It is challenging to stimulate gamma motor neurons, which are important regulators of muscle spindle afferent function, without also recruiting alpha motor neurons. Here, we test the feasibility of stimulating gamma motor neuron axons using optogenetics in two transgenic mouse lines. We used an ex vivo muscle-nerve preparation in adult mice to monitor muscle spindle afferent firing, which should increase in response to gamma motor neuron-induced lengthening of the sensory region of the muscle spindle. A force transducer measured alpha motor neuron-mediated twitch contractions. Blue LED light (470 nm; 1-5 mW) was delivered via a light guide to the sciatic nerve. We confirmed that the more slowly conducting gamma motor neurons were recruited first in mice expressing channelrhodopsin 2 in choline acetyltransferase-positive motor neurons, whereas alpha motor neurons required higher optical intensities, enabling co-activation of alpha and gamma motor neurons depending on light intensity. However, this approach cannot isolate gamma motor neuron activity completely. Cre-dependent channelrhodopsin 2 optoactivation using the putative gamma motor neuron marker neuronal PAS domain protein 1 (Npas1) also increased muscle spindle afferent firing rates and caused only small twitch contractions. This provides functional validation that Npas1 is present primarily in gamma motor neurons and can be used to manipulate gamma motor neurons independently. We propose optogenetic stimulation as a promising tool to manipulate gamma motor neuron activity.

Animals requiring purposeful movement for survival are endowed with mechanoreceptors, called proprioceptors, that provide essential sensory feedback from muscles and joints to spinal cord circuits, which modulates motor output. Despite the essential nature of proprioceptive signaling in daily life, the mechanisms governing proprioceptor activity are poorly understood. Here, we identified nonredundant roles for two voltage-gated sodium channels (Na V s), Na V 1.1 and Na V 1.6, in mammalian proprioception. Deletion of Na V 1.6 in somatosensory neurons (Na V 1.6 cKO mice) causes severe motor deficits accompanied by loss of proprioceptive transmission, which contrasts with our previous findings using similar mouse models to target Na V 1.1 (Na V 1.1 cKO ). In Na V 1.6 cKO animals, we observed impairments in proprioceptor end-organ structure and a marked reduction in skeletal muscle myofiber size that were absent in Na V 1.1 cKO mice. We attribute the differential contributions of Na V 1.1 and Na V 1.6 to distinct cellular localization patterns. Collectively, we provide evidence that Na V s uniquely shape neural signaling within a somatosensory modality.

Voltage gated sodium channels (NaVs) are crucial for the generation and propagation of action potentials. There are 9 different isoforms found in mammals (NaV1.1-1.9), and NaV1.1, NaV1.6, and NaV1.7 are found in the muscle proprioceptors that sense muscle stretch, the muscle spindle afferents. This sensory information is critical for motor reflexes and the sense of body and limb position in space. We previously found that mice lacking one or both copies of NaV1.1 in peripheral sensory neurons displayed motor deficits, including intention tremors. Muscle spindle afferents in these mice also had unstable static stretch sensitivity but normal dynamic sensitivity, suggesting a specific deficit in the maintenance of proprioceptor excitability when the Piezo2 channel was likely to be closed (Espino, et al., 2022). We hypothesized that NaV1.6 would also be necessary for normal motor behavior and muscle spindle afferent function. We generated sensory neuron specific deletions of NaV1.6 by crossing NaV1.6fl/fl mice with mice expressing PirtCre (NaV1.6cko) and observed severe motor deficits on the open field and rotarod test only in NaV1.6cko (n=15) and not NaV1.6Het (n=12) mice compared to NaV1.6fl/fl controls (n=8). In contrast to mice lacking NaV1.1, we did not observe intention tremors. We used an ex vivo muscle nerve preparation consisting of the extensor digitorum longus muscle and peroneal branch of the sciatic nerve. The muscle was maintained at the length of maximal twitch contraction (Lo) and given a battery of 9 ramp-and-hold stretches (2.5, 5, and 7.5% Lo repeated 3 times each) and 16 sinusoidal vibrations (5-100 μm amplitude; 10-100 Hz frequency). Preliminary results include recordings from 18 adult mice of both sexes (NaV1.6fl/fl n=8; NaV1.6cko n=10). We were only able to find stretch sensitive responses in 3 of 10 NaV1.6cko mice, and those were severely impaired with firing rates less than 5 Hz. In contrast, we found robust slowly adapting responses to stretch in all 8 NaV1.6fl/fl mice. Only 1 of 10 NaV1.6cko mice had any firing in response to vibration, in contrast to the normal dynamic sensitivity observed in NaV1.6fl/fl and NaV1.1cko afferents. This suggests that NaV1.6 plays a critical role in initiating muscle spindle afferent transmission, whereas NaV1.1 is essential for maintaining proprioceptor excitability during static stretch. Future studies will determine whether loss of NaV1.7, which does not result in motor deficits, alters muscle spindle afferent function. This work was supported by NIH Grant 5SC3GM127195 (KAW) a RISE Fellowship 5R25GM71381 (SO), NIH 5T32GM099608-10 and 1T32GM1144303-01A1 (CE). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PIEZO2 is an essential mechanosensitive ion channel for touch discrimination, vibration, interoception, and proprioception. Mechanosensitive ion channels rely on membrane composition to transduce physical stimuli into electrical signals. Dietary fatty acids are among the membrane lipid components that dynamically regulate ion channel function. We have previously shown that when part of the plasma membrane, the polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (C20:5, EPA) decreases membrane rigidity and bending stiffness, while enhancing PIEZO1 inactivation. To determine if EPA could also modulate PIEZO2 channel function, we used electrophysiology, fatty acid supplementation, dietary intervention, and lipidomics in vitro and ex vivo. We found that EPA, when incorporated into the membrane, increased the inactivation of heterologously expressed PIEZO2 without significantly affecting the current magnitude or apparent activation threshold. Similarly, we measured an increase in PIEZO2 inactivation in rat and mouse dorsal root ganglia (DRG) neurons after EPA supplementation. Importantly, cultured DRG neurons from mice carrying the C. elegans FAT-1 enzyme (which converts ω-6 to ω-3 PUFAs) or fed for 4-12 weeks with an EPA-enriched diet displayed enhanced PIEZO2 inactivation when compared to neurons from control mice. Moreover, using a consecutive mechanical step protocol, we found that PIEZO2 slowly recovered from inactivation in DRG neurons supplemented with EPA, from fat-1 mice, or from mice fed with an EPA-enriched diet, when compared with the control. These results support the notion that EPA favors PIEZO2 non-conductive states. Gain-of-function PIEZO2 mutations causing Distal Arthrogryposis type 5 display decreased PIEZO2 inactivation and enhanced recovery from inactivation. We show that EPA supplementation restored the gating properties of Distal Arthrogryposis causing mutations to wild-type levels in vitro. Overall, our findings show that dietary fatty acids modulate PIEZO2 function and could be an effective strategy to ameliorate GOF PIEZO2-mediated diseases.

Distal arthrogryposis (DA) is a collection of rare disorders that are characterized by congenital joint contractures. Most DA mutations are in muscle- and joint-related genes, and the anatomical defects originate cell-autonomously within the musculoskeletal system. However, gain-of-function mutations in PIEZO2, a principal mechanosensor in somatosensation, cause DA subtype 5 (DA5) through unknown mechanisms. We show that expression of a gain-of-function PIEZO2 mutation in proprioceptive sensory neurons that mainly innervate muscle spindles and tendons is sufficient to induce DA5-like phenotypes in mice. Overactive PIEZO2 causes anatomical defects through increased activity within the peripheral nervous system during postnatal development. Furthermore, botulinum toxin (Botox) and a dietary fatty acid that modulates PIEZO2 activity reduce DA5-like deficits. This reveals a role for somatosensory neurons: Excessive mechanosensation within these neurons disrupts musculoskeletal development.

The voltage-gated sodium channel (NaV), NaV1.1, is well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we identify a new role for NaV1.1 in mammalian proprioception. RNAscope analysis and in vitro patch-clamp recordings in genetically identified mouse proprioceptors show ubiquitous channel expression and significant contributions to intrinsic excitability. Notably, genetic deletion of NaV1.1 in sensory neurons caused profound and visible motor coordination deficits in conditional knockout mice of both sexes, similar to conditional Piezo2-knockout animals, suggesting that this channel is a major contributor to sensory proprioceptive transmission. Ex vivo muscle afferent recordings from conditional knockout mice found that loss of NaV1.1 leads to inconsistent and unreliable proprioceptor firing characterized by action potential failures during static muscle stretch; conversely, afferent responses to dynamic vibrations were unaffected. This suggests that while a combination of Piezo2 and other NaV isoforms is sufficient to elicit activity in response to transient stimuli, NaV1.1 is required for transmission of receptor potentials generated during sustained muscle stretch. Impressively, recordings from afferents of heterozygous conditional knockout animals were similarly impaired, and heterozygous conditional knockout mice also exhibited motor behavioral deficits. Thus, NaV1.1 haploinsufficiency in sensory neurons impairs both proprioceptor function and motor behaviors. Importantly, human patients harboring NaV1.1 loss-of-function mutations often present with motor delays and ataxia; therefore, our data suggest that sensory neuron dysfunction contributes to the clinical manifestations of neurological disorders in which NaV1.1 function is compromised. Collectively, we present the first evidence that NaV1.1 is essential for mammalian proprioceptive signaling and behaviors.

The primary sensory input for proprioception, or the sense of body position in space, comes from muscle spindle afferents, which are mechanoreceptors that relay information about changes in muscle length. The muscle spindle is also innervated by sympathetic neurons, but the role of this sympathetic innervation on muscle spindle function is not well understood. Here we test the hypothesis that muscle spindle afferents will exhibit decreased firing in response to muscle stretch following exposure to the sympathetic neurotransmitters norepinephrine and epinephrine. To test this hypothesis we used an ex vivo mouse muscle‐nerve preparation. We recorded muscle spindle afferent firing activity during ramp‐and‐hold stretch and sinusoidal vibration before and after the addition of norepinephrine, epinephrine, or adrenergic receptor agonists. We observed significantly decreased firing during the end of stretch after both norepinephrine (100 µm, n=6) and epinephrine (30 µm, n=6). To identify the adrenergic receptor(s) involved, we also tested two ɑ2 adrenergic receptor agonists, Clonidine and Dexmedetomidine, and the ɑ1 receptor agonist phenylephrine. Both ɑ2 receptor agonists caused a decrease in muscle spindle afferent firing (1 mM clonidine, n=8 decreased firing; 100 µm dexmedetomidine, n=3). Phenylephrine had no significant effect on muscle spindle afferent firing (100 µm n=2; 30 µm n=6). These results show direct effects of sympathetic neurotransmitters on muscle spindle afferent stretch sensitivity and further support the hypothesis that sympathetic innervation of the muscle spindle plays an important role in modulating muscle spindle afferent activity and therefore motor control. Future studies will investigate the location of the adrenergic receptor(s) mediating this effect as well as the mechanism of action.

The muscle spindle (MS) provides essential sensory information for motor control and proprioception. The Group Ia and II MS afferents are low threshold slowly-adapting mechanoreceptors and report both static muscle length and dynamic muscle movement information. The exact molecular mechanism by which MS afferents transduce muscle movement into action potentials is incompletely understood. This short review will discuss recent evidence suggesting that PIEZO2 is an essential mechanically sensitive ion channel in MS afferents and that vesicle-released glutamate contributes to maintaining afferent excitability during the static phase of stretch. Other mechanically gated ion channels, voltage-gated sodium channels, other ion channels, regulatory proteins, and interactions with the intrafusal fibers are also important for MS afferent mechanosensation. Future studies are needed to fully understand mechanosensation in the MS and whether different complements of molecular mediators contribute to the different response properties of Group Ia and II afferents.

The muscle spindle is a sensory organ located in the skeletal muscle that is critical for motor control and proprioception. The muscle spindle is innervated by stretch sensitive muscle spindle afferents and gamma motor neurons that control the length of the intrafusal muscle fibers and therefore the sensitivity of the stretch sensitive afferents. However, it has been challenging to study the gamma motor neurons since it is hard to specifically stimulate the gamma but not the alpha motor neurons that control the force generating extrafusal fibers. A previous study showed that alpha motor neurons were recruited from small diameter to large diameter with increasing optical intensities in mice expressing the blue light gated Channelrhodopsin2 (ChR2) in motor neurons (Llewellyn, et al., 2010). This is the reverse recruitment pattern of electrical stimulation. We hypothesized that gamma motor neurons, which are smaller than even the smallest alpha motor neurons, will be recruited first using low optical stimuli. The extensor digitorum longus muscle and sciatic nerve were dissected and placed in a tissue bath with oxygenated synthetic interstitial fluid. The sciatic nerve was attached to an extracellular suction electrode that recorded muscle spindle afferent activity. We used a light guide to deliver blue LED light (470nm; 0.5mW-5 mW) to the end of the nerve. We found that at lower levels of optical stimulation we recruited the gamma motor neurons as evidenced by an increased muscle spindle afferent firing rate and a pause after stimulation, which is typically observed following the release of stretch. Higher optical intensities were required to recruit alpha motor neurons to produce a twitch muscle contraction. Higher frequencies of optical stimulation led to greater increases in muscle spindle afferent firing rates as expected. We are currently testing additional stimulation frequencies and increasing our sample size to understand individual variability. We are also verifying that ChR2 is found in motor neurons only using immunohistochemistry. We will use this technique to study gamma motor neuron function and to produce a more physiological relevant system to study muscle spindle afferents. We can also use this method to screen for intrafusal fiber dysfunction in disease models.

Key points Muscle spindle afferents are slowly adapting low threshold mechanoreceptors that report muscle length and movement information critical for motor control and proprioception. The rapidly adapting cation channel PIEZO2 has been identified as necessary for muscle spindle afferent stretch sensitivity, although the properties of this channel suggest that additional molecular elements are necessary for mediating the complex slowly adapting response of muscle spindle afferents. We report that glutamate increases muscle spindle afferent static sensitivity in an ex vivo mouse muscle nerve preparation, although blocking glutamate packaging into vesicles by the sole vesicular glutamate transporter, VGLUT1, either pharmacologically or by transgenic knockout of one allele of VGLUT1 decreases muscle spindle afferent static but not dynamic sensitivity. Our results confirm that vesicle‐released glutamate is an important contributor to maintained muscle spindle afferent excitability and may suggest a therapeutic target for normalizing muscle spindle afferent function. Abstract Muscle spindle afferents are slowly adapting low threshold mechanoreceptors that have both dynamic and static sensitivity to muscle stretch. The exact mechanism by which these neurons translate muscle movement into action potentials is not well understood, although the PIEZO2 mechanically sensitive cation channel is essential for stretch sensitivity. PIEZO2 is rapidly adapting, suggesting the requirement for additional molecular elements to maintain firing during stretch. Spindle afferent sensory endings contain glutamate‐filled synaptic‐like vesicles that are released in a stretch‐ and calcium‐dependent manner. Previous work has shown that glutamate can increase and a phospholipase‐D coupled metabotropic glutamate antagonist can abolish firing during static stretch. Here, we test the hypothesis that vesicle‐released glutamate is necessary for maintaining muscle spindle afferent excitability during static but not dynamic stretch. To test this hypothesis, we used a mouse muscle‐nerve ex vivo preparation to measure identified muscle spindle afferent responses to stretch and vibration. In C57BL/6 adult mice, bath applied glutamate significantly increased the firing rate during the plateau phase of stretch but not during the dynamic phase of stretch. Blocking the packaging of glutamate into vesicles by the sole vesicular glutamate transporter, VGLUT1, either with xanthurenic acid or by using a transgenic mouse with only one copy of the VGLUT1 gene (VGLUT1 +/– ), decreased muscle spindle afferent firing during sustained stretch but not during vibration. Our results suggest a model of mechanotransduction where calcium entering the PIEZO2 channel can cause the release of glutamate from synaptic‐like vesicles, which then helps to maintain afferent depolarization and firing.

The muscle spindle is an important sense organ for motor control and proprioception. Specialized intrafusal fibers are innervated by both stretch sensitive afferents and γ motor neurons that control the length of the spindle and tune the sensitivity of the muscle spindle afferents to both dynamic movement and static length. γ motor neurons share many similarities with other skeletal motor neurons, making it challenging to identify and specifically record or stimulate them. This short review will discuss recent advances in genetic and molecular biology techniques, electrophysiological recording, optical imaging, computer modelling, and stem cell culture techniques that have the potential to help answer important questions about fusimotor function in motor control and disease.

The molecular mechanisms by which Group Ia and II muscle spindle (MS) afferents transduce muscle movement into action potentials is not fully understood, although the rapidly adapting PIEZO2 mechanically sensitive cation channel is essential. Mechanosensation in many types of sensory neurons relies on PIEZO2, suggesting that unique response properties are mediated by the complement of molecular mediators found in the different sensory neurons. Synaptic-like vesicles containing glutamate are released from the MS afferent receptor ending in a stretch and calcium dependent manner, suggesting a way to couple depolarization and increased intracellular calcium via PIEZO2 with additional depolarizing current via glutamate. Here we test the hypothesis that vesicle-released glutamate is necessary for maintaining MS afferent excitability especially during the static phase of stretch. We isolated the extensor digitorum longus muscle and the sciatic nerve from adult mice (8-12 wks old) and compared identified MS afferent firing rates during ramp-and-hold stretch and vibration before and after the addition of glutamate and an inhibitor of glutamate packaging into vesicles (xanthurenic acid; XA). Glutamate addition led to a significant increase in MS afferent firing rate during the end of stretch (1mM; n=12 in control and glutamate groups), while blocking glutamate release with XA significantly decreased firing (3mM; n=17), with 5 afferents exhibiting a complete absence of firing. Firing rates during the dynamic phase of stretch and sinusoidal vibrations were not as affected by drug treatment. However, we noticed heterogeneity in MS afferent responses, with some afferents not responding to the drug treatments. To confirm our pharmacological findings, we used a transgenic mouse line with 1 or 2 copies of the vesicular glutamate 1 transporter gene (VGLUT1; B6.129X1-Sic17a7tm1Edw/MmcD; Fremeau, 2004). VGLUT1+/- afferents (n=14) had significantly lower firing rates during the end of stretch than afferents from wildtype littermates (WT; n=14), however firing rates during vibration were not significantly different. Importantly, 3 of 14 VGLUT1+/- afferents could not maintain firing throughout the entire 4 s stretch, something never seen in WT afferents. These results support our hypothesis that glutamate is critical to maintaining MS afferent firing during maintained stretch. Future studies will explore which glutamate receptor subtype and signaling pathway mediates this effect. A better understanding of MS afferent mechanotransduction could define targets for therapeutic intervention during diseases with abnormal afferent excitability.

The muscle spindle is an important sense organ for motor control and proprioception. Specialized intrafusal fibers are innervated by both stretch sensitive afferents and gamma motor neurons that control the length of the spindle and tune the sensitivity of the muscle spindle afferents to both dynamic movement and static length. gamma motor neurons share many similarities with other skeletal motor neurons, making it challenging to identify and specifically record or stimulate them. This short review will discuss recent advances in genetic and molecular biology techniques, electrophysiological recording, optical imaging, computer modelling, and stem cell culture techniques that have the potential to help answer important questions about fusimotor function in motor control and disease.

Proprioception is the ability to sense body position and motion in space. The primary proprioceptors are the Group Ia and II muscle spindle afferents that innervate the muscle spindle and comprise the sensory arm of the muscle stretch reflex. In‐vivo animal studies have shown that increased sympathetic outflow decreases dynamic and static stretch sensitivity of muscle spindle afferents. However, the direct effect of sympathetic neurotransmitters on muscle spindle afferent stretch sensitivity is not well understood. We hypothesized that the addition of sympathetic neurotransmitters would cause a decrease in responsiveness of muscle spindle afferents via direct action in the muscle spindle. To test this hypothesis, we used an in‐vitro mouse muscle nerve preparation to record muscle spindle afferent firing rates during stretch. The muscle was stretched once every three minutes to 5% resting length for 4 s. Muscle spindle afferent firing rates were determined during the plateau phase of stretch and compared before and after drug addition. Instantaneous firing frequency during a static muscle stretch was measured at different time intervals: beginning of stretch (0.5 seconds into the hold phase or Initial Static Time (IST)) and end of stretch (3.5 seconds into the hold phase or Final Static Time (FST)). All values are expressed as a percentage of the average of 6 pre‐drug baseline responses (BL) and compared to no drug control mice stretched for the equivalent time. Exposure to 100μM norepinephrine (n=6, IST=54.18% BL, FST=37.70% BL) significantly decreased the response to stretch as compared to no drug control animals (n=12, IST=90.82% BL, FST=91.50% BL; p<0.05 One‐way ANOVA w Tukey’s post‐hoc to control). The addition of epinephrine decreased muscle spindle afferent firing, however only significantly at the lower dose of 30μM (n=6, IST=76.90% BL, FST=73.20% BL; p<0.05). The α1 adrenergic receptor agonist, phenylephrine, was used to test the hypothesis that the decrease seen following norepinephrine was due to binding at the α1 adrenergic receptor. No statistically significant decrease was found following phenylephrine administration, however, the sample size used was small (n=3, IST=96.70% BL, FST=96.98% BL). These results show direct effects of sympathetic neurotransmitters on muscle spindle afferent stretch sensitivity and support the hypothesis that sympathetic innervation of the muscle spindle plays an important role in modulating stretch responsiveness. Future studies will use optogenetic techniques to stimulate the sympathetic neurons to determine whether physiologically relevant release of sympathetic neurotransmitters controls muscle spindle afferent sensitivity.Support or Funding InformationThis work was supported by NIH SC3 GM127195 (KAW) and NIH R25 GM071381 (AS and SV).

Group Ia and II muscle spindle afferents are sensory neurons critical to detecting muscle movement and length, which is important in proprioception and motor control. Glutamate is released by synaptic‐like vesicles (SLVs) at muscle spindle afferent nerve endings and exogenous glutamate increases sensory neuron firing rate during stretch (Bewick, et al., 2005). However, the activity of individual muscle spindle afferents was not measured, allowing for the possibility that this increased firing also included other sensory neurons known to have glutamate receptors, like nociceptors. Here we test the hypothesis that glutamate increases the firing rate of individual muscle spindle afferents. We isolated the extensor digitorum longus muscle and the sciatic nerve from adult mice (8–12 wks old) and recorded the activity of individual muscle spindle afferents during exogenous glutamate treatment. The muscle‐nerve preparation was perfused in an oxygenated synthetic interstitial fluid tissue bath, an extracellular recording electrode was placed on the nerve to measure the activity of individual muscle spindle afferents, and a battery of 4s ramp‐and‐hold stretches were performed on the muscle. Glutamate addition led to a significant increase in muscle spindle afferent firing rate during stretch (1mM; n=12, 18.8%). We also decreased glutamate release by blocking the vesicular glutamate transporter 1 (VGLUT1) with the inhibitor xanthurenic acid (XA). We found a significant decrease in muscle spindle afferent firing rates (3mM; n=17, −39%), with 5 afferents exhibiting a complete absence of firing. However, we noticed heterogeneity in muscle spindle afferent responses to glutamate and XA treatment, with some afferents not responding at all to the drug treatments. To confirm our pharmacological findings, we obtained a transgenic mouse line lacking a single copy of vesicular glutamate transporter 1 (VGLUT1; B6.129X1‐Sic17a7tm1Edw/MmcD; Fremeau, 2004). We observed two muscle spindle afferents responding normally to stretch, while another two failed to sustain firing at the end of stretch, something that is only rarely seen in wild type animals. This heterogeneity may be due to differences in VGLUT1 protein levels in individual afferents. We are currently increasing our sample size, but our preliminary results support our hypothesis that glutamate is critical to maintaining muscle spindle afferent firing during maintained stretch. These studies will lead to a better understanding of how muscle spindle afferent stretch sensitivity is regulated.Support or Funding InformationThis work was supported by NIH SC3 GM127195 (KAW) and NIH R25 GM071381 (SO and NV).

Obesity is associated with balance and motor control deficits. We have recently shown that Group Ia muscle spindle afferents, the sensory arm of the muscle stretch reflex, are less responsive in mice fed a high-fat diet. Here we test the hypothesis that reflex excitability to sensory information from Group Ia muscle spindle afferents is altered in a mouse model of diet-induced obesity. We measured the anesthetized Hoffmann's or H-reflex, the electrical analog of the muscle stretch reflex. Adult mice of both sexes were fed a control diet (CD; 10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) for 5, 10, or 15 weeks. We used three quantitative measures of H-reflex excitability: (1) H-reflex latency; (2) the percentage of motor neurons recruited from electrical stimulation of Group Ia muscle spindle afferents (H-max/M-max); and (3) rate-dependent depression (RDD), the decrease in H-reflex amplitude to high frequency stimulation (20 stimuli at 5 Hz). A HFD did not significantly alter H latency (P = 0.16) or H-max/M-max ratios (P = 0.06), but RDD was significantly lower in HFD compared to CD groups (P < 0.001). Interestingly, HFD males exhibited decreased RDD compared to controls only after 5 and 10 weeks of feeding, but females showed progressive decreases in RDD that were only significant at 10 and 15 weeks on the HFD. These results suggest that high-fat feeding increases H-reflex excitability. Future studies are needed to determine whether these changes alter muscle stretch reflex strength and/or balance and to determine the underlying mechanism(s).

Key points Acetylcholine receptors are aggregated in the central regions of intrafusal muscle fibres. Single unit muscle spindle afferent responses from isolated mouse extensor digitorum longus muscle were recorded in the absence of fusimotor input to ramp and hold stretches as well as to sinusoidal vibrations in the presence and absence of the acetylcholine receptor blockers d‐tubocurarine and α‐bungarotoxin. Proprioceptive afferent responses to both types of stretch were enhanced in the presence of either blocker. Blocking acetylcholine uptake and vesicular acetylcholine release by hemicholinium‐3 also enhanced stretch‐evoked responses. These results represent the first evidence that acetylcholine receptors negatively modulate muscle spindle responses to stretch. The data support the hypothesis that the sensory nerve terminal is able to release vesicles to fine‐tune proprioceptive afferent sensitivity. AbstractMuscle spindles are complex stretch‐sensitive mechanoreceptors. They consist of specialized skeletal muscle fibres, called intrafusal fibres, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ‐motoneurons. Previously it was shown that acetylcholine receptors (AChR) are concentrated in the equatorial region at the contact site between the sensory neuron and the intrafusal muscle fibre. To address the function of these AChRs, single unit sensory afferents were recorded from an isolated mouse extensor digitorum longus muscle in the absence of γ‐motoneuron activity. Specifically, we investigated the responses of individual sensory neurons to ramp‐and‐hold stretches and sinusoidal vibrations before and after the addition of the competitive and non‐competitive AChR blockers d‐tubocurarine and α‐bungarotoxin, respectively. The presence of either drug did not affect the resting action potential discharge frequency. However, the action potential frequencies in response to stretch were increased. In particular, frequencies of the dynamic peak and dynamic index to ramp‐and‐hold stretches were significantly higher in the presence of either drug. Treatment of muscle spindle afferents with the high‐affinity choline transporter antagonist hemicholinium‐3 similarly increased muscle spindle afferent firing frequencies during stretch. Moreover, the firing rate during sinusoidal vibration stimuli at low amplitudes was higher in the presence of α‐bungarotoxin compared to control spindles also indicating an increased sensitivity to stretch. Collectively these data suggest a modulation of the muscle spindle afferent response to stretch by AChRs in the central region of intrafusal fibres possibly fine‐tuning muscle spindle sensitivity.

Inflammation is known to alter nervous system function, but its effect on muscle spindle afferent mechanosensation and sensory integration in the spinal cord has not been well studied. We tested the hypothesis that systemic inflammation induced by an intraperitoneal injection of the endotoxin lipopolysaccharide (LPS; 7.5x10(5) endotoxin units/kg 18 h before experiment) would alter muscle spindle afferent mechanosensation and spinal cord excitability to Group Ia input in male and female adult C57Bl/6 mice. LPS injection caused a systemic immune response, evidenced by decreased white blood cell, monocyte, and lymphocyte concentrations in the blood, increased blood granulocyte concentration, and body weight loss. The immune response in both sexes was qualitatively similar. We used an invitro muscle-nerve preparation to assay muscle spindle afferent response to stretch and vibration. LPS injection did not significantly change the response to stretch or vibration, with the exception of small decreases in the ability to entrain to high-frequency vibration in male mice. Similarly, LPS injection did not alter spinal cord excitability to Group Ia muscle spindle afferent input as measured by the Hoffman's reflex test in anesthetized mice (100 mg/kg ketamine, 10 mg/kg xylazine). Specifically, there were no changes in M or H wave latencies nor in the percentage of motor neurons excited by electrical afferent stimulation (H-max/M-max). Overall, we found no major alterations in muscle proprioceptor function or sensory integration following exposure to LPS at a dose and time course that causes changes in nociceptor function and central processing.

The SJSU Biological Sciences Department recently redesigned the core introductory biology sequence to align with the best practices in biology pedagogy as described in the Vision and Change in Undergraduate Biology Education report . We now emphasize depth not breadth of information and focus on understanding how to interpret experimental results. We have incorporated more inquiry‐based labs and active learning strategies, including out of class reading quizzes and in‐class clicker questions. Our goal is to improve student performance in this 2 semester‐long lecture and lab course and improve student preparation for success in the major. To assess the success of our redesign and aid in our understanding of the student population that takes these courses, we administer two validated concept inventories, the Biology Concept Inventory (BCI) and the Test of Scientific Literacy Skills (TOSLS) , and a demographic survey at the beginning of the first course and the end of the second course. We have finished the first full year of the redesigned series and have seen modest improvements in failure rates (old version: BIOL 1A: 22%, BIOL 1B: 24% failure rate; new version: BIOL 30: 19%, BIOL 31: 13%). We also saw improvements in the performance of first generation college students and students from underrepresented minority groups in the new courses as well as increased retention into the second semester. Students in both versions of the courses scored significantly higher on the BCI following the second semester, suggesting improved mastery of the course content. Students in the old version of the course scored significantly worse on the TOSLS in the second semester, while students in the new version of the course had post‐test TOSLS scores that were trending higher, suggesting the new version was more effective in improving scientific literacy skills. We are continuing to track student success in future courses and are investigating changes in student attitudes about science with the Colorado Learning Attitudes about Science Survey (CLASS). Overall, the redesign has had positive effects on student success. Support or Funding Information We are supported by a California State University Program in Education and Research in Biotechnology (CSUPERB) Curriculum Grant. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Populations with obesity are more likely to fall and exhibit balance instability. The reason for this is likely multifactorial, but there is some evidence that sensory function is impaired during obesity. We tested the hypothesis that muscle proprioceptor function is compromised in a mouse model of diet induced obesity. An in vitro muscle-nerve preparation was used to record muscle spindle afferent responses to physiological stretch and sinusoidal vibration. We compared the responses of C57/Bl6 male and female mice on a control diet (10% kcal fat) with those eating a high fat diet (HFD; 60% kcal fat) for 10 weeks (final age 14-15 weeks old). Following HFD feeding, adult mice of both sexes exhibited decreased muscle spindle afferent responses to muscle movement. Muscle spindle afferent firing rates during the plateau phase of stretch were significantly lower in both male and female HFD animals as were two measures of dynamic sensitivity (dynamic peak and dynamic index). Muscle spindle afferents in male mice on a HFD were also significantly less likely to entrain to vibration. Due to the importance of muscle spindle afferents to proprioception and motor control, decreased muscle spindle afferent responsiveness may contribute to balance instability during obesity.