Episodic acidification and its associated aluminum (Al) toxicity has been identified as a possible cause of Atlantic salmon decline in the northeastern United States including Maine where several salmon rivers are listed as endangered. During precipitation events such as snowmelts and storms, rivers and streams in this region experience episodic pulses of low pH and elevated inorganic Al which can damage the gill epithelium of fish leading to ion regulatory disturbances. To date, the impacts of episodic acid/Al on the physiology of Atlantic salmon undergoing critical life-stage transitions such as the parr-smolt transformation remain largely unknown. In this dissertation, I have used both laboratory and field studies to demonstrate that Atlantic salmon smolts are particulary vulnerable to ion regulatory disturbances during episodic acid/Al exposure. In particular, short-duration (days) exposures to acid and low levels of inorganic Al can impair the seawater tolerance of smolts in the absence of detectable impacts on freshwater ion regulation demonstrating the extreme sensitivity of the smolt hypoosmoregulatory system. I have also presented evidence that loss of seawater tolerance occurs through alterations in gill ion transporter expression, chloride cell dynamics, and several endocrine systems including the growth hormone-insulin-like growth factor I, interrenal and thyroid systems. Many of these alterations are likely involved in the upregulation of ion uptake mechanisms as part of acclimation to acid/Al in freshwater which may come as a direct cost to the ability to maintain ion homeostasis in seawater. The results presented here have important implications for salmon populations in regions affected by episodic acidification. Smolts with compromised seawater tolerance may experience delayed migration, decreased seawater preference and increased susceptibility to predation. This is likely to increase mortality during downstream migration, seawater entry, and marine residence ultimately leading to population level effects. Furthermore, these results support the idea that the freshwater experience of smolts may have a direct impact on survival in the marine environment.

Most recent phylogenetic analyses of snakes have recognized two major clades within Serpentes: Alethinophidia and Scolecophidia. Alethinophidians feed predominantly on relatively large vertebrate prey, which they transport into and through the mouth via reciprocating ratcheting movements of the toothed palatopterygoid jaw arches. In contrast, scolecophidians are small-prey specialists, feeding almost exclusively on small arthropods. In addition, these diminutive, fossorial snakes lack many of the key morphological features which underlie the feeding mechanisms of alethinophidians, such as toothed palatopterygoid jaw arches and a distensible lower jaw. However, the functional significance of these morphological differences has remained poorly understood because there have been no detailed descriptions of feeding behavior in Scolecophidia. I used magnified high-speed videography, videofluoroscopy, and standard histological and gross morphological preparations to study the functional morphology of the feeding apparatus in representatives of two families of Scolecophidia, Leptotyphlopidae and Typhlopidae. In Leptotyphlops (Leptotyphlopidae), a mandibular raking mechanism is used to capture, ingest and transport prey. In this mechanism, the toothed anterior portions of the mandibular rami are rotated medially about the intramandibular joints in a bilaterally synchronous fashion. In contrast, Typhlops and Rhinotyphlops (Typhlopidae) feed via a maxillary raking mechanism, in which asynchronous rotations of the toothed maxillae are used to drag prey into and through the mouth. Both mandibular raking and maxillary raking involve exceptionally rapid (3–5 Hz) movements of the tooth-bearing elements of the jaws, thereby facilitating the ingestion of large numbers of small prey within relatively brief periods of time.

Canids have long been considered to be conservative in their postcranial anatomy, so there are few studies examining individual canid taxa for locomotor adaptations. Canids are generally considered to be the most cursorial of the carnivorans. The limbs of large canids are generally adapted for rapid terrestrial locomotion, as these animals frequently rely on speed for prey capture. The prey animal is captured and killed using the jaws and teeth. Smaller canids, such as the red fox Vulpes and gray fox Urocyon, do not use their limbs primarily for fast running. The red fox appears to have many adaptations for running, including long slender legs, but these foxes do not run in their daily activities except when chased. The red fox uses its forelimbs to help in prey capture and its hind legs for leaping. The gray fox is an unusual canid since it regularly climbs trees. The limbs of the gray fox, especially the forelimb, are utilized in climbing. This dissertation contains a detailed description of the postcranial osteology and myology Vulpes and Urocyon cinercoargenteus and includes an analysis of these anatomical features in a functional framework. An examination of both the osteology and myology of the fore and hind limbs of these two foxes reveals that their behavior is reflected in a number of anatomical characters. Adaptations for leaping in the red fox include the presence of unusually long hind legs relative to the front legs, and an increase in the length of the distal bony limb elements relative to more proximal ones. In addition, the limb bones are very slender. Muscle bellies of tarsal and digital flexors and extensors are restricted to a proximal position on the limb, and muscles in general are emphasized that act along the long axis of the limbs. Adaptations of the gray fox for climbing include the presence of relatively short legs, a greater ability to rotate the radius on the ulna relative to other canids, and a relatively greater ability to abduct the hind limb. In addition, both red and gray foxes are able to retract their claws, an ability that is not generally associated with canids.

Most larval and permanently aquatic salamanders use undulatory swimming as their primary mode of steady aquatic locomotion. These swimming movements are powered by the segmented axial musculature. The hypaxial region of each segment consists of distinct muscle layers, which have a simple planar geometry and have varying architectural features. In an aquatic salamander Siren lacertina, the morphological features of the lateral hypaxial layers allow the shortening of muscle fibers to be amplified during contraction. The angled muscle fibers in these layers function to allow fiber shortening to be accompanied by substantial rotation of muscle fibers during contraction. The connective tissue sheets separating adjacent muscle segments (myosepta), allow the segment to bulge in a way that further amplifies muscle fiber rotation. The combined effect of architectural and moment arm variation ensures that muscle fibers from different layers undergo similar shortening patterns during swimming to allow for the generation of optimal tension during locomotion. In addition to steady swimming many salamanders respond to a predatory stimulus by performing a “C-start” aquatic escape response. This unsteady maneuver involves two kinematic stages, which function to propel the salamander away from the perceived threat. During metamorphosis, the tailfin of salamanders is resorbed and is thought to result in a substantial decrease in escape performance. However, in a stream salamander Eurycea bislineata , adults spend significant time in the water and behaviorally compensate for metamorphic changes in tail morphology by increasing the amplitude of escape responses. Aquatic locomotion in salamanders is not limited to axial swimming. Some salamanders also utilize their limbs to move along the substrate at slow speeds, while submerged. Structures used during aquatic walking face dramatically different mechanical loads compared to limbs used on land. The greater hydrodynamic resistance associated with water lowers the effective weight and can act to stabilize an organism throughout its gait. Therefore structures, such as the reduced limbs of S. lacertina, which would be considered ineffective on land, can be fully functional during aquatic walking.

Immunocytochemistry was used to study the distribution of gamma-aminobutyric acid (GABA) throughout the central visual nuclei and retina in Rana pipiens. In the diencephalon, intensely-labeled GABA immunoreactive neurons and nerve fibers were observed within the neuropil of Bellonci (nB) and corpus geniculatum (CG), while only immunoreactive puncta were found in the rostral visual nucleus (RVN). In the pretectal region, the posterior thalamic nucleus (nPT) contained the most intensely-labeled GABA immunoreactive perikarya and nerve fibers in the entire brain. Lightly immunoreactive perikarya were also found in the large-celled nucleus lentiformis mesencephali (nLM), as well as in the pretectal gray which contains neurons postsynaptic to the retinal terminal zones within nLM. In the optic tectum (OT), both immunoreactive perikarya and fibers were found within superficial layers 8 and 9; whereas only densely-packed immunoreactive perikarya were evident in the deep tectal layers (i.e. 2, 4, 6). The nucleus of the basal optic root (nBOR) contained a small number of lightly-labeled GABA immunoreactive perikarya mostly located in the dorsal half of the nucleus. A large number of perikarya within the nucleus isthmi (NI) were also lightly immunostained. In the retina, GABA immunoreactivity (both somata and fibers) was observed in all layers except the outer nuclear layer (ONL). Besides GABAergic putative horizontal and amacrine cells in the inner nuclear layer (INL), about 30% of total neurons within ganglion cell layer (GCL) expressed GABA immunoreactivity. Double-labeling studies indicated that about half of the GABA-containing perikarya in the GCL were retinal ganglion cells (RGCs). In addition, three GABAergic projection pathways existing in the visual system of Rana pipiens were demonstrated: (1) from RGCs to the contralateral OT; (2) from nBOR to the pretectal nLM; and (3) bilaterally from the NI to the OT. These results indicate that GABA is an important neurotransmitter in the frog visual system.

In rodents, there exists a strong interaction between the reproductive and circadian systems. For this thesis the female hamster was used as a model for the study of this interaction. Studies described in chapter II investigated whether the circadian regulation of reproductive processes may be through direct input of the suprachiasmatic nucleus (SCN) to neurons containing estrogen receptor (ER) and/or to neurons containing luteinizing hormone releasing hormone (LHRH). The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was applied to the SCN and double label immunocytochemistry for PHA-L and either ER or LHRH was carried out. Both ER- and LHRH-immunoreactive cells show appositions with SCN efferents or with efferents of the subparaventricular nucleus and the retrochiasmatic area. Results suggest that the circadian system can regulate reproductive processes via input to LHRH- and/or ER-containing neurons. Studies described in chapter 111 investigated whether effects of estrogen on circadian rhythms may be exerted through estrogen-binding systems afferent to the SCN. Immunocytochemistry for ER and the retrograde tracer cholera toxin B subunit, after its application to the SCN, demonstrated that some areas contain relatively high percentages of SCN afferent neurons which show ER immunoreactivity. Retrograde tracing results were compared with results of anterograde tracing from some of the sites containing SCN afferents. Furthermore, using a combined retrograde and anterograde tracing technique, SCN input to some SCN afferent neurons was demonstrated. However, no evidence of reciprocity between single ER-immunoreactive cells and the SCN was found. Results indicate the existence of estrogen binding systems afferent to the SCN which might mediate the effects of gonadal steroid hormones on circadian rhythms. Studies in chapter IV analyze the effects of blockade of SCN axonal output by local unilateral application of tetrodotoxin (TTX) on the LH surge. Injections of TTX on either the morning or the afternoon of proestrus were unable to block the LH surge. Results favor the interpretation that the SCN output signal responsible of the circadian gating of the LH surge occurs before the onset of the light period on the day of proestrus.

The skeletal elements of cartilaginous fishes are composed of a thin layer of mineralized tissue, ‘prismatic cartilage’, overlaying a hyaline cartilage core. Cartilage, even with a surface layer of mineralization, is far less stiff and strong than bone. Nevertheless, several species of stingray, including Rhinoptera and Aetobatus, subsist by crushing hard-shelled mollusks and crustaceans in their cartilaginous jaws. The jaws of these stingrays are composed of a previously undescribed form of cartilage. This tissue, ‘trabecular cartilage’, has mineralized struts which run through the central hyaline core. The struts, or trabeculae, are hollow tubes, made of calcified blocks, arranged as in a brick chimney. They serve to prevent buckling and bending of the jaws while prey is being crushed. The struts are present in late term embryos, indicating that feeding on hard prey does not cause them to form. As the animal grows the struts lengthen and thicken though they do not appear to become more numerous. Trabecular cartilage appears to have evolved at the base of the clade containing the hard prey specialists. This clade also includes Manta, a planktivorous species, which retains trabecular cartilage.

One of the key aspects of the immune system is the ability to prime cells by vaccination. The γδ T lymphocytes represent a significant population of cells in the peripheral blood of cattle that have largely been ignored in this regard. Here we have explored the potential for WC1+ γδ T cells to be primed by vaccination. We hypothesize that the γδ T cells that exhibit a recall response in vitro following in vivo priming represent a unique population. Utilizing a Leptospira borgpetersenii vaccine, we examined the recall responses of WC1+ and CD4+ T cells to leptospiral antigen. The WC1+ γδ cells were the major responding population to antigen for the first few weeks following in vivo priming, with the CD4 T cell response only began to overtake them after a booster dose was administered. The primed WC1+ γδ T cells displayed a unique pattern of surface marker expression when stimulated with antigen compared to mitogen-stimulated cells, and which paralleled that observed on the CD4 T cells that responded to antigen. Additionally, chemokine receptor expression was assessed in both ex vivo and antigen-stimulated WC1 and CD4T cells. Ex vivo CD4+ and WC1+ T cells differed with regard to chemokine receptor transcript expression while the antigen-activated cells had very similar patterns of expression. Both subsets expressed genes typical of TH1-polarized cells, but differed with regard to transcripts for co-stimulatory molecules expressed. TCR usage by the antigen-responsive WC1+ γδ T cells from vaccinated animals was evaluated. The antigen-responsive cells had transcripts for several different Vγ and Vδ gene segments with very limited usage of J genes and highly variable CDR3 sequences. These results did not differ greatly from those obtained with nondividing cells or with ex vivo peripheral blood mononuclear cells suggesting little if any enrichment for a specific TCR type. These results support the hypothesis that WC1 + γδ T cells and CD4 αβ T cells that respond to leptospira antigen are likely to differ mainly with regard to how they are activated and the pathogen molecules that activate them, rather than with regard to their effector functions.

Assessments of stress typically involve either point samples (blood or saliva) reflecting a moment in time or state samples (urine of feces) reflecting several hours or a day. Currently, there is no way to assess chronic levels of stress without using repeated sampling procedures which are both time consuming, expensive, and possibly stressful. The purpose of this dissertation was three fold: (1) to develop a more chronic estimate of stress by measuring cortisol concentrations in hair, (2) to determine using this measure, how rhesus macaques responded to the prolonged stress of relocation, and (3) to determine the role of a proposed modulatory protein corticosteroid binding globulin (CBG) in measuring stress. Results demonstrate that cortisol can be quantified in hair and that stress reactivity can also be measured in hair. Rhesus monkeys responded to a major life stress (relocation) with a significant increase in cortisol in both hair and serum and with substantial behavioral changes. CBG concentrations failed to show alteration initially following relocation, however a significant increase was observed one year later. The findings demonstrate the importance of the cortisol/CBG relationship during a prolonged stress paradigm. Our data also provide further evidence that the free cortisol index (cortisol:CBG ratio) may be a better indicator of stress reactivity when compared to the commonly used serum cortisol concentrations.

The frog neuromuscular junction is a unique model that allowed us to selectively remove cellular components from the neuromuscular junction and create preparations with varying degrees of nerve terminal stability. We found further evidence that frog terminal Schwann cells communicate with their cellular partners, as terminal Schwann cells responded with changes in number or morphology as a result of changes in synaptic integrity. Terminal Schwann cells divided at synaptic sites in response to a regenerating nerve terminal. Terminal Schwann cells also had morphological changes in response to changes in status of their cellular partners; they extended processes in response to removal of the nerve terminal. Orientation and length of these processes was profoundly affected by the presence or absence of muscle fiber and nerve terminal. Similar to observations at the mammalian neuromuscular junction, terminal Schwann cells appear to play a role in reinnervation, as frequently regenerating nerve terminals were within the confines of terminal Schwann cells and their processes. I also investigated the organization of actin within preparations with varying amounts of nerve terminal stability, including developing nerve terminals and regenerating adult nerve terminals that were forming either stable or unstable connections. Previously, F-actin stained target-deprived nerve terminals in a ladder-like pattern and was concentrated in the nonrelease domains (Dunaevsky and Connor 2000). I found that β-actin was similarly distributed and localized to the nonrelease domains of nerve terminals at intact neuromuscular junctions. Further, association of actin with these particular domains appeared to be important for nerve terminal stability. As nerve terminals acquired increasing stability during development, they acquired this domain specific distribution of F-actin. Additionally, although synaptic sites with stable regenerating nerve terminal acquired this ladder-like pattern of F-actin, it was very rare for unstable regenerating nerve terminals to do so. I also tested the dynamic nature of F-actin with pharmacological perturbation. F-actin at nonrelease domains was found to be very stable. This stability of the F-actin based cytoskeleton further suggests that F-actin at the nonrelease domains of nerve terminals may play a role in the stability of motor nerve terminals.