Src-NADH dehydrogenase subunit 2 complex and recognition memory of imprinting in domestic chicks.

Src is a non-receptor tyrosine kinase participating in a range of neuronal processes, including synaptic plasticity. We have recently shown that the amounts of total Src and its two phosphorylated forms, at tyrosine-416 (activated) and tyrosine-527 (inhibited), undergoes time-dependent, region-specific learning-related changes in the domestic chick forebrain after visual imprinting. These changes occur in the intermediate medial mesopallium (IMM), a site of memory formation for visual imprinting, but not the posterior pole of the nidopallium (PPN), a control brain region not involved in imprinting. Src interacts with mitochondrial genome-coded NADH dehydrogenase subunit 2 (NADH2), a component of mitochondrial respiratory complex I. This interaction occurs at brain excitatory synapses bearing NMDA glutamate receptors. The involvement of Src-NADH2 complexes in learning and memory is not yet explored. We show for the first time that, independently of changes in total Src or total NADH2, NADH2 bound to Src immunoprecipitated from the P2 plasma membrane-mitochondrial fraction: (i) is increased in a learning-related manner in the left IMM 1 h after the end of training; (ii), is decreased in the right IMM in a learning-related way 24 h after training. These changes occurred in the IMM but not the PPN. They are attributable to learning occurring during training rather than a predisposition to learn. Learning-related changes in Src-bound NADH2 are thus time- and region-dependent.

Presence of sibling during the learning phase of imprinting affects escape behavior from a new object in chicks.

Imprinting is a crucial learning behavior by the hatchlings of precocious birds. In nature, hatchlings in a group environment imprint on a hen, but the effect of siblings on the imprinting process remains largely unknown. To investigate this issue, we examined how the social context modulated visual imprinting in domestic chicks. One-day-old postnatal chicks in isolation (RS chicks) or with siblings (RD chicks), were first exposed to an imprinting stimulus, and subsequently the responses to the imprinting stimulus as well as a new stimulus were examined and compared. The experiment constituted three types of siblings: a 20-min pre-trained tutor, a 60-min pre-trained tutor, and a naïve chick. A multiple comparison test revealed that the preference score (PS) to the new stimulus of RD chicks trained with a 60-min pre-trained tutor was significantly lower than that of RS chicks. Multiple linear regression analysis revealed that the length of the tutor's pre-training significantly correlated negatively with the PS to the new stimulus, but this variable did not correlate with the PS to the imprinting stimulus. These results revealed that the presence of highly imprinted siblings could enhance the escape response to the new stimulus. We discussed the possible involvement of the chick's medial amygdala in the social aspect of imprinting.

What can a worm learn in a bacteria-rich habitat?

With a nervous system that has only a few hundred neurons, Caenorhabditis elegans was initially not regarded as a model for studies on learning. However, the collective effort of the C. elegans field in the past several decades has shown that the worm displays plasticity in its behavioral response to a wide range of sensory cues in the environment. As a bacteria-feeding worm, C. elegans is highly adaptive to the bacteria enriched in its habitat, especially those that are pathogenic and pose a threat to survival. It uses several common forms of behavioral plasticity that last for different amounts of time, including imprinting and adult-stage associative learning, to modulate its interactions with pathogenic bacteria. Probing the molecular, cellular and circuit mechanisms underlying these forms of experience-dependent plasticity has identified signaling pathways and regulatory insights that are conserved in more complex animals.

Natal imprinting to the Earth's magnetic field in a pelagic seabird.

In migratory animals for whom post-natal care is limited, it is essential that there are inherited mechanisms whereby an individual can navigate-first, to the terminus of their migration, and second, back to a suitable breeding site. In birds, empirical evidence suggests that orientation on first migration is controlled by an inherited navigational vector, a direction and a distance in which to move (the "clock and compass" model) [1-5]. The mechanism and information that underlie the return to the natal breeding site are, however, almost entirely unknown. A potential solution to this problem would be for an animal to learn the values for spatially and temporally stable gradient cues that specifically indicate the location of the natal site [6-16]. One potential cue for latitude is magnetic inclination. Here, we use ringing recoveries made over the last 80 years to investigate whether magnetic inclination might be used as a navigational cue to control the latitude of recruitment in a trans-global migrant, the Manx shearwater (Puffinus puffinus). We find that small changes in inclination between when a bird fledges and when it returns from first migration correlate with probabilistic changes in latitude at recruitment, in doing so quantitatively fulfilling a priori predictions as to the magnitude and direction of latitudinal shift. This, we believe, suggests that (1) natal magnetic inclination is learnt prior to fledging and (2) is used to provide latitudinal information when making the first return trip from the wintering grounds.

Chicken cognin interactome and the memory of filial imprinting.

Visual imprinting is a learning process whereby young animals come to prefer a visual stimulus after exposure to it (training). The intermediate medial mesopallium in the domestic chick forebrain is critical for visual imprinting and contributes to molecular regulation of memory formation. Criteria used to infer that a change following training is learning-related have been formulated and published. Cognin (protein disulphide isomerase) is one of several identified plasma membrane and mitochondrial proteins that are upregulated in a learning-related way 24 hours after training. Since virtually nothing is known about the cognin interactome, we have used immunoaffinity chromatography and mass spectrometry to identify proteins that interact with cognin in the cytoplasmic and plasma membrane-mitochondrial fractions. As the learning-related upregulation of cognin has been shown to occur in the plasma membrane-mitochondrial fraction and not in the cytoplasmic fraction, we studied the effect of training on three cognin-interacting partners in the plasma membrane-mitochondrial fraction: the b5 subunit of mitochondrial ATP synthase and the alpha-2 and alpha-3 subunits of sodium-potassium ATPase. Learning-related upregulation was found in the left intermediate medial mesopallium 24 hours after training for the b5 subunit of mitochondrial ATP synthase and the alpha-2 subunit of sodium-potassium ATPase. The hemispheric asymmetry revealed here is consistent with the predominance of many other learning-related effects in the left intermediate medial mesopallium. The alpha-2 subunit of sodium-potassium ATPase is mainly expressed in astrocytes, supporting a role for these glial cells in memory.

Behavioral evidence for geomagnetic imprinting and transgenerational inheritance in fruit flies.

Certain long-distance migratory animals, such as salmon and sea turtles, are thought to imprint on the magnetic field of their natal area and to use this information to help them return as adults. Despite a growing body of indirect support for such imprinting, direct experimental evidence thereof remains elusive. Here, using the fruit fly as a magnetoreceptive model organism, we demonstrate that exposure to a specific geographic magnetic field during a critical period of early development affected responses to a matching magnetic field gradient later in life. Specifically, hungry flies that had imprinted on a specific magnetic field from 1 of 3 widely separated geographic locations responded to the imprinted field, but not other magnetic fields, by moving downward, a geotactic behavior associated with foraging. This same behavior occurred spontaneously in the progeny of the next generation: female progeny moved downward in response to the field on which their parents had imprinted, whereas male progeny did so only in the presence of these females. These results represent experimental evidence that organisms can learn and remember a magnetic field to which they were exposed during a critical period of development. Although the function of the behavior is not known, one possibility is that imprinting on the magnetic field of a natal area assists flies and their offspring in recognizing locations likely to be favorable for foraging and reproduction.

Role of sexual imprinting in assortative mating and premating isolation in Darwin's finches.

Global biodiversity is being degraded at an unprecedented rate, so it is important to preserve the potential for future speciation. Providing for the future requires understanding speciation as a contemporary ecological process. Phylogenetically young adaptive radiations are a good choice for detailed study because diversification is ongoing. A key question is how incipient species become reproductively isolated from each other. Barriers to gene exchange have been investigated experimentally in the laboratory and in the field, but little information exists from the quantitative study of mating patterns in nature. Although the degree to which genetic variation underlying mate-preference learning is unknown, we provide evidence that two species of Darwin's finches imprint on morphological cues of their parents and mate assortatively. Statistical evidence of presumed imprinting is stronger for sons than for daughters and is stronger for imprinting on fathers than on mothers. In combination, morphology and species-specific song learned from the father constitute a barrier to interbreeding. The barrier becomes stronger the more the species diverge morphologically and ecologically. It occasionally breaks down, and the species hybridize. Hybridization is most likely to happen when species are similar to each other in adaptive morphological traits, e.g., body size and beak size and shape. Hybridization can lead to the formation of a new species reproductively isolated from the parental species as a result of sexual imprinting. Conservation of sufficiently diverse natural habitat is needed to sustain a large sample of extant biota and preserve the potential for future speciation.

Wnt-2b in the intermediate hyperpallium apicale of the telencephalon is critical for the thyroid hormone-mediated opening of the sensitive period for filial imprinting in domestic chicks (Gallus gallus domesticus).

Filial imprinting is the behavior observed in chicks during the sensitive or critical period of the first 2-3 days after hatching; however, after this period they cannot be imprinted when raised in darkness. Our previous study showed that temporal augmentation of the endogenous thyroid hormone 3,5,3'-triiodothyronine (T3) in the telencephalon, by imprinting training, starts the sensitive period just after hatching. Intravenous injection of T3 enables imprinting of chicks on days 4 or 6 post-hatching, even when the sensitive period has ended. However, the molecular mechanism of how T3 acts as a determinant of the sensitive period is unknown. Here, we show that Wnt-2b mRNA level is increased in the T3-injected telencephalon of 4-day old chicks. Pharmacological inhibition of Wnt signaling in the intermediate hyperpallium apicale (IMHA), which is the caudal area of the telencephalon, blocked the recovery of the sensitive period following T3 injection. In addition, injection of recombinant Wnt-2b protein into the IMHA helped chicks recover the sensitive period without the injection of T3. Lastly, we showed Wnt signaling to be involved in imprinting via the IMHA region on day 1 during the sensitive period. These results indicate that Wnt signaling plays a critical role in the opening of the sensitive period downstream of T3.

Regulation of filial imprinting and structural plasticity by mTORC1 in newborn chickens.

Dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling leads to memory deficits and abnormal social behaviors in adults. However, whether mTORC1 is involved in critical periods of early learning remains largely unexplored. Our study addressed this question by investigating imprinting, a form of learning constrained to a sensitive period that supports filial attachment, in newborn chickens. Imprinting to virtual objects and sounds was assessed after acute manipulations of mTORC1. To further understand the role of mTORC1 during the critical period, structural plasticity was analyzed using DiOlistic labeling of dendritic spines. We found that mTORC1 is required for the emergence of experience-dependent preferences and structural plasticity within brain regions controlling behavior. Furthermore, upon critical period closure, pharmacological activation of the AKT/mTORC1 pathway was sufficient to rescue imprinting across sensory modalities. Thus, our results uncover a novel role of mTORC1 in the formation of imprinted memories and experience-dependent reorganization of neural circuits during a critical period.

Gene expression of Dio2 (thyroid hormone converting enzyme) in telencephalon is linked with predisposed biological motion preference in domestic chicks.

Filial imprinting leads to the formation of social attachment if training is performed during a brief sensitive period after hatching. We found that thyroid hormone (3,5,3'-triiodothyronine, T3) acts as a critical determining factor of the sensitive period in domestic chicks. Imprinting upregulates gene expression of the converting enzyme (Dio2, type 2 iodothyronine deiodinase) in the telencephalon, leading to increased brain T3 content. If systemically applied, T3 facilitates imprinting in aged chicks even after the sensitive period is over. Imprinting is also associated with the rapid development of visual perception. Exposure to motion pictures induces a predisposed preference to Johansson's biological motion (BM), and those individuals with higher BM preference are more easily imprinted. Here, we examined whether Dio2 expression is also linked with BM predisposition. Chicks were trained by a rotating red block, and tested for imprinting (experiment 1) and BM preference (experiment 2). To examine the time courses of behavioural and physiological processes, Dio2 expression in telencephalon was compared among three groups: naïve control chicks, and chicks trained for a short (0.5 h) or long period (2 h). In experiment 1, higher Dio2 expression appeared in the 2-h group than in the 0.5-h/control groups, but it was not correlated with the individual imprinting score. In experiment 2, a significant positive correlation appeared between Dio2 expression and BM preference in 2-h-trained chicks. Memory priming by T3 is therefore functionally linked to BM preference induction, leading to successful imprinting to natural objects even when they are initially exposed to artificial objects.

Evidence that Magnetic Navigation and Geomagnetic Imprinting Shape Spatial Genetic Variation in Sea Turtles.

The canonical drivers of population genetic structure, or spatial genetic variation, are isolation by distance and isolation by environment. Isolation by distance predicts that neighboring populations will be genetically similar and geographically distant populations will be genetically distinct [1]. Numerous examples also exist of isolation by environment, a phenomenon in which populations that inhabit similar environments (e.g., same elevation, temperature, or vegetation) are genetically similar even if they are distant, whereas populations that inhabit different environments are genetically distinct even when geographically close [2-4]. These dual models provide a widely accepted conceptual framework for understanding population structure [5-8]. Here, we present evidence for an additional, novel process that we call isolation by navigation, in which the navigational mechanism used by a long-distance migrant influences population structure independently of isolation by either distance or environment. Specifically, we investigated the population structure of loggerhead sea turtles (Caretta caretta) [9], which return to nest on their natal beaches by seeking out unique magnetic signatures along the coast-a behavior known as geomagnetic imprinting [10-12]. Results reveal that spatial variation in Earth's magnetic field strongly predicts genetic differentiation between nesting beaches, even when environmental similarities and geographic proximity are taken into account. The findings provide genetic corroboration of geomagnetic imprinting [10, 13]. Moreover, they provide strong evidence that geomagnetic imprinting and magnetic navigation help shape the population structure of sea turtles and perhaps numerous other long-distance migrants that return to their natal areas to reproduce [13-17].

Early social experience alters transcriptomic responses to species-specific song stimuli in female songbirds.

Amongst an array of stimuli from countless species, animals must recognize salient signals, including those of their own species. In songbirds, behavioral tests have demonstrated that preferences for conspecific male songs are determined by both preexisting biases and social experience with a male 'tutor' during the sensitive period for learning. Although immediate early gene expression (e.g. ZENK) and electrophysiological experiments generally find greater neural responses for conspecific songs, it remains unclear whether distinct mechanisms, such as sensory gating, are engaged to filter out irrelevant heterospecific songs. Here we compare the transcriptomic profiles, via RNA-seq, of non-singing females of a songbird, the zebra finch (Taeniopygia guttata), by focusing on the auditory forebrain, a region known to be critical in the processing of conspecific vs. heterospecific songs. Gene expression profiles demonstrate that different neural mechanisms are involved in the processing of conspecific versus heterospecific Bengalese finch (Lonchura striata) songs. In particular, one gene known to mediate sensory gating, the alpha 3 subunit member of nicotinic cholinergic receptors (CHRNA3), was significantly downregulated in response to hearing Bengalese finch song, but not when young females were tutored by a Bengalese male during early development. Overall, our results confirm previous behavioral and physiological studies, such that heterospecific-tutored individuals processed both conspecific and tutor songs similarly. Using transcriptomic profiling of peripheral blood samples, we also demonstrate the methodological potential of non-terminal sampling to identify transcriptomic biomarkers for conspecific auditory recognition. These results show how experience and inherited preferences facilitate the neural processing of salient songs by female songbirds.

Mitochondrial fusion and fission proteins and the recognition memory of imprinting in domestic chicks.

Visual imprinting is a learning process through which young, visually naive animals come to recognize a visual stimulus by being exposed to it (training) and subsequently approach the stimulus in preference to others. A large body of evidence indicates that a restricted part of the forebrain, the intermediate medial mesopallium (IMM), is a memory region for visual imprinting in the domestic chick. Previous studies have shown learning-related up-regulation of several mitochondrial proteins in the IMM 24 h after training. Learning-related increases in transcription factors involved in mitochondrial biogenesis were found without significant change in mitochondrial DNA copy number, but the issue of whether mitochondrial fusion or fission processes change with learning was unresolved. The present study enquired whether proteins involved in mitochondrial fusion and fission contribute to memory following imprinting. Tissue was sampled from the left and right IMM, and the left and right posterior pole of the nidopallium (a control brain region not involved in imprinting). The amounts of the following proteins were measured by Western immunoblotting 24 h after training: mitochondrial mitofusin-1 (MTF-1, as indicator of mitochondrial fusion), membrane dynamin-related protein-1 (DRP-1, as indicator of mitochondrial fission) and cytoplasmic DRP-1. Learning-related increases in MTF-1 and DRP-1 were observed bilaterally in the IMM, but not in either side of the posterior pole of the nidopallium. Cytoplasmic DRP-1 was not changed significantly in any region studied. The results implicate increased, balanced levels of mitochondrial fusion and fission in memory formation up to 24 h after training.Supplementary Video Abstract (Supplemental digital content 1, http://links.lww.com/WNR/A446).

Feeding Imprinting: The Extreme Test Case of Premature Infants Born With Very Low Birth Weight.

Feeding imprinting, considered a survival-enabling process, is not well understood. Infants born very preterm, who first feed passively, are an effective model for studying feeding imprinting. Retrospective analysis of neonatal intensive care unit (NICU) records of 255 infants (Mgestational age  = 29.98 ± 1.64) enabled exploring the notion that direct breastfeeding (DBF) during NICU stay leads to consumption of more mother's milk and earlier NICU discharge. Results showed that DBF before the first bottle feeding is related to shorter transition into oral feeding, a younger age of full oral feeding accomplishment and earlier discharge. Furthermore, the number of DBF meals before first bottle feeding predicts more maternal milk consumption and improved NICU outcomes. Improved performance in response to initial exposure to DBF at the age of budding feeding abilities supports a feeding imprinting hypothesis.

Spontaneous generalization of abstract multimodal patterns in young domestic chicks.

From the early stages of life, learning the regularities associated with specific objects is crucial for making sense of experiences. Through filial imprinting, young precocial birds quickly learn the features of their social partners by mere exposure. It is not clear though to what extent chicks can extract abstract patterns of the visual and acoustic stimuli present in the imprinting object, and how they combine them. To investigate this issue, we exposed chicks (Gallus gallus) to three days of visual and acoustic imprinting, using either patterns with two identical items or patterns with two different items, presented visually, acoustically or in both modalities. Next, chicks were given a choice between the familiar and the unfamiliar pattern, present in either the multimodal, visual or acoustic modality. The responses to the novel stimuli were affected by their imprinting experience, and the effect was stronger for chicks imprinted with multimodal patterns than for the other groups. Interestingly, males and females adopted a different strategy, with males more attracted by unfamiliar patterns and females more attracted by familiar patterns. Our data show that chicks can generalize abstract patterns by mere exposure through filial imprinting and that multimodal stimulation is more effective than unimodal stimulation for pattern learning.

Regulation of visual Wulst cell responsiveness by imprinting causes stimulus-specific activation of rostral cells.

Imprinting behaviour in chicks can be induced exclusively during a short period after hatching. During this period, visual information on the imprinting stimulus is conveyed to the visual Wulst (VW) in the telencephalon, which corresponds to the visual cortex of mammals, and then to the memory-storing region known as the intermediate medial mesopallium. These two regions are indispensable for imprinting. We previously showed that imprinting training altered the response pattern of the VW to the imprinting stimulus; however, the precise distribution of cells and the mechanism involved with this altered response remains unclear. Here we showed that a specific population of rostral VW cells responded to the imprinting stimulus by analysing the subcellular localization of Arc/arg3.1 transcripts in VW cells. GABAergic parvalbumin (PV) cells are abundant in the dorsal region of this area, and imprinting training doubled the number of activated PV-positive neurons. An injection of bicuculline, a GABA(A) receptor antagonist, in the dorsal VW disturbed the rostral distribution of responsive cells and thus resulted in a lack of imprinting. These results suggest that activated PV cells restrict VW cells response to dorsal area to form a specific imprinting pathway.

Enhanced sensitivity to drugs of abuse and palatable foods following maternal overnutrition.

Epidemiological studies have shown an association between maternal overnutrition and increased risk of the progeny for the development of obesity as well as psychiatric disorders. Animal studies have shown results regarding maternal high-fat diet (HFD) and a greater risk of the offspring to develop obesity. However, it still remains unknown whether maternal HFD can program the central reward system in such a way that it will imprint long-term changes that will predispose the offspring to addictive-like behaviors that may lead to obesity. We exposed female dams to either laboratory chow or HFD for a period of 9 weeks: 3 weeks before conception, during gestation and lactation. Offspring born to either control or HFD-exposed dams were examined in behavioral, neurochemical, neuroanatomical, metabolic and positron emission tomography (PET) scan tests. Our results demonstrate that HFD offspring compared with controls consume more alcohol, exhibit increased sensitivity to amphetamine and show greater conditioned place preference to cocaine. In addition, maternal HFD leads to increased preference to sucrose as well as to HFD while leaving the general feeding behavior intact. The hedonic behavioral alterations are accompanied by reduction of striatal dopamine and by increased dopamine 2 receptors in the same brain region as evaluated by post-mortem neurochemical, immunohistochemical as well as PET analyses. Taken together, our data suggest that maternal overnutrition predisposes the offspring to develop hedonic-like behaviors to both drugs of abuse as well as palatable foods and that these types of behaviors may share common neuronal underlying mechanisms that can lead to obesity.

Social predisposition dependent neuronal activity in the intermediate medial mesopallium of domestic chicks (Gallus gallus domesticus).

Species from phylogenetically distant animal groups, such as birds and primates including humans, share early experience-independent social predispositions that cause offspring, soon after birth, to attend to and learn about conspecifics. One example of this phenomenon is provided by the behaviour of newly-hatched visually-naïve domestic chicks that preferentially approach a stimulus resembling a conspecific (a stuffed fowl) rather than a less naturalistic object (a scrambled version of the stuffed fowl). However, the neuronal mechanisms underlying this behaviour are mostly unknown. Here we analysed chicks' brain activity with immunohistochemical detection of the transcription factor c-Fos. In a spontaneous choice test we confirmed a significant preference for approaching the stuffed fowl over a texture fowl (a fowl that was cut in small pieces attached to the sides of a box in scrambled order). Comparison of brain activation of a subgroup of chicks that approached either one or the other stimulus revealed differential activation in an area relevant for imprinting (IMM, intermediate medial mesopallium), suggesting that a different level of plasticity is associated with approach to naturalistic and artificial stimuli. c-Fos immunoreactive neurons were present also in the intermediate layers of the optic tectum (a plausible candidate for processing early social predispositions) showing a trend similar to the results for the IMM.

Imprinting: When Early Life Memories Make Food Smell Bad.

A recent study has found that pathogen exposure early in the life of the nematode Caenorhabditis elegans leads to a long-lasting aversion that requires distinct sets of neurons for the formation and retrieval of the imprinted memory.