Anterograde labeling from lumbosacral DRG in vivo reveals spinal afferent nerve endings identified in internodal strands that run between myenteric ganglia.

<p>A, shows an anterogradely labeled single axon and varicose nerve ending terminating in an internodal strand. B, shows the CGRP image of the panel shown in A. This anterogradely labeled ending is CGRP positive. The arrow in panel B indicates the same spinal afferent axon as in panel A. This CGRP positive axon exits the myenteric ganglion briefly before returning back into the internodal strand. The arrow in panel C, shows a superimposed image of panels A & B. The arrow in panel C indicates a superimposed image of the CGRP positive axon in panel B and the anterogradely labeled axon in panel A. Panel D, shows a second example from a different animal of a single spinal afferent axon that terminates in an internodal strand. E, shows CGRP immunoreactivity of the same region in panel A. The three arrows in panel A indicate three discrete varicosities which are also CGRP positive (see arrows) in panel E. F, shows a superimposed image of panels D & E. The arrows in panel F indicate in this superimposed image of anterogradely labeled varicosities and CGRP immunoreactivity.</p

Anterograde labeling from lumbosacral DRG in vivo reveals two distinct types of spinal afferent nerve ending identified in the submucosa.

<p>A, shows a “branching-type” spinal afferent nerve ending in the submucosa. This ending ramifies extensively with many branching varicose axonal processes that align preferentially in the rostral-caudal axis, within the submucosa. The region indicated by the arrow in panel A is shown on expanded scale in panel B. The nerve endings indicated by the arrow in panel B are not CGRP positive (compare with arrow in panel C). Panel C shows CGRP immunoreactivity of the region shown in panel B. The arrow shows a lack of CGRP immunoreactivity of the ending indicated by the arrow in panel B. Panel D, shows a superimposed image of panels B & C. Panel E, shows a “complex-type” spinal afferent ending in the submucosa at the same level as the base of the Crypts of Lieberkhun. This complex type ending arises from a single spinal afferent axon and then ramifies into a complex structure with multiple varicose axons that branch in no apparent preferential orientation. The arrow in panel E indicates a varicose axonal ending that is not CGRP positive (compare with arrow at the same region in panel F). F, shows CGRP immunoreactivity of the same region shown in panel D. The arrow in panel F shows a lack of CGRP immunoreactivity of the ending in panel E. G, shows a superimposed image of E & F. It is clear that this complex-type spinal afferent ending is not CGRP immunoreactive - compare arrows in panels E, F & G.</p

Anterograde labeling from lumbosacral DRG in vivo reveals intraganglionic varicose endings (IGVEs) in a myenteric ganglion.

<p>A, shows a single axon of an anterogradely labeled spinal afferent that enters a myenteric ganglion then bifurcates into multiple other axon terminals each of which ramifies extensively throughout the ganglion, but lacks any complex nerve terminal specialization. The single axon that enters the ganglion subdivides to form 6 different endings that all terminate at different sites within the same ganglion. B, shows the upper segment of this myenteric ganglion and the spinal afferent that ramifies throughout this region. The arrow indicates a single axon that exits the ganglion briefly before returning back into the same ganglion. C, shows CGRP immunoreactivity of the region shown in panel B. The arrow in C indicates the axon in panel B (indicated by the arrow) is CGRP immunoreactive. Panel D, shows a superimposed image of panels B & C. This IGVE is CGRP immunoreactive (compare arrows in panels B, C & D).</p

Anterograde labeling from lumbosacral DRG in vivo reveals spinal afferent nerve endings in the mucosa of three different mice.

<p>A, a single nerve axon ending is shown consisting of a bare unspecialized terminal with few varicosities. The arrows indicate the trajectory of this axon that is initially out of focus as it projects through the submucosa, then becomes in focus at the level of the mucosa. B, shows CGRP immunoreactivity of the region shown in panel A. The arrows indicate the same region of the axon that is in panel A. C, shows a superimposed image of panels B & A. The arrows indicate the anterogradely labeled axon is CGRP immunoreactive. D, shows a mucosal ending in a different animal consisting of a bifurcating axon, again with very few varicosities along its axon (see arrow). E, shows CGRP immunoreactivity of the region shown in panel D, see arrow. F, shows a superimposed image of panels E and D. This ending is CGRP positive, see arrow. G, shows another example from a different mouse of a varicose spinal afferent ending in the mucosa. The arrow shows a fine varicose ending. H, shows CGRP immunoreactivity of the region shown in G. The arrow indicates a lack of CGRP immunoreactivity of the ending shown by the arrow in panel G. I, shows a superimposed image of panels G and H. It can be seen that this mucosal ending is not CGRP positive (see arrows).</p

Anterograde labeling from lumbosacral DRG in vivo reveals spinal afferent endings and axons in blood vessels.

<p>A, shows a single axon and fine nerve ending that terminates without any complex morphology and few varicosities along its axon. The arrows in A indicate the trajectory of single anterogradely labeled axons. B, shows CGRP immunoreactivity of the same region shown in panel A. The arrows in B show positive CGRP immunoreactivity of the anterogradely labeled axons in panel A (indicated by arrows). Many other CGRP immunoreactive nerve fibres project along this blood vessel but were not identified after anterograde labeling. Panel C, shows a superimposed image of panels B & C, with the arrows indicating colocalization between anterogradely labeled axons and CGRP. Panel D, shows a blood vessel from a different animal that bifurcates and branches into two other vessels. Spinal afferent axons ramify along and around these blood vessels. A single axon (see arrow) can be seen ramifying over one of the blood vessels. E, shows CGRP immunoreactivity of the same region shown in panel D. The arrow indicates the same axon as in panel D (see arrow) and this axon is CGRP positive. F, shows a superimposed image of panels D & E and the arrow shows colocalization of the anterogradely labeled axons with CGRP.</p

Anterograde labeling from lumbosacral DRG in vivo reveals two different types of spinal afferent nerve endings that innervate the circular muscle layer.

<p>A, shows a “simple class” of ending that consists of a single axon that does not branch, but rather projects out of a myenteric ganglion and then a short distance into the CM, terminating in a varicose ending (see arrow). B, shows CGRP immunoreactivity of the image shown in panel A. The arrow in panel B indicates the same varicose ending as shown by the arrow in panel A. This nerve ending is CGRP positive. Panel C, shows a superimposed image of A & B with the arrow indicating the same varicosity as in panels A & B. Panel D shows a “branching-type” ending in the CM layer. This ending arose from a single parent axon, that subdivided into multiple other varicose axons running parallel to the CM fibres. The region indicated by the arrow is shown in expanded scale in panel E. The arrow in panel E indicates a varicose ending in the CM layer. Panel F, shows a CGRP image of the region shown in panel E. The arrow in panel F indicates that the same ending as shown in panel E (see arrow) which is CGRP positive. Panel G, shows a superimposed overlay of panels E & F. All the anterogradely labeled axons can be seen to be CGRP positive. There are other CGRP immunoreactive axons that were not identified by anterograde labeling.</p

Anterograde labeling from lumbosacral DRG in vivo reveals a “complex-type” spinal afferent nerve ending in the circular muscle layer.

<p>A, shows a montage of a single spinal afferent axon that traverses through many myenteric ganglia, then projects into the CM layer forming a highly complex axonal arbor with multiple varicose branch points. These axon terminals do not align in any preferential orientation with the CM layer. Panel B, shows an expanded portion of the region indicated by the arrow b in panel A. Arrow C in panel A indicates part of the region shown in panel C on expanded scale. Panel D, shows the CGRP immunoreactivity of the region shown in panel C. Panel E, shows a superimposed image of panels C & D. This complex type ending is CGRP positive.</p

Diagrammatic representation of the different types of spinal afferent endings identified in the mouse large intestine, following minute injections of dextran biotin into lumbosacral DRGs in vivo.

<p>A, shows a schematic of the nerve pathways utilized to transport dextran biotin to the distal colon. B, shows that 13 distinct types of nerve endings were identified. 1 shows varicose endings in longitudinal muscle which occurred extremely rarely. Numbers 2 & 3, refer two types of endings identified in myenteric ganglia. 2 refers to intraganglionic varicose endings (IGVEs) which are common, while 3 refers to rectal intraganglionic laminar endings (rIGLEs) which are rare, 4 refers to varicose endings in internodal strands. 5 refers to “branching-type” endings in the CM layer that run parallel to the CM fibres, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g006" target="_blank">Figure 6D</a>. 6 refers to “simple-type” afferent endings in the CM that consist of a single non-branching varicose axon, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g006" target="_blank">Figure 6A</a>. 7 refers to the “complex-type” endings in the CM that arise from a single axon and ramify extensively throughout the CM layer in no preferential orientation, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g005" target="_blank">Figure 5A</a>. 8, refers to “branching-type” spinal endings in the submucosa that ramify extensively in the rostral-caudal axis, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g009" target="_blank">figure 9A</a>. 9, refers to endings on blood vessels. 10, refers to rIGLEs in submucosal ganglia. 11, refers to “complex-type” endings at the level of the Crypts of Lieberkhun, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g009" target="_blank">Figure 9E</a>. 12, refers to simple type endings in the submucosa that consist of axon terminals that encircle the base of the Crypts with few or no varicosities, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g010" target="_blank">Figure 10A & 10D</a>. 13, refers to nerve endings that innervate into the mucosa. KEY: LM refers to longitudinal muscle, MG = myenteric ganglion, CM = circular muscle, SM = submucosa, SG = submucosal ganglion and CL = Crypts of Lieberkuhn.</p

Characteristics of different classes of spinal afferent nerve endings identified in mouse large intestine following anterograde labeling from DRG in vivo.

<p>Characteristics of different classes of spinal afferent nerve endings identified in mouse large intestine following anterograde labeling from DRG in vivo.</p

Graphical representation of the relative proportion of spinal afferent nerve endings that innervate distinct anatomical sites within the large intestine.

<p>A, shows that the majority of nerve endings were identified in the circular muscle layer (3 distinct types present), submucosa (3 distinct types present) and myenteric ganglia including internodal strands (3 distinct types). Very few endings were identified in submucosal ganglia and longitudinal muscle. 11% of all anterogradely labeled spinal afferent endings innervated the mucosa. B, shows the percentage of labeled spinal afferent endings that were immunoreactive (positive and negative) for CGRP. Some classes of spinal afferent nerve endings such as simple endings in the CM layer, those in internodal strands and blood vessels were always CGRP immunoreactive. However, rIGLEs in myenteric ganglia, complex-type endings in submucosa and branching-type endings in submucosa were never found to be CGRP positive.</p