Developmental-Based Classification of Enkephalin and Somatostatin Containing Neurons of the Chicken Central Extended Amygdala

The central extended amygdala, including the lateral bed nucleus of the stria terminalis and the central amygdala, plays a key role in stress response. To understand how the central extended amygdala regulates stress it is essential to dissect this structure at molecular, cellular and circuit levels. In mammals, the central amygdala contains two distinct cell populations that become active (on cells) or inactive (off cells) during the conditioned fear response. These two cell types inhibit each other and project mainly unidirectionally to output cells, thus providing a sophisticated regulation of stress. These two cell types express either protein kinase C-delta/enkephalin or somatostatin, and were suggested to originate in different embryonic domains of the subpallium that respectively express the transcription factors Pax6 or Nkx2.1 during development. The regulation of the stress response by the central extended amygdala is poorly studied in non-mammals. Using an evolutionary developmental neurobiology approach, we previously identified several subdivisions in the central extended amygdala of chicken. These contain Pax6, Islet1 and Nkx2.1 cells that originate in dorsal striatal, ventral striatal or pallidopreoptic embryonic divisions, and also contain neurons expressing enkephalin and somatostatin. To know the origin of these cells, in this study we carried out multiple fluorescent labeling to analyze coexpression of different transcription factors with enkephalin or somatostatin. We found that many enkephalin cells coexpress Pax6 and likely derive from the dorsal striatal division, resembling the off cells of the mouse central amygdala. In contrast, most somatostatin cells coexpress Nkx2.1 and derive from the pallidal division, resembling the on cells. We also found coexpression of enkephalin and somatostatin with other transcription factors. Our results show the existence of multiple cell types in the central extended amygdala of chicken, perhaps including on/off cell systems, and set the basis for studying the role of these cells in stress regulation.Funded by grants from the Spanish Ministerio de Ciencia e Innovación (Agencia Estatal de Investigación, Grant no. PID2019-108725RB-100) and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 812777 (H2020-MSCA-ITN-2018-812777). AHM and AP have contracts as Early-Stage Researchers paid by the H2020- MSCA-ITN-2018-812777 project. The authors declare that the funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit It for publication

Distinct Subdivisions in the Transition Between Telencephalon and Hypothalamus Produce Otp and Sim1 Cells for the Extended Amygdala in Sauropsids

Based on the coexpression of the transcription factors Foxg1 and Otp, we recently identified in the mouse a new radial embryonic division named the telencephalon-opto-hypothalamic (TOH) domain that produces the vast majority of glutamatergic neurons found in the medial extended amygdala. To know whether a similar division exists in other amniotes, we carried out double labeling of Foxg1 and Otp in embryonic brain sections of two species of sauropsids, the domestic chicken (Gallus gallus domesticus), and the long-tailed lacertid lizard (Psammodromus algirus). Since in mice Otp overlaps with the transcription factor Sim1, we also analyzed the coexpression of Foxg1 and Sim1 and compared it to the glutamatergic cell marker VGLUT2. Our results showed that the TOH domain is also present in sauropsids and produces subpopulations of Otp/Foxg1 and Sim1/Foxg1 cells for the medial extended amygdala. In addition, we found Sim1/Foxg1 cells that invade the central extended amygdala, and other Otp and Sim1 cells not coexpressing Foxg1 that invade the extended and the pallial amygdala. These different Otp and Sim1 cell subpopulations, with or without Foxg1, are likely glutamatergic. Our results highlight the complex divisional organization of telencephalon-hypothalamic transition, which contributes to the heterogeneity of amygdalar cells. In addition, our results open new venues to study further the amygdalar cells derived from different divisions around this transition zone and their relationship to other cells derived from the pallium or the subpallium.This study was funded by grants from the Spanish Ministerio de Ciencia e Innovación (Agencia Estatal de Investigación, Grant No: PID2019-108725RB-100) and the European Union’s Horizon 2020 Research and Innovation Programme under Marie Skłodowska-Curie grant agreement no. 812777 (H2020-MSCA-ITN-2018-812777). AM and AP have contracts as early-stage researchers paid by the H2020-MSCA-ITN-2018-812777 project. JF had a training fellowship for undergraduate students from the Spanish Ministry of Education, Culture and Sports. The authors declare that the funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication

Embryonic Exposure to Valproic Acid Affects Social Predispositions for Dynamic Cues of Animate Motion in Newly-Hatched Chicks

Early predispositions to preferentially orient towards cues associated with social partners have been documented in several vertebrate species including human neonates and domestic chicks. Human newborns at high familiar risk of Autism Spectrum Disorder (ASD) show differences in their attention toward these predisposed stimuli, suggesting potential impairments in these social-orienting mechanisms in ASD. Using embryonic exposure to valproic acid (VPA) we modelled ASD behavioural deficits in domestic chicks. To investigate social predispositions towards animate motion in domestic chicks, we focused on self-propulsion, using two video-animations representing a simple red circle moving at constant speed (speed-constant) or one that was changing its speed (accelerating and decelerating; speed-change). Using a six minutes spontaneous choice test for the two stimuli, we compared unlearned preferences for stimuli that autonomously change speed between VPA- and vehicle-injected chicks. We found that the preference for speed changes was abolished in VPA-injected chicks compared to vehicle-injected controls. These results add to previous findings indicating similar impairments for static social stimuli and suggest a specific effect of VPA on the development of mechanisms that enhance orienting towards animate stimuli. These findings strengthen the hypothesis of an early impairment of predispositions in the early development of ASD. Hence, early predispositions are a potentially useful tool to detect early ASD symptoms in human neonates and to investigate the molecular and neurobiological mechanisms underlying the onset of this neurodevelopmental disorder

Amygdalar neurons regulating the stress response in chicken

Els trastorns causats per l'estrès són un greu problema per als animals de granja, ja que repercuteixen negativament tant en el seu benestar, com en la productivitat de les granges. En mamífers, l'amígdala central estesa (EAce, que inclou l'amígdala central i el nucli del llit lateral de l'estria terminal o BSTL) exerceix un paper clau en la regulació de l'estrès, en controlar els aspectes fisiològics (neuroendocrins, autonòmics) i conductuals de la resposta d'estrès. En aus, la regulació de la resposta a l'estrès per EAce a penes es coneix, en part a causa de la dificultat d'identificar una "amígdala central" per la divergència evolutiva del telencèfal. Aquest problema va ser abordat pel nostre grup utilitzant l'enfocament de la biologia evolutiva del desenvolupament, que ens va portar a proposar diverses subdivisions i tipus cel·lulars de l'amígdala central estesa d’aus. En aquesta tesi, hem volgut aprofundir en l'anàlisi de l’EAce del pollastre estudiant: (1) la diversitat de tipus cel·lulars en funció del seu diferent origen embrionari; (2) les interconnexions entre les seves diferents subdivisions, mitjançant experiments de traçat de connexions; i (3) la implicació de les diferents subdivisions en la regulació de l'estrès, estudiant l'expressió de c-FOS mitjançant anàlisi de qPCR després d'induir estrès agut, utilitzant un paradigma d'aïllament social. Quant al primer objectiu, hem estudiat tres poblacions neuropeptidèrgiques, que expressen encefalina (ENK), somatostatina (SST) o factor alliberador de corticotropina (CRF). Moltes cèl·lules ENK coexpressen Pax6 i probablement s'originen en la divisió embrionària estriatal dorsal, les cèl·lules SST expressen principalment el factor de transcripció Nkx2.1 i tenen origen palidopreòptic, mentre que les cèl·lules CRF inclouen dos subtipus sobre la base de la coexpressió amb Pax6 (origen estriatal dorsal) o Islet1 (origen estriatal ventral). Aquestes cèl·lules són presents tant en el BSTL com en les parts laterals de la EAce del pollastre, globalment comparable a l'amígdala central d'altres amniotes. Les cèl·lules SST i ENK de l'amígdala central del pollastre semblen comparables a les cèl·lules "on" i "off" de l'amígdala central del ratolí, que s'activen o desactiven durant l'expressió de la por condicionada. Les cèl·lules CRF s'assemblen a les trobades en l'amígdala central i el BSTL de mamífers, relacionades amb les respostes a l'estrès crònic. Els nostres resultats de connectivitat van demostrar projeccions des de diferents subdivisions de l'amígdala central de pollastre al BSTL, que inclouen vies directes i indirectes, que probablement impliquen cèl·lules ENK i CRF. Finalment, els nostres resultats van mostrar que, com ocorre en mamífers, el BSTL i una part lateral de l'amígdala central de pollastre s'activen en l'aïllament social agut. Els nostres resultats donen suport a que las EAce de pollastre i ratolí tenen una organització i funció similars, obrint noves vies per a futures recerques sobre com les diferents cèl·lules i circuits regulen l'estrès.Los trastornos causados por el estrés son un grave problema para los animales de granja, ya que repercuten negativamente tanto en su bienestar, como en la productividad de las granjas. En mamíferos, la amígdala central extendida (EAce, que incluye la amígdala central y el núcleo del lecho lateral de la estría terminal o BSTL) desempeña un papel clave en la regulación del estrés, al controlar los aspectos fisiológicos (neuroendocrinos, autonómicos) y conductuales de la respuesta de estrés. En aves, la regulación de la respuesta al estrés por EAce apenas se conoce, en parte debido a la dificultad de identificar una "amígdala central" por la divergencia evolutiva del telencéfalo. Este problema fue abordado por nuestro grupo utilizando el enfoque de la biología evolutiva del desarrollo, que nos llevó a proponer varias subdivisiones y tipos celulares de la amígdala central extendida de aves. En esta tesis, hemos querido profundizar en el análisis de la EAce del pollo estudiando: (1) la diversidad de tipos celulares en función de su diferente origen embrionario; (2) las interconexiones entre sus diferentes subdivisiones, mediante experimentos de trazado de conexiones; y (3) la implicación de las diferentes subdivisiones en la regulación del estrés, estudiando la expresión de c-FOS mediante análisis de qPCR tras inducir estrés agudo, utilizando un paradigma de aislamiento social. En cuanto al primer objetivo, estudiamos tres poblaciones neuropeptidérgicas, que expresan encefalina (ENK), somatostatina (SST) o factor liberador de corticotropina (CRF). Muchas células ENK coexpresan Pax6 y probablemente se originan en la división embrionaria estriatal dorsal, las células SST expresan principalmente el factor de transcripción Nkx2.1 y tienen origen palidopreóptico, mientras que las células CRF incluyen dos subtipos en base a su coexpresión con Pax6 (origen estriatal dorsal) o Islet1 (origen estriatal ventral). Estas células están presentes tanto en el BSTL como en las partes laterales de la EAce del pollo, globalmente comparable a la amígdala central de otros amniotas. Las células SST y ENK de la amígdala central del pollo parecen comparables a las células "on" y "off" de la amígdala central del ratón, que se activan o desactivan durante el expresion del miedo condicionado. Las células CRF se asemejan a las encontradas en la amígdala central y el BSTL de mamíferos, relacionadas con las respuestas al estrés crónico. Nuestros resultados de conectividad demostraron proyecciones desde diferentes subdivisiones de la amígdala central de pollo al BSTL, que incluyen vías directas e indirectas, que probablemente implican células ENK y CRF. Finalmente, nuestros resultados mostraron que, como ocurre en mamíferos, el BSTL y una parte lateral de la amígdala central de pollo se activan en el aislamiento social agudo. Nuestros resultados apoyan que las EAce de pollo y ratón tienen una organización y función similares, abriendo nuevas vías para futuras investigaciones sobre cómo las diferentes células y circuitos regulan el estrés.Stress disorders are a major problem for farm animals, having a negative impact in both animals’ welfare and farm production. In mammals, the central extended amygdala (EAce, including central amygdala and lateral bed nucleus of the stria terminalis or BSTL), plays a key role in stress regulation, by controlling the physiological (neuroendocrine, autonomic) and behavioral aspects of the stress response. The regulation of the stress response by EAce is poorly understood in birds, in part because of problems in the identification of a 'central amygdala' due to the great evolutionary divergence of the telencephalon. This problem was previously addressed by our group using an evolutionary developmental biology approach, resulting in a proposal of several subdivisions and cell types of the avian central extended amygdala. In this thesis, we wanted to deepen in the analysis of the chicken EAce by studying: (1) the diversity of cell types based on their different developmental origin; (2) the interconnections between its different subdivisions, by way of tract-tracing experiments; and (3) the implication of the different subdivisions in stress regulation, studying c-FOS expression by qPCR analysis after inducing acute stress, using a social isolation paradigm. Regarding the first aim, we focused on three major neuropeptidergic populations, expressing enkephalin (ENK), somatostatin (SST) or corticotropin-releasing factor (CRF). We found that many ENK cells coexpress Pax6 and likely originate in the dorsal striatal embryonic division, SST cells mainly express the transcription factor Nkx2.1 and have pallidopreoptic origin, while CRF cells include two subtypes based on their coexpression with Pax6 (dorsal striatal origin) or Islet1 (ventral striatal origin). These different cells are present in both BSTL and lateral parts of chicken EAce, globally comparable to the central amygdala of other amniotes. SST and ENK cells of the chicken central amygdala appear comparable to the 'on' and 'off' cells of the mouse central amygdala, which become active or inactive during conditioned fear expression. CRF cells resemble those found in the central amygdala and BSTL of mammals, related to chronic stress responses. Our connectivity results showed complex projections from different subdivisions of the chicken central amygdala to BSTL, which include direct and indirect pathways likely involving ENK and CRF cells that may lead to inhibition or disinhibition of BSTL outputs. Finally, our results showed that chicken BSTL and a lateral part of the chicken central amygdala become active during acute social isolation, resembling results in mammals. Overall, we provide evidence for similar organization and function of chicken and mouse EAce, opening new venues for further investigations on how different cells and circuits regulate stress