A novel FGF23 mutation in hyperphosphatemic familial tumoral calcinosis and its deleterious effect on protein O-glycosylation.

Background: Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare disease characterized by hyperphosphatemia and ectopic calcification, predominantly at periarticular locations. This study was performed to characterize the clinical profile of tumoral calcinosis and to identify gene mutations associated with HFTC and elucidated its pathogenic role. Methods: The three subjects (two male and one female) were aged 30, 25 and 15 years, respectively. The clinical features, histopathological findings, and outcomes of three subjects with HFTC were retrospectively reviewed. The three subjects were analyzed for FGF23, GALNT3 and KL mutations. Function of mutant gene was analyzed by western blotting and wheat germ agglutinin affinity chromatography. Results: All subjects had hyperphosphatemia and elevated calcium-phosphorus product. Calcinosis positions included the left shoulder, left index finger, and right hip. Bone and joint damage were present in two cases and multiple foci influenced body growth in one case. The histopathological features were firm, rubbery masses comprising multiple nodules of calcified material bordered by the proliferation of mononuclear or multinuclear macrophages, osteoclastic-like giant cells, fibroblasts, and chronic inflammatory cells. The novel mutation c.484A>G (p.N162D) in exon 3 of FGF23 was identified in one subject and his family members. Measurement of circulating FGF23 in the subject confirmed low intact FGF23 and increased C-terminal fragment. In vitro experiments showed that the mutant FGF23 proteins had defective O-glycosylation and impaired protein proteolysis protection. Conclusion: We identified a novel FGF23 missense mutation, and confirmed its damaging role in FGF23 protein O-glycosylation. Our findings expand the current spectrum of FGF23 variations that influence phosphorus metabolism.

Bacterial expression, purification and biophysical characterization of wheat germ agglutinin and its four hevein-like domains.

Wheat germ agglutinin (WGA), a chitin binding lectin, has attracted increasing interest because of its unique characteristics such as conformational stability, binding specificity and transcytosis capacity. To pave the way for the study of the molecular basis of WGA's structural stability and binding capacity, as well as to facilitate its use in biomedical and biotechnological developments, we produced recombinant WGA and its 4 isolated hevein-like domains in a bacterial system. All the proteins were expressed as fusion constructs linked to a thioredoxin domain, which was enzymatically or chemically released. The structural and ligand-binding properties of recombinant WGA were similar to the wild lectin. The 4 isolated domains folded and were ligand-binding competent, indicating that each domain constitutes an independent folding unity. The biophysical characterization of the recombinant domains sheds new light on the intricate folding and binding behavior of this emblematic lectin.

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Homotypic endothelial nanotubes induced by wheat germ agglutinin and thrombin.

Endothelial barrier formation is maintained by intercellular communication through junctional proteins. The mechanisms involved in maintaining endothelial communication subsequent to barrier disruption remain unclear. It is known that low numbers of endothelial cells can be interconnected by homotypic actin-driven tunneling nanotubes (TNTs) which could be important for intercellular transfer of information in vascular physiology. Here we sought insight into the triggers for TNT formation. Wheat germ agglutinin, a C-type lectin and known label for TNTs, unexpectedly caused striking induction of TNTs. A succinylated derivative was by contrast inactive, suggesting mediation by a sialylated protein. Through siRNA-mediated knockdown we identified that this protein was likely to be CD31, an important sialylated membrane protein normally at endothelial cell junctions. We subsequently considered thrombin as a physiological inducer of endothelial TNTs because it reduces junctional contact. Thrombin reduced junctional contact, redistributed CD31 and induced TNTs, but its effect on TNTs was CD31-independent. Thrombin-induced TNTs nevertheless required PKCα, a known mediator of thrombin-dependent junctional remodelling, suggesting a necessity for junctional proteins in TNT formation. Indeed, TNT-inducing effects of wheat germ agglutinin and thrombin were both correlated with cortical actin rearrangement and similarly Ca2+-dependent, suggesting common underlying mechanisms. Once formed, Ca2+ signalling along TNTs was observed.

Characterization of the antifungal functions of a WGA-Fc (IgG2a) fusion protein binding to cell wall chitin oligomers.

The majority of therapeutic strategies for mycosis require the protracted administration of antifungals, which can result in significant toxicities and have unacceptable failure rates. Hence, there is an urgent need for the development of improved therapeutic approaches, and monoclonal antibody-based drugs are potentially a powerful alternative to standard antifungals. To develop a broad antibody-like reagent against mycosis, wheat germ agglutinin (WGA) was linked to the effector Fc region of murine IgG2a. The resultant WGA-Fc displayed high affinity to purified chitin and bound efficiently to fungal cell walls, co-localizing with chitin, in patterns ranging from circular (Histoplasma capsulatum) to punctate (Cryptococcus neoformans) to labeling at the bud sites (Candida albicans and Saccharomyces cerevisiae). WGA-Fc directly inhibited fungal growth in standard cultures. WGA-Fc opsonization increased fungal phagocytosis, as well augmented the antifungal functions by macrophages. Prophylactic administration of WGA-Fc fully protected mice against H. capsulatum, correlating with a reduction in lung, spleen and liver fungal burdens. Administration of WGA-Fc also dramatically diminished pulmonary inflammation. Hence, the opsonic activity of WGA-Fc effectively modulates fungal cell recognition and promotes the elimination of fungal pathogens. Therefore, we propose WGA-Fc as a potential "pan-fungal" therapeutic that should be further developed for use against invasive mycoses.

Loss of Cln3 impacts protein secretion in the social amoeba Dictyostelium.

Neuronal ceroid lipofuscinosis (NCL), also referred to as Batten disease, is the most common form of childhood neurodegeneration. Mutations in CLN3 cause the most prevalent subtype of the disease, which manifests during early childhood and is currently untreatable. The precise function of the CLN3 protein is still not known, which has inhibited the development of targeted therapies. In the social amoeba Dictyostelium discoideum, loss of the CLN3 homolog, Cln3, reduces adhesion during early development, which delays streaming and aggregation. The results of the present study indicate that this phenotype may be at least partly due to aberrant protein secretion in cln3- cells. It is well-established that Cln3 localizes primarily to the contractile vacuole (CV) system in Dictyostelium, and to a lesser extent, compartments of the endocytic pathway. Intriguingly, the CV system has been linked to the secretion of proteins that do not contain a signal peptide for secretion (i.e., unconventional protein secretion). Proteins that do contain a signal peptide are secreted via a conventional mechanism involving the endoplasmic reticulum, transport through the Golgi, and secretion via vesicle release. In this study, Cln3 was observed to co-localize with the Golgi marker wheat germ agglutinin suggesting that Cln3 participates in both secretion mechanisms. Chimeras of wild-type (WT) and cln3- cells displayed delayed streaming and aggregation, and interestingly, cln3- cells starved in conditioned media (CM) harvested from starving WT cells showed near normal timing of streaming and aggregation suggesting aberrant protein secretion in Cln3-deficient cells. Based on these observations, LC-MS/MS was used to reveal the protein content of CM from starved cells (mass spectrometry data are available via ProteomeXchange with identifier PXD004897). A total of 450 proteins were detected in WT and cln3- CM, of which 3 were absent in cln3- CM. Moreover, 12 proteins that were present in cln3- CM were absent in WT CM. Label-free quantification identified 42 proteins that were present in significantly higher amounts in cln3- CM compared to WT, and 3 proteins that were present in significantly reduced amounts. A GO term enrichment analysis showed that a majority of the affected proteins are linked to endocytosis, vesicle-mediated transport, proteolysis, and metabolism. In total, the results of this study indicate that Cln3 functions in both conventional and unconventional protein secretion and that loss of Cln3 results in deregulated secretion during early development. Importantly, this is the first evidence in any system linking CLN3 function to protein secretion.

Evaluation of WGA-Cre-dependent topological transgene expression in the rodent brain.

Novel neuromodulation techniques in the field of brain research, such as optogenetics, prompt to target specific cell populations. However, not every subpopulation can be distinguished based on brain area or activity of specific promoters, but rather on topology and connectivity. A fascinating tool to detect neuronal circuitry is based on the transsynaptic tracer, wheat germ agglutinin (WGA). When expressed in neurons, it is transported throughout the neuron, secreted, and taken up by synaptically connected neurons. Expression of a WGA and Cre recombinase fusion protein using a viral vector technology in Cre-dependent transgenic animals allows to trace neuronal network connections and to induce topological transgene expression. In this study, we applied and evaluated this technology in specific areas throughout the whole rodent brain, including the hippocampus, striatum, substantia nigra, and the motor cortex. Adeno-associated viral vectors (rAAV) encoding the WGA-Cre fusion protein under control of a CMV promoter were stereotactically injected in Rosa26-STOP-EYFP transgenic mice. After 6 weeks, both the number of transneuronally labeled YFP+/mCherry- cells and the transduced YFP+/mCherry+ cells were quantified in the connected regions. We were able to trace several connections using WGA-Cre transneuronal labeling; however, the labeling efficacy was region-dependent. The observed transneuronal labeling mostly occurred in the anterograde direction without the occurrence of multi-synaptic labeling. Furthermore, we were able to visualize a specific subset of newborn neurons derived from the subventricular zone based on their connectivity.

[Construction of a lentivirus vector expressing wheat germ agglutinin and its infection in human adipose-derived stem cells].

OBJECTIVE: To construct a lentivirus vector carrying wheat germ agglutinin (WGA) and evaluate its ability of tracing WGA in the brain of mice with ischemic brain injury. METHODS: WGA gene was inserted into the lentiviral vector Plvx IRES-ZsGreen1 using genetic engineering methods. 293T cells were transfected with the vector and 3 packaging plasmids (RPEV, PRRE, and VSVG) to obtain the recombinant lentivirus for infection of human adipose-derived stem cells (hADSCs). The infected hADSCs were injected into the damaged brain area by in situ injection in a mouse model of middle cerebral artery occlusion (MCAO) and the expression of GFP was traced. RESULTS: Immunofluorescence identification detected WGA protein expression in the infected hADSCs, which survived in the infarct area of mice with MCAO. CONCLUSION: Packaging WGA gene in lentivirus is a reliable approach to allow efficient neuroanatomical tracing of various cells.

Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner.

The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR.

The PDZ-domain protein Whirlin facilitates mechanosensory signaling in mammalian proprioceptors.

Mechanoreception is an essential feature of many sensory modalities. Nevertheless, the mechanisms that govern the conversion of a mechanical force to distinct patterns of action potentials remain poorly understood. Proprioceptive mechanoreceptors reside in skeletal muscle and inform the nervous system of the position of body and limbs in space. We show here that Whirlin/Deafness autosomal recessive 31 (DFNB31), a PDZ-scaffold protein involved in vestibular and auditory hair cell transduction, is also expressed by proprioceptive sensory neurons (pSNs) in dorsal root ganglia in mice. Whirlin localizes to the peripheral sensory endings of pSNs and facilitates pSN afferent firing in response to muscle stretch. The requirement of Whirlin in both proprioceptors and hair cells suggests that accessory mechanosensory signaling molecules define common features of mechanoreceptive processing across sensory systems.

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations.

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics.

Gustatory neural pathways revealed by genetic tracing from taste receptor cells.

Taste receptor cells encounter chemicals in foods and transmit this information to the gustatory neurons, which convey it further to the gustatory relay nuclei in the lower brainstem. Characterizing neurons involved in the transmission of gustatory information in the peripheral and central nervous systems helps us better understand how we perceive and discriminate tastes. However, it is difficult to anatomically identify them. Using cell-type-specific promoters/enhancers and a transneuronal tracer, we generated transgenic mice to visualize neurons in the gustatory neural pathways. We observed the tracer in the neurons of cranial sensory ganglia and the nucleus of the solitary tract in the medulla where gustatory neurons project. The tracer was also distributed in the reticular formation and several motor nuclei in the medulla that have not been recognized as gustatory ascending pathways. These transgenic mice revealed gustatory relay neurons in the known gustatory ascending pathway and an unexpected, thus presumably novel, neural circuit of gustatory system.

Transgenic labeling of higher order neuronal circuits linked to phospholipase C-ß2-expressing taste bud cells in medaka fish.

The sense of taste plays a pivotal role in the food-selecting behaviors of vertebrates. We have shown that the fish ortholog of the phospholipase C gene (plc-ß2) is expressed in a subpopulation of taste bud cells that transmit taste stimuli to the central nervous system to evoke favorable and aversive behaviors. We generated transgenic medaka expressing wheat germ agglutinin (WGA) under the control of a regulatory region of the medaka plc-ß2 gene to analyze the neuronal circuit connected to these sensory cells. Immunohistochemical analysis of the transgenic fish 12 days post fertilization revealed that the WGA protein was transferred to cranial sensory ganglia and several nuclei in the hindbrain. WGA signals were also detected in the secondary gustatory nucleus in the hindbrain of 3-month-old transgenic fish. WGA signals were observed in several diencephalic and telencephalic regions in 9-month-old transgenic fish. The age-dependent increase in the labeled brain regions strongly suggests that labeling occurred at taste bud cells and progressively extended to cranial nerves and neurons in the central nervous system. These data are the first to demonstrate the tracing of higher order gustatory neuronal circuitry that is associated with a specific subpopulation of taste bud cells. These results provide insight into the basic neuronal architecture of gustatory information processing that is common among vertebrates.

"Small axonless neurons": postnatally generated neocortical interneurons with delayed functional maturation.

GABAergic interneurons of the mouse cortex are generated embryonically in the ventral telencephalon. Recent evidence, however, indicated that a subset of cortical cells expressing interneuronal markers originate in the neonatal subventricular zone. This has raised interest in the functional development and incorporation of these postnatally generated cells into cortical circuits. Here we demonstrate that these cells integrate in the cortex, and that they constitute two distinct GABAergic interneuronal classes. Whereas one class reflects the tail end of embryonic interneuron genesis, the other class comprises interneurons that are exclusively generated perinatally and postnatally. The latter constitute a novel subclass of interneurons. They are preferentially located in the deeper layers of the olfactory and orbital cortices, exhibit a unique firing pattern and slow functional maturation. Based on their distinct morphology we termed them "small axonless neurons" and indeed, unlike other cortical neurons, they communicate with their neuronal partners via dendrodendritic synapses. Finally, we provide evidence that the number of small axonless neurons is enhanced by odor enrichment, a further indication that they integrate into neural circuits and participate to olfactory processing.

Genetic tracing of the gustatory neural pathway originating from Pkd1l3-expressing type III taste cells in circumvallate and foliate papillae.

Polycystic kidney disease 1-like 3 (Pkd1l3) is expressed specifically in sour-sensing type III taste cells that have synaptic contacts with afferent nerve fibers in circumvallate (CvP) and foliate papillae (FoP) located in the posterior region of the tongue, although not in fungiform papillae (FuP) or the palate. To visualize the gustatory neural pathways that originate from type III taste cells in CvP and FoP, we established transgenic mouse lines that express the transneuronal tracer wheat germ agglutinin (WGA) under the control of the mouse Pkd1l3 gene promoter/enhancer. The WGA transgene was accurately expressed in Pkd1l3-expressing type III taste cells in CvP and FoP. Punctate WGA protein signals appeared to be detected specifically in type III taste cells but not in other types of taste cells. WGA protein was transferred primarily to a subset of neurons located in close proximity to the glossopharyngeal (GL) nerve bundles in the nodose/petrosal ganglion (NPG). WGA signals were also observed in a small population of neurons in the geniculate ganglion (GG). This result demonstrates the anatomical connection between taste receptor cells (TRCs) in the FoP and the chorda tympani (CT) nerves. WGA protein was further conveyed to neurons in a rostro-central subdivision of the nucleus of the solitary tract (NST). These findings demonstrate that the approximately 10 kb 5'-flanking region of the mouse Pkd1l3 gene functions as a type III taste cell-specific promoter/enhancer. In addition, experiments using the pkd1l3-WGA transgenic mice reveal a sour gustatory pathway that originates from TRCs in the posterior region of the tongue.

A bicistronic lentiviral vector-based method for differential transsynaptic tracing of neural circuits.

We developed a bicistronic HIV1-derived lentiviral vector system co-expressing green fluorescent protein (AcGFP1) and wheat germ agglutinin (WGA) mediated by picornaviral 2A peptide. This system was first applied to the analysis of the rat cerebellar efferent pathways. When the lentiviral vector was injected into a specific lobule, the local Purkinje cell population (first-order neurons) was efficiently infected and co-expressed both AcGFP1 and WGA protein. In the second-order neurons in the cerebellar and vestibular nuclei, WGA but not AcGFP1 protein was differentially detected, demonstrating that the presence of AcGFP1 protein enables discrimination of first-order neurons from second-order neurons. Furthermore, WGA protein was detected in the contralateral ventrolateral thalamic nucleus (third-order nucleus). This system also successfully labeled rat cortical pathways from the primary somatosensory cortex and monkey cerebellar efferent pathways. Thus, this bicistronic lentiviral vector system is a useful tool for differential transsynaptic tracing of neural pathways originating from local brain regions.

Genetic tracing of the neural pathway for bitter taste in t2r5-WGA transgenic mice.

To visualize the neural pathways originating from bitter taste receptor cells (TRCs), we generated transgenic mice expressing the transneuronal tracer wheat germ agglutinin (WGA) under the control of the mouse T2R5 gene promoter/enhancer (t2r5-WGA mice). WGA mRNA was specifically expressed in bitter TRCs. The WGA protein was detected in bitter TRCs and nerve processes in taste buds, but not in sweet, umami, or sour TRCs. The WGA protein was transferred to a subset of sensory neurons in the geniculate and nodose/petrosal ganglia. These results suggest that bitter TRCs, which are devoid of synaptic structures, are innervated by gustatory neurons and that bitter sensory information is directly transmitted to specific gustatory neurons. The t2r5-WGA mice provide a useful tool for identifying gustatory relay neurons in the peripheral sensory ganglia responsible for aversive sensations.

Gene transfer in the nervous system and implications for transsynaptic neuronal tracing.

IMPORTANCE OF THE FIELD: Neuronal circuitries are determined by specific synaptic connections and they provide the cellular basis of cognitive processes and behavioral functions. To investigate neuronal circuitries, tracers are typically used to identify the original neurons and their projection targets. AREAS COVERED IN THIS REVIEW: Traditional tracing methods using chemical tracers have major limitations such as non-specificity. In this review, we highlight novel genetic tracing approaches that enable visualization of specific neuronal pathways by introducing cDNA encoding a transsynaptic tracer. In contrast to conventional tracing methods, these genetic approaches use cell-type-specific promoters to express transsynaptic tracers such as wheat germ agglutinin and C-terminal fragment of tetanus toxin, which allows labeling of either the input or output populations and connections of specific neuronal type. WHAT THE READER WILL GAIN: Specific neuronal circuit information by these genetic approaches will allow more precise, comprehensive and novel information about individual neural circuits and their function in normal and diseased brains. TAKE HOME MESSAGE: Using tracer gene transfer, neuronal circuit plasticity after traumatic injury or neurodegenerative diseases can be visualized. Also, this can provide a good marker for evaluation of therapeutic effects of neuroprotective or neurotrophic agents.

[Visualization of selective transsynaptic neural pathways using a genetic method].

Functional logic employed by the nervous system for coding and processing information is determined by the wiring patterns among specific types of neurons. Therefore, detailed knowledge about neuronal circuits is essential for understanding the wide range of brain functions. In our opinion, an effective and long-awaited method for analyzing the neuronal connectivity patterns would be to selectively deliver tracers to specific types of neurons and simutaneously performing transsynaptic labeling of the target neurons. A unique property of plant lectins, such as wheat germ agglutinin (WGA) have the unique property of traveling across synapses and this property has been applied in classical neuroanatomical studies in order to label various neural pathways. In 1999, we developed a novel strategy that employs WGA cDNA as a transgene in order to visualize selective and functional neural pathways. Over the last decade, this method has gained popularity; further with substantial technical improvement and refinement, this method has been successfully employed for the analysis of various neural systems such as the olfactory, visual, gustatory, somatosensory, motor, and serotonergic systems. However, a few studies that employed this method failed because of the misinterpretation of the results. In this review, I summarize the principle, application, recent progress, and caveats of the WGA transgene technology.

Triggering genetically-expressed transneuronal tracers by peripheral axotomy reveals convergent and segregated sensory neuron-spinal cord connectivity.

To better understand the mechanisms through which non-painful and painful stimuli evoke behavior, new resources to dissect the complex circuits engaged by subsets of primary afferent neurons are required. This is especially true to understand the consequences of injury, when reorganization of central nervous system circuits likely contributes to the persistence of pain. Here we describe a transgenic mouse line (ZWX) in which there is Cre-recombinase-dependent expression of a transneuronal tracer, wheat germ agglutinin (WGA), in primary somatic or visceral afferent neurons, but only after transection of their peripheral axons. The latter requirement allows for both regional and temporal control of tracer expression, even in the adult. Using a variety of Cre lines to target WGA transport to subpopulations of sensory neurons, here we demonstrate the extent to which myelinated and unmyelinated "pain" fibers (nociceptors) engage different spinal cord circuits. We found significant convergence (i.e., manifest as WGA-transneuronal labeling) of unmyelinated afferents, including the TRPV1-expressing subset, and myelinated afferents to NK1-receptor-expressing neurons of lamina I. By contrast, PKCgamma interneurons of inner lamina II only receive a myelinated afferent input. This differential distribution of WGA labeling in the spinal cord indicates that myelinated and unmyelinated sensory neurons target different and spatially segregated populations of postsynaptic neurons. On the other hand, we show that neurons of deeper laminae (III-V) receive direct (i.e., monosynaptic) inputs from myelinated afferents and polysynaptic input from unmyelinated afferents. Taken together, our results indicate that peripheral sensory information is transmitted to the central nervous system both through segregated and convergent pathways.