suggest a distinct way of cortical development in which neurons with a common molecular phenotype are generated concurrently and migrate toward their eventual positions, which are not necessarily located in a single lamina. In addition, our results suggest some kind of tangential heterogeneity in the mechanism involved in neocortical histogenesis, supporting the concept of early regional specification within the neocortex.

Early regional specification for a molecular neuronal phenotype in the rat neocortex.

The timing of neocortical regional specification was examined using a monoclonal antibody, designated PC3.1, that binds a 29-kDa polypeptide and recognizes a neuronal subpopulation located in the lateral but not dorsomedial neocortex in the rat. When lateral cortical tissue fragments at embryonic days 12 and 16 were maintained in an organotypic culture system, a substantial number of neurons became PC3.1-immunopositive. In marked contrast, considerably fewer, if any, PC3.1-positive neurons were observed in cultures of dorsal cortical tissue. The selective appearance of PC3.1-immunopositive neurons was also observed in dissociated cultures derived from the lateral, but not dorsal, cortical primordium at embryonic day 13 and later. In light of previous reports showing that the interactions between developing neocortical neurons and cortical afferents begin at embryonic day 14 or later, our findings imply that some regional specification occurs well before these interactions and suggest the importance of elements intrinsic to the neocortex in establishing neocortical regional specificity. Furthermore, [3H]thymidine birth-dating experiments revealed that the majority of presumptive PC3.1-immunopositive neurons underwent their final mitosis around embryonic day 15, suggesting that the regional specification events for these neurons occur before their neurogenesis.

An epidemiological study with risk analysis of liver diseases in the general population living in a methyl mercury polluted area.

STUDY OBJECTIVE: The aim was to determine the actual prevalence of liver disease and to investigate the contribution of various risk factors to liver disease among the population in a methyl mercury polluted area. DESIGN: The study was a population based cross sectional mass screening survey. A case-control study was designed to estimate the role of various risk factors for liver diseases. SETTING: The study was confined to a small rural town 10 km north of Minamata City. SUBJECTS: 1406 persons aged 50 to 69 years were examined (78.3% of the total population of this age in the locality). MEASUREMENTS AND MAIN RESULTS: Measurements of liver disease were made on the basis of haematological, physical, and ultrasonographic examinations. Data on liver risk factors were collected by questionnaire, and by measurement of body height, weight (obesity), and hepatitis B surface antigen (HBsAg). The prevalence rate of liver tumour was 0.5% in males, liver cirrhosis was found in 0.5% of males and 0.1% of females, and hepatitis was seen in 5.4% of males and 1.0% of females. Frequency rates of risk factors for liver disease among subjects with obesity were significantly higher in the female patient group, and the frequency rate among subjects with alcoholic drinking habits was significantly higher in the male patient group. The odds ratio of past history of blood transfusion showed the highest value among other related factors (7.73) and the attributable risk for this was very high (87.1%); HBsAg was next in rank (odds ratio 3.04; attributable risk 67.1%). CONCLUSIONS: The prevalence of liver disease in this methyl mercury polluted area was not increased, contrary to what was expected based on the standard mortality ratios. The main risk factors for liver disease in this area appear to be alcoholic drinking habits and a history of blood transfusion.