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Biology subjects

Bell, A. M.

Publications and source records attributed to Bell, A. M..

At least 19 recordsLinked to original sources

Neurons in the lateral spinal nucleus give rise to the spinotectal component of the anterolateral system in the mouse

The anterolateral system (ALS) originates from neurons in various regions of the spinal cord and conveys somatosensory information related to pain, skin temperature and itch to several brain targets. One component of the ALS is the spinotectal tract, which projects to the superior colliculus (SC) and is thought to underlie directed movements of the head towards a painful stimulus. Using anterograde and retrograde tracing, we demonstrate that the input from the spinal enlargements to the SC originates predominantly from neurons in the lateral spinal nucleus (LSN), many of which express calbindin. This input, which mainly targets the caudal and lateral parts of the SC, is somatotopically organised, and is associated with the clusters of Pitx2-expressing neurons that have been identified in previous studies. Since activation of Pitx2 neurons drives directed head movements, input from LSN neurons to the Pitx2 cells is likely to be a critical component of orientation and attention towards a noxious stimuli. We also provide evidence that spinotectal LSN cells receive excitatory synaptic input from neurons in superficial dorsal horn that express substance P or the gastrin-releasing peptide receptor (GRPR). Since GRPR neurons are strongly implicated in itch, our findings raise the possibility that the SC has a role in coordinating movements in response to pruritic stimuli.

neuroscience↗

Deep FLASH-seq profiling of purified canine sensory neurons uncovers species-specific signatures relevant to pain and itch

Naturally occurring pain and itch disorders in the domestic dog represent an important and underexploited opportunity for translational sensory neuroscience. These conditions largely mirror human disease, highlighting the need for detailed comparative understanding of canine somatosensory neurobiology. Here, we present a single-cell transcriptomic characterisation of the canine dorsal root ganglion (DRG), providing molecular insights into sensory neuron diversity in a species of direct veterinary and biomedical relevance. We develop a novel mechanical dissociation and fluorescence-activated cell sorting strategy enabling purification of intact whole neurons from adult canine DRG, followed by deep, full-length RNA sequencing using FLASH-seq. This approach yields high-quality transcriptional profiles with molecular depth analogous to deep neuronal profiling in human DRG, enabling resolution of neuronal identities and subtype-specific gene programs. Using these data, we identify canine sensory neuron clusters conforming to conserved principles of DRG molecular organization observed across species, including peptidergic and non-peptidergic nociceptors, low-threshold mechanoreceptors, proprioceptors, and thermosensory populations. Cross-species comparisons with human and mouse DRG datasets reveal broad conservation of pain- and itch-relevant pathways and therapeutic targets, alongside biologically meaningful divergence. We further identify species-specific differences in subtype-restricted expression of the pharmacologically relevant receptors IL31RA and SSTR2, which we validate using in situ hybridization and contextualize with human spatial transcriptomic data. Finally, we provide evidence that domestication-associated genes are non-randomly enriched in specific sensory neuron populations, suggesting that evolutionary history may have shaped somatosensory function. These data represent a resource for comparative sensory neuroscience and inform translational interpretation of pain and itch therapeutics across species.

neuroscience↗

Characterisation of cold-selective lamina I spinal projection neurons

Skin cooling is detected by primary afferents that express the Trpm8 channel, but how this information is conveyed to the brain remains poorly understood. We have previously identified a population of lamina I projection neurons belonging to the anterolateral system (ALS) that receive numerous contacts from Trpm8-expressing primary afferents. Here, using a semi-intact somatosensory preparation, we provide evidence that these cells correspond to the cold-selective ALS neurons identified in previous physiological studies. We also confirm the presence of synapses from Trpm8 afferents onto these cells at the ultrastructural level and with optogenetics. Based on our previous transcriptomic findings, we identify calbindin as a molecular marker, and show that this can be used to target the cold-selective ALS neurons for anterograde tracing studies. We provide evidence that they project to brain regions that have been implicated in thermosensation: the rostralmost part of the lateral parabrachial area, the caudal part of the periaqueductal grey matter, and the posterior triangular and ventral posterolateral nuclei of the thalamus. Our findings provide important insights into the organisation of neuronal circuits that underlie thermoregulation and the perception of cold stimuli applied to the skin.

neuroscience↗

Strong but diffuse genetic divergence underlies differentiation in an incipient species of marine stickleback

Understanding how lineages proceed along the "speciation continuum" and how species boundaries are maintained over time remain central questions in evolutionary biology. Populations early in the speciation process can give us detailed insight into the reproductive barriers that first initiate speciation. In this study, we explore the nature of genomic divergence between two sympatric marine stickleback ecotypes from Atlantic Canada, "whites" and "commons". Males of each ecotype exhibit distinct nuptial colorations, nesting habits, and parental care strategies. Using population genomic analyses of SNPs and copy number variants (CNVs; deletions and duplications) we show that whites and commons consistently form distinct populations. We uncover genomic differentiation in the white ecotype characteristic of an incipient species, showing extremely low genome-wide differentiation (FST) and very recent divergence ([~]1 kya). Demographic analysis detected very low levels of ongoing gene flow between populations. Our results and prior genomic studies suggest that reproductive isolation is being maintained between ecotypes despite recent evidence that hybridization in nature does occur. Contrary to other systems, we found many small, but dispersed regions of high differentiation throughout the genome rather than explicitly within chromosomal inversions or the sex chromosomes. On chromosomes VII and XVI, we identified CNVs overlapping genes enriched for olfaction, which may play a role in differences in reproductive strategies between ecotypes. Ultimately, our results demonstrate that genome-wide rather than localized differences can underlie the early stages of divergence, and that this pattern is corroborated by both SNPs and CNVs.

evolutionary biology↗

Discrete genetic modules underlie divergent reproductive strategies in three-spined stickleback

A central challenge in biology is to understand how complex behaviors evolve. Given their importance to fitness, complex behavioral traits often evolve as an integrated package, but it is unclear whether suites of traits evolve through a few pleiotropic genetic changes, each affecting many behaviors, or by accumulating several changes that, when combined, give rise to an entire package of correlated traits. Typically, three-spined stickleback exhibit paternal care, a behavior that characterizes the entire Gasterosteidae family. However, an unusual "white" three-spined stickleback ecotype exhibits a suite of traits associated with the evolutionary loss of paternal care. In the white ecotype, males disperse embryos from their nests rather than care for them, build loose nests, exhibit high rates of courtship, and are relatively small in body size. These behavioral differences are apparent in stickleback reared in a common garden environment, suggesting the differences have a heritable basis. In an F2 intercross, we show that these traits are genetically uncorrelated and map to different genomic regions, suggesting that components of the white reproductive strategy segregate independently and evolved through the addition of multiple genetic changes. These results contribute to the growing body of evidence that the behavioral diversity observed in nature may often evolve by accumulating and combining alleles, each with modular effects, and show that this principle applies to a suite of behavioral traits that together form an integrated and adaptive strategy.

evolutionary biology↗

Evidence of parental care as a novel reproductive isolating mechanism

Variation in behavior can contribute to reproductive isolation by preventing gene flow among populations. Here, we tested the novel hypothesis that parental care, when dysregulated, can function as a reproductive isolating mechanism in three-spined stickleback fish (Gasterosteus aculeatus). In the typical "common" stickleback ecotype, males provide care to their offspring by fanning with their pectoral fins and defending their nest. In contrast, a divergent "white" stickleback ecotype has evolutionarily lost care and disperses embryos into the surrounding environment. We examined how paternal care from common, white, and F1 hybrid fathers influenced offspring survival. We detected no intrinsic incompatibilities in embryos, but F1 hybrid fathers exhibited dysregulated parental care that coincided with decreased survival of parented offspring. The increased offspring mortality may be explained by further dysregulation of feeding and parenting circuitry, as F2 hybrids exhibited significantly higher rates of post-fertilization filial cannibalism than common or white fathers. Additionally, despite strong divergence in nesting-building and courtship behavior, F1 hybrids achieved mating success at a similar rate as male commons and whites, suggesting that prezygotic barriers against hybrids may be weak. The observed postzygotic isolation, and potentially weak prezygotic isolation in lab-based studies, suggest that hybridization is likely occurring at low rates in the wild. Population genetic analysis supported this, as low proportions of putative hybrids were detected in sympatric sites. Together, these results may help explain why genetic divergence between these phenotypically distinct ecotypes is low and provide evidence that dysregulated parental behavior can act as a newly discovered postzygotic reproductive isolating barrier.

evolutionary biology↗

An evolutionary shift to prioritizing mating over care is associated with consistently high androgen levels in male threespine stickleback

Steroid hormones play a role in regulating social behaviors in vertebrates, but how they mediate the evolution of these traits remains an open question. Here, we use liquid chromatography-mass spectrometry (LC-MS/MS) to quantify a panel of steroids in breeding males of two recently diverged populations of threespine stickleback. The common ecotype provides paternal care, while the white ecotype has evolutionarily lost paternal care. Hormone levels were quantified in both ecotypes at three reproductive stages: (1) after completing a nest, (2) soon after mating, when commons start to provide care and whites disperse the embryos, and (3) four days after mating, when commons are performing parental care and no longer courting females while whites are not providing care and are courting females. Androgens declined in the common ecotype as they began providing care but remained elevated in the white ecotype across stages, possibly to maintain the production of "spiggin," the androgen-dependent glue males use to construct nests. Progestogen levels were low in whites and were lowest in commons after mating, suggesting an antagonistic relationship between progestogens and sexual behavior. Both ecotypes showed elevated glucocorticoids after mating, suggesting the stress axis may not have diverged between ecotypes. Altogether, these results provide evidence that the ecotypes regulate steroid levels differently to support the ways they balance mating and parental effort. Our data suggest a variety of mechanisms by which steroid signaling and regulation can change during the early stages of divergence between behaviorally distinct populations.

animal behavior and cognition↗

Expression of Tacr1 and Gpr83 by spinal projection neurons

Anterolateral system (ALS) projection neurons underlie perception of pain, itch and skin temperature. These cells are heterogeneous, and there have therefore been many attempts to define functional populations. A recent study identified two classes of ALS neuron in mouse superficial dorsal horn (SDH) based on expression of the G protein-coupled receptors Tacr1 or Gpr83. It was reported that cells expressing these receptors formed largely non-overlapping populations, and that [~]60% of ALS cells in SDH expressed Tacr1. An additional finding was that while Tacr1- and Gpr83-expressing ALS cells projected to several brain nuclei, their axons did not reach the ventral posterolateral (VPL) thalamic nucleus, which is reciprocally connected to the primary somatosensory cortex. These results were surprising, because we had reported that [~]90% of SDH ALS neurons in the mouse possess the neurokinin 1 receptor (NK1r), which is encoded by Tacr1, and in addition the VPL is thought to receive input from lamina I ALS cells. Here we use retrograde and anterograde labelling in Tacr1CreERT2 and Gpr83CreERT2 mice to reinvestigate the expression of the receptors by ALS neurons and to reassess their projection patterns. We find that [~]90% of ALS neurons in SDH express Tacr1, with 40-50% expressing Gpr83. We also show that axons of both Tacr1- and Gpr83-expressing ALS neurons reach the VPL. These results suggest that ALS neurons in the SDH that express these GPCRs show considerable overlap, and that they are likely to contribute to the sensory-discriminative dimension of pain through their projections to VPL.

neuroscience↗

Anatomical characterisation of somatostatin-expressing neurons belonging to the anterolateral system

Anterolateral system (ALS) spinal projection neurons are essential for pain perception. However, these cells are heterogeneous, and there has been extensive debate about the roles of ALS populations in the different pain dimensions. We recently performed single-nucleus RNA sequencing on a developmentally-defined subset of ALS neurons, and identified 5 transcriptomic populations. One of these, ALS4, consists of cells that express Sst, the gene coding for somatostatin, and we reported that these were located in the lateral part of lamina V. Here we use a SstCre mouse line to characterise these cells and define their axonal projections. We find that their axons ascend mainly on the ipsilateral side, giving off collaterals throughout their course in the spinal cord. They target various brainstem nuclei, including the parabrachial internal lateral nucleus, and the posterior triangular and medial dorsal thalamic nuclei. We also show that in the L4 segment Sst is expressed by [~]75% of ALS neurons in lateral lamina V and that there are around 120 Sst-positive lateral lamina V cells on each side. Our findings indicate that this is a relatively large population, and based on projection targets we conclude that they are likely to contribute to the affective-motivational dimension of pain.

neuroscience↗

Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons

The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify 3 clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 & ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons. Significance StatementThe anterolateral system (ALS) is a major ascending pathway from the spinal cord that underlies perception of pain, itch and skin temperature. It is therefore an important target for the development of new treatments for chronic pain. Our understanding of this system has been hampered by the considerable diversity of its constituent cells. Here we dissect the complex heterogeneity of these cells by using high-resolution RNA sequencing. We reveal five distinct types of ALS neurons, which are differentially distributed within the spinal cord, and probably represent functional populations. Our data provide novel insights into the molecular architecture of the ALS, and will be important for future studies to define the roles of different ALS cell types in sensory processing.

neuroscience↗

Calretinin-expressing islet cells: a source of pre- and post-synaptic inhibition of non-peptidergic nociceptor input to the mouse spinal cord

Unmyelinated non-peptidergic nociceptors (NP afferents) arborise in lamina II of the spinal cord and receive GABAergic axoaxonic synapses, which mediate presynaptic inhibition. However, until now the source of this axoaxonic synaptic input was not known. Here we provide evidence that it originates from a population of inhibitory calretinin-expressing interneurons (iCRs), which correspond to lamina II islet cells. The NP afferents can be assigned to 3 functionally distinct classes (NP1-3). NP1 afferents have been implicated in pathological pain states, while NP2 and NP3 afferents also function as pruritoceptors. Our findings suggest that all 3 of these afferent types innervate iCRs and receive axoaxonic synapses from them, providing feedback inhibition of NP input. The iCRs also form axodendritic synapses, and their targets include cells that are themselves innervated by the NP afferents, thus allowing for feedforward inhibition. The iCRs are therefore ideally placed to control the input from non-peptidergic nociceptors and pruritoceptors to other dorsal horn neurons, and thus represent a potential therapeutic target for the treatment of chronic pain and itch.

neuroscience↗

Neuropeptide Y-expressing dorsal horn inhibitory interneurons gate spinal pain and itch signalling

Somatosensory information is processed by a complex network of interneurons in the spinal dorsal horn. It has been reported that inhibitory interneurons that express neuropeptide Y (NPY), either permanently or during development, suppress mechanical itch, with no effect on pain. Here we investigate the role of interneurons that continue to express NPY (NPY-INs) in adulthood. We find that chemogenetic activation of NPY-INs reduces behaviours associated with acute pain and pruritogen-evoked itch, whereas silencing them causes exaggerated itch responses that depend on cells expressing the gastrin-releasing peptide receptor. As predicted by our previous studies, silencing of another population of inhibitory interneurons (those expressing dynorphin) also increases itch, but to a lesser extent. Importantly, NPY- IN activation also reduces behavioural signs of inflammatory and neuropathic pain. These results demonstrate that NPY-INs gate pain and itch transmission at the spinal level, and therefore represent a potential treatment target for pathological pain and itch.

neuroscience↗

How basic components of information-updating interact to encourage variation in the results of empirical studies of within and transgenerational plasticity

Experiences of parents and/or offspring are often assumed to affect the development of trait values in offspring because they provide information about the external environment, but it is currently unclear how information from different sources and times might combine to affect the information-states that provide the foundation for the patterns observed in empirical studies of developmental plasticity in response to environmental cues. We analyze Bayesian models designed to mimic fully-factorial experimental studies of within and transgenerational plasticity (TWP), in which parents, offspring, neither or both are exposed to cues from predators, to determine how different durations of cue exposure for parents and offspring, the devaluation of information from parents or the degradation of information from parents would affect offspring estimates of environmental states related to risk of predation at the end of such experiments. We show that the effects of different cue durations, the devaluation of information from parents, and the degradation of information from parents on offspring estimates are all expected to vary as a function of interactions with two other key parameters of information-based models of TWP: parental priors and the relative cue reliability in the different treatments. Our results suggest empiricists should expect to observe considerable variation across experimental studies of TWP based on simple principles of information-updating, without needing to invoke additional assumptions about costs, tradeoffs, development constraints, the fitness consequences of different trait values, or other factors.

animal behavior and cognition↗

Three neurotransmitters regulate diverse inhibitory and excitatory Parvalbumin interneuron circuits in the dorsal horn

Parvalbumin-expressing interneurons (PVINs) in the spinal dorsal horn are found primarily in laminae II inner and III. Inhibitory PVINs (iPVINs) play an important in segregating innocuous tactile input from pain-processing circuits, achieved through presynaptic inhibition of myelinated low-threshold mechanoreceptors and postsynaptic inhibition of distinct spinal circuits. By comparison, relatively little is known of the role of excitatory PVINs (ePVINs) in sensory processing. Here we use neuroanatomical and optogenetic approaches to show that ePVINs comprise a larger proportion of the PVIN population than previously reported, and that both ePVIN and iPVIN populations form synaptic connections amongst (and between) themselves. We find that these cells contribute to neuronal networks that influence activity within several functionally distinct circuits, and that aberrant activity of ePVINs under pathological conditions contributes to the development of mechanical hypersensitivity.

neuroscience↗

The specificity of sperm-mediated paternal effects in threespined sticklebacks

Parental effects can help offspring cope with challenging environments, but whether these effects are unique to specific environmental conditions is largely unknown. Parental effects may evolve via a core pathway that generally prepares offspring for risky environments or could be stimuli-specific, with offspring developing phenotypes that are tailored to specific environmental challenges. We exposed threespined sticklebacks (Gasterosteus aculeatus) fathers to a potentially threatening stimulus (net) versus native predator (sculpin). Offspring of sculpin-exposed fathers were more responsive (greater change in activity) to a simulated predator attack, while offspring of net-exposed fathers were less responsive (lower plasma cortisol and fewer antipredator behaviors). To evaluate offspring response to native and non-native stimuli, we sequentially exposed offspring of net-exposed, sculpin-exposed or control fathers to a net, native sculpin model, or non-native trout model. Paternal treatment did not influence offspring response to stimuli; instead, offspring were more responsive to the native sculpin predator compared to nets or non-native trout predator. Collectively, we demonstrate that sperm-mediated paternal effects in response to different, potentially stressful stimuli result in distinct offspring phenotypes. This specificity may be key to understanding the evolution of adaptive parental effects and how parents prime offspring for encountering both evolved and novel environmental stimuli.Competing Interest StatementThe authors have declared no competing interest.View Full Text

animal behavior and cognition↗

Viral-mediated transgenesis of MAOA and AVP increases territorial aggression in stickleback

Behavioral genetics in non-model organisms is currently gated by technological limitations. However, with the growing availability of genome editing and functional genomic tools, complex behavioral traits such as social behavior can now be explored in diverse organisms. Here we present a minimally invasive neurosurgical procedure for a classic behavioral, ecological and evolutionary system: threespine stickleback (Gasterosteus aculeatus). This method of direct brain injection enables viral-mediated transgenesis and pharmaceutical delivery which bypasses the blood-brain barrier. This method is flexible, fast, and amenable to statistically powerful within-subject experimental designs, making it well-suited for use in genetically diverse animals such as those collected from natural populations. Viral-mediated transgenesis in the brain allows for a direct examination of the genetic mechanisms underlying behavior in wild-caught animals from natural populations. Using this method, we were able to detect changes in aggression from the knockdown of either of two different genes, arginine vasopressin (AVP) and monoamine oxidase (MAOA), in outbred animals in less than one month. In addition, we demonstrate that widely available mammalian plasmids work with this method, lowering the barrier of entry to the technique.

genetics↗

Grandpaternal effects are lineage- and sex-specific in threespined sticklebacks

O_LITransgenerational plasticity (TGP) occurs when the environment encountered by one generation (F0) alters the phenotypes of one or more future generations (e.g. F1 and F2). Sex selective TGP, via specific lineages or to only male or female descendants, has been underexplored in natural systems, and may be adaptive if it allows past generations to fine-tune the phenotypes of future generations in response to sex-specific life history strategies. C_LIO_LIWe sought to understand if exposing males to predation risk can influence grandoffspring via sperm in threespined stickleback (Gasterosteus aculeatus). We specifically tested the hypothesis that grandparental effects are transmitted in a sex-specific way down the male lineage, from paternal grandfathers to F2 males. C_LIO_LIWe reared F1 offspring of unexposed and predator-exposed F0 males under control conditions and used them to generate F2s with control grandfathers, a predator-exposed maternal grandfather (i.e., predator-exposed F0 males to F1 daughters to F2 offspring), a predator-exposed paternal grandfather (i.e., predator-exposed F0 males to F1 sons to F2 offspring), or two predator-exposed grandfathers. We then assayed male and female F2s for a variety of traits related to antipredator defense. C_LIO_LIWe found little evidence that transgenerational effects were mediated to only male descendants via the paternal lineage. Instead, grandpaternal effects depended on lineage and were mediated largely across sexes, from F1 males to F2 females and from F1 females to F2 males. When their paternal grandfather was exposed to predation risk, female F2s were heavier and showed a reduced change in behavior in response to a simulated predator attack relative to offspring of control, unexposed grandparents. In contrast, male F2s showed reduced antipredator behavior when their maternal grandfather was exposed to predation risk. However, these patterns were only evident when one grandfather, but not both grandfathers, was exposed to predation risk, suggesting the potential for non-additive interactions across lineages. C_LIO_LIIf sex-specific and lineage effects are common, then grandparental effects are likely underestimated in the literature. These results draw attention to the importance of sex-selective inheritance of environmental effects and raise new questions about the proximate and ultimate causes of selective transmission across generations. C_LI

evolutionary biology↗

Sex-specific transgenerational plasticity in threespined sticklebacks

O_LITransgenerational plasticity (TGP) or parental effects - when parental environments alter the phenotype of future generations - can influence how organisms cope with environmental change. An intriguing, underexplored possibility is that sex -of both the parent and the offspring - plays an important role in driving the evolution of transgenerational plasticity in both adaptive and nonadaptive ways. C_LIO_LIHere, we evaluate the potential for sex-specific parental effects in a freshwater population of threespined sticklebacks (Gasterosteus aculeatus) by independently and jointly manipulating maternal and paternal experiences and separately evaluating their phenotypic effects in sons versus daughters. We tested the adaptive hypothesis that daughters are more responsive to cues from their mother, while sons are more responsive to cues from their father. C_LIO_LIWe exposed mothers, fathers, or both parents to visual cues of predation risk and measured offspring antipredator traits and brain gene expression. C_LIO_LIPredator-exposed fathers produced sons that were more risk-prone, while predator-exposed mothers produced more anxious sons and daughters. Further, maternal and paternal effects on offspring survival were nonadditive: offspring with a predator-exposed father, but not two predator-exposed parents, had lower survival against live predators. There were also strong sex-specific effects on brain gene expression: exposing mothers versus fathers to predation risk activated different transcriptional profiles in their offspring, and sons and daughters strongly differed in the ways in which their brain gene expression profiles were influenced by parental experience. C_LIO_LIWe found little evidence to support the hypothesis that offspring prioritize their same-sex parents experience. Parental effects varied with both the sex of the parent and the offspring in complicated and nonadditive ways. Failing to account for these sex-specific patterns (e.g., by pooling sons and daughters) would have underestimated the magnitude of parental effects. Altogether, these results draw attention to the potential for sex to influence patterns of TGP and raise new questions about the interface between transgenerational plasticity and sex-specific selective pressures, sexual conflict, and sexual selection. C_LI

evolutionary biology↗