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INTEGRATIVE |
1 Department of Clinical Neuroscience, Division of Neuroscience and Mental Health, Imperial College London, Charing Cross Campus, St. Dunstans Road, London W6 8RP, UK
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(Received 13 December 2006;
accepted after revision 24 January 2007;
first published online 25 January 2007)
Corresponding author R. Durbaba: Department of Clinical Neuroscience, Division of Neuroscience & Mental Health, Imperial College London, Charing Cross Campus, St. Dunstans Road, London W6 8RP, UK. Email: r.durbaba{at}imperial.ac.uk
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Introduction |
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Another consideration is that, in a number of studies of juvenile scoliosis (e.g. Trontelj et al. 1979; Dimitrijevic et al. 1980), of spinal cord injury (Kuppuswamy et al. 2006) and of back pain mechanisms (Zedka et al. 1999), use has been made of tests of the phasic stretch reflex of the paraspinal muscles evoked by mechanical tapping. Interpretation of such observations has been based on simple extrapolation from data from other muscles, notably hindlimb extensors in the cat. It is therefore useful to make specific observations on the stretch reflex mechanisms of the back muscles, to check the validity of such extrapolation.
The information previously available has concerned population effects obtained by graded electrical stimulation of muscle nerves or by minute brief muscle stretches (Jankowska & Odutola, 1980; Gorska & Johannisson, 1986). These methods are limited with regard to the identification of different afferent types, and do not permit distinction to be made between b1b2c and b2c primary spindle afferents. For this to be possible it is necessary to isolate and identify individual afferents in continuity, and to assess their synaptic effects by spike-triggered averaging (STA). Ideally, intracellular recordings would be made from identified motoneurons, but this proves very difficult because the motoneurons of the longissimus are irregularly scattered in a thin column (Holstege et al. 1987). Therefore in this paper we record the excitatory effects of individual spindle afferents by extracellular STA of focal synaptic potentials (FSPs). The spindle afferent characteristics are assessed with vibration to distinguish primary from secondary afferents, and with succinylcholine (SCh) to identify bag1 and bag2 connections. This leaves the identification of target cells to some extent uncertain, but permits the comparison of the projection strengths of the different spindle afferent types to particular regions of the spinal cord.
The longissimus muscles constitute the intermediate part of the dorsal axial muscles having fascial origins from the sacrum, lower lumbar vertebrae and iliac crest. A number of distinct heads are identifiable in their more rostral parts, where they insert via distinct tendons onto the accessory processes of the lumbar and lower thoracic vertebrae (Bogduk, 1980). They are convenient for study as examples of axial muscles because these distinct tendons can be separated for mechanical stretching, and the muscles protected from damage during laminectomy. Their action is normally to bend the vertebral column dorsally and laterally. In this work we designate the individual heads according to the vertebra of insertion, but it must be appreciated that their innervation is derived from posterior primary rami of several nerve roots caudal to this. Also, a given spinal nerve passes laterally to innervate parts of several overlapping heads.
Part of this work has been published in abstract form (Taylor et al. 2004).
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Methods |
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The lumbar fascia was opened, and the sacrocaudalis muscle on the left dissected away and removed, avoiding damage to the longissimus (see Bogduk, 1980). The tendons of insertion of four or five heads of the longissimus were isolated from the multifidus. Each tendon was then tied securely to braided silk thread and detached from the accessory process of the vertebra. In some experiments, pairs of enamelled silver wire (125 µm diameter) stimulating electrodes were implanted around intact muscle nerve branches of appropriate posterior primary rami. Nerve stimuli were 0.1 ms duration repeated at 2 s–1, and responses were recorded as averages of 20 or more repetitions. The animal was secured in a spinal frame with a clamp on the T10 vertebra and on the sacrum and sometimes with an additional support on L4. The remaining multifidus muscles were removed from vertebrae from T13 to L4, and laminectomy performed in this region. The dura was opened and arachnoid removed where possible. Usually, the dorsal roots and dorsal root ganglia (drg) of three segments were exposed. Recording from the surface of the nerve roots or spinal cord was arranged by means of a ball (0.5 mm diameter) fused at the end of a 0.1 mm silver wire. To facilitate the isolation of single dorsal root afferents, the unwanted afferent input was reduced by cutting the ventral segmental nerves L1 to L4, where they emerged laterally to innervate the abdominal muscles and skin. It was not possible to cut the ventral primary rami close to their origin because of risk of damage to the muscle nerve branches. With the surgery completed, a skin pool was formed and filled with 2% agar in saline at 40°C. When this had set, agar was scooped out to expose the spinal cord and to free the muscle heads. The cavity formed was filled with mineral oil kept close to 37°C by a radiant heat lamp.
The threads tied to the longissimus tendons were attached via wire hooks to the servo-controlled electromagnetic puller, so that each could be tensioned independently and thereby stretched singly or in groups. To provide a specific spindle primary afferent volley, the muscle heads were stretched with a minute brief pull (see Lundberg & Winsbury, 1960), which is referred to subsequently as a quick stretch (QS). This was an approximately triangular pulse of the order of 50 µm, with rising and falling phases lasting 3 ms. The justification for QS as a selective means of exciting primary afferents has been well reviewed by Lucas & Willis (1974). The ramp-and-hold stretches used for characterizing the response to SCh were as described recently (Durbaba et al. 2006). For the purposes of STA of field potentials, it was necessary to record from single afferents in continuity. This was achieved by inserting glass-coated tungsten electrodes into a drg or, more successfully, by a floating electrode just touching the surface of a dorsal rootlet close to the cord entry point. This type of electrode consisted of an 8 mm segment cut from the tip of a tungsten electrode and provided with a flexible microwire connection. Methods for the recording and characterization of the back muscle spindle afferents have been described recently in some detail (Durbaba et al. 2006), and therefore are outlined here only briefly. Spindle afferents were detected by their response to small 1 Hz sinusoidal muscle stretches, by their silencing by muscle twitches and by the excitatory effects of SCh (as described below). The site of each spindle was defined by the response to stretching individual longissimus heads and by probing the muscle surface. In the absence of reliable peripheral conduction velocity measurements, the distinction of primary from secondary afferents depended chiefly on the maximum frequency of longitudinal muscle vibration which they could follow one-to-one. Those following above 100 Hz were referred to as ‘primary-like’, and those below as ‘secondary-like’. Additional information was provided by the effects of a standardized dose of SCh on the spindle responses to ramp-and-hold stretches (see Taylor et al. 1992a,b). The change in initial frequency (
IF) was taken to indicate the strength of the bag2 influence on an afferent. The change in the dynamic response to stretch (DD) was originally taken to indicate the strength of the bag1 influence, but in the case of the back muscles (Durbaba et al. 2006) it was found more appropriate to take the maximum value of DD in the presence of SCh (SChDD) for this purpose. Thus, a high value of SChDD (>50 Hz) indicated that the afferent made significant contacts on the bag1 fibre, consistent with its classification as primary-like. However, for 41% of units responding to vibration above 100 Hz, there was a low value of SChDD. These units were regarded as b2c primaries. Secondary-like afferents had relatively low values of SChDD and a wide range of IF values.
For recording field potentials in the spinal cord, glass-coated tungsten electrodes were used as described by Merrill & Ainsworth (1972). Tracks were made vertically through the spinal cord using a stepping motor drive. For single electrode recordings the amplifier (AM Systems Inc. 1800; Caborg, WA, USA) was set to a gain of 104 and bandwidth 10 Hz to 5 kHz. In later experiments, linear arrays of six electrodes were used spaced at 2 mm or 5 mm intervals. In this case six custom-built amplifiers were used with characteristics similar to the above. Data were gathered using a CED 1401 interface and PC with Signal and Spike2 software (Cambridge Electronic Design, Cambridge, UK). The sampling rate for field potentials was 20 or 40 kHz on each channel. In all the records shown, positivity is indicated upwards. Contour plots of field potential amplitudes were computed using Sigmaplot 8.2. (SPSS Inc., Chicago Ill, USA).
The location of recording sites was based primarily on the distance from the midline and the depth of penetration from the surface, and these measurements were related to a complete set of sections held in the laboratory. Although histology for individual experiments was not available, the location of the observed sites for maximal antidromic potentials from longissimus nerve stimulation agreed closely with those expected from horseradish peroxidose (HRP) studies of the distribution of longissimus motoneurons (Holstege et al. 1987; Vanderhorst & Holstege, 1997).
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Results |
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Initially, observations were made to determine the conduction velocity (CV) of the lowest-threshold muscle afferents in the spinal cord. The nerve branch to each head of the longissimus was stimulated with graded stimuli of 0.1 ms duration. Interference from the EMG was eliminated by temporary paralysis with gallamine. Recordings were made with a silver ball electrode lightly contacting the cord surface at a series of points along a line just medial to the entry of the dorsal roots. Figure 1A shows averaged records with recording positions spaced at 4 mm intervals. The stimulus to the muscle nerve entering at L2 was made just strong enough to yield a maximal short-latency response. The latency of the peak negative deflection was measurable in eight cases and plotted against the rostro-caudal position in Fig. 1B and the best fit straight line drawn for the rostral axon branch. The inverse slope of this represents the conduction velocity of the fastest action potentials in the rostral axon branch. It is notable that the CV was essentially constant over a distance of 25 mm. Data were obtained for seven measurements each of ascending and descending CV, giving means of 44.7 and 30.9 m s–1, respectively, which are significantly different (t = 2.59, degrees of freedom (d.f.) = 12, P < 0.025).
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The usefulness of the above data is limited because it provides only mean CV values of afferents of unknown functionality. More information is provided by estimating CV for single afferents of known function, using afferent spike-triggered averaging as described and discussed in detail by Kirkwood & Sears (1980, 1982). Conduction along the spinal cord was recorded by a silver ball electrode moved along the dorsal root entry zone in 2.5 or 5.0 mm steps, and computing spike-triggered averages of 1024 sweeps at each point. Figure 1C shows an example of the records obtained in this way. The latencies measured to the most prominent negative deflection are plotted against the rostro-caudal displacement along the cord (Fig. 1D). Straight lines have been fitted to the data in the plot below, and the inverse of the slopes taken to measure CV. There appear to be three distinct regions. The region rostral to the axon entry point has a CV of 74.2 m s–1, while the caudal part has a CV of 42.6 m s–1, leading to a part starting at 15 mm from the entry point with a CV of 19.9 m s–1. This slowing of axon conduction presumably reflects a thinning of the descending axon branch.
Additional data on afferent conduction velocity were obtained using an electrode array of six tungsten recording electrodes, spaced at 2.0 mm intervals. The tips were placed in contact with the cord surface along a line immediately medial to the dorsal root entry zone. By moving the array longitudinally by 1.0 mm, a complete set of recording points were sampled, spaced at 1.0 mm intervals spanning 11.0 mm. The whole array could then be moved longitudinally by 12.0 mm and then again by 1.0 mm. In total, CV values were obtained for 23 spindle afferents of which 16 were for the ascending branch only, 2 for the descending branch and 5 for both. The distributions of conduction velocities are shown in Fig. 2A, with the ascending values shown shaded above the line and descending as open bars below the line. Mean values for all were: ascending, 56.5 m s–1 (S.D. 17.3); descending: 40.8 m s–1 (S.D. 13.5) and the difference was significant (unpaired t = 2.19, P = 0.038). For the five units with both ascending and descending estimates, the respective means were 64.0 m s–1 and 43.0 m s–1 and the difference was clearly significant (paired t = 7.41, P = 0.0018).
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IF (r2
= 0.51, P
= 0.009, n
= 12), a measure which is taken to indicate the strength of the bag2 influence. There is also a suggestion in Fig. 2D of a positive correlation with the maximum vibration frequency for one-to-one following, but this does not quite reach the accepted level for significance (r2
= 0.27, P
= 0.067). The mean CV measured for 11 primary-like ascending axons was 67.1 m s–1 (S.E.M. 3.3) and for 4 secondary-like axons was 56.5 m s–1 (S.E.M. 4.4). The difference did not reach significance (P
= 0.10). Conduction in axon collaterals
During the course of recording average unitary field potentials along vertical tracks as described below, it was usually possible to distinguish single fibre action potentials, as described by Munson & Sypert (1979). By plotting the latency of these against electrode depth, CV in collaterals could be estimated as in Fig. 3. In Fig. 3A the plot was fitted with three straight line segments, indicating successively slower collateral branches. In Fig. 3B, the data suggest a descending collateral (13.1 m s–1) dividing at 2.0 mm below the surface into two branches with conduction velocities of 1.5 and 4.3 m s–1. CVs of 77 distinct collateral segments could be measured, and Fig. 3C shows the distributions for primary- and secondary-like afferents. They were not significantly different.
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Initially, the recording electrode entered the cord vertically 0.6 mm lateral to the midline and recordings were made at 100 µm intervals usually in the interval 2000–3000 µm, thus covering the intermediate region (INT, laminae V, VI and VII) and the ventral horn (VR, laminae VIII and IX). The principal objective was to pass through the longissimus motoneuron column as identified by antidromic activation or inferred by the location of the maximal responses to QS. Excitatory synaptic effects of muscle spindle afferents were studied by the FSPs produced by nerve stimulation, by QS and by STA from single afferents. Figure 4A shows the result of stimulation of a branch of the L4 posterior primary ramus which supplied part of one head of the longissimus. The stimulus at 1.5 x motor threshold would be expected to excite most of the 
-motor axons together with group Ia, Ib and some group II fibres. A negative field potential first appears at depth 1500 µm. At depths 2000 and 2100 µm this field potential is larger and has superimposed high-frequency spikes, which are regular enough to persist in these averaged records and probably represent firing of interneurons in the INT region. The afferents responsible for this are, at least in part, group Ia because excitatory effects are also seen at this point in response to QS (Fig. 4B), but Ib and group II may also contribute. At depths 2300 and 2400 µm in Fig. 4A, the record is of a form characteristic of a compound excitatory FSP with a latency of 0.4 ms, which indicates a monosynaptic excitation due to fast afferents. At greater depths the first part of the deflection is dominated by the very rapid negative phase of the antidromic spike potential (arrow), as the electrode reaches the motoneuron cell bodies and then the grouped motor axons (3000 µm). In Fig. 4B the Ia volley evoked by QS produces negative synaptic fields starting at 1500 µm and growing to a maximum at 2500 µm without contamination by motoneuron firing. Figure 4A and B illustrate the means employed for locating the longissimus motoneuron pool. Figure 4C demonstrates by stimulation of the L3 branch to the longissimus that an early antidromic potential (arrow) dominates the recording amongst the motoneurons in that same segment, but in L1 and L2, purely synaptic field potentials are seen preceded by afferent volleys at greater latencies because of the greater conduction distance. Thus, it is evident that afferent input at one level can project to at least two segments more rostral. Figure 4D again shows that when there is antidromic invasion of cells in the segment of the stimulated nerve (lower trace, arrow), there is very little sign of a synaptic field potential. In the next most rostral segment (upper trace) there is no antidromic spike, but a clear afferent volley spike followed by a negative synaptic field.
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These population responses can be made more specific by extracellular spike-triggered averaging from a single spindle afferent. Figure 6 shows examples of records obtained by averaging from a microelectrode at 100 µm intervals along the vertical track indicated on the transverse section in the central panel. The negative FSP in L3 spinal cord segment in response to QS of the L1 muscle head (left column) is seen to reach a maximum amplitude at a depth of 2800 µm from the cord dorsum, a region known to contain the longissimus and related motoneurones. It is immediately preceded by a positive wave, which is thought to arise from the activation of the presynaptic afferent collaterals and terminals (Munson & Sypert, 1979). The negative deflection commences at 3.2 ms after the initiation of the QS and has two peaks of amplitude, 34.4 µV and 52.5 µV, respectively. The second peak occurs at 1.5 ms after the first and is likely to have arisen from double firing of some of the afferents. This was seen to be the case in experiments on cat jaw muscle spindles (Appenteng et al. 1978). Nevertheless, it seems wise to consider only the first peak as being certainly due to the spindle primary volley, and the maximum amplitudes observed for it in 20 separate experiments ranged from 6.2 to 34.4 µV (mean: 17.2, S.E.M. 1.8 µV).
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Distribution of unitary focal synaptic potentials
A total of 154 single afferents were studied by the above method. Definite signs of excitatory projections were detected for 60 of these. In the course of tracking through the cord, 122 regions of maximal negative FSPs were found, typical of recordings from a group of cells or their dendrites receiving monosynaptic excitation. The mean amplitude was 7.51 µV (S.E.M. = 0.90). Additionally, in 10 cases a positive FSP with the same latency was found, indicating that the electrode tip was in a region adjacent to the site of excitatory projection, acting as a current source. Studies were made of the distribution, form and amplitude of the unitary negative FSPs, and some examples are illustrated in Fig. 7. In panel A the left and right hand columns show the averages from two different primary-like afferents in the same track. On the left, the FSP is maximal at a depth of 3200 µm, whereas on the right the maximum is at a depth of 2300 µm. Thus these two afferents from the same muscle appear to project to quite distinct regions. In panel B the two columns show FSPs, again at 100 µm intervals, resulting from triggering from another two different spindle afferents along the same electrode track, but different to that of panel A. On the left, the afferent (secondary-like) shows a strong projection at the level of the motoneurons (depth 3000 µm) and a weak one more dorsally in lamina VII (depth 2400 µm). In between these two points the negative FSP is replaced by a small positive wave, which probably represents the source for the current sink in the region of the excitatory terminals. This effect is more pronounced in the right-hand column of Fig. 7B where records were taken along the same track, but with a different afferent trigger. Here we see a well-developed positive wave following the afferent spike at depths 2600–2900 µm. This implies that this second afferent projects to a region just off the track, and that the afferents project to subgroups of motoneurons (or other cells), rather than generally to the whole population.
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More compact and complete visualizations of the sites of excitatory projections are provided by colour contour plots as illustrated in Figs 8 and 9, constructed from the maximum FSP amplitudes recorded at 100 or 200 µm intervals from several linear medio-lateral or rostro-caudal tracks. In Fig. 8A, the main negative FSP site is seen to coincide with the tip of the ventral horn of grey matter and to give an appearance of two closely related regions. The HRP study by Holstege et al. (1987) showed that the motoneurons of the longissimus lumborum are located in this region. Another, smaller and quite separate FSP site is seen in the intermediate grey matter, which could have involved Clarke's column. In Fig. 8B, from another experiment, we again see clear evidence of an excitatory projection into the ventral tip of grey matter, but in this case there is a clearer division into two foci, with a small positive region between them. The longitudinal distribution of unitary FSPs is illustrated by data from two experiments in Fig. 9. The electrode tracks were made in a line 0.6 mm lateral to the midline. The negative FSPs are arranged at unequal intervals of from 1.5 to 3.5 mm. They appear to be elongated rostro-caudally, but this is probably a consequence of the wider spacing of the sampling points in the horizontal than in the vertical direction. The more ventral of the FSPs are seen to be at a depth appropriate for the longissimus motoneurons, but in addition there are negative fields in a more dorsal situation, possibly in lamina VII. These are generally less prominent than the ventral ones, except for the most rostral FSP in the lower panel. In some cases, as in Figs 8 and 9, regions of positivity are seen adjacent to the negative FSPs. These are thought to represent the regions constituting the sources from which current is passing into the postsynaptic dendrites and cell bodies. The positive potential amplitudes were generally less than 5 µV. There is a very prominent area of positivity in the ventral region in the lower panel of Fig. 9 between 3 and 4.5 mm caudal. In another series of tracks made 200 µm further lateral (not shown), there was a strongly negative site in this region.
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In Fig. 10A and B, the sites of negative FSP maxima are shown for primary-like and secondary-like afferents, respectively. Both types are seen to project into both INT and VH regions. Since intracellular studies have shown generally that hindlimb primary spindle afferents have stronger monosynaptic projections to motoneurons than do secondary afferents, it is possible that this would be reflected in the relative sizes of FSPs. The distinction of primary and secondary afferents was based not on conduction velocity, as is usual in hindlimb muscles, but on vibration sensitivity and on the response to succinylcholine (see Durbaba et al. 2006), and consequently we refer to the afferents as ‘primary-like’ and ‘secondary-like’. In Fig. 10C the distributions of the maximum amplitudes (taking all areas together) of the negative FSPs generated by the two types are compared. For the 37 primary-like units, giving 93 maxima, the mean amplitude was 5.4 µV (S.E.M. = 0.49), and for the six secondary-like units, giving 20 maxima, 9.1 µV (S.E.M. = 1.3). The difference is significant by the unpaired t test (P = 0.003) and implies a stronger projection for the secondary-like than for the primary-like afferents. In Fig. 10D the mean FSP amplitudes are compared not only for afferent type, but also for VH versus INT targets. It is evident that the amplitude of FSPs of secondary-like afferents to the INT area (10.4 µV; S.E.M. 2.1) is significantly stronger (P = 0.006) than that of primary-like afferents to that area (4.6 µV; S.E.M. 0.65). The same comparison applied to the VH projection again suggests that the secondary-like projection (8.1 µV; S.E.M. 1.7) is stronger than that of the primary-like afferents (5.5 µV; S.E.M. 0.56), but this difference does not achieve statistical significance (P = 0.12).
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Discussion |
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Axonal conduction velocities
The conduction velocities of the fast rostral and caudal axonal branches were first measured by recording compound action potentials on the cord surface following just-suprathreshold stimulation of the muscle nerves. The observed mean velocities were 44.7 and 30.9 m s–1, respectively, and the difference was significant. Qualitatively, this result is comparable with data from unspecified dorsal column afferents in the rat (Wall, 1994) in which the ascending branches were twice as fast as descending branches. More specificity was achieved in the present experiments with spike-triggered averaging (STA) from individual spindle afferents, characterized by physiological tests. This showed that conduction at essentially constant velocities occurred for at least 20 mm rostrally and caudally of the entry point, with mean velocities of 56.5 m s–1 and 40.8 m s–1, respectively. Very little information about descending branches of spindle afferents has previously been published, but rostral projections of cat hindlimb afferents have been reported in some detail (Fern et al. 1988). In this case, stimuli were applied at successive points along the cord dorsum and unit spikes recorded in a muscle nerve filament. Though the afferents were not specifically identified as originating in spindles, their peripheral conduction velocities identified them as group I or group II. Group I fibres from hamstring muscles had ascending velocities ranging from 56 to 60 m s–1 within segments L6 to L7. Group II fibre velocities ranged from 22 to 40 m s–1. The measurements by Ishizuka et al. (1979) on horseradish peroxidase (HRP)-filled hindlimb Ia afferents in the cord gave axon diameters of 4.5–7.5 µm for ascending branches, and 2.5–3.5 for descending branches. Using the ratio of axon diameter to total diameter for myelinated fibres of 0.7 (Arbuthnott et al. 1977), and the factor for conduction velocity of 5.7 m s–1 (µm total diameter)–1 (Boyd & Kalu, 1979), it would be expected to find conduction velocities of 36–61 m s–1 and 20–28 m s–1, respectively, for ascending and descending branches. These values are reasonably close to those found here for the back muscle afferents. Perhaps more relevant are the HRP studies of intercostals Ia afferents (Nakayama et al. 1998). In this case the ascending and descending axon branches were generally restricted to a single segment. The diameter of the caudal branch fell more steeply than that of the rostral branch, with distance from the bifurcation point. The finding in the present work of a positive correlation between conduction velocity of the ascending afferent branches and the value of IF may be related to a similar observation for hindlimb muscles (Taylor et al. 1995), though in that case it was the peripheral nerve conduction velocity that was measured.
Focal synaptic field potentials
The most detailed and specific information regarding the projection of muscle spindle afferents is to be obtained by recording intracellularly the postsynaptic potentials due to single identified afferents by STA (see, e.g. Mendell & Henneman, 1971; Kirkwood & Sears, 1974; Watt et al. 1976). However, because of the difficulty of obtaining and retaining simultaneously good intracellular recordings and well-isolated and -characterized single afferent recordings, relatively few large-scale studies have been undertaken with this method (e.g. Kirkwood & Sears, 1974; Watt et al. 1976; Munson & Sypert, 1979; Harrison & Taylor, 1981). In the case of the longissimus, the density of motoneurons is low and the cell column narrow (Holstege et al. 1987; Vanderhorst & Holstege, 1997), making it particularly difficult to record intracellularly. Consequently, in this study we have sacrificed detailed knowledge of the projection target by recording extracellular FSPs while retaining good identification of the spindle afferent units. Previous examples of the use of this approach were studies of spindle afferent projections to the fifth cranial nerve motor nucleus and the supra-trigeminal region (Appenteng et al. 1978; Taylor et al. 1978, 1993), and detection of the excitatory effects on thoracic motoneurons of respiratory interneurons (Schmid et al. 1993) and of expiratory bulbospinal axons (Kirkwood, 1995).
Though extracellular recording does not allow for certain identification of the target cells, it is nevertheless possible to make some useful deductions. In the VH region, the majority of maximal negative FSPs were found near the tip of the ventral horn, the region in which antidromic field potentials from longissimus nerves were recorded and in which QS FSPs were maximal. This is also the region in which HRP studies have revealed cell bodies of longissimus, iliocostalis and levator costae motoneurons (Holstege et al. 1987). It is therefore very likely that the FSPs from spindle afferents recorded in this region were predominantly due to excitatory projections to these cells. When a maximum in the FSP was observed towards the lateral margin of the VH (as in Fig. 8), this may well have represented the effects of excitation of motoneurons of other muscle groups such as internal oblique (Holstege et al. 1987), or to cells of the ventral spino-cerebellar tract (VSCT), which have been clearly identified on the dorso-lateral border of the VH in segments L1 to L4 (Matsushita et al. 1979), but not extending to the tip of the VH. The latter authors showed that there were also some VSCT cells located more medially in the L4 and more caudal segments, but they were in the INT rather than the VH, and so could not have given rise to confusion with motoneurons.
It could be argued that FSPs may be due to current flow into dendrites of more distant cells. However, if this were the case, unitary FSPs would not have been expected to be so highly localized (see, e.g. Figs 6 and 7). Though the majority of the synaptic current flows into dendrites of a motoneuron, rather than into the soma, the synapses are concentrated within a sphere of 500 µm diameter around the soma (Burke & Glenn, 1996). Since it is the local current density that determines the FSP amplitude, one must also take account of the cubic increase in the volume conductor with increasing distance from the soma, which would cause a rapid fall in current density and hence of the FSP amplitude. An appropriate modelling of FSP distribution does not appear to be available, but the unitary study of hindlimb Ia FSPs by Munson & Sypert (1979) found very localized potentials (as in the present study), and these were shown to be close to the motoneurons. Related studies of the projection of jaw muscle spindle afferents to the trigeminal motor nucleus (Appenteng et al. 1978; Taylor et al. 1993) also showed similarly localized FSPs, with the additional signs that the FSP maximum was slightly dorsal to the region of the maximal antidromic potential. Thus, on balance, it appears that the peak value of FSP is to be found close to the target cells, but displaced towards the centre of concentration of the synapses.
In the INT region it is not possible to identify the target cells with any certainty, due to fact that there are various types of interneuron in that region, which receive input from group Ia, Ib and II muscle afferents either alone or convergently (see Jankowska & Lindstrõm, 1972; Edgley & Jankowska, 1987; Cabaj et al. 2006). It is also possible that the more dorso-medial projections seen in L2 could be projections to dorsal spino-cerebellar tract cells (DSCT), which are known to be located here (Matsushita et al. 1979) and receive inputs from group Ia and II muscle afferents (Eccles et al. 1961; Tracey & Walmsley, 1984; Walmsley & Nicol, 1990).
HRP studies (see Ishizuka et al. 1979; Brown, 1981; Hongo, 1992; Burke & Glenn, 1996) show that muscle spindle primary and secondary afferents project to the dorsal, intermediate and ventral regions of the spinal cord grey matter. Therefore it is to be expected that extracellular FSPs should be observed in these regions. The data presented above were predominantly gathered from cord depths 
2000 µm, because our principal interest was in projections to the motoneurons. The region covered included laminae VII to IX, that is the two regions designated as ventral by Edgley & Jankowska (1987) in their study of field potentials elicited by group II volleys in hindlimb nerves, but excluded their dorsal region (laminae IV to VI). The unitary FSPs recorded in lamina VII in the present work were generated by both primary-like and secondary-like afferents. The mean amplitudes of those due to secondary-like afferents were approximately twice as large as those due to primary-like afferents, a finding similar to that made by Edgley & Jankowska (1987) for hindlimb afferents. In the trigeminal region also (Taylor et al. 1993; Dessem et al. 1997), spindle secondary afferents were found to project more strongly to the supra-trigeminal interneuron region than to the motoneurons.
An important consideration is that, whereas in the trigeminal system the motor nucleus is a compact cell mass about 1 mm in diameter, the longissimus cells are spread thinly. Nevertheless, the mean amplitudes of unitary FSPs were similar in the two cases (7.55 µV for trigeminal and 7.51 µV for longissimus cells). This implies that in this method of unitary recording, the FSP arises predominantly from excitatory effects upon a small group of cells close to the electrode tip. On the other hand, when a near-maximal volley of Ia impulses is generated by a minute transient muscle stretch (‘quick stretch’ QS; see Watt et al. 1976), the compound FSP in the trigeminal motor nucleus is much larger (214 µV in Taylor et al. 1993) than the mean of 17.2 µV in the longissimus recordings. Studies of FSPs generated by medial gastrocnemius spindles (mainly Ia) in the homonymous motor nucleus (Collins et al. 1986) gave a mean amplitude of 9 µV, while an estimate of the QS response is 260 µV (Watt et al. 1976). These observations taken together imply either that the projection of longissimus muscle spindle primaries may be relatively weak or that the motoneurons largely responsible for the FSPs recorded here are thinly scattered. An unusually large FSP of 30.7 µV amplitude was observed on one occasion (Fig. 6C). Two similarly large values have previously been recorded in the trigeminal motor nucleus (Taylor et al. 1993). Presumably, such large FSPs indicate that the electrode tip is amidst a close grouping of motoneurons.
When the projection of single afferents was examined by a series of electrode tracks at 0.5 or 1 mm intervals along the cord, it was found that excitatory FSPs in the ventral horn occurred at irregular spacings of 1.5–3.5 mm (see, e.g. Fig. 9). Assuming that the FSPs were generated predominantly by contacts on motoneurons, this could mean either that each afferent might project to only a proportion of the motoneurons, or that the motoneurons are arranged in corresponding discontinuous clusters. The latter seems the more likely in view of the studies of Mendell & Henneman (1971) by intracellular STA, in which it was concluded that each Ia afferent sent terminals to all or nearly all of its homonymous motoneurons. Also, the HRP labelling of the motoneurons of the longissimus in lower thoracic segments in the rat indicated that they were clustered, rather than in a continuous column (Smith & Hollyday, 1983). An advantage of the technique used here is that by recording from an array of extracellular electrodes, the rostro-caudal distribution of the excitatory projection of each afferent could be assessed rapidly, though with a loss of specificity regarding target cell type.
A related feature of interest is that the mean unitary FSP observed here for secondary-like afferents into the VH region is at least as large as that of the primary-like afferents. It has been found in a recent study of the properties of longissimus muscle spindles (Durbaba et al. 2006) that 41% of the primary-like afferents were of the b2c type when tested with succinylcholine. Having no contacts on bag1 intrafusal muscle fibres, they would not be under the control of the dynamic fusimotor system and would therefore have much the same function as muscle spindle secondary afferents. Consequently, it appears that the dominant reflex effect of longissimus spindles is likely to be tonic rather than dynamic. This may reflect the obvious importance of the maintenance of static posture in the vertebral column. Though it is unjustifiable to make a simple extrapolation of this finding to man, it may mean that assessing stretch reflex effects in human back muscles by responses to brief phasic stretches would underestimate the true importance of the reflex connections (Dimitrijevic et al. 1980; Skotte et al. 2005; Kuppuswamy et al. 2005).
The evidence of the quick stretch experiments presented here is that spindle primary afferent inputs from several adjacent overlapping heads of the longissimus converge upon motoneurons in any given related spinal segment. Unitary FSP recordings also indicated that VH regions in a given segment received input from both primary-like and secondary-like afferents from muscle heads inserting on vertebrae rostral and caudal to the recording point. Thus, the stretch reflex of longissimus muscles appears to act in unison across several segments. This is supported by other work in which electrical stimulation of longissimus nerves at group I strength produced intracellular EPSPs in motoneurons at L4 (Akatani et al. 2004). The EPSP amplitudes were progressively smaller on moving stimulation from L4 to L1. One of the objectives of the present study was to try to relate the strength of excitatory projections from spindle afferents to their properties, especially the relative strength of their contacts with bag1 and bag2 fibres. It had been found previously (Taylor et al. 1993) for the trigeminal system that there was a clear positive correlation of projection strength with the strength of bag2 contacts, but not with the strength of bag1 contacts. This correlation has not been found for the longissimus, but the method of estimation of projection strength in this situation may not be appropriate, because the longissimus motoneurons are not in a compact group, as is the case for trigeminal motoneurons. To follow up this question further would require a large-scale intracellular study.
Problems associated with defective spinal posture are very important in human medicine, but relatively few basic studies have been made previously of the control of back muscles. The present study, limited though it is to back muscles in the cat, points to some features of specialization, which may be of importance in man.
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