Adult Neural Plasticity in Naked Mole-Rats: Implications of Fossoriality, Longevity and Sociality on the Brain's Capacity for Change.

Naked mole-rats (Heterocephalus glaber) are small African rodents that have many unique behavioral and physiological adaptations well-suited for testing hypotheses about mammalian neural plasticity. In this chapter, we focus on three features of naked mole-rat biology and how they impact neural plasticity in this species: (1) their fossorial lifestyle, (2) their extreme longevity with a lack of demonstrable senescence, and (3) their unusual social structure. Critically, each of these features requires some degree of biological flexibility. First, their fossorial habitat situates them in an environment with characteristics to which the central nervous system is particularly sensitive (e.g., oxygen content, photoperiod, spatial complexity). Second, their long lifespan requires adaptations to combat senescence and declines in neural functioning. Finally, their extreme reproductive skew and sustained ability for release from reproductive suppression indicates remarkable neural sensitivity to the sociosexual environment that is distinct from chronological age. These three features of naked mole-rat life are not mutually exclusive, but they do each offer unique considerations for the possibilities, constraints, and mechanisms associated with adult neural plasticity.

The stress of being alone: Removal from the colony, but not social subordination, increases fecal cortisol metabolite levels in eusocial naked mole-rats.

In many social species, hierarchical status within the group is associated with differences in basal adrenocortical activity. We examined this relationship in naked mole-rats (Heterocephalus glaber), eusocial rodents with arguably the most extreme social hierarchies of all mammals. This species lives in colonies where breeding is restricted to one socially dominant 'queen' and her male consorts, and all other individuals are reproductively suppressed 'subordinates'. The relationship between cortisol and social status in naked mole-rats has not fully been elucidated, as prior results on this topic have been contradictory. We used non-invasive feces sampling to measure baseline cortisol levels in eight laboratory colonies of naked mole-rats, to either replicate or reject rank differences. First, we successfully validated an assay to measure fecal cortisol metabolites (FCMs). Removal from the colony for the validation experiment, either alone or with an opposite sex conspecific, induced prolonged elevation of FCM levels on a scale of days to weeks. This increase in cortisol did not prevent the removed animals from sexually maturing. In colony-housed animals, we found no relationship between rank in the social hierarchy and FCM levels. Further, queens, breeding males, and reproductively suppressed subordinates all had equivalent FCM levels. We conclude that this species shows little evidence of the 'stress of dominance' or 'stress of subordination' and that reproductive suppression in naked mole-rats is not driven by elevated cortisol levels.

Subcaste differences in neural activation suggest a prosocial role for oxytocin in eusocial naked mole-rats.

The neuropeptide oxytocin (OT) influences prosocial behavior(s), aggression, and stress responsiveness, and these diverse effects are regulated in a species- and context-specific manner. The naked mole-rat (Heterocephalus glaber) is a unique species with which to study context-dependent effects of OT, exhibiting a strict social hierarchy with behavioral specialization within the subordinate caste: soldiers are aggressive and defend colonies against unfamiliar conspecifics while workers are prosocial and contribute to in-colony behaviors such as pup care. To determine if OT is involved in subcaste-specific behaviors, we compared behavioral responses between workers and soldiers of both sexes during a modified resident/intruder paradigm, and quantified activation of OT neurons in the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) using the immediate-early-gene marker c-fos co-localized with OT neurons. Resident workers and soldiers were age-matched with unfamiliar worker stimulus animals as intruders, and encounters were videorecorded and scored for aggressive behaviors. Colony-matched controls were left in their home colony for the duration of the encounters. Brains were extracted and cell counts were conducted for OT immunoreactive (ir), c-fos-ir, and percentage of OT-c-fos double-labeled cells. Results indicate that resident workers were less aggressive but showed greater OT neural activity than soldiers. Furthermore, a linear model including social treatment, cortisol, and subcaste revealed that subcaste was the only significant predictor of OT-c-fos double-labeled cells in the PVN. These data suggest that in naked mole-rats OT promotes prosocial behaviors rather than aggression and that even within subordinates status exerts robust effects on brain and behavior.

Peripheral administration of oxytocin increases social affiliation in the naked mole-rat (Heterocephalus glaber).

The neuropeptide oxytocin regulates a wide variety of social behaviors across diverse species. However, the types of behaviors that are influenced by this hormone are constrained by the species in question and the social organization that a particular species exhibits. Therefore, the present experiments investigated behaviors regulated by oxytocin in a eusocial mammalian species by using the naked mole-rat (Heterocephalus glaber). In Experiment 1, adult non-breeding mole-rats were given intraperitoneal injections of either oxytocin (1mg/kg or 10mg/kg) or saline on alternate days. Animals were then returned to their colony and behavior was recorded for minutes 15-30 post-injection. Both doses of oxytocin increased huddling behavior during this time period. In Experiment 2, animals received intraperitoneal injections of either oxytocin (1mg/kg), an oxytocin-receptor antagonist (0.1mg/kg), a cocktail of oxytocin and the antagonist, or saline across 4 testing days in a counterbalanced design. Animals were placed in either a 2-chamber arena with a familiar conspecific or in a small chamber with 1week old pups from their home colony and behaviors were recorded for minutes 15-30 post-injection. Oxytocin increased investigation of, and time spent in close proximity to, a familiar conspecific; these effects were blocked by the oxytocin antagonist. No effects were seen on pup-directed behavior. These data suggest that oxytocin is capable of modulating affiliative-like behavior in this eusocial species.

Oxytocin Manipulation Alters Neural Activity in Response to Social Stimuli in Eusocial Naked Mole-Rats.

The social decision-making network (SDMN) is a conserved neural circuit that modulates a range of social behaviors via context-specific patterns of activation that may be controlled in part by oxytocinergic signaling. We have previously characterized oxytocin's (OT) influence on prosociality in the naked mole-rat, a eusocial mammalian species, and its altered neural distribution between animals of differing social status. Here, we asked two questions: (1) do patterns of activation in the SDMN vary by social context and (2) is functional connectivity of the SDMN altered by OT manipulation? Adult subordinate naked mole-rats were exposed to one of three types of stimuli (three behavioral paradigms: familiar adult conspecific, unfamiliar adult conspecific, or familiar pups) while manipulating OT (three manipulations: saline, OT, or OT antagonist). Immediate early gene c-Fos activity was quantified using immunohistochemistry across SDMN regions. Network analyses indicated that the SDMN is conserved in naked mole-rats and functions in a context-dependent manner. Specific brain regions were recruited with each behavioral paradigm suggesting a role for the nucleus accumbens in social valence and sociosexual interaction, the prefrontal cortex in assessing/establishing social dominance, and the hippocampus in pup recognition. Furthermore, while OT manipulation was generally disruptive to coordinated neural activity, the specific effects were context-dependent supporting the hypothesis that oxytocinergic signaling promotes context appropriate social behaviors by modulating co-ordinated activity of the SDMN.

Antidepressant effects of focused ultrasound induced blood-brain-barrier opening.

In many cases, hippocampal neurogenesis appears to be a hallmark of antidepressant treatments. One novel technique for inducing this type of neurogenesis is using focused ultrasound waves, in conjunction with circulating microbubbles, to open the blood-brain-barrier. The present experiment aimed to test whether this technique has antidepressant effects in a rodent model. Rats were subjected to 1, 2 or 3 weekly treatments of magnetic resonance-guided focused ultrasound in order to open the blood-brain-barrier in the hippocampal region. Before and after treatments, animals went through modified forced swim tests. 1 week after the final treatment, animals that received 2 weekly treatments showed antidepressant-like effects on behavioural measures in comparison to untreated controls. This was not the case for animals that received 1 or 3 weekly treatments. Effects had disappeared by 5 weeks following the first ultrasound treatment. These results suggest that focused ultrasound may be used for inducing short-term antidepressant effects.

Retinal ganglion cell survival and axon regeneration after optic nerve injury in naked mole-rats.

In the adult mammalian central nervous system (CNS), axonal damage often triggers neuronal cell death and glial activation, with very limited spontaneous axon regeneration. In this study, we performed optic nerve injury in adult naked mole-rats, the longest living rodent, with a maximum life span exceeding 30 years, and found that injury responses in this species are quite distinct from those in other mammalian species. In contrast to what is seen in other mammals, the majority of injured retinal ganglion cells (RGCs) survive with relatively high spontaneous axon regeneration. Furthermore, injured RGCs display activated signal transducer and activator of transcription-3 (STAT3), whereas astrocytes in the optic nerve robustly occupy and fill the lesion area days after injury. These neuron-intrinsic and -extrinsic injury responses are reminiscent of those in "cold-blooded" animals, such as fish and amphibians, suggesting that the naked mole-rat is a powerful model for exploring the mechanisms of neuronal injury responses and axon regeneration in mammals. J. Comp. Neurol. 525:380-388, 2017. © 2016 Wiley Periodicals, Inc.

Focused Ultrasound-Induced Neurogenesis Requires an Increase in Blood-Brain Barrier Permeability.

Transcranial focused ultrasound technology used to transiently open the blood-brain barrier, is capable of stimulating hippocampal neurogenesis; however, it is not yet known what aspects of the treatment are necessary for enhanced neurogenesis to occur. The present study set out to determine whether the opening of blood-brain barrier, the specific pressure amplitudes of focused ultrasound, and/or the intravenous administration of microbubbles (phospholipid microspheres) are necessary for the enhancement of neurogenesis. Specifically, mice were exposed to burst (10ms, 1Hz burst repetition frequency) focused ultrasound at the frequency of 1.68MHz and with 0.39, 0.78, 1.56 and 3.0MPa pressure amplitudes. These treatments were also conducted with or without microbubbles, at 0.39 + 0.78MPa or 1.56 + 3.0MPa, respectively. Only focused ultrasound at the ~0.78 MPa pressure amplitude with microbubbles promoted hippocampal neurogenesis and was associated with an increase in blood-brain barrier permeability. These results suggest that focused ultrasound -mediated neurogenesis is dependent upon the opening of the blood-brain barrier.

Sex, social status, and CRF receptor densities in naked mole-rats.

Naked mole-rats (Heterocephalus glaber) live in groups that are notable for their large size and caste structure, with breeding monopolized by a single female and a small number of males. Recent studies have demonstrated substantial differences between the brains of breeders and subordinates induced by changes in social standing. Corticotropin-releasing factor (CRF) receptors-which bind the hormone CRF as well as related peptides-are important regulators of stress and anxiety, and are emerging as factors affecting social behavior. We conducted autoradiographic analyses of CRF1 and CRF2 receptor binding densities in female and male naked mole-rats varying in breeding status. Both globally and in specific brain regions, CRF1 receptor densities varied with breeding status. CRF1 receptor densities were higher in subordinates across brain regions, and particularly in the piriform cortex and cortical amygdala. Sex differences were present in CRF2 receptor binding densities, as is the case in multiple vole species. CRF2 receptor densities were higher in females, both globally and in the cortical amygdala and lateral amygdalar nucleus. These results provide novel insights into the neurobiology of social hierarchy in naked mole-rats, and add to a growing body of work that links changes in the CRF system with social behavior.

Successful intracerebroventricular cannulation of a eusocial mammal.

BACKGROUND: Manipulating neural activity in live animals within a colony would allow researchers to more fully explore the neurobiology of complex social behaviors. However, some colony-living animals like the naked mole-rat (Heterocephalus glaber) cannot be reintroduced to a colony after the extended recovery time required following cranial surgery. Furthermore, the colony setting creates increased risk of infection and interruption of cranial surgical sites. NEW METHOD: A protocol for intracerebroventricular cannulations was developed for securing and minimizing exposure of the intracranial apparatus. We tested whether animals could be reintroduced to the colony immediately following surgery and whether they showed full recovery and expression of normal behavior a week later, after intracerebroventricular infusion of saline. RESULTS: Animals were successfully reincorporated into their home colony and showed normal behavior. No animals lost guide cannulae within their colony and loss of dummy cannulae was minimized. Any loss of animals was due to surgical complications or multiple intracerebroventricular infusions of saline rather than recovery in the colony, per se. COMPARISON WITH EXISTING METHODS: Standard cranial cannulation methods for small rodents were used with the addition of implanting a shortened guide cannula under the skin for limited exposure of cannulae to the external environment. Furthermore, dummy cannulae were sealed to guides to avoid loss in-colony. CONCLUSION: The use of intracranial cannulations is a viable option for colony-living rodents when the proper care is taken to minimize cannula exposure and when animals are carefully and promptly reintroduced to the colony setting after surgery.

A game of thrones: neural plasticity in mammalian social hierarchies.

Social status is a key regulator of health and reproduction in mammals, including humans. Despite this, relatively little is known about how social status influences the mammalian brain. Furthermore, the extent to which status is an independent construct, i.e., not simply acting as a psychosocial stressor, is yet to be determined. Research to date reveals several promising mechanisms and/or systems associated with social status, including monoamine systems, hypothalamic neuroendocrine axes, and the hippocampus, though whether these differences are the cause or effect of status is often unclear. We review these candidates and propose how best to approach this research question in the future.

Inadequate early social experience increases the incentive salience of reward-related cues in adulthood.

The mechanisms by which childhood abuse and/or neglect become risk factors for the development of drug addiction, problem gambling, and other disorders of behavioral inhibition are unknown. The loss of behavioral inhibition is often triggered by reward-related cues that acquire incentive salience. This study examined whether inadequate early-life social experience in rats affects the incentive salience of reward-related cues. Rats were deprived of early-life social experience with the mother and litter through artificial-rearing (AR). A group of AR rats (AR+STM) received additional tactile stimulation that mimicked maternal licking, a critical component of rat maternal care. Control rats were maternally reared (MR). The incentive salience attributed to a food cue was measured in adult rats using a conditioned approach task, where a conditional stimulus (CS; lever) was paired with food delivery, and in a conditional reinforcement task. The dependent measures were approach towards the CS (sign-tracking) versus approach towards the place of food delivery (goal-tracking) and instrumental responding for the CS. AR rats made significantly more sign-tracking responses than MR rats. AR rats also made more instrumental responses when reinforced with the CS. AR+STM rats' responses were intermediate to MR and AR rats. Thus, inadequate early-life social experience enhanced the incentive salience of a reward-related cue in adulthood. Replacement of maternal licking partially reversed this effect. These results highlight a potential link between early-life social adversity and susceptibility to disorders of behavioral inhibition.