Manatee cognition in the wild: an exploration of the manatee mind and behavior through neuroanatomy, psychophysics, and field observations.

Cognition refers to the mechanisms for acquiring, processing, storing, and acting on information, all of which are critical to understanding the behavior of animals. These mechanisms are poorly known in manatees, especially how they are expressed in the wild. To expand our understanding of manatee cognition, we gathered information from behavioral experimentation in the laboratory, neuroanatomical research, controlled field studies, integrated laboratory and field measurement, and natural history observations (published reports, written surveys, and interviews with knowledgeable observers). Laboratory research, both neuroanatomical and behavioral, provided the most empirical data, primarily on sensory/perceptual capacities. Inferences from these data and narratives from surveys and interviews illuminated possibilities for higher order cognition. Evidence from field measurements was sparse, although substantial amounts of information have been collected from tracking data and to a lesser extent vessel impact studies, which can be used to infer cognitive attributes. Manatees are tactile-auditory specialists with complementary visual and chemosensory abilities. They demonstrate learning characteristics typical of vertebrates. Movement tracking data plus direct observations suggest that they have good spatial cognition, indicated by their ability to traverse complicated water networks and memory for foraging and warm water sites. They engage in a wide range of play-like, object manipulation, and mimetic behaviors, which suggests cognitive capacities beyond basic associative learning. Understanding manatee cognition beyond the laboratory will be necessary for conservation of manatees as they face challenges such as habitat degradation and threats from water-borne vessel traffic. There is a clear need for more direct research in natural settings.

Parcellation in the dorsal column nuclei of Florida manatees (Trichechus manatus latirostris) and rock hyraxes (Procavia capensis) indicates the presence of body barrelettes.

Florida manatees (Trichechus manatus latirostris) and rock hyraxes (Procavia capensis) exhibit expanded tactile arrays of vibrissae that are distributed not only on the face but also on the entire postfacial body. In contrast, the vibrissae of most mammals are principally restricted to the face. These facial vibrissae may be associated with central nervous system representations known as barrels in the cerebral cortex, barreloids in the thalamus, and barrelettes in the trigeminal nuclei of the brainstem. To date, vibrissae representations found within the brainstem have been principally limited to facial vibrissae representations in the trigeminal nuclei. We hypothesized that the tactile specializations of the manatee and rock hyrax would produce a unique modification of typical mammalian central nervous system organization, with postfacial vibrissae representations appearing in the cuneate and gracile nuclei as "body barrelettes." Using histological and histochemical methods, including cresyl violet, myelin, and cytochrome oxidase processing, we first delineated the rostral, middle, and caudal zones of the cuneate and gracile nuclei. Within the middle zone, divisions were present, including extensive parcellation in the cluster region, particularly in manatees. These clusters were particularly densely distributed and distinguishable in the presumptive postfacial body representations in the cuneate and gracile nuclei but otherwise shared many attributes with the barrelettes found in the trigeminal nuclei of other species. This study represents the first characterization of postfacial body vibrissae representations, or "body barrelettes," in the brainstem of any species. Previous studies have predominantly focused on facial vibrissae representations, which have served for decades as a model for sensory organization and plasticity. Our results extend what is known about vibrissae representations in the central nervous system to include expansions related to peripheral specializations of the postfacial body. Unusual somatosensory adaptations in the manatee and rock hyrax are highly informative regarding how mammalian brain organization responds to evolutionary pressures on sensory systems.

Thromboelastography in wild Florida manatees (Trichechus manatus latirostris).

BACKGROUND: Thromboelastography (TEG) provides a comprehensive evaluation of blood clot formation. This test can be used to identify abnormalities in coagulation by assessing multiple aspects of the clotting cascade, including the speed of clot initiation and formation, clot strength, and ultimately fibrinolysis. Thromboembolic disease has been hypothesized to play a role in the pathophysiology of cold stress syndrome (CSS), an important cause of mortality in the threatened Florida manatee (Trichechus manatus latirostris). OBJECTIVES: The objective of this study was to establish thromboelastography RIs using the TEG 5000 with citrated whole blood samples and kaolin activation in wild, healthy manatees. METHODS: In December 2014 and January 2015, 29 wild manatees (17 male and 12 female) were blood sampled as part of the annual wild manatee health assessments organized by United States Geological Survey (USGS). TEG was performed using citrated kaolin-activated samples. RESULTS: The samples were obtained from manatees caught in Crystal River, Citrus County, and used to identify the mean ± SD of normal TEG parameters: R = reaction time 2.1 minutes (0.8), K = clot formation time 0.8 min (0), α angle = 83.1° (2), MA = maximum amplitude 75 mm (7.6), and LY30 = clot lysis 0.41% (0.68). No significant differences were found between manatee size, sex, or time between sampling and running the test. CONCLUSIONS: Manatee TEG parameters demonstrate a relatively hypercoagulable condition when compared to other mammals. This information will facilitate detection of changes in hemostasis during injury and disease and provide a valuable reference range.

Identifying coagulopathies in the pathophysiology of cold stress syndrome in the Florida manatee Trichechus manatus latirostris.

Cold stress syndrome (CSS) in the Florida manatee Trichechus manatus latirostris has been defined as morbidity and mortality resulting from prolonged exposure to water temperatures <20°C. The pathophysiology is described as multifactorial, involving nutritional, immunological and metabolic disturbances; however, the exact mechanisms are unknown. We hypothesized that thromboembolic complications contribute to the pathophysiology of CSS in addition to the previously described factors. During the winter of 2014-2015, 10 Florida manatees with clinical signs of CSS were presented to Lowry Park Zoo, Tampa, FL, USA. Thromboelastography (TEG) and coagulation panels were performed at admission. In addition, coagulation panel data from 23 retrospective CSS cases were included in the analyses. There were numerous differences between mean values of TEG and coagulation parameters for healthy manatees and those for CSS cases. Among TEG parameters, reaction time (R), clot formation time (K) and percentage of clot lysed after 30 min (LY30) values were significantly different (p < 0.05) between the 2 groups. CSS cases also had significantly higher mean D-dimer concentration and coagulation factor XI activity, prolonged mean activated partial thromboplastin time (aPTT) and significantly decreased mean antithrombin activity. These combined abnormalities include clinicopathologic criteria of disseminated intravascular coagulation, indicating an increased risk of thromboembolic disease associated with manatee CSS.

PROPOSAL AND APPLICATION OF A NOVEL DISSEMINATED INTRAVASCULAR COAGULATION SCORING SYSTEM IN THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS).

Disseminated intravascular coagulopathy (DIC) is an acquired disorder of hemostasis resulting in activation of the coagulation and fibrinolytic pathways. It is reported secondarily to multiple disease processes and can be associated with increased mortality. Previous research at Tampa's Lowry Park Zoo (LPZ) demonstrated that Florida manatees ( Trichechus manatus latirostris) with cold stress syndrome (CSS) demonstrated thromboembolic disease. The object of this retrospective study was to establish the presence and clinical relevance of DIC in Florida manatees admitted to LPZ for rehabilitation from 07 March 2010 to 15 August 2015. A coagulation panel, including prothrombin time, partial thromboplastin time, platelet count, fibrinogen level, and D-dimer level was used to diagnose DIC. There were 100 cases identified in the study period: 35 trauma, 43 CSS, 17 secondary to harmful algae blooms (HAB), and five miscellaneous. Manatees with CSS had the highest incidence of DIC with 24 of 43 cases (56%) affected, followed by trauma with 18 of 35 cases (52%) affected. None of the manatees with HAB were found to have DIC. Manatees that developed DIC during rehabilitation or when DIC progressed did not survive. Due to the clinical implications of DIC, identifying its presence and recognizing its severity could improve clinical outcomes by enabling more intensive treatment protocols.

Detection of hydrodynamic stimuli by the postcranial body of Florida manatees (Trichechus manatus latirostris).

Manatees live in shallow, frequently turbid waters. The sensory means by which they navigate in these conditions are unknown. Poor visual acuity, lack of echolocation, and modest chemosensation suggest that other modalities play an important role. Rich innervation of sensory hairs that cover the entire body and enlarged somatosensory areas of the brain suggest that tactile senses are good candidates. Previous tests of detection of underwater vibratory stimuli indicated that they use passive movement of the hairs to detect particle displacements in the vicinity of a micron or less for frequencies from 10 to 150 Hz. In the current study, hydrodynamic stimuli were created by a sinusoidally oscillating sphere that generated a dipole field at frequencies from 5 to 150 Hz. Go/no-go tests of manatee postcranial mechanoreception of hydrodynamic stimuli indicated excellent sensitivity but about an order of magnitude less than the facial region. When the vibrissae were trimmed, detection thresholds were elevated, suggesting that the vibrissae were an important means by which detection occurred. Manatees were also highly accurate in two-choice directional discrimination: greater than 90% correct at all frequencies tested. We hypothesize that manatees utilize vibrissae as a three-dimensional array to detect and localize low-frequency hydrodynamic stimuli.

Golgi Analysis of Neuron Morphology in the Presumptive Somatosensory Cortex and Visual Cortex of the Florida Manatee (Trichechus manatus latirostris).

The current study investigates neuron morphology in presumptive primary somatosensory (S1) and primary visual (V1) cortices of the Florida manatee (Trichechus manatus latirostris) as revealed by Golgi impregnation. Sirenians, including manatees, have an aquatic lifestyle, a large body size, and a relatively large lissencephalic brain. The present study examines neuron morphology in 3 cortical areas: in S1, dorsolateral cortex area 1 (DL1) and cluster cortex area 2 (CL2) and in V1, dorsolateral cortex area 4 (DL4). Neurons exhibited a variety of morphological types, with pyramidal neurons being the most common. The large variety of neuron types present in the manatee cortex was comparable to that seen in other eutherian mammals, except for rodents and primates, where pyramid-shaped neurons predominate. A comparison between pyramidal neurons in S1 and V1 indicated relatively greater dendritic branching in S1. Across all 3 areas, the dendritic arborization pattern of pyramidal neurons was also similar to that observed previously in the afrotherian rock hyrax, cetartiodactyls, opossums, and echidnas but did not resemble the widely bifurcated dendrites seen in the large-brained African elephant. Despite adaptations for an aquatic environment, manatees did not share specific neuron types such as tritufted and star-like neurons that have been found in cetaceans. Manatees exhibit an evolutionarily primitive pattern of cortical neuron morphology shared with most other mammals and do not appear to have neuronal specializations for an aquatic niche.

Evolution of cytoarchitectural landscapes in the mammalian isocortex: Sirenians (Trichechus manatus) in comparison with other mammals.

The isocortex of several primates and rodents shows a systematic increase in the number of neurons per unit of cortical surface area from its rostrolateral to caudomedial border. The steepness of the gradient in neuronal number and density is positively correlated with cortical volume. The relative duration of neurogenesis along the same rostrocaudal gradient predicts a substantial fraction of this variation in neuron number and laminar position, which is produced principally from layers II-IV neurons. However, virtually all of our quantitative knowledge about total and laminar variation in cortical neuron numbers and neurogenesis comes from rodents and primates, leaving whole taxonomic groups and many intermediate-sized brains unexplored. Thus, the ubiquity in mammals of the covariation of longer cortical neurogenesis and increased cortical neuron number deriving from cortical layers II-IV is undetermined. To begin to address this gap, we examined the isocortex of the manatee using the optical disector method in sectioned tissue, and also assembled partial data from published reports of the domestic cat brain. The manatee isocortex has relatively fewer neurons per total volume, and fewer II-IV neurons than primates with equivalently sized brains. The gradient in number of neurons from the rostral to the caudal pole is intermediate between primates and rodents, and, like those species, is observed only in the upper cortical layers. The cat isocortex (Felis domesticus) shows a similar structure. Key species for further tests of the origin, ubiquity, and significance of this organizational feature are discussed.

Neuron Types in the Presumptive Primary Somatosensory Cortex of the Florida Manatee (Trichechus manatus latirostris).

Within afrotherians, sirenians are unusual due to their aquatic lifestyle, large body size and relatively large lissencephalic brain. However, little is known about the neuron type distributions of the cerebral cortex in sirenians within the context of other afrotherians and aquatic mammals. The present study investigated two cortical regions, dorsolateral cortex area 1 (DL1) and cluster cortex area 2 (CL2), in the presumptive primary somatosensory cortex (S1) in Florida manatees (Trichechus manatus latirostris) to characterize cyto- and chemoarchitecture. The mean neuron density for both cortical regions was 35,617 neurons/mm(3) and fell within the 95% prediction intervals relative to brain mass based on a reference group of afrotherians and xenarthrans. Densities of inhibitory interneuron subtypes labeled against calcium-binding proteins and neuropeptide Y were relatively low compared to afrotherians and xenarthrans and also formed a small percentage of the overall population of inhibitory interneurons as revealed by GAD67 immunoreactivity. Nonphosphorylated neurofilament protein-immunoreactive (NPNFP-ir) neurons comprised a mean of 60% of neurons in layer V across DL1 and CL2. DL1 contained a higher percentage of NPNFP-ir neurons than CL2, although CL2 had a higher variety of morphological types. The mean percentage of NPNFP-ir neurons in the two regions of the presumptive S1 were low compared to other afrotherians and xenarthrans but were within the 95% prediction intervals relative to brain mass, and their morphologies were comparable to those found in other afrotherians and xenarthrans. Although this specific pattern of neuron types and densities sets the manatee apart from other afrotherians and xenarthrans, the manatee isocortex does not appear to be explicitly adapted for an aquatic habitat. Many of the features that are shared between manatees and cetaceans are also shared with a diverse array of terrestrial mammals and likely represent highly conserved neural features. A comparative study across manatees and dugongs is necessary to determine whether these traits are specific to one or more of the manatee species, or can be generalized to all sirenians.

Elaboration and Innervation of the Vibrissal System in the Rock Hyrax (Procavia capensis).

Mammalian tactile hairs are commonly found on specific, restricted regions of the body, but Florida manatees represent a unique exception, exhibiting follicle-sinus complexes (FSCs, also known as vibrissae or tactile hairs) on their entire body. The orders Sirenia (including manatees and dugongs) and Hyracoidea (hyraxes) are thought to have diverged approximately 60 million years ago, yet hyraxes are among the closest relatives to sirenians. We investigated the possibility that hyraxes, like manatees, are tactile specialists with vibrissae that cover the entire postfacial body. Previous studies suggested that rock hyraxes possess postfacial vibrissae in addition to pelage hair, but this observation was not verified through histological examination. Using a detailed immunohistochemical analysis, we characterized the gross morphology, innervation and mechanoreceptors present in FSCs sampled from facial and postfacial vibrissae body regions to determine that the long postfacial hairs on the hyrax body are in fact true vibrissae. The types and relative densities of mechanoreceptors associated with each FSC also appeared to be relatively consistent between facial and postfacial FSCs. The presence of vibrissae covering the hyrax body presumably facilitates navigation in the dark caves and rocky crevices of the hyrax's environment where visual cues are limited, and may alert the animal to predatory or conspecific threats approaching the body. Furthermore, the presence of vibrissae on the postfacial body in both manatees and hyraxes indicates that this distribution may represent the ancestral condition for the supraorder Paenungulata.

In contrast to many other mammals, cetaceans have relatively small hippocampi that appear to lack adult neurogenesis.

The hippocampus is essential for the formation and retrieval of memories and is a crucial neural structure sub-serving complex cognition. Adult hippocampal neurogenesis, the birth, migration and integration of new neurons, is thought to contribute to hippocampal circuit plasticity to augment function. We evaluated hippocampal volume in relation to brain volume in 375 mammal species and examined 71 mammal species for the presence of adult hippocampal neurogenesis using immunohistochemistry for doublecortin, an endogenous marker of immature neurons that can be used as a proxy marker for the presence of adult neurogenesis. We identified that the hippocampus in cetaceans (whales, dolphins and porpoises) is both absolutely and relatively small for their overall brain size, and found that the mammalian hippocampus scaled as an exponential function in relation to brain volume. In contrast, the amygdala was found to scale as a linear function of brain volume, but again, the relative size of the amygdala in cetaceans was small. The cetacean hippocampus lacks staining for doublecortin in the dentate gyrus and thus shows no clear signs of adult hippocampal neurogenesis. This lack of evidence of adult hippocampal neurogenesis, along with the small hippocampus, questions current assumptions regarding cognitive abilities associated with hippocampal function in the cetaceans. These anatomical features of the cetacean hippocampus may be related to the lack of postnatal sleep, causing a postnatal cessation of hippocampal neurogenesis.

Eight-choice sound localization by manatees: performance abilities and head related transfer functions.

Two experiments investigated the ability and means by which two male Florida manatees (Trichechus manatus latirostris) may determine the direction of a sound source. An eight-choice discrimination paradigm was used to determine the subjects' sound localization abilities of five signal conditions covering a range of frequencies, durations, and levels. Subjects performed above the 12.5% chance level for all broadband frequencies and were able to localize sounds over a large level range. Errors were typically located to either side of the signal source location when presented in the front 180° but were more dispersed when presented from locations behind the subject. Front-to-back confusions were few and accuracy was greater when signals originated from the front 180°. Head-related transfer functions were measured to determine if frequencies were filtered by the manatee body to create frequency-specific interaural level differences (ILDs). ILDs were found for all frequencies as a function of source location, although they were largest with frequencies above 18 kHz and when signals originated to either side of the subjects. Larger ILDs were found when the signals originated behind the subjects. A shadowing-effect produced by the body may explain the relatively low occurrence of front-back confusions in the localization study.

Comparative neuronal morphology of the cerebellar cortex in afrotherians, carnivores, cetartiodactyls, and primates.

Although the basic morphological characteristics of neurons in the cerebellar cortex have been documented in several species, virtually nothing is known about the quantitative morphological characteristics of these neurons across different taxa. To that end, the present study investigated cerebellar neuronal morphology among eight different, large-brained mammalian species comprising a broad phylogenetic range: afrotherians (African elephant, Florida manatee), carnivores (Siberian tiger, clouded leopard), cetartiodactyls (humpback whale, giraffe) and primates (human, common chimpanzee). Specifically, several neuron types (e.g., stellate, basket, Lugaro, Golgi, and granule neurons; N = 317) of the cerebellar cortex were stained with a modified rapid Golgi technique and quantified on a computer-assisted microscopy system. There was a 64-fold variation in brain mass across species in our sample (from clouded leopard to the elephant) and a 103-fold variation in cerebellar volume. Most dendritic measures tended to increase with cerebellar volume. The cerebellar cortex in these species exhibited the trilaminate pattern common to all mammals. Morphologically, neuron types in the cerebellar cortex were generally consistent with those described in primates (Fox et al., 1967) and rodents (Palay and Chan-Palay, 1974), although there was substantial quantitative variation across species. In particular, Lugaro neurons in the elephant appeared to be disproportionately larger than those in other species. To explore potential quantitative differences in dendritic measures across species, MARSplines analyses were used to evaluate whether species could be differentiated from each other based on dendritic characteristics alone. Results of these analyses indicated that there were significant differences among all species in dendritic measures.

Detection of hydrodynamic stimuli by the Florida manatee (Trichechus manatus latirostris).

Florida manatees inhabit the coastal and inland waters of the peninsular state. They have little difficulty navigating the turbid waterways, which often contain obstacles that they must circumnavigate. Anatomical and behavioral research suggests that the vibrissae and associated follicle-sinus complexes that manatees possess over their entire body form a sensory array system for detecting hydrodynamic stimuli analogous to the lateral line system of fish. This is consistent with data highlighting that manatees are tactile specialists, evidenced by their specialized facial morphology and use of their vibrissae during feeding and active investigation/manipulation of objects. Two Florida manatees were tested in a go/no-go procedure using a staircase method to assess their ability to detect low-frequency water movement. Hydrodynamic vibrations were created by a sinusoidally oscillating sphere that generated a dipole field at frequencies from 5 to 150 Hz, which are below the apparent functional hearing limit of the manatee. The manatees detected particle displacement of less than 1 µm for frequencies of 15-150 Hz and of less than a nanometer at 150 Hz. Restricting the facial vibrissae with various size mesh openings indicated that the specialized sensory hairs played an important role in the manatee's exquisite tactile sensitivity.

Audiogram and auditory critical ratios of two Florida manatees (Trichechus manatus latirostris).

Manatees inhabit turbid, shallow-water environments and have been shown to have poor visual acuity. Previous studies on hearing have demonstrated that manatees possess good hearing and sound localization abilities. The goals of this research were to determine the hearing abilities of two captive subjects and measure critical ratios to understand the capacity of manatees to detect tonal signals, such as manatee vocalizations, in the presence of noise. This study was also undertaken to better understand individual variability, which has been encountered during behavioral research with manatees. Two Florida manatees (Trichechus manatus latirostris) were tested in a go/no-go paradigm using a modified staircase method, with incorporated 'catch' trials at a 1:1 ratio, to assess their ability to detect single-frequency tonal stimuli. The behavioral audiograms indicated that the manatees' auditory frequency detection for tonal stimuli ranged from 0.25 to 90.5 kHz, with peak sensitivity extending from 8 to 32 kHz. Critical ratios, thresholds for tone detection in the presence of background masking noise, were determined with one-octave wide noise bands, 7-12 dB (spectrum level) above the thresholds determined for the audiogram under quiet conditions. Manatees appear to have quite low critical ratios, especially at 8 kHz, where the ratio was 18.3 dB for one manatee. This suggests that manatee hearing is sensitive in the presence of background noise and that they may have relatively narrow filters in the tested frequency range.

Manatee vibrissae: evidence for a "lateral line" function.

Aquatic mammals use vibrissae to detect hydrodynamic stimuli over a range from 5 to 150 Hz, similar to the range detected by lateral line systems in fishes and amphibians. Manatees possess ∼5,300 vibrissae distributed over the body, innervated by ∼209,000 axons. This extensive innervation devoted to vibrissae follicles is reflected in enlarged, elaborate somatosensory regions of the gracile, cuneate, and Bischoff's brain-stem nuclei, ventrobasal thalamus, and presumptive somatosensory cortex. Our preliminary psychophysical testing indicates that in Florida and Antillean manatees the Weber fraction for detection thresholds for grating textures ranges from 0.025 to 0.14. At the lower end of this range, sensitivity is comparable to human index finger thresholds. For hydrodynamic stimuli of 5-150 Hz, detection threshold levels for manatees using facial or postfacial vibrissae were substantially lower than those reported for harbor seals and similar to reports of sensitivity for the lateral line systems of some fish. Our findings suggest that the facial and postfacial vibrissae are used to detect hydrodynamic stimuli, whereas only the facial vibrissae are used for direct contact investigation.

Mammalian tactile hair: divergence from a limited distribution.

Mammalian species use tactile hairs to address a variety of perceptual challenges in detecting and responding appropriately to environmental stimuli. With a wide range of functional roles that range from object detection, to fine texture discrimination, to hydrodynamic trail perception, tactile hairs have been adapted for a variety of environmental niches to enhance survival through optimizing detection of somatosensory cues. Because the high level of innervation associated with tactile hairs requires a commensurately high dedication of neural resources, their distribution is restricted to specific regions of the body that encounter stimuli of interest--commonly, the face. However, several species--namely bats, naked mole-rats, hyraxes, manatees, and dugongs--are rare exceptions, with tactile hair distribution that has expanded to cover the entire body. This review examines the behavioral advantages conferred by this unusual trait, the neuroanatomical adaptations that accompany it, and how this pattern might have evolved.

Developmental uterine anomalies in cats and dogs undergoing elective ovariohysterectomy.

OBJECTIVE: To describe the characteristics and frequency of gross uterine anomalies in cats and dogs undergoing elective ovariohysterectomy. DESIGN: Prospective and retrospective case series. ANIMALS: 53,258 cats and 32,660 dogs undergoing elective ovariohysterectomy at 26 clinics in the United States and Canada during 2007. PROCEDURES: Clinics prospectively reported gross anomalies and submitted tissues from abnormal reproductive tracts identified during surgery. Records from a feral cat spay-neuter clinic were evaluated retrospectively. RESULTS: Suspected congenital anomalies of the uterus were identified in 0.09% (49/53,258) of female cats and 0.05% (15/32,660) of female dogs. Uterine anomalies identified included unicornuate uterus (33 cats and 11 dogs), segmental agenesis of 1 uterine horn (15 cats and 3 dogs), and uterine horn hypoplasia (1 cat and 1 dog). Ipsilateral renal agenesis was present in 29.4% (10/34) of cats and 50.0% (6/12) of dogs with uterine anomalies in which kidneys were evaluated. Mummified ectopic fetuses were identified in 4 cats with uterine anomalies. Both ovaries and both uterine tubes were present in most animals with uterine anomalies. CONCLUSIONS AND CLINICAL RELEVANCE: Urogenital anomalies were twice as common in cats as in dogs. Identification of uterine developmental anomalies in dogs and cats should trigger evaluation of both kidneys and both ovaries because ipsilateral renal agenesis is common, but both ovaries are likely to be present and should be removed during ovariohysterectomy.

Phrenic nerve afferent activation of neurons in the cat SI cerebral cortex.

Stimulation of respiratory afferents elicits neural activity in the somatosensory region of the cerebral cortex in humans and animals. Respiratory afferents have been stimulated with mechanical loads applied to breathing and electrical stimulation of respiratory nerves and muscles. It was hypothesized that stimulation of the phrenic nerve myelinated afferents will activate neurons in the 3a and 3b region of the somatosensory cortex. This was investigated in cats with electrical stimulation of the intrathoracic phrenic nerve and C(5) root of the phrenic nerve. The somatosensory cortical response to phrenic afferent stimulation was recorded from the cortical surface, contralateral to the phrenic nerve, ispilateral to the phrenic nerve and with microelectrodes inserted into the cortical site of the surface dipole. Short-latency, primary cortical evoked potentials (1 degrees CEP) were recorded with stimulation of myelinated afferents of the intrathoracic phrenic nerve in the contralateral post-cruciate gyrus of all animals (n = 42). The mean onset and peak latencies were 8.5 +/- 5.7 ms and 21.8 +/- 9.8 ms, respectively. The rostro-caudal surface location of the 1 degrees CEP was found between the rostral edge of the post-cruciate dimple (PCD) and the rostral edge of the ansate sulcus, medio-lateral location was between 2 mm lateral to the sagittal sulcus and the lateral end of the cruciate sulcus. Histological examination revealed that the 1 degrees CEP sites were recorded over areas 3a and 3b of the SI somatosensory cortex. Intracortical activation of 16 neurons with two patterns of neural activity was recorded: (1) short-latency, short-duration activation of neurons and (2) long-latency, long-duration activation of neurons. Short-latency neurons had a mean onset latency of 10.4 +/- 3.1 ms and mean burst duration of 10.1 +/- 3.2 ms. The short-latency units were recorded at an average depth of 1.7 +/- 0.5 mm below the cortical surface. The long-latency neurons had a mean onset latency of 36.0 +/- 4.2 ms and mean burst duration of 32.2 +/- 8.4 ms. The long-latency units were recorded at an average depth of 2.4 +/- 0.2 mm below the cortical surface. The results of the study demonstrated that phrenic nerve afferents have a short-latency central projection to the SI somatosensory cortex. The phrenic afferents activated neurons in lamina III and IV of areas 3a and 3b. The cortical representation of phrenic nerve afferents is medial to the forelimb, lateral to the hindlimb, similar to thoracic loci, hence the phrenic afferent SI site in the cat homunculus is consistent with body position (thoracic region) rather than spinal segment (C(5)-C(7)). The phrenic afferent activation of the somatosensory cortex is bilateral, with the ipsilateral cortical activation occurring subsequent to the contralateral. These results support the hypothesis that phrenic afferents provide somatosensory information to the cerebral cortex which can be used for diaphragmatic proprioception and somatosensation.

Multiple neuroanatomical tract-tracing using fluorescent Alexa Fluor conjugates of cholera toxin subunit B in rats.

Cholera toxin subunit B (CTB) is a highly sensitive retrograde neuroanatomical tracer. With the new availability of fluorescent Alexa Fluor (AF) conjugates of CTB, multiple neuroanatomical connections can be reliably studied and compared in the same animal. Here we provide a protocol that describes the use of AF-CTB for studying connections in the central nervous system of rats. The viscous properties of CTB allow small and discreet injection sites yet still show robust retrograde labeling. Furthermore, the AF conjugates are extremely bright and photostable, compared with other conventional fluorescent tracers. This protocol can also be adapted for use with other neuroanatomical tracers. Including a 7-d survival period, this protocol takes approximately 11 to 12 d to complete in its entirety.