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Mice lacking DIX domain containing-1 (DIXDC1), an intracellular Wnt/β-catenin signal pathway protein, have abnormal measures of anxiety, depression and social behavior. Pyramidal neurons in these animals' brains have reduced dendritic spines and glutamatergic synapses. Treatment with lithium or a glycogen synthase kinase-3 (GSK3) inhibitor corrects behavioral and neurodevelopmental phenotypes in these animals. Analysis of DIXDC1 in over 9000 cases of autism, bipolar disorder and schizophrenia reveals higher rates of rare inherited sequence-disrupting single-nucleotide variants (SNVs) in these individuals compared with psychiatrically unaffected controls. Many of these SNVs alter Wnt/β-catenin signaling activity of the neurally predominant DIXDC1 isoform; a subset that hyperactivate this pathway cause dominant neurodevelopmental effects. We propose that rare missense SNVs in DIXDC1 contribute to psychiatric pathogenesis by reducing spine and glutamatergic synapse density downstream of GSK3 in the Wnt/β-catenin pathway.

Abstract Background Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. At present no reliable biomarkers are available to guide the management of this condition. Microarray technology may allow appropriate biomarkers to be identified but present platforms are lacking disease focus and are thus likely to miss potentially vital information contained in patient tissue samples. Methods A combination of large-scale in-house sequencing, gene expression profiling and public sequence and gene expression data mining were used to characterise the transcriptome of NSCLC and the data used to generate a disease-focused microarray – the Lung Cancer DSA research tool. Results Built on the Affymetrix GeneChip platform, the Lung Cancer DSA research tool allows for interrogation of ~60,000 transcripts relevant to Lung Cancer, tens of thousands of which are unavailable on leading commercial microarrays. Conclusion We have developed the first high-density disease specific transcriptome microarray. We present the array design process and the results of experiments carried out to demonstrate the array's utility. This approach serves as a template for the development of other disease transcriptome microarrays, including non-neoplastic diseases.

Bisphenol F (BPF) is a potential neurotoxicant used as a replacement for bisphenol A (BPA) in polycarbonate plastics and epoxy resins. We investigated the neurodevelopmental impacts of BPF exposure using Drosophila melanogaster as a model. Our transcriptomic analysis indicated that developmental exposure to BPF caused the downregulation of neurodevelopmentally relevant genes, including those associated with synapse formation and neuronal projection. To investigate the functional outcome of BPF exposure, we evaluated neurodevelopmental impacts across two genetic strains of Drosophila- w1118 (control) and the Fragile X Syndrome (FXS) model-by examining both behavioral and neuronal phenotypes. We found that BPF exposure in w1118 Drosophila caused hypoactive larval locomotor activity, decreased time spent grooming by adults, reduced courtship activity, and increased the severity but not frequency of β-lobe midline crossing defects by axons in the mushroom body. In contrast, although BPF reduced peristaltic contractions in FXS larvae, it had no impact on other larval locomotor phenotypes, grooming activity, or courtship activity. Strikingly, BPF exposure reduced both the severity and frequency of β-lobe midline crossing defects in the mushroom body of FXS flies, a phenotype previously observed in FXS flies exposed to BPA. This data indicates that BPF can affect neurodevelopment and its impacts vary depending on genetic background. Further, BPF may elicit a gene-environment interaction with Drosophila fragile X messenger ribonucleoprotein 1 (dFmr1)-the ortholog of human FMR1, which causes fragile X syndrome and is the most common monogenetic cause of intellectual disability and autism spectrum disorder., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kimberly Mulligan reports financial support was provided by National Institute of General Medical Sciences., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neurodevelopmental disorders are a heterogeneous group of behaviorally defined disorders with both genetic and environmental risk factors. Given that many neurodevelopmental disorders are characterized by impaired learning and/or intellectual abilities, behavioral paradigms that assess cognition in animal models have been effective tools in delineating underlying genetic variants that impact disease pathophysiology. For example, learning and memory paradigms in the common fruit fly Drosophila melanogaster have been successfully used to study risk genes and biological pathways associated with several neurodevelopmental disorders, including fragile X syndrome, autism spectrum disorder, and CHARGE syndrome. While these established Drosophila behavioral paradigms have historically been used to investigate genetic risk factors, they also have many other applications, including use in developmental neurotoxicology studies to determine environmental risk factors for neurodevelopmental disorders. There is, however, a deficit of step-by-step protocols that describe how to apply learning and memory assays in fruit flies to developmental neurotoxicology studies. Here, we describe two quantitative behavioral paradigms for Drosophila-predator-induced oviposition and courtship conditioning-that can be used to measure different forms of learning and memory in the context of a developmental neurotoxicology study. Non-associative learning and memory are measured here by examining female Drosophila oviposition behavior in response to endoparasitoid wasps, while associative learning and memory are measured in males using courtship conditioning. Our protocols outline procedures for oral toxicant exposure of developing fruit flies, culturing of endoparasitoid wasps, measuring Drosophila oviposition activity, and assessing conditioned courtship in order to identify the impact of toxicants on learning and memory in both females and males. As an example, we present the protocols using bisphenol A, a chemical utilized in the synthesis of polycarbonate plastics, to determine its impacts on learning and memory. These protocols are inexpensive and relatively simple to perform, and can be used by labs that are new to Drosophila behavioral research to evaluate how toxicant exposure influences learning and memory in male and female flies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of toxicant-containing food and developmental exposure Basic Protocol 2: Predator-induced oviposition assay Support Protocol: Culture of Leptopilina heterotoma Basic Protocol 3: Conditioned courtship assay., (© 2022 Wiley Periodicals LLC.)

Triple-negative breast cancer (TNBC) remains the most lethal breast cancer subtype with poor response rates to the current chemotherapies and a lack of additional effective treatment options. We have identified deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumour DNA from the genotoxic misincorporation of uracil during treatment with standard chemotherapeutic agents commonly used in the FEC regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP), providing dUMP for thymidylate synthase as part of the thymidylate biosynthesis pathway and maintaining low intracellular dUTP concentrations. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporation into DNA. Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by fluoropyrimidines or anthracyclines represents an effective strategy to induce cell lethality. dUTPase inhibition significantly sensitised TNBC cell lines to fluoropyrimidines and anthracyclines through imbalanced nucleotide pools and increased DNA damage leading to decreased proliferation and increased cell death. These results suggest that repair of treatment-mediated DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a promising strategy to enhance the efficacy of TNBC chemotherapy.

Mounting evidence indicates that Wnt signaling is relevant to pathophysiology of diverse mental illnesses including schizophrenia, bipolar disorder, and autism spectrum disorder. In the 35 years since Wnt ligands were first described, animal studies have richly explored how downstream Wnt signaling pathways affect an array of neurodevelopmental processes and how their disruption can lead to both neurological and behavioral phenotypes. Recently, human induced pluripotent stem cell (hiPSC) models have begun to contribute to this literature while pushing it in increasingly translational directions. Simultaneously, large-scale human genomic studies are providing evidence that sequence variation in Wnt signal pathway genes contributes to pathogenesis in several psychiatric disorders. This article reviews neurodevelopmental and postneurodevelopmental functions of Wnt signaling, highlighting mechanisms, whereby its disruption might contribute to psychiatric illness, and then reviews the most reliable recent genetic evidence supporting that mutations in Wnt pathway genes contribute to psychiatric illness. We are proponents of the notion that studies in animal and hiPSC models informed by the human genetic data combined with the deep knowledge base and tool kits generated over the last several decades of basic neurodevelopmental research will yield near-term tangible advances in neuropsychiatry.

TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.

The therapeutic potential of Wnt proteins has long been recognized but challenges associated with in vivo stability and delivery have hindered their development as drug candidates. By exploiting the hydrophobic nature of the protein we provide evidence that exogenous Wnt3a can be delivered in vivo if it is associated with a lipid vesicle. Recombinant Wnt3a associates with the external surface of the lipid membrane; this association stabilizes the protein and leads to prolonged activation of the Wnt pathway in primary cells. We demonstrate the consequences of Wnt pathway activation in vivo using a bone marrow engraftment assay. These data provide validation for the development of WNT3A as a therapeutic protein.

Members of the Wnt family of secreted signaling proteins influence many aspects of neural development and function. Wnts are required from neural induction and axis formation to axon guidance and synapse development, and even help modulate synapse activity. Wnt proteins activate a variety of downstream signaling pathways and can induce a similar variety of cellular responses, including gene transcription changes and cytoskeletal rearrangements. This review provides an introduction to Wnt signaling pathways and discusses current research on their roles in vertebrate neural development and function.

Lipid-modified Wnt/Wingless (Wg) proteins can signal to their target cells in a short- or long-range manner. How these hydrophobic proteins travel through the extracellular environment remains an outstanding question. Here, we report on a Wg binding protein, Secreted Wg-interacting molecule (Swim), that facilitates Wg diffusion through the extracellular matrix. Swim, a putative member of the Lipocalin family of extracellular transport proteins, binds to Wg with nanomolar affinity in a lipid-dependent manner. In quantitative signaling assays, Swim is sufficient to maintain the solubility and activity of purified Wg. In Drosophila, swim RNAi phenotypes resemble wg loss-of-function phenotypes in long-range signaling. We propose that Swim is a cofactor that promotes long-range Wg signaling in vivo by maintaining the solubility of Wg.

Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. At present no reliable biomarkers are available to guide the management of this condition. Microarray technology may allow appropriate biomarkers to be identified but present platforms are lacking disease focus and are thus likely to miss potentially vital information contained in patient tissue samples., Methods: A combination of large-scale in-house sequencing, gene expression profiling and public sequence and gene expression data mining were used to characterise the transcriptome of NSCLC and the data used to generate a disease-focused microarray - the Lung Cancer DSA research tool., Results: Built on the Affymetrix GeneChip platform, the Lung Cancer DSA research tool allows for interrogation of ~60,000 transcripts relevant to Lung Cancer, tens of thousands of which are unavailable on leading commercial microarrays., Conclusion: We have developed the first high-density disease specific transcriptome microarray. We present the array design process and the results of experiments carried out to demonstrate the array's utility. This approach serves as a template for the development of other disease transcriptome microarrays, including non-neoplastic diseases.

Background: A novel glycoprotein, pMQ1, is positively correlated with increasing histological grade in malignant astrocytomas. Cerebral metastases from breast cancer have also been found to contain pMQ1-positive cells. This study aimed to determine the role of pMQ1 in primary breast cancer., Methods: Breast cancer specimens were analysed for pMQ1 by immunohistochemistry. The expression of pMQ1 was correlated with conventional prognostic indicators. Kaplan-Meier analyses were performed to compare clinical outcome between pMQ1-positive and pMQ1-negative tumours., Results: pMQ1 was expressed in most of the breast cancer specimens. The surrounding normal tissue margins and benign breast tissues always lacked pMQ1 expression. A significant positive correlation was observed between pMQ1 expression and histological grade, the presence of lymphovascular invasion and Nottingham Prognostic Index. Cancers that were pMQ1 positive were significantly more likely to develop a local recurrence., Conclusion: pMQ1 appears to be a tumour-associated protein. The positive correlation of pMQ1 with histological grade, presence of lymphovascular invasion and Nottingham Prognostic Index suggests that it confers an adverse prognosis., (Copyright (c) 2005 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

Although possessing a long history of use, the therapeutic use of epidural steroid injections still needs substantiation. Refinements in our understanding of the pathophysiology of radicular pain and in the techniques used to deliver depo-steroids to the target tissue will lead to improved clinical outcomes and fewer technique and drug-related side effects. Administration of epidural steroids at lumbar spine sites is more common than at cervical spine levels, although the same pain management concepts are applicable. Comparative studies are necessary to clearly define the advantages and disadvantages of the use of fluoroscopy and the transforaminal technique.

CD44/hyaluronan interactions and epidermal growth factor (EGF) stimulation are both known to enhance tumour invasion in vitro. The frequent amplification of the EGF receptor (EGFR) in high-grade astrocytomas led to the examination of the hypothesis that CD44-dependent astrocytoma invasion is regulated by EGF. It has been shown that human astrocytoma cells express only the standard (haemopoietic) form of CD44 (CD44s) and that EGF up-regulates CD44 mRNA and protein in a time- and dose-dependent (10-100 ng/ml) manner. EGF stimulation did not result in induction of additional splice variants. No EGF-induced increase in CD44s was observed after treatment of cells with the wild-type EGFR tyrosine kinase inhibitor Tyrphostin AG1478 (30 nM). Up-regulation of CD44 by EGF is also prevented by the transcriptional inhibitor actinomycin D (5 microg/ml) and by blocking the MAP kinase (MAPK) pathway using the MEK inibitor U0126 (100 microM). CD44 up-regulation was associated with a 50% increase in invasion through hyaluronan-supplemented Matrigel(trade mark), which was abrogated by ligating CD44 with the specific antibody KM201. These results suggest that increased CD44 expression in response to EGF stimulation plays a significant role in astrocytoma invasion., (Copyright 2000 John Wiley & Sons, Ltd.)

Objectives: This study was designed to assess whether intermittent impedance of inspiratory gas exchange improves the efficiency of standard cardiopulmonary resuscitation (CPR)., Background: Standard CPR relies on the natural elastic recoil of the chest to transiently decrease intrathoracic pressures and thereby promote venous blood return to the heart. To further enhance the negative intrathoracic pressures during the "relaxation" phase of CPR, we tested the hypothesis that intermittent impedance to inspiratory gases during standard CPR increases coronary perfusion pressures and vital organ perfusion., Methods: CPR was performed with a pneumatically driven automated device in a porcine model of ventricular fibrillation. Eight pigs were randomized to initially receive standard CPR alone, while seven pigs initially received standard CPR plus intermittent impedance to inspiratory gas exchange with a threshold valve set to -40 cm H2O. The compression:ventilation ratio was 5:1 and the compression rate was 80/min. At 7-min intervals the impedance threshold valve (ITV) was either added or removed from the ventilation circuit such that during the 28 min of CPR, each animal received two 7-min periods of CPR with the ITV and two 7-min periods without the valve., Results: Vital organ blood flow was significantly higher during CPR performed with the ITV than during CPR performed without the valve. Total left ventricular blood flow (mean+/-SEM) (mL/min/g) was 0.32+/-0.04 vs 0.23+/-0.03 without the ITV (p<0.05). Cerebral blood flow (mL/min/g) was 20% higher with the ITV (+ITV, 0.23+/-0.02; -ITV, 0.19+/-0.02; p<0.05). Each time the ITV was removed, there was a statistically significant decrease in the vital organ blood flow and coronary perfusion pressure., Conclusions: Intermittent impedance to inspiratory flow of respiratory gases during standard CPR significantly improves CPR efficiency during ventricular fibrillation. These studies underscore the importance of lowering intrathoracic pressures during the relaxation phase of CPR.

Both epinephrine (Epi) and vasopressin (VP) increase coronary perfusion pressure (CPP) when administered during cardiac arrest. Given their different mechanisms of action we tested the hypothesis that during cardiopulmonary resuscitation (CPR) a combination of VP plus Epi would be superior to either agent alone. Epi(40 microg/kg), VP(0.3 U/kg) and the combination of both agents were assessed in a porcine model of ventricular fibrillation (VF). Maximum CPP (diastolic aortic-right atrial pressures) during CPR was similar among the groups but the time course of action was different in each group: with Epi + VP the increase in CPP was significantly more rapid than with VP alone whereas the CPP remained significantly higher for a longer periods of time with VP or VP + Epi versus Epi alone. Left ventricular blood flow (ml/min per g) determined during CPR two min after drug administration was similar between groups: Epi 1.06 +/- 0.16; VP 0.82 +/- 0.26; Epi + VP 0.83 +/- 0.14 (P = N.S.). Post drug administration. 2 min, cerebral blood flow (ml/min per g) in the VP group (0.76 +/- 0.15) was more than two times higher compared with Epi alone (Epi:0.30 +/- 0.08, P < 0.01 versus VP) and Epi plus VP (Epi + VP:0.23 +/- 0.03, P < 0.01 versus VP). We conclude that combination of VP + Epi during cardiac arrest results in a more rapid rise in CPP when compared with VP alone and a more sustained elevation in CPP than observed with Epi alone. Thus, the synergistic effects of these two potent vasopressor agents may be of benefit during CPR.

In an effort to reduce energy requirements for atrial defibrillation to a level low enough to perform painless electrical cardioversion with an implantable atrial defibrillator, we tested the hypothesis that drug therapy with the class III agent d-sotalol, when used concurrently with a low-energy shock, reduces atrial defibrillation threshold. In a nonthoracotomy canine model of atrial fibrillation, intracardiac shocks were delivered between the distal coronary sinus and the mid-right atrium. Based on a step-up energy delivery protocol the atrial defibrillation threshold was defined as the least amount of energy that resulted in a >10% and <90% rate of successful defibrillation. At a dose associated with class III antiarrhythmic effects (5 mg/kg), d-sotalol significantly reduced atrial defibrillation threshold from 1.72 +/- 1.12 J to 0.59 +/- 0.60 J (p < 0.01). These results support the feasibility of using antiarrhythmic drug therapy with d-sotalol to minimize energy requirements for intracardiac electrical cardioversion of atrial fibrillation.

Three morphological types of axons have been recognized in previous studies of the serotoninergic (5-HT) innervation of the cat cerebral cortex (Mulligan and Törk, 1988; DeFelipe et al., 1991): thick, nonvaricose axons, fine axons with small varicosities, and beaded axons with large, spherical varicosities. In the present study, the laminar density and distribution of the three 5-HT fiber types in area 17 are characterized. In both coronal and sagittal immunostained sections, 5-HT axons exhibit an overall gradient of decreasing density from layer I to the white matter. The three 5-HT axon types exhibit distinctive innervation patterns. (1) Fine axons with small varicosities comprise the greatest number of fibers in each layer (56-98%). They usually have oblique or radial trajectories, but some horizontally oriented fibers run through layers I, III, VI, and the white matter. (2) Non-varicose axons, the preterminal portions of the beaded axons with the large varicosities (Mulligan and Törk, 1988), comprise only about 7% of the total 5-HT fiber population in area 17, and are found mainly in layer I and in the white matter where they form a horizontally oriented plexus. (3) Large varicose axons ("beaded" axons) are rare (about 3% of the total 5-HT population) and are restricted to layers I and V. Although large varicose axons often form elaborate pericellular basketlike arbors around the soma and dendrites of neurons in other parts of the cat cerebral cortex (Mulligan and Törk, 1987, 1988), such arrays are rare in area 17. When observed in area 17, pericellular arrays are typically simple structures with a few large varicosities apparently contacting only the somata of a small population of layer I neurons. Comparison of these results with reports of 5-HT innervation of area 17 in other species suggests that the 5-HT innervation of the cortex is highly species specific.

Lateral suprasylvian visual cortex in the cat has been studied extensively, but its retinotopic organization remains controversial. Although some investigators have divided this region into many distinct areas, others have argued for a simpler organization. A clear understanding of the region's retinotopic organization is important in order to define distinct areas that are likely to subserve unique visual functions. We therefore reexamined the map of the lower visual field in the striate-recipient region of lateral suprasylvian cortex, a region we refer to as the lateral suprasylvian area, LS. A dual mapping approach was used. First, receptive fields were plotted at numerous locations along closely spaced electrode penetrations; second, different anterograde tracers were injected at retinotopically identified sites in area 17, yielding patches of label in LS. To visualize the resulting data, suprasylvian cortex was flattened with the aid of a computer. Global features of the map reported in many earlier studies were confirmed. Central visual field was represented posteriorly, and elevations generally shifted downward as one moved anteriorly. Often (though not always) there was a progression from peripheral locations towards the vertical meridian as the electrode moved down the medial suprasylvian bank. The map had some remarkable characteristics not previously reported in any map in the cat. The vertical meridian's representation was split into two pieces, separated by a gap, and both pieces were partially internalized within the map. Horizontal meridian occupied the gap. The area centralis usually had a dual representation along the posterior boundary of the lower field representation, and other fragments of visual field were duplicated as well. Finally, magnification appeared to change abruptly and unexpectedly, so that compressed regions of representation adjoined expanded regions. Despite its complexity, we found the map to be more orderly than previously thought. There was no clearcut retinotopic basis on which to subdivide LS's lower field representation into distinct areas.

The staining patterns produced by the lectin Vicia villosa and by a commercially available polyclonal antibody generated to substance P were analysed and compared in monkey visual cortex at the light and electron microscopic levels. Vicia villosa lectin labels the cell surface of a subpopulation of cortical cells, producing a meshlike pattern over the soma and proximal dendrites. The polyclonal antibody labels three distinct elements in the cortex: a pericellular epitope present on a subpopulation of non-pyramidal cells, and putative intracellular sites in a type of small pyramidal cell located at the layer 5/6 border, and in a small number of non-pyramidal cells in the underlying white matter. Because of the similarity of the appearance of the Vicia villosa lectin labelling and the pericellular labelling produced by the polyclonal antibody, further experiments were conducted to determine the relationship between the cell surface sites recognized by these markers. Double-labelling experiments show that both sites are present on the same population of cells, and at the ultrastructural level both markers appear to outline the intersynaptic cell membrane, sometimes extending around presynaptic elements. However, preadsorption experiments indicate that the markers recognize different sites on the cell membrane. Preadsorption experiments also show that the pericellular epitope recognized by the polyclonal antibody is unlikely to be substance P, but it may be structurally similar to keyhole limpet haemocyanin. Comparison of cortical and subcortical staining patterns produced with the polyclonal antibody and with a commonly used monoclonal antibody to substance P reveal that one of the putative intracellular epitopes recognized by the polyclonal antibody is likely to be substance P.

The retinotopic map in the striate-recipient region of the cat's lateral suprasylvian cortex (referred to here as the lateral suprasylvian area (LS)) has generally been described as quite disorderly. The disorder is commonly attributed to receptive field scatter within cell columns, reflecting the very large size of receptive fields. However, scatter within columns has never been investigated. In the experiments reported here, we examined the receptive field scatter of cells in columns, and also the scatter of a limited sample of their afferents arising from areas 17 and 18. To measure post-synaptic receptive field scatter, electrode penetrations were made parallel to columns in LS, with the electrode approaching from the medial side, traversing the suprasylvian gyrus and emerging into the suprasylvian sulcus. In all 13 such penetrations, receptive fields were clustered together despite their large size. Their centers were scattered over a region that occupied on average less than 20% of the largest field in the column. In contrast, in columns in areas 17 and 18 receptive field centers reportedly are dispersed over regions about equal to the largest of the fields (Hubel and Wiesel 1962, 1965, 1974). The scatter of afferents' receptive fields was assessed anatomically by measuring the overlap between patches of different anterograde tracers in LS. These patches represented terminal labeling from two adjacent or overlapping tracer injections in area 17. While a large degree of overlap would be predicted if afferents have substantial scatter, we found the overlap to be small unless the two injection sites themselves were highly overlapping. Scatter in afferents' receptive fields was measured more directly by physiological recording. In previous experiments, cells in LS were silenced by the local injection of kainic acid, and responses were recorded from axon terminals arising from areas 17 and 18 (Sherk 1989). We examined the receptive field scatter in three penetrations made approximately normal to the cortical surface. Scatter was modest, much less than predicted by the size of post-synaptic receptive fields. Because the degree of receptive field scatter for postsynaptic cells in LS was similar to that of inputs from areas 17 and 18, the scatter of these inputs might be entirely responsible for that seen postsynaptically. Postsynaptic receptive field scatter, on the other hand, was too small to explain the reported disorder in the map in LS.

The distribution, morphology, and ionic conductances of Vicia villosa agglutinin (VVA)-labeled cells were examined in the rat hippocampal formation. The heaviest labeling and highest density of labeled neurons were found in the subicular complex. Lighter VVA-labeling and fewer labeled cells were found in hippocampal strata pyramidale, oriens, and alveus. VVA-labeled cells were found to be heterogeneous morphologically, including multipolar, bipolar, and basket-like shapes. The majority of VVA-labeled cells contained GABA and parvalbumin immunoreactivity; thus VVA-labeled cells in the hippocampal formation resemble previously described VVA-labeled neurons in cerebral cortex. Electrophysiological properties of subicular VVA-labeled cells were studied in an acutely dissociated neuron preparation. Dissociated cells were labeled in vitro with VVA coupled either to a fluorescent marker or to small beads. The viability of labeled dissociated cells was confirmed, and identified cells were partially characterized electrophysiologically using whole-cell voltage clamp recording. VVA-labeled cells were electrophysiologically similar to pyramidal cells from the same region, except that the VVA-labeled cells showed only small transient outward currents.

The development of immunoreactivity for the calcium-binding proteins parvalbumin (PV) and calbindin-D28K (Cal) was studied in Macaca nemestrina striate cortex from fetal (F) 60 days to postnatal (P) 5 + years. We correlated changes in PV and Cal staining patterns with the well-documented developmental sequence for primate striate cortex neuron generation and maturation, synaptogenesis, and thalamocortical axon interactions in an attempt to deduce a functional role for these proteins. Our major findings is that Cal and PV have diametrically opposed developmental patterns except in layer 1. At F60 days both are present only in neurons of layer 1 and the number of labeled cell bodies and processes increases up to F125 days. Almost all Cal+ and PV+ cells in layer 1 disappear by P12 weeks. Cal is present by F113 days in pyramidal and stellate neurons, particularly layers 4-6. The numbers and staining density of cells in layers 2-6 increases up to birth and then both decline by P9-12 weeks. Supragranular layers show a second increase in Cal labeling from P20-36 weeks, and then there is a slow decline to the adult pattern which is reached by P1-2 years. Cell bodies in layers 4A, 4C alpha, and deep 4C beta are heavily Cal+ during pre- and early post-natal periods, but upper 4C beta remains unlabeled. PV is not seen until F155-162 days in layers 2-6. Large stellate and a few pyramidal cells appear first in layers 5/6 and 4C alpha, but PV+ stellate neurons are found in all layers except 4C beta by P6 weeks. Layer 4C beta contains a few PV+ cell bodies at P3 weeks, and light neuropile staining at P6 weeks, but then PV labeling rapidly increases so that by P12 weeks the density of 4C beta exceeds that of 4C alpha. Striate cortex has an adult pattern of cell number and neuropile density by P20 weeks. These developmental patterns suggest that the highest density of Cal cell body staining does not correlate with synaptogenesis, or the postnatal critical period of visually driven, binocular interactions. Rather Cal appears when lateral geniculate axons arrive in cortex, persists over the entire span of thalamocortical interactions, and disappears during the decline of cortical plasticity. The appearance of PV is highly correlated with the onset of complex visually driven activity at birth, while both the number of PV+ cell bodies and the density of PV+ neuropile reach adult levels coincident with the completion of thalamocortical connections.

Recent studies have shown that the presence of immunoreactivity for parvalbumin (PV-IR) and calbindin-D 28k (Cal-IR) can be used as markers for certain types of gamma-aminobutyric acid (GABA) immunoreactive interneurons in monkey cerebral cortex. Little quantitative information is available regarding the features that distinguish these two subpopulations, however. Therefore, in this study we localized PV-IR and Cal-IR neurons in Macaca monkey striate cortex and analyzed quantitatively their laminar distribution, cell morphology, and co-localization with GABA by double-labeling immunocytochemistry. PV-IR was found in nonpyramidal cells in all layers of the cortex, although PV-IR cells in layer 1 were rare. In contrast, Cal-IR was found mainly in nonpyramidal cells in two bands corresponding to layers 2-3 and 5-6. We found very few double-labeled PV-IR/Cal-IR cells but confirmed that almost all PV-IR and Cal-IR cells are GABAergic. Overall, 74% of GABA neurons in striate cortex displayed PV-IR compared to only 12% that displayed Cal-IR and 14% that were GABA-IR only. Quantitative analysis indicated that the relative proportion of GABA cells that displayed PV-IR or Cal-IR showed conspicuous laminar differences, which were often complementary. Cell size measurements indicated that PV-IR/GABA cells in layers 2-3 and 5-6 were significantly larger than Cal-IR/GABA cells. Analysis of the size, shape, and orientation of stained cell bodies and proximal dendrites further demonstrated that each subpopulation contained several different types of smooth stellate cells, suggesting that Cal-IR and PV-IR are found in functionally and morphologically heterogeneous subpopulations of GABA neurons. There was a thick bundle of PV-IR axons in the white matter underlying the striate but not prestriate cortex. PV-IR punctate labeling matched the cytochrome oxidase staining pattern in layers 4A and 4C, suggesting that PV-IR is present in geniculocortical afferents as well as intrinsic neurons. Cal-IR neuropil staining was high in layers 1, 2, 4B, and 5, where cytochrome oxidase staining is relatively low. We did not find a preferential localization of either PV-IR or Cal-IR cell bodies in any cytochrome oxidase compartments in layers 2-3 of the cortex. These findings indicate that PV and Cal are distributed into different neuronal circuits.

The projection from the dorsal lateral geniculate complex to the visual cortex in Pseudemys and Chrysemys turtles was examined by using the anterograde transport of horseradish peroxidase (HRP) in vitro and the retrograde transport of HRP in vivo. In vitro HRP injections into the lateral forebrain bundle were used to fill geniculocortical axons anterogradely, which were then analyzed in cortical wholemount preparations. Geniculocortical axons gain access to the visual cortex along its entire rostral-caudal extent. They course in slightly curved trajectories for up to 2 mm from the lateral edge of the cortex through both the lateral (or pallial thickening) and medial parts of Desan's cortical area D2. Single axons are of fine caliber. They tend to cross each other and sometimes branch in the pallial thickening, but are generally unbranched in the medial part of D2. They bear small, fusiform varicosities at irregular intervals along their lengths. Although axons show small variations in the number of varicosities per 100 microns segment, no consistent variation in varicosity number as a function of distance could be detected. These results indicate that geniculocortical axons project to the visual cortex in an orderly pattern. The retrograde transport experiments provide some clue as to the significance of this pattern. Small, ionotophoretic injections of HRP in the visual cortex retrogradely labeled neurons in the dorsal lateral geniculate complex. Injections in the rostral visual cortex retrogradely labeled neurons in the caudal pole of the geniculate complex. Injections at progressively more caudal loci within the visual cortex labeled neurons at progressively more rostral loci within the geniculate complex. Thus, there is a representation of the rostral-caudal axis of the geniculate complex along the caudal-rostral axis of the visual cortex. Consistent with the anterograde transport experiments that showed individual geniculocortical axons coursing through both lateral and medial parts of the visual cortex, HRP injections restricted to the medial edge of the visual cortex retrogradely labeled neurons along the entire dorsal-ventral axis of the geniculate complex at the appropriate rostral-caudal position. The neurophysiological studies of Mazurskaya ('72: J. Evol. Biochem. Physiol. 8:550-555; respond to a small, moving stimulus anywhere in visual space, implying a convergence of inputs from all points in visual space somewhere along the retinogeniculocortical pathway. The experiments reported here suggest a convergence in the geniculocortical projections of information along the vertical meridians, or azimuth lines, of visual space onto neurons lying along lateral to medial transects through the visual cortex.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonergic axons in the posterior cerebral cortex of the cat were demonstrated immunohistochemically using a monoclonal antibody to serotonin (5-HT). This technique reveals the presence of a dense serotonergic innervation of single cortical neurons at the light microscopic level. 5-HT axons with large varicosities (1-6 microns in diameter) form distinct, basket-like arrays around counterstained somata principally in layer I. In each basket one or more axons encircle and make repeated contact with the soma. Some axons extend from the soma and apparently climb along the dendrites of the target neuron. The climbing 5-HT axons form a stellate or horizontal pattern suggesting that the target cells are non-pyramidal neurons of the supragranular layers.

The morphology and distribution of neurons labeled specifically by the lectin, Vicia villosa (VVA), were examined in striate cortex of adult macaque monkeys. Following incubation with VVA conjugated to histochemical markers, fine punctate reaction product appears to cover the surface of the soma and proximal dendrites of a population of cortical neurons. Although a small number of VVA-labeled cells are located in layers 2, 3A, 5, and 6, approximately 75% are located in a strip of cortex overlying layers 3B through 4Ca. Layers 1 and 4C beta are virtually devoid of labeled cells. The morphology of labeled cells varies throughout the layers. In the supragranular layers, the labeled cells generally display a round or multipolar soma with a small number of radially disposed dendrites. In deeper layers, labeled cells are multipolar or horizontal, and their proximal dendrites are often more densely labeled. There is no clear correlation between the distribution of labeled cells and the pattern of cytochrome oxidase staining in supragranular layers. Double labeling of single sections for VVA and for GABA (gamma-aminobutyric acid) immunoreactivity revealed that most VVA-labeled cells are also immunoreactive for GABA. The double-labeled cells comprise approximately 30% of all GABA immunoreactive cells. Soma size analysis of double-labeled cells shows that medium-to-large GABA cells in each layer are labeled by VVA. The soma size, laminar distribution, and morphology of the VVA-labeled GABA cells suggest that they include the large basket cells originally observed in Golgi preparations.

Serotoninergic axons in the cat cerebral cortex were demonstrated immunohistochemically with a monoclonal antibody to serotonin (5-HT). Three types of 5-HT axons are distinguished at the light microscopic level by differences in their morphology. Small varicose axons are fine (less than 0.5 micron) and bear fusiform varicosities that are generally less than 1 micron in diameter. These axons extend throughout the width of the cortex and branch frequently, giving rise to widely spreading collaterals. Nonvaricose axons are smooth, show a relatively large and constant caliber (about 1 micron), travel in straight, horizontal trajectories, and branch infrequently. Large varicose axons are distinguished by large round or oval varicosities (1 micron or more in diameter) borne on fine-caliber fibers. These axons often form basket-like arbors around the somata of single neurons. In the simplest basket-like arbors, several large, round varicosities from a small number of axons contact the soma. In complex baskets intertwining collaterals contact the soma and apparently climb along and outline the cell's major dendrites. The patterns revealed by the climbing axons suggest that a variety of nonpyramidal cell types selectively receive dense 5-HT innervation. Serial reconstructions of the 5-HT axons within the cortex show that the large varicose axons arise as infrequent collaterals from the nonvaricose axons. A single nonvaricose parent axon gives rise to several large varicose axon collaterals that may contribute to different basket-like arbors. Conversely, a single basket-like arbor may be formed by large varicose axon collaterals from more than one nonvaricose parent axon. The small varicose axons do not appear to be related within the cortex to either the nonvaricose or large varicose axon types. The results support the hypothesis that the 5-HT projection to the cortex is organized into two subsystems, one of which may exert widespread influence in the cortex via highly divergent branches, while the other, with a more restricted distribution, acts on specific classes of cortical neurons.