Guy S. Bewick

Autogenic modulation of mechanoreceptor excitability by glutamate release from synaptic‐like vesicles: evidence from the rat muscle spindle primary sensory ending

Fifty‐nanometre diameter, clear, synaptic‐like vesicles (SLVs) are found in primary mechanosensory nerve terminals of vertebrate and invertebrate animals. We have investigated their role in mechanosensory function using the muscle spindle primary endings of rat Ia afferents as a model. Uptake and release of the synaptic vesicle marker FM1‐43 indicated that SLVs recycle like synaptic vesicles and do so in a Ca2+‐sensitive manner. Mechanical stimulation increased SLV recycling, increasing both dye uptake and release. Immunogold/electronmicroscopy showed that, like the central synaptic endings, Ia peripheral endings are enriched with glutamate. Moreover, exogenous glutamate enhanced stretch‐induced Ia excitability. Enhanced excitability persisted in the presence of antagonists to the commonest ionotropic and metabotropic glutamate receptors (kynurenate, MCPG, CPPG and MAP4). However, excitation by glutamate was abolished by (R,S)‐3,5‐dihydroxyphenylglycine (DHPG), and rather more effectively by (2R,1′‐S,2′‐R,3′‐S)‐2‐(2′‐carboxy‐3′‐phenylcyclopropyl) glycine (PCCG‐13). PCCG‐13 also significantly reduced stretch‐activated excitability in the absence of exogenous glutamate. These data indicate that SLVs recycle at rest, releasing glutamate, and that mechanical activity increases this process. The blockade with DHPG and PCCG‐13 suggests that endogenous glutamate release acts, at least in part, through the recently described phospholipase D‐linked metabotropic Glu receptor to maintain the excitability of the sensory endings.

Amiloride-sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles

We investigated whether channels of the epithelial sodium/amiloride‐sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch‐evoked afferent discharge was reduced in a dose‐dependent manner by amiloride and three analogues – benzamil, 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching ≥85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC δ subunit agonists (icilin and capsazepine). HMAs profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P < 0.01 each) at 1 μm, while 10 μm HMA was required for highly significant inhibition (P < 0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low‐affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC α, β and γ subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC α, β or γ relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P < 0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1α) were also highly significantly greater (P < 0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic‐like vesicles which regulate afferent excitability in these mechanosensitive endings.

Mechanotransduction in the muscle spindle

The focus of this review is on the principal sensory ending of the mammalian muscle spindle, known as the primary ending. The process of mechanosensory transduction in the primary ending is examined under five headings: (i) action potential responses to defined mechanical stimuli—representing the endings input–output properties; (ii) the receptor potential—including the currents giving rise to it; (iii) sensory-terminal deformation—measurable changes in the shape of the primary-ending terminals correlated with intrafusal sarcomere length, and what may cause them; (iv) putative stretch-sensitive channels—pharmacological and immunocytochemical clues to their identity; and (v) synaptic-like vesicles—the physiology and pharmacology of an intrinsic glutamatergic system in the primary and other mechanosensory endings, with some thoughts on the possible role of the system. Thus, the review highlights spindle stretch-evoked output is the product of multi-ionic receptor currents plus complex and sophisticated regulatory gain controls, both positive and negative in nature, as befits its status as the most complex sensory organ after the special senses.

TGF-β2 alters the characteristics of the neuromuscular junction by regulating presynaptic quantal size

The amount of neurotransmitter released from a presynaptic terminal is the product of the quantal content (number of vesicles) and the presynaptic quantal size (QSpre, amount of transmitter per vesicle). QSpre varies with synaptic use, but its regulation is poorly understood. The motor nerve terminals at the neuromuscular junction (NMJ) contain TGF-β receptors. We present evidence that TGF-β2 regulates QSpre at the NMJ. Application of TGF-β2 to the rat diaphragm NMJ increased the postsynaptic response to both spontaneous and evoked release of acetylcholine, whereas antibodies to TGF-β2 or its receptor had the converse effect. L-vesamicol and bafilomycin blocked the actions of TGF-β2, indicating that TGF-β2 acts by altering the extent of vesicular filling. Recordings of the postsynaptic currents from the diaphragm were consistent with TGF-β2 having this presynaptic action and a lesser postsynaptic effect on input resistance. TGF-β2 also decreased quantal content by an atropine-sensitive pathway, indicating that this change is secondary to cholinergic feedback on vesicular release. Consequently, the net actions of TGF-β2 at the NMJ were to amplify the postsynaptic effects of spontaneous transmission and to diminish the number of vesicles used per evoked stimulus, without diminishing the amount of acetylcholine released.

Evidence for the involvement of ASIC3 in sensory mechanotransduction in proprioceptors

Acid-sensing ion channel 3 (ASIC3) is involved in acid nociception, but its possible role in neurosensory mechanotransduction is disputed. We report here the generation of Asic3-knockout/eGFPf-knockin mice and subsequent characterization of heterogeneous expression of ASIC3 in the dorsal root ganglion (DRG). ASIC3 is expressed in parvalbumin (Pv+) proprioceptor axons innervating muscle spindles. We further generate a floxed allele of Asic3 (Asic3f/f) and probe the role of ASIC3 in mechanotransduction in neurite-bearing Pv+ DRG neurons through localized elastic matrix movements and electrophysiology. Targeted knockout of Asic3 disrupts spindle afferent sensitivity to dynamic stimuli and impairs mechanotransduction in Pv+ DRG neurons because of substrate deformation-induced neurite stretching, but not to direct neurite indentation. In behavioural tasks, global knockout (Asic3−/−) and Pv-Cre::Asic3f/f mice produce similar deficits in grid and balance beam walking tasks. We conclude that, at least in mouse, ASIC3 is a molecular determinant contributing to dynamic mechanosensitivity in proprioceptors.

Postnatal emergence of mature release properties in terminals of rat fast- and slow-twitch muscles

Motor nerve terminals in adult mammalian slow‐twitch muscles have lower levels of spontaneous and evoked neurotransmitter release than terminals in fast‐twitch muscles. These reflect adaptive differences, allowing terminals in slow (postural) muscles to sustain release during the prolonged firing trains experienced in vivo. Here we ask whether these differences in terminal release properties in Sprague–Dawley rat extensor digitorum longus (EDL, fast) and soleus (slow) muscles reflect their early cytodifferentiation in the embryo or whether they might be adaptations to their distinct mature activity patterns, which emerge around two weeks postnatally. We find that the mature pattern of differences in release arise through co‐ordinated increases in presynaptically dependent release properties (quantal content, spontaneous release frequency and evoked potential amplitude), beginning at three weeks, which are particularly substantial in EDL. In contrast, other synaptic properties are either consistently greater in the same muscle throughout development (evoked potential kinetics, muscle fibre diameter) or display no systematic muscle type‐dependent differences (terminal area, input resistance, spontaneous release amplitude). Thus, the emergence of adaptive differences in terminal release properties correlates with the differentiation of locomotor activity patterns in postnatal rat hindlimb muscles.

Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles

•  The lanceolate sensory nerve ending of hair follicles is known to contain small (∼50 nm), clear vesicles similar to those of presynaptic terminals, but of unknown function. •  We show that the sensory terminals spontaneously take up and release the fluorescent styryl dye FM1‐43, and also provide other evidence that the dye flux is primarily by recycling of these synaptic‐like vesicles (SLVs). •  FM1‐43 labelling is Ca2+ dependent, and its release is sensitive to α‐latrotoxin, which is known to deplete synaptic vesicles at neuromuscular junctions. •  Responses of hair follicle afferents are not significantly affected by FM1‐43 at a concentration (10 μm) sufficient to label the endings, so the mechanotransduction channel that has previously been shown to be blocked by FM1‐43 permeation in hair cells of the inner ear and in cultured dorsal root ganglion cells is either not responsible for sensory transduction in the lanceolate ending or is in some way protected from exposure to the dye. •  The sensory terminals are relatively enriched in glutamate, presumably within the vesicles. •  Exogenous glutamate increases FM1‐43 labelling, whereas the labelling is strongly inhibited by PCCG‐13, a specific blocker of a non‐canonical phospholipase D‐linked metabotropic glutamate receptor, but not by canonical ionotropic or metabotropic glutamate receptor blockers. It is also inhibited by FIPI, a novel phospholipase D inhibitor. •  The system of SLVs is closely similar to that we have previously described in the muscle spindle, and where we further demonstrated the regulatory action of glutamate on the sensory response to maintained stretch. •  We conclude that an SLV‐mediated glutamatergic system is present in the mechanosensory endings of the primary afferents of lanceolate endings, and it appears to function in a similar way to the autoregulatory system of the muscle spindle.

The PDZ-Domain Protein Whirlin Facilitates Mechanosensory Signaling in Mammalian Proprioceptors

Mechanoreception is an essential feature of many sensory modalities. Nevertheless, the mechanisms that govern the conversion of a mechanical force to distinct patterns of action potentials remain poorly understood. Proprioceptive mechanoreceptors reside in skeletal muscle and inform the nervous system of the position of body and limbs in space. We show here that Whirlin/Deafness autosomal recessive 31 (DFNB31), a PDZ-scaffold protein involved in vestibular and auditory hair cell transduction, is also expressed by proprioceptive sensory neurons (pSNs) in dorsal root ganglia in mice. Whirlin localizes to the peripheral sensory endings of pSNs and facilitates pSN afferent firing in response to muscle stretch. The requirement of Whirlin in both proprioceptors and hair cells suggests that accessory mechanosensory signaling molecules define common features of mechanoreceptive processing across sensory systems.

Piezo is essential for amiloride-sensitive stretch-activated mechanotransduction in larval Drosophila dorsal bipolar dendritic sensory neurons.

Stretch-activated afferent neurons, such as those of mammalian muscle spindles, are essential for proprioception and motor co-ordination, but the underlying mechanisms of mechanotransduction are poorly understood. The dorsal bipolar dendritic (dbd) sensory neurons are putative stretch receptors in the Drosophila larval body wall. We have developed an in vivo protocol to obtain receptor potential recordings from intact dbd neurons in response to stretch. Receptor potential changes in dbd neurons in response to stretch showed a complex, dynamic profile with similar characteristics to those previously observed for mammalian muscle spindles. These profiles were reproduced by a general in silico model of stretch-activated neurons. This in silico model predicts an essential role for a mechanosensory cation channel (MSC) in all aspects of receptor potential generation. Using pharmacological and genetic techniques, we identified the mechanosensory channel, DmPiezo, in this functional role in dbd neurons, with TRPA1 playing a subsidiary role. We also show that rat muscle spindles exhibit a ruthenium red-sensitive current, but found no expression evidence to suggest that this corresponds to Piezo activity. In summary, we show that the dbd neuron is a stretch receptor and demonstrate that this neuron is a tractable model for investigating mechanisms of mechanotransduction.

A study of the expression of small conductance calcium-activated potassium channels (SK1-3) in sensory endings of muscle spindles and lanceolate endings of hair follicles in the rat

Processes underlying mechanotransduction and its regulation are poorly understood. Inhibitors of Ca2+-activated K+ channels cause a dramatic increase in afferent output from stretched muscle spindles. We used immunocytochemistry to test for the presence and location of small conductance Ca2+-activated K+ channels (SK1-3) in primary endings of muscle spindles and lanceolate endings of hair follicles in the rat. Tissue sections were double immunolabelled with antibodies to one of the SK channel isoforms and to either synaptophysin (SYN, as a marker of synaptic like vesicles (SLV), present in many mechanosensitive endings) or S100 (a Ca2+-binding protein present in glial cells). SK channel immunoreactivity was also compared to immunolabelling for the Na+ ion channel ASIC2, previously reported in both spindle primary and lanceolate endings. SK1 was not detected in sensory terminals of either muscle spindles or lanceolate endings. SK2 was found in the terminals of both muscle spindles and lanceolate endings, where it colocalised with the SLV marker SYN (spindles and lanceolates) and the satellite glial cell (SGC) marker S100 (lanceolates). SK3 was not detected in muscle spindles; by contrast it was present in hair follicle endings, expressed predominantly in SGCs but perhaps also in the SGC: terminal interface, as judged by colocalisation statistical analysis of SYN and S100 immunoreactivity. The possibility that all three isoforms might be expressed in pre-terminal axons, especially at heminodes, cannot be ruled out. Differential distribution of SK channels is likely to be important in their function of responding to changes in intracellular [Ca2+] thereby modulating mechanosensory transduction by regulating the excitability of the sensory terminals. In particular, the presence of SK2 throughout the sensory terminals of both kinds of mechanoreceptor indicates an important role for an outward Ca2+-activated K+ current in the formation of the receptor potential in both types of ending.