Hydrogen sulfide (H2S) is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS), an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration.

Although somatosensation in multiple whisker systems has been studied in considerable detail, relatively little information is available regarding whisker usage and movement patterns during natural behaviors. The Etruscan shrew, one of the smallest mammals, relies heavily on its whisker system to detect and kill its highly mobile insect prey. Here, we tracked whisker and body motion during prey capture. We found that shrews made periodic whisker movements (whisking) with frequencies ranging from 12 to 17 Hz. We compared shrew and rat whisking and found that shrew whisking was smaller amplitude and higher frequency than rat whisking, but that the shrew and rat whisking cycle were similar in that the velocity was higher during retraction than protraction. We were able to identify four phases during the shrew hunting behavior: (i) an immobile phase often preceding hunting, (ii) a search phase upon the initiation of hunting, (iii) a contact phase defined by whisker-to-cricket contact, and (iv) an attack phase, characterized by a rapid head movement directed toward the cricket. During the searching phase, whisking was generally rhythmic and whiskers were protracted forward. After prey contact, whisking amplitude decreased and became more variable. The final strike was associated with an abrupt head movement toward the prey with high head acceleration. Prey capture proceeded extremely fast and we obtained evidence that shrews can initiate corrective maneuvers with a minimal latency <30 ms. While the shrew's rostrum is straight and elongated during most behaviors, we show for the first time that shrews bend their rostrum during the final strike and grip their prey with a parrot beak shaped snout.

A crucial role of tactile experience for the maturation of neural response properties in the somatosensory system is well established, but little is known about the role of tactile experience in the development of tactile behaviors. Here we study how tactile experience affects prey capture behavior in Etruscan shrews, Suncus etruscus. Prey capture in adult shrews is a high-speed behavior that relies on precise attacks guided by tactile Gestalt cues. We studied the role of tactile experience by three different approaches. First, we analyzed the hunting skills of young shrews' right after weaning. We found that prey capture in young animals in most, but not all, aspects is similar to that of adults. Second, we performed whisker trimming for 3-4 weeks after birth. Such deprivation resulted in a lasting disruption of prey capture even after whisker re-growth: attacks lacked precise targeting and had a lower success rate. Third, we presented adult shrews with an entirely novel prey species, the giant cockroach. The shape of this roach is very different from the shrew's normal (cricket) prey and the thorax-the preferred point of attack in crickets-is protected by a heavy cuticle. Initially shrews attacked giant roaches the same way they attack crickets and targeted the thoracic region. With progressive experience, however, shrews adopted a new attack strategy targeting legs and underside of the roaches while avoiding other body parts. Speed and efficiency of attacks improved. These data suggest that tactile experience shapes prey capture behavior.

Many insects use the pattern of polarized light in the sky for spatial orientation and navigation. We have investigated the polarization vision system in the desert locust. To create a common platform for anatomical studies on polarization vision pathways, Kurylas et al. (2008) have generated a three-dimensional (3D) standard brain from confocal microscopy image stacks of 10 male brains, using two different standardization methods, the Iterative Shape Averaging (ISA) procedure and the Virtual Insect Brain (VIB) protocol. Comparison of both standardization methods showed that the VIB standard is ideal for comparative volume analysis of neuropils, whereas the ISA standard is the method of choice to analyze the morphology and connectivity of neurons. The central complex is a key processing stage for polarization information in the locust brain. To investigate neuronal connections between diverse central-complex neurons, we generated a higher-resolution standard atlas of the central complex and surrounding areas, using the ISA method based on brain sections from 20 individual central complexes. To explore the usefulness of this atlas, two central-complex neurons, a polarization-sensitive columnar neuron (type CPU1a) and a tangential neuron that is activated during flight, the giant fan-shaped (GFS) neuron, were reconstructed 3D from brain sections. To examine whether the GFS neuron is a candidate to contribute to synaptic input to the CPU1a neuron, we registered both neurons into the standardized central complex. Visualization of both neurons revealed a potential connection of the CPU1a and GFS neurons in layer II of the upper division of the central body.

Anatomical, stimulation and lesion data implicate vibrissa motor cortex in whisker motor control. Work on motor cortex has focused on movement generation, but correlations between vibrissa motor cortex activity and whisking are weak. The exact role of vibrissa motor cortex remains unknown. We recorded vibrissa motor cortex neurons during various forms of vibrissal touch, which were invariably associated with whisker protraction and movement. Free whisking, object palpation and social touch all resulted in decreased cortical activity. To understand this activity decrease, we performed juxtacellular recordings, nanostimulation and in vivo whole-cell recordings. Social touch resulted in decreased spiking activity, decreased cell excitability and membrane hyperpolarization. Activation of vibrissa motor cortex by intracortical microstimulation elicited whisker retraction, as if to abort vibrissal touch. Various vibrissa motor cortex inactivation protocols resulted in contralateral protraction and increased whisker movements. These data collectively point to movement suppression as a prime function of vibrissa motor cortex activity.

Synaptic transmission and plasticity in the hippocampus are integral factors in learning and memory. While there has been intense investigation of these critical mechanisms in the brain of rodents, we lack a broader understanding of the generality of these processes across species. We investigated one of the smallest animals with conserved hippocampal macroanatomy-the Etruscan shrew, and found that while synaptic properties and plasticity in CA1 Schaffer collateral synapses were similar to mice, CA3 mossy fiber synapses showed striking differences in synaptic plasticity between shrews and mice. Shrew mossy fibers have lower long term plasticity compared to mice. Short term plasticity and the expression of a key protein involved in it, synaptotagmin 7 were also markedly lower at the mossy fibers in shrews than in mice. We also observed similar lower expression of synaptotagmin 7 in the mossy fibers of bats that are evolutionarily closer to shrews than mice. Species specific differences in synaptic plasticity and the key molecules regulating it, highlight the evolutionary divergence of neuronal circuit functions.

The naked mole rat (Heterocephalus glaber, NMR) is a rodent with exceptional longevity, low rates of age-related diseases and spontaneous carcinogenesis. The NMR represents an attractive animal model in longevity and cancer research, but there are no NMR-specific antibodies available to study its immune system with respect to age- and cancer-related questions. Substantial homology of major NMR immune cell markers with those of Guinea pig, human and, to a lesser extent, mouse and rat origin are implicated for the existence of immunological cross-reactivity. We identified 10 antibodies recognising eight immunophenotypic markers expressed on the NMR's T and B lymphocytes, macrophages/monocytes and putative haematopoietic precursors and used them for an immunophenotyping of leukocyte subsets of peripheral blood, spleen and bone marrow samples. Overall, we found that the leukocyte composition of NMR peripheral blood is comparable to that of mice. Notably, the frequency of cytotoxic T cells was found to be lower in the NMR compared to corresponding mouse tissues and human blood. Antibodies used in the present paper are available either commercially or from the scientific community and will provide new opportunities for the NMR as a model system in ageing- and cancer-related research areas.

The relationship between tickling, sensation, and laughter is complex. Tickling or its mere anticipation makes us laugh, but not when we self-tickle. We previously showed rat somatosensory cortex drives tickling-evoked vocalizations and now investigated self-tickle suppression and tickle anticipation. We recorded somatosensory cortex activity while tickling and touching rats and while rats touched themselves. Allo-touch and tickling evoked somatotopic cortical excitation and vocalizations. Self-touch induced wide-ranging inhibition and vocalization suppression. Self-touch also suppressed vocalizations and cortical responses evoked by allo-touch or cortical microstimulation. We suggest a global-inhibition model of self-tickle suppression, which operates without the classically assumed self versus other distinction. Consistent with this inhibition hypothesis, blocking cortical inhibition with gabazine abolished self-tickle suppression. We studied anticipation in a nose-poke-for-tickling paradigm. Although rats nose poked for tickling, they also showed escaping, freezing, and alarm calls. Such ambivalence ("Nervenkitzel") resembles tickle behaviors in children. We conclude that self-touch-induced GABAergic cortical inhibition prevents self-tickle, whereas anticipatory layer 5 activity drives anticipatory laughter. VIDEO ABSTRACT.

In classical neuroscience experiments, neural activity is measured across many identical trials of animals performing simple tasks and is then analyzed, associating neural responses to pre-defined experimental parameters. This type of analysis is not suitable for patterns of behavior that unfold freely, such as play behavior. Here, we attempt an alternative approach for exploratory data analysis on a single-trial level, applicable in more complex and naturalistic behavioral settings in which no two trials are identical. We analyze neural population activity in the prefrontal cortex (PFC) of rats playing hide-and-seek and show that it is possible to discover what aspects of the task are reflected in the recorded activity with a limited number of simultaneously recorded cells (≤ 31). Using hidden Markov models, we cluster population activity in the PFC into a set of neural states, each associated with a pattern of neural activity. Despite high variability in behavior, relating the inferred states to the events of the hide-and-seek game reveals neural states that consistently appear at the same phases of the game. Furthermore, we show that by applying the segmentation inferred from neural data to the animals' behavior, we can explore and discover novel correlations between neural activity and behavior. Finally, we replicate the results in a second dataset and show that population activity in the PFC displays distinct sets of states during playing hide-and-seek and observing others play the game. Overall, our results reveal robust, state-like representations in the rat PFC during unrestrained playful behavior and showcase the applicability of population analyses in naturalistic neuroscience.

Touch is a fundamental aspect of social, parental and sexual behavior. In contrast to our detailed knowledge about cortical processing of non-social touch, we still know little about how social touch impacts cortical circuits. We investigated neural activity across five frontal, motor and sensory cortical areas in rats engaging in naturalistic social facial touch. Information about social touch and the sex of the interaction partner (a biologically significant feature) is a major determinant of cortical activity. 25.3% of units were modulated during social touch and 8.3% of units displayed 'sex-touch' responses (responded differently, depending on the sex of the interaction partner). Single-unit responses were part of a structured, partner-sex- and, in some cases, subject-sex-dependent population response. Spiking neural network simulations indicate that a change in inhibitory drive might underlie these population dynamics. Our observations suggest that socio-sexual characteristics of touch (subject and partner sex) widely modulate cortical activity and need to be investigated with cellular resolution.

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.

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, β, 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 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.

Extracellular recordings in medial entorhinal cortex have revealed the existence of spatially-modulated firing patterns, which are thought to contribute to a cognitive map of external space. Previous work indicated that during exploration of novel environments, spiking activity in deep entorhinal layers is much sparser than in superficial layers. In the present report, we ask whether this laminar activity profile is a consequence of environmental novelty. We report on a large dataset of juxtacellularly-recorded neurons (n = 70) whose spiking activity was monitored while rats explored either a novel or a familiar environment, or both within the same session. Irrespective of previous knowledge of the environment, deep layer activity was very low during exploration (median firing rate 0.4 Hz for non-silent cells), with a large fraction of silent cells (n = 19 of a total 37), while superficial layer activity was several times higher (median firing rate 2.4 Hz; n = 33). The persistence of laminar differences in firing activity both under environmental novelty and familiarity, and even in head-restrained stationary animals, suggests that sparse coding might be a constitutive feature of deep entorhinal layers.

We investigated the structural development of superficial-layers of medial entorhinal cortex and parasubiculum in rats. The grid-layout and cholinergic-innervation of calbindin-positive pyramidal-cells in layer-2 emerged around birth while reelin-positive stellate-cells were scattered throughout development. Layer-3 and parasubiculum neurons had a transient calbindin-expression, which declined with age. Early postnatally, layer-2 pyramidal but not stellate-cells co-localized with doublecortin - a marker of immature neurons - suggesting delayed functional-maturation of pyramidal-cells. Three observations indicated a dorsal-to-ventral maturation of entorhinal cortex and parasubiculum: (i) calbindin-expression in layer-3 neurons decreased progressively from dorsal-to-ventral, (ii) doublecortin in layer-2 calbindin-positive-patches disappeared dorsally before ventrally, and (iii) wolframin-expression emerged earlier in dorsal than ventral parasubiculum. The early appearance of calbindin-pyramidal-grid-organization in layer-2 suggests that this pattern is instructed by genetic information rather than experience. Superficial-layer-microcircuits mature earlier in dorsal entorhinal cortex, where small spatial-scales are represented. Maturation of ventral-entorhinal-microcircuits - representing larger spatial-scales - follows later around the onset of exploratory behavior.

In medial entorhinal cortex, layer 2 principal cells divide into pyramidal neurons (mostly calbindin positive) and dentate gyrus-projecting stellate cells (mostly calbindin negative). We juxtacellularly labeled layer 2 neurons in freely moving animals, but small sample size prevented establishing unequivocal structure-function relationships. We show, however, that spike locking to theta oscillations allows assigning unidentified extracellular recordings to pyramidal and stellate cells with ∼83% and ∼89% specificity, respectively. In pooled anatomically identified and theta-locking-assigned recordings, nonspatial discharges dominated, and weakly hexagonal spatial discharges and head-direction selectivity were observed in both cell types. Clear grid discharges were rare and mostly classified as pyramids (19%, 19/99 putative pyramids versus 3%, 3/94 putative stellates). Most border cells were classified as stellate (11%, 10/94 putative stellates versus 1%, 1/99 putative pyramids). Our data suggest weakly theta-locked stellate border cells provide spatial input to dentate gyrus, whereas strongly theta-locked grid discharges occur mainly in hexagonally arranged pyramidal cell patches and do not feed into dentate gyrus.

African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds.

In many species, polyploidy, in which an increase in nuclear DNA content is accompanied by an increase in cell size, contributes to cellular diversity. In the rat visual cortex, most neurons are small and homogeneous in size, while layer 5 cells are heterogeneous, containing some very large neurons. To measure DNA content, we quantified nuclear chromocenters and integrated DNA/DAPI fluorescence. The results suggest that most cortical neurons, non-neuronal cells, parvalbumin-positive interneurons, and large entorhinal layer 2 stellate projection neurons are diploid. In contrast, chromocenter counts and integrated fluorescence are ∼2-fold higher for some excitatory neurons in layer 5, suggesting that large Ctip2-negative and Ctip2-positive layer 5 neurons might be tetraploid. The distribution of putatively tetraploid neurons differed between areas and showed sharp borders aligned with functional subdivisions of the somatosensory cortex. Telomere counting and flow cytometry supported layer 5 polyploidy. We conclude that polyploidy contributes to cellular and areal diversity of rat cortex.

Rat somatosensory cortex contains a large sexually monomorphic genital representation. Genital cortex undergoes an unusual 2-fold expansion during puberty. Here, we investigate genital cortex development and female rat sexual maturation. Ovariectomies and estradiol injections suggested sex hormones cause the pubertal genital cortex expansion but not its maintenance at adult size. Genital cortex expanded by thalamic afferents invading surrounding dysgranular cortex. Genital touch was a dominant factor driving female sexual maturation. Raising female rats in contact with adult males promoted genital cortex expansion, whereas contact to adult females or nontactile (audio-visual-olfactory) male cues did not. Genital touch imposed by human experimenters powerfully advanced female genital cortex development and sexual maturation. Long-term blocking of genital cortex by tetrodotoxin in pubescent females housed with males prevented genital cortex expansion and decelerated vaginal opening. Sex hormones, sexual experience, and neural activity shape genital cortex, which contributes to the puberty promoting effects of sexual touch.

The genetics of lifespan determination is poorly understood. Most research has been done on short-lived animals and it is unclear if these insights can be transferred to long-lived mammals like humans. Some African mole-rats (Bathyergidae) have life expectancies that are multiple times higher than similar sized and phylogenetically closely related rodents. To gain new insights into genetic mechanisms determining mammalian lifespans, we obtained genomic and transcriptomic data from 17 rodent species and scanned eleven evolutionary branches associated with the evolution of enhanced longevity for positively selected genes (PSGs). Indicating relevance for aging, the set of 250 identified PSGs showed in liver of long-lived naked mole-rats and short-lived rats an expression pattern that fits the antagonistic pleiotropy theory of aging. Moreover, we found the PSGs to be enriched for genes known to be related to aging. Among these enrichments were "cellular respiration" and "metal ion homeostasis", as well as functional terms associated with processes regulated by the mTOR pathway: translation, autophagy and inflammation. Remarkably, among PSGs are RHEB, a regulator of mTOR, and IGF1, both central components of aging-relevant pathways, as well as genes yet unknown to be aging-associated but representing convincing functional candidates, e.g. RHEBL1, AMHR2, PSMG1 and AGER. Exemplary protein homology modeling suggests functional consequences for amino acid changes under positive selection. Therefore, we conclude that our results provide a meaningful resource for follow-up studies to mechanistically link identified genes and amino acids under positive selection to aging and lifespan determination.