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Objective: To determine the utility of detecting a-synuclein (aSyn) in colonic mucosal biopsy tissue as a potential diagnostic biomarker for Parkinson disease (PD)., Methods: We used the paraffin-embedded tissue (PET) blot, which degrades physiologic nonaggregated aSyn using proteinase K and enhances antigen retrieval allowing sensitive and selective detection of remaining protein aggregates, to detect aSyn in colonic mucosal biopsies from 15 patients with early PD (,3 years), 7 patients with later PD (.5 years), and 11 individuals without PD. aSyn and serine 129–phosphorylated aSyn (Ser129p-aSyn) were assessed by PET blot and conventional immunohistochemistry., Results: PET blot–resistant aggregated aSyn and Ser129p-aSyn was present in 12 of 15 individuals with early PD, 7 of 7 individuals with later PD, and 11 of 11 control subjects. The number of biopsies positive by PET blot relative to conventional immunohistochemistry was significantly lower in both PD groups compared with the control group for both aSyn and Ser129p-aSyn,whereas routine immunohistochemistry was positive more often in PD, but was positive in as many as 9 of 11 control individuals., Conclusion: Strong evidence of the presence of aggregated hyperphosphorylated aSyn in individuals with and without PD, using such a sensitive and specific method as the PET blot, suggests that colonic deposition of aSyn is not a useful diagnostic test for PD. The utility of detecting aSynin the colon as a biomarker in combination with other assessments remains to be determined.

An accurate early diagnostic test for Parkinson's disease (PD) is a critical unmet need. Recently, independent groups using different histological techniques have reported that the presence of alpha-synuclein (α-syn) in colonic biopsy tissue is able to distinguish living patients with PD from those without the disease. In addition, a further study has suggested that the presence of α-syn in colonic biopsy tissue may be evident in early or even prodromal PD. However, several questions remain regarding the translation of these findings into using the assessment of α-syn deposition in the enteric nervous system as a diagnostic biomarker for prodromal PD. Here we address critical issues related to the location and quantification of enteric α-syn, detection of α-syn with currently available histological techniques, timing of detection of α-syn deposition, and, most crucially, whether enteric α-syn can distinguish those with PD from both healthy individuals and individuals with other related diseases. We conclude that, although enteric α-syn is a very exciting prospect, further studies will be vital to determine whether enteric α-syn deposition has the potential to be the biomarker for prodromal PD that the field so desperately seeks., (© 2013 International Parkinson and Movement Disorder Society.)

Diabetes affects many aspects of gastrointestinal motility, in part due to changes in interstitial cells of Cajal (ICC). The effect of diabetes on the colon, however, is not well characterized, and the aim of the present study was to investigate possible relationships between altered colonic motility as a consequence of streptozotocin-induced diabetes and injury to ICC. Physiological, immunohistochemical, and ultrastructural techniques were employed. The motor pattern of the rat colon was dominated by rhythmic high-amplitude, low-frequency contractions that were primarily myogenic in origin. These rhythmic contractions were induced by stretch associated with increased tension; the amplitude of the superimposed rhythmic contractions increased with increasing applied tension. In diabetic rats, the stretch-induced rhythmic contractile activity remained robust and of similar frequency but was significantly higher in amplitude compared with that in control rats. At 700 mg of applied tension, the force of contraction in circular colonic muscle strips of the diabetic rats was 370% of control values. This robust presence of low-frequency contractions is consistent with the unaffected pacemaker, the ICC associated with Auerbach's plexus, and the increased amplitude correlates with loss of and injury to ICC of the submuscular plexus and intramuscular ICC. Loss of inhibitory nitrergic nerves does not appear to be a factor based on unaltered nNOS immunoreactivity.

Muscarinic receptor mediated membrane currents and contractions were studied in isolated canine colon circular smooth muscle cells. Carbachol (10(-5) M) evoked a slow transient inward current that was superimposed by a transient outward current at holding potentials greater than -50 mV. Carbachol contracted the cells by 70 +/- 2%. The effects of carbachol were blocked by atropine (10(-6) M), tetraethyl ammonium (20 mM), and BAPTA-AM (25 mM applied for 20 min). The inward current and contraction were not sensitive to diltiazem (10(-5) M), nitrendipine (3 x 10(-7) M), niflumic acid (10(-5) M), or N-phenylanthranilic acid (10(-4) M), but were gradually inhibited after repetitive stimulations in Ca2+ free solution. Ni2+ (2 mM) inhibited the inward current by 67 +/- 4%. The inward current reversed at +15 mV. The outward component could be selectively inhibited by iberiotoxin (20 nM) or by intracellular Cs+. Repeated stimulation in the presence of cyclopiazonic acid (CPA, 3 microM) inhibited the carbachol-induced outward current and partially inhibited contraction. CPA did not inhibit the inward current. In conclusion, muscarinic receptor stimulation evoked a CPA-sensitive calcium release that caused contraction and a CPA-insensitive transient inward current was activated that is primarily carried by Ca2+ ions and is sensitive to Ni2+.

The network of interstitial cells of Cajal associated with Auerbach's (myenteric) plexus in the canine colon was investigated to determine its role in facilitating communication between circular and longitudinal muscle layers. Electrical coupling between the muscle layers was demonstrated by propagating extracellularly evoked electrotonic pulses from circular muscle cells to nearby longitudinal muscle cells. The likelihood of cytoplasmic continuity across Auerbach's plexus was further demonstrated by the ability of neurobiotin to spread between the interstitial cells and the circular and longitudinal muscle cells. Importantly, direct neurobiotin spread between circular and longitudinal muscle cells was not observed even when they were in close proximity as determined by confocal microscopy. When neurobiotin did spread across the two muscle layers, the intervening interstitial cells were always neurobiotin-positive. In regions where circular and longitudinal muscle cells approach each other closely, electron microscopy revealed the presence of close appositions between interstitial cells and smooth muscle cells. Gap junctions between interstitial cells and smooth muscle cells of both layers, as judged by electron microscopy, were extremely rare. Neither gap junctions nor close appositions were observed between longitudinal and circular muscle cells. The special arrangement for electrotonic coupling across Auerbach's plexus through interstitial cells of Cajal suggests controlled coupling between the two muscle layers, explaining the preservation of their distinct electrical activities.

The circular muscle (CM) layer of canine colon consists of circumferentially oriented lamellae separated by connective tissue septa. These lamellae facilitate circumferential ring contractions. Communication between CM lamellae is necessary to generate coordinated, propulsive phasic contractions to create peristaltic movement. Potential roles of the submuscular network of interstitial cells of Cajal and branching smooth muscle cells (ICC-bSM), the myenteric interstitial cells of Cajal network (ICC-AP), and the longitudinal muscle (LM) layer in mediating communication between the CM lamellae were studied by simultaneously recording with three surface electrodes, using different types of muscle strip preparations. When the ICC-bSM network was intact, slow waves were observed to be entrained both along and across CM lamellae. In contrast, the CM layer devoid of the ICC-bSM network, the myenteric plexus, and the longitudinal muscle (CM preparation) was spontaneously quiescent. Spike-like action potentials, evoked in the CM preparations by Ba2+ (0.5 mM), were entrained within CM lamellae but were not coordinated between the CM lamellae. In the LM-CM preparations, in which the longitudinal muscle and the ICC-AP network were intact, the Ba(2+)-evoked action potentials were again not coordinated across septa but entrained within CM lamellae. In a step preparation, in which the ICC-bSM network was removed from part of the muscle strip, slow waves were observed to be entrained in areas with and without the ICC-bSM network when electrodes were positioned along septa. When electrodes were positioned across CM lamellae, synchronized slow wave activity was observed only in areas with the intact ICC-bSM network and quiescent activity was recorded in areas devoid of the ICC-bSM network. These results demonstrate that CM cells are electrically coupled within a CM lamella, but not between CM lamellae. The submuscular ICC-bSM network, but not longitudinal or circular muscle cells, nor the ICC-AP, mediates communication between CM lamellae.

Incubation with 50 microM methylene blue (MB) and subsequent intense illumination resulted in abolition of the slow-wave activity in the submuscular interstitial cells of Cajal-circular muscle (ICC-CM) preparations of canine colon. This was often accompanied by a decrease in resting membrane potential. Repolarization of cells back to -70 mV did not restore the slow-wave activity, indicating that MB plus light directly interrupted the generation mechanism of slow waves. After MB incubation, a 2-min illumination consistently changed the mitochondrial conformation in ICCs from very condensed to orthodox, without inducing any obvious changes in smooth muscle cells. After 4- to 10-min illumination, ICCs became progressively more damaged with swollen and ruptured mitochondria, loss of cytoplasmic contrast and detail, loss of caveolae, and rupture of the plasma membrane. No damage was seen in smooth muscle cells or nerves. Gap junctional ultrastructure was preserved. Intense illumination without preincubation with MB left the slow waves and the ultrastructure of ICC-CM preparations unaffected. In CM preparations, without the submuscular ICC-smooth-muscle network, MB plus light induced no changes in electrical activity. We conclude that the correlation between selective damage to the submuscular ICCs (relative to smooth muscle) and selective loss of the slow-wave activity (relative to other electrical activity of the CM) strongly indicates that the ICCs play an essential role in the generation of slow waves.

Two dominant types of action potentials in canine colon are slow wave type action potentials (slow waves) and spike-like action potentials (SLAPs). The slow waves, originating at the submuscular surface where a network of interstitial cells of Cajal (ICCs) is found, possess a pacemaker component. Activation of the pacemaker component is insensitive to voltage changes and L-type calcium channel blockers, and is postulated to involve a metabolic clock sensitive to cyclic AMP. SLAPs are more prominent in the longitudinal muscle. To understand the contribution circular muscle cells make to the generation of these action potentials, a circular muscle preparation (devoid of the submuscular ICC-smooth muscle network, longitudinal muscle, and myenteric plexus) was developed. Circular muscle preparations were spontaneously quiescent, with a resting membrane potential of -62.9 +/- 0.6 mV. Ba2+ (0.5 mM) depolarized the cells to -51.8 +/- 0.6 mV and induced electrical oscillations with a frequency, duration, amplitude, and rate of rise equal to 6.6 +/- 0.4 cpm, 2.2 +/- 0.2 s, 19.4 +/- 0.9 mV, and 21.8 +/- 1.7 mV/s, respectively. In most cases, Ba(2+)-induced oscillations were preceded by a prepotential of 4.4 +/- 0.3 mV, with a rate of rise of 1.1 +/- 0.1 mV/s. Ba(2+)-induced oscillations were abolished by 1 microM D600 as well as by repolarization of 6-12 mV. Addition of 0.1 microM Bay K8644 in the presence of Ba2+ further depolarized circular muscle cells to -42.4 +/- 0.8 mV and increased the oscillation frequency to 16.8 +/- 1.8 cpm. The electrical oscillations induced in circular muscle preparations by Ba2+ and Bay K8644 were similar to the SLAPs exhibited by the isolated longitudinal muscle layer, indicating that generation of SLAPs is an intrinsic property of smooth muscle cells. Forskolin (1 microM), previously shown to dramatically decrease the frequency but not the amplitude of slow waves in preparations including the submuscular ICC network, decreased the amplitude of the Ba(2+)-induced oscillations in circular muscle preparations without changing the frequency. These results provide strong evidence for the hypothesis that the submuscular ICC-smooth muscle network is essential for the initiation of the pacemaker component of the colonic slow waves. The mechanism for regulating the frequency of slow waves is different from that responsible for the Ba(2+)-induced oscillations in circular muscle preparations. Circular muscle cells are shown to be excitable and capable of generating oscillatory activity dominated by L-type calcium channel activity, which is regulated by K+ conductance.

1. Electrical communication between circular muscle, longitudinal muscle and interstitial cells of Cajal (ICC) was investigated; the hypothesis was tested that the resting membrane potential (RMP) gradient in the circular muscle of canine colon is caused by electrical coupling to neighbouring cells. 2. Isolated longitudinal muscle exhibited spike-like action potentials at a RMP of -45 mV with a frequency and amplitude of 20 cycles/min and 12 mV, respectively. 3. The circular muscle (CM), devoid of longitudinal muscle, myenteric plexus and submuscular ICC-smooth-muscle network, was electrically quiescent at a uniform RMP of -62 mV across the entire circular muscle layer. 4. Preparations consisting of only the submuscular ICC network and a few adjacent layers of circular muscle cells exhibited slow wave-type action potentials at a RMP of about -80 mV. 5. In ICC-CM preparations, consisting of the submuscular ICC network and circular muscle, a RMP gradient of 10 mV was observed near the submucosal border, whereas the RMP was constant at -62 mV in the myenteric half of the circular muscle. 6. In full thickness (FT) preparations, a RMP gradient of 23 mV was observed. The RMP decreased gradually from -71 mV at the submucosal border to -48 mV at the myenteric border of the circular muscle. 7. Coupling of longitudinal muscle to circular muscle caused circular muscle cells at the myenteric surface to depolarize by 14 mV and longitudinal muscle cells to hyperpolarize by 3 mV. 8. In the ICC-CM preparations, the slow wave amplitudes did not decay exponentially away from the ICC network indicating that slow waves propagated actively into the circular muscle; in the FT preparations there was an apparent exponential decay but this was due to the RMP gradient. 9. Spike-like action potentials (SLAPs) superimposed on the plateau phase of slow waves did not decay exponentially away from the myenteric border suggesting that SLAPs were generated within the circular muscle layer. 10. In summary, circular muscle cells possess a uniform intrinsic RMP of -62 mV. The RMP gradient in situ is caused by electrical coupling of circular muscle cells to longitudinal muscle cells and the submuscular network of ICC. In situ, slow wave-type action potentials propagate actively into the circular muscle layer, and, dependent on the level of excitation, circular muscle cells actively generate spikes.

The network of interstitial cells of Cajal (ICC) at the submucosal surface of the canine colon was selectively stained by incubation with 15-50 microM methylene blue for 30-45 min. The network was composed of regularly scattered ICC cell bodies interconnected by long processes. Circular muscle cells were unstained. Staining of neurons was limited to one or two axons within bundles. The ICC network had a thickness of a single cell, since no overlapping of ICC cell bodies was observed. The ICC network connected the circular muscle cells at the submucosal surface across the septa which circumferentially divided the circular muscle into lamellae. Methylene blue at 50 microM slightly decreased the resting membrane potential and increased the duration of slow waves, leading to an increase in the force of phasic contractions, with no significant influence on other slow-wave parameters. Methylene blue produced neither electrophysiological nor mechanical effects on circular muscle preparations from which the submuscular ICC network was removed, indicating that the excitatory effects of methylene blue on the full-thickness circular muscle layer were mediated by ICC. In summary, the three-dimensional aspects of the submuscular ICC network can be visualized after selective staining by methylene blue. This staining does not affect physiological characteristics of smooth muscle cells.