Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo MouseEar Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given

In lanceolate endings of rat hair follicles the small conductance Ca2+-activated K+ channel SK3 is found mainly in glial cells.

Autogenic modulation of mechanoreceptor excitability by Ca2+-dependent glutamate release from synaptic-like vesicles (SLVs) has been shown using the muscle spindle as a model (1). The excitability of lanceolate endings of hair follicles may be similarly regulated (2). In spindles Ca2+ entry through P/Q type channels activates KCa channels (BK or SK) to regulate afferent firing (3). In excitatory synapses of mouse hippocampus SK3 is a presynaptic channel (4) where it is probably involved in regulating neurotransmitter release. SK3 might therefore play a role in modulating glutamate release from SLVs. We have now studied SK3 expression in spindles and lanceolate endings, plus their associated satellite glial cells (SLGs) by immunocytochemistry. Synaptophysin (SYN, a marker of SLVs) was used to label sensory endings while the Ca2+-binding protein, S100 was used to identify SLGs. Adult rats (2) were deeply anaesthetized with sodium pentobarbitone (45 mg kg−1, I.P.) and fixed by transcardial perfusion (4% (w/v) paraformaldehyde in 0.1M phosphate buffer, pH 7.4; all procedures in accordance with ASPA 1986). Immunofluorescence labelling was carried out on 10µm cryosections of pinna skin. Sections were double stained with one of four antibody combinations: 1) anti-SK3 (5µg/ml, goat polyclonal Santa Cruz Biotechnology) + anti-SYN (1μg/ml, mouse monoclonal Millipore); 2) anti-SK3 + anti-S100 (1:400, mouse monoclonal Santa Cruz Biotechnology); 3) anti-ASIC2 (5µg/ml, goat polyclonal Santa Cruz Biotechnology) + anti-SYN; 4) anti-ASIC2 + anti-S100. Secondary antibodies were Alexa Fluor (AF) conjugated (AF 594 goat anti-rabbit and AF 488 goat anti-mouse, Invitrogen). Images were taken with a Leica SP5 Laser Scanning Microscope. Colocalisation of SK3 and ASIC2 reactivity with either S100 or SYN labelling was assessed by Pearson’s correlation coefficient (r) using LAS AF Lite software (Leica Microsystems CMS GmbH). We were unable to demonstrate SK3 in spindle terminals but it was present in the SLGs of lanceolate endings and, at lower levels, in the terminals themselves. Correlation of SK3 with S100 (r = 0.34 + 0.05, mean + SE) was greater than ASIC2 with S100 (0.15 + 0.03), P < 0.01. SK3 correlation with SYN (0.24 + 0.02) was less than that of ASIC2 with SYN (0.42 + 0.03), P < 0.01. SK3 correlation with S100 was greater than with SYN, but this did not reach statistical significance. Thus, SK3 was expressed predominantly in SLGs. By contrast immunolabelling for the Na+ ion channel ASIC2, previously reported in lanceolate endings (5) and SYN were largely coincident. Our data suggest that in lanceolate endings SK3 is expressed predominantly in SLG’s. SK3 channels could play a role in shaping SLG responses to fluctuations in intracellular Ca2+ and thereby indirectly influence afferent excitability of the terminals

A role for glutamate in regulating mechanosensory sensitivity: Further evidence from rat muscle spindle primary afferent terminals.

Muscle spindles are proprioceptive sensory organs that constantly report the length and movements of skeletal muscle. The complicated process of mechanotransduction underlying their function is still poorly understood. The stretch-sensitive annulospiral terminals have populations of small (50 nm), clear synaptic-like vesicles (SLVs). We have shown (Bewick et al., 2005) SLVs contain glutamate, which when released, increases spindle firing by acting on a non-canonical, phospholipase D-coupled metabotropic glutamate receptor (PLD-mGluR). For a better understanding of how glutamate modulates afferent firing, we examined the effects of DL-TBOA, a non-selective excitatory amino acid transporter (EAAT) inhibitor and Rose Bengal, a blocker of the vesicular glutamate transporters (VGLUTs). Adult Sprague-Dawley rats (male, 300-370g) were killed and 4th lumbrical nerve-muscle preparations excised from both hind legs and stored under gassed (95%O2-5%CO2) saline. Spindle discharges were recorded at room temperature during 1 mm stretch-and-hold cycles (~10% muscle length). Data are expressed as mean ± SD. Differences between the pre-drug control and with-drug mean firing frequencies (impulses per second, imp/sec) were evaluated by paired t-test, with a significance threshold of P = 0.05. 100 µM DL-TBOA increased afferent firing from 199.0 ± 48.3 to 322.9 ± 48.9 imp/sec (n = 8; P < 0.0001) after 3 hr incubation, indicating that EAATs are important regulators of the effects of endogenously-released glutamate. This excitation involved activation of PLD-mGluRs, since it could be counteracted by PCCG-13, a specific PLD-mGluR blocker. Thus, when lumbricals were incubated in DL-TBOA with 10 µM PCCG-13, firing initially increased, from 214.6 ± 19.4 to 273.6 ± 27.7 imp/sec (2 hr, n = 2; P < 0.002), but then decreased to 123.5 ± 28.6 imp/sec (3 hr, n = 2; P < 0.003). All effects were fully reversible on washing. However, the non-selective VGLUT inhibitor Rose Bengal had no significant effect on the afferent discharge at 100 nM (n = 2; P < 0.51) or 1 µM (n = 2; P < 0.11). These data indicate tonic endogenous glutamate release from SLV recycling constantly modulates firing in muscle spindle afferents, with EAATs limiting its effects by re-uptake. The absence of perturbation from the VGLUT inhibitor, despite the presence of VGLUT1 (Wu et al., 2004), requires further study. It may indicate a large pool of pre-loaded SLVs must first be emptied before glutamate depletion effects are observed

Modulation of afferent excitability and reflex responses by phospholipase D-coupled metabotropic glutamate receptors in the peripheral terminals of rat arterial baroreceptors.

Vertebrate primary mechanosensory nerve terminals contain populations of clear synaptic-like vesicles (SLVs). We have demonstrated glutamate released from SLVs produces autogenic modulation of mechanoreceptor excitability in Ia primary afferent endings of rat muscle spindles and guard hair follicles (Bewick et al., 2005; Singh et al., 2009), acting on phospholipase D-linked metabotropic glutamate receptors (PLD-mGluRs). Our aim here was to test whether the SLVs in baroreceptor endings (Krauhs, 1979) indicate a similar mechanism can modulate afferent discharge and reflex-evoked responses. Male Wistar rats (60-100g) were prepared for the working heart-brainstem preparation (Paton, 1996) by deeply anaesthetising with halothane (by inhalation) until loss of paw and tail withdrawal reflex then bisected sub-diaphragmatically. The animals were decerebrated at the precollicular level to ensure insentience before anaesthesia was discontinued. Perfusion pressure (PP), heart rate (HR) and thoracic chain sympathetic nerve activity (SNA) were recorded. Baroreceptors were stimulated by ramp increases in PP while either afferent discharge or reflex changes in HR and SNA were measured after superfusion of the aortic arch with either the PLD-mGluR antagonist PCCG-13 (20 μM) or glutamate (10 mM). Afferent activity data were normalised as μV/mmHg and expressed as % change and the gain of the cardiac baroreceptor reflex response expressed as beats/min(bpm)/mmHg. Data are from 4 rats and expressed as mean ± SEM; statistical significance was determined using a Student’s t-test. PCCG-13 decreased baroreceptor activity by 35.4 ± 5.0% (P<0.05) whereas glutamate increased discharge by 42.0 ± 6.0% (P<0.05) relative to control levels; both effects were reversible. Control cardiac baroreceptor reflex gain was 2.8 ± 0.4 bpm/mmHg but was attenuated reversibly following application of PCCG-13 (-50.7%; P<0.05). The baroreceptor reflex-mediated sympathoinhibition was also depressed (n = 1). Finally, glutamate applied to the aortic arch mimicked a baroreflex response. These data indicate autogenic glutamate release from baroreceptor terminals can effectively regulate afferent activity through PLD-mGluRs, to modulate reflex cardiac and sympathomotor responses. Together with similar systems in hair follicle and muscle spindle afferent terminals, this is consistent with a general role for SLVs in glutamatergic modulation of peripheral mechanosensory terminals

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

KCa channels regulate stretch-evoked afferent firing from muscle spindles.

Muscle spindles constantly report skeletal muscle length and movement. Mechanosensory transduction occurs at annulospiral terminals around intrafusal muscle fibres. We reported that Ca2+-dependent glutamate release from synaptic-like vesicles in these terminals modulates spindle firing (Bewick et al., 2005). To test if voltage-gated Ca2+ channels (VGCCs) or Ca2+-dependent K+ channels (KCa) are also functionally important, we tested whether channel-selective neurotoxins affected spindle afferent discharge. Adult Sprague-Dawley rats (male, 300-370 g) were killed (Schedule 1, Animal (Scientific Procedures) Act, 1986) and 4th lumbrical nerve-muscle preparations excised and placed in gassed (95%O2-5%CO2) Liley`s saline. Spindle afferent discharges were recorded en passant with Ag wire electrodes and firing frequency (mean ± SE, n) determined for the first 0.5 s of the “hold” phase of stretch-and-hold cycles (~10% muscle length) for ‘n’ preparations. The significance of differences between pre-drug and with-drug means was evaluated by paired t-test, with a threshold of P < 0.05. Unlike inorganic Ca2+ channel blockers Co2+ and Ni2+/Cd2+, which abolish responses (Bewick et al., 2005), P/Q type VGCC inhibitors enhanced firing. ω-Agatoxin-IVA (200 nM, P/Q type) increased firing to 301% of control (92.7 ± 15.8 imp/s vs 278.6 ± 23.9 imp/s, 6; P < 0.0001). ω-Conotoxin-MVIIC (1 μM, Q type) enhanced firing to 202% (98.40 ± 21.83 imp/s vs 199.60 ± 24.62 imp/s, 5; P < 0.002). Conversely, ω-conotoxin-GVIA (1 μM; N type) had no significant effect (176.92 ± 14.77 imp/s vs 196.92 ± 10.47 imp/s, 6; P = 0.1). L-type blockers Taicatoxin (50 nM, 4) and Nifedipine (10 μM, 5) increased afferent firing (131.88 ± 10.05 imp/s vs 302.13 ± 25.17 imp/s and 192.00 ± 13.09 imp/s vs 271.70 ± 9.56 imp/s, respectively) but also caused spontaneous muscle twitching, suggesting actions on skeletal muscle dihydropyridine receptors. Thus, only P/Q type channel blockers increased afferent discharge in the absence of other effects. P/Q type channels often regulate KCa (BK or SK) channel opening (Edgerton & Reinhart, 2003). Therefore, selective KCa channel blockers were applied. Charybdotoxin (200 nM; SK & BK), iberiotoxin (200 nM; BK selective) and apamin (200 nM; SK selective) all increased afferent discharge (to 195%, 4; 224%, 4 & 160%, 6 of control, respectively; all P < 0.02). Conversely, NS1619 (BK channel activator) inhibited firing (1 μM, 179.75 ± 14.35 imp/s vs 33.25 ± 18.44 imp/s, 6). This was not due to voltage-gated potassium channel or action potential conduction block, since NS1619 did not affect Nervus saphenus compound action potential amplitude (100 μM, 4). The data suggest Ca2+ entry through P/Q type channels activates KCa channels to regulate firing frequency in spindle 1a afferents