A bird's eye view of the hippocampus beyond space: Behavioral, neuroanatomical, and neuroendocrine perspectives.

Although the hippocampus is one of the most-studied brain regions in mammals, research on the avian hippocampus has been more limited in scope. It is generally agreed that the hippocampus is an ancient feature of the amniote brain, and therefore homologous between the two lineages. Because birds and mammals are evolutionarily not very closely related, any shared anatomy is likely to be crucial for shared functions of their hippocampi. These functions, in turn, are likely to be essential if they have been conserved for over 300 million years. Therefore, research on the avian hippocampus can help us understand how this brain region evolved and how it has changed over evolutionary time. Further, there is a strong research foundation in birds on hippocampal-supported behaviors such as spatial navigation, food caching, and brood parasitism that scientists can build upon to better understand how hippocampal anatomy, network circuitry, endocrinology, and physiology can help control these behaviors. In this review, we summarize our current understanding of the avian hippocampus in spatial cognition as well as in regulating anxiety, approach-avoidance behavior, and stress responses. Although there are still some questions about the exact number of subdivisions in the avian hippocampus and how that might vary in different avian families, there is intriguing evidence that the avian hippocampus might have complementary functional profiles along the rostral-caudal axis similar to the dorsal-ventral axis of the rodent hippocampus, where the rostral/dorsal hippocampus is more involved in cognitive processes like spatial learning and the caudal/ventral hippocampus regulates emotional states, anxiety, and the stress response. Future research should focus on elucidating the cellular and molecular mechanisms - including endocrinological - in the avian hippocampus that underlie behaviors such as spatial navigation, spatial memory, and anxiety-related behaviors, and in so doing, resolve outstanding questions about avian hippocampal function and organization.

Sex-specific effects of neonatal paternal deprivation on microglial cell density in adult California mouse (Peromyscus californicus) dentate gyrus.

Adverse early-life experiences are risk factors for psychiatric disease development, resulting in stress-related neuronal modeling and neurobehavioral changes. Stressful experiences modulate the immune system, contributing to neuronal damage in higher cortical regions, like the hippocampus. Moreover, early-life stressors dysregulate the function of microglia, the resident immune cells of the brain, in the developing hippocampus. Paternal deprivation, an early-life stressor in many biparental species, facilitates sex-dependent inhibitions in hippocampal plasticity, but parental contributors to these sex-specific outcomes are unknown. Also, neurobiological mechanisms contributing to impairments in hippocampal neuroplasticity are less known. Thus, our goals were to 1) determine whether parental behavior is altered in maternal females following removal of the paternal male, 2) assess the effects of paternal deprivation on dentate gyrus (DG) volume and microglia proliferation, and 3) determine if early-life experimental handling mitigates sex-specific reductions in DG cell survival. California mice were born to multiparous breeders and reared by both parents (biparental care) or by their mother alone (i.e., father removed on postnatal day 1; paternal deprivation). One cohort of offspring underwent offspring retrieval tests for eight days beginning on postnatal day 2. On PND 68, these offspring (and a second cohort of mice without behavioral testing) were euthanized and brains visualized for bromodeoxyuridine (BrdU) and neuron-specific class III beta-tubulin (TuJ-1) or ionized calcium binding adaptor molecule 1 (Iba1). While mate absence did not impair maternal retrieval, paternal deprivation reduced DG volume, but Iba1+ cell density was only higher in paternally-deprived females. Neither sex or paternal deprivation significantly altered the number of BrdU+ or Tuj1+ cells in the DG - an absence of a reduction in cell survival may be related to daily handing during early offspring retrieval tests. Together, these data suggest that paternal deprivation impairs hippocampal plasticity; however, sex and early environment may influence the magnitude of these outcomes.

The neural distribution of the avian homologue of oxytocin, mesotocin, in two songbird species, the zebra finch and the canary: A potential role in song perception and production.

The avian homologue of oxytocin (OT), formerly called mesotocin, influences social behaviors in songbirds and potentially song production. We sought to characterize the distribution of OT peptide in the brain of two songbird species: canaries (Serinus canaria) and zebra finches (Taeniopygia guttata). To visualize OT, we performed immunocytochemistry using an antibody previously shown to identify OT in avian species. In both canaries and zebra finches, dense OT-ir perikarya were located in the paraventricular nucleus (PVN), preoptic area (POA), supraoptic nucleus (SON), and medial bed nucleus of the stria terminalis (BNSTm). We also observed morphologically distinct OT-ir cells scattered throughout the mesopallium. OT-ir fibers were observed in the PVN, ventral medial hypothalamus (VMH), periaqueductal gray (PAG), intercollicular nucleus (ICo), and ventral tegmental area (VTA). We also observed punctate OT-ir fibers in the song control nucleus HVC. In both male and female canaries, OT-ir fibers were present in the lateral septum (LS), but innervation was greater in males. We did not observe this sex difference in zebra finches. Much of the OT staining observed is consistent with general distributions within the vertebrate hypothalamus, indicating a possible conserved function. However, some extra-hypothalamic distributions, such as perikarya in the mesopallium, may be specific to songbirds and play a role in song perception and production. The presence of OT-ir fibers in HVC and song control nuclei projecting dopaminergic regions provides anatomical evidence in support of the idea that OT can influence singing behavior-either directly via HVC or indirectly via the PAG, VTA, or POA.

Neuroendocrine and behavioral response to testosterone-induced female song in canaries (Serinus canaria).

Male song in songbirds is a critical and elaborate signal for mate attraction. In many species female listeners respond to male song both behaviorally and physiologically (e.g. copulation solicitation displays and production of the immediate early gene ZENK in auditory regions). It is becoming increasingly well known that females in many species also sing. However, in common lab species, such as canaries (Serinus canaria), female song is limited and has been primarily studied in the context of administering of exogenous testosterone (T) to increase song rate and length. In this study we addressed to what extent female canary songs are masculinized by the administration of exogenous T based on the behavioral and physiological responses of avian receivers. Specifically, are T induced female songs sufficient to elicit courtship behaviors and auditory ZENK expression in female listeners? We found that female songs after 3 weeks of exogenous T were significantly longer and more complex than female songs after 12 weeks of exogenous T. Additionally, we found that playback of 3-week T song significantly increased sexual response behaviors and the expression of ZENK in the auditory brain regions of female listeners. Finally, we conclude that extended periods of T do not necessarily maintain the masculinization of female song.

Intraspecific variation in testosterone-induced neuroplasticity in two canary strains.

Temperate zone songbird species, such as the canary (Serinus canaria), can serve as model systems to investigate adult seasonal plasticity in brain and behavior. An increase in day length, experienced by canaries in the early spring stimulates gonadal recrudescence and an associated increase in circulating testosterone concentrations. This increase in plasma testosterone results in marked morphological changes in well-defined neural circuitry regulating reproductive behaviors including birdsong as well as behavioral changes such as increases in song length and complexity. An obvious measure of plasticity in neural morphology can be assessed via changes in brain nuclei volume and testosterone actions on a number of cellular features including the integration and incorporation of new neurons in the adult canary brain. Previous work in our lab suggests that there may be systematic intraspecific variability within canaries in testosterone-induced adult neuroplasticity. For example, the song nucleus HVC increases in size in response to testosterone in male canaries but we found that males of the American Singer strain exhibited minimal and variable responses as compared to other canary strains such as the Border canary strain, which is thought to be closer to wild type canaries. In this study, we systematically compared the effects of testosterone on the volume of song nuclei and the number of new neurons as assessed with the neurogenesis marker doublecortin in American Singer and Border canaries. We found more pronounced testosterone-induced neuroplasticity in the Border strain than the American Singer. These data suggest that the process of selection for certain strain phenotypes is also associated with significant changes in hormone-regulated brain plasticity.

How does testosterone act to regulate a multifaceted adaptive response? Lessons from studies of the avian song system.

In male songbirds, song functions to attract a mate or to defend a territory; it is therefore often produced in the context of reproduction. Testosterone of gonadal origin increases during the reproductive phase of the annual cycle and significantly enhances song production, as well as song development, via effects on song crystallisation. The neural control of birdsong production and learning is highly modular. We implanted testosterone or androgen antagonists into specific brain regions or in the periphery of castrated male canaries and, in this way, identified how androgen signalling in specific locations regulates a variety of birdsong features. For example, castrated male canaries treated with testosterone in the preoptic area only and exposed to long days sing at high rates compared to castrated male canaries not treated with testosterone. However, these birds with testosterone in the preoptic area still produce songs with substantially lower song stereotypy and amplitude; these features are controlled by testosterone acting in the song control nuclei HVC and robust nucleus of the arcopallium. Specific aspects of the learned singing behaviour are thus regulated by androgens acting at multiple levels in the brain in a non-redundant fashion. The action of testosterone in the preoptic area is related to the hormonal regulation of the motivation to sing but not to various aspects of song performance. Multiple aspects of song quality are instead precisely regulated by steroids acting in distinct song control nuclei. Females exert a strong choice for specific features of male song in canaries and this choice is influenced by the endocrine state of the female. The female song system is also involved in song production, as well as song perception, although the specificity of this hormone action has not yet been investigated.

Strain differences in hearing in song canaries.

Belgian Waterslager song canaries, bred for hundreds of years for a low-pitched song, have also acquired an inherited high-frequency hearing loss associated with hair cell abnormalities. Here, auditory thresholds measured using auditory brainstem responses and psychophysical methods in three different strains of canaries are compared: Belgian Waterslagers, American Singers, and Borders. Border canaries have not been bred for song characteristics while American Singer canaries have been bred for song only since the 1930s. Results show that American Singer canaries also have elevated high frequency thresholds that are similar to those of the Belgian Waterslager, while Border canaries have normal thresholds. These results strengthen the case that song canary breeders in selecting for song characteristics may have inadvertently selected for hearing abnormalities.

Effects of Song Experience and Song Quality on Immediate Early Gene Expression in Female Canaries (Serinus canaria).

Female songbirds are thought to make mate choices based on aspects of male song quality. Male canaries (Serinus canaria) produce songs with "special" syllables that have been shown to be highly salient to female listeners - eliciting high rates of sexual displays and enhanced immediate early gene (IEG) expression. Immunohistochemistry for the IEG ZENK was used to examine the effects of experience with these syllables on activity in the caudal mesopallium (CMM) and nidocaudal mesopallium (NCM), two auditory areas important in processing conspecific song. Photostimulated female canaries were housed in sound attenuated chambers and played pseudosongs containing either three special syllables or three non-special syllables, an intro, and an outro sequence. Females that heard special syllable pseudosongs exhibited higher ZENK expression in CMM. To assess the effects of experience, photostimulated females were pair housed and exposed to playback of song with or without special syllables for 14 days. After transfer to individual housing, birds were played one of the aforementioned stimuli or silence. ZENK expression in CMM and NCM was equivalent for song with and without special syllables, but significantly lower for silence. Females who experienced song with special syllables had lower plasma estradiol concentrations after final song playback. This study indicates that CMM exhibits an IEG response bias to special syllables in limited acoustic contexts, but not in full song, which may contain additional biologically relevant information. Furthermore, estradiol concentrations may mediate changes in song responses, serving as a mechanism for modulating mate choice in differing song environments.

Acute stress-induced cortisol elevation during memory consolidation enhances pattern separation.

Stress is a potent modulator of brain function and particularly mnemonic processes. While chronic stress is associated with long-term deficits in memory, the effects of acute stress on mnemonic functions are less clear as previous reports have been inconsistent. Some studies suggest that cortisol, a stress hormone that modulates biological changes in response to stress, may enhance memory consolidation and impair memory retrieval. However, other studies report no effect of cortisol on either memory consolidation or retrieval. These discrepancies could be due to differences in the timing and sequencing of the experimental procedures or individual differences in participants' stress response. In the present study, we examined the effect of increased cortisol levels due to acute stress, induced by the Trier Social Stress Test (TSST), on a pattern separation memory task while differentiating the distinct stages of memory processing and controlling for the effects of diurnal variation. Sixty-nine young adults completed a 2-d study in which subjects either underwent the TSST immediately following the encoding part of the memory task, targeting memory consolidation, or immediately prior to the recognition part of the memory task on the second day, targeting memory retrieval. Control subjects completed the same study procedures but underwent a control version of the TSST that did not induce a stress response. Mnemonic discrimination of highly similar stimuli was enhanced by stress induced during consolidation with better discrimination showing a significant correlation with increased cortisol responses. Stress induced during memory retrieval showed no significant effect on memory performance. These findings suggest that stress induced changes in cortisol differentially affect the consolidation and retrieval stages of memory function.

Sex differences in hippocampal mineralocorticoid and glucocorticoid receptor mRNA expression in response to acute mate pair separation in zebra finches (Taeniopygia guttata).

Mate separation has been shown to mediate changes in physiological and behavioral processes via activation of the hypothalamo-pituitary-adrenal (HPA) axis in both mammalian and avian species. To elucidate the neural mechanisms associated with changes in the HPA axis in response to social stress, we investigated the effects of mate pair separation on circulating corticosterone concentrations as well as gene expression levels of mineralocorticoid receptor (MR), glucocorticoid receptor (GR), and corticotropin releasing hormone (CRH) in the hypothalamus and hippocampus of both male and female zebra finches, a species that forms strong pair bonds. Zebra finches (Taeniopygia guttata) were housed three to a cage (a mated pair plus a stimulus female), and were assigned to one of three new housing treatment groups: (1) male or female removed from their respective mate and placed in a cage with a new opposite sex conspecific and stimulus female (2) male or female that remained with their mate, but a new stimulus female was introduced, or (3) the subjects were handled but not separated from their mate or the stimulus female. After 48 hr in the new housing condition, we observed significant increases in plasma corticosterone concentrations in response to both mate pair and stimulus female separation. No significant differences in MR, GR, or CRH mRNA expression in the hypothalamus were observed in response to any treatment for both males and females. Females exhibited a significant up regulation in hippocampal MR, but not GR mRNA, whereas males exhibited a significant down regulation of both hippocampal MR and GR mRNA in response to mate pair separation. Thus, the hippocampus appears to play a key role in regulating sex specific responses to social stressors.

Estrogen-dependent modifications to hippocampal plasticity in paternal California mice (Peromyscus californicus).

In many biparental species, mothers and fathers experience similar modifications to circulating hormones. With these modifications come alterations in neural structure and function suggesting that neuroendocrine mechanisms may underlie postpartum plasticity in both males and females. In the biparental California mouse (Peromyscus californicus), adult neurogenesis is maintained and anxiety-like behavior is attenuated in fathers during the mid-postpartum period. Given a causal relationship between estrogen and regulation of both adult neurogenesis and anxiety, we aimed to elucidate the role of estrogen-dependent mechanisms in paternal experience-related modifications to hippocampal neuroplasticity in California mice. In Experiment 1, hippocampal estrogen receptor beta (ERß) mRNA expression, along with circulating estradiol concentrations, were determined throughout the postpartum period. An upregulation in ERß expression was observed in postnatal day 16 males compared to virgins. Additionally, a rise in circulating estradiol concentrations was detected on postnatal day 2 compared to virgins; levels began to decline toward virgin levels on postnatal day 16 and postnatal day 30. In Experiment 2, we determined the role of estrogen-dependent mechanisms in adult neurogenesis and anxiety-like behavior by treating virgin and paternal males with saline or the selective estrogen receptor modulator, tamoxifen (TMX), during the time of axon extension (i.e., one week after bromodeoxyuridine injection). While TMX failed to alter elevated plus maze performance, TMX treatment inhibited survival of adult born neurons but only in paternal mice. These findings highlight the potential for estrogen-dependent pathways to mediate hippocampal adult neurogenesis in paternal mice.

Perineuronal nets and vocal plasticity in songbirds: A proposed mechanism to explain the difference between closed-ended and open-ended learning.

Perineuronal nets (PNN) are aggregations of chondroitin sulfate proteoglycans surrounding the soma and proximal processes of neurons, mostly GABAergic interneurons expressing parvalbumin. They limit the plasticity of their afferent synaptic connections. In zebra finches PNN develop in an experience-dependent manner in the song control nuclei HVC and RA (nucleus robustus arcopallialis) when young birds crystallize their song. Because songbird species that are open-ended learners tend to recapitulate each year the different phases of song learning until their song crystallizes at the beginning of the breeding season, we tested whether seasonal changes in PNN expression would be found in the song control nuclei of a seasonally breeding species such as the European starling. Only minimal changes in PNN densities and total number of cells surrounded by PNN were detected. However, comparison of the density of PNN and of PNN surrounding parvalbumin-positive cells revealed that these structures are far less numerous in starlings that show extensive adult vocal plasticity, including learning of new songs throughout the year, than in the closed-ended learner zebra finches. Canaries that also display some vocal plasticity across season but were never formally shown to learn new songs in adulthood were intermediate in this respect. Together these data suggest that establishment of PNN around parvalbumin-positive neurons in song control nuclei has diverged during evolution to control the different learning capacities observed in songbird species. This differential expression of PNN in different songbird species could represent a key cellular mechanism mediating species variation between closed-ended and open-ended learning strategies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 975-994, 2017.

Pleiotropic Control by Testosterone of a Learned Vocal Behavior and Its Underlying Neuroplasticity(1,2,3).

Steroid hormones coordinate multiple aspects of behavior and physiology. The same hormone often regulates different aspects of a single behavior and its underlying neuroplasticity. This pleiotropic regulation of behavior and physiology is not well understood. Here, we investigated the orchestration by testosterone (T) of birdsong and its neural substrate, the song control system. Male canaries were castrated and received stereotaxic implants filled with T in select brain areas. Implanting T solely in the medial preoptic nucleus (POM) increased the motivation to sing, but did not enhance aspects of song quality such as acoustic structure and stereotypy. In birds implanted with T solely in HVC (proper name), a key sensorimotor region of the song control system, little or no song was observed, similar to castrates that received no T implants of any sort. However, implanting T in HVC and POM simultaneously rescued all measures of song quality. Song amplitude, though, was still lower than what was observed in birds receiving peripheral T treatment. T in POM enhanced HVC volume bilaterally, likely due to activity-dependent changes resulting from an enhanced song rate. T directly in HVC, without increasing song rate, enhanced HVC volume on the ipsilateral side only. T in HVC enhanced the incorporation and recruitment of new neurons into this nucleus, while singing activity can independently influence the incorporation of new neurons into HVC. These results have broad implications for how steroid hormones integrate across different brain regions to coordinate complex social behaviors.

Cocaine-induced sensitization correlates with testosterone in male Japanese quail but not with estradiol in female Japanese quail.

Research has indicated that gonadal hormones may mediate behavioral and biological responses to cocaine. Estrogen, in particular, has been shown to increase behavioral responding to cocaine in female rats relative to male rats. The current study investigated the effect of cocaine on locomotor activity and hormonal correlates in male and female Japanese quail (Coturnix japonica). In Japanese quail, circulating hormone levels can be manipulated without surgical alterations via modifying the photoperiod. Male and female quail were housed on either 8L:16D (light:dark) or 16L:8D (light:dark) cycle for 21days. Blood samples were taken prior to the beginning of the experiment and assays were performed to determine the levels of testosterone (T) and estradiol (E2). Quail were given injections of saline or cocaine (10 or 20mg/kg) once a day for 10days. Immediately after each injection, birds were placed in open field arenas and distance traveled was measured for 30min. Results showed that male quail housed under long-light conditions exhibited cocaine-induced sensitization to 10mg/kg cocaine which was correlated with the high levels of plasma T. Female quail housed under short-light conditions demonstrated sensitization to 10mg/kg cocaine, but this was not correlated with the levels of plasma E2. The current findings suggest that cocaine-induced locomotor activity was associated with T in males but not with E2 in females.

Reversing song behavior phenotype: Testosterone driven induction of singing and measures of song quality in adult male and female canaries (Serinus canaria).

In songbirds, such as canaries (Serinus canaria), the song control circuit has been shown to undergo a remarkable change in morphology in response to exogenous testosterone (T). It is also well established that HVC, a telencephalic nucleus involved in song production, is significantly larger in males than in females. T regulates seasonal changes in HVC volume in males, and exposure to exogenous T in adult females increases HVC volume and singing activity such that their song becomes more male-like in frequency and structure. However, whether there are sex differences in the ability of T to modulate changes in the song system and song behavior has not been investigated in canaries. In this study, we compared the effects of increasing doses of T on singing and song control nuclei volumes in adult male and female American Singer canaries exposed to identical environmental conditions. Males were castrated and all birds were placed on short days (8L:16D) for 8 weeks. Males and females were implanted either with a 2, 6 or 12 mm long Silastic™ implant filled with crystalline T or an empty 12 mm implant as control. Birds were then housed individually in sound-attenuated chambers. Brains were collected from six birds from each group after 1 week or 3 weeks of treatment. Testosterone was not equally effective in increasing singing activity in both males and females. Changes in song quality and occurrence rate took place after a shorter latency in males than in females; however, females did undergo marked changes in a number of measures of song behavior if given sufficient time. Males responded with an increase in HVC volume at all three doses. In females, T-induced changes in HVC volume only had limited amplitude and these volumes never reached male-typical levels, suggesting that there are sex differences in the neural substrate that responds to T.

Alarin stimulates food intake in male rats and LH secretion in castrated male rats.

Alarin is a newly identified member of the galanin family of neuropeptides that includes galanin-like peptide (GALP) and galanin. Alarin was discovered as an alternate transcript of the GALP gene in neuroblastoma cells, and subsequently alarin mRNA was detected in the brain of rodents. GALP and galanin are important central regulators of both feeding and reproductive behavior. We hypothesized, that, as a member of the galanin family of peptides, alarin would also have central effects on feeding and reproduction. To test this hypothesis, we treated male rats with alarin intracerebroventricularly (i.c.v.) and measured its effects on food intake and energy homeostasis as well as sexual behavior and luteinizing hormone (LH) secretion. We observed that i.c.v. injection of 1.0 nmol alarin significantly increased food intake (p<0.01) and body weight (p<0.05). Alarin did not affect sexual behavior in male rats; however, alarin did significantly (p<0.01) increase LH levels in castrated, but not intact, male rats. Alarin immunoreactive cell bodies were detected within the locus coeruleus and locus subcoeruleus of the midbrain, which is a brainstem nucleus involved in coordinating many physiological activities, including food intake and reproduction. Lastly, alarin stimulated Fos induction in hypothalamic nuclei, such as the paraventricular nucleus and the nucleus of the tractus solitarious. Our studies demonstrate that alarin, like other members of the galanin family, is a neuromediator of food intake and body weight.