Long range synchronization within the enteric nervous system underlies propulsion along the large intestine in mice.

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.

A novel method for quantifying periodicity and time delay in dynamic neural networks using unstable subaction potential threshold depolarizations.

Techniques to identify and correlate the propagation of electrical signals (like action potentials) along neural networks are well described, using multisite recordings. In these cases, the waveform of action potentials is usually relatively stable and discriminating relevant electrical signals straightforward. However, problems can arise when attempting to identify and correlate the propagation of signals when their waveforms are unstable (e.g., fluctuations in amplitude or time course). This makes correlation of the degree of synchronization and time lag between propagating electrical events across two or more recording sites problematic. Here, we present novel techniques for the determination of the periodicity of electrical signals at individual sites. When recording from two independent sites, we present novel analytical techniques for joint determination of periodicity and time delay. The techniques presented exploit properties of the cross-correlation function, rather than utilizing the time lag at which the cross-correlation function is maximized. The approach allows determination of directionality of the spread of excitation along a neural network based on measurements of the time delay between recording sites. This new method is particularly applicable to analysis of signals in other biological systems that have unstable characteristics in waveform that show dynamic variability.NEW & NOTEWORTHY The determination of frequency(s) at which two sources are synchronized, and relative time delay between them, is a fundamental problem for a wide a range of signal-processing applications. In this methodology paper, we present novel procedures for periodicity estimation for single time series and joint periodicity and time delay estimation for two time series. The methods use properties of the cross-correlation function rather than the cross-correlation function explicitly.

Identification of a Rhythmic Firing Pattern in the Enteric Nervous System That Generates Rhythmic Electrical Activity in Smooth Muscle.

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.

Techniques to identify and temporally correlate calcium transients between multiple regions of interest in vertebrate neural circuits.

Calcium imaging is commonly used to record dynamic changes in excitability from axons or cell bodies in the nervous system of vertebrates. These recordings often reveal discrete calcium transients that have variable amplitudes, durations, and rates of rise and decay, all of which can arise from an unstable or "noisy" baseline. This often leads to considerable ambiguity about how to discriminate and quantify calcium transients. We describe an analytical methodology that objectively identifies multiple calcium transients from multiple recording sites and quantifies the degree of temporal synchrony between each event. The methodology consists of multiple steps. The first step involves baselining, to either preserve the underlying shape of calcium transients or remove unwanted frequency components and transform the peaks of calcium transients into more easily detectable patterns. The second step is the application of at least one of two different spike detection algorithms, one based on a gradient estimate and the other on template matching. The third step is the quantification of synchrony between pairs of recordings using at least one of two time lag correlation measures. The fourth step is the identification of statistically significant coincident firing patterns. This allows discrimination of neuronal firing patterns between different sites that appear to occur simultaneously and that statistically could not be attributed to chance. The analytical methods we have demonstrated can be applied not only to calcium imaging but also to many other physiological recordings, where discrimination and temporal correlation of biological signals from multiple sites is required, particularly when arising from unstable baselines, with variable signal-to-noise ratios.NEW & NOTEWORTHY Dynamic imaging of intracellular calcium is commonly used to record changes in excitability in central and peripheral neurons. We describe a novel analytical methodology that objectively discriminates calcium transients from low signal-to-noise recordings from multiple sites and quantifies the degree of temporal synchrony between events. These new methods can be applied not only to calcium imaging but also to many other physiological recordings where discrimination and temporal correlation of biological signals from multiple sites is required.

Imaging activation of peptidergic spinal afferent varicosities within visceral organs using novel CGRPα-mCherry reporter mice.

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.