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Plants are resilient to transient limitations in the availability of oxygen for efficient energy production. The highly reversible response to oxygen deprivation (hypoxia) is characterized by dynamics in accumulation and differential translation of a subset of genes in the model plant Arabidopsis thaliana. Transcriptional upregulation of genes associated with survival in response to hypoxia are mediated by group VII ETHYLENE RESPONSIVE FACTOR (ERFVII) transcription factors via binding to a conserved Hypoxia Responsive Promoter Element (HRPE) cis-element. Nonetheless, there is little knowledge of the effects of hypoxia on nuclear processes, including histone modification, chromatin accessibility, RNA polymerase II (RNAPII) elongation or RNA export. Additionally, in vivo ERFVII binding sites and dynamics are largely unexplored at the global scale.In this dissertation, the modulation of chromatin features, RNAPII elongation and nascent transcripts were contrasted with the total polyadenylated and ribosome-associated sub-populations of mRNA of seedlings exposed to non-stress, hypoxic, or re-oxygenation conditions. The technologies utilized in this study to generate datasets included Chromatin Immunoprecipitation (ChIP-seq), Isolation of Nuclei Tagged in Specific Cell Types (INTACT), Assay for Transposase Accessible Chromatin (ATAC-seq), RNA sequencing (RNA-seq), and Translating Ribosome Affinity Purification (TRAP-seq). In vivo binding sites were mapped for the Arabidopsis ERFVII HYPOXIA RESPONSIVE ERF2 (HRE2), and the rice (Oryza sativa) ERFVIIs SUBMERGENCE 1A and C (SUB1A/C). Integrated bioinformatic analyses were performed to investigate coordination in regulation from chromatin to translation. This led to the identification of multiple nuclear-regulated processes that contribute to dynamics in gene activity. The ~50 hypoxia-responsive genes that display coordinate transcript accumulation and translation were characterized by increased chromatin accessibility, pronounced elevation of Histone 3-lysine 9 acetylation and depletion of Histone 2A.Z under hypoxic stress. HRE2 bound just 5’ of the transcription start site for many of these genes. Universal stress response genes including those associated with heat stress displayed an early increase in RNAPII engagement along the transcription unit with elevation of mRNA only after the stress was prolonged. This study revealed genes and nascent transcripts poised for expression in anticipation of prolonged stress or reoxygenation that were previously unrecognized in widely utilized whole-cellular RNA-seq analyses.

Major sensory innervation to the uterus is provided by spinal afferent nerves, whose cell bodies lie predominantly in thoracolumbar dorsal root ganglia (DRG). While the origin of the cell bodies of uterine spinal afferents is clear, the identity of their sensory endings has remained unknown. Hence, our major aim was to identify the location, morphology, and calcitonin gene-related peptide (CGRP)-immunoreactivity of uterine spinal afferent endings supplied by thoracolumbar DRG. We also sought to determine the degree of uterine afferent innervation provided by the vagus nerve. Using an anterograde tracing technique, nulliparous female C57BL/6 mice were injected unilaterally with biotinylated dextran into thoracolumbar DRG (T13-L3). After 7-9 days, uterine horns were stained to visualize traced nerve axons and endings immunoreactive to CGRP. Whole uteri from a separate cohort of animals were injected with retrograde neuronal tracer (DiI) and dye uptake in nodose ganglia was examined. Anterogradely labeled axons innervated each uterine horn, these projected rostrally or caudally from their site of entry, branching to form varicose endings in the myometrium and/or vascular plexus. Most spinal afferent endings were CGRP-immunoreactive and morphologically classified as "simple-type." Rarely, uterine nerve cell bodies were labeled in nodose ganglia. Here, we provide the first detailed description of spinal afferent nerve endings in the uterus of a vertebrate. Distinct morphological types of spinal afferent nerve endings were identified throughout multiple anatomical layers of the uterine wall. Compared to other visceral organs, uterine spinal afferent endings displayed noticeably less morphological diversity. Few neurons in nodose ganglia innervate the uterus.

The mechanisms underlying electrical rhythmicity in smooth muscle of the proximal colon are incompletely understood. Our aim was to identify patterns of electrical rhythmicity in smooth muscle of the proximal region of isolated whole mouse colon and characterize their mechanisms of origin. Two independent extracellular recording electrodes were used to record the patterns of electrical activity in smooth muscle of the proximal region of whole isolated mouse colon. Cross-correlation analysis was used to quantify spatial coordination of these electrical activities over increasing electrode separation distances. Four distinct neurogenic patterns of electrical rhythmicity were identified in smooth muscle of the proximal colon, three of which have not been identified and consisted of bursts of rhythmic action potentials at 1–2 Hz that were abolished by hexamethonium. These neurogenic patterns of electrical rhythmicity in smooth muscle were spatially and temporally synchronized over large separation distances (≥2 mm rosto-caudal axis). Myogenic slow waves could be recorded from the same preparations, but they showed poor spatial and temporal coordination over even short distances (≤1 mm rostro-caudal axis). It is not commonly thought that electrical rhythmicity in gastrointestinal smooth muscle is dependent upon the enteric nervous system. Here, we identified neurogenic patterns of electrical rhythmicity in smooth muscle of the proximal region of isolated mouse colon, which are dependent on synaptic transmission in the enteric nervous system. If the whole colon is studied in vitro, recordings can preserve novel neurogenic patterns of electrical rhythmicity in smooth muscle. NEW & NOTEWORTHY Previously, it has not often been thought that electrical rhythmicity in smooth muscle of the gastrointestinal tract is dependent upon the enteric nervous system. We identified patterns of electrical rhythmicity in smooth muscle of the mouse proximal colon that were abolished by hexamethonium and involved the temporal synchronization of smooth muscle membrane potential over large spatial fields. We reveal different patterns of electrical rhythmicity in colonic smooth muscle that are dependent on the ENS.

While sensory and sympathetic neurons are known to innervate bone, previous studies have found it difficult to unequivocally identify and characterize only those that are of sensory origin. In this study, we have utilized an in vivo anterograde tracing technique to selectively label spinal afferent (sensory) nerve endings that innervate the periosteum and marrow cavity of murine long bones. Unilateral injections of dextran-biotin (anterograde tracer; 20% in saline, 50-100 nl) were made into L3-L5 dorsal root ganglia. After a 10-day recovery period to allow sufficient time for selective anterograde transport of the tracer to nerve terminal endings in bone, the periosteum (whole-mount) and underlying bone were collected, processed to reveal anterograde labeling, and immuno-labeled with antibodies directed against protein gene product (pan-neuronal marker; PGP9.5), tyrosine hydroxylase (sympathetic neuron marker; TH), calcitonin gene-related protein (peptidergic nociceptor marker; CGRP), and/or neurofilament 200 (myelinated axon marker; NF200). Anterograde-labeled nerve endings were dispersed throughout the periosteum and marrow cavity and could be identified in close apposition to blood vessels and at sites distant from them. The periosteum and the marrow cavity were each innervated by myelinated (NF200+) sensory neurons, and unmyelinated (NF200-) sensory neurons that were either peptidergic (CGRP+) or nonpeptidergic (CGRP-). Spinal afferent nerve endings did not express TH, and lacked the cylindrical morphology around blood vessels characteristic of sympathetic innervation. This approach to selective labeling of sensory nerve terminal endings will help to better identify how different sub-populations of sensory neurons, and their peripheral nerve terminal endings, interact with bone.

Cross talk between the gastrointestinal tract and brain is of significant relevance for human health and disease. However, our understanding of how the gut and brain communicate has been limited by a lack of techniques to identify the precise spatial relationship between extrinsic nerve endings and their proximity to specific cell types that line the inner surface of the gastrointestinal tract. We used an in vivo anterograde tracing technique, previously developed in our laboratory, to selectively label single spinal afferent axons and their nerve endings in mouse colonic mucosa. The closest three-dimensional distances between spinal afferent nerve endings and axonal varicosities to enterochromaffin (EC) cells, which contain serotonin (5-hydroxytryptamine; 5-HT), were then measured. The mean distances (± standard deviation) between any varicosity along a spinal afferent axon or its nerve ending, and the nearest EC cell, were 5.7 ± 6.0 μm (median: 3.6 μm) and 26.9 ± 18.6 μm (median: 24.1 μm), respectively. Randomization of the spatial location of EC cells revealed similar results to this actual data. These distances are ∼200-1,000 times greater than those between pre- and postsynaptic membranes (15-25 nm) that underlie synaptic transmission in the vertebrate nervous system. Our findings suggest that colonic 5-HT-containing EC cells release substances to activate centrally projecting spinal afferent nerves likely via diffusion, as such signaling is unlikely to occur with the spatial fidelity of a synapse.

How the enteric nervous system determines the pacing and propagation direction of neurogenic contractions along the colon remains largely unknown. We used a chemogenetic strategy to ablate enteric neurons expressing calretinin (CAL). Mice expressing human diphtheria toxin receptor (DTR) in CAL neurons were generated by crossing CAL-ires-Cre mice with Cre-dependent ROSA26-DTR mice. Immunohistochemical analysis revealed treatment with diphtheria toxin incurred a 42% reduction in counts of Hu-expressing colonic myenteric neurons (P = 0.036), and 57% loss of CAL neurons (comprising ∼25% of all Hu neurons; P = 0.004) compared to control. As proportions of Hu-expressing neurons, CAL neurons that contained nitric oxide synthase (NOS) were relatively spared (control: 15 ± 2%, CAL-DTR: 13 ± 1%; P = 0.145), while calretinin neurons lacking NOS were significantly reduced (control: 26 ± 2%, CAL-DTR: 18 ± 5%; P = 0.010). Colonic length and pellet sizes were significantly reduced without overt inflammation or changes in ganglionic density. Interestingly, colonic motor complexes (CMCs) persisted with increased frequency (mid-colon interval 111 ± 19 vs. 189 ± 24 s, CAL-DTR vs. control, respectively, P < 0.001), decreased contraction size (mid-colon AUC 26 ± 24 vs. 59 ± 13 gram/seconds, CAL-DTR vs. control, respectively, P < 0.001), and lacked preferential anterograde migration (P < 0.001). The functional effects of modest calretinin neuron ablation, particularly increased neurogenic motor activity frequencies, differ from models that incur general enteric neuron loss, and suggest calretinin neurons may contribute to pacing, force, and polarity of CMCs in the large bowel.

How the Enteric Nervous System (ENS) coordinates propulsion of content along the gastrointestinal (GI)-tract has been a major unresolved issue. We reveal a mechanism that explains how ENS activity underlies propulsion of content along the colon. We used a recently developed high-resolution video imaging approach with concurrent electrophysiological recordings from smooth muscle, during fluid propulsion. Recordings showed pulsatile firing of excitatory and inhibitory neuromuscular inputs not only in proximal colon, but also distal colon, long before the propagating contraction invades the distal region. During propulsion, wavelet analysis revealed increased coherence at ~2 Hz over large distances between the proximal and distal regions. Therefore, during propulsion, synchronous firing of descending inhibitory nerve pathways over long ranges aborally acts to suppress smooth muscle from contracting, counteracting the excitatory nerve pathways over this same region of colon. This delays muscle contraction downstream, ahead of the advancing contraction. The mechanism identified is more complex than expected and vastly different from fluid propulsion along other hollow smooth muscle organs; like lymphatic vessels, portal vein, or ureters, that evolved without intrinsic neurons., Nick Spencer et al. made simultaneous multi-site electrophysiological recordings with video imaging of colonic wall movements from ex vivo mouse colon, in order to correlate propulsion of content with underlying electrical signals from the smooth muscle. Their results demonstrate that excitatory and inhibitory junction potentials are synchronized in both the proximal and distal colon, suggesting that the enteric nervous system network communicates over a longer range than previously expected.

The dynamic changes in uterine contractility in response to distension are incompletely understood. Rhythmic, propagating contractions of nonpregnant uterine smooth muscle occur in the absence of nerve activity (i.e., myogenic), events that decline during pregnancy and reemerge at parturition. We therefore sought to determine how myogenic contractions of the nonpregnant uterus are affected by distension, which might provide mechanistic clues underlying distension-associated uterine conditions such as preterm birth. Uteri isolated from nulliparous adult female mice in proestrus were video imaged to generate spatiotemporal maps, and myoelectrical activity simultaneously recorded using extracellular suction electrodes. Motility patterns were examined under basal conditions and following ramped intraluminal distension with fluid to 5 and 10 cmH2O. Intraluminal distension caused pressure-dependent changes in the frequency, amplitude, propagation speed, and directionality of uterine contractions, which reversed upon pressure release. Altered burst durations of underlying smooth muscle myoelectric events were concurrently observed, although action potential spike intervals were unchanged. Voltage-gated sodium channel blockade [tetrodotoxin (TTX); 0.6 µM] attenuated both the amplitude of contractions and burst duration of action potentials, whereas all activity was abolished by L-type calcium channel blockade (nifedipine; 1 µM). These data suggest that myogenic motility patterns of the nonpregnant mouse uterus are sensitive to changes in intraluminal pressure and, at high pressures, may be modulated by voltage-gated sodium channel activity. Future studies may investigate whether similar distension-evoked changes occur in the pregnant uterus and the possible pathophysiological role of such activity in the development of preterm birth.

Electrical stimulation of the enteric nervous system (ENS) is an attractive approach to modify gastrointestinal transit. Colonic motor complexes (CMCs) occur with a periodic rhythm, but the ability to elicit a premature CMC depends, at least in part, upon the intrinsic refractory properties of the ENS, which are presently unknown. The objectives of this study were to record myoelectric complexes (MCs, the electrical correlates of CMCs) in the smooth muscle and 1) determine the refractory periods of MCs, 2) inform and evaluate closed-loop stimulation to repetitively evoke MCs, and 3) identify stimulation methods to suppress MC propagation. We dissected the colon from male and female C57BL/6 mice, preserving the integrity of intrinsic circuitry while removing the extrinsic nerves, and measured properties of spontaneous and evoked MCs in vitro. Hexamethonium abolished spontaneous and evoked MCs, confirming the necessary involvement of the ENS for electrically evoked MCs. Electrical stimulation reduced the mean interval between evoked and spontaneous CMCs (24.6 ± 3.5 vs. 70.6 ± 15.7 s, P = 0.0002, n = 7). The absolute refractory period was 4.3 s (95% confidence interval (CI) = 2.8–5.7 s, R(2) = 0.7315, n = 8). Electrical stimulation applied during fluid distention-evoked MCs led to an arrest of MC propagation, and following stimulation, MC propagation resumed at an increased velocity (n = 9). The timing parameters of electrical stimulation increased the rate of evoked MCs and the duration of entrainment of MCs, and the refractory period provides insight into timing considerations for designing neuromodulation strategies to treat colonic dysmotility. NEW & NOTEWORTHY Maintained physiological distension of the isolated mouse colon induces rhythmic cyclic myoelectric complexes (MCs). MCs evoked repeatedly by closed-loop electrical stimulation entrain MCs more frequently than spontaneously occurring MCs. Electrical stimulation delivered at the onset of a contraction temporarily suppresses the propagation of MC contractions. Controlled electrical stimulation can either evoke MCs or temporarily delay MCs in the isolated mouse colon, depending on timing relative to ongoing activity.

In electrophysiology, many methods have been proposed for the analysis of action potential firing frequencies. The aim of this study was to present an algorithm developed for a continuous wavelet transform that enables the filtering out of frequencies contributing to the shapes of action potentials (spikes), whilst retaining the frequencies that encode the periodicity of spike trains. The continuous wavelet transform allows us to decompose a signal into its constituent frequencies. A signal with a single event, such as a spike, is composed of frequencies that characterize the shape of the spike. A signal with two spikes will also be composed of frequencies characterizing the shape of the action potential, but in addition will include a substantial portion of its power at the frequency corresponding to the time-difference between the two spikes. This is achieved by clipping peaks from the wavelet amplitudes that are narrower than a given minimum number of phase cycles. We present some application examples in both synthetic signals and electrophysiological recordings. This new approach can provide a major new analytical tool for analysis of electrophysiological signals.

The enteric nervous system (ENS) contains millions of neurons essential for organization of motor behavior of the intestine. It is well established that the large intestine requires ENS activity to drive propulsive motor behaviors. However, the firing pattern of the ENS underlying propagating neurogenic contractions of the large intestine remains unknown. To identify this, we used high-resolution neuronal imaging with electrophysiology from neighboring smooth muscle. Myoelectric activity underlying propagating neurogenic contractions along murine large intestine [also referred to as colonic migrating motor complexes, (CMMCs)] consisted of prolonged bursts of rhythmic depolarizations at a frequency of ∼2 Hz. Temporal coordination of this activity in the smooth muscle over large spatial fields (∼7 mm, longitudinally) was dependent on the ENS. During quiescent periods between neurogenic contractions, recordings from large populations of enteric neurons, in mice of either sex, revealed ongoing activity. The onset of neurogenic contractions was characterized by the emergence of temporally synchronized activity across large populations of excitatory and inhibitory neurons. This neuronal firing pattern was rhythmic and temporally synchronized across large numbers of ganglia at ∼2 Hz. ENS activation preceded smooth muscle depolarization, indicating rhythmic depolarizations in smooth muscle were controlled by firing of enteric neurons. The cyclical emergence of temporally coordinated firing of large populations of enteric neurons represents a unique neural motor pattern outside the CNS. This is the first direct observation of rhythmic firing in the ENS underlying rhythmic electrical depolarizations in smooth muscle. The pattern of neuronal activity we identified underlies the generation of CMMCs. SIGNIFICANCE STATEMENT How the enteric nervous system (ENS) generates neurogenic contractions of smooth muscle in the gastrointestinal (GI) tract has been a long-standing mystery in vertebrates. It is well known that myogenic pacemaker cells exist in the GI tract [called interstitial cells of Cajal (ICCs)] that generate rhythmic myogenic contractions. However, the mechanisms underlying the generation of rhythmic neurogenic contractions of smooth muscle in the GI tract remains unknown. We developed a high-resolution neuronal imaging method with electrophysiology to address this issue. This technique revealed a novel pattern of rhythmic coordinated neuronal firing in the ENS that has never been identified. Rhythmic neuronal firing in the ENS was found to generate rhythmic neurogenic depolarizations in smooth muscle that underlie contraction of the GI tract.

In vertebrates, visceral pain from internal organs is detected by spinal afferents, whose cell bodies lie in dorsal root ganglia (DRG). Until now, all recordings from spinal afferents have been restricted to recording transmission of action potentials along axons, or from cell bodies lying outside their target organ, which is not where sensory transduction occurs. Our aim was to record directly from a major class of spinal afferent within visceral organs, where transduction of sensory stimuli into action potentials occurs. Using novel calcitonin gene-related peptide (CGRP)α reporter mice, DRG neurons expressed mCherry, including nerve axons within viscera. In colon, a minority of total CGRP immunoreactivity was attributed CGRPα. In isolated unstretched colon, calcium imaging from CGRPα-expressing varicose axons did not detect resolvable calcium transients. However, noxious levels of maintained circumferential stretch to the colon induced repetitive calcium transients simultaneously in multiple neighboring varicosities along single mCherry-expressing axons. Discrete varicosities could generate unitary calcium transients independently of neighboring varicosities. However, axons expressing mCherry only generated coordinated calcium transients when accompanied by simultaneous activation of multiple varicosities along that axon. Simultaneous imaging from different classes of myenteric neurons at the same time as mCherry-expressing axons revealed coordinated calcium transients in multiple myenteric neurons, independent of activity in mCherry-expressing axons. CGRPα-expressing axon terminals preferentially responded to heat, capsaicin, and low pH. We show that direct recordings can be made from the major class of peptidergic spinal afferent that contributes to visceral nociception. This approach can provide powerful insights into transduction of stimuli in viscera.

Background and aims Recently, it was demonstrated that optogenetics could be used to stimulate enteric calretinin neurons, leading to increased colonic transit in vitro and in vivo. The aim of the current study was to determine if similar approaches could be used to stimulate the isolated mouse small intestine, with the aim of potentially also improving transit in the small bowel. Methods Cre-Lox recombination was used to generate transgenic mice expressing the light-sensitive ion channel channelrhodopsin-2 (ChR2) in calretinin neurons. Results Spontaneous migrating motor complexes were recorded from isolated terminal small intestine in both CalCre+ mice expressing ChR2 in calretinin-expressing neurons and experimental controls, CalCre−. Trains of blue light pulses (20 ms, 5 Hz, 20s) evoked a brief local contraction of circular muscle, but never a premature MMC, irrespective of light intensity (1–40 mV/mm2) or the region of ileum stimulated. However, MMCs were readily evoked by calretinin neuron activation in colon, consistent with our previous study. Light-evoked contractions of the terminal small bowel were hexamethonium-resistant (300 μM), but blocked by tetrodotoxin (0.6 μM). Light-evoked smooth muscle contraction did not change the pacemaker frequency underlying MMCs. Conclusion Focal illumination of the small intestine does not appear as effective at inducing propulsive motor activity as has been demonstrated in the colon of the same colony mice. This study suggests caution should be exercised when assuming optogenetic technology is equally effective at increasing GI transit in the small intestine as in the large intestine of mice.

The gastrointestinal tract contains its own independent population of sensory neurons within the gut wall. These sensory neurons have been referred to as intrinsic primary afferent neurons (IPANs) and can be identified by immunoreactivity to calcitonin gene-related peptide (CGRP) in mice. A common feature of IPANs is a paucity of fast synaptic inputs observed during sharp microelectrode recordings. Whether this is observed using different recording techniques is of particular interest for understanding the physiology of these neurons and neural circuit modeling. Here, we imaged spontaneous and evoked activation of myenteric neurons in isolated whole preparations of mouse colon and correlated recordings with CGRP and nitric oxide synthase (NOS) immunoreactivity, post hoc. Calcium indicator fluo 4 was used for this purpose. Calcium responses were recorded in nerve cell bodies located 5–10 mm oral to transmural electrical nerve stimuli. A total of 618 recorded neurons were classified for CGRP or NOS immunoreactivity. Aboral electrical stimulation evoked short-latency calcium transients in the majority of myenteric neurons, including ~90% of CGRP-immunoreactive Dogiel type II neurons. Activation of Dogiel type II neurons had a time course consistent with fast synaptic transmission and was always abolished by hexamethonium (300 μM) and by low-calcium Krebs solution. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (during synaptic blockade) directly activated Dogiel type II neurons. The present study suggests that murine colonic Dogiel type II neurons receive prominent fast excitatory synaptic inputs from hexamethonium-sensitive neural pathways.NEW & NOTEWORTHY Myenteric neurons in isolated mouse colon were recorded using calcium imaging and then neurochemically defined. Short-latency calcium transients were detected in >90% of calcitonin gene-related peptide-immunoreactive neurons to electrical stimulation of hexamethonium-sensitive pathways. Putative sensory Dogiel type II calcitonin gene-related peptide-immunoreactive myenteric neurons may receive widespread fast synaptic inputs in mouse colon.

Neurons in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus pars compacta (NAc) innervate the subdiaphragmatic oesophagus in rodents but little is known about the central circuitry that controls the activity of these vagal motor neurons. We assessed the monoamine innervation of these two groups of esophageal motor neurons with immunohistochemistry, conventional retrograde tracing and anterograde tracing using lentiviral vectors that drive GFP expression in either catecholamine or 5-HT neurons. Close appositions from axons immunoreactive for 5-HT or catecholamine synthetic enzymes (tyrosine hydroxylase or phenylethanolamine N-methyltransferase) occurred on many esophageal motor neurons in DMV that had been retrogradely labelled with cholera toxin B subunit (CTB) from the oesophagus. In contrast, all three types of axons very rarely apposed CTB-labelled motor neurons in NAc. In rats with lentiviral-induced selective GFP expression in C1, C3 or midline 5-HT neurons, GFP-immunoreactive axons w...

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Spinal afferent neurons play a major role in detecting noxious and innocuous stimuli from visceral organs, such as the gastrointestinal tract. However, all our understanding about spinal afferents has been obtained from recordings of spinal afferent axons, or cell bodies that lie outside the gut wall, or peripheral organ they innervate. No recordings have been made directly from spinal afferent nerve endings, which is where sensory transduction occurs. We developed a preparation whereby recordings could be made from rectal afferent nerve endings in the colon, to characterize mechanisms underlying sensory transduction. Dorsal root ganglia (L6-S2) were removed from mice, whilst retaining neural continuity with the colon. Fluo-4-AM was used to record from rectal afferent nerve endings in myenteric ganglia and circular muscle at 36°C. In slack (unstretched) preparations of colon, no calcium transients were recorded from spinal afferent endings. However, in response to a maintained increase in circumferential diameter, a maintained discharge of calcium transients occurred simultaneously in multiple discrete varicosities along single axons of rectal afferents in myenteric ganglia and circular muscle. Stretch-activated calcium transients were resistant to hexamethonium and nifedipine, but were abolished by tetrodotoxin, CPA, BAPTA-AM, cobalt, gadolinium, or replacement of extracellular Na+ with NMDG. In summary, we present a novel preparation in which stretch-activated firing of spinal afferent nerve endings can be recorded, using calcium imaging. We show that circumferential stretch of the colon activates a maintained discharge of calcium transients simultaneously in varicosities along single rectal afferent endings in myenteric ganglia and circular muscle. Non-selective cation channels, TTX-sensitive Na+ channels and both extracellular calcium influx and intracellular Ca2+ stores are required for stretch-activated calcium transients in rectal afferent endings.

Using a model of O-ring production function, the paper demonstrates how certain communities can get caught in a low-literacy trap in which each individual finds it not worthwhile investing in higher skills because others are not high-skilled. The model sheds light on educational policy. It is shown that policy for promoting human capital has to take the form of a mechanism for solving the coordination failure in people's choice of educational strategy.

By now the importance of literacy in the process of development is widely accepted. However, unlike measuring welfare or inequality, the problem of literacy measurement remains largely unexamined. Alternatives to the standard literacy rate, R, equal to the number of literate adults as a percentage of the adult population, are not well known, but this measure has its deficiencies. In particular, several authors have identified the externality accruing to proximate illiterates, that is, illiterate people with access to a literate person. The standard literacy rate ignores this externality; measures of effective literacy are sensitive to it. The present note offers two effective literacy rates (measures) and a set of axioms characterizing each. Nearly all measures of effective literacy appearing in the literature are greater than or equal to R. In fact, the best known of these, the Basu-Foster measure is strictly greater in virtually every case (see Basu and Foster [1]). Although the inequality L R is an unintended consequence of their construction, it amounts to setting a benchmark for the effective literacy rate. This notes examines Basu and Foster's framework and offers an alternative benchmark.