Inhibitory Axons Research Articles

Characterization of β-Hydroxybutyrate as a Cell Autonomous Fuel for Active Excitatory and Inhibitory Neurons: β-Hydroxybutyrate as a Fuel for Active Neurons.

The ketogenic diet is an effective treatment for drug-resistant epilepsy, but the therapeutic mechanisms are poorly understood. Although ketones are able to fuel the brain, it is not known whether ketones are directly metabolized by neurons on a time scale sufficiently rapid to fuel the bioenergetic demands of sustained synaptic transmission. Here, we show that nerve terminals can use the ketone β-hydroxybutyrate in a cell- autonomous fashion to support neurotransmission in both excitatory and inhibitory nerve terminals and that this flexibility relies on Ca2+ dependent upregulation of mitochondrial metabolism. Using a genetically encoded ATP sensor, we show that inhibitory axons fueled by ketones sustain much higher ATP levels under steady state conditions than excitatory axons, but that the kinetics of ATP production following activity are slower when using ketones as fuel compared to lactate/pyruvate for both excitatory and inhibitory neurons.

Neuroanatomical Substrates of Circuit-Specific Cholinergic Modulation across the Primate Anterior Cingulate Cortex.

Acetylcholine is a robust neuromodulator of the limbic system and a critical regulator of arousal and emotions. The anterior cingulate cortex (ACC) and the amygdala (AMY) are key limbic structures that are both densely innervated by cholinergic afferents and interact with each other for emotional regulation. The ACC is composed of functionally distinct dorsal (A24), rostral (A32), and ventral (A25) areas that differ in their connections with the AMY. The structural substrates of cholinergic modulation of distinct ACC microcircuits and outputs to AMY are thought to depend on the laminar and subcellular localization of cholinergic receptors. The present study examines the distribution of muscarinic acetylcholine receptors, m1 and m2, on distinct excitatory and inhibitory neurons and on AMY-targeting projection neurons within ACC areas, via immunohistochemistry and injections of neural tracers into the basolateral AMY in adult rhesus monkeys of both sexes. We found that laminar densities of m1+ and m2+ expressing excitatory and inhibitory neurons depended on area and cell type. Among the ACC areas, ventral subgenual ACC A25 exhibited greater m2+ localization on presynaptic inhibitory axon terminals and greater density of m1+ and m2+ expressing AMY-targeting (tracer+) pyramidal neurons. These patterns suggest robust cholinergic disinhibition and potentiation of amygdalar outputs from the limbic ventral ACC, which may be linked to the hyperexcitability of this subgenual ACC area in depression. These findings reveal the anatomical substrate of diverse cholinergic modulation of specific ACC microcircuits and amygdalar outputs that mediate cognitive-emotional integration and dysfunctions underlying stress and affective disorders.

Retrograde endocannabinoid signaling at inhibitory synapses in vivo.

Endocannabinoid (eCB)-mediated suppression of inhibitory synapses has been hypothesized, but this has not yet been demonstrated to occur in vivo because of the difficulty in tracking eCB dynamics and synaptic plasticity during behavior. In mice navigating a linear track, we observed location-specific eCB signaling in hippocampal CA1 place cells, and this was detected both in the postsynaptic membrane and the presynaptic inhibitory axons. All-optical in vivo investigation of synaptic responses revealed that postsynaptic depolarization was followed by a suppression of inhibitory synaptic potentials. Furthermore, interneuron-specific cannabinoid receptor deletion altered place cell tuning. Therefore, rapid, postsynaptic, activity-dependent eCB signaling modulates inhibitory synapses on a timescale of seconds during behavior.

Excitatory synchronization of rat hippocampal interneurons during network activation in vitro.

Hippocampal interneurons (INs) are known to synchronize their electrical activity via mechanisms, which are poorly defined due to immense complexity of neural tissue but seem to depend on local cell interactions and intensity of network activity. Here, synchronization of INs was studied using paired patch-clamp recordings in a simplified culture model with intact glutamate transmission. The level of network activity was moderately elevated by field electric stimulation, which is probably an analogue of afferent processing in situ. Even in baseline conditions, ∼45% of spontaneous inhibitory postsynaptic currents (sIPSCs) resulting from firing of individual presynaptic INs coincided between cells within ±1 ms due to simple divergence of inhibitory axons. Brief network activation induced an appearance of 'hypersynchronous' (∼80%) population sIPSCs occurring in response to coherent discharges of several INs with jitter ±4 ms. Notably, population sIPSCs were preceded by transient inward currents (TICs). Those were excitatory events capable to synchronize firing of INs, in this respect being reminiscent of so-called fast prepotentials observed in studies on pyramidal neurons. TICs also had network properties consisting of heterogeneous components: glutamate currents, local axonal and dendritic spikelets, and coupling electrotonic currents likely via gap junctions; putative excitatory action of synaptic gamma-aminobutyric acid (GABA) was not involved. The appearance of population excitatory-inhibitory sequences could be initiated and reproduced by firing of a single excitatory cell reciprocally connected with one IN. Our data demonstrate that synchronization of INs is initiated and dominated by glutamatergic mechanisms, which recruit, in a whole-sale manner, into supporting action other excitatory means existing in a given neural system.

Difference in axon diameter and myelin thickness between excitatory and inhibitory callosally projecting axons in mice.

Synchronization of network oscillation in spatially distant cortical areas is essential for normal brain activity. Precision in synchronization between hemispheres depends on the axonal conduction velocity, which is determined by physical parameters of the axons involved, including diameter, and extent of myelination. To compare these parameters in long-projecting excitatory and inhibitory axons in the corpus callosum, we used genetically modified mice and virus tracing to separately label CaMKIIα expressing excitatory and GABAergic inhibitory axons. Using electron microscopy analysis, we revealed that (i) the axon diameters of excitatory fibers (myelinated axons) are significantly larger than those of nonmyelinated excitatory axons; (ii) the diameters of bare axons of excitatory myelinated fibers are significantly larger than those of their inhibitory counterparts; and (iii) myelinated excitatory fibers are significantly larger than myelinated inhibitory fibers. Also, the thickness of myelin ensheathing inhibitory axons is significantly greater than for excitatory axons, with the ultrastructure of the myelin around excitatory and inhibitory fibers also differing. We generated a computational model to investigate the functional consequences of these parameter divergences. Our simulations indicate that impulses through inhibitory and excitatory myelinated fibers reach the targetalmost simultaneously, whereas action potentials conducted by nonmyelinated axons reach target cells with considerable delay.

Interlayer Connectivity Affects the Coherence Resonance and Population Activity Patterns in Two-Layered Networks of Excitatory and Inhibitory Neurons.

The firing patterns of neuronal populations often exhibit emergent collective oscillations, which can display substantial regularity even though the dynamics of individual elements is very stochastic. One of the many phenomena that is often studied in this context is coherence resonance, where additional noise leads to improved regularity of spiking activity in neurons. In this work, we investigate how the coherence resonance phenomenon manifests itself in populations of excitatory and inhibitory neurons. In our simulations, we use the coupled FitzHugh-Nagumo oscillators in the excitable regime and in the presence of neuronal noise. Formally, our model is based on the concept of a two-layered network, where one layer contains inhibitory neurons, the other excitatory neurons, and the interlayer connections represent heterotypic interactions. The neuronal activity is simulated in realistic coupling schemes in which neurons within each layer are connected with undirected connections, whereas neurons of different types are connected with directed interlayer connections. In this setting, we investigate how different neurophysiological determinants affect the coherence resonance. Specifically, we focus on the proportion of inhibitory neurons, the proportion of excitatory interlayer axons, and the architecture of interlayer connections between inhibitory and excitatory neurons. Our results reveal that the regularity of simulated neural activity can be increased by a stronger damping of the excitatory layer. This can be accomplished with a higher proportion of inhibitory neurons, a higher fraction of inhibitory interlayer axons, a stronger coupling between inhibitory axons, or by a heterogeneous configuration of interlayer connections. Our approach of modeling multilayered neuronal networks in combination with stochastic dynamics offers a novel perspective on how the neural architecture can affect neural information processing and provide possible applications in designing networks of artificial neural circuits to optimize their function via noise-induced phenomena.

Stepwise disassembly of GABAergic synapses during pathogenic excitotoxicity.

SUMMARYGABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABAARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABAAR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.

Kv4.2-Positive Domains on Dendrites in the Mouse Medial Geniculate Body Receive Ascending Excitatory and Inhibitory Inputs Preferentially From the Inferior Colliculus.

The medial geniculate body (MGB) is the thalamic center of the auditory lemniscal pathway. The ventral division of MGB (MGV) receives excitatory and inhibitory inputs from the inferior colliculus (IC). MGV is involved in auditory attention by processing descending excitatory and inhibitory inputs from the auditory cortex (AC) and reticular thalamic nucleus (RTN), respectively. However, detailed mechanisms of the integration of different inputs in a single MGV neuron remain unclear. Kv4.2 is one of the isoforms of the Shal-related subfamily of potassium voltage-gated channels that are expressed in MGB. Since potassium channel is important for shaping synaptic current and spike waveforms, subcellular distribution of Kv4.2 is likely important for integration of various inputs. Here, we aimed to examine the detailed distribution of Kv4.2, in MGV neurons to understand its specific role in auditory attention. We found that Kv4.2 mRNA was expressed in most MGV neurons. At the protein level, Kv4.2-immunopositive patches were sparsely distributed in both the dendrites and the soma of neurons. The postsynaptic distribution of Kv4.2 protein was confirmed using electron microscopy (EM). The frequency of contact with Kv4.2-immunopositive puncta was higher in vesicular glutamate transporter 2 (VGluT2)-positive excitatory axon terminals, which are supposed to be extending from the IC, than in VGluT1-immunopositive terminals, which are expected to be originating from the AC. VGluT2-immunopositive terminals were significantly larger than VGluT1-immunopositive terminals. Furthermore, EM showed that the terminals forming asymmetric synapses with Kv4.2-immunopositive MGV dendritic domains were significantly larger than those forming synapses with Kv4.2-negative MGV dendritic domains. In inhibitory axons either from the IC or from the RTN, the frequency of terminals that were in contact with Kv4.2-positive puncta was higher in IC than in RTN. In summary, our study demonstrated that the Kv4.2-immunopositive domains of the MGV dendrites received excitatory and inhibitory ascending auditory inputs preferentially from the IC, and not from the RTN or cortex. Our findings imply that time course of synaptic current and spike waveforms elicited by IC inputs is modified in the Kv4.2 domains.

Anatomy embroiders function in Purkinje cells

The anatomy of the human cerebellar Purkinje cell, as captured by Ramón y Cajal in a drawing, inspired the spectacular cover embroidery by Lisa Stoneman (Roanoke College, Roanoke, VA, USA) in the October issue of The Lancet Neurology. 1 Mehta AR Abbott CM Chandran S Haley JE The Cajal Embroidery Project: celebrating neuroscience. Lancet Neurol. 2020; 19: 979 Summary Full Text Full Text PDF Scopus (6) Google Scholar Like contemporary confocal microscopy images do, this anatomical representation also raises fascinating questions about the function served by the complex array of dendrites in this cell type. Each Purkinje cell, in rodents, receives about 174 000 synaptic inputs from the parallel fibre axons of the far tinier cerebellar granule cells, along with a single powerful input from the climbing fibre arising from the inferior olivary nucleus. 2 Howarth C Gleeson P Attwell D Updated energy budgets for neural computation in the neocortex and cerebellum. J Cereb Blood Flow Metab. 2012; 32: 1222-1232 Crossref PubMed Scopus (337) Google Scholar Even though a large fraction of those synapses can become inactive with learning, 3 Isope P Barbour B Properties of unitary granule cell→Purkinje cell synapses in adult rat cerebellar slices. J Neurosci. 2002; 22: 9668-9678 Crossref PubMed Google Scholar it is remarkable that the information processed from such a large number of inputs, which are even more numerous in human beings, is ultimately channelled down a single inhibitory axon to the deep cerebellar nuclei, as denoted by the label a in Cajal's original 1894 drawing (appendix).

Primate neuronal connections are sparse in cortex as compared to mouse.

Detailing how primate and mouse neurons differ is critical for creating generalized models of how neurons process information. We reconstruct 15,748 synapses in adult Rhesus macaques and mice and ask how connectivity differs on identified cell types in layer 2/3 of primary visual cortex. Primate excitatory and inhibitory neurons receive 2-5 times fewer excitatory and inhibitory synapses than similar mouse neurons. Primate excitatory neurons have lower excitatory-to-inhibitory (E/I) ratios than mouse but similar E/I ratios in inhibitory neurons. In both species, properties of inhibitory axons such as synapse size and frequency are unchanged, and inhibitory innervation of excitatory neurons is local and specific. Using artificial recurrent neural networks (RNNs) optimized for different cognitive tasks, we find that penalizing networks for creating and maintaining synapses, as opposed to neuronal firing, reduces the number of connections per node as the number of nodes increases, similar to primate neurons compared with mice.

Aberrant hippocampal mossy fibers in temporal lobe epilepsy target excitatory and inhibitory neurons.

The pathoanatomical correlate of temporal lobe epilepsy is hippocampal sclerosis, characterized by selective neuronal death of mossy cells in the hilus and of pyramidal cells in cornu ammonis 1. Although granule cells survive, they lose mossy cells as a target and redirect their axons (mossy fibers) backward into the molecular cell layer. It has been assumed that this process results in excitatory circuits. We therefore examined whether sprouted mossy fibers form synaptic connection not only with excitatory granule cells but also with inhibitory interneurons, such as basket cells. Resected hippocampal specimens of patients with hippocampal sclerosis were compared to controls of patients with extrahippocampal lesions with only mild sclerosis. Mossy fibers were traced with Neurobiotin or labeled against synaptoporin; inhibitory interneurons were labeled against parvalbumin. Synapses were examined with electron microscopy, labeled with γ-aminobutyric acid immunogold. Sprouted mossy fibers of epileptic hippocampi innervate not only excitatory granule cells but also inhibitory parvalbuminergic interneurons. Despite neuronal death in hippocampal sclerosis, the axonal plexus of inhibitory parvalbuminergic interneurons surrounding the granule cells is preserved. Connections of sprouted mossy fibers and inhibitory axon terminals were quantified, showing that the number of inhibitory axon terminals significantly exceeds the number of sprouted excitatory mossy fiber terminals (.03boutons/µm vs. .11 boutons/µm; p < .001). Although no definite conclusions regarding the function of our findings may be derived from this anatomical study, the observed aberrant connectivity might lead to an increased inhibition and synchronization of granule cells, because the preserved inhibitory interneurons show an additional innervation through sprouted mossy fibers. This might result in the instability of a previously balanced network.

Axonal CB1 Receptors Mediate Inhibitory Bouton Formation via cAMP Increase and PKA.

Experience-dependent formation and removal of inhibitory synapses are essential throughout life. For instance, GABAergic synapses are removed to facilitate learning, and strong excitatory activity is accompanied by the formation of inhibitory synapses to maintain coordination between excitation and inhibition. We recently discovered that active dendrites trigger the growth of inhibitory synapses via CB1 receptor-mediated endocannabinoid signaling, but the underlying mechanism remained unclear. Using two-photon microscopy to monitor the formation of individual inhibitory boutons in hippocampal organotypic slices from mice (both sexes), we found that CB1 receptor activation mediated the formation of inhibitory boutons and promoted their subsequent stabilization. Inhibitory bouton formation did not require neuronal activity and was independent of Gi/o-protein signaling, but was directly induced by elevating cAMP levels using forskolin and by activating Gs-proteins using DREADDs. Blocking PKA activity prevented CB1 receptor-mediated inhibitory bouton formation. Our findings reveal that axonal CB1 receptors signal via unconventional downstream pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels. Our results demonstrate an unexpected role for axonal CB1 receptors in axon-specific, and context-dependent, inhibitory synapse formation.SIGNIFICANCE STATEMENT Coordination between excitation and inhibition is required for proper brain function throughout life. It was previously shown that new inhibitory synapses can be formed in response to strong excitation to maintain this coordination, and this was mediated by endocannabinoid signaling via CB1 receptors. As activation of CB1 receptors generally results in the suppression of synaptic transmission, it remained unclear how CB1 receptors can mediate the formation of inhibitory synapses. Here we show that CB1 receptors on inhibitory axons signal via unconventional intracellular pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels and requires PKA activity. Our findings point to a central role for axonal cAMP signaling in activity-dependent inhibitory synapse formation.

Keratan sulfate proteoglycan as an axonal insulating barrier in the forebrain of fetuses with alobar/semi-lobar holoprosencephaly.

Keratan sulfate (KS) is an abundant proteoglycan in the developing human CNS where it functions as an extracellular axonal guidance molecule, repelling glutamatergic while facilitating GABAergic axons. It ensheaths axonal fascicles. In fetal brain maturation, KS acts as a barrier to axonal penetration. Its possible role in the pathogenesis of fetal holoprosencephaly (HPE) was studied. Forebrains of 6 human fetuses with HPE identified by prenatal ultrasound were examined at autopsy with KS immunoreactivity and other markers of cellular maturation and synaptogenesis, with age-matched controls. KS was strongly expressed in astrocytes in the thalamus from 13 weeks gestational age (GA) and in globus pallidus but not corpus striatum. Cortical plate reactivity was limited to the molecular zone, where KS was excessive, ensheathing individual transverse molecular zone axons. Axonal envelopment preceding myelination also was seen in the internal capsule and thalamocortical projections, but perifascicular KS was diminished. KS was not expressed in hippocampus in either HPE or controls. Glutamate receptor-2 (GluR2) was evident in hippocampal granular and pyramidal neurons at mid-gestation. KS distribution did not, however, correlate with synaptophysin. Excessive ensheathment of axons by KS provides additional protection of GABAergic inhibitory axons and synapses that may help suppress epileptogenesis. Though involved in selection of excitatory and inhibitory synaptogenesis, KS does not follow a developmental sequence corresponding to synaptophysin or GluR2 reactivities in either HPE or in normal fetal brain.

Modular and Distinct Plexin-A4/FARP2/Rac1 Signaling Controls Dendrite Morphogenesis

Diverse neuronal populations with distinct cellular morphologies coordinate the complex function of the nervous system. Establishment of distinct neuronal morphologies critically depends on signaling pathways that control axonal and dendritic development. The Sema3A-Nrp1/PlxnA4 signaling pathway promotes cortical neuron basal dendrite arborization but also repels axons. However, the downstream signaling components underlying these disparate functions of Sema3A signaling are unclear. Using the novel PlxnA4KRK-AAA knock-in male and female mice, generated by CRISPR/cas9, we show here that the KRK motif in the PlxnA4 cytoplasmic domain is required for Sema3A-mediated cortical neuron dendritic elaboration but is dispensable for inhibitory axon guidance. The RhoGEF FARP2, which binds to the KRK motif, shows identical functional specificity as the KRK motif in the PlxnA4 receptor. We find that Sema3A activates the small GTPase Rac1, and that Rac1 activity is required for dendrite elaboration but not axon growth cone collapse. This work identifies a novel Sema3A-Nrp1/PlxnA4/FARP2/Rac1 signaling pathway that specifically controls dendritic morphogenesis but is dispensable for repulsive guidance events. Overall, our results demonstrate that the divergent signaling output from multifunctional receptor complexes critically depends on distinct signaling motifs, highlighting the modular nature of guidance cue receptors and its potential to regulate diverse cellular responses.SIGNIFICANCE STATEMENT The proper formation of axonal and dendritic morphologies is crucial for the precise wiring of the nervous system that ultimately leads to the generation of complex functions in an organism. The Semaphorin3A-Neuropilin1/Plexin-A4 signaling pathway has been shown to have multiple key roles in neurodevelopment, from axon repulsion to dendrite elaboration. This study demonstrates that three specific amino acids, the KRK motif within the Plexin-A4 receptor cytoplasmic domain, are required to coordinate the downstream signaling molecules to promote Sema3A-mediated cortical neuron dendritic elaboration, but not inhibitory axon guidance. Our results unravel a novel Semaphorin3A-Plexin-A4 downstream signaling pathway and shed light on how the disparate functions of axon guidance and dendritic morphogenesis are accomplished by the same extracellular ligand in vivo.

Mining the jewels of the cortex's crowning mystery.

Neocortical Layer 1 consists of a dense mesh of excitatory and inhibitory axons, dendrites of pyramidal neurons, as well as neuromodulatory inputs from diverse brain regions. Layer 1 also consists of a sparse population of inhibitory interneurons, which are appropriately positioned to receive and integrate the information from these regions of the brain and modulate cortical processing. Despite being among the sparsest neuronal population in the cortex, Layer 1 interneurons perform powerful computations and have elaborate morphologies. Here we review recent studies characterizing their origin, morphology, physiology, and molecular profiles, as well as their connectivity and in vivo response properties.

Interneuron hypomyelination is associated with cognitive inflexibility in a rat model of schizophrenia

Impaired cognitive functioning is a core feature of schizophrenia, and is hypothesized to be due to myelination as well as interneuron defects during adolescent prefrontal cortex (PFC) development. Here we report that in the apomorphine-susceptible (APO-SUS) rat model, which has schizophrenia-like features, a myelination defect occurred specifically in parvalbumin interneurons. The adult rats displayed medial PFC (mPFC)-dependent cognitive inflexibility, and a reduced number of mature oligodendrocytes and myelinated parvalbumin inhibitory axons in the mPFC. In the developing mPFC, we observed decreased myelin-related gene expression that persisted into adulthood. Environmental enrichment applied during adolescence restored parvalbumin interneuron hypomyelination as well as cognitive inflexibility. Collectively, these findings highlight that impairment of parvalbumin interneuron myelination is related to schizophrenia-relevant cognitive deficits.

Effects of levetiracetam on axon excitability and synaptic transmission at the crayfish neuromuscular junction.

Levetiracetam (LEV) is a widely prescribed antiepileptic drug, but its actions on neuronal function are not fully characterized. Since this drug is believed to enter neurons by binding to a vesicular protein during endocytosis, we used motor axons of the crayfish opener neuromuscular junction to examine potential impacts of LEV on axon excitability. Two electrode current clamp from the inhibitory axon of the opener showed that LEV reduced action potential (AP) amplitude (APamp ) and suppressed synaptic transmission, although the latter occurred with a longer delay than the reduction in APamp . Comparison of antidromic and orthodromic conducting APs in LEV suggested that this drug preferentially reduced excitability of the proximal axon, despite the expectation that it entered the axon at the terminals and should affect the distal branches first. Results presented here suggest that LEV modulates axonal excitability, which may in turn contribute to its antiepileptic effects.

Cell-type specific innervation of cortical pyramidal cells at their apical dendrites.

We investigated the synaptic innervation of apical dendrites of cortical pyramidal cells in a region between layers (L) 1 and 2 using 3-D electron microscopy applied to four cortical regions in mouse. We found the relative inhibitory input at the apical dendrite's main bifurcation to be more than 2-fold larger for L2 than L3 and L5 thick-tufted pyramidal cells. Towards the distal tuft dendrites in upper L1, the relative inhibitory input was at least about 2-fold larger for L5 pyramidal cells than for all others. Only L3 pyramidal cells showed homogeneous inhibitory input fraction. The inhibitory-to-excitatory synaptic ratio is thus specific for the types of pyramidal cells. Inhibitory axons preferentially innervated either L2 or L3/5 apical dendrites, but not both. These findings describe connectomic principles for the control of pyramidal cells at their apical dendrites and support differential computational properties of L2, L3 and subtypes of L5 pyramidal cells in cortex.

A132 SMOOTH MUSCLE BIOPSIES FROM POEM PROCEDURES FOR ACHALASIA SHOW LACK OF FUNCTIONAL INNERVATION

Abstract Background Idiopathic achalasia is a disease of the esophagus causing impaired peristalsis and absent lower esophageal sphincter relaxation. The etiopathogenesis of the disease is unclear but most histopathologic studies from surgical resections show an absence of nitric oxide-producing neurons in the myenteric plexus. The peroral endoscopic myotomy (POEM) procedure has evolved in the last decade to treat achalasia and has provided a unique way to sample diseased tissue from an otherwise inaccessible tissue compartment. Aims To study the effects of achalasia on innervation of smooth muscle and smooth muscle phenotype. Methods Patients with a diagnosis of achalasia based on high resolution manometry undergoing a POEM at Kingston general hospital were approached for enrollment between June 2017 to September 2018. Demographic information including age, symptom duration, previous treatments, and Eckhardt score was collected. Intraoperatively, biopsies of the circular smooth muscle (CSM) layer were taken at the proximal and distal extents of the myotomy and placed in formalin. Tissue was embedded in wax, sectioned and stained using immunocytochemistry (ICC) for neuronal, axonal and smooth muscle cell markers. This was compared to control tissue from patients undergoing gastroesophagectomy for adjacent malignancy. Results Control tissue from 3 separate esophagectomies were obtained. Samples were obtained from a total of 25 patients (13 males), with a median age of 50 (IQR 38–67). Most patients had Type 2 achalasia (19 [76%]) followed by Type 1 and 3 (2, [8%] respectively). Two cases had conflicting manometric findings. The median duration of symptoms was 3 years (IQR 1.5–10.5) and the median Eckhardt score was 6.5 (IQR 5–9). In sample tissues, no neuronal cell bodies were detected in the CSM layer. ICC for the axonal marker PGP9.5 showed that CSM of achalasia samples were almost completely devoid of axon structure, independent of the subtype of achalasia, compared to abundant axon presence in control tissues. In parallel, ICC showed that cholinergic (ChAT) or nitrergic (nNOS) axonal subtypes were absent in biopsy CSM while abundant in controls. CSM cells displayed hypertrophy with no detection of proliferation by ICC (KI67) or alterations in the phenotypic marker SM-22. Conclusions Preliminary results from advanced immunohistochemical techniques show the absence of functional innervation of the CSM layer of all patients with achalasia. This is characterized by a depleted excitatory and inhibitory axon population. Further studies are focused on defining differences in smooth muscle phenotype and the presence or absence of inflammatory cells within the CSM. Funding Agencies None

Primate Neuronal Connections are Sparse as Compared to Mouse

Mouse and macaque primary visual cortices (V1) are foundational models of the cortex. Detailing differences in how V1 neurons connect across these species would inform models of cortical functioning and brain evolution. However, existing comparisons are limited, measuring synapse density without regard to where synapses are made. We use large volume electron microscopy to address this gap and find that primate connections are broadly sparse: primate excitatory and inhibitory neurons received 3-5 times fewer spine and somatic synapses with lower excitatory to inhibitory synapse ratios than mouse equivalents. Excitatory outputs differ between species with more primate neurons forming local axon collaterals than mouse. However, inhibitory axons innervated neighboring excitatory neurons at similar rates and proportions across species. Finally, we discover that when optimizing for a cognitive task, artificial recurrent neural networks, constrained by a metabolic cost for creating/maintaining synapses, reduces the number of connections/node as the number of nodes increases.