Detection of novel orthoparamyxoviruses, orthonairoviruses and an orthohepevirus in European white-toothed shrews.

While the viromes and immune systems of bats and rodents have been extensively studied, comprehensive data are lacking for insectivores (order Eulipotyphla) despite their wide geographic distribution. Anthropogenic land use and outdoor recreational activities, as well as changes in the range of shrews, may lead to an expansion of the human-shrew interface with the risk of spillover infections, as reported for Borna disease virus 1. We investigated the virome of 45 individuals of 4 white-toothed shrew species present in Europe, using metagenomic RNA sequencing of tissue and intestine pools. Moderate to high abundances of sequences related to the families Paramyxoviridae, Nairoviridae, Hepeviridae and Bornaviridae were detected. Whole genomes were determined for novel orthoparamyxoviruses (n=3), orthonairoviruses (n=2) and an orthohepevirus. The novel paramyxovirus, tentatively named Hasua virus, was phylogenetically related to the zoonotic Langya virus and Mòjiang virus. The novel orthonairoviruses, along with the potentially zoonotic Erve virus, fall within the shrew-borne Thiafora virus genogroup. The highest viral RNA loads of orthoparamyxoviruses were detected in the kidneys, in well-perfused organs for orthonairoviruses and in the liver and intestine for orthohepevirus, indicating potential transmission routes. Notably, several shrews were found to be coinfected with viruses from different families. Our study highlights the virus diversity present in shrews, not only in biodiversity-rich regions but also in areas influenced by human activity. This study warrants further research to characterize and assess the clinical implications and risk of these viruses and the importance of shrews as reservoirs in European ecosystems.

Macrovibrissae and microvibrissae inversion and lateralization in elephants.

Elephants are known for strongly lateralized trunk behaviors, but the mechanisms driving elephant lateralization are poorly understood. Here, we investigate features of elephant mouth organization that presumably promote lateralization. We find the lower jaw of elephants is of narrow width, but is rostrally strongly elongated even beyond the jaw bone. Elephant lip vibrissae become progressively longer rostrally. Thus, elephants have two lateral dense, short microvibrissae arrays and central, less dense long macrovibrissae. This is an inversion of the ancestral mammalian facial vibrissae pattern, where central, dense short microvibrissae are flanked by two lateral macrovibrissae arrays. Elephant microvibrissae have smaller follicles than macrovibrissae. Similar to trunk-tip vibrissae, elephant lip microvibrissae show laterally asymmetric abrasion. Observations on Asian zoo elephants indicate lateralized abrasion results from lateralized feeding. It appears that the ancestral mammalian mouth (upper and lower lips, incisors, frontal microvibrissae) is shaped by oral food apprehension. The elephant mouth organization radically changed, however, because trunk-mediated feeding replaced oral apprehension. Such elephant mouth changes include the upper lip-nose fusion to the trunk, the super-flexible elongated lower jaw, the loss of incisors, and lateral rather than frontal microvibrissae. Elephants' specialization for lateral food insertion is reflected by the reduction in the centering effects of oral food apprehension and lip vibrissae patterns.

Social play behavior is driven by glycine-dependent mechanisms.

Social play is pervasive in juvenile mammals, yet it is poorly understood in terms of its underlying brain mechanisms. Specifically, we do not know why young animals are most playful and why most adults cease to social play. Here, we analyze the synaptic mechanisms underlying social play. We found that blocking the rat periaqueductal gray (PAG) interfered with social play. Furthermore, an age-related decrease of neural firing in the PAG is associated with a decrease in synaptic release of glycine. Most importantly, modulation of glycine concentration-apparently acting on the glycinergic binding site of the N-methyl-D-aspartate (NMDA) receptor-not only strongly modulates social play but can also reverse the age-related decline in social play. In conclusion, we demonstrate that social play critically depends on the neurotransmitter glycine within the PAG.

Chromosome level genome assembly of the Etruscan shrew Suncus etruscus.

Suncus etruscus is one of the world's smallest mammals, with an average body mass of about 2 grams. The Etruscan shrew's small body is accompanied by a very high energy demand and numerous metabolic adaptations. Here we report a chromosome-level genome assembly using PacBio long read sequencing, 10X Genomics linked short reads, optical mapping, and Hi-C linked reads. The assembly is partially phased, with the 2.472 Gbp primary pseudohaplotype and 1.515 Gbp alternate. We manually curated the primary assembly and identified 22 chromosomes, including X and Y sex chromosomes. The NCBI genome annotation pipeline identified 39,091 genes, 19,819 of them protein-coding. We also identified segmental duplications, inferred GO term annotations, and computed orthologs of human and mouse genes. This reference-quality genome will be an important resource for research on mammalian development, metabolism, and body size control.

Rat Wetness Response: Sensory Cues, Behavior & Fur-based Drying.

It never rains in standard lab-confinements; thus we have limited understanding of animal reactions to water and wetness. To address this issue, we sprayed water on different body parts of rats and measured drying and fur temperature by thermal imaging while manipulating behavior, sensory cues and fur. Spraying water on rats resulted in fur changes (hair clumping, apex formation), grooming, shaking, and scratching. Anesthesia abolished behavioral responses, interfered with fur changes, and slowed drying. Spraying water on different body parts resulted in differential behavioral drying responses. Spraying the head resulted in grooming and shaking responses; water evaporated twice as fast as water sprayed on the animal's back or belly. We observed no effect of whisker removal on post-water-spraying behavior. In contrast, local anesthesia of dorsal facial skin reduced post-water-spraying behavioral responses. Shaving of head fur drastically enhanced post-water-spraying behaviors, but reduced water loss during drying; indicating that fur promotes evaporation, acting in tandem with behavior to mediate drying. Excised wet fur patches dried and cooled faster than shaved excised wet skin. Water was sucked into distal hair tips, where it evaporated. We propose the wet-fur-heat-pump-hypothesis; fur might extract heat required for drying by cooling ambient air.

Dense reconstruction of elephant trunk musculature.

The elephant trunk operates as a muscular hydrostat1,2 and is actuated by the most complex musculature known in animals.3,4 Because the number of trunk muscles is unclear,5 we performed dense reconstructions of trunk muscle fascicles, elementary muscle units, from microCT scans of an Asian baby elephant trunk. Muscle architecture changes markedly across the trunk. Trunk tip and finger consist of about 8,000 extraordinarily filigree fascicles. The dexterous finger consists exclusively of microscopic radial fascicles pointing to a role of muscle miniaturization in elephant dexterity. Radial fascicles also predominate (at 82% volume) the remainder of the trunk tip, and we wonder if radial muscle fascicles are of particular significance for fine motor control of the dexterous trunk tip. By volume, trunk-shaft muscles6 comprise one-third of the numerous, small radial muscle fascicles; two-thirds of the three subtypes of large longitudinal fascicles (dorsal longitudinals, ventral outer obliques, and ventral inner obliques);7,8,9 and a small fraction of transversal fascicles. Shaft musculature is laterally, but not radially, symmetric. A predominance of dorsal over ventral radial muscles and of ventral over dorsal longitudinal muscles may result in a larger ability of the shaft to extend dorsally than ventrally10 and to bend inward rather than outward. There are around 90,000 trunk muscle fascicles. While primate hand control is based on fine control of contraction by the convergence of many motor neurons on a small set of relatively large muscles, evolution of elephant grasping has led to thousands of microscopic fascicles, which probably outnumber facial motor neurons.

DiI-CT-A bimodal neural tracer for X-ray and fluorescence imaging.

Here, we present an X-ray-visible neural tracer, referred to as DiI-CT, which is based on the well-established lipophilic indocarbocyanine dye DiI, to which we conjugated two iodine atoms. The tracer is visible with microfocus computed tomography (microCT) imaging and shares the excellent fluorescent tracing properties of DiI. We document the discovery potential of DiI-CT by analyzing the vibrissa follicle-sinus complex, a structure where visual access is poor and 3D tissue structure matters and reveal innervation patterns of the intact follicle in unprecedented detail. In the brain, DiI-CT tracing holds promise for verification evaluation of indirect connectivity measures, such as diffusion tensor imaging. We conclude that the bimodal dye DiI-CT opens new avenues for neuroanatomy.

Play and tickling responses map to the lateral columns of the rat periaqueductal gray.

The persistence of play after decortication points to a subcortical mechanism of play control. We found that global blockade of the rat periaqueductal gray with either muscimol or lidocaine interfered with ticklishness and play. We recorded vocalizations and neural activity from the periaqueductal gray of young, playful rats during interspecific touch, play, and tickling. Rats vocalized weakly to touch and more strongly to play and tickling. Periaqueductal gray units showed diverse but strong modulation to tickling and play. Hierarchical clustering based on neuronal responses to play and tickling revealed functional clusters mapping to different periaqueductal gray columns. Specifically, we observed play-neutral/tickling-inhibited and tickling/play-neutral units in dorsolateral and dorsomedial periaqueductal gray columns. In contrast, strongly play/tickling-excited units mapped to the lateral columns and were suppressed by anxiogenic conditions. Optogenetic inactivation of lateral periaqueductal columns disrupted ticklishness and play. We conclude that the lateral periaqueductal gray columns are decisive for play and laughter.

Supra-orbital whiskers act as wind-sensing antennae in rats.

We know little about mammalian anemotaxis or wind sensing. Recently, however, Hartmann and colleagues showed whisker-based anemotaxis in rats. To investigate how whiskers sense airflow, we first tracked whisker tips in anesthetized rats under low (0.5 m/s) and high (1.5 m/s) airflow. Whisker tips showed increasing movement from low to high airflow conditions, with all whisker tips moving during high airflow. Low airflow conditions-most similar to naturally occurring wind stimuli-engaged whisker tips differentially. Most whiskers moved little, but the long supra-orbital (lSO) whisker showed maximal displacement, followed by the α, ß, and A1 whiskers. The lSO whisker differs from other whiskers in its exposed dorsal position, upward bending, length and thin diameter. Ex vivo extracted lSO whiskers also showed exceptional airflow displacement, suggesting whisker-intrinsic biomechanics mediate the unique airflow-sensitivity. Micro computed tomography (micro-CT) revealed that the ring-wulst-the follicle structure receiving the most sensitive afferents-was more complete/closed in the lSO, and other wind-sensitive whiskers, than in non-wind-sensitive whiskers, suggesting specialization of the supra-orbital for omni-directional sensing. We localized and targeted the cortical supra-orbital whisker representation in simultaneous Neuropixels recordings with D/E-row whisker barrels. Responses to wind-stimuli were stronger in the supra-orbital whisker representation than in D/E-row barrel cortex. We assessed the behavioral significance of whiskers in an airflow-sensing paradigm. We observed that rats spontaneously turn towards airflow stimuli in complete darkness. Selective trimming of wind-responsive whiskers diminished airflow turning responses more than trimming of non-wind-responsive whiskers. Lidocaine injections targeted to supra-orbital whisker follicles also diminished airflow turning responses compared to control injections. We conclude that supra-orbital whiskers act as wind antennae.

The functional anatomy of elephant trunk whiskers.

Behavior and innervation suggest a high tactile sensitivity of elephant trunks. To clarify the tactile trunk periphery we studied whiskers with the following findings. Whisker density is high at the trunk tip and African savanna elephants have more trunk tip whiskers than Asian elephants. Adult elephants show striking lateralized whisker abrasion caused by lateralized trunk behavior. Elephant whiskers are thick and show little tapering. Whisker follicles are large, lack a ring sinus and their organization varies across the trunk. Follicles are innervated by ~90 axons from multiple nerves. Because elephants don't whisk, trunk movements determine whisker contacts. Whisker-arrays on the ventral trunk-ridge contact objects balanced on the ventral trunk. Trunk whiskers differ from the mobile, thin and tapered facial whiskers that sample peri-rostrum space symmetrically in many mammals. We suggest their distinctive features-being thick, non-tapered, lateralized and arranged in specific high-density arrays-evolved along with the manipulative capacities of the trunk.

Fiber counts and architecture of the human dorsal penile nerve.

The human penis transmits behaviorally important sensory information via the dorsal penile nerve, which is required for initiation and maintenance of erection. The human penis differs from the penes of other hominids. The lack of a baculum makes the human penis dependent on erectile tissue, which is under control of neural signals activated by tactile stimulation. Accordingly, the penile sensory innervation is crucial for human sexual behavior. To clarify penile innervation, we analyzed the architecture of the dorsal penile nerve of five male subjects who donated their body. We stained the sensory fibers in the penile dorsal nerve with anti-neurofilament H antibody, and identified myelinated axons with Luxol fast blue staining. Furthermore, we visualized nerve bundles as they travel along the shaft of the penis by performing microfocus computed tomography scans after counterstaining penes with iodine. Our results show that the dorsal penile nerve is organized in 25-45 loosely packed nerve bundles, running mediodorsally in the shaft of the penis. This organization corresponds to that in penes of other mammalian species, but differs from the organization of the other peripheral sensory nerves. Around half of the dorsal penile nerve fibers were myelinated and a human hemipenis contained a total of 8290 ± 2553 (mean ± SD) axons. Thus, the number of sensory axons in the human dorsal penile nerve is higher than in other species described so far. The large fraction of unmyelinated nerve fibers suggests that the conduction speed is not a crucial aspect of penile sensory transmission.

Elephant banana peeling.

VIDEO ABSTRACT.

Coevolution of rostrum and brain in pig species.

Domestic pigs have a prominent cortical gyrus (the rostrum gyrus) isomorphic to the contralateral hemirostrum. It is unclear, however, if the size and shape of the rostrum gyrus are of evolutionary/functional relevance. Here, we address this question by assessing the relationship of rostrum and rostrum gyrus across eight pig species. To this end, we quantified rostrum morphology in fresh and alcohol-preserved pig specimens by surface scans, microfocus computed tomography scans, and photography. We establish that the size and shape of the rostrum gyrus can be precisely inferred from endocasts. We then took advantage of the accessibility of pig skulls and endocasts to assess features of the rostrum gyrus across species. Our investigation led to the following results: (i) The rostra of pig species show basic similarities. (ii) A cortical rostrum gyrus is apparent in all pigs. (iii) The size of the rostrum gyrus differs across species and outgroups of the evolutionary dominant suinae (i.e., peccaries and the babirusa) have a noticeably smaller rostrum gyrus. (iv) Warthogs have a derived rostrum morphology with an extra fold and a very wide rostrum; the warthog rostrum gyrus recapitulates these rostrum features. (v) Domestic pigs have relatively smaller rostrum gyrus than wild boars. We also provide indications for a conserved cytoarchitectonic patterning of the rostrum gyrus. We conclude that the rostrum gyrus is a neural module that was putatively present in the common ancestor of pigs and that this neural module is conserved across pig species. Natural selection maintains the rostrum gyrus' size and its exact isomorphism to the rostrum.

Cortical synapses of the world's smallest mammal: An FIB/SEM study in the Etruscan shrew.

The main aim of the present study was to determine if synapses from the exceptionally small brain of the Etruscan shrew show any peculiarities compared to the much larger human brain. We analyzed the cortical synaptic density and a variety of structural characteristics of 7,239 3D reconstructed synapses, using using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM). We found that some of the general synaptic characteristics are remarkably similar to those found in the human cerebral cortex. However, the cortical volume of the human brain is about 50,000 times larger than the cortical volume of the Etruscan shrew, while the total number of cortical synapses in human is only 20,000 times the number of synapses in the shrew, and synaptic junctions are 35% smaller in the Etruscan shrew. Thus, the differences in the number and size of synapses cannot be attributed to a brain size scaling effect but rather to adaptations of synaptic circuits to particular functions.

Tickle contagion in the rat somatosensory cortex.

The cellular mechanisms of emotional contagion are unknown. We investigated tickle contagion and the underlying neuronal representations in playful rats. We recorded trunk somatosensory cortex activity of observer rats while they received tickling and audiovisual playback of tickling footage and while they witnessed tickling of demonstrator rats. Observers vocalized and showed "Freudensprünge" ("joy jumps") during witnessing live tickling, while they showed little behavioral responses to playbacks. Deep layers in the trunk somatosensory neurons showed a larger correlation between direct and witnessed tickling responses compared to superficial layers. Trunk somatosensory neurons discharged upon emission of own and demonstrator's vocalizations and might drive contagious "laughter". We conclude that trunk somatosensory cortex might represent ticklishness contagion.

Relative enlargement of the medial preoptic nucleus in the Etruscan shrew, the smallest torpid mammal.

Endothermy is a key feature of mammalian biology and enables mammals to maintain stable body temperature and homeostatic functions in the face of a rapidly changing environment. However, when faced with harsh environmental conditions, certain mammalian species enter torpor, a state characterized by reduced metabolism, body temperature, and activity, to minimize energy loss. Etruscan shrews are the smallest mammals, with a surface-to-volume ratio that is very unfavorable for endothermic animals. As a result, Etruscan shrews have an extremely high metabolic rate and are known to enter torpor frequently, presumably as an energy-saving measure. Despite the recent identification of medial preoptic area (MPA) as a key brain region to regulate torpor in mouse, little is known about neural control of torpor in other endothermic animals, including the Etruscan shrew. Here, we confirmed that Etruscan shrews readily enter torpor even in the absence of strong physiological triggers. We then compared the medial preoptic nucleus (MPN) within the MPA of Etruscan shrew and rat, a mammal that does not enter torpor under physiological conditions. While rats have roughly 100 times the body weight and 33 times the brain weight of Etruscan shrews, we find that the male rat MPN exhibits only 6.7 times the volume of that of the male Etruscan shrew. Accordingly, the relative brain volume of the MPN was 6.5-fold larger in shrews, a highly significant difference. Moreover, MPN neuron counts were only roughly twofold lower in shrews than in rats, an astonishing observation considering the interspecies size difference and that neocortical neurons are ~ 20 × more numerous in rats than in shrews. We suggest that the extraordinary enlargement of the Etruscan shrew MPN is a specialization for orchestrating torpor in a mammal with an exceptional metabolism.

Elephant facial motor control.

We studied facial motor control in elephants, animals with muscular dexterous trunks. Facial nucleus neurons (~54,000 in Asian elephants, ~63,000 in African elephants) outnumbered those of other land-living mammals. The large-eared African elephants had more medial facial subnucleus neurons than Asian elephants, reflecting a numerically more extensive ear-motor control. Elephant dorsal and lateral facial subnuclei were unusual in elongation, neuron numerosity, and a proximal-to-distal neuron size increase. We suggest that this subnucleus organization is related to trunk representation, with the huge distal neurons innervating the trunk tip with long axons. African elephants pinch objects with two trunk tip fingers, whereas Asian elephants grasp/wrap objects with larger parts of their trunk. Finger "motor foveae" and a positional bias of neurons toward the trunk tip representation in African elephant facial nuclei reflect their motor strategy. Thus, elephant brains reveal neural adaptations to facial morphology, body size, and dexterity.

Maternity: Oxytocin circuits during birth and lactation.

Maternity transforms body, brain and behavior. A new study analyzing the activity of oxytocin neurons across birth and lactation revealed strengthening of suckling responses in mice. Although this did not involve major rewiring of inputs to oxytocin neurons, inhibition from the stria terminalis was found to pattern the suckling responses.

The human tickle response and mechanisms of self-tickle suppression.

A tickle is a complex sensation: it occurs in response to touch but not unequivocally so, and makes us laugh albeit not when we self-tickle. We quantified human ticklishness by means of physiological, visual and acoustic measures alongside subjective reports, and assessed mechanisms of self-tickle suppression. Tickle responses arose faster than previously reported as changes in thoracic circumference and joyous facial expressions co-emerge approximately 300 ms after tickle onset and are followed by vocalizations starting after an additional 200 ms. The timing and acoustic properties of vocalizations tightly correlated with subjective reports: the faster, louder and higher-pitched participants laughed, the stronger they rated the experienced ticklishness. Externally evoked ticklishness is reduced by simultaneous self-tickling, whereby self-touch evokes stronger suppression than sole self-tickle movement without touch. We suggest that self-tickle suppression can be understood as broad attenuation of sensory temporally coincident inputs. Our study provides new insight on the nature of human ticklishness and the attenuating effects of self-tickling. This article is part of the theme issue 'Cracking the laugh code: laughter through the lens of biology, psychology and neuroscience'.

Large-Scale Mapping of Vocalization-Related Activity in the Functionally Diverse Nuclei in Rat Posterior Brainstem.

The identity and location of vocalization pattern generating (VPG) circuits in mammals is debated. Based on physiological experiments, investigators suggested anterior brainstem circuits in the reticular formation, and anatomic evidence suggested the nucleus retroambiguus (NRA) in the posterior brainstem, or combinations of these sites as the putative mammalian VPG. Additionally, vocalization loudness is a critical factor in acoustic communication. However, many of the underlying neuronal mechanisms are still unknown. Here, we evoked calls by stimulation of the periaqueductal gray in anesthetized male rats, performed a large-scale mapping of vocalization-related activity using the activity marker c-fos, and high-density recordings of brainstem circuits using Neuropixels probes. Both c-fos expression and recording of vocalization-related activity point to a participation of the NRA in vocalization. More important, among our recorded structures, we found that the NRA is the only brainstem area showing a strong correlation between unit activity and call intensity. In addition, we observed functionally diverse patterns of vocalization-related activity in a set of regions around NRA. Dorsal to NRA, we observed activity specific to the beginning and end of vocalizations in the posterior level of the medullary reticular nucleus, dorsal part, whereas medial and lateral to the NRA, we observed activity related to call initiation. No clear vocalization-related activity was observed at anterior brainstem sites. Our findings suggest a set of functionally heterogeneous regions around the NRA contribute to vocal pattern generation in rats.SIGNIFICANCE STATEMENT Vocalization patterns are shaped in the mammalian brainstem, but the identity and location of the circuits involved is debated. Additionally, the neuronal mechanisms of vocal intensity control are still unknown. This study consisted of a large-scale mapping of brainstem vocalization circuits based on the activity marker c-fos and high-density recordings with Neuropixels probes. The results confirm the role of nucleus retroambiguus in call production and point to a key role of neurons in this nucleus in loudness control. Dorsal to the nucleus retroambiguus and in the posterior medulla, the authors identify neurons with activity specific to the beginning and end of vocalizations. The results point to specific neural dials for various aspects of rat vocalization control in the posterior brainstem.