Faculty Bibliography

This ethopharmacological investigation comprised a long-term field study that examined the function of serotonergic and vasotonergic systems in territoriality. Adult territorial and non-territorial (silent) male coquí frogs (Eleutherodactylus coqui) were injected (IP) with either arginine vasotocin (AVT) or one of two serotonin agonists, 5-HT_2A/2C selective agonist, (±) DOI - [(±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane], or 2) the 5-HT_1A selective agonist, 8-OH-DPAT - [(±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene]. Control groups received saline injections. Each male received two injections. Following the first injection, whether AVT or a 5HT agonist, the male was observed so that behavior could be documented prior to the second injection, which consisted of the other drug class. All frogs were marked, placed back in the exact location as captured, and observed for all behaviors and vocalizations. Territoriality in E. coqui includes several behavioral components: movement into a calling site, presentation of dominant physical displays, emitting advertisement calls, and defense a territory (including the use of physical force and/or aggressive vocalizations). This investigation found that particular territorial behaviors were significantly influenced by 5HT and AVT action. Initiation of advertisement calling is activated by AVT and suppressed by 5HT, calling rate is affected by 5HT activation, presentation of dominant physical displays are activated by AVT and repressed by 5HT activation, and movement associated with activation of territorial behavior is stimulated by AVT. These data suggested that both 5HT and AVT have a profound impact on territoriality and are two fundamental neuroendocrine systems that govern territorial behavior in social systems.

This is a case of both congenital left ventricular (LV) free wall submitral aneurysm and left atrial appendage aneurysm with 6 years of clinical follow-up. Each lesion is a rare entity, and to the best of our knowledge, this is the first case in medical literature of both lesions occurring in the same patient, raising the likelihood of a common etiology. The workup was initiated in the third trimester of fetal life with irregular heart rate and abnormal fetal ultrasound and echocardiogram at that time. The patient required emergent atrial appendage plication due to blood clot formation and suffered from multiple other complications including ventricular ectopy and surgically induced pseudoaneurysm. Follow-up interval echocardiograms have revealed continued good LV function with persistent LV aneurysm. In review of the case, there were several potential in utero causes including maternal viral upper respiratory infection and bacteriuria with exposure to amoxicillin. These as well as other considerations are discussed along with a brief review of these rare lesions, usual presentation, and known associations.

Arginine vasopressin (VP) and oxytocin (OT) are two nonapeptides present in mammals and execute a wide array of physiological and behavioral functions. In amphibians arginine vasotocin (VT) is hypothesized as a homologous nonapeptide for VP and also performs physiological and behavioral tasks. Studies have demonstrated that the structural and functional relationships between VP, OT, and VT receptor families are similar; however, little behavioral data has complimented these studies. The objective of this investigation was to determine if the mammalian nonapeptides VP and OT would activate behavioral manifestations naturally activated by VT. Frogs are particularly attractive for such an investigation because it is well documented that VT activates advertisement calling and territorial behavior. This investigation was a large sample size field study that utilized the territorial frog, Eleutherodactylus coqui. Fieldwork occurred on the Islands of Puerto Rico and Hawai'i and focused on territorial (calling) and non-territorial (silent) males. Frogs were administered exogenous injections of VP, OT, VT (positive control), or saline (control) in the field, placed back in their original locations, and were observed for behaviors. Exogenous injections of VP and OT significantly activated silent males to emit advertisement calls and exhibit territorial behavior. Additionally, silent males moved into new areas prior to calling whereas territorial males remained in their own territories. Control (saline) males displayed normal behaviors. This is the first study to demonstrate that mammalian nonapeptides activate calling and territorial behaviors in frogs and corroborates the close evolutionary relationships within the nonapeptide family.

INTRODUCTION/BACKGROUND:This report introduces a new vertebrate whole-embryo culture model for the direct application of pharmaceuticals and/or toxins into developing embryos. This method uses a terrestrial amphibian system and therefore has eliminated the problem of mammalian placental and uterine concerns. To test the utility and effectiveness of this method, we investigated the effect of fluoxetine on craniofacial development. Fluoxetine is one of the most commonly prescribed selective serotonin reuptake inhibitor (SSRI) on the market and treatment of depression during pregnancy is commonly deemed necessary. Previous studies have shown that SSRIs may promote developmental defects and congenital malformations of the heart. METHODS:This model utilized the egg/embryos of the directly developing Puerto Rican coquí frog, Eleutherodactylus coqui. The E. coqui embryo clutches were placed on filter paper in a Petri dish and were directly exposed (chronically) to fluoxetine concentrations ranging from 0.10mM to 1.0mM. Traditional whole-mount bone (Alizarin red) and cartilage (Alcian blue) staining was utilized to show the effect of fluoxetine on craniofacial development. RESULTS:Whole-mount staining revealed profound defects in cartilage development, particularly in the nasal capsule, mandible, and the brain case. Further, fluoxetine-treated embryos developed significantly slower compared to control animals. DISCUSSION/CONCLUSIONS:We found that the E. coqui culture model was an effective and sensitive technique for pharmaceutical studies, particularly since it allows the direct application of drugs and toxins into the developing embryo without the hindrance of the uterus and placenta. Chromatographic analysis revealed that fluoxetine infiltrated and penetrated embryonic tissue. It was found that altering serotonergic activity during development, via fluoxetine, stunted craniofacial development and organization.

Serotonin (5-HT) is an essential neurotransmitter for many physiological and behavioral processes. Clinically and experimentally 5-HT metabolism can be altered using a class of drugs known as selective serotonin reuptake inhibitors (SSRIs). SSRIs such as fluoxetine have proven effective tools for elevating 5-HT activity and are routinely utilized to study the role of 5-HT in aggressive behavior. The Puerto Rican coquí frog, Eleutherodactylus coqui, is a terrestrial amphibian that exhibits territoriality and paternal care. E. coqui have three different male behavioral modes, territorial (calling), non-calling, and paternal. Territorial male E. coqui aggressively maintain and defend territories by emitting advertisement calls. The objective of this study was to determine if the SSRI, fluoxetine, influences the establishment of the dominant (territorial)-subordinate (non-calling) relationship in male E. coqui. Wild captured adult territorial male E. coqui were grouped into 16 pairs in semi-natural terraria. Pharmacological treatment consisted of injecting (IP) experimental frogs with 25 μl (10mg/kg fluoxetine) of a fluoxetine/saline solution while control males received IP injections of saline. Injections were administered every other day for 20 days while observations continued until 40 days. Results indicated significantly higher numbers of advertisement calls emitted from control males after 20, 30, and 40 days. Additional, 13 of 16 control males emitted significantly more advertisement calls and became the territorial male. Fluoxetine treatment significantly reduced advertisement calling and territorial behavior in E. coqui males. These findings demonstrate that in E. coqui the 5-HT system has profound influence on male territorial and social behavior.

The Puerto Rican coquí frog, Eleutherodactylus coqui, is a directly developing frog that exhibits male territoriality and paternal behaviors. Male frogs also produce advertisement and aggressive vocalizations or calls. Territorial males emit advertisement calls to delineate territories and attract mates. Paternal males guard and brood the directly developing embryos during embryogenesis and up to five days after hatching; advertisement calling is normally absent or infrequent during paternal care. Territorial and paternal males commonly produce aggressive calls during agonistic situations. The neuropeptide, arginine vasotocin (AVT), has been shown to promote calling in anurans, including E. coqui. The objective of this study was to determine if exogenous AVT promotes calling and territorial behavior in paternal males and if it promotes males to abandon their offspring. Injections (IP) of AVT were given to paternal males immediately before the scotoperiod. Frogs were monitored for at least four hours after the injection and the following morning for calling activity and abandonment of egg clutches. AVT-injected males showed a dramatic and significant increase in aggressive calls compared to control males (saline injections). Exogenous AVT did induce advertisement calling in some paternal males but did not significantly elevate paternal males to territorial status nor did it significantly induce abandonment of eggs/embryos. In conclusion, the type of vocalization that AVT activates in E. coqui depends upon the reproductive state of the male and the social environment that surrounds the male.

PURPOSE: The present study assessed damage to the inferior alveolar nerve (IAN) following nerve lateralization and implant placement surgery through optical and transmission electron microscopy (TEM). MATERIALS AND METHODS: IAN lateralization was performed in 16 adult female rabbits (Oryctolagus cuniculus). During the nerve lateralization procedure, one implant was placed through the mandibular canal, and the IAN was replaced in direct contact with the implant. The implant was placed in the right mandible, and the left side was used as a control (no surgical procedure). After 8 weeks, the animals were sacrificed and samples were prepared for optical and TEM analysis of IAN structural damage. Histomorphometric analysis was performed to determine the number and cross-sectional dimensions of nerve fascicles and myelin sheath thickness between experimental and control groups. The different parameters were compared by one-way analysis of variance at the 95% significance level. RESULTS: Alterations in the perineural and endoneural regions of the IAN, with higher degrees of vascularization, were observed in the experimental group. TEM showed that the majority of the myelinated nerve fibers were not affected in the experimental samples. No significant variation in the number of fascicles was observed, significantly larger fascicle height and width were observed in the control group, and significantly thicker myelin sheaths were observed in the experimental samples. CONCLUSION: IAN lateralization resulted in substantial degrees of tissue disorganization at the microstructural level because of the presence of edema. However, at the ultrastructural level, small amounts of fiber degeneration were observed.

It is well established that the serotonergic system modulates social and aggressive behaviors. This study employed field experiments to examine the effects of serotonin on male social structure and behavior in the Puerto Rican coqui frog, Eleutherodactylus coqui. This frog displays three types of male behavioral phenotypes: territorial, satellite, and paternal males. Territorial males produce advertisement calls that delineate territories and actively defend these areas. Satellite males typically do not call or defend given areas but are commonly within given conspecific territories. Paternal males brood and defend developing embryos typically in isolation. The objective of this investigation was to focus on territorial and satellite males to determine the impact of the serotonergic system on territorial behavior, specifically with regard to advertisement calling. Serotonin receptor subtype systems 5-HT(1A) and 5-HT(2A/C) were chosen due to their known function in behavioral regulation. Fifteen territorial and satellite males were injected with the 5-HT(1A) agonist 8-OH-DPAT and 15 territorial and satellite males were injected with the 5-HT(2A/C) agonist DOI. Control territorial and satellite males received saline injections. Results indicated that a significant number of territorial males injected with 8-OH-DPAT and DOI failed to emit territorial vocalizations and did not display dominant postural behaviors. It is hypothesized that 8-OH-DPAT and DOI activate 5-HT(1A) and 5-HT(2A/C) receptor systems, respectively, and this activation results in the elimination of territorial behavior resulting in subordinate status. It is concluded that the serotonergic system is essential for the manifestation of male social behavior in E. coqui

Central serotonin (5-HT) metabolism during embryogenesis and a 3-day post-hatching period was analyzed using high performance liquid chromatography in the directly developing frog, Eleutherodactylus coqui. This anuran bypasses the free-swimming larval stage and embryos hatch as miniature frogs in the adult phenotype. During embryogenesis and for a short time immediately after hatching, male E. coqui provide paternal care by brooding and guarding eggs/embryos to prevent desiccation and predation. Serotonin and its catabolite, 5-HIAA, were measured from whole brain during embryogenesis and at 3 days post-hatch to identify critical periods in 5-HT development and to determine the relationship between 5-HT and life history events such as hatching and frog dispersal from the nest site. Serotonergic activity was highest during the early-mid embryonic stages as indicated by the ratio of 5-HIAA/5-HT, a general indicator of turnover and metabolism. There were significant increases in tissue concentrations of 5-HT during the latest or terminal embryonic stage, just prior to hatching, and also at 3 days post-hatch, shortly before neonates disperse into the rainforest. These two increases probably represent different functional requirements during development. The first may occur as a result of the surge of development in the 5-HT system during late embryogenesis that occurs in E. coqui and the second may be from the increase demand in sensory and motor neural development required before dispersal from the nest site

Embryonic development of the central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui, was determined by using immunocytochemistry. The majority of anuran amphibians (frogs) possess a larval stage (tadpole) that undergoes metamorphosis, a dramatic post-embryonic event, whereby the tadpole transforms into the adult phenotype. Directly developing frogs have evolved a derived life-history mode where the tadpole stage has been deleted and embryos develop directly into the adult bauplan. Embryonic development in E. coqui is classified into 15 stages (TS 1-15; 1 = oviposition/15 = hatching). Serotonergic immunoreactivity was initially detected at TS 6 in the raphe nuclei in the developing rhombencephalon. At TS 7, immunopositive perikarya were observed in the paraventricular organ in the hypothalamus and reticular nuclei in the hindbrain. Development of the serotonergic system was steady and gradual during mid-embryogenesis. However, starting at TS 13 there was a substantial increase in the number of serotonergic neurons in the paraventricular, raphe, and reticular nuclei, a large increase in the number of varicose fibers, and a differentiation of the reticular nuclei in the hindbrain. Consequentially, E. coqui displayed a well-developed central serotonergic system prior to hatching (TS 15). In comparison, the serotonergic system in metamorphic frogs typically starts to develop earlier but the surge of development that transpires in this system occurs post-embryonically, during metamorphosis, and not in the latter stages of embryogenesis, as it does in E. coqui. Overall, the serotonergic development in E. coqui is similar to the other vertebrates