Pathogenic role of delta 2 tubulin in bortezomib-induced peripheral neuropathy

The pathogenesis of chemotherapy-induced peripheral neuropathy (CIPN) is poorly understood. Here, we report that the CIPN-causing drug bortezomib (Bort) promotes delta 2 tubulin (D2) accumulation while affecting microtubule stability and dynamics in sensory neurons in vitro and in vivo and that the accumulation of D2 is predominant in unmyelinated fibers and a hallmark of bortezomib-induced peripheral neuropathy (BIPN) in humans. Furthermore, while D2 overexpression was sufficient to cause axonopathy and inhibit mitochondria motility, reduction of D2 levels alleviated both axonal degeneration and the loss of mitochondria motility induced by Bort. Together, our data demonstrate that Bort, a compound structurally unrelated to tubulin poisons, affects the tubulin cytoskeleton in sensory neurons in vitro, in vivo, and in human tissue, indicating that the pathogenic mechanisms of seemingly unrelated CIPN drugs may converge on tubulin damage. The results reveal a previously unrecognized pathogenic role for D2 in BIPN that may occur through altered regulation of mitochondria motility.

Chemotherapy-induced peripheral neuropathy (CIPN) is a “dying back” neuropathy that features a distal-to-proximal peripheral nerve degeneration that frequently occurs in cancer patients undergoing chemotherapy (1). Modification of the chemotherapy treatment of a patient is sometimes required to limit the severity of CIPN, preventing patients from receiving effective cancer treatment (2). The pathogenesis of CIPN is largely unknown, and this incomplete knowledge is a main reason for the absence of effective neuroprotection strategies while maintaining chemotherapy drug anticancer activities (2–5). Several classes of anticancer drugs with different antineoplastic mechanisms can induce CIPN (6–8). However, sensory impairment is the predominant adverse effect associated with each class, suggesting the existence of a common mechanism of pathogenesis (2).Tubulin and microtubules (MTs) are well-established targets for multiple anticancer drugs that induce CIPN (9). The contribution of the MT changes to the onset of CIPN is not well understood but is strongly implicated as the determining factor. MT hyper-stabilization plays a direct role in paclitaxel neurotoxicity (10), and a single nucleotide polymorphism in TUBB2a, a gene encoding a tubulin isoform, is associated with an enhanced risk of CIPN (11). Moreover, MT dynamics and stability are influenced by nicotinamide adenine dinucleotide (NAD+) levels through sirtuin modulation (12), suggesting a tubulin-mediated mechanism also for the NAD+-consuming activity of Sterile Alpha and Tir Motifs–containing protein 1 (SARM1) in driving axonal degeneration in CIPN models (13).Tubulin, its posttranslational modifications (PTMs), MT dynamics, and stability play critical roles in neurons via the regulation of long-distance transport, MT severing, Ca²⁺ homeostasis, and mitochondrial energetics (14–20). Each of these functions provides a potential mechanism that could result in the axonal degeneration in CIPN (21). Furthermore, tubulin and MTs functionally interact with Transient Receptor Potential Cation Channel Subfamily V (TRPV) members 1 and 4, two nonselective cation channels that are implicated in both sporadic and familial cases of peripheral neuropathy (22–24). Importantly, anomalies in MT dynamics and tubulin PTMs can drive axon regeneration failure and neurodegeneration (25–32), and α-tubulin acetylation is a critical component of the mammalian mechanotransduction machinery and a pathological hallmark in the neuropathy induced by the drug vincristine and genetically inherited forms of peripheral neuropathy (13, 31, 33–36).In addition to taxanes and vinca alkaloids, tubulin changes have been reported downstream of the reversible 26S proteasomal subunit inhibitor bortezomib (Bort), a widely employed drug with anti-tumor activity in hematological malignancies (37). Bortezomib-induced peripheral neuropathy (BIPN) is a painful axonal sensory–predominant and length-dependent peripheral neuropathy that affects ∼40% of Bort-treated patients (3). Bort has been shown to increase MT polymer without disrupting MT ultrastructure and to affect MT-dependent axonal transport (37, 38). However, the in vivo and in vitro effects of Bort on tubulin PTMs and MT behavior as a potential BIPN pathogenic pathway have not been characterized. Furthermore, whether the perturbation of one or more tubulin PTMs may be sufficient and necessary to induce CIPN has not been determined.Herein, we report that delta 2 tubulin (D2), an irreversible tubulin PTM residing on hyper-stable MTs, was increased in dorsal root ganglia (DRG) and sciatic nerves (SNs) from rats treated with Bort and in the sural nerve from a patient suffering from BIPN. D2 is a tubulin PTM that accumulates on very long-lived MTs and is promoted in vitro by MT stabilizers and drugs that cause CIPN, such as paclitaxel and vinca alkaloids (39, 40). The function of D2 remains unknown, although it is abundant in neurons as well as in long-lasting MT conformations in cilia. D2 is produced by the sequential action of a tubulin carboxypeptidase (VASH1/2) that cleaves the last residue of tyrosine from an α-tubulin subunit residing on stable MTs followed by the irreversible cleavage of a residue of glutamic acid by carboxypeptidase 1 (CCP1), a member of a family of tubulin de-glutamylases that is also expressed in neurons (42–45). Tubulin tyrosine ligase (TTL) is a rate-limiting enzyme that re-tyrosinates tubulin and so controls the level of de-tyrosinated tubulin, a reversible short-lived D2 precursor (46). Interestingly, constitutive suppression of TTL in mice results in perinatal death linked to a disorganization of CNS neuronal networks, underscoring a critical role for de-tyrosination and D2 in neuronal development (47, 48) A pathogenic role for D2 in neurodegeneration, however, has never been demonstrated.We found that Bort affects MT dynamics in vitro and in vivo and that D2 accumulation in sensory neurons may result from Bort-induced MT stabilization and increased tubulin heterodimer stability. Importantly, D2 was sufficient and necessary for both the axonopathy and disrupted mitochondria motility caused by Bort. These findings reveal a previously unrecognized role for the disruption of the tubulin tyrosination/de-tyrosination cycle in the onset of axonal injury that may occur through D2-dependent regulation of mitochondria transport.     

Multisensory interactions regulate feeding behavior in Drosophila

The integration of two or more distinct sensory cues can help animals make more informed decisions about potential food sources, but little is known about how feeding-related multimodal sensory integration happens at the cellular and molecular levels. Here, we show that multimodal sensory integration contributes to a stereotyped feeding behavior in the model organism Drosophila melanogaster. Simultaneous olfactory and mechanosensory inputs significantly influence a taste-evoked feeding behavior called the proboscis extension reflex (PER). Olfactory and mechanical information are mediated by antennal Or35a neurons and leg hair plate mechanosensory neurons, respectively. We show that the controlled delivery of three different sensory cues can produce a supra-additive PER via the concurrent stimulation of olfactory, taste, and mechanosensory inputs. We suggest that the fruit fly is a versatile model system to study multisensory integration related to feeding, which also likely exists in vertebrates.

Feeding is one of the most important animal behaviors for survival. Taste allows animals to select and ingest nutritious foods and prevents them from accidentally ingesting toxic substances (1). It is not just taste, however, that animals use when selecting foods. All of us, in our daily experiences with food, have noticed that our perceptions of a food do not solely depend on its taste. Instead, our perceptions are shaped by cross-modal combinations of taste, smell, texture, temperature, sights, and sounds (2). The most obvious example of cross-modal combination is the interaction between taste and olfaction. Typically tasteless odorants, such as those that smell of strawberry or vanilla, increase the perceived sweetness of sugar solutions (3). In contrast, the loss of retronasal olfactory inputs due to the common cold causes a reduced ability to distinguish various tastes (4, 5).Despite the obvious importance of multisensory integration in food perception and feeding, many studies have focused instead on discrete sensory channels (1, 6). This is mainly due to technical difficulties in measuring the relative contributions of each sensory channel. Drosophila is an excellent model system for investigating the role multimodal interactions play in food perception and feeding (7–15). Powerful neurogenetic tools developed for use in Drosophila allow us to parse the contributions of each individual sensory channel in various aspects of fly feeding using simple behavioral assays combined with the selective silencing or activation of distinct sensory modalities.Indeed, flies utilize multiple sensory modalities while searching for, evaluating, and ingesting various foods. Food-derived odorants are important cues in long- and short-range food searching (7, 8, 16) and also promote feeding initiation and increase food intake (10, 12). The mechanical properties of foods (e.g., hardness, viscosity, etc.) also affect food preference. Flies prefer a range of soft and viscous foods, detecting a food’s texture via taste sensilla-associated mechanosensory neurons (MNs) and multidendritic neurons in the fly labellum (9, 11, 13–15). It is noteworthy that a fly’s preference for a specific texture depends on the presence of appetitive taste cues, suggesting cross-modal interactions drive food perception and preference (11).Here, we report that three distinct sensory channels—taste, olfaction, and mechanosensation—cooperate to enhance fly feeding initiation. In flies, the proboscis extension reflex (PER) is a proxy for food palatability and feeding (17). Activation of olfactory receptor neurons enhances the PER evoked by low concentrations of sugars, but either an increase in food viscosity or genetic perturbation of mechanosensation abolish this PER enhancement. We found that the silencing of hair plate MNs abolishes odor-induced enhancement of PER, while optogenetic activation of hair plate MNs in mechanosensory mutants recapitulates PER enhancement. Finally, we found that only concurrent inputs from the three different sensory modalities enhance PER, indicating that multisensory integration guides feeding behavior.     

Stac protein regulates release of neuropeptides

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca²⁺. However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca²⁺ channels (CaChs) and excitation–contraction coupling in vertebrate skeletal muscles. There is a small family of stac genes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood. Drosophila melanogaster contain a single stac gene, Dstac, which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca²⁺ imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) in Drosophila motor neurons and this, in turn, controls the release of neuropeptides.

Neuropeptides are required for a myriad of brain functions, such as regulation of complex social behaviors, including emotional behavior (1). The activities of many neuropeptides within the brain are now well-established, but, despite the important role neuropeptides play, our understanding of the mechanisms by which neuropeptides are released by neurons and how release is regulated is not as advanced as that of classical neurotransmitters. A greater understanding of neuropeptide release could help untangle the mechanisms of complex behaviors as well as reveal therapeutic targets that could modulate aberrant behaviors.Release of neuropeptides, which are packaged in dense core vesicles (DCVs) rather than in small synaptic vesicles that contain neurotransmitters, often involves activation of L-type Ca²⁺ channels (CaChs) and Ca²⁺-induced Ca²⁺ release (CICR) (2). Much of what is known about DCV exocytosis is from the study of nonneural cells. For example, in adrenal chromaffin cells, the exocytosis of DCVs containing catecholamines and/or peptide hormones involves CICR either via the ryanodine receptor (RyR) or inositol trisphosphate receptor (iP₃R) (3, 4) initiated by an influx of Ca²⁺ through voltage-gated CaChs, such as L-type CaChs. In neurons, the release of neuropeptides is more complex. Neuropeptides can be released from dendrites, cell bodies, axons, and presynaptic terminals (5–7), and release can involve a kiss-and-run mechanism (8). DCVs are not tightly clustered as are small synaptic vesicles, and DCVs are generally not associated with presynaptic specializations for release of neurotransmitters, such as active zones (9). Furthermore, the DCVs within the central nervous system (CNS) are not as numerous nor as large as they are in chromaffin cells and neurohypophyseal terminals. In many neurons, the release of neuropeptides requires bursts of action potentials (10, 11). Based primarily upon pharmacological experiments, it appears that an influx of Ca²⁺ via L-type CaChs is necessary for release of neuropeptides from some neurons (8, 12–18), with some cases also involving Ca²⁺ release from internal stores while others not. This suggests the possibility that, in neurons, neuropeptide release may be initiated by an influx of Ca²⁺ via L-type CaCh.Drosophila provide an ideal system to study neuropeptide release due to the vast array of molecular and genetic tools available for manipulating them. Numerous neuropeptides are expressed by the Drosophila nervous system, including proctolin by motor neurons (19, 20). Furthermore, larval motor neurons fire bursts of high frequency action potentials (21, 22) so motor boutons are likely to have the long-lasting increases in Ca²⁺ transients in motor boutons that are thought to be required for the release of neuropeptides. Motor boutons contain a network of endoplasmic reticulum (ER) (23), and there is a RyR-dependent release of Ca²⁺ from the ER presumably via CICR, which is required for sustained release of neuropeptides at the neuromuscular junction (NMJ) of third instar larvae (24). Elegant dynamic examination of DCVs in boutons with fluorescence recovery after photobleaching showed that they are immobile in the resting state, but they move randomly and release neuropeptides for several minutes following activity-dependent Ca²⁺ increases (25). Furthermore, simultaneous photobleaching and imaging of DCVs suggest that DCVs release neuropeptides via multiple rounds of kiss-and-run events (26). Thus, in Drosophila, neuropeptide release at the NMJ appears to involve CICR from the ER.One way to regulate the release of neuropeptides is to control changes in cytoplasmic Ca²⁺ levels. In mammals, stac3 is a member of a small family of genes, along with stac1 and stac2, which are expressed by subsets of neurons (27–29). Stac3 was identified as a regulator of L-type CaChs and excitation–contraction (EC) coupling in zebrafish skeletal muscles (30, 31). Stac3 also regulates EC coupling in murine skeletal muscles (32) and is causal for the congenital Native American myopathy (30). EC coupling is the process that transduces changes in muscle membrane voltage to initiate release of Ca²⁺ from the sarcoplasmic reticulum (SR) and contraction. In vertebrate skeletal muscles, EC coupling is mediated by the L-type CaCh DHPR, which is in the transverse tubule membrane (t-tubules) and is the voltage sensor for EC coupling, and the RyR, which is the Ca²⁺ release channel in the SR (33–36). In zebrafish, Stac3 regulates EC coupling by colocalizing with DHPR and RyR, and by regulating DHPR stability and functionality, including the response to voltage of DHPRs, but not trafficking of DHPRs (31, 37).The in vivo function of the stac1 and stac2 genes expressed by neurons is, however, unknown. Recently, a stac-like gene, Dstac, was identified in Drosophila (38). There is a single stac gene in Drosophila, and it is expressed both by muscles and a subset of neurons, including in the lateral ventral neurons (LN_Vs) that express the neuropeptide, pigment-dispersing factor (PDF), in the brain. Previously, genetic manipulation of PDF demonstrated the necessity of PDF for circadian rhythm (39). Interestingly, knocking down Dstac selectively in the PDF neurons disrupted circadian rhythm, suggesting the hypothesis that Dstac regulates the release of neuropeptides such as PDF. Since Stac3 regulates the L-type CaCh in vertebrate skeletal muscle, Dstac might control neuropeptide release via regulation of the single L-type CaCh in Drosophila neurons (Dmca1D) (40). We tested this hypothesis by examining the role of Dstac for neuropeptide release by the more accessible presynaptic boutons of motor neurons at the NMJs of third instar larvae.     

MrgprC11+ sensory neurons mediate glabrous skin itch

Itch arising from glabrous skin (palms and soles) has attracted limited attention within the field due to the lack of methodology. This is despite glabrous itch arising from many medical conditions such as plantar and palmar psoriasis, dyshidrosis, and cholestasis. Therefore, we developed a mouse glabrous skin behavioral assay to investigate the contribution of three previously identified pruriceptive neurons in glabrous skin itch. Our results show that MrgprA3⁺ and MrgprD⁺ neurons, although key mediators for hairy skin itch, do not play important roles in glabrous skin itch, demonstrating a mechanistic difference in itch sensation between hairy and glabrous skin. We found that MrgprC11⁺ neurons are the major mediators for glabrous skin itch. Activation of MrgprC11⁺ neurons induced glabrous skin itch, while ablation of MrgprC11⁺ neurons reduced both acute and chronic glabrous skin itch. Our study provides insights into the mechanisms of itch and opens up new avenues for future glabrous skin itch research.

Chronic itch is a debilitating disease that arises from a multitude of etiologies. It is the most common reason for visiting the dermatologist and has few effectual treatments (1, 2). Although itch sensation can arise from any area of the skin, itchy palms and soles (hairless glabrous skin) are considered the most debilitating and are associated with many dermatological and systemic conditions (3–5). A few examples include palmoplantar pustulosis (or palmar and plantar psoriasis), a chronic skin disease characterized by inflamed scaly skin and intense itch on the palms and soles that is reported to affect 0.01 to 0.05% of the US population (4); dyshidrosis, a skin condition causing itchy blisters to develop only on the palm and soles that results in an estimated 200,000 US cases per year (5); and cholestatic itch, an intense itching sensation felt in the limbs, and particularly the palms and soles of feet, frequently reported with hepatobiliary disorders (3). In recent years, considerable progress has been made in our understanding of itch with the identification of key molecules and neuronal populations mediating itch in both the peripheral and central nervous system (2, 6). However, the majority of established itch behavioral assays are performed with pruritogens applied to the hairy skin including the back, cheek, and hindlimb (7). Since hairy and glabrous skin are anatomically and physiologically distinct and are innervated by different cutaneous nerves (8, 9), it is unclear whether different mechanisms are employed for itch arising from the two types of skin.Recent functional studies and single-cell RNA-sequencing analysis have characterized three subtypes of itch-sensing neurons in the dorsal root ganglia (10–14). Two of them are labeled by MrgprA3 and MrgprD, respectively, both of which are members of the Mrgpr (mas-related G protein-coupled receptor) itch receptor family (15). MrgprA3⁺ neurons can respond to multiple pruritogens and play a key role in both acute and chronic itch (16–19). MrgprD⁺ neurons mediate β-alanine–induced itch and are defined as itch-sensing neurons (20). Additionally, Sst⁺/Nppb⁺ neurons express a distinct array of itch receptors and are key mediators for mast cell-induced itch (12, 21). However, it is unknown whether these defined pruriceptive neuronal populations project to the glabrous skin and whether they play a role in glabrous skin itch.In this study, we analyzed skin innervation patterns of three subtypes of itch-sensing neurons and found that MrgprA3⁺ neurons only sparsely innervate the glabrous skin. This result raises the question if the itch mechanisms in hairy skin and glabrous skin are different. This is a critical question, as different itch mechanisms implies the need for different clinical treatment strategies. Therefore, to investigate the mechanisms of glabrous skin itch, we developed a mouse behavioral assay to examine itch sensation arising from the glabrous skin of the plantar hindpaw. Using this itch assay, combined with genetic, physiological, and chemogenetic approaches, we demonstrate that glabrous skin itch is mainly mediated by MrgprC11⁺ neurons, but not MrgprA3⁺ or MrgprD⁺ neurons.     

Spontaneous activity competes with externally evoked responses in sensory cortex

The interaction between spontaneous and externally evoked neuronal activity is fundamental for a functional brain. Increasing evidence suggests that bursts of high-power oscillations in the 15- to 30-Hz beta-band represent activation of internally generated events and mask perception of external cues. Yet demonstration of the effect of beta-power modulation on perception in real time is missing, and little is known about the underlying mechanism. Here, we used a closed-loop stimulus-intensity adjustment system based on online burst-occupancy analyses in rats involved in a forepaw vibrotactile detection task. We found that the masking influence of burst occupancy on perception can be counterbalanced in real time by adjusting the vibration amplitude. Offline analysis of firing rates (FRs) and local field potentials across cortical layers and frequency bands confirmed that beta-power in the somatosensory cortex anticorrelated with sensory evoked responses. Mechanistically, bursts in all bands were accompanied by transient synchronization of cell assemblies, but only beta-bursts were followed by a reduction of FR. Our closed loop approach reveals that spontaneous beta-bursts reflect a dynamic state that competes with external stimuli.

The brain is constantly active, even at resting states in the absence of external stimuli (1). Spontaneously active resting-state networks (RSNs) were found in memory, visual, auditory, tactile, and sensorimotor regions, with activity patterns similar to task-evoked responses (2, 3). Functional connectivity studies in humans suggest that a default network, spontaneously activated at resting states and deactivated upon increased cognitive demands, antagonizes a network involved in active attention to external sensory input (4–8). However, whether the activity in different networks is anticorrelated is under debate, and their antagonizing mechanisms and influence on local circuits remain unknown (9).Here, we utilized the occupancy of high-power bursts in the beta-band (15 to 30 Hz) of local field potentials (LFPs) as an indicator of spontaneous activity to investigate its influence on detection in real time. Several lines of evidence relate the RSN to beta-bursts. First, spontaneous correlated oscillatory activity in beta (termed “beta-connectome”) (10) was reported in anatomical regions corresponding to the RSN (11, 12). A recent study derived this beta-connectome from burst occupancy (10). Second, beta-oscillations are dominant during the resting state (13) and bursts are responsible for virtually all beta-band power modulation (14). Third, task-dependent desynchronization of beta was observed in the somatosensory (15, 16), visual (17), auditory (18), and motor (19) cortices, resembling RSN deactivation (4, 7). The task-dependent averaged power modulation was attributed to changes in burst rates in rodents, nonhuman primates, and humans (14, 20, 21). Fourth, the burst duration (50 to a few hundred milliseconds) (22) is similar to “packets” of neural activity, which are conceived as messages initiated in a particular cortical region and spread as a wave over the cortex. Most of these packets are generated spontaneously, and spontaneous and sensory-evoked packets are remarkably similar (23).We found that bursts in all bands indicate transient synchronization of flexible neuronal networks, but only beta-bursts were followed by a reduction in population firing rate (FR). High occupancies of beta-bursts predicted reduced detection, and this effect can be counterbalanced bidirectionally in real time by adjusting the stimulus intensity according to burst occupancy.     

Cerebellar Purkinje cells can differentially modulate coherence between sensory and motor cortex depending on region and behavior

Activity of sensory and motor cortices is essential for sensorimotor integration. In particular, coherence between these areas may indicate binding of critical functions like perception, motor planning, action, or sleep. Evidence is accumulating that cerebellar output modulates cortical activity and coherence, but how, when, and where it does so is unclear. We studied activity in and coherence between S1 and M1 cortices during whisker stimulation in the absence and presence of optogenetic Purkinje cell stimulation in crus 1 and 2 of awake mice, eliciting strong simple spike rate modulation. Without Purkinje cell stimulation, whisker stimulation triggers fast responses in S1 and M1 involving transient coherence in a broad spectrum. Simultaneous stimulation of Purkinje cells and whiskers affects amplitude and kinetics of sensory responses in S1 and M1 and alters the estimated S1–M1 coherence in theta and gamma bands, allowing bidirectional control dependent on behavioral context. These effects are absent when Purkinje cell activation is delayed by 20 ms. Focal stimulation of Purkinje cells revealed site specificity, with cells in medial crus 2 showing the most prominent and selective impact on estimated coherence, i.e., a strong suppression in the gamma but not the theta band. Granger causality analyses and computational modeling of the involved networks suggest that Purkinje cells control S1–M1 phase consistency predominantly via ventrolateral thalamus and M1. Our results indicate that activity of sensorimotor cortices can be dynamically and functionally modulated by specific cerebellar inputs, highlighting a widespread role of the cerebellum in coordinating sensorimotor behavior.

Integration of sensory feedback into motor control is of obvious importance for the learning and execution of skilled movements. This integration is particularly relevant when subjects explore their environment via active touch, requiring sensory input to be directly related to the momentary position and movement of eyes, fingertips, antennae, whiskers, or other sense organs (1–3). In vertebrates, adapting movement based upon somatosensory feedback requires collaborative action of primary somatosensory (S1) and motor (M1) cortices (4–6), which are directly and reciprocally connected (7–12). The reciprocal connections between S1 and M1 imply that each region can differentially affect the amplitude of neuronal responses in the other region. These connections can also create coherent activity patterns in S1 and M1 (13–17).Coherence can add an extra layer of neuronal integration, as it has been suggested to bind different brain areas by affecting susceptibility of neurons to synaptic input and providing a timing mechanism for generating a common dynamical frame for cortical operations (18–22). As such, coherence can create a temporal framework for concerted neural activity that facilitates integration of the activity of sensory and motor areas (23–25). Coherence often occurs in specific frequency bands that can be associated with different functions. In the field of sensorimotor integration, skilled movements rely on intercortical coherence between sensory and motor areas that occur in the theta range (4 to 8 Hz) during force generation, while coherence at higher bands is engaged during the preparation thereof (26). Likewise, within the field of visual perception, coherence in the alpha (8 to 12 Hz) and gamma (30 to 100 Hz) bands have been found to contribute to feedback and feedforward processing, respectively (27, 28).Cortical coherence can be modulated by subcortical activity, as has been particularly well established for the thalamus (29–32). Accordingly, the cerebellum, one of the main inputs to the thalamus, has a strong impact in organizing cortical coherence (33–35). Indeed, disruption of cerebellar function, whether inflicted pharmacologically in rats (34) or by stroke in patients (33), affects cortico-cortical coherence.Given the strong cerebello-thalamo-cortical projections (36, 37), it is not surprising that cerebellar activity affects the amplitude and coherence of S1 and M1 (20, 34, 38, 39). However, it is unclear to what extent cerebellar activity can modulate the kinetics of the fast sensory responses in S1 that are propagated by the canonical lemniscal and paralemniscal pathways (40), whether it can differentially influence individual frequency bands, to what extent such potentially different impacts depend on the behavioral context, and whether these differential effects are mediated through different cerebellar modules (41–43). Here, we set out to address these questions by investigating the impact of Purkinje cell activity on the response amplitude and the estimated coherence between the whisker areas of the primary somatosensory (wS1) and motor cortex (wM1) during stimulation of the whiskers in awake behaving mice. Stimulating the whiskers results in transient sensory responses in wS1 and wM1, which were affected by optogenetic stimulation of Purkinje cells at different intervals with respect to air-puff stimulation of the whiskers. The estimated coherence of the sensory response in wS1 and wM1 was differentially modulated by Purkinje cell stimulation, affecting mainly the activity in the theta and gamma bands, depending on ongoing behavior and their precise site in the cerebellar hemispheres.     

The neuropeptide allatostatin C from clock-associated DN1p neurons generates the circadian rhythm for oogenesis

The link between the biological clock and reproduction is evident in most metazoans. The fruit fly Drosophila melanogaster, a key model organism in the field of chronobiology because of its well-defined networks of molecular clock genes and pacemaker neurons in the brain, shows a pronounced diurnal rhythmicity in oogenesis. Still, it is unclear how the circadian clock generates this reproductive rhythm. A subset of the group of neurons designated “posterior dorsal neuron 1” (DN1p), which are among the ∼150 pacemaker neurons in the fly brain, produces the neuropeptide allatostatin C (AstC-DN1p). Here, we report that six pairs of AstC-DN1p send inhibitory inputs to the brain insulin-producing cells, which express two AstC receptors, star1 and AICR2. Consistent with the roles of insulin/insulin-like signaling in oogenesis, activation of AstC-DN1p suppresses oogenesis through the insulin-producing cells. We show evidence that AstC-DN1p activity plays a role in generating an oogenesis rhythm by regulating juvenile hormone and vitellogenesis indirectly via insulin/insulin-like signaling. AstC is orthologous to the vertebrate neuropeptide somatostatin (SST). Like AstC, SST inhibits gonadotrophin secretion indirectly through gonadotropin-releasing hormone neurons in the hypothalamus. The functional and structural conservation linking the AstC and SST systems suggest an ancient origin for the neural substrates that generate reproductive rhythms.

To maximize reproductive fitness, every species on earth optimizes the timing of its reproduction (1). Large mammals, such as goats and sheep, which have a gestation period of ∼6 mo, breed in the fall so they can give birth in the spring. Birds and small mammals with shorter gestation periods breed in both the spring and summer. Biological clocks monitor the changes in day length that occur as the seasons progress so they can better direct behavior, but it remains unclear how clocks direct seasonal changes in reproduction.In the fruit fly Drosophila melanogaster, ∼150 brain pacemaker neurons that express the highly conserved circadian clock genes work together to generate a robust diurnal rhythmicity in locomotion and many other biological processes (2–4). Reproductive activity in Drosophila is also under the control of the circadian clock. Males lacking clock genes, such as period (per) or timeless (tim), release significantly fewer sperm from the testes to the seminal vesicles (5). Female mating activity also shows a robust circadian rhythmicity that is lost in per or tim mutants (6). In addition to mating activity, oogenesis shows a diurnal rhythmicity that is maintained in the absence of environmental circadian cues (i.e., in constant darkness) (7). It remains unclear, though, how the biological clock regulates reproductive behaviors and their associated physiological processes.In Drosophila, insulin/insulin-like signaling (IIS) from insulin-producing cells (IPCs) located in the pars intercerebralis region of the brain plays a crucial role in oogenesis. The IPCs are a set of 14 median neurosecretory cells that produce Drosophila insulin-like peptides (Dilps) 2, 3, and 5. Brain IIS directly stimulates germline stem cell (GSC) division in the germarium and promotes germline cyst and oocyte development (8). In addition to its direct roles, IIS also regulates the synthesis of the steroid hormone ecdysteroid (20E) and the sesquiterpenoid juvenile hormones (JHs), both of which constitute complex endocrine networks essential for oocyte production (9–11). 20E signaling is critical for the stimulation of GSC proliferation that occurs upon mating (12), and for the maintenance of GSCs in aging females (13, 14). With 20E, the JHs promote vitellogenesis by stimulating yolk protein synthesis by the fat body and then its uptake by developing oocytes (15).It is clear that JHs are essential for vitellogenesis; ablation of the corpora allata (CA), the sole endocrine organ that produces and secretes JHs, results in a marked loss of vitellogenesis that is reversible upon treatment with JH mimics (16). The many central and peripheral signals that regulate JH can be divided into two groups: Allatotropins activate JH biosynthesis, while allatostatins inhibit it. In Drosophila, IIS promotes JH biosynthesis and secretion through insulin receptor (lnR) in the CA (11). Ecdysis triggering hormones from the peritracheal gland Inka cells stimulate JH production during eclosion and in stress conditions (17). The neuropeptide allatostatin C (AstC) inhibits JH biosynthesis in several species. AstC was first identified in the hawkmoth Manduca sexta (where it was designated Manse-AST) based on its allatostatic activity on isolated CAs (18). Genes encoding AstC have since been identified in the true armyworm Pseudaletia unipuncta (19) and in D. melanogaster (20). The AstC peptide is highly conserved across insect species, with AstC from M. sexta and D. melanogaster differing by only one amino acid residue. AstC expression occurs mainly in the central nervous system and in the enteroendocrine cells of the gastrointestinal system (20, 21). In adult Drosophila, although knockdown of either AstC or its two G protein-coding receptor (GPCR) receptors (star1 and AICR2) increase JH levels (22, 23), the natural contexts in which AstC inhibits JH levels in adults remain unclear.A subset of posterior dorsal neurons (DN1p) in the brain clock neuron network produces AstC (AstC-DN1p) (24). In this study, we report that AstC from AstC-DN1p generates a circadian oogenesis rhythm. This AstC acts through its two GPCR receptors to suppress vitellogenesis by blocking Dilp secretion from the IPCs. Our results suggest that as AstC-DN1p activity peaks at dawn, it represses IPC activity. As AstC-DN1p activity reaches its lowest point in the afternoon, IPC activity is derepressed. It is this modulation of IPC activity that then modulates vitellogenesis initiation. In addition, AstC is orthologous to the vertebrate somatostatins (SST) that inhibit gonadotrophin releasing hormone (GnRH) secretion. Thus, we expect our discovery of a causal association between AstC-producing clock neurons and oogenesis rhythms in Drosophila will also offer insight into the neural and molecular mechanisms that determine reproduction timing in seasonal breeding animals.     

Neural and behavioral control in Caenorhabditis elegans by a yellow-light–activatable caged compound

Caenorhabditis elegans is used as a model system to understand the neural basis of behavior, but application of caged compounds to manipulate and monitor the neural activity is hampered by the innate photophobic response of the nematode to short-wavelength light or by the low temporal resolution of photocontrol. Here, we develop boron dipyrromethene (BODIPY)-derived caged compounds that release bioactive phenol derivatives upon illumination in the yellow wavelength range. We show that activation of the transient receptor potential vanilloid 1 (TRPV1) cation channel by spatially targeted optical uncaging of the TRPV1 agonist N-vanillylnonanamide at 580 nm modulates neural activity. Further, neuronal activation by illumination-induced uncaging enables optical control of the behavior of freely moving C. elegans without inducing a photophobic response and without crosstalk between uncaging and simultaneous fluorescence monitoring of neural activity.

Caged compounds that release bioactive molecules upon light irradiation have been widely used for photocontrol of cell signaling (1). Various molecules such as neurotransmitters, nucleotides, ions, drugs, fluorophores, and proteins can be rendered biologically inert by using photoreactive caging groups (2). Light irradiation induces photolysis of the caging groups to restore the bioactivity of these molecules. Since the initial applications of caged cyclic adenosine monophosphate (cAMP) (3) and caged adenosine triphosphate (ATP) (4) in biological experiments, caged compounds have been applied to cultured cells (1), brain slices (5, 6), and living animals (7). Although the light-mediated delivery of chemical probes in vivo is challenging, there are reports of photoactivation of neurons in Drosophila by caged ATP (8, 9) and photo-mediated gene activation in zebrafish by caged RNA/DNA (10, 11). The optical transparency of these species makes them particularly suitable targets for photochemical probes.The nematode Caenorhabditis elegans is also amenable to optical manipulation using photocontrollable tools owing to its transparent body, compact nervous system, and ease of genetic manipulation (12–15). Nevertheless, few reports describe the application of caged compounds to C. elegans. One reason for this may be that most conventional caged compounds have the major limitation that uncaging requires short-wavelength (ultraviolet to blue) light, which induces an innate photophobic response, as well as causing cell damage or even death of the nematode (16, 17). Thus, it would be preferable to achieve photocontrol by using longer-wavelength visible light.Methods for uncaging with longer-wavelength visible light (green to near infrared) include photorelease via metal–ligand photocleavage (18), via photooxidative C-C cleavage and hydrolysis (19), and by using a photosensitizer (20). However, these strategies have disadvantages: The photocages can only release the fluorophore upon irradiation at over 550 nm (18), the caged compounds show relatively poor temporal resolution of neural control due to the multistep nature of the photoreaction (19), and they generate toxic levels of reactive oxygen species under normoxic conditions (20). Therefore, they are unsuitable for neurophysiological experiments in C. elegans, a model system that is widely used to elucidate the neuronal basis of behavior. Another method for releasing bioactive molecules with visible/near-infrared light is to use two-photon excitation, wherein the excitation wavelength can be twice that of the one-photon counterpart (21–23). Two-photon excitation is particularly useful for studies that require spatially high-resolution uncaging, such as functional mapping of receptors along dendrites (6) and single-spine stimulation (24), although the amount of the photorelease is very small (25).Here, we aimed to develop practically useful caged compounds that can be uncaged by one-photon excitation in C. elegans without the disadvantages described above and that would be suitable for neurophysiological experiments. We confirmed that our compounds exhibit sharp absorption spectra at around 580 nm that do not overlap with those of GFP-based probes and show simple, single-step photorelease of the caged molecules in response to light irradiation at over 550 nm. We also validated the application of caged N-vanillylnonanamide for neurophysiological studies. Uncaging by illumination at 580 nm with simultaneous monitoring of neuronal activity using GCaMP demonstrated that the uncaging triggers responses in sensory neurons, body wall muscles, motor neurons, and interneurons that are associated with behavioral changes of freely moving C. elegans.     

Collagen IV differentially regulates planarian stem cell potency and lineage progression

The extracellular matrix (ECM) provides a precise physical and molecular environment for cell maintenance, self-renewal, and differentiation in the stem cell niche. However, the nature and organization of the ECM niche is not well understood. The adult freshwater planarian Schmidtea mediterranea maintains a large population of multipotent stem cells (neoblasts), presenting an ideal model to study the role of the ECM niche in stem cell regulation. Here we tested the function of 165 planarian homologs of ECM and ECM-related genes in neoblast regulation. We identified the collagen gene family as one with differential effects in promoting or suppressing proliferation of neoblasts. col4-1, encoding a type IV collagen α-chain, had the strongest effect. RNA interference (RNAi) of col4-1 impaired tissue maintenance and regeneration, causing tissue regression. Finally, we provide evidence for an interaction between type IV collagen, the discoidin domain receptor, and neuregulin-7 (NRG-7), which constitutes a mechanism to regulate the balance of symmetric and asymmetric division of neoblasts via the NRG-7/EGFR pathway.

Across the animal kingdom, stem cell function is regulated by the microenvironment in the surrounding niche (1), where the concentration of molecular signals for self-renewal and differentiation can be precisely regulated (2). The niche affects stem cell biology in many processes, such as aging and tissue regeneration, as well as pathological conditions such as cancer (3). Most studies have been done in tissues with large stem cell populations, such as the intestinal crypt (4) and the hair follicle (5) in mice. Elucidation of the role of the stem cell niche in tissue regeneration requires the study of animals with high regenerative potential, such as freshwater planarians (flatworms) (6). Dugesia japonica and Schmidtea mediterranea are two well-studied species that possess the ability to regenerate any missing body part (6, 7).Adult S. mediterranea maintain a high number of stem cells (neoblasts)—∼10 to 30% of all somatic cells in the adult worm—with varying potency, including pluripotent cells (8–14). Neoblasts are the only proliferating somatic cells: they are molecularly heterogeneous, but all express piwi-1 (15–18). Lineage-committed neoblasts are “progenitors” that transiently express both piwi-1 and tissue-specific genes (15, 19). Examples include early intestinal progenitors (γ neoblast, piwi-1⁺/hnf4⁺) (8, 10, 15, 19–21) and early epidermal progenitors (ζ neoblast, piwi-1⁺/zfp-1⁺) (8, 15). Other progenitor markers include collagen for muscles (22), ChAT for neurons (23), and cavII for protonephridia (24, 25). During tissue regeneration, neoblasts are recruited to the wound site, where they proliferate then differentiate to replace the missing cell types (16, 26). Some neoblasts express the pluripotency marker tgs-1, and are designated as clonogenic neoblasts (cNeoblasts) (10, 11). cNeoblasts are located in the parenchymal space adjacent to the gut (11).Neoblasts are sensitive to γ-irradiation and can be preferentially depleted in the adult planarian (27). After sublethal γ-irradiation, remaining cNeoblasts can repopulate the stem cell pool within their niche (10, 11). The close proximity of neoblasts to the gut suggests gut may be a part of neoblast niche (28, 29). When gut integrity was impaired by silencing gata4/5/6, the egfr-1/nrg-1 ligand-receptor pair, or wwp1, maintenance of non–γ-neoblasts were also disrupted (20, 30, 31), but whether that indicates the gut directly regulates neoblast remains unclear. There is evidence indicating the dorsal-ventral (D/V) transverse muscles surrounding the gut may promote neoblast proliferation and migration, with the involvement of matrix metalloproteinase mt-mmpB (32, 33). The central nervous system has also been implicated in influencing neoblast maintenance through the expression of EGF homolog neuregulin-7 (nrg-7), a ligand for EGFR-3, affecting the balance of neoblast self-renewal (symmetric or asymmetric division) (34).In other model systems, an important component of the stem-cell niche is the extracellular matrix (ECM) (35). Germline stem cells in Drosophila are anchored to niche supporting cells with ECM on one side, while the opposite side is exposed to differentiation signals, allowing asymmetric cell fate outcomes for self-renewal or differentiation following division (36–38). Few studies have addressed the ECM in planarians, largely due to the lack of genetic tools to manipulate the genome, the absence of antibodies to specific planarian ECM homologs, or the tools required to study cell fate changes. However, the genomes of D. japonica (39–41) and S. mediterranea (41–45), and single-cell RNA-sequencing (scRNA-seq) datasets for S. mediterranea are now available (11, 46–50). A recent study of the planarian matrisome demonstrated that muscle cells are the primary source of many ECM proteins (51), which, together with those produced by neoblasts and supporting parenchymal cells, may constitute components of the neoblast niche. For example, megf6 and hemicentin restrict neoblast’s localization within the parenchyma (51, 52). Functional studies also implicate ECM-modifiers, such as matrix metalloproteases (MMPs) in neoblast migration and regeneration. For example, reducing the activity of the ECM-degrading enzymes mt-mmpA (26, 33), mt-mmpB (53), or mmp-1 (33) impaired neoblast migration, proliferation, or overall tissue growth, respectively. Neoblasts are also likely to interact with ECM components of the niche via cell surface receptors, such as β1 integrin, inactivation of which impairs brain regeneration (54, 55).Here, we identified planarian ECM homologs in silico, followed by systematic functional assessment of 165 ECM and ECM-related genes by RNA interference (RNAi), to determine the effect on neoblast repopulation in planarians challenged by a sublethal dose of γ-irradiation (10). Surprisingly, multiple classes of collagens were shown to have the strongest effects. In particular, we show that the type IV collagens (COLIV) of basement membranes (BMs), were required to regulate the repopulation of neoblasts as well as lineage progression to progenitor cells. Furthermore, our data support an interaction between COLIV and the discoidin domain receptor (DDR) in neurons that activates signaling of NRG-7 in the neoblasts to regulate neoblast self-renewal versus differentiation. Together, these data demonstrate multifaceted regulation of planarian stem cells by ECM components.