Innervation Patterns of Harp Seal (Pagophilus groenlandicus) Vibrissal Sensory Systems

Vibrissae, or whiskers, are largest among pinnipeds and are specialized hairs that potentially evolved to serve sensory, thermoregulatory or protective functions. Behavioral data from pinniped and rodent vibrissa studies indicate that functional differences exist between medial microvibrissae and lateral macrovibrissae. However, comparative data are lacking, and current pinniped studies only focus on the largest ventrolateral macrovibrissae. Consequently, we investigated the medial-to-lateral innervation and microanatomy of harp seal (Pagophilus groenlandicus) vibrissal Follicle-Sinus Complexes (F-SCs). F-SCs were sectioned either longitudinally or in cross-section. Sections remained unstained or were stained with a modified Bodian silver stain (innervation) or Masson’s trichrome stain (microanatomy). Harp seals possessed 88-105 F-SCs and each exhibited a tripartite blood organization system. Hair shafts were more circular medially but became more elliptical laterally. Medial F-SCs had more symmetrical dermal capsule thicknesses and distributions of major branches of the deep vibrissal nerve, but these symmetries diminished as F-SCs became more lateral. Medial-to-lateral axon counts ranged from 550 ± 97.4 axons/F-SC (medial) to 1632 ± 173.2 axons/F-SC (lateral). Overall, axon counts averaged 1221 ± 422.3 axons/F-SC (n=146 cross-sections), indicating a total of 117,235 axons/snout. Lateral F-SCs alone possessed a mean of 1533 ± 192.9 axons (n=82 cross-sections), which is similar to counts reported in other pinniped vibrissal innervation studies. These data suggest that conventional studies that only examine lateral vibrissae overestimate total innervation by ~20%. Moreover, we counted axon bundles with and without silver staining (n=834) and determined that unstained sections yielded more accurate and ~10% greater axon counts. Consequently, conventional analyses are likely only overestimating innervation by ~10% overall. The relationship between axon count and F-SC surface area was non-linear (p<<0.01; n=24 cross-sections), presumably from mechanoreceptors reaching carrying capacity, and axon densities were consistent across the snout. Presumptive Merkel-Neurite complexes and lanceolate endings were observed at the glassy membrane and outer root sheath interface. Our data agree well with behavioral research on pinnipeds and rodents that documents functional compartmentalization between micro-(medial) and macrovibrissae (lateral). Furthermore, our results support that vibrissal innervation variation observed among extant mammals initially diverged as a result of phylogeny and then environment (i.e., terrestrial, semi-aquatic, fully aquatic)

Active touch sensing in pinnipeds

Active touch sensing in humans is characterised by making purposive movements with their fingertips. These movements are task-specific to maximise the relevant information gathered from an object. In whisker-touch sensing, previous research has suggested that whisker movements are purposive, but no one has ever examined task-specific whisker movements in any animal. Pinnipeds are whisker specialists, with long, mobile, sensitive whiskers and diverse whisker morphologies. The aim of this PhD is to investigate active touch sensing in Pinnipeds (seals, sea lions and walrus), by: i) describing whisker morphology; ii) comparing and quantifying whisker movements; and iii) characterising task-dependency of whisker movements during texture, size and luminance discrimination tasks. Pinnipeds with long, numerous whiskers, such as California sea lions (Zalophus californianus) and Stellar sea lions (Eumetopias jubatus) have larger infraorbital foramen (IOF) sizes and therefore, more sensitive whiskers. The IOF being a small hole in the skull, allowing the infraorbital nerve (ION) to pass through, which supplies sensation to the whiskers. Comparing whisker movements in Harbor seals (Phoca vitulina), California sea lions and Pacific walrus (Odobenidae rosmarus), showed these species all protracted their whiskers forwards and oriented their head towards a moving fish stimulus. However, California sea lions moved their whiskers more than the other species, and independently of the head. Due to the movement capabilities and sensitivity of whiskers in California sea lions, this species was used to investigate whether whiskers can be moved in a task-specific way. Results suggested that California sea lions make task-specific movements, by feeling around the edge of different-sized shapes, and focussing and spreading their whiskers on the centre of different-textured shapes. Therefore, California sea lion whiskers are controlled like a true active touch sensory system, similar to human fingertips. I suggest that active touch sensing is likely to efficiently guide foraging and prey capture in dark, murky waters in these animals. Moreover, the complexity of California sea lion whisker movements and their subsequent behaviours makes them a good candidate from which to further investigate animal decision-making, perception and cognition

Molecular-Genetic and Behavioral Analysis of the Functionality of Patterning in the Trigeminal Neuraxis

A striking feature of the vibrissal representation in rodents is the presence; at brainstem (barrellettes), thalamic (barrelloids) and cortical levels (barrels) of a somatotopically organized pattern of neurons which is isomorphic, both morphologically and physiologically, to the pattern of vibrissae on the snout. The vibrissal system is required for several classes of behavior, including feeding and active vibrissal sensing, but the functional role of the patterning in these behaviors is unknown. We used two mutant animals lacking patterning in two areas of the vibrissal neuraxis to examine the functional role of patterning. We examined feeding behavior using a knockout of Prxxl, which abolishes somatotopic barrellette patterning in the lemniscal brainstem nucleus. Null animals were significantly smaller than littermates by postnatal day 5, but reached developmental landmarks at appropriate times, and survived to adulthood on liquid diet. A careful analysis of infant and adult ingestive behavior revealed subtle impairments in suckling, increases in time spent feeding and the duration of feeding bouts, feeding during inappropriate times of day, and difficulties in the mechanics of feeding. During liquid diet feeding, null mice displayed abnormal behaviors including extensive use of the paws to move food into the mouth, submerging the snout in the diet, changes in licking, and also had difficulty consuming solid chow pellets. We suggest that barrellette patterning is necessary for normal ingestive behavior. To examine the role of patterning in active sensing, we used the BRL mouse, an Adenylyl Cyclase 1 mutant in which TCAs enter the cortex but do not cluster into barrels. Prior studies lesioning or chemically silencing barrel cortex suggests that vibrissal active sensing tasks such as texture discrimination are barrel-cortex dependent. However, these studies confound the functional role of the somatotopic barrel patterning with the function of barrel cortex cell activity. Use of the BRL mouse allowed us to dissociate these two. We found that BRL mice are impaired in a texture discrimination task relative to wildtype mice, suggesting a functional role for cortical barrel patterning. We discuss the role of patterning versus topographical organization of afferents

Molecular and cellular basis of Exogen and Anagen induction

The hair follicle has the unique capacity to pass through periods of growth, regression and rest before regenerating itself to restart the cycle. This dynamic cycling capacity enables animals to change their coats, and for hair length to be controlled at different body sites. While progress has been made on some of the outstanding questions in hair follicle biology, many of the cellular and molecular mechanisms involved in driving hair follicle cycling remain undiscovered. The first section of work in this thesis reports on the dermal papilla, an essential component of the hair follicle with a key role in follicle regeneration. Using hanging drop cultures spheres of human dermal papilla cells were created, and the expression of cytoskeletal and extracellular matrix components characteristic of intact papillae were analysed using immunohistochemistry and real time PCR. I found evidence that over time the gene and protein expression profiles of the papilla cell spheres became more representative of intact dermal papillae, although differences were seen between papilla cells strains derived from different individuals. I then demonstrated the inductive capability of human dermal papilla cells using this spherical model in an amputated follicle assay. This data provides some of the first evidence that cultured human dermal papilla cells can retain inductive capacity without having to be combined with other cell types. This induction phenomenon may also have relevance to anagen initiation and cycling. The second section of this thesis was concentrated on the process of club fibre shedding, now widely recognised as a phase of the follicle cycle known as exogen. The vibrissa follicle, with its predictable timing of club fibre loss, was first developed as a new model for exogen, and the structural and adhesive changes associated with the progression towards club fibre release were analysed using SEM and ТЕМ. Combined with plucking experiments these showed club loss to be gradual process. The expression of proteins involved in cell adhesion, differentiation, communication and digestion, all potential mediators of fibre loss were examined around "young “and "old" club fibres using immunohistochemistry. To further assess the mechanism of exogen, and to search for signalling molecules microarray technology was utilised, enabling the identification of 75 genes that were associated with the process. Group analysis highlighted a particular role for proteases and their inhibitors in the retention and final release of the club fibre. Moreover, immunofluorescent analysis of the results from the microarray identified a process of differentiation, specific to the cell layer surrounding the club fibre prior to club fibre release. This data provides evidence that the process of exogen is progressive, associated with maturation of the cells surrounding the club fibre, and terminating with the release of the club fibre. The processes involved in club fibre release are becoming more widely recognised as an important aspect of the hair cycle and this thesis is one of the first comprehensive pieces of work that analyses club retention and release as an active phase

The comparative myology of four dipodoid rodents (genera Zapus, Napaeozapus, Sicista, and Jaculus).

http://deepblue.lib.umich.edu/bitstream/2027.42/56368/1/MP124.pd

Hair follicle dermal cells: a morphological, behavioural and molecular study

Anatomy and smooth muscle a-actin expression in hair follicles from a variety of animal species (Mink, Polecat, Meerkat, Grey squirrel and stoat) was investigated. Smooth muscle ɑ-actin expression was related to follicle activity. Expression was greatest during anagen, with a marked reduction in expression during telogen. Follicular dermal cells were cultured from the above animal species. In vitro grey squirrel dermal papilla (DP) and dermal sheath (DS) cells both expressed smooth muscle ɑ -actin. Rat dermal papilla cell in vitro aggregative behaviour was characterized, by proliferation, chemotactic and molecular studies. Aggregation behaviour in these cells was not attributed to focal proliferation. However, fluctuations in cell motility correlated with the aggregation process, with the greatest motility between subconfluent and clumped cells. Motility was terminated within the clumped cells. Furthermore, DP cells in vitro secreted molecules that enhancer! motility in subconfluent DP cells and a variety of other cell types. As yet the type and specificity of this medium borne component is unknown. TGFP, bFGF and aFGF were all used as a comparison to the unknown molecule, however migration was rarely similar in magnitude to the response of the DP cell medium. Mouse cDNA probes of BMP 2 and BMP 4 were used to isolate rat homologues from a cDNA library. Wholemount in situ hybridization of BMP 4 expression was consistent with the data in the mouse, however BMP 4 was also expressed in adult rat telogen vibrissae. Molecular expression within in vitro DP cells was studied using a differential screen of a cDNA library. A number of clumped DP specific clones were differentially expressed, one of which having high homology with migration inhibitory factor. These results are discussed, and a hypothetical model is proposed to describe how DP aggregation occurs with reference to dermal condensation in vivo

Telemetry Controlled Brain Machine Interface To Train Cortical Circuits

The goal of this dissertation is to document functional reorganization in rat primary somatosensory (SI) cortex. This work proposes to strengthen the interhemispheric connection between homotopic sites in forelimb barrel cortex (FBC) through intracortical microstimulation (ICMS) and induce functional reorganization whereby neurons in the FBC respond to new input from the ipsilateral forelimb. Furthermore, a wireless microstimulation and recording device was developed for producing enhancement and functional reorganization of cortical circuits in FBC. The goal of Experiment One was to test the hypothesis that layer V neurons projected to homotopic sites in contralateral layer V FBC. Retrograde or anterograde neuronal tracer injections were made to characterize the distribution of callosal projecting neurons in contralateral SI that terminate in layer VFBC and where layer V callosal projecting neurons terminate in contralateral SI. The results showed a differential pattern of interhemispheric connectivity between homotopic forelimb representations in layer V FBC. The goal of Experiment Two was to test the hypothesis that ICMS enhances the interhemispheric pathway and leads to functional reorganization. ICMS was delivered in vivo to the interhemispheric pathway between homotopic layer V barrel cortices and multiunit recordings were made to assess changes in firing rate. The results showed ICMS strengthens interhemispheric connectivity and leads to functional reorganization in rat FBC. The goal of Experiment Three was to develop an interactive telemetry-based neural interface device for the controlled delivery of ICMS and recording response activity in rodent. The device successfully delivered microstimulation to a single electrode in SIand recorded evoked responses from a separate electrode in contralateral SI. Its performance was shown to be comparable to commercial stimulating and recording systems. This system serves as a prototype of a wearable compact device. The data suggest that neurons in rat FBC can be induced to respond to new input from the ipsilateral forelimb by enhancing the interhemispheric pathway with ICMS. An interactive system for the controlled delivery of telemetry-based microstimulation and real-time recordings has been demonstrated in vivo. These studies provide the framework for subsequent studies of interhemispheric pathway enhancement and functional reorganization in freely moving rats

Whisker touch sensing guides locomotion in small, quadrupedal mammals

All small mammals have prominent facial whiskers that they employ as tactile sensors to guide navigation and foraging in complex habitats. Nocturnal, arboreal mammals tend to have the longest and most densely packed whiskers, and semi-aquatic mammals have the most sensitive. Here we present evidence to indicate that many small mammals use their whiskers to tactually guide safe foot positioning. Specifically, in 11, small, non-flying mammal species, we demonstrate that forepaw placement always falls within the ground contact zone of the whisker field and that forepaw width is always smaller than whisker span. We also demonstrate commonalities of whisker scanning movements (whisking) and elements of active control, associated with increasing contact with objects of interest, across multiple small mammal species that have previously only been shown in common laboratory animals. Overall, we propose that guiding locomotion, alongside environment exploration, is a common function of whisker touch sensing in small, quadrupedal mammals

Comparative myology and phylogenetic systematics of the Heteromyidae (Mammalia, Rodentia).

http://deepblue.lib.umich.edu/bitstream/2027.42/56420/1/MP176.pd