Morphology of cat phrenic motoneurons as revealed by intracellular injection of horseradish peroxidase

The morphology of phrenic motoneurons (PMs) of adult cat was examined by utilizing the technique of intracellular injection of horseradish peroxidase. Twenty-one cells were reconstructed from serial sections in transverse, sagittal, and horizontal planes. The cell bodies were ellipsoid, with the major diameter oriented parallel to the longitudinal axis of the spinal cord. The dendrites of PMs are not distributed in a radially symmetric fashion, but rather project to four separate fields. The field containing the greatest number of dendrites extends rostrocaudally within the phrenic motor column. This collection of dendrites forms a rostrocaudal bundle in which the dendrites from neighboring PMs lie in close association with one another. The remaining dendrites project dorsolaterally, dorsomedially, and to a lesser extent, ventrally. The dorsolaterally directed dendrites form bundles upon entering the lateral funiculus with the dendrites from other PMs. Several of the dorsomedially directed dendrites cross to the contralateral spinal cord via the anterior commissure or central gray. A wide variety of dendritic spines and appendages was observed. There were no instances in which axon collaterals were observed for the 11 well-stained axons examined. The length of the initial segment of the axon was a function of the distance of the cell body from the ventral funiculus.     

The ultrastructure of group Ia afferent fiber synapses in the lumbosacral spinal cord of the cat

Ia synapses in laminae VI and IX of the cat's spinal cord were examined in the electron microscope following iontophoretic injection of horseradish peroxidase (HRP) into single, identified, Ia afferent fibers from gastrocnemius muscles. Ia boutons contacting motoneuron dendrites in lamina IX contained spherical synaptic vesicles and generally contracted only one postsynaptic profile. The Ia boutons were often postsynaptic to smaller P-type axonal terminal. Consequently Ia boutons may be classified as S-boutons with axo-axonic contacts.     

An ultrastructural study of serially sectioned Renshaw cells. II. Synaptic types

Boutons and synaptic contacts on 17 presumed Renshaw cells were studied ultrastructurally. All 17 neurons were postsynaptic to axon collateral boutons of intracellularly HRP-stained triceps surae α-motoneurons and located in lamina VII, ventromedially to the main motor nuclei.The boutons and synaptic contacts could be classified into two main categories on the basis of the synaptic vesicles: S-type boutons with spherical synaptic vesicles and F-type boutons with flattened vesicles, the α-motoraxon collateral boutons falling into the S-category. In addition, some S-type boutons containing neurofilaments and some being apposed by small presynaptic boutons were observed. The results are discussed in relation to earlier observations on the synaptology of central neurons, particularly spinal α-motoneurons.     

Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). II. A parametric study in the peripheral motor system of the cat

Fluorescent compounds which are currently used as retrograde tracers were tested in the cat peripheral motor system and compared with horseradish peroxidase (HRP). The tracers were either injected into forelimb muscles or applied to the proximal end of transected forelimb nerves. The remaining muscles of the limb has been carefully denervated. Following intramuscular injection all fluorescent compounds labeled spinal cord motoneurons, the DAPI compounds labeled endothelial cells in addition. In the nerve application mode tracer positive motoneurons were only observed when propidium iodide (PI) and the DAPI compounds were used, whereas bisbenzimide (BB), nuclear yellow (NY) and primuline did not label any cells. The fluorescence of BB labeled motoneurons were predominantly located in the cytoplasma. NY positive motoneurons showed a different localization of the fluorescent label between the different neurons of the same motornucleus: in some neurons it was exclusively located in the nucleus, in others predominantly in the cytoplasma, in the majority in both compartments. The intracellular distribution of the BB and the NY label was independent of the pH of the fixation fluid. The fluorescent tracers labeled the motoneuronal cell columns in their complete rostrocaudal extent and in a position identical to the one obtained with HRP. However, some substances (PI, fast blue) labeled less motoneurons of a motornucleus than did HRP, none of the fluorescent tracers labeled more. The results are discussed under several aspects: use of the investigated fluorescent compounds as single tracers; use of several tracers in the same animal to map collateral projections of one and the same neuron; use of several tracers in the same animal to establish the topographical relation between several independent neuronal populations.     

An ultrastructural study of serially sectioned renshaw cells. III. Quantitative distribution of synaptic boutons

The quantitative distribution of synaptic boutons on 17 presumed Renshaw cells has been studied ultrastructurally. All 17 neurons were postsynaptic to axon collateral boutons of intracellular HRP-stained triceps surae α-motoneuorons and were located in lamina VII, ventromedially to the main motor nuclei.In each of the presumed Renshaw cells, the values for mean length and mean area of apposition, percentage synaptic covering, and packing density of S-type, F-type, and S+F-type boutons were estimated on the cell body and in two dendritic compartments. The F/S percentage synaptic covering ratio was also calculated. The previously demonstrated differences within the present group of neurons, with respect to the site of axonal origin, were not accompanied by any corresponding differences in the quantitative distribution of synaptic boutons. However, it is suggested that the presumed Renshaw cells may possibly fall into two categories with respect to the F/S percentage synaptic covering ratio. The results are discussed in relation to previous studies on the neuronal architecture and synaptic types on the same presumed Renshaw cells, as well as in relation to earlier observations on the quantitative distribution of boutons on central neurons, particularly spinal α-motoneurons.     

Light microscopic observations on cat Renshaw cells after intracellular staining with horseradish peroxidase. I. The axonal systems

Five intracellularly HRP-stained Renshaw cells were subjected to light microscopic analysis of the trajectories, branching patterns, and projections of the axonal systems. The cell bodies were located ventrally in lamina VII. In three neurons the axon originated from the cell body and in the remaining two cells from a dendrite. After a 600-870-microns distance the axons entered the ventral funiculus, where all of them continued rostrally. Two axons also gave off a caudal branch in the funiculus. The diameters of the main axons varied between 2.1 and 10.0 microns. The main axons gave off one to four first-order collaterals before entering the ventral funiculus and up to three collaterals could be seen to originate from the same node of Ranvier. In the ventral funiculus up to five first-order collaterals could be traced from the same main axon. The axon collateral trees were often very extensive and daughter branches up to the 22nd order were observed. The distance between two successive branching points varied between 4 and 410 microns. A large number of boutonlike swellings were found along (59%) or at the ends of the collateral branches. At the most, 1,278 swellings originated from a single axon collateral tree. Most of the swellings were located in lamina IX, but they also appeared ventrally and dorsolaterally in lamina VII.     

Light microscopic observations on cat Renshaw cells after intracellular staining with horseradish peroxidase. II. The cell bodies and dendrites

The cell bodies and dendritic trees of five lumbosacral Renshaw cells of adult cats were studied in the light microscope (LM) after intracellular injection with horseradish peroxidase (HRP). The cell bodies were all located in the ventral part of lamina VII. The dendrites extended up to 0.7 mm from the cell body into the neighbouring parts of laminae VIII and IX as well as into more dorsal parts of lamina VII. The dendritic branching was sparse and about half the dendrites were unbranched. The mean diameter of the cell body was positively correlated to both the combined and mean diameters of the first-order dendrites. Between four and eight dendrites originated from the cell bodies. The number of dendritic end-branches, the combined dendritic length, the mean dendritic length from the cell body to the termination of the end branches, the distance from the cell body to the termination of the most remote end-branch, the dendritic surface area, and the dendritic volume all correlated positively with the diameter of the parent first-order dendrite. The dendritic tapering was somewhat more pronounced in the Renshaw cells than previously observed in alpha- and gamma-motoneurons. The present data are discussed in relation to previous morphological observations on Renshaw cells and alpha- and gamma-motoneurons.     

Decreased horseradish peroxidase labeling in deafferented spinal motoneurons of the rat

It has been suggested^29,50 that the incorporation and retrograde transport of horseradish peroxidase (HRP) were linked to the level of neuronal activity. Therefore one could postulate that the motor impairment resulting from dorsal rhizotomy affects the HRP labeling of spinal motoneurons in the absence of morphological damage to the motor system. This hypothesis was tested in the adult rat by sectioning bilaterally the L₃-L₅ dorsal roots. 2–18 months after surgery, the L₄ radicular nerve was immersed in a solution of HRP. Labeled motoneurons were counted together with the motor axons of the L₄ ventral root and results were compared with values obtained in paired controls. Deafferentation resulted in a crippling deficit of lower movements with disuse atrophy of muscle fibers but had no effect on the fiber population of the sciatic nerve and the L₄ ventral root. Whereas in normal animals the L₄ HRP-labeled motoneurons represented 71.9–98.3% (average 85.4) of the motor axonal counts, in animals studied 4, 12 and 18 months after dorsal rhizotomy, the number of motoneurons containing HRP granules constituted only 20.1–55.7% (average 46.2) of the number of motor axons and many of the labeled cells were faintly stained. These findings, which may reflect either a decreased retrograde transport of HRP in deafferented motoneurons or an increased turnover of the enzyme in the cell body, call attention to the possibility that the degree of activity in neuronal pathways influences HRP labeling.     

Neurons at the origin of the medical component of the bulbopontine spinoreticular tract in the rat: An anatomical study using horseradish peroxidase retrograde transport

An anatomical technique based on the retrograde transport of horseradish perodidase (HRP) was used to investigate the progections of sponal cord neurions to the reticular formations in the rat. Both large and restricted injections were staggered all along the bulbar and pontine levels, involving the nucleus gigantocellularis, the nuclei reticularis pontis, pars oralis and cauladis and in some cases the nucleus raphé magnus. Labeled cells we constantly encountered in the reticular part of the neck of the dorsal horn throughout the whole length of the cord, mainly contralateral to the central core of the injection iste. This area was taken as the equivalent of lamina V in the cat. Other labeled cells were observed in the medial arts of the intermediate and ventral horns, in areas considered similar to laminae VII and VIII in the cat. The two most rostral cervical nating form the dorsolateral part of ventral horns. Thus, this study is a clear confirmation that the bulbopontine reticular formations constitute a target for various somatosensory inputs originating in spinal cord. It demonstrates that the medial spinoreticular tract (mSRT) differs from the other main ascending tracts by the absence of projections from (1) superficial layers and nucleus of the dorsolateral funiculus contrary to the spinomesencephalic tract; (2)ventromedial zone of the lumber dorsal horn unlike the spinothalamic tact; (3) the neck of the doral hor in its medial portuion contrary to the spinoreticular component reaching the lateral reticular nucleus; and (4) central cervical nucleus and Clarke's columns, unlike the spinocerebellar tracts. The difficulty in demonstrating retrograde labeling from discrete injections coulde result from the fact that mSRT neu-rons have sparsely ramified collaterals on their terminal zones.     

Quantitative analysis of the dendrites of cat phrenic motoneurons stained intracellularly with horseradish peroxidase

All the dendrites (N = 37) generated by four phrenic motoneurons were analyzed following intracellular injection of horseradish peroxidase. The dendritic arbors produced from each of these stem dendrites were studied in detail. The mean number of stem dendrites produced by a phrenic motoneuron was 9.7, their mean diameter was 6.0 micron, and their mean combined diameter was 58.3 micron. The length at which a phrenic motoneuronal dendrite terminated was 1,236 micron, with several end terminals extending more than 2 mm from the cell body. The mean value for the combined lengths of all segments originating from a single stem dendrite was 5.3 mm. A full spectrum of dendritic branching patterns was observed from simple (five unbranched) to complex, the latter producing up to ninth-order branches. Most terminal and nonterminal dendritic segments tapered, producing a mean diameter reduction of 34%, or approximately 9% per 100-micron length. All phrenic motoneurons exhibited a steady decrease in the combined dendritic parameter (sigma d3/2) with distance from the soma as a result of tapering and end-branch termination. The mean surface area and volume of a phrenic motoneuronal dendrite were 35.3 X 10(3) micron 2 and 25.9 X 10(3) micron 3, respectively. The dendrites constituted greater than 97% of the total phrenic motoneuronal surface area, with 75% of this area lying outside of a 300-micron radius from the cell body. The diameter of a stem dendrite was positively correlated with its combined dendritic length, number of terminal branches, dendritic surface area, and volume. Despite this strong correlation, the value of total dendritic surface area calculated using the power equation derived from the dendritic surface area versus stem dendritic diameter plot was not a consistent estimator of the total dendritic surface area directly measured for these four phrenic motoneurons. It is suggested that this inconsistency may be the result of a heterogeneity in the phrenic motoneuronal population and/or in the dendrites projecting to the different terminal fields.     

An examination of intraspinal sprouting in dorsal root axons with the tracer horseradish peroxidase

Postdeafferentation reorganization in the central terminal fields of spared dorsal root axons was evaluated by examining the intraspinal distribution of horseradish peroxidase-labeled sciatic nerve afferent fibers at various intervals following the removal of several lumbar dorsal root ganglia. The sciatic projection to the spinal cord, as determined by the pattern and density of intraspinal reaction product, was remarkably stable following the ganglionectomies. For as long as 3 months later, there was no evidence that sciatic afferent fibers had formed anomalous connections either with new spinal segments or in denervated areas within normal segments of entry. These findings cast doubt upon the existence of anatomic reorganization within the spinal cord following its partial deafferentation and suggest that physiological processes other than new axonal growth underlie observations such as postdenervation alterations in the response properties of dorsal horn neurons and the recovery of behavioral function.     

Factors determining the variation of the afterhyperpolarization duration in cat lumbar α-motoneurons

The duration of the afterhyperpolarization (AHP) in cat spinal α-motoneurons varies systematically with motoneurone type, being shorter in motoneurones projecting to fast-contracting muscle units. Recent experiments have shown that the AHP duration is correlated with the amount of sag found in the voltage response to injected constant current pulses. Using a model of the sag process, the present study shows that this correlated is likely to be causal to a substantial extent. Short AHP durations in fast motoneurones may thus be as much, or more, a consequence of a more developed sag process than of faster kinetics of the K conductance process underlying the AHP. This notion is also supported by the experimental observation of a decreased amount of sag and a prolonged AHP duration after axotomy.     

Segmental distribution and central projectionsof renal afferent fibers in the cat studied by transganglionic transport of horseradish peroxidase

The segmental and central distributions of renal nerve afferents in adult cats and kittens were studied by using retrograde and transganglionic transport of horseradish peroxidase (HRP). Transport of HRP from the central cut ends of the left renal nerves labeled afferent axons in the ipsilateral minor splanchnic nerves and sensory perikarya in the dorsal root ganglia from T12 to L4. The majority of labeled cells (85%) were located between L1 and L3. A few neurons in the contralateral dorsal root ganglia were also labeled. Labeled cells were not confined to any particular region within a dorsal root ganglion. Some examples of bifurcation of the peripheral and central processes within the ganglion were noted. A small number of preganglionic neurons, concentrated in the intermediolateral nucleus, were also identified in some experiments. In addition, many sympathetic postganglionic neurons were labeled in the renal nerve ganglia, the superior mesenteric ganglion, and the ipsilateral paravertebral ganglia from T12 to L3 Transganglionic transport of HRP labeled renal afferent projections to the spinal cord of kittens from T1 1 to L6, with the greatest concentrations between Ll and L3. These afferents extended rostrocaudally in Lissauer's tract and sent collaterals into lamina I. In the transverse plane, a major lateral projection and a minor medial projection were observed along the outer and inner margins of the dorsal horn, respectively. From the lateral projection many fibers extended medially in laminae V and VI forming dorsal and ventral bundles around Clarke's nucleus. The dorsal bundle was joined by collaterals from the medial afferent projection and crossed to the contralateral side. The ventral bundle extended into lamina VII along the lateroventral border of Clarke's nucleus. Some afferents in the lateral projection could be followed ventrally into the dorsolateral portion of lamina VII in the vicinity of the intermediolateral nucleus. In the contralateral spinal cord, labeled afferent fibers were mainly seen in laminae V and VI These results provide the first anatomical evidence for sites of central termination of renal afferent axons. Renal inputs to regions (laminae I, V, and VI) containing spinoreticular and spinothajamic tract neurons may be important in the mediation of supraspinal cardiovascular reflexes as well as in the transmission of activity from nociceptors in the kidney. In addition, the identification of a bilateral renal afferent projection in close proximity to the thoracolumbar autonomic nuclei is consistent with the demonstration in physiological experiments of a spinal pathway for the renorenal sympathetic reflexes.     

Electron microscopic observations on the synaptic contacts of group Ia muscle spindle afferents in the cat lumbosacral spinal cord

After intra-axonal injection of horseradish peroxidase (HRP) into afferent fibers originating from muscle spindle primary endings of the cat gastrocnemius, group Ia boutons located in the ventral horn of the spinal cord were identified and studied electron microscopically. The Ia boutons were invariably found to contain spherical synaptic vesicles (S-type boutons), and a number of them were also postsynaptic to smaller P-type boutons (large S-type boutons with axo-axonic contacts). None of the present Ia- boutons belonged to the previously described M-type⁸. The vast majority of the studied boutons were considered to be located at less than 500 μm distance from the α-motoneuron soma. The results are discussed in relation to previous light and electron microscopic data.     

Activity of motoneurons during fictitious scratch reflex in the cat

1. (1) Intracellular recording of motoneurons of different hindlimb muscles: tibialis anterior (TA), gastrocnemius and soleus (GS), vastus crureus (Vast), posterior biceps and semitendinosus (PBSt), was carried out during the fictitious scratch reflex⁵ in decerebrate cats.

2. (2) During the postural stage of the reflex, a depolarization (3.8 mV on average) was observed in TA motoneurons accompanied by tonic discharge. No change of the membrane potential (MP) and no discharge were observed during this stage in GS, Vast and PBSt motoneurons.

3. (3) In the rhythmical stage of the reflex, the MP of TA motoneurons changed only slightly during the ‘long’ (L) phase⁵ of the scratch cycle and remained at approximately the same level as during the postural stage. In this phase, motoneurons discharged at frequencies of 20–100 pps. In the ‘short’ (S) phase⁵ of the scratch cycle a strong repolarization occurred, the MP reached the same level as observed during resting conditions (MP₀), and the discharge discontinued.

4. (4) GS motoneurons were gradually depolarized during the second half of the L-phase. The depolarization reached its maximum (5.5 mV on average in relation to the MP₀) in the S-phase, and several action potentials were generated with intervals of 5–10 msec. Then, at the beginning of the L-phase, the motoneurons were repolarized and the MP reached the level of the MP₀. The behavior of Vast motoneurons was essentially similar to that of GS motoneurons.

5. (5) The PBSt motoneurons usually had two peaks of depolarization per cycle — in the S-phase and at the beginning of the L-phase. The maximal depolarization was 3.5 mV (on average). The motoneurons generated action potentials at one or both peaks of depolarization.

6. (6) The possible organization of the central influences upon motoneurons of different muscles during scratching is discussed.

Brainstem afferents to the omnipause region in the cat: a horseradish peroxidase study

"Omnipause" neurons (OPNs), located in the nucleus raphe pontis and the reticular formation, actively suppress saccadic eye movements during intersaccadic intervals. To determine the brainstem afferents that may inhibit the OPNs and thereby allow a saccade to occur, we injected horseradish peroxidase into the raphe pontis of four cats at the site of physiologically identified OPNs. Labeled neurons were found in a number of brainstem nuclei. The greatest concentrations, composed of small to medium-sized neurons, were located in a group of nuclei around the habenulopeduncular tract, in the rostral mesencephalic reticular formation, in the deep layers of the superior colliculus, and in parts of the subjacent cuneiform and subcuneiform reticular nuclei. Smaller numbers were found in the nucleus reticularis pontis oralis. Caudal to the injection site, labeled neurons were scattered in parts of the nuclei reticularis gigantocellularis, paragigantocellularis dorsalis, and paragigantocellularis lateralis. A few neurons were labeled in a restricted region of the causal part of the nucleus prepositus hypoglossi and in the nucleus reticularis medullaris ventralis. Larger numbers of neurons were labeled in the dorsal column nuclei and in parts of the cochlear nuclei. Smaller numbers were found in the spinal trigeminal nucleus, the lateral nucleus of the superior olive, and the fastigial nucleus of the cerebellum. The nonreticular brainstem projections may contribute sensory information in a number of modalities since OPNs respond to visual, somesthetic, and auditory stimuli. Our findings indicate a number of regions that may contain neural elements impinging on the OPNs. The best prospects for a saccade initiation signal from one of the labeled populations appear to be the meso-diencephalic reticular formation and/or the superior colliculus.     

Ultrastructural study of large efferent neurons in the superior colliculus of the cat after retrograde labeling with horseradish peroxidase

The ultrastructure of large neurons in the stratum griseum intermedium of the cat superior colliculus was examined following injections of horseradish peroxidase (HRP) into the dorsal tegmental decussation. Four HRP-labeled cells were selected, and the synaptology of their cell bodies and selected regions of proximal and distal dendrites was examined. The four neurons represent four morphologically distinct cell types: multipolar radiating, tufted, large vertical, and medium-sized trapezoid radiating. These four neurons correspond with cell types X1, X2, X3, and T1 respectively, according to the recent classification of neurons in the superior colliculus of the cat by Moschovakis and Karabelas (J. Comp Neurol. 239:276-308, '85). The three X type neurons are similar in having 83% of their somata and over 74% of their proximal dendrites contacted by synaptic profiles. Distal dendrites of the X type neurons, however, receive fewer synaptic contacts. In contrast, in the T1 cell, only 69% of the soma membrane is contacted by synaptic profiles, and the synaptic coverage on proximal and distal dendrites does not vary much from this. Of the eight types of synaptic terminals described in the stratum griseum intermedium of the cat superior colliculus by Norita (J. Comp. Neurol. 190:29-48, '80), only five are found in contact with the X and T type efferent neurons described here. There are some regional differences in terminal distribution, although each terminal is represented on each cell. Type III terminals (small, contain mostly pleomorphic vesicles, and make symmetrical contacts) are the most abundant on cell bodies and dendrites of all four cell types. Terminal types II (medium-sized, containing round and flattened vesicles, and making asymmetrical contacts), and IV (medium to large in size, containing flattened vesicles, and making symmetrical contacts) are well represented. In general, terminal types I (small, containing densely packed round vesicles, and making asymmetrical contacts) and VI (small and irregular in shape, containing flattened vesicles and making symmetrical contacts) are found infrequently. The identity of different types of synaptic terminal is discussed.     

Spinal neurons reaching the lateral reticular nucleus as studied in the rat by retrograde transport of horseradish peroxidase

An anatomical technique based on the retrograde transport of horseradish peroxidase (HRP) was used to investigate the projections of spinal cord neurons to the lateral reticular nucleus (LRN). Labeled cells were found at all spinal levels and in particular large numbers in cervical and lumbar segments. Various spinal areas gave rise to cells of origin of this tract, which appears to be more prominent than any other tract previously studied with a similar approach. Labeling common to all spinal segments was observed in (1) ventromedial parts of both intermediate zone and ventral horn (laminae VII, VIII and X), mainly contralaterally; (2) the reticular extension of the neck of the dorsal horn, partly bilateral; and (3) superficial layers of the dorsal horn and nucleus of the dorsolateral funiculus (NDLF), mainly contralateral and projecting essentially to the lateral zone of the LRN. Additional labeling was observed at cervical and lumbar levels, each with specific qualities: (1) the cervical enlargement, which displayed labeling in the central part of the ipsilateral intermediate zone (lamina VII); (2) the rostral lumbar levels, which had projections from the contralateral median portion of the neck of the dorsal horn. These latter projections appear to be specific to pathways reaching the lateral reticular nucleus and the inferior olive. Control injections in neighboring structures demonstrated the similarity between the afferents to the lateral reticular nucleus and the inferior olive. Control injections in neighboring structures demonstrated the similarity between the afferents to the lateral reticular nucleus and the inferior olive (except lamina I and NDLF projections) and the differences between these afferents and those projecting to the dorsal reticular formation, i.e., the nucleus reticularis ventralis.     

Morphological relationships among extensor digitorum longus, tibialis anterior, and semitendinosus motor nuclei of the cat: an investigation employing the retrograde transport of multiple fluorescent tracers.

To determine the morphological relationships among extensor digitorum longus (EDL), tibialis anterior (TA), and semitendinosus (St) motor nuclei in the spinal cord of the cat, these nuclei were retrogradely labeled with three different fluorescent tracers. The fluorochromes--bisbenzimide, nuclear yellow, and propidium iodide--were applied by intramuscular injection or soaking the muscle nerve. The positions of the labeled motor nuclei were bilaterally symmetrical. The EDL and TA motoneurons were located in close proximity to one another, in the lateral regions of lamina IX in spinal segments L6 and L7. Although the boundaries of each nucleus were tightly opposed, the EDL and TA motor nuclei overlapped minimally, with the somata of EDL motoneurons positioned dorsal to those of TA. The St motor nucleus was located ventromedial to that of EDL and extended from the caudal portion of L6 through S1. Supplemental studies of the reflex effects evoked in EDL, TA, and St muscles by cutaneous nerve stimulation provided physiological observations that may be related to these anatomical results.