Synaptic‐like vesicles and candidate transduction channels in mechanosensory terminals

This article summarises progress to date over an exciting and very enjoyable first 15 years of collaboration with Bob Banks. Our collaboration began when I contacted him with (to me) an unexpected observation that a dye used to mark recycling synaptic vesicle membrane at efferent terminals also labelled muscle spindle afferent terminals. This observation led to the re‐discovery of a system of small clear vesicles present in all vertebrate primary mechanosensory nerve terminals. These synaptic‐like vesicles (SLVs) have been, and continue to be, the major focus of our work. This article describes our characterisation of the properties and functional significance of these SLVs, combining our complementary skills: Bob's technical expertise and encyclopaedic knowledge of mechanosensation with my experience of synaptic vesicles and the development of the styryl pyridinium dyes, of which the most widely used is FM1‐43. On the way we have found that SLVs seem to be part of a constitutive glutamate secretory system necessary to maintain the stretch‐sensitivity of spindle endings. The glutamate activates a highly unusual glutamate receptor linked to phospholipase D activation, which we have termed the PLD‐mGluR. It has a totally distinct pharmacology first described in the hippocampus nearly 20 years ago but, like the SLVs that were first described over 50 years ago, has since been little researched. Yet, our evidence and literature searches suggest this glutamate/SLV/PLD‐mGluR system is a ubiquitous feature of mechanosensory endings and, at least for spindles, is essential for maintaining mechanosensory function. This article summarises how this system integrates with the classical model of mechanosensitive channels in spindles and other mechanosensory nerve terminals, including hair follicle afferents and baroreceptors controlling blood pressure. Finally, in this time when there is an imperative to show translational relevance, I describe how this fascinating system might actually be a useful therapeutic drug target for clinical conditions such as hypertension and muscle spasticity. This has been a fascinating 15‐year journey in collaboration with Bob who, as well as having an astute scientific mind, is also a great enthusiast, motivator and friend. I hope this exciting and enjoyable journey will continue well into the future.     

A `late supernormal period' in the recovery of excitability following an action potential in muscle spindle and tendon organ receptors

1. Discharge patterns have been recorded from five types of stretch receptor; frog muscle spindles, lizard tendon organs, cat soleus tendon organs and primary and secondary endings of cat soleus muscle spindles.2. The fully adapted discharge of each type of receptor is irregular, especially for frog spindles and primary endings of cat spindles as compared with the other three types (the ;regularly firing' receptors). Frog spindles and some cat spindle primary endings would maintain a discharge at very low mean rates (1/sec or less) while the remaining receptors would stop suddenly, as soon as their rate of discharge fell below a critical value characteristic for each individual ending.3. This pattern of discharge suggests that there is a peak in the excitability of ;regularly firing' receptors at a time following a preceding impulse, which corresponds to the intervals between impulses at each particular receptor's slowest rate of maintained firing, and that the excitability subsequently falls again. Primary endings of cat muscle spindles also showed some evidence of such a ;late supernormal period', but frog spindles did not.4. Direct evidence for the ;late supernormal period' was obtained from experiments in which a maintained discharge was restarted by an antidromic action potential in a receptor which had stopped firing, and to which had been applied a stretch just too small to restart the discharge.5. It is shown in an Appendix that a model receptor in which the recovery of excitability following an impulse has a hyperbolic time course, and in which Gaussian distributed noise is superimposed on the generator potential, can have a discharge pattern very closely resembling that of a frog spindle (cf. Buller, 1965).6. After addition of a late supernormal period to the model, its discharge pattern could mimic closely that of a lizard or cat tendon organ, or of a secondary ending of a cat spindle.     

Repetitive activation of glutamatergic inputs evokes a long-lasting excitation in rat globus pallidus neurons in vitro

External globus pallidus (GPe) neurons express abundant metabotropic glutamate receptor 1 (mGluR1) in their somata and dendrites and receive glutamatergic inputs mainly from the subthalamic nucleus. We investigated whether synaptically released glutamate could activate mGluR1s using whole cell and cell-attached recordings in rat brain slice preparations. Repetitive internal capsule stimulation evoked EPSPs followed by a slow depolarizing response (sDEPO) lasting 10-20 s. Bath application of both GABA(A) and GABA(B) receptor antagonists increased the amplitude of sDEPOs. A mixture of AMPA/kainate and N-methyl-d-aspartate receptor antagonists did not alter sDEPOs. The induction of sDEPOs was only partially mediated by mGluR1 because mGluR1 antagonists reduced but failed to completely block the responses. Voltage-clamp recordings revealed that slow inward currents sensitive to mGluR1 antagonist were larger at -60 than at -100 mV, whereas the currents insensitive to mGluR1 antagonist were larger at -100 than at -60 mV. In cell-attached recordings, repetitive internal capsule stimulation evoked long-lasting excitations in GPe neurons, which were also partially suppressed by mGluR1 antagonists. Application of a glutamate uptake inhibitor or an mGluR1 agonist significantly increased the spontaneous firing rate but decreased the excitations to repetitive stimulation. These results suggest that synaptically released glutamate can activate mGluR1, contributing to the induction of long-lasting excitation in GPe neurons and that background mGluR1 activation suppresses the slow mGluR1 responses. Thus mGluR1 may play important roles in the control of GPe neuronal activity.     

Stretching the imagination beyond muscle spindles – stretch‐sensitive mechanisms in arthropods

Much attention has been given to mammalian muscle spindles and their role in stretch‐mediated muscle proprioception. Recent studies, particularly, have sought to determine the molecular mediators of stretch‐evoked mechanotransduction, which these endings rely upon for functionality. Nonetheless, much about these endings remains unknown. Opportunities may be presented from consideration of extensive parallel research in stretch receptor mechanisms in arthropods. Such systems may provide a useful source of additional data and powerful tools for dissecting the complex systems of stretch transduction apparatus. At the least, such systems provide tractable exemplars of how organisms solve the problem of converting stretch stimuli to electrical output. Potentially, they may even provide molecular mechanisms and candidate molecular mediators of direct relevance to mammalian muscle spindles. Here we provide a brief overview of research on arthropod stretch receptors.     

Receptor potential and spike initiation in two varieties of snake muscle spindles.

1. Receptor potentials, in response to ramp-and-hold stretch, have been recorded from two varieties of snake muscle spindles. 2. The two types of spindles have a similar sensitivity of impulse discharge to amplitude of receptor potential during the static phase of stretch. 3. Receptor potentials from short-capsule spindles show a high dynamic sensitivity to velocity of stretch. Amplitude of dynamic receptor potentials is well related to frequency of dynamic discharge except beyond a certain velocity of stretch where the frequency deviates progressively more than expected from linearity. 4. Receptor potentials from long-capsule spindles show a low dynamic sensitivity to velocity of stretch and amplitude of dynamic receptor potentials is well correlated with dynamic firing frequency. 5. The threshold level of receptor potential for initiating spike discharge varies with the velocity of stretch, the relation being similar for the two types of spindles. 6. It is concluded that the basis for functional differentiation of snake spindles may lie in the mechanism by which deformation of sensory endings is transformed into receptor potential. 7. Late adaptation of impulse discharge, a characteristic feature of the response of the short-capsule spindle to maintained stretch, has been related to length changes of the sensory region measured directly with Nomarski optics. The linear relation found between the slow adaptive fall of impulse discharge and the simultaneous shortening of the sensory region strongly suggests a mechanical basis for the late adaptation.     

Physiological properties of muscle spindles in dorsal neck muscles of the cat.

1. Single-fiber recording was used to examine the properties of 107 spindle endings in cat biventer cervicis (BC) and complexus (CM) muscles. Responses of receptors were examined following muscle contraction and ramp and hold stretch. Twenty-two endings in splenius (SP) were also examined, but their responses could not be quantitated because the anatomy of SP prevented the application of appropriate stretches. 2. Conduction velocitites of spindle afferents ranged from 13 to 90 m/s. Endings with primary response patterns usually had faster conduction velocities than secondary endings, but there was overlap in the conduction velocity ranges of the two subgroups. 3. Most neck spindle afferents could be classified as either primary or secondary by a constellation of physiological criteria including dynamic response pattern, dynamic index, and variability of resting discharge frequency. However, 22 of 107 endings from BC and CM had responses with characteristics intermediate between primary and secondary responses. The possible sources of these characteristics are discussed. 4. Despite the similarity in properties between spindles of different neck muscles, the length sensitivities of CM spindles were high compared to those of BC spindles. CM spindles showed length-related modulation of firing frequency over a more restricted range of initial muscle lengths than did BC spindles. 5. Eight Golgi tendon organs (GTO) were identified by their characteristics responses. Conduction velocities obtained for five GTO afferent nerves ranged from 50 to 67 m/s. Recordings were also made from receptros in deep muscles surrounding the vertebrae. These receptors had properties characteristic of muscle spindles.     

Responses of primary and secondary endings of isolated mammalian muscle spindles to sinusoidal length changes.

1. Responses of primary and secondary endings of isolated cat spindles to sinusoidal length changes have been recorded before and after block of impulse activity by tetrodotoxin. 2. Primary endings may discharge with each cycle of sinusoidal stretch at 25-50 Hz, with stretch amplitudes applied to the spindle poles as small as 1 micron. Thresholds are higher at lower frequencies. 3. In primary endings, amplitude of the receptor potential varies with frequency and magnitude of sinusoidal stretch. At a given stretch amplitude, the receptor-potential response increases markedly between 1 and 10 Hz. At a fixed frequency, for example, at Hz, the response to graded amplitude of sinusoidal stretch is highly nonlinear, sensitivity decreasing with large amplitudes. 4. Secondary endings show a much higher threshold than primary endings to sinusoidal stretch. Thus, at 25 Hz, secondary endings required stretch amplitudes of 50-100 micron to evoke discharge. Relatively large amplitudes of stretch were also required to evoked detectable receptor potentials. Over the range studied, the receptor potential varied more linearly with stretch amplitude in secondary than in primary endings.     

Two kinds of resting discharge in cat muscle spindles.

1. The behavior of primary endings of cat soleus muscle spindles was studied during shortening steps carried out at different muscle lengths. 2. Spindles were of two kinds: one, silent spindles, whose afferents fell silent after the shortening, at least over part of the range of lengths tested. The second, spontaneous spindles, resumed firing at all lengths. 3. For silent spindles, the duration of the silent period, measured at lengths where they did recover a resting rate, depended directly on muscle length and became shorter at longer lengths. This is what would be expected if the slack introduced in the spindle by the shortening step was removed more rapidly at longer lengths by the higher passive tension. For spontaneous spindles, on the other hand, the duration of the silent period after the shortening was largely independent of muscle length and depended on the spindle's rate of firing immediately before the shortening. 4. At intermediate lengths the discharge of slack spontaneous spindles remained unaffected by an isometric muscle contraction. It was therefore not possible to produce a pause in the discharge, behavior normally taken as typical of spindles. The discharge could be interrupted by the contraction if this was combined with a large shortening movement. 5. It is proposed that when intrafusal fibers are slackened by a shortening step, the resting discharge in spontaneous spindles is generated by a maintained depolarization of the annulospiral ending resulting from extension of the terminal coils by forces from within the receptor. A shortening contraction compresses the spirals to interrupt the discharge. The sensory endings of silent spindles remain below threshold until the spirals have been opened out sufficiently by external stretch.     

Unilateral suppression of pharyngeal motor cortex to repetitive transcranial magnetic stimulation reveals functional asymmetry in the hemispheric projections to human swallowing

Age-related physiological and morphological changes of muscle spindles were examined in rats (male Fischer 344/DuCrj: young, 4–13 months; middle-aged, 20–22 months; old, 28–31 months). Single afferent discharges of the muscle spindles in gastrocnemius muscles were recorded from a finely split dorsal root during ramp-and-hold (amplitude, 2.0 mm; velocity, 2–20 mm s⁻¹) or sinusoidal stretch (amplitude, 0.05–1.0 mm; frequency, 0.5–2 Hz). Respective conduction velocities (CVs) were then measured. After electrophysiological experimentation, the muscles were dissected. The silver-impregnated muscle spindles were teased and then analysed using a light microscope. The CV and dynamic response to ramp-and-hold stretch of many endings were widely overlapped in old rats because of the decreased CV and dynamic response of primary endings. Many units in old rats showed slowing of discharge during the release phase under ramp-and-hold stretch and continuous discharge under sinusoidal stretch, similarly to secondary endings in young and middle-aged rats. Morphological studies revealed that primary endings of aged rat muscle spindles were less spiral or non-spiral in appearance, but secondary endings appeared unchanged. These results suggest first that primary muscle spindles in old rats are indistinguishable from secondary endings when determined solely by previously used physiological criteria. Secondly, these physiological results reflect drastic age-related morphological changes in spindle primary endings.     

Effects of changes in preoptic temperature on stretch response of muscle spindle endings in the cat's soleus muscle

1. The effects of changing the temperature in the preoptic region on the stretch responses of primary and secondary endings of the muscle spindle in the soleus muscle was investigated in urethane anesthetized cats. The local temperature of the preoptic region was controlled by implanting water perfused thermodes (32–42.5°C).
2. A standard ramp and hold stretch stimulus was repeatedly applied to the soleus muscle during changes of preoptic temperature from normal to hypo-or hyperthermic values. During each stretch the instantaneous firing rate was recorded and the static firing frequency and the dynamic index were electronically determined according to Crowe and Matthews (1964).
3. Of a total of 76 investigated muscle spindle afferents, the stretch responses of 49 (65%) were altered by changing preoptic temperature. The static stretch response was increased in all cases during cooling, whereas during preoptic heating the static stretch response increased in about half of the afferents and decreased in the rest. Increase in the static stretch response during both cooling and heating was usually combined with the appearence of spontaneous discharges at rest.
4. With regard to the static stretch response, primary and secondary muscle spindle endings responded similarly to cooling and heating.
5. Two types of preoptic temperature effects on the stretch responses of primary endings were observed. In the majority of afferents only the static stretch response was augmented during cooling, whereas the peak response was little changed resulting in a decrease of the dynamic index (‘static’ response type). In a minority of primary afferents the effect of preoptic cooling consisted of a greater increase of the peak response than of the static stretch response. i.e., the dynamic index also increased (‘dynamic-static’ response type). In secondary muscle spindle endings only the static type of response to preoptic cooling was observed.
6. It is concluded that changes of preoptic temperature influence the stretch response of muscle spindle afferents mainly by altering the activity of the static fusimotor innervation, its activation by preoptic cooling being the most consistent finding. In a minority of muscle spindles the dynamic fusimotor innervation appears to be additionally activated by preoptic cooling.

     

Functional similarities and differences of AMPA and kainate receptors expressed by cultured rat sensory neurons

Dorsal root ganglion neurons express functional AMPA and kainate receptors near their central terminals. Activation of these receptors causes a decrease in glutamate release during action potential evoked synaptic transmission. Due to differences in kinetic properties and expression patterns of these two families of glutamate receptors in subpopulations of sensory neurons, AMPA and kainate receptors are expected to function differently. We used embryonic dorsal root ganglion (DRG) neurons maintained in culture to compare functional properties of kainate and AMPA receptors. Most DRG neurons in culture expressed kainate receptors and about half also expressed AMPA receptors. Most AMPA and kainate receptor-expressing DRG neurons were sensitive to capsaicin, suggesting involvement of these glutamate receptors in nociception. When activated by kainate, AMPA receptors were capable of driving a sustained train of action potentials while kainate receptors tended to activate action potential firing more transiently. Glutamate elicited more action potentials and a larger steady-state depolarization in neurons expressing both AMPA and kainate receptors than in neurons expressing only kainate receptors. Adding to their more potent activation properties, AMPA receptors recovered from desensitization much more quickly than kainate receptors. Activation of presynaptic receptors by low concentrations of kainate, but not ATPA, caused a tetrodotoxin-sensitive increase in the frequency of spontaneous EPSCs recorded in dorsal horn neurons. By recording synaptic pairs of DRG and dorsal horn neurons, we found that activation of presynaptic kainate and AMPA receptors decreased evoked glutamate release from terminals of DRG neurons in culture. Our data suggest that the endogenous ligand, glutamate, will cause a different physiological impact when activating these two types of non-NMDA glutamate receptors at central or peripheral nerve endings of sensory neurons.     

Initial burst of primary endings of isolated mammalian muscle spindles.

The initial burst has been studied in primary endings of isolated mammalian muscle spindles subject to controlled ramp-and-hold stretch. Near the onset of ramp stretch the primary ending discharges at a frequency dependent on stretch velocity. The initial burst is reduced or abolished by repetitive stretch. After block of impulse activity by tetrodotoxin, the receptor potential of primary endings shows an initial component, a rapid depolarization which occurs near the onset of ramp stretch at the same time as the initial burst. This initial component depends, in rate of rise and amplitude, on stretch velocity. It is also reduced or abolished by repetitive stretch. Recording of tension development by the isolated spindle in response to ramp-and-hold stretch shows an early rise in tension associated with the initial burst and the initial component of the receptor potential. This tension rise is also dependent on stretch velocity and is reduced or abolished by repetitive stretch. The results provide direct evidence that the initial burst results from mechanical factors, probably from cross bridge formation between thick and thin filaments as has been suggested (3).     

Facilitation effect of auxiliary noise stimuli on response of isolated frog muscle spindle to sinusoidal movements

Receptor potentials and impulse patterns were recorded from isolated frog muscle spindles using sinusoidal and superimposed random stretches as stimuli with different sinus-to-noise ratios. The entire dynamic amplitude range of the spindle receptor was evaluated by measuring the sensory response at different levels of static stretch. Auxiliary random stimuli provoked rectified fast depolarizing receptor potential transients; their amplitude and slope grew larger with increasing intensity of the noise stimulus and with increasing prestretch level. Due to this strongly nonlinear behavior of the transducing site the frequency and size of the receptor potentials evoked by the auxiliary input signal increased during the stretching phase of the sinusoidal movement. Since the fast depolarizing receptor potential transients provided a powerful trigger for the action potential encoding site, auxiliary random stimuli effectively enhanced the afferent discharge rate, especially during the stretching phase of the sinusoidal movement. Auxiliary noise stimuli could even activate the afferent discharge to an otherwise subthreshold sinusoidal stretch. It is assumed that by the same mechanism the transfer characteristic of the receptor is broadened towards higher frequencies. Since auxiliary random stimuli increased the nonlinear properties of all receptor response components, a "linearizing" approximation technique only partially describes the receptor's transfer properties. The facilitation effect recorded in the differentiated muscle spindle when random stimuli were superimposed on sinusoidal displacements closely resembled the excitation of afferent firing when passive stretching interacted with active fusimotor innervation. A hypothesis is proposed to explain both effects by the same mechanism acting upon the transducing sensory endings: Since passive random stretches as well as active twitching of the intrafusal muscle fibers exhibited almost the same range of frequency components, we propose that both stimuli also generate the same kind of receptor potentials; namely, those fast-rising depolarization transients of the receptor potential, which vigorously drive the encoding site. In general, these experiments explain how the specific response of a neuron can be facilitated by an additional unspecific (noisy) input.     

A high-affinity presynaptic kainate-type glutamate receptor facilitates glutamate exocytosis from cerebral cortex nerve terminals (synaptosomes)

Ionotropic glutamate receptor agonists, kainate, -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate, all facilitated 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerve terminals (synaptosomes). The non-selective, non-N-methyl- -aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked kainate facilitation of glutamate release. AMPA responses were non-desensitizing and insensitive to the AMPA receptor desensitization inhibitor, cyclothiazide. The AMPA receptor antagonist GYKI 52466 failed to block ionotropic glutamate receptor-mediated facilitation, but the ionotropic glutamate receptor 6 kainate receptor subunit antagonist NS-102 was a potent blocker. Furthermore, kainate and AMPA responses were not additive. Taken together, our results indicate that, in the cerebral cortex, both kainate and AMPA may be facilitating glutamate release through the activation of a high-affinity kainate receptor containing glutamate receptor 6 kainate subunits. Kainate enhanced 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential, indicating that a ligand-gated ion channel that conducts cations may underlie the mechanism by which kainate mediates facilitation of glutamate release. While the facilitatory effect of kainate on glutamate release is consistent with a classical ionotropic action of ionotropic glutamate receptors, our observation that kainate inhibits GABA release suggests that alternative presynaptic mechanisms may operate in cerebrocortical nerve terminals to mediate the ionotropic glutamate receptor modulation of glutamate and GABA release.

We conclude that high-affinity kainate-type glutamate autoreceptors represent a positive feed-forward system for potentiating the release of glutamate from cerebrocortical nerve terminals.     

Static γ-motoneurones couple group Ia and II afferents of single muscle spindles in anaesthetised and decerebrate cats

Ideas about the functions of static γ-motoneurones are based on the responses of primary and secondary endings to electrical stimulation of single static γ-axons, usually at high frequencies. We compared these effects with the actions of spontaneously active γ-motoneurones. In anaesthetised cats, afferents and efferents were recorded in intramuscular nerve branches to single muscle spindles. The occurrence of γ-spikes, identified by a spike shape recognition system, was linked to video-taped contractions of type-identified intrafusal fibres in the dissected muscle spindles. When some static γ-motoneurones were active at low frequency (< 15 Hz) they coupled the firing of group Ia and II afferents. Activity of other static γ-motoneurones which tensed the intrafusal fibres appeared to enhance this effect. Under these conditions the secondary ending responded at shorter latency than the primary ending. In another series of experiments on decerebrate cats, responses of primary and secondary endings of single muscle spindles to activation of γ-motoneurones by natural stimuli were compared with their responses to electrical stimulation of single γ-axons supplying the same spindle. Electrical stimulation mimicked the natural actions of γ-motoneurones on either the primary or the secondary ending, but not on both together. However, γ-activity evoked by natural stimuli coupled the firing of afferents with the muscle at constant length, and also when it was stretched. Analysis showed that the timing and tightness of this coupling determined the degree of summation of excitatory postsynaptic potentials (EPSPs) evoked by each afferent in α-motoneurones and interneurones contacted by terminals of both endings, and thus the degree of facilitation of reflex actions of group II afferents.     

Involvement of group I metabotropic glutamate receptors and glutamate transporters in the slow excitatory synaptic transmission in the spinal cord dorsal horn

Our experiments demonstrate a novel role for group I metabotropic glutamate receptor (mGluR) subtypes 1 and 5 in generating a long-lasting synaptic excitation in the substantia gelatinosa (SG) and deep dorsal horn (DH) neurons of the rat spinal cord. In the present study we have investigated a slow excitatory postsynaptic current (EPSC), elicited by a brief high intensity (at Adelta/C fiber strength) and high frequency (20 or 100 Hz) stimulation of primary afferent fibers (PAFs) using whole-cell patch-clamp recordings from neurons located in the DH (laminae II-V) in spinal cord slices of young rats and wild-type and gene-targeted mice lacking mGluR1 subtype. The results shown here suggest that the activation of both mGluR1 and mGluR5 along with NK1 receptors, may be involved in the generation of the slow EPSC in the spinal cord DH. Inhibition of glial and neuronal glutamate transporters by dl-threo-beta-benzyloxyaspartate (TBOA) enhanced the group I mGluR-dependent slow EPSC about eightfold. Therefore, we conclude, that glutamate transporters strongly influence the group I mGluR activation by PAFs possibly at sensory synapses in the DH. Overall these data indicate that stimulus trains can generate a sustained and widespread glutamate signal that can further elicit prolonged EPSCs predominantly mediated by the group I mGluRs. These slow excitatory synaptic currents may have important functional implications for DH cell firing and synaptic plasticity of sensory transmission, including nociception.     

The muscle spindles in slow and twitch skeletal muscle of the lizard

1. Responses from stretch receptors, identified as muscle spindles, were recorded in filaments of the nerve supplying a twitch muscle, semimembranosus, and a slow muscle, semitendinosus in the lizard Tiliqua.2. While recording afferent discharges in one filament of the motor nerve, several adjacent filaments were each in turn stimulated repetitively until one was encountered which on stimulation produced a powerful increase in spindle firing. Such an effect of the motor stimulus was interpreted as resulting from intrafusal contraction. Any interference with spindle firing patterns from extrafusal contraction produced by the motor stimulation was removed by differentially blocking the contraction with the drug curare.3. Discharge patterns of spindles in response to a slow stretch of the muscle were compared with the response to the same stretch, but during repetitive stimulation of the motor nerve filament which produced an intrafusal contraction.4. At the initial length, the firing rate of spindles in the twitch muscle was greatly increased by the motor tetanus. There was little further increase in the response during and following stretch of the muscle.5. While the spindles in the slow muscle were only moderately excited by the motor tetanus at the initial length of the muscle, a large increase was recorded during the dynamic component of the stretch. At the new length, the steady-state firing continued at a rate well above that for the initial length.6. The effect of the motor tetanus on the response to stretch of muscle spindles in the slow muscle could be mimicked by adding succinyl choline (5 mug/ml.) to the perfusion solution. Spindles in the twitch muscle did not show a sustained sensitivity to the drug.7. It is suggested that while the different effects of motor stimulation on the responses to stretch of spindles in slow and twitch muscle can be explained by propositions based on the sliding filament theory of contraction, the sustained elevation, at the new length, of firing frequencies of spindles in slow muscle might require an additional explanation.     

Responses of muscle spindles following a series of eccentric contractions

To investigate the effects of eccentric exercise on the signalling properties of muscle spindles, experiments were done using the medial gastrocnemius muscle of cats anaesthetised with 40 mg/kg sodium pentobarbitone, i.p. Responses were recorded from single afferent nerve fibres in filaments of dorsal root during slow stretch of the passive muscle and during intrafusal contractions at a range of lengths, before and after a series of eccentric contractions. The sensitivity to slow stretch was measured as the average firing rate between muscle lengths 10.5 and 9.5 mm shorter than the physiological maximum (L_m), during stretch at 1 mm/s over the whole physiological range. The mean sensitivity of both primary and secondary spindle endings increased slightly, but not significantly, after a series of 20–150 eccentric contractions consisting of a 6 mm stretch, at 50 mm/s, to a final length of between L_m –7 mm and L_m, during stimulation of the whole muscle or sometimes of single fusimotor fibres. Discharges were recorded from primary endings during fusimotor stimulation at 100–150 pulses/s, and from secondary endings during static bag intrafusal contractures produced by i.v. injection of 0.2 mg/kg succinyl choline. Spindle responses were recorded, over a range of muscle lengths, in steps covering the whole physiological range. About half of the responses showed a peak in the relation between length and net increase in firing rate, while the remainder either progressively increased or progressively decreased over the physiological range. No large or consistent changes were seen after the eccentric contractions. It is concluded that the intrafusal fibres of muscle spindles are not prone to damage of the kind seen in extrafusal fibres after a series of eccentric contractions.     

Structures in sensory region of snake spindles and their displacment during stretch.

1. Structures within the sensory region of short- and long-capsule snake muscle spindles have been visualized using differential interference contrast microscopy. Profiles seen with Nomarski microscopy have been identified by electron microscopy of the same preparations. 2. Sensory nerve terminals, nuclei and other cytoplasmic inclusions in the intrafusal fiber, collagen bands, and capsular cells may be seen in the living preparation. 3. The length changes of various elements within the sensory region in response to stretch of the spindle have been measured using high-speed ciné photomicrography. This has been corrleated with the impulse response from sensory endings in short-and long-capsule spindles. 4. Short-capsule spindles, which have a high dynamic sensitivity, show length changes in the sensory region in response to ramp-and-hold stretch, which are not parallel to the changes in impulse frequency. The implications for mechanical models of spindle behavior are discussed.     

Effects of glutamate receptor activation on NG2-glia in the rat optic nerve

NG2‐glia are a substantial population of cells in the central nervous system (CNS) that can be identified by their specific expression of the NG2 chondroitin sulphate (CSPG). NG2‐glia can generate oligodendrocytes, but it is unlikely this is their only function; indeed, they may be multipotent neural stem cells. Moreover, NG2‐glia are a highly reactive cell type and a major function is to help form the axon growth inhibitory glial scar in response to CNS injury. The factors that regulate these diverse behaviours of NG2‐glia are not fully resolved, but NG2‐glia express receptors to the neurotransmitter glutamate, which has known potent effects on other glia. Here, we have examined the actions of glutamate receptor activation on NG2‐glia in the rat optic nerve, a typical CNS white matter tract that does not contain neuronal cell bodies. Glutamate induces an increase in [Ca²⁺]_i in immuno‐identified NG2‐glia in situ and in vitro. In addition, we examined the effects of glutamate receptor activation in vivo by focal injection of the glutamate receptor agonist kainate into the optic nerve; saline was injected in controls. Changes in glial and axonal function were determined at 7 days post injection (dpi), by immunohistochemistry and electrophysiological measurement of the compound action potential (CAP). Injection of kainate resulted in a highly localized ‘injury response’ in NG2‐glia, marked by dense labelling for NG2 at the lesion site, as compared to astrocytes, which displayed a more extensive reactive astrogliosis. Furthermore, injection of kainate resulted in an axonal conduction block. These glial and axonal changes were not observed following injection of saline vehicle. In addition, we provide evidence that endogenous glutamate induces calcium‐dependent phosphorylation of extracellular signal‐regulated kinases (ERK1/2), which may provide a potential mechanism by which glutamate‐mediated changes in raised intracellular calcium could regulate the observed gliosis. The results provide evidence that activation of AMPA‐kainate type ionotropic glutamate receptors evoke raised calcium in NG2‐glia and induces an injury response in NG2‐glia.