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To bridge the gap between bench and bedside, there is a need for more faithful models of human cancers that can recapitulate key features of the human tumor microenvironment (TME) and simultaneously facilitate large-scale drug tests. Our recently developed microdissection method optimizes the yield of large numbers of cuboidal microtissues (″cuboids″, ~(400 µm) 3 ) from a tumor biopsy. Here we demonstrate that cuboids from syngeneic mouse tumor models and human tumors retain a complex TME, making them amenable for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as cellular architecture, cytokine secretion, proteomics profiles, and response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong cytokine expression and drug responses, including to immunotherapy. Overall, our results suggest that cuboids could provide essential therapeutic information for personalized oncology applications and could help the development of TME-dependent therapeutics and cancer disease models, including for clinical trials.

The microvasculature within the tumor microenvironment (TME) plays an essential role in cancer signaling beyond nutrient delivery. However, it has been challenging to control the generation and/or maintenance of microvasculature in ex vivo systems, a critical step for establishing cancer models of high clinical biomimicry. There have been great successes in engineering tissues incorporating microvasculature de novo ( e.g ., organoids and organs-on-chip), but these reconstituted tissues are formed with non-native cellular and molecular components that can skew certain outcomes such as drug efficacy. Microdissected tumors, on the other hand, show promise in preserving the TME, which is key for creating cancer models that can bridge the gap between bench and bedside. However, microdissected tumors are challenging to perfuse. Here, we developed a microfluidic platform that allows for perfusing the microvasculature of microdissected tumors. We demonstrate that, compared to diffusive transport, microfluidically perfused tissues feature larger and longer microvascular structures, with a better expression of CD31, a marker for endothelial cells, as analyzed by 3D imaging. This study also explores the effects of nitric oxide pathway-related drugs on endothelial cells, which are sensitive to shear stress and can activate endothelial nitric oxide synthase, producing nitric oxide. Our findings highlight the critical role of controlled perfusion and biochemical modulation in preserving tumor microvasculature, offering valuable insights for developing more effective cancer treatments., Competing Interests: Conflict of Interest. LH and AF are the founders of OncoFluidics, a startup that seeks to commercialize microfluidic drug tests using intact tissues. All the authors declare no conflict of interest.

Functional assays on intact tumor biopsies can complement genomics-based approaches for precision oncology, drug testing, and organs-on-chips cancer disease models by capturing key therapeutic response determinants, such as tissue architecture, tumor heterogeneity, and the tumor microenvironment. Most of these assays rely on fluorescent labeling, a semiquantitative method best suited for single-time-point assays or labor-intensive immunostaining analysis. Here, we report integrated aptamer electrochemical sensors for on-chip, real-time monitoring of cytochrome C, a cell death indicator, from intact microdissected tissues with high affinity and specificity. The platform features a multi-well sensor layout and a multiplexed electronic setup. The aptasensors measure increases in cytochrome C in the supernatant of mouse or human microdissected tumors after exposure to various drug treatments. Because of the sensor's high affinity, it primarily tracks rising concentrations of cytochrome C, capturing dynamic changes during apoptosis. This approach could help develop more advanced cancer disease models and apply to other complex in vitro disease models, such as organs-on-chips and organoids.

The scarcity of human biopsies available for drug testing is a paramount challenge for developing new therapeutics, disease models, and personalized treatments. Microtechnologies that combine the microscale manipulation of tissues and fluids offer the exciting possibility of miniaturizing both disease models and drug testing workflows on scarce human biopsies. Unfortunately, these technologies presently require microfluidic devices or robotic dispensers that are not widely accessible. We have rapidly-prototyped an inexpensive platform based on an off-the-shelf robot that can microfluidically manipulate live microtissues into/out of culture plates without using complicated accessories such as microscopes or pneumatic controllers. The robot integrates complex functions with a simple, cost-effective and compact construction, allowing placement inside a tissue culture hood for sterile workflows. We demonstrated a proof-of-concept cancer drug evaluation workflow of potential clinical utility using patient tumor biopsies with multiple drugs on 384-well plates. Our user-friendly, low-cost platform promises to make drug testing of microtissues broadly accessible to pharmaceutical, clinical, and biological laboratories., Teaser: A low-cost robot for handling microtissues and catalyzing their use in cancer drug evaluation and personalized oncology.

Cancer drug testing in animals is an extremely poor predictor of the drug's safety and efficacy observed in humans. Hence there is a pressing need for functional testing platforms that better predict traditional and immunotherapy responses in human, live tumor tissue or tissue constructs, and at the same time are compatible with the use of mouse tumor tissue to facilitate building more accurate disease models. Since many cancer drug actions rely on mechanisms that depend on the tumor microenvironment (TME), such platforms should also retain as much of the native TME as possible. Additionally, platforms based on miniaturization technologies are desirable to reduce animal use and sensitivity to human tissue scarcity. Present high-throughput testing platforms that have some of these features, e.g. based on patient-derived tumor organoids, require a growth step that alters the TME. On the other hand, microdissected tumors (μDTs) or "spheroids" that retain an intact TME have shown promising responses to immunomodulators acting on native immune cells. However, difficult tissue handling after microdissection has reduced the throughput of drug testing on μDTs, thereby constraining the inherent advantages of producing numerous TME-preserving units of tissue for drug testing. Here we demonstrate a microfluidic 96-well platform designed for drug treatment of hundreds of similarly-sized, cuboidal μDTs ("cuboids") produced from a single tumor sample. The platform organizes a monodisperse array of four cuboids per well in 384 hydrodynamic traps. The microfluidic device, entirely fabricated in thermoplastics, features 96 microvalves that fluidically isolate each well after the cuboid loading step for straightforward multi-drug testing. Since our platform makes the most of scarce tumor tissue, it can potentially be applied to human biopsies that preserve the human TME while minimizing animal testing.

Metastasis is the leading cause of mortality in cancer patients. To migrate to distant sites, cancer cells would need to adapt their behaviour in response to different tissue environments. Thus, it is essential to study this process in models that can closely replicate the tumour microenvironment. Here, we evaluate the use of organotypic liver and brain slices to study cancer metastasis. Morphological and viability parameters of the slices were monitored daily over 3 days in culture to assess their stability as a realistic 3D tissue platform for in vitro metastatic assays. Using these slices, we evaluated the invasion of MDA-MB-231 breast cancer cells and of a subpopulation that was selected for increased motility. We show that the more aggressive invasion of the selected cells likely resulted not only from their lower stiffness, but also from their lower adhesion to the surrounding tissue. Different invasion patterns in the brain and liver slices were observed for both subpopulations. Cells migrated faster in the brain slices (with an amoeboid-like mode) compared to in the liver slices (where they migrated with mesenchymal or collective migration-like modes). Inhibition of the Ras/MAPK/ERK pathway increased cell stiffness and adhesion forces, which resulted in reduced invasiveness. These results illustrate the potential for organotypic tissue slices to more closely mimic in vivo conditions during cancer cell metastasis than most in vitro models., (Copyright © 2021 Elsevier Inc. All rights reserved.)

As preclinical animal tests often do not accurately predict drug effects later observed in humans, most drugs under development fail to reach the market. Thus there is a critical need for functional drug testing platforms that use human, intact tissues to complement animal studies. To enable future multiplexed delivery of many drugs to one small biopsy, we have developed a multi-well microfluidic platform that selectively treats cuboidal-shaped microdissected tissues or "cuboids" with well-preserved tissue microenvironments. We create large numbers of uniformly-sized cuboids by semi-automated sectioning of tissue with a commercially available tissue chopper. Here we demonstrate the microdissection method on normal mouse liver, which we characterize with quantitative 3D imaging, and on human glioma xenograft tumors, which we evaluate after time in culture for viability and preservation of the microenvironment. The benefits of size uniformity include lower heterogeneity in future biological assays as well as facilitation of their physical manipulation by automation. Our prototype platform consists of a microfluidic circuit whose hydrodynamic traps immobilize the live cuboids in arrays at the bottom of a multi-well plate. Fluid dynamics simulations enabled the rapid evaluation of design alternatives and operational parameters. We demonstrate the proof-of-concept application of model soluble compounds such as dyes (CellTracker, Hoechst) and the cancer drug cisplatin. Upscaling of the microfluidic platform and microdissection method to larger arrays and numbers of cuboids could lead to direct testing of human tissues at high throughput, and thus could have a significant impact on drug discovery and personalized medicine.

The intricate microarchitecture of tissues - the "tissue microenvironment" - is a strong determinant of tissue function. Microfluidics offers an invaluable tool to precisely stimulate, manipulate, and analyze the tissue microenvironment in live tissues and engineer mass transport around and into small tissue volumes. Such control is critical in clinical studies, especially where tissue samples are scarce, in analytical sensors, where testing smaller amounts of analytes results in faster, more portable sensors, and in biological experiments, where accurate control of the cellular microenvironment is needed. Microfluidics also provides inexpensive multiplexing strategies to address the pressing need to test large quantities of drugs and reagents on a single biopsy specimen, increasing testing accuracy, relevance, and speed while reducing overall diagnostic cost. Here, we review the use of microfluidics to study the physiology and pathophysiology of intact live tissues at sub-millimeter scales. We categorize uses as either in vitro studies - where a piece of an organism must be excised and introduced into the microfluidic device - or in vivo studies - where whole organisms are small enough to be introduced into microchannels or where a microfluidic device is interfaced with a live tissue surface (e.g. the skin or inside an internal organ or tumor) that forms part of an animal larger than the device. These microfluidic systems promise to deliver functional measurements obtained directly on intact tissue - such as the response of tissue to drugs or the analysis of tissue secretions - that cannot be obtained otherwise., Competing Interests: Conflict of interestL.F.H. and A.F. are the founders of OncoFluidics, a startup that seeks to commercialize microfluidic drug tests using intact tissues. L.F.H and A.F. are inventors in U.S. patent No. US 9,518,977 (filed 13 Dec 2016) related to this work. T.R. is an inventor on patents related to this work filed by the U.S. Patent and Trademark Office (no. PCT/US2018/035738, filed 1 June 2018 and no. PCT/US2018/035661, filed 1 June 2018)., (© The Author(s) 2020.)

Current methods to assess the drug response of individual human cancers are often inaccurate, costly, or slow. Functional approaches that rapidly and directly assess the response of patient cancer tissue to drugs or small molecules offer a promising way to improve drug testing, and have the potential to identify the best therapy for individual patients. We developed a digitally manufactured microfluidic platform for multiplexed drug testing of intact cancer slice cultures, and demonstrate the use of this platform to evaluate drug responses in slice cultures from human glioma xenografts and patient tumor biopsies. This approach retains much of the tissue microenvironment and can provide results rapidly enough, within days of surgery, to guide the choice of effective initial therapies. Our results establish a useful preclinical platform for cancer drug testing and development with the potential to improve cancer personalized medicine., Competing Interests: Competing interestsThe microfluidic device is covered under US patent 20140113838A1, on which L.F.H., R.J.M., A.F., and R.C.R. are inventors. L.F.H. and A.F. are founders of OncoFluidics, LLC., (© The Author(s) 2020.)

Present approaches to assess cancer treatments are often inaccurate, costly, and/or cumbersome. Functional testing platforms that use live tumor cells are a promising tool both for drug development and for identifying the optimal therapy for a given patient, i.e. precision oncology. However, current methods that utilize patient-derived cells from dissociated tissue typically lack the microenvironment of the tumor tissue and/or cannot inform on a timescale rapid enough to guide decisions for patient-specific therapy. We have developed a microfluidic platform that allows for multiplexed drug testing of intact tumor slices cultured on a porous membrane. The device is digitally-manufactured in a biocompatible thermoplastic by laser-cutting and solvent bonding. Here we describe the fabrication process in detail, we characterize the fluidic performance of the device, and demonstrate on-device drug-response testing with tumor slices from xenografts and from a patient colorectal tumor.

Concerns over biosafety, cost, and carrying capacity of viral vectors have accelerated research into physical techniques for gene delivery such as electroporation and mechanoporation. Advances in microfabrication have made it possible to create high electric fields over microscales, resulting in more efficient DNA delivery and higher cell viability. Continuous-flow microfluidic methods are typically more suitable for cellular therapies where a large number of cells need to be transfected under sterile conditions. However, the existing continuous-flow designs used to generate multiple pulses either require expensive peripherals such as high-voltage (>400 V) sources or function generators, or result in reduced cell viability due to the proximity of the cells to the electrodes. In this paper, we report a continuous-flow microfluidic device whose channel geometry reduces instrumentation demands and minimizes cellular toxicity. Our design can generate multiple pulses of high DC electric field strength using significantly lower voltages (15-60 V) than previous designs. The cells flow along a serpentine channel that repeatedly flips the cells between a cathode and an anode at high throughput. The cells must flow through a constriction each time they pass from an anode to a cathode, exposing them to high electric field strength for short durations of time (the "pulse-width"). A conductive biocompatible poly-aniline hydrogel network formed in situ is used to apply the DC voltage without bringing the metal electrodes close to the cells, further sheltering cells from the already low voltage electrodes. The device was used to electroporate multiple cell lines using electric field strengths between 700 and 800 V/cm with transfection efficiencies superior than previous flow-through designs.

The advent of soft lithography allowed for an unprecedented expansion in the field of microfluidics. However, the vast majority of PDMS microfluidic devices are still made with extensive manual labor, are tethered to bulky control systems, and have cumbersome user interfaces, which all render commercialization difficult. On the other hand, 3D printing has begun to embrace the range of sizes and materials that appeal to the developers of microfluidic devices. Prior to fabrication, a design is digitally built as a detailed 3D CAD file. The design can be assembled in modules by remotely collaborating teams, and its mechanical and fluidic behavior can be simulated using finite-element modeling. As structures are created by adding materials without the need for etching or dissolution, processing is environmentally friendly and economically efficient. We predict that in the next few years, 3D printing will replace most PDMS and plastic molding techniques in academia., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Microfluidic automation - the automated routing, dispensing, mixing, and/or separation of fluids through microchannels - generally remains a slowly-spreading technology because device fabrication requires sophisticated facilities and the technology's use demands expert operators. Integrating microfluidic automation in devices has involved specialized multi-layering and bonding approaches. Stereolithography is an assembly-free, 3D-printing technique that is emerging as an efficient alternative for rapid prototyping of biomedical devices. Here we describe fluidic valves and pumps that can be stereolithographically printed in optically-clear, biocompatible plastic and integrated within microfluidic devices at low cost. User-friendly fluid automation devices can be printed and used by non-engineers as replacement for costly robotic pipettors or tedious manual pipetting. Engineers can manipulate the designs as digital modules into new devices of expanded functionality. Printing these devices only requires the digital file and electronic access to a printer.

The mammalian olfactory system detects a plethora of environmental chemicals that are perceived as odors or stimulate instinctive behaviors. Studies using odorant receptor (OR) genes have provided insight into the molecular and organizational strategies underlying olfaction in mice. One important unanswered question, however, is whether these strategies are conserved in primates. To explore this question, we examined the macaque, a higher primate phylogenetically close to humans. Here we report that the organization of sensory inputs in the macaque nose resembles that in mouse in some respects, but not others. As in mouse, neurons with different ORs are interspersed in the macaque nose, and there are spatial zones that differ in their complement of ORs and extend axons to different domains in the olfactory bulb of the brain. However, whereas the mouse has multiple discrete band-like zones, the macaque appears to have only two broad zones. It is unclear whether the organization of OR inputs in a rodent/primate common ancestor degenerated in primates or, alternatively became more sophisticated in rodents. The mouse nose has an additional small family of chemosensory receptors, called trace amine-associated receptors (TAARs), which may detect social cues. Here we find that TAARs are also expressed in the macaque nose, suggesting that TAARs may also play a role in human olfactory perception. We further find that one human TAAR responds to rotten fish, suggesting a possible role as a sentinel to discourage ingestion of food harboring pathogenic microorganisms., (Copyright © 2014 the authors 0270-6474/14/3412241-12$15.00/0.)

The mammalian olfactory system is able to discriminate among tens of thousands of odorant molecules. In mice, each odorant is sensed by a small subset of the approximately 1000 odorant receptor (OR) types, with one OR gene expressed by each olfactory sensory neuron (OSN). However, the sum of the large repertoire of OR-OSN types and difficulties with heterologous expression have made it almost impossible to analyze odorant-responsiveness across all OR-OSN types. We have developed a microfluidic approach that allowed us to screen over 20,000 single cells at once in microwells. By using calcium imaging, we were able to detect and analyze odorant responses of about 2900 OSNs simultaneously. Importantly, this technique allows for both the detection of rare responding OSNs as well as the identification of OSN populations broadly responsive to odorants of unrelated structures. This technique is generally applicable for screening large numbers of single cells and should help to characterize rare cell behaviors in fields such as toxicology, pharmacology, and cancer research.

The identification of receptors that detect environmental stimuli lays a foundation for exploring the mechanisms and neural circuits underlying sensation. The mouse vomeronasal organ (VNO), which detects pheromones and other semiochemicals, has 2 known families of chemoreceptors, V1Rs and V2Rs. Here, we report a third family of mouse VNO receptors comprising 5 of 7 members of the formyl peptide receptor (FPR) family. Unlike other FPRs, which function in the immune system, these FPRs are selectively expressed in VNO neurons in patterns strikingly similar to those of V1Rs and V2Rs. Each FPR is expressed in a different small subset of neurons that are highly dispersed in the neuroepithelium, consistently coexpress either G alpha(i2) or G alpha(o), and lack other chemoreceptors examined. Given the presence of formylated peptides in bacteria and mitochondria, possible roles for VNO FPRs include the assessment of conspecifics or other species based on variations in normal bacterial flora or mitochondrial proteins.

We have further tested the hypothesis that receptor-mediated modulation of KCNQ channels involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC). We used four parallel assays to characterize the agonist-induced PLC response of cells (tsA or CHO cells) expressing M1 muscarinic receptors: translocation of two fluorescent probes for membrane lipids, release of calcium from intracellular stores, and chemical measurement of acidic lipids. Occupation of M1 receptors activates PLC and consumes cellular PIP2 in less than a minute and also partially depletes mono- and unphosphorylated phosphoinositides. KCNQ current is simultaneously suppressed. Two inhibitors of PLC, U73122 and edelfosine (ET-18-OCH3), can block the muscarinic actions completely, including suppression of KCNQ current. However, U73122 also had many side effects that were attributable to alkylation of various proteins. These were mimicked or occluded by prior reaction with the alkylating agent N-ethylmaleimide and included block of pertussis toxin-sensitive G proteins and effects that resembled a weak activation of PLC or an inhibition of lipid kinases. By our functional criteria, the putative PLC activator m-3M3FBS did stimulate PLC, but with a delay and an irregular time course. It also suppressed KCNQ current. The M1 receptor-mediated activation of PLC and suppression of KCNQ current were stopped by lowering intracellular calcium well below resting levels and were slowed by not allowing intracellular calcium to rise in response to PLC activation. Thus calcium release induced by PLC activation feeds back immediately on PLC, accelerating it during muscarinic stimulation in strong positive feedback. These experiments clarify important properties of receptor-coupled PLC responses and their inhibition in the context of the living cell. In each test, the suppression of KCNQ current closely paralleled the expected fall of PIP2. The results are described by a kinetic model.

We studied modulation of current in human embryonic kidney tsA-201 cells coexpressing rat erg1 channels with M(1) muscarinic receptors. Maximal current was inhibited 30% during muscarinic receptor stimulation, with a small positive shift of the midpoint of activation. Inhibition was attenuated by coexpression of the regulator of G-protein signalling RGS2 or of a dominant-negative protein, G(q), but not by N-ethylmaleimide or C3 toxin. Overexpression of a constitutively active form of G(q) (but not of G(13) or of G(s)) abolished the erg current. Hence it is likely that G(q/11), and not G(i/o) or G(13), mediates muscarinic inhibition. Muscarinic suppression of erg was attenuated by chelating intracellular Ca(2+) to < 1 nm free Ca(2+) with 20 mm BAPTA in the pipette, but suppression was normal if internal Ca(2+) was strongly clamped to a 129 nm free Ca(2+) level with a BAPTA buffer and this was combined with numerous other measures to prevent intracellular Ca(2+) transients (pentosan polysulphate, preincubation with thapsigargin, and removal of extracellular Ca(2+)). Hence a minimum amount of Ca(2+) was necessary for the inhibition, but a Ca(2+) elevation was not. The ATP analogue AMP-PCP did not prevent inhibition. The protein kinase C (PKC) blockers staurosporine and bisindolylmaleimide I did not prevent inhibition, and the PKC-activating phorbol ester PMA did not mimic it. Neither the tyrosine kinase inhibitor genistein nor the tyrosine phosphatase inhibitor dephostatin prevented inhibition by oxotremorine-M. Hence protein kinases are not needed. Experiments with a high concentration of wortmannin were consistent with recovery being partially dependent on PIP(2) resynthesis. Wortmannin did not prevent muscarinic inhibition. Our studies of muscarinic inhibition of erg current suggest a role for phospholipase C, but not the classical downstream messengers, such as PKC or a calcium transient.

Receptor-mediated modulation of KCNQ channels regulates neuronal excitability. This study concerns the kinetics and mechanism of M1 muscarinic receptor-mediated regulation of the cloned neuronal M channel, KCNQ2/KCNQ3 (Kv7.2/Kv7.3). Receptors, channels, various mutated G-protein subunits, and an optical probe for phosphatidylinositol 4,5-bisphosphate (PIP2) were coexpressed by transfection in tsA-201 cells, and the cells were studied by whole-cell patch clamp and by confocal microscopy. Constitutively active forms of Galphaq and Galpha11, but not Galpha13, caused a loss of the plasma membrane PIP2 and a total tonic inhibition of the KCNQ current. There were no further changes upon addition of the muscarinic agonist oxotremorine-M (oxo-M). Expression of the regulator of G-protein signaling, RGS2, blocked PIP2 hydrolysis and current suppression by muscarinic stimulation, confirming that the Gq family of G-proteins is necessary. Dialysis with the competitive inhibitor GDPbetaS (1 mM) lengthened the time constant of inhibition sixfold, decreased the suppression of current, and decreased agonist sensitivity. Removal of intracellular Mg2+ slowed both the development and the recovery from muscarinic suppression. When combined with GDPbetaS, low intracellular Mg2+ nearly eliminated muscarinic inhibition. With nonhydrolyzable GTP analogs, current suppression developed spontaneously and muscarinic inhibition was enhanced. Such spontaneous suppression was antagonized by GDPbetaS or GTP or by expression of RGS2. These observations were successfully described by a kinetic model representing biochemical steps of the signaling cascade using published rate constants where available. The model supports the following sequence of events for this Gq-coupled signaling: A classical G-protein cycle, including competition for nucleotide-free G-protein by all nucleotide forms and an activation step requiring Mg2+, followed by G-protein-stimulated phospholipase C and hydrolysis of PIP2, and finally PIP2 dissociation from binding sites for inositol lipid on the channels so that KCNQ current was suppressed. Further experiments will be needed to refine some untested assumptions.

The olfactory system translates myriad chemical structures into diverse odour perceptions. To gain insight into how this is accomplished, we prepared mice that coexpressed a transneuronal tracer with only one of about 1,000 different odorant receptors. The tracer travelled from nasal neurons expressing that receptor to the olfactory bulb and then to the olfactory cortex, allowing visualization of cortical neurons that receive input from a particular odorant receptor. These studies revealed a stereotyped sensory map in the olfactory cortex in which signals from a particular receptor are targeted to specific clusters of neurons. Inputs from different receptors overlap spatially and could be combined in single neurons, potentially allowing for an integration of the components of an odorant's combinatorial receptor code. Signals from the same receptor are targeted to multiple olfactory cortical areas, permitting the parallel, and perhaps differential, processing of inputs from a single receptor before delivery to the neocortex and limbic system.

Mammalian nervous system function involves billions of neurons which are interconnected in a multitude of neural circuits. Here we describe a genetic approach to chart neural circuits. By using an olfactory-specific promoter, we selectively expressed barley lectin in sensory neurons in the olfactory epithelium and vomeronasal organ of transgenic mice. The lectin was transported through the axons of those neurons to the olfactory bulb, transferred to the bulb neurons with which they synapse, and transported through the axons of bulb neurons to the olfactory cortex. The lectin also was retrogradely transported from the bulb to neuromodulatory brain areas. No evidence could be obtained for adverse effects of the lectin on odorant receptor gene expression, sensory axon targeting in the bulb, or the generation or transmission of signals by olfactory sensory neurons. Transneuronal transfer was detected prenatally in the odor-sensing pathway, but only postnatally in the pheromone-sensing pathway, suggesting that odors, but not pheromones, may be sensed in utero. Our studies demonstrate that a plant lectin can serve as a transneuronal tracer when its expression is genetically targeted to a subset of neurons. This technology can potentially be applied to a variety of vertebrate and invertebrate neural systems and may be particularly valuable for mapping connections formed by small subsets of neurons and for studying the development of connectivity as it occurs in utero.

In mammals, odors are detected by approximately 1000 different types of odorant receptors (ORs), each expressed by a fraction of neurons in the olfactory epithelium. Neurons expressing a given OR are confined to one of four spatial zones but are distributed randomly throughout that zone. In the olfactory bulb, the axons of neurons expressing different ORs synapse at different sites, giving rise to a highly organized and stereotyped information map. An important issue is whether the epithelial and bulbar maps evolve independently or are linked, for example, by retrograde influences of the bulb on the epithelium. Here we examined the onset of expression and patterning of genes encoding ORs and sensory transduction molecules during mouse embryogenesis and in mice lacking olfactory bulbs. Our results argue for an independent development of epithelial and bulbar maps and an early functional development that may be pertinent to pattern development in the olfactory bulb.

The prognostic significance for residual or recurrent disease of cervical intraepithelial neoplasia Grade III in endocervical glands by cone biopsy was examined in 341 consecutive patients diagnosed from 1979 through 1983 and followed through 1988. Treatment by hysterectomy, within 8 weeks of cone biopsy, was done in 96 patients. The only variable that could predict residual disease at hysterectomy was positive margins (P = 0.059). However, both positive margins and positive glands were (independently of one another and after the effects of length of follow-up, hospital of admission, and age at time of first diagnosis were held constant) highly significant predictors of residual or recurrent disease in the 245 women who did not undergo a hysterectomy (P = 0.000 for each). The authors therefore conclude that information concerning gland involvement on cone biopsy specimens should influence patient management.