259
J. Anat. (1990), 171, pp. 259-263 With 2 figures Printed in Great Britain
Comment J. M. WALRO AND J. KUCERA*
Department of Anatomy, Northeastern Ohio Universities College of Medicine, 4209 State Rt 44, PO Box 95, Rootstown, Ohio 44272, USA and *Department of Neurology, School of Medicine, Boston University, Boston, MA 02130, USA
A recent paper by Arbuthnott and her colleagues in the Journal of Anatomy (163, 183-190, 1989) re-examined some of our previous work on rat soleus muscle spindles (Walro & Kucera, Exp. Brain Res. 56, 187-192, 1984; Walro & Kucera, Am. J. Anat. 173, 55-68, 1985) that showed distinct differences in the neural organisation of rat soleus and cat tenuissimus spindles. Their paper cast doubt on the validity of our observations concerning the fusimotor innervation to rat spindles. We feel that many of their observations are either inaccurate or inconsistent with previously published data on rat spindles. We feel obliged to point out these inaccuracies or inconsistencies because the issues raised in these two papers are important to understanding muscle spindle function in mammals. Arbuthnott et al. (1989) questioned the reliability of tracing fusimotor axons from 1 Ium plastic sections stained with toluidine blue by light microscopy alone, yet they proceeded to replicate our study by this same technique. They claim that they were able to trace accurately the distribution of motor axons to intrafusal fibres in the rat soleus muscle only because all fusimotor axons were myelinated to within 10 l,m of their termination on the fibres. However, as we reported previously (Walro & Kucera, 1984, 1985) and as we show in Figures 1A and B, some rat fusimotor axons had unmyelinated preterminal segments up to several hundred ,um in length. Thus, Arbuthnott et al. (1989) apparently omitted some unmyelinated axons that terminate on intrafusal fibres from their sample. Arbuthnott et al. (1989) implied that we might have misidentified the nuclear bag, and the nuclear bag2 fibres in our sample, thereby invalidating the patterns of innervation that we observed. Initially, we used the criterion of size, the bag2 being larger than the bag1 fibre, to discriminate between the two types of nuclear bag fibres. Subsequently, size and distribution of granules (mitochondria) and the number and distribution of motor endplates, on the fibre we assumed to be the bag1 in the 1 um sections, correlated with patterns of staining for oxidative enzymes such as NADHTR and the distribution of sites of acetylcholinesterase (AChE) activity in fibres identified as bag1 by their staining for mATPase after preincubation in an acid or alkaline medium (Kucera et al. J. Histochem. Cytochem. 26, 973-988, 1978). Alternatively, they proposed that dissociation of the bag, fibre from the rest of the intrafusal bundle in the sensory region, a feature of cat spindles, is the most reliable criterion for identifying the bag1 fibre in rat spindles. The bag1 fibre occupies a separate compartment and does not share sensory terminals with the bag2 fibre in cat spindles (Banks et al. Phil. Trans. R. Soc. London B 299, 329-364, 1984). However, the bag1 and bag2 fibres share a compartment formed by inner capsule cells throughout most of the sensory region (Fig. 2 A) and even share a common basal lamina at sites where they share sensory terminals in rat soleus and extensor digitorum longus spindles (Diwan & Milburn, J. Embryol. exp. Morphol. 92, 223-254, 1986; Walro & Kucera, Brain Res. 425, 311-318, 1987). Thus the assertion by Arbuthnott et al. that the bag, fibre dissociates itself from the remainder of the intrafusal bundle in rat
260
J. M. WALRO AND J. KUCERA
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Comment 261 spindles conflicts directly with observations made by light and electron microscopy of spindles in two different rat muscles by two independent laboratories. Arbuthnott et al. (1989) reported that all motor axons that innervated intrafusal fibres were myelinated to within 10,m of their termination on intrafusal fibres. This observation conflicts with our observation that some of the axons to the bag, fibre were unmyelinated at entry into the spindle or were unmyelinated for considerable distances as they coursed within the spindle capsule prior to their termination on intrafusal fibres (Walro & Kucera, 1984, 1985). We classified these axons as fusimotor because we observed their terminations on intrafusal fibres in 1 ,um sections. Moreover, we were convinced that these axons were fusimotor rather than autonomic, because the number and location of motor endings observed on the three types of intrafusal fibres in 1 ,um sections stained with toluidine blue correlated with the number and location of sites of AChE on the three types of intrafusal fibres identified by their patterns of staining for mATPase (Kucera et al. 1978). Several unmyelinated fusimotor axons or long unmyelinated branches of myelinated fusimotor axons similar to those we described previously by light microscopy are visible in Figures 1 and 2. These axons are clearly somatic because they are larger than autonomic axons (Fig. 1 A) and terminate in identifiable endings on intrafusal fibres (Figs. 1, 2). We are unable to explain why these axons were not reported by Arbuthnott et al. (1989). They either did not see these axons in their preparations or might have assumed that these axons were autonomic. At any rate, they did not check the identity of these axons by electron microscopy or reconcile their data with the distributions of sites of AChE activity on different types of intrafusal fibres (Kucera et al. 1978). Exclusion of these axons that terminate predominantly on the bag, fibre might also explain why they reported that 75 % of bag1 fibre poles had zero or one motor ending, whereas we observed all bag1 fibre poles to be innervated by motoneurons at multiple sites, and why they observed fewer motor axons to rat soleus spindles than we did. Arbuthnott et al. (1989) concur with our observation that the dynamic bag1 and static bag2 and/or chain fibres are co-innervated by fusimotor axons, but claim that the frequency of axons distributed to dynamic and static intrafusal fibres in their sample was significantly less than the frequency we reported (Walro & Kucera, 1985). Had they performed a statistical analysis of the data in the two studies, they would have found that the 5 out of 40 (12-5 %) fusimotor axons which co-innervated dynamic and static intrafusal fibres in their sample is equivalent to the 12 out of 53 (22-5 %) fusimotor axons that we observed in our sample (R x C test of independence using the G-statistic with Yate's correction, x2 = 0X98, D.F. = 1, P > 0-10). Fig. 1 (A-B). Electron micrographs of one rat soleus muscle spindle at two different levels in the motor region. These micrographs illustrate features of unmyelinated fusimotor axons reported previously (Walro & Kucera, 1984, 1985). Sections shown in (A) and (B) are separated by a gap of 80 ,um. One nuclear bag, (b,), one nuclear bag2 (b2) and two nuclear chain (c) fibres comprise the intrafusal bundle of this spindle. A primary sensory (ps), a fusimotor (f) and a cluster of autonomic (a) axons are visible in the spindle nerve at the right of (A). Two fusimotor axons, one myelinated and the other unmyelinated (open arrowheads), are visible coursing within the periaxial space. A third fusimotor axon, unmyelinated as it courses through the periaxial space (long arrows) terminates at multiple sites on the bag, fibre. Two different terminations of this axon on the bag, fibre (short filled arrowheads) are shown in (A) and (B). Note the great disparity in size between unmyelinated fusimotor axons in the periaxial space and the autonomic axons in the spindle nerve. Arbuthnott et al. (1989) reported that all fusimotor axons in rat soleus spindles were myelinated to within 10 ,um of their terminations on intrafusal fibres. These micrographs show that some fusimotor axons which terminate predominantly on the nuclear bag fibres are unmyelinated within the periaxial space far from their terminations on intrafusal fibres. Scale bars, 5 4rm.
262
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263 Comment We reported that several features such as the extent of myelination, distribution of axons to dynamic and static intrafusal fibres, and the number of sites of motor innervation on different types of intrafusal fibres differed between rats and cats. The major purported contribution of the paper by Arbuthnott et al. (1989) was to dispute these points by questioning the validity of our technique and the accuracy of our data. As we have shown in Figures 1 and 2, the unmyelinated axons in rat spindles, which we classified as fusimotor, are indeed fusimotor and not autonomic, and do terminate at multiple sites along nuclear bag fibres. Therefore, we feel our previously published data concerning the fusimotor innervation of rat spindles (Walro & Kucera, 1984, 1985) is accurate. After having examined more than 15 spindle poles (sensory and motor region) in three different hindlimb muscles of rats by light and electron microscopy (unpublished data), more than 50 rat spindle poles by light microscopy alone and more than 40 poles in cat tenuissimus spindles (Kucera & Walro, Am. J. Anat. 176, 97-117, 1986) by light and electron microscopy, we feel that current anatomical data do not support the contention of Arbuthnott et al. (1989) that the fusimotor organisation in rats and cats is 'essentially similar'.
Fig. 2(A-B). Motor ending on the bag, (b1) fibre of a rat soleus spindle. Sensory terminals (st) on the chain (c) fibres indicate that the section is cut through the outer sensory region of the spindle. Note that the bag, fibre is not dissociated from the intrafusal bundle as proposed by Arbuthnott et al. (1989) and even occupies a compartment formed by inner capsule (ic) cells with the bag2 (b2) fibre. Two myelinated fusimotor axons are visible within the spindle capsule. One of the fusimotor axons (*), which terminated on the bag2 fibre at a site more distal from the equator, gave off an unmyelinated branch (I) that terminated on the bag1 fibre (arrows). (B) Enlargement of the bag1 motor ending shown in (A). The postsynaptic folds on the bag1 fibre and the basal lamina in the synaptic cleft (arrows) beneath the axon terminals (at) confirm the fusimotor nature of this axon. Labels in (B) are the same as those in (A). Scale bars, 5 ,um.
J. Anat. (1990), 171, pp. 259-263 With 2 figures Printed in Great Britain
Comment J. M. WALRO AND J. KUCERA*
Department of Anatomy, Northeastern Ohio Universities College of Medicine, 4209 State Rt 44, PO Box 95, Rootstown, Ohio 44272, USA and *Department of Neurology, School of Medicine, Boston University, Boston, MA 02130, USA
A recent paper by Arbuthnott and her colleagues in the Journal of Anatomy (163, 183-190, 1989) re-examined some of our previous work on rat soleus muscle spindles (Walro & Kucera, Exp. Brain Res. 56, 187-192, 1984; Walro & Kucera, Am. J. Anat. 173, 55-68, 1985) that showed distinct differences in the neural organisation of rat soleus and cat tenuissimus spindles. Their paper cast doubt on the validity of our observations concerning the fusimotor innervation to rat spindles. We feel that many of their observations are either inaccurate or inconsistent with previously published data on rat spindles. We feel obliged to point out these inaccuracies or inconsistencies because the issues raised in these two papers are important to understanding muscle spindle function in mammals. Arbuthnott et al. (1989) questioned the reliability of tracing fusimotor axons from 1 Ium plastic sections stained with toluidine blue by light microscopy alone, yet they proceeded to replicate our study by this same technique. They claim that they were able to trace accurately the distribution of motor axons to intrafusal fibres in the rat soleus muscle only because all fusimotor axons were myelinated to within 10 l,m of their termination on the fibres. However, as we reported previously (Walro & Kucera, 1984, 1985) and as we show in Figures 1A and B, some rat fusimotor axons had unmyelinated preterminal segments up to several hundred ,um in length. Thus, Arbuthnott et al. (1989) apparently omitted some unmyelinated axons that terminate on intrafusal fibres from their sample. Arbuthnott et al. (1989) implied that we might have misidentified the nuclear bag, and the nuclear bag2 fibres in our sample, thereby invalidating the patterns of innervation that we observed. Initially, we used the criterion of size, the bag2 being larger than the bag1 fibre, to discriminate between the two types of nuclear bag fibres. Subsequently, size and distribution of granules (mitochondria) and the number and distribution of motor endplates, on the fibre we assumed to be the bag1 in the 1 um sections, correlated with patterns of staining for oxidative enzymes such as NADHTR and the distribution of sites of acetylcholinesterase (AChE) activity in fibres identified as bag1 by their staining for mATPase after preincubation in an acid or alkaline medium (Kucera et al. J. Histochem. Cytochem. 26, 973-988, 1978). Alternatively, they proposed that dissociation of the bag, fibre from the rest of the intrafusal bundle in the sensory region, a feature of cat spindles, is the most reliable criterion for identifying the bag1 fibre in rat spindles. The bag1 fibre occupies a separate compartment and does not share sensory terminals with the bag2 fibre in cat spindles (Banks et al. Phil. Trans. R. Soc. London B 299, 329-364, 1984). However, the bag1 and bag2 fibres share a compartment formed by inner capsule cells throughout most of the sensory region (Fig. 2 A) and even share a common basal lamina at sites where they share sensory terminals in rat soleus and extensor digitorum longus spindles (Diwan & Milburn, J. Embryol. exp. Morphol. 92, 223-254, 1986; Walro & Kucera, Brain Res. 425, 311-318, 1987). Thus the assertion by Arbuthnott et al. that the bag, fibre dissociates itself from the remainder of the intrafusal bundle in rat
260
J. M. WALRO AND J. KUCERA
9
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Comment 261 spindles conflicts directly with observations made by light and electron microscopy of spindles in two different rat muscles by two independent laboratories. Arbuthnott et al. (1989) reported that all motor axons that innervated intrafusal fibres were myelinated to within 10,m of their termination on intrafusal fibres. This observation conflicts with our observation that some of the axons to the bag, fibre were unmyelinated at entry into the spindle or were unmyelinated for considerable distances as they coursed within the spindle capsule prior to their termination on intrafusal fibres (Walro & Kucera, 1984, 1985). We classified these axons as fusimotor because we observed their terminations on intrafusal fibres in 1 ,um sections. Moreover, we were convinced that these axons were fusimotor rather than autonomic, because the number and location of motor endings observed on the three types of intrafusal fibres in 1 ,um sections stained with toluidine blue correlated with the number and location of sites of AChE on the three types of intrafusal fibres identified by their patterns of staining for mATPase (Kucera et al. 1978). Several unmyelinated fusimotor axons or long unmyelinated branches of myelinated fusimotor axons similar to those we described previously by light microscopy are visible in Figures 1 and 2. These axons are clearly somatic because they are larger than autonomic axons (Fig. 1 A) and terminate in identifiable endings on intrafusal fibres (Figs. 1, 2). We are unable to explain why these axons were not reported by Arbuthnott et al. (1989). They either did not see these axons in their preparations or might have assumed that these axons were autonomic. At any rate, they did not check the identity of these axons by electron microscopy or reconcile their data with the distributions of sites of AChE activity on different types of intrafusal fibres (Kucera et al. 1978). Exclusion of these axons that terminate predominantly on the bag, fibre might also explain why they reported that 75 % of bag1 fibre poles had zero or one motor ending, whereas we observed all bag1 fibre poles to be innervated by motoneurons at multiple sites, and why they observed fewer motor axons to rat soleus spindles than we did. Arbuthnott et al. (1989) concur with our observation that the dynamic bag1 and static bag2 and/or chain fibres are co-innervated by fusimotor axons, but claim that the frequency of axons distributed to dynamic and static intrafusal fibres in their sample was significantly less than the frequency we reported (Walro & Kucera, 1985). Had they performed a statistical analysis of the data in the two studies, they would have found that the 5 out of 40 (12-5 %) fusimotor axons which co-innervated dynamic and static intrafusal fibres in their sample is equivalent to the 12 out of 53 (22-5 %) fusimotor axons that we observed in our sample (R x C test of independence using the G-statistic with Yate's correction, x2 = 0X98, D.F. = 1, P > 0-10). Fig. 1 (A-B). Electron micrographs of one rat soleus muscle spindle at two different levels in the motor region. These micrographs illustrate features of unmyelinated fusimotor axons reported previously (Walro & Kucera, 1984, 1985). Sections shown in (A) and (B) are separated by a gap of 80 ,um. One nuclear bag, (b,), one nuclear bag2 (b2) and two nuclear chain (c) fibres comprise the intrafusal bundle of this spindle. A primary sensory (ps), a fusimotor (f) and a cluster of autonomic (a) axons are visible in the spindle nerve at the right of (A). Two fusimotor axons, one myelinated and the other unmyelinated (open arrowheads), are visible coursing within the periaxial space. A third fusimotor axon, unmyelinated as it courses through the periaxial space (long arrows) terminates at multiple sites on the bag, fibre. Two different terminations of this axon on the bag, fibre (short filled arrowheads) are shown in (A) and (B). Note the great disparity in size between unmyelinated fusimotor axons in the periaxial space and the autonomic axons in the spindle nerve. Arbuthnott et al. (1989) reported that all fusimotor axons in rat soleus spindles were myelinated to within 10 ,um of their terminations on intrafusal fibres. These micrographs show that some fusimotor axons which terminate predominantly on the nuclear bag fibres are unmyelinated within the periaxial space far from their terminations on intrafusal fibres. Scale bars, 5 4rm.
262
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263 Comment We reported that several features such as the extent of myelination, distribution of axons to dynamic and static intrafusal fibres, and the number of sites of motor innervation on different types of intrafusal fibres differed between rats and cats. The major purported contribution of the paper by Arbuthnott et al. (1989) was to dispute these points by questioning the validity of our technique and the accuracy of our data. As we have shown in Figures 1 and 2, the unmyelinated axons in rat spindles, which we classified as fusimotor, are indeed fusimotor and not autonomic, and do terminate at multiple sites along nuclear bag fibres. Therefore, we feel our previously published data concerning the fusimotor innervation of rat spindles (Walro & Kucera, 1984, 1985) is accurate. After having examined more than 15 spindle poles (sensory and motor region) in three different hindlimb muscles of rats by light and electron microscopy (unpublished data), more than 50 rat spindle poles by light microscopy alone and more than 40 poles in cat tenuissimus spindles (Kucera & Walro, Am. J. Anat. 176, 97-117, 1986) by light and electron microscopy, we feel that current anatomical data do not support the contention of Arbuthnott et al. (1989) that the fusimotor organisation in rats and cats is 'essentially similar'.
Fig. 2(A-B). Motor ending on the bag, (b1) fibre of a rat soleus spindle. Sensory terminals (st) on the chain (c) fibres indicate that the section is cut through the outer sensory region of the spindle. Note that the bag, fibre is not dissociated from the intrafusal bundle as proposed by Arbuthnott et al. (1989) and even occupies a compartment formed by inner capsule (ic) cells with the bag2 (b2) fibre. Two myelinated fusimotor axons are visible within the spindle capsule. One of the fusimotor axons (*), which terminated on the bag2 fibre at a site more distal from the equator, gave off an unmyelinated branch (I) that terminated on the bag1 fibre (arrows). (B) Enlargement of the bag1 motor ending shown in (A). The postsynaptic folds on the bag1 fibre and the basal lamina in the synaptic cleft (arrows) beneath the axon terminals (at) confirm the fusimotor nature of this axon. Labels in (B) are the same as those in (A). Scale bars, 5 ,um.
