Association of connexin36 with adherens junctions at mixed synapses and distinguishing electrophysiological features of those at mossy fiber terminals in rat ventral hippocampus.

BACKGROUND: Granule cells in the hippocampus project axons to hippocampal CA3 pyramidal cells where they form large mossy fiber terminals. We have reported that these terminals contain the gap junction protein connexin36 (Cx36) specifically in the stratum lucidum of rat ventral hippocampus, thus creating morphologically mixed synapses that have the potential for dual chemical/electrical transmission. METHODOLOGY: Here, we used various approaches to characterize molecular and electrophysiological relationships between the Cx36-containing gap junctions at mossy fiber terminals and their postsynaptic elements and to examine molecular relationships at mixed synapses in the brainstem. RESULTS: In rat and human ventral hippocampus, many of these terminals, identified by their selective expression of vesicular zinc transporter-3 (ZnT3), displayed multiple, immunofluorescent Cx36-puncta representing gap junctions, which were absent at mossy fiber terminals in the dorsal hippocampus. In rat, these were found in close proximity to the protein constituents of adherens junctions (i.e., N-cadherin and nectin-1) that are structural hallmarks of mossy fiber terminals, linking these terminals to the dendritic shafts of CA3 pyramidal cells, thus indicating the loci of gap junctions at these contacts. Cx36-puncta were also associated with adherens junctions at mixed synapses in the brainstem, supporting emerging views of the structural organization of the adherens junction-neuronal gap junction complex. Electrophysiologically induced long-term potentiation (LTP) of field responses evoked by mossy fiber stimulation was greater in the ventral than dorsal hippocampus. CONCLUSIONS: The electrical component of transmission at mossy fiber terminals may contribute to enhanced LTP responses in the ventral hippocampus.

Patterns of connexin36 and eGFP reporter expression among motoneurons in spinal sexually dimorphic motor nuclei in mouse.

BACKGROUND: Sexually dimorphic spinal motoneurons (MNs) in the dorsomedial nucleus (DMN) and dorsolateral nucleus (DLN) as well as those in the cremaster nucleus are involved in reproductive behaviours, and the cremaster nucleus additionally contributes to testicular thermoregulation. It has been reported that MNs in DMN and DLN are extensively linked by gap junctions forming electrical synapses composed of connexin36 (Cx36) and there is evidence that subpopulation of MNs in the cremaster nucleus are also electrically coupled by these synapses. METHODOLOGY: We used immunofluorescence methods to detect enhanced green fluorescent protein (eGFP) reporter for Cx36 expression in these motor nuclei. RESULTS: We document in male mice that about half the MNs in each of DMN and DLN express eGFP, while the remaining half do not. Further, we found that the eGFP+ vs. eGFP- subsets of MNs in each of these motor nuclei innervate different target muscles; eGFP+ MNs in DMN and DLN project to sexually dimorphic bulbocavernosus and ischiocavernosus muscles, while the eGFP- subsets project to sexually non-dimorphic anal and external urethral sphincter muscles. Similarly, eGFP+ vs. eGFP- cremaster MNs were found to project to anatomically distinct portions of the cremaster muscle. By immunofluorescence, nearly all motoneurons in both DMN and DLN displayed punctate labelling for Cx36, including at eGFP+/eGFP+, eGFP+/eGFP- and eGFP-/eGFP- cell appositions. CONCLUSIONS: Most if not all motoneurons in DMN and DLN are electrically coupled, including sexually dimorphic and non-dimorphic motoneurons with each other, despite absence of eGFP reporter in the non-dimorphic populations in these nuclei that have selective projections to sexually non-dimorphic target muscles.

Connexin-36-positive gap junctions in ventral tegmental area GABA neurons sustain opiate dependence.

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 fl(CFP)/fl(CFP)) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.

Ambiguity in Solving Imaging Inverse Problems with Deep-Learning-Based Operators.

In recent years, large convolutional neural networks have been widely used as tools for image deblurring, because of their ability in restoring images very precisely. It is well known that image deblurring is mathematically modeled as an ill-posed inverse problem and its solution is difficult to approximate when noise affects the data. Really, one limitation of neural networks for deblurring is their sensitivity to noise and other perturbations, which can lead to instability and produce poor reconstructions. In addition, networks do not necessarily take into account the numerical formulation of the underlying imaging problem when trained end-to-end. In this paper, we propose some strategies to improve stability without losing too much accuracy to deblur images with deep-learning-based methods. First, we suggest a very small neural architecture, which reduces the execution time for training, satisfying a green AI need, and does not extremely amplify noise in the computed image. Second, we introduce a unified framework where a pre-processing step balances the lack of stability of the following neural-network-based step. Two different pre-processors are presented. The former implements a strong parameter-free denoiser, and the latter is a variational-model-based regularized formulation of the latent imaging problem. This framework is also formally characterized by mathematical analysis. Numerical experiments are performed to verify the accuracy and stability of the proposed approaches for image deblurring when unknown or not-quantified noise is present; the results confirm that they improve the network stability with respect to noise. In particular, the model-based framework represents the most reliable trade-off between visual precision and robustness.

Effect of high fat diet on maternal behavior, brain-derived neurotrophic factor and neural stem cell proliferation in mice expressing human placental lactogen during pregnancy.

Maternal obesity is a serious health concern because it increases risks of neurological disorders, including anxiety and peripartum depression. In mice, a high fat diet (HFD) in pregnancy can negatively affect placental structure and function as well as maternal behavior reflected by impaired nest building and pup-retrieval. In humans, maternal obesity in pregnancy is associated with reduced placental lactogen (PL) gene expression, which has been linked to a higher risk of depression. PL acting predominantly through the prolactin receptor maintains energy homeostasis and is a marker of placenta villous trophoblast differentiation during pregnancy. Impaired neurogenesis and low serum levels of brain-derived neurotrophic factor (BDNF) have also been implicated in depression. Augmented neurogenesis in brain during pregnancy was reported in the subventricular zone (SVZ) of mice at gestation day 7 and linked to increased prolactin receptor signaling. Here, we used transgenic CD-1 mice that express human (h) PL during pregnancy to investigate whether the negative effects of diet on maternal behavior are mitigated in these (CD-1[hGH/PL]) mice. Specifically, we examined the effect of a HFD on nest building prepartum and pup retrieval postpartum, as well as on brain BDNF levels and neurogenesis. In contrast to wild-type CD-1[WT]mice, CD-1[hGH/PL] mice displayed significantly less anxiety-like behavior, and showed no impairment in prepartum nest building or postpartum pup-retrieval when fed a HFD. Furthermore, the HFD decreased prepartum and increased postpartum BDNF levels in CD-1[WT] but not CD-1[hGH/PL] mice. Finally, neurogenesis in the SVZ as well as phosphorylated mitogen-activated protein kinase, indicative of lactogenic signaling, appeared unaffected by pregnancy and diet at gestation day 7 in CD-1[hGH/PL] mice. These observations indicate that CD-1[hGH/PL] mice are resistant to the negative effects of HFD reported for CD-1[WT] mice, including effects on maternal behaviors and BDNF levels, and potentially, neurogenesis. This difference probably reflects a direct or indirect effect of the products of the hGH/PL transgene.

Numerical methods for CT reconstruction with unknown geometry parameters.

Computed tomography (CT) techniques are well known for their ability to produce high-quality images needed for medical diagnostic purposes. Unfortunately, standard CT machines are extremely large, heavy, require careful and regular calibration, and are expensive, which can limit their availability in point-of-care situations. An alternative approach is to use portable machines, but parameters related to the geometry of these devices (e.g., distance between source and detector, orientation of source to detector) cannot always be precisely calibrated, and these parameters may change slightly when the machine is adjusted during the image acquisition process. In this work, we describe the non-linear inverse problem that models this situation, and discuss algorithms that can jointly estimate the geometry parameters and compute a reconstructed image. In particular, we propose a hybrid machine learning and block coordinate descent (ML-BCD) approach that uses an ML model to calibrate geometry parameters, and uses BCD to refine the predicted parameters and reconstruct the imaged object simultaneously. We show using numerical experiments that our new method can efficiently improve the accuracy of both the image and geometry parameters.

Minimizing L 1 over L 2 norms on the gradient.

In this paper, we study the L 1 /L 2 minimization on the gradient for imaging applications. Several recent works have demonstrated that L 1 /L 2 is better than the L 1 norm when approximating the L 0 norm to promote sparsity. Consequently, we postulate that applying L 1 /L 2 on the gradient is better than the classic total variation (the L 1 norm on the gradient) to enforce the sparsity of the image gradient. Numerically, we design a specific splitting scheme, under which we can prove subsequential and global convergence for the alternating direction method of multipliers (ADMM) under certain conditions. Experimentally, we demonstrate visible improvements of L 1 /L 2 over L 1 and other nonconvex regularizations for image recovery from low-frequency measurements and two medical applications of MRI and CT reconstruction. Finally, we reveal some empirical evidence on the superiority of L 1 /L 2 over L 1 when recovering piecewise constant signals from low-frequency measurements to shed light on future works.

Iodine quantification in limited angle tomography.

PURPOSE: To develop and test the feasibility of a two-pass iterative reconstruction algorithm with material decomposition designed to obtain quantitative iodine measurements in digital breast tomosynthesis. METHODS: Contrast-enhanced mammography has shown promise as a cost-effective alternative to magnetic resonance imaging for imaging breast cancer, especially in dense breasts. However, one limitation is the poor quantification of iodine contrast since the true three-dimensional lesion shape cannot be inferred from the two-dimensional (2D) projection. Use of limited angle tomography can potentially overcome this limitation by segmenting the iodine map generated by the first-pass reconstruction using a convolutional neural network, and using this segmentation to restrict the iodine distribution in the second pass of the reconstruction. To evaluate the performance of the algorithms, a set of 2D digital breast phantoms containing targets with varying iodine concentration was used. In each breast phantom, a single simulated lesion with a random size (4 to 8 mm) was placed in a random location within each phantom, with the iodine distribution defined as either homogeneous or rim-enhanced and blood iodine concentration set between 1.4 and 5.6 mg/mL. Limited angle projection data of these phantoms were simulated for wide and narrow angle geometries, and the proposed reconstruction and segmentation algorithms were applied. RESULTS: The median Dice similarity coefficient of the segmented masks was 0.975 for the wide angle data and 0.926 for the narrow angle data. Using these segmentations during the second reconstruction pass resulted in an improvement in the concentration estimates (mean estimated-to-true concentration ratio, before and after second pass: 48% to 73% for wide angle; 30% to 73% for narrow angle), and a reduction in the coefficient of variation of the estimates (55% to 27% for wide angle; 54% to 35% for narrow angle). CONCLUSION: We demonstrate that the proposed two-pass reconstruction can potentially improve accuracy and precision of iodine quantification in contrast-enhanced tomosynthesis.

Gap junction connexin43 is a key element in mediating phagocytosis activity in human trabecular meshwork cells.

Human trabecular meshwork (TM) cells play pivotal roles in maintaining homeostasis of intraocular pressure via regulation of aqueous humor outflow. These cells are capable of phagocytosis, which is considered to be essential for their regulatory function. In addition, there is a strong expression of the gap junction protein connexin43 (Cx43) in the TM. Here, we investigated functional relationships between phagocytosis activity of TM cells and their expression of Cx43. Phagocytosis was measured by showing the ability of TM cells to engulf inert fluorescent particles consisting of pHrodo. We found that internalized pHrodo was partially co-localized with Cx43 and that the phagocytic activity was dramatically reduced after knockdown of Cx43 using lentiviral Cx43 shRNA. These results suggest that Cx43 is involved in the regulation of phagocytosis by TM cells.

ZO-1 associates with α3 integrin and connexin43 in trabecular meshwork and Schlemm's canal cells.

Cellular structures that perform essential homeostatic functions include tight junctions, gap junctions, desmosomes and adherens junctions. The aqueous humor, produced by the ciliary body, passes into the anterior chamber of the eye and is filtered by the trabecular meshwork (TM), a tiny tissue found in the angle of the eye. This tissue, along with Schlemm's canal (SC) inner wall cells, is thought to control intraocular pressure (IOP) homeostasis for normal, optimal vision. The actin cytoskeleton of the tissue plays a regulatory role in maintaining IOP. One of the key risk factors for primary open angle glaucoma is persistent elevation of IOP, which compromises the optic nerve. The ZO-1 (Zonula Occludens-1), extracellular matrix protein integrins, and gap junction protein connexin43 (Cx43) are widely expressed in many different cell populations. Here, we investigated the localization and interactions of ZO-1, α3 integrin, ß1 integrin, and Cx43 in cultured porcine TM and SC cells using RT-PCR, western immunoblotting and immunofluorescence labeling with confocal microscopy, along with co-immunoprecipitation. ZO-1 partially co-localized with α3 integrin, but not with ß1 integrin, and co-immunoprecipitated with Cx43, as well as with α3 integrin. The association of ZO-1 with α3 integrin and Cx43 suggests that these proteins may form a multiple protein complex in porcine TM and SC cells. Since integrins interact with the actin cytoskeleton via scaffolding proteins, these results implicate junctional and scaffolding protein ZO-1 as a potential control point in regulation of IOP to normal levels for glaucoma therapy.

Connexin36 localization along axon initial segments in the mammalian CNS.

Electrical synapses formed by gap junctions occur at a variety of neuronal subcellular sites in the mammalian central nervous system (CNS), including at somatic, dendritic and axon terminal compartments. Numerous electrophysiological studies using mice and rats, as well as computer modelling approaches, have predicted the additional occurrence of electrical synapses between axons near their emergence from neuronal somata. Here, we used immunofluorescence methods to search for localization of the neuronal gap junction-forming protein connexin36 (Cx36) along axon initial segments (AISs) labelled for the AIS marker ankyrinG. Immunofluorescent Cx36-puncta were found to be associated with AISs in several CNS regions of mice, including the spinal cord, inferior olive and cerebral cortex. Localization of Cx36-puncta at AISs was confirmed by confocal single scan and 3D imaging, immunofluorescence intensity profiling and high resolution structured illumination microscopy (SIM). AISs measuring up to 30 µm in length displayed typically a single Cx36-punctum and the incidence of these long AISs displaying Cx36-puncta ranged from 3% to 7% in the inferior olive and in various layers of the cerebral cortex. In the inferior olive, the gap junction associated protein zonula occludens-1 (ZO-1) was found to be co-localized with Cx36-puncta on AISs, indicating that these puncta have some of the molecular constituents of gap junctions. Our results add to the neuronal subcellular locations at which Cx36 is deployed, and raise possibilities for its involvement in novel functions in the AIS compartment.

Could electrical coupling contribute to the formation of cell assemblies?

Cell assemblies and central pattern generators (CPGs) are related types of neuronal networks: both consist of interacting groups of neurons whose collective activities lead to defined functional outputs. In the case of a cell assembly, the functional output may be interpreted as a representation of something in the world, external or internal; for a CPG, the output 'drives' an observable (i.e. motor) behavior. Electrical coupling, via gap junctions, is critical for the development of CPGs, as well as for their actual operation in the adult animal. Electrical coupling is also known to be important in the development of hippocampal and neocortical principal cell networks. We here argue that electrical coupling - in addition to chemical synapses - may therefore contribute to the formation of at least some cell assemblies in adult animals.

Semi-blind sparse affine spectral unmixing of autofluorescence-contaminated micrographs.

MOTIVATION: Spectral unmixing methods attempt to determine the concentrations of different fluorophores present at each pixel location in an image by analyzing a set of measured emission spectra. Unmixing algorithms have shown great promise for applications where samples contain many fluorescent labels; however, existing methods perform poorly when confronted with autofluorescence-contaminated images. RESULTS: We propose an unmixing algorithm designed to separate fluorophores with overlapping emission spectra from contamination by autofluorescence and background fluorescence. First, we formally define a generalization of the linear mixing model, called the affine mixture model (AMM), that specifically accounts for background fluorescence. Second, we use the AMM to derive an affine nonnegative matrix factorization method for estimating fluorophore endmember spectra from reference images. Lastly, we propose a semi-blind sparse affine spectral unmixing (SSASU) algorithm that uses knowledge of the estimated endmembers to learn the autofluorescence and background fluorescence spectra on a per-image basis. When unmixing real-world spectral images contaminated by autofluorescence, SSASU greatly improved proportion indeterminacy as compared to existing methods for a given relative reconstruction error. AVAILABILITY AND IMPLEMENTATION: The source code used for this paper was written in Julia and is available with the test data at https://github.com/brossetti/ssasu.

On the occurrence and enigmatic functions of mixed (chemical plus electrical) synapses in the mammalian CNS.

Electrical synapses with diverse configurations and functions occur at a variety of interneuronal appositions, thereby significantly expanding the physiological complexity of neuronal circuitry over that provided solely by chemical synapses. Gap junctions between apposed dendritic and somatic plasma membranes form "purely electrical" synapses that allow for electrical communication between coupled neurons. In addition, gap junctions at axon terminals synapsing on dendrites and somata allow for "mixed" (dual chemical+electrical) synaptic transmission. "Dual transmission" was first documented in the autonomic nervous system of birds, followed by its detection in the central nervous systems of fish, amphibia, and reptiles. Subsequently, mixed synapses have been detected in several locations in the mammalian CNS, where their properties and functional roles remain undetermined. Here, we review available evidence for the presence, complex structural composition, and emerging functional properties of mixed synapses in the mammalian CNS.

Astrocytes drive cortical vasodilatory signaling by activating endothelial NMDA receptors.

Astrocytes express neurotransmitter receptors that serve as sensors of synaptic activity and initiate signals leading to activity-dependent local vasodilation and increases in blood flow. We previously showed that arteriolar vasodilation produced by activation of cortical astrocytes is dependent on endothelial nitric oxide synthase (eNOS) and endogenous agonists of N-methyl-D-aspartate (NMDA) receptors. Here, we tested the hypothesis that these effects are mediated by NMDA receptors expressed by brain endothelial cells. Primary endothelial cultures expressed NMDA receptor subunits and produced nitric oxide in response to co-agonists, glutamate and D-serine. In cerebral cortex in situ, immunoelectron microscopy revealed that endothelial cells express the GluN1 NMDA receptor subunit at basolateral membrane surfaces in an orientation suitable for receiving intercellular messengers from brain cells. In cortical slices, activation of astrocytes by two-photon flash photolysis of a caged Ca2+ compound or application of a metabotropic glutamate receptor agonist caused endothelial NO generation and local vasodilation. These effects were mitigated by NMDA receptor antagonists and conditional gene silencing of endothelial GluN1, indicating at least partial dependence on endothelial NMDA receptors. Our observations identify a novel astrocyte-endothelial vasodilatory signaling axis that could contribute to endothelium-dependent vasodilation in brain functional hyperemia.

E3 ubiquitin ligases LNX1 and LNX2 localize at neuronal gap junctions formed by connexin36 in rodent brain and molecularly interact with connexin36.

Electrical synapses in the mammalian central nervous system (CNS) are increasingly recognized as highly complex structures for mediation of neuronal communication, both with respect to their capacity for dynamic short- and long-term modification in efficacy of synaptic transmission and their multimolecular regulatory and structural components. These two characteristics are inextricably linked, such that understanding of mechanisms that contribute to electrical synaptic plasticity requires knowledge of the molecular composition of electrical synapses and the functions of proteins associated with these synapses. Here, we provide evidence that the key component of gap junctions that form the majority of electrical synapses in the mammalian CNS, namely connexin36 (Cx36), directly interacts with the related E3 ubiquitin ligase proteins Ligand of NUMB protein X1 (LNX1) and Ligand of NUMB protein X2 (LNX2). This is based on immunofluorescence colocalization of LNX1 and LNX2 with Cx36-containing gap junctions in adult mouse brain versus lack of such coassociation in LNX null mice, coimmunoprecipitation of LNX proteins with Cx36, and pull-down of Cx36 with the second PDZ domain of LNX1 and LNX2. Furthermore, cotransfection of cultured cells with Cx36 and E3 ubiquitin ligase-competent LNX1 and LNX2 isoforms led to loss of Cx36-containing gap junctions between cells, whereas these junctions persisted following transfection with isoforms of these proteins that lack ligase activity. Our results suggest that a LNX protein mediates ubiquitination of Cx36 at neuronal gap junctions, with consequent Cx36 internalization, and may thereby contribute to intracellular mechanisms that govern the recently identified modifiability of synaptic transmission at electrical synapses.

Electrical synapses in mammalian CNS: Past eras, present focus and future directions.

Gap junctions provide the basis for electrical synapses between neurons. Early studies in well-defined circuits in lower vertebrates laid the foundation for understanding various properties conferred by electrical synaptic transmission. Knowledge surrounding electrical synapses in mammalian systems unfolded first with evidence indicating the presence of gap junctions between neurons in various brain regions, but with little appreciation of their functional roles. Beginning at about the turn of this century, new approaches were applied to scrutinize electrical synapses, revealing the prevalence of neuronal gap junctions, the connexin protein composition of many of those junctions, and the myriad diverse neural systems in which they occur in the mammalian CNS. Subsequent progress indicated that electrical synapses constitute key elements in synaptic circuitry, govern the collective activity of ensembles of electrically coupled neurons, and in part orchestrate the synchronized neuronal network activity and rhythmic oscillations that underlie fundamental integrative processes. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Electrical Synapses: New Rules for Assembling an Old Structure Asymmetrically.

Electrical synapses are emerging as complex structures, consisting of gap junction-forming connexin proteins and also multiple scaffolding and regulatory protein components, which were assumed to be symmetrically organized across these synapses; however, new findings reveal their synaptic asymmetry.

High-resolution speckle imaging through strong atmospheric turbulence.

We demonstrate that high-resolution imaging through strong atmospheric turbulence can be achieved by acquiring data with a system that captures short exposure ("speckle") images using a range of aperture sizes and then using a bootstrap multi-frame blind deconvolution restoration process that starts with the smallest aperture data. Our results suggest a potential paradigm shift in how we image through atmospheric turbulence. No longer should image acquisition and post processing be treated as two independent processes: they should be considered as intimately related.

KV1 channels identified in rodent myelinated axons, linked to Cx29 in innermost myelin: support for electrically active myelin in mammalian saltatory conduction.

Saltatory conduction in mammalian myelinated axons was thought to be well understood before recent discoveries revealed unexpected subcellular distributions and molecular identities of the K(+)-conductance pathways that provide for rapid axonal repolarization. In this study, we visualize, identify, localize, quantify, and ultrastructurally characterize axonal KV1.1/KV1.2 channels in sciatic nerves of rodents. With the use of light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling electron microscopy, KV1.1/KV1.2 channels are localized to three anatomically and compositionally distinct domains in the internodal axolemmas of large myelinated axons, where they form densely packed "rosettes" of 9-nm intramembrane particles. These axolemmal KV1.1/KV1.2 rosettes are precisely aligned with and ultrastructurally coupled to connexin29 (Cx29) channels, also in matching rosettes, in the surrounding juxtaparanodal myelin collars and along the inner mesaxon. As >98% of transmembrane proteins large enough to represent ion channels in these specialized domains, ∼500,000 KV1.1/KV1.2 channels define the paired juxtaparanodal regions as exclusive membrane domains for the voltage-gated K(+)conductance that underlies rapid axonal repolarization in mammals. The 1:1 molecular linkage of KV1 channels to Cx29 channels in the apposed juxtaparanodal collars, plus their linkage to an additional 250,000-400,000 Cx29 channels along each inner mesaxon in every large-diameter myelinated axon examined, supports previously proposed K(+)conductance directly from juxtaparanodal axoplasm into juxtaparanodal myeloplasm in mammalian axons. With neither Cx29 protein nor myelin rosettes detectable in frog myelinated axons, these data showing axon-to-myelin linkage by abundant KV1/Cx29 channels in rodent axons support renewed consideration of an electrically active role for myelin in increasing both saltatory conduction velocity and maximum propagation frequency in mammalian myelinated axons.