Wayne J. Kuenzel

1974). Upon completing a PhD at UGA, my major professor encouraged me to pursue an academic career. I still remember his kind words.An important obligation remained. I was in ROTC at Bucknell University. I therefore entered the U.S. Army for two years and was sent to the Medical Service Corps (MSC), Ft. Sam Houston, TX. I and others served as military instructors and taught human anatomy and physiology to soldiers. Soldiers remaining at Ft. Sam Houston would be trained as combat medics. During the second year at the MSC, at Ft. Sam, I was asked to serve as Chief, Basic Science Branch and was promoted from first lieutenant to captain. Thereafter, I obtained a National Institutes of Health (NIH) Postdoctoral Fellowship and spent three years at Cornell University experimenting with chickens under the direction of the neuroendocrine expert, Dr. Ari van Tienhoven. Additionally, the positive experience in the laboratory of Dr. Ari van Tienhoven, encouraged me to apply for a faculty position and was fortunate to spend half of my career (26 years) in the Poultry Science (POSC) Department (Dept.) at the University of Maryland, College Park, MD and am currently working in the POSC Dept. at the University of Arkansas, Fayetteville that began in 2000.Academic Roles at the Univ. of Maryland (UMD) and Univ. of Arkansas, Fayetteville (UAF).At both the UMD and UAF, I was hired primarily to (a) produce original research and (b) teach courses (three courses at UMD, two courses at UAF). Additionally, a third role was to serve on committees, from international ones down to university committees. The order of importance for the 3 roles at both UMD and UAF was to build a reputation in a particular research area, followed by teaching and service on committees. In research, I have focused on the avian brain, specifically its anatomy and physiology. What was so valuable at both institutions was the opportunity to teach courses in the disciplines that were relevant to my research interests. The major academic discipline I wished to address was an overall knowledge of avian neuroendocrine physiology. The specific research area was to develop knowledge of neural structures involved with the anterior pituitary gland. This would include contributing new, anatomical and functional knowledge of the regulation of specific neuroendocrine processes. At both UMD and UAF, I have contributed new knowledge to three avian neuroendocrine areas: 1) the location of structures regulating the reproductive system, 2) stress pathways and 3) seasonal regulation of food intake.Value of (1) Academic Support at UMD/UAF, (2) Two Group Meetings, (3) Sabbatical Years, (4) Funding.There were four processes/events experienced that enabled me to contribute a few advancements associated with avian brain function: 1) Academic structure and support at UMD and UAF provided support for a technical assistant person, 2) two group meetings that focused on avian neuroanatomy, 3) sabbatical leaves were encouraged by UMD and UAF, 4) both universities have an Agricultural Experiment Station that has an annual, competitive grants program providing funding for research programs involving crops, and/or agricultural animals designed to improve food production or agribusiness. Success in this program has provided opportunities for faculty to be successful in national grants programs due to the successful data collection from a previous experiment station grant. Funding I received was also used to support presentations at scientific meetings and publications in journals. Additionally, one objective throughout my career was to train graduate students to become knowledgeable and comfortable in pursuing a specific research area and develop an ability to communicate their research results not only to specific scientific audiences, but also to their local public community when appropriate.Note that the four processes/events emphasized previously were not sequential. In fact, all four were interactive throughout my career. In my chosen research field, avian neuroanatomy, I have always been intrigued by determining the specific location of structures within the brain, finding their appropriate name and abbreviation, their product and receptors, and critical tests to complete for demonstrating their specific function/s.During my career, I was invited to join two groups of scientists. There was a significant overlap of specific individuals in different named groups. The first one was the Thinktank, organized by Dr. Anton (Tony) Reiner at the University of Tennessee Health Science Center, Memphis, TN. Years later, the Nomenclature Forum was initiated by Dr. Eric Jarvis, Howard Hughes Medical Institute, The Rockefeller University, NY, NY; the latter was supported by NSF and NIH. Both groups addressed critical issues in the avian literature. A persistent use of inappropriate terms for brain structures had occurred, based upon outdated assumptions of homology to mammalian structures, particularly in the forebrain that needed to be changed. I have kept in touch with Dr. Reiner and Dr. Jarvis over the years with issues that have surfaced regarding the use of appropriate terms and acronyms for avian neural structures.In 1988 I published a book with Manju Masson titled 'A Stereotaxic Atlas of the Brain of the Chick, Gallus domesticus' that showed the accurate location, name and its abbreviation (acronym) for all known neural structures throughout the brain of a chick. It was published by The Johns Hopkins University Press (Kuenzel and Masson, 1988). It was the first stereotaxic atlas of an avian brain displaying complete sets of images in three planes: coronal, sagittal and horizontal sections. A total of 500 copies of the book were produced by The Johns Hopkins University Press (TJHUP). Two or three years ago I received a letter from TJHUP that they released all rights to me and Manju for that book. I therefore copied the book and placed it on my website with the...

The location of corticotropin-releasing hormone receptor 2 (CRH-R2) on thyrotropes within the avian anterior pituitary (APit) and its activation by different stressors indicate a possible communication between hypothalamo-pituitary-adrenal (HPA) and thyroid (HPT) axes. Therefore, an experiment was designed to 1) compare the timing of major components of the HPT axis to those of the HPA axis; 2) address whether stressors activating the HPA axis may simultaneously upregulate components of the HPT axis. Blood, brain, and APit were sampled from chicks prior to stress (control) and 15, 30, 60, 90, and 120 min following immobilization (IM) stress. The nucleus of the hippocampal commissure (NHpC) and paraventricular nucleus (PVN) were cryo-dissected from brains for RT-qPCR. Gene expression of thyrotropin-releasing hormone (TRH) and its receptors (TRH-R1 and TRH-R3), urocortin3 (UCN3), deiodinase 2 (D2), and the second type of corticotropin-releasing hormone (CRH2) within the NHpC and PVN was measured. Additionally, gene expression of TRH receptors, thyroid stimulating hormone subunit beta (TSHβ), and D2 was determined in the APit and corticosterone assayed in blood. In brains, a significant upregulation in examined genes occurred at different times of IM. Specifically, UCN3 and CRH2 which have a high affinity to CRH-R2 showed a rapid increase in their mRNA levels that were accompanied by an early upregulation of TRHR1 in the NHpC. In the APit, a significant increase in gene expression of TSHβ and TRH receptors was observed. Therefore, results supported concurrent activation of major brain and APit genes associated with the HPA and HPT axes following IM. The initial neural gene expression originating within the NHpC resulted in the increase of TSHβ mRNA in the APit. Specifically, the rapid upregulation of UCN3 in the NHpC appeared responsible for the early activation of TSHβ in the APit. While sustaining TSHβ activation appeared to be due to both CRH2 and TRH. Therefore, data indicate that CRH-producing neurons and corticotropes as well as CRH- and TRH-producing neurons and thyrotropes are activated to produce the necessary energy required to maintain homeostasis in birds undergoing stress. Overall, data support the inclusion of the NHpC in the classical avian HPA axis and for the first time show the concurrent activation of the HPA axis and components of the HPT axis following a psychogenic stressor.

Corticotropin releasing hormone (CRH) neurons located in the nucleus of hippocampal commissure (NHpC) have been proposed to be involved in the avian neuroendocrine regulation of stress and appeared to respond prior to CRH neurons in the hypothalamic paraventricular nucleus (PVN) when food deprivation stress was applied. Since the response of the NHpC was rapid and short-lived, was it regulated differentially from CRH neurons in the PVN? We, therefore, applied immobilization stress to test whether the NHpC response was stressor specific. Gene expression of CRH and stress-related genes in the NHpC, PVN, anterior pituitary (APit) as well as plasma corticosterone (CORT) were determined. Furthermore, brain derived neurotrophic factor (BDNF) and glucocorticoid receptor (GR) were examined regarding their possible roles in the regulation of CRH neurons. Data showed that rapid activation of CRH mRNA in the NHpC occurred and preceded a slower gene activation in the PVN, upregulation of proopiomelanocortin (POMC) transcripts in the APit and significant increases of CORT concentrations. Results suggested BDNF’s role in negative feedback between CRH and CRHR1 in the NHpC and positive feedback between CRH and CRHR1 in the PVN. In the APit, V1bR activation appeared responsible for sustaining CORT release when stress persisted. Overall, data suggest that the NHpC functions as part of the HPA axis of birds and perhaps a comparable extra-hypothalamic structure occurs in other vertebrates.

Mitigation of stress is of great importance in poultry production, as chronic stress can affect the efficiency of production traits. Selective breeding with a focus on stress responses can be used to combat the effects of stress. To better understand the genetic mechanisms driving differences in stress responses of a selectively bred population of Japanese quail, we performed genomic resequencing on 24 birds from High Stress (HS) and Low Stress (LS) lines of Japanese quail using Illumina HiSeq 2 × 150 bp paired end read technology in order to analyze Single Nucleotide Polymorphisms (SNPs) within the genome of each line. SNPs are common mutations that can lead to genotypic and phenotypic variations in animals. Following alignment of the sequencing data to the quail genome, 6,364,907 SNPs were found across both lines of quail. 10,364 of these SNPs occurred in coding regions, from which 2886 unique, non-synonymous SNPs with a SNP% ≥ 0.90 and a read depth ≥ 10 were identified. Using Ingenuity Pathway Analysis, we identified genes affected by SNPs in pathways tied to immune responses, DNA repair, and neurological signaling. Our findings support the idea that the SNPs found within HS and LS lines of quail could direct the observed changes in phenotype.

Vasotocin 1a and 1b receptors (V1aR and V1bR) have been shown to play important roles in the neuroendocrine regulation of stress responses via the anterior pituitary (AP) of birds. To identify effective subtype-specific antagonists for the chicken V1aR (cV1aR) and cV1bR, potential antagonists to the mammalian V1R were screened against the cV1aR and cV1bR 3D structural models by molecular docking analysis with determination of binding pocket/amino acid residues involved in the interaction. The antagonistic effects of the selected ligands were examined by measuring pro-opiomelanocortin (POMC) heteronuclear RNA (hnPOMC) levels following the in vitro stress administration to primary chicken AP cells. Results of in silico analysis showed that the Manning compound and several other antagonists were bound to cV1bR with higher affinity than the natural agonist, arginine vasotocin (AVT). Similarities and differences in the antagonist-receptor binding interface with receptors were characterized for each ligand. Non-peptide mammalian V1bR antagonists, SSR-149415 and L-368899, were shown to be effective and had an additive effect in blocking POMC hnRNA expression in pituitary cell culture studies. SR-49059 antagonized the effect(s) of AVT/CRH on the downregulation of the cV1aR and the upregulation of the cCRH-R2 expression but not the cV1bR and cCRH-R1. The Manning compound antagonized the downregulation of cV1aR, cV1bR and cCRH-R1 and the upregulation of cCRH-R2 expression. The specificity of antagonists apparently resulted from unique differences in the interacting residues and their binding affinities. Collectively, these results provide valuable leads for future development of novel compounds capable of blocking or attenuating the AP stress response of avian species and perhaps other non-mammalian vertebrates as well.

Previously, we showed that corticotrophin‐releasing hormone immunoreactive (CRH‐IR) neurones in a septal structure are associated with stress and the hypothalamic‐pituitary‐adrenal axis in birds. In the present study, we focused upon CRH‐IR neurones located within the septal structure called the nucleus of the hippocampal commissure (NHpC). Immunocytochemical and gene expression analyses were used to identify the anatomical and functional characteristics of cells within the NHpC. A comparative morphometry analysis showed that CRH‐IR neurones in the NHpC were significantly larger than CRH‐IR parvocellular neurones in the paraventricular nucleus of the hypothalamus (PVN) and lateral bed nucleus of the stria terminalis. Furthermore, these large neurones in the NHpC usually have more than two processes, showing characteristics of multipolar neurones. Utilisation of an organotypic slice culture method enabled testing of how CRH‐IR neurones could be regulated within the NHpC. Similar to the PVN, CRH mRNA levels in the NHpC were increased following forskolin treatment. However, dexamethasone decreased forskolin‐induced CRH gene expression only in the PVN and not in the NHpC, indicating differential inhibitory mechanisms in the PVN and the NHpC of the avian brain. Moreover, immunocytochemical evidence also showed that CRH‐IR neurones reside in the NHpC along with the vasotocinergic system, comprising arginine vasotocin (AVT) nerve terminals and immunoreactive vasotocin V1a receptors (V1aR) in glia. Hence, we hypothesised that AVT acts as a neuromodulator within the NHpC to modulate activity of CRH neurones via glial V1aR. Gene expression analysis of cultured slices revealed that AVT treatment increased CRH mRNA levels, whereas a combination of AVT and a V1aR antagonist treatment decreased CRH mRNA expression. Furthermore, an attempt to identify an intercellular mechanism in glial‐neuronal communication in the NHpC revealed that brain‐derived neurotrophic factor (BDNF) and its receptor (TrkB) could be involved in the signalling mechanism. Immunocytochemical results further showed that both BDNF and TrkB receptors were found in glia of the NHpC. Interestingly, in cultured brain slices containing the NHpC, the use of a selective TrkB antagonist decreased the AVT‐induced increase in CRH gene expression levels. The results from the present study collectively suggest that CRH neuronal activity is modulated by AVT via V1aR involving BDNF and TrkB glia in the NHpC.