A series of experiments was undertaken to test the model that SLVs undergo rounds of endo- and exocytosis in an activity- and Ca2+-modulated manner, releasing a neuroactive substance which is important for mechanosensory function.
Fluorescent staining with FM1-43: evidence for activity- and Ca2+-dependent recycling of SLVs
Styryl dyes such as FM1-43 have been introduced as fluorescent markers of recycling synaptic vesicles (Betz & Bewick, 1992; Betz et al. 1992). Spindle primary endings label brightly with FM1-43 (Fig. 1A and B; 5 μm FM1-43) when the muscles containing them are pinned at resting length. The phenomenon was first seen during the development of these dyes as synaptic vesicle markers (Betz et al. 1992). Dye uptake occurred even if muscles were not pinned out but left free-floating in the dye and in the presence of TTX to block spontaneous action potential generation (G. S. Bewick & W. J. Betz, unpublished observations). It was subsequently described in more detail by Chua & Hunt (1995). This labelling, together with the many similarities of SLVs to synaptic vesicles noted above, suggested that dye uptake might occur by SLV endocytosis. If SLV recycling was occurring and is important for mechanosensory function, mechanical activity might be expected to affect it. Consistent with this, we found that repeatedly stretching muscles to maximal in situ length during dye incubation increased the labelling intensity to 275.4 ± 91.0% (mean ±s.e.m., n= 5) above controls (n= 10; P < 0.03; Fig. 1C). Thus static muscle stretch increased dye uptake, consistent with a stretch-activated increase in endocytosis.
Figure 1. FM1-43 labelling in muscle spindle primary afferent terminals is enhanced by stretch Low- (A) and high-magnification fluorescence images (B) of rat lumbrical muscle spindles labelled by exposure to 5 μm FM1-43 for 2 h at resting in situ length. C, labelling by FM1-43 is approximately 4-fold brighter if preparations are repeatedly stretched to maximal in situ length during labelling (means ±s.e.m., Student's t test). a.u., arbitrary units of grey level.
Download figure to PowerPoint
To test whether dye labelling, like synaptic vesicle recycling, is Ca2+ dependent, we examined the effects of cobalt, a voltage-gated Ca2+ channel blocker, on dye uptake. When 3 mm CoCl2 was substituted for 2 mm CaCl2 in the physiological saline, the amount of dye uptake was markedly reduced (n= 4, P < 0.01; Fig. 2A–C). In a previous study, Gale et al. (2001) reported that labelling of some mechanosensory cells (hair cells) occurs by styryl dye permeation of mechanically sensitive channels. This labelling is inhibited by 10 mm extracellular Ca2+ (Gale et al. 2001). In our preparations, 10 mm Ca2+ had no effect on fluorescence intensity (Fig. 2C), suggesting that little, if any, dye internalization occurs by channel permeation. Together, these observations indicate that FM1-43 uptake occurs tonically at rest, is by vesicle endocytosis in a Ca2+-dependent manner and that this endocytosis increases with mechanical activity.
Figure 2. Labelling is blocked by extracellular cobalt but not by high calcium concentrations, suggesting that labelling is by uptake into recycling SLVs rather than direct channel permeation A, B and C (upper panel), the voltage-gated Ca2+ channel blocker Co2+ substantially reduced FM1-43 uptake, indicating that Ca2+ influx through voltage-gated Ca2+ channels is important for dye internalization. C (lower panel), conversely, 10 mm Ca2+ extracellularly does not block labelling, suggesting that labelling is not due to dye permeation directly through mechanically sensitive channels.
Download figure to PowerPoint
The uptake of FM1-43 by endocytosis implies a preceding exocytosis and local recycling. However, a more direct test would be the presence of dye loss, since dye internalized by endocytosis is released again during vesicle recycling. If such local SLV recycling was occurring tonically, as implied by the dye uptake at resting length, labelled spindle primary endings should destain spontaneously when transferred to normal saline. Conversely, if dye uptake is by channel permeation, labelling should be irreversible (Gale et al. 2001). When labelled spindles were repeatedly imaged, significant destaining occurred even at rest (2.7 ± 0.9% of initial intensity over 5 min, n= 6; Fig. 3A–C), after background subtraction for each image to correct for photobleaching and dye washout. This observation is consistent with an on-going tonic exocytosis, similar to that observed previously for the endocytosis-mediated labelling of SLVs at rest. In order to test whether the rate of destaining, and hence SLV exocytosis, was at all dependent on the mechanosensory transducing function of the primary ending, we made use of the well-known sensitivity of the primary ending to small-amplitude sinusoidal stretch. Over a range of frequencies from about 100 to 300 Hz, the primary ending of the cat spindle can be ‘driven’ or caused to fire an impulse for each stretch cycle (for review see Matthews, 1972). Similarly, in the rat soleus muscle, afferents identified as arising from the spindle primary endings can be driven up to at least 150 Hz by vibration of 0.25 mm amplitude applied to the muscle spindle (De-Doncker et al. 2003). If the proposed model is correct, such a strong mechanical stimulus should lead to significant SLV exocytosis and hence destaining. As predicted, when we applied a vibrating probe (200 Hz, ∼50 μm amplitude) to the spindle pole in the isolated lumbrical preparation, destaining increased 10-fold (27.6 ± 6.4% of initial intensity in 5 min, P < 0.01) during vibration. Importantly, on cessation of stimulation, destaining returned to prestimulation basal levels (3.4 ± 2.7%; P > 0.8versus prestimulation destain rate; (Fig. 3B and C). Thus, dye uptake and release, consistent with SLV endo- and exocytosis, occur tonically at rest and both processes are markedly increased in parallel by mechanical activity.
Figure 3. Destaining of labelled spindles increases with mechanical activity and is Ca2+-modulated A, images of a living terminal acquired between alternating 5 min periods of rest and stimulation, beginning with a rest. Upper row, Start = initial image; Rest = 5 min from Start, no vibration applied; Stim1 = 5 min from Rest, during which 5 min vibration was applied. Lower row, Rest = 5 min from Stim1, no vibration applied; Stim2 = 5 min from Rest, during which a further 5 min of vibration was applied. B, comparison of dye loss in the presence, as seen in A, and absence of Ca2+ (0 Ca2+ and 500 μm EGTA). Destaining increased markedly during stimulation (5 min vibration) in each case but was significantly reduced in the absence of Ca2+. Data are means ±s.e.m. for 8 terminal regions. C, mean destain for 6 labelled preparations stimulated in the presence of 2 mm Ca2+. Spindles destained by only 2.7 ± 0.9 and 3.4 ± 2.7% in the 5 min before and after stimulation, but by 27.6 ± 6.4% during 5 min of stimulation (P < 0.01versus prestimulation and P < 0.005versus poststimulation destain periods).
Download figure to PowerPoint
We next tested whether destaining, like dye uptake, was Ca2+ sensitive, consistent with SLV exocytosis being Ca2+ mediated. The mechanical enhancement of destaining was at least partly Ca2+ dependent, since vibration-induced destaining in Ca2+-free saline (0 CaCl2 and 500 μm EGTA, n= 4) was significantly reduced (P < 0.03, n= 4). A representative example is shown in Fig. 3B, where destaining is reduced by approximately 50% over the course of two stimulation periods (1 terminal analysed per spindle, fluorescence intensity of 5 regions analysed per terminal).
These observations therefore extend the parallels between synaptic vesicles and SLVs in four ways. First, significant amounts of dye are internalized via endocytosis (though a small amount of dye entry by channel permeation cannot be excluded, see Gale et al. 2001). Second, the re-release of previously internalized dye suggests that SLVs maintain their functional integrity throughout the cycle, consistent with endocytosis followed by exocytosis, i.e. localized SLV recycling. Third, they demonstrate that activity increases both dye internalization (endocytosis) and release (exocytosis), i.e. activity increases the total number of SLVs recycling. Finally, both internalization and release of dye exhibit a marked Ca2+ dependence.
Immunogold labelling for glutamate in spindle Ia afferent endings
This localized SLV recycling presumably releases vesicular contents during exocytosis, to perform a physiological role. In order to test what such a role might be, it was important to identify what the SLV contents might be. According to Dale's Principle (Dale, 1935), all terminals of a particular neurone release the same neurotransmitter(s). The similarity of SLVs and synaptic vesicles led us to postulate that SLVs might therefore contain the same neuroactive substance as the central presynaptic endings of the spindle primary-ending afferent (the Ia afferent). Since the central terminals of Ia afferents are glutamatergic (Engberg et al. 1993; Walmsley & Bolton, 1994), we used EM immunocytochemistry to test for glutamate-like immunoreactivity (glutamate-LI) in the sensory terminals (Fig. 4A). No attempt was made to determine absolute amounts of glutamate, but relative comparisons were made with components of the cerebellar cortex, treating this as a positive control. Negative controls, in which the primary antibody was omitted, showed very low particle densities. Nevertheless, this background labelling varied from one component to another, ranging from 0.23 gold particles μm−2 in sensory terminals to 1.46 gold particles μm−2 in axonal terminals of Golgi cells (Fig. 4B). In principle, these mean background values could be subtracted from each item of data from similar components in the positively stained sections. However, since the lowest value was that of the sensory terminals themselves, background subtraction would only serve to increase any observed differences between the sensory terminals and other components, and so was not carried out.
Figure 4. Elevated glutamate-like immunoreactivity in muscle spindle primary afferents terminals Aa, electron micrograph of muscle spindle primary afferent terminals (t) enclosing an intrafusal muscle fibre (note dark-staining nucleus in the centre) processed for glutamate-like immunogold labelling. Scale bar = 1 μm; box indicates region seen in higher power in b. Ab, higher power view, showing labelling (arrows) in a terminal region containing synaptic-like vesicles (V). Vesicular membranes are less clear than in conventionally prepared material. Note that particle density is much lower in the adjacent intrafusal muscle fibre. Scale = 0.2 μm B, quantitative comparison of gold particle density with other tissues. Data are expressed as means ±s.e.m., up to 3 columns per tissue. Left-hand (light grey) and centre (black) columns represent 2 samples treated with antiglutamate primary antibodies. The right-hand (dark grey) column represents the labelling density for control samples with no primary antibody, treated in parallel with left-hand or centre column specimens (mean for pooled data if 2 samples were processed).
Download figure to PowerPoint
Sections of muscle and cerebellum from one animal, and of muscle, cerebellum and lumbosacral spinal cord from a second, were processed using identical protocols but on different days. Reproducibility of the procedure is demonstrated both by the very similar labelling density data obtained from the cerebellar cortical components in each case (Fig. 4B) and also by their similarity to those found by Somogyi et al. (1986) in the same region of the cat brain. A spindle sensory ending from each muscle was sampled at multiple sites for glutamate-LI and though the average particle densities for the two endings differed (7.65 ± 0.15 gold particles μm−2, n= 22 and 24.58 ± 1.71 gold particles μm−2, n= 20), the densities of both sensory endings were at least 300% of the intrafusal (2.08 ± 0.22 gold particles μm−2, n= 22) or extrafusal (non-spindle) muscle fibre labelling (1.21 ± 0.16 gold particles μm−2, n= 9; P < 0.001 in each case). In one of the spindle sensory endings the particle density was similar to that shown by components of glutamatergic cerebellar neurones (parallel fibres, 26.29 ± 4.19 gold particles μm−2, n= 8; mossy fibres, 27.86 ± 1.55 gold particles μm−2, n= 20; and granule cell dendrites, 18.36 ± 1.87 gold particles μm−2, n= 20) and significantly higher than non-neuronal, non-synaptic, cerebellar glial cell processes (6.60 ± 1.95 gold particles μm−2, n= 8; P < 0.001).
In the sample of lumbosacral spinal cord, two components from lamina IX were quantitatively analysed: large, type S boutons in synaptic contact with proximal dendrites of motoneurones; and the dendrites themselves. The axonal source of the individual type S boutons could not be identified, but Ia afferents have been shown to have similar central terminals (Conradi et al. 1983), and only the largest dendritic profiles were selected to ensure that they were most probably of motoneuronal origin. While the postsynaptic dendrites themselves expressed very little glutamate-LI (3.01 ± 0.52 gold particles μm−2, n= 19), glutamate-LI was significantly greater in the synaptic boutons (6.48 ± 0.54 gold particles μm−2, n= 20; P < 0.001). Interestingly, these presumed glutamatergic terminals showed only as much glutamate LI as the cerebellar glial cell processes, which was much less than the (peripheral) Ia sensory terminals processed at the same time (Fig. 4B).
In summary, therefore, peripheral sensory terminals have elevated levels of glutamate-LI. Furthermore, the level of glutamate-LI in spindle sensory endings is at least comparable to that in central Ia synaptic terminals and is significantly elevated above that in either adjacent muscle fibres or central non-glutamatergic cellular components. Thus, these data indicate that there is a high glutamate content in these endings.
Effect of exogenous glutamate on spindle afferent excitability
If SLVs contain glutamate, it is presumably released during exocytosis to elicit a physiological response. We next asked, therefore, what role glutamate release might have. To assess the effect of glutamate on spindle responsiveness, exogenous glutamate (0.01–1 mm) was added to the bathing solution and spindle discharge frequency monitored during repeated 1 mm stretch-and-hold cycles (resting muscle length, ∼10 mm). Glutamate at 0.1–1 mm significantly increased the discharge frequency during the ‘hold’ phase of such cycles (Fig. 5A and B), over a period of 30–60 min. For example, 100 μm glutamate increased the number of spikes to 137.4 ± 14.4% (mean ±s.e.m. of 4 preparations; P < 0.05) of the control value. There may also have been some increase in afferent discharge activity at resting length, but this was not quantified. These effects reversed fully upon washing, over a similar time scale.
To test whether the enhancement was receptor mediated and identify the type of receptor involved, a range of glutamate (Glu) receptor antagonists was applied. Kynurenic acid (1 mm), a non-selective ionotropic Glu receptor antagonist, had little effect on the glutamate-mediated enhancement (data not shown). Somewhat surprisingly, neither did broad-spectrum antagonists of the group I–III metabotropic Glu receptors (MCPG, 0.5–1.0 mm, groups I and II; CPPG, 10–100 nm, groups II and III; and MAP4, 1 mm, groups II and III), even when applied in combination, with or without kynurenate. For example, the presence of 500 μm MCPG and 100 nm CPPG failed to prevent enhancement in the response to stretch by 100 μm glutamate, the number of spikes increasing to 156.8 ± 13.1% (mean ±s.e.m. of 4 preparations; P < 0.03, Student's paired t test) of the predrug control value (Fig. 5C). Thus, exogenous glutamate increased spindle excitability, but this effect was not mediated through the best-characterized Glu receptors.
Recently, however, a metabotropic Glu receptor coupled to phospholipase D (PLD) has been described (Pellegrini-Giampietro et al. 1996) that is currently designated outside the standard group I–III metabotropic Glu receptor categories. This receptor is inhibited by (R,S)3,5-dihydroxyphenylglycine (DHPG), a group I agonist (Ito et al. 1992), and rather more effectively by (2R,1′-S,2′-R,3′-S)-2-(2′-carboxy-3′-phenylcyclopropyl) glycine (PCCG-13; Albani-Torregrossa et al. 1999). Accordingly, when recording in the presence of 200 μm DHPG, 100 μm glutamate produced only a slight, insignificant enhancement of the mean discharge rate. The number of spikes increased to 111.3 ± 10.0% of the control value (mean ±s.e.m. of 4 preparations; P > 0.2, Student's paired t test), indicating that DHPG effectively blocked the expected enhancement by glutamate (Fig. 6A). Further experiments applying the selective antagonist PCCG-13 (1 μm) confirmed the involvement of PLD-coupled receptors (Fig. 6B). Thus, when glutamate (100 μm) was applied in the presence of PCCG-13 (1 μm) the mean number of spikes fell significantly below (71.8 ± 8.7%) that of the control value (mean ±s.e.m. of 6 preparations; P < 0.05, Student's paired t test). Spindle endings were therefore sensitive to the application of exogenous glutamate and only application of antagonists of PLD-coupled metabotropic Glu receptors effectively blocked this action.
Figure 6. Antagonists of PLD-coupled metabotropic Glu receptors inhibit glutamate-mediated increases in spindle excitability and reduce spindle excitability when applied alone A, the afferent discharge in the presence of both glutamate and DHPG (200 μm) was not significantly different from predrug levels. Thus, DHPG blocked the effect of exogenous glutamate, indicating that glutamate-mediated excitability requires a PLD-metabotropic Glu receptor. B, because DHPG can also act as an agonist at type I metabotropic Glu receptors, a more specific PLD-metabotropic Glu receptor antagonist, PCCG-13, was tested. It too abolished the effects of exogenous glutamate at 1 μm.C and D, PCCG-13 also reduced spindle discharge frequency in the absence of exogenous glutamate, but required higher concentrations (10 μm). This is consistent with PCCG-13 blocking receptor activation through tonic release of endogenous glutamate. E, representative experiment showing the time course and profound effect of PCCG-13 applied alone on the responsiveness of a spindle to stretch. F, time-matched controls showed that stretch-evoked responses are well maintained in the absence of drugs. Values in A–D and F are means ±s.e.m.; Student's paired t test comparison of predrug and with drug responses.
Download figure to PowerPoint
Effect of blocking endogenous glutamate on spindle afferent excitability
While the preceding experiments indicate that exogenous glutamate can activate metabotropic receptors, they do not test directly whether there is endogenous glutamate release or, if so, that it activates the same receptors. To test more directly for endogenous glutamate release (i.e. exocytosis from glutamatergic SLVs) activating this same pathway, PCCG-13 was applied in the absence of exogenous glutamate (Fig. 6C and D). At 1 μm, PCCG-13 did tend to reduce the mean number of spikes in the responses to stretch of seven preparations to 80.2 ± 12.1% of the control value, but this was not significant (P < 0.1, Student's paired t test). However, at 10 μm, PCCG-13 in the bathing medium profoundly inhibited the response, the number of spikes falling to just 24.7 ± 7.9% (mean ±s.e.m.) of the predrug control values, the effect being highly significant (P < 0.01, Student's paired t test, n= 6). An example of such an experiment is shown in Fig. 6E. Figure 6F shows that afferent discharges are otherwise well maintained in time-matched controls in the absence of PCCG-13. Thus, endogenous glutamate release is indeed occurring during normal stretch activity and, furthermore, it does act through the same receptors as exogenously applied glutamate.
Finally, we investigated the effect of blocking Ca2+-mediated SLV recycling on spindle function. Previous experiments showed that CoCl2 markedly inhibits vesicle recycling (see above). Cobalt chloride is also reported to inhibit spindle afferent discharge (Kruse & Poppele, 1991). We therefore tested the affect on afferent output of CoCl2 concentrations that profoundly reduce SLV recycling. We found that after 1 h of 3 mm CoCl2 application spindle activity was completely blocked in two preparations and was just detectable (<4% of control values) in three others (mean number of spikes in 3 mm CoCl2 was 2.4 ± 1.0% of predrug control values, P < 0.002, n= 5, Student's paired t test).