Comparative MRI analysis of the forebrain of three sauropsida models.

The study of the brain by magnetic resonance imaging (MRI) allows to obtain detailed anatomical images, useful to describe specific encephalic structures and to analyze possible variabilities. It is widely used in clinical practice and is becoming increasingly used in veterinary medicine, even in exotic animals; however, despite its potential, its use in comparative neuroanatomy studies is still incipient. It is a technology that in recent years has significantly improved anatomical resolution, together with the fact that it is non-invasive and allows for systematic comparative analysis. All this makes it particularly interesting and useful in evolutionary neuroscience studies, since it allows for the analysis and comparison of brains of rare or otherwise inaccessible species. In the present study, we have analyzed the prosencephalon of three representative sauropsid species, the turtle Trachemys scripta (order Testudine), the lizard Pogona vitticeps (order Squamata) and the snake Python regius (order Squamata) by MRI. In addition, we used MRI sections to analyze the total brain volume and ventricular system of these species, employing volumetric and chemometric analyses together. The raw MRI data of the sauropsida models analyzed in the present study are available for viewing and downloading and have allowed us to produce an atlas of the forebrain of each of the species analyzed, with the main brain regions. In addition, our volumetric data showed that the three groups presented clear differences in terms of total and ventricular brain volumes, particularly the turtles, which in all cases presented distinctive characteristics compared to the lizards and snakes.

Nature or Nurture: Is the Digestive System of the Pontoporia blainvillei Influenced or Determined by Its Diet?

The Franciscana (also known as the La Plata River Dolphin) is a small dolphin that lives in the coastal waters of Brazil, Uruguay, and Argentina. This species is considered the most endangered marine mammal in the western South Atlantic Ocean. Anatomic dissection of the digestive system of 19 animals of different ages, including 2 neonates, 12 juveniles, and 5 adults, was performed. Parameters related to length, breadth, weight, and diameter of the digestive viscera were considered in each case. Our results show that the Franciscana dolphin presents differential characteristics in relation to several parts of the digestive system, including, specifically, the tongue, the teeth, the stomach, and the small intestine. Thus, this paper add precious information to the actual knowledge of this vulnerable marine mammal species in order to improve conservation efforts.

The Application of 3D Anatomy for Teaching Veterinary Clinical Neurology.

Neuroanatomy is always a challenging topic for veterinary students. It is widely accepted that understanding the anatomy of the central nervous system (CNS) is essential to explain many of the pathological processes that affect the brain. Although its study has varied over time to achieve this goal, in human and veterinary medicine it is difficult to find a teaching method that associates normal anatomy with pathological alterations of the brain. For the first time, we have created an educational tool that combines neuroanatomy and neuropathology, using different magnetic resonance (MR) images as a basis and EspINA software as analyzer, to obtain segmented structures and 3D reconstructions of the dog brain. We demonstrate that this combination is an optimal tool to help anatomists to understand the encephalon, and additionally to help clinicians to recognize illness including a multitude of neurological problems. In addition, we have tried to see whether photogrammetry, which is a common technique in other sciences, for example geology, could be useful to teach veterinary neuroanatomy. Although we still need further investigations, we have been able to generate 3D reconstructions of the whole brain, with very promising results to date.

The Respiratory System of the Arctocephalus australis in Comparison to the Dog as a Land-Carnivore: Are There Adaptations to Marine Life?

Marine mammals are divided into three groups, with similar adaptations resulting from their aquatic lifestyle: sirenians, pinnipeds, and cetaceans. The present work focused on the South American fur seal, or Arctocephalus australis, a carnivore included in the pinnipeds group. We assessed whether the anatomical features of the Arctocephalus australis' respiratory system are comparable to those of other land-carnivores or whether these individuals show anatomical adaptations related to their ability to dive or their breath-holding capacities. We studied 11 cadavers of Arctocephalus australis, which included adult (n = 2) and juvenile (n = 9) individuals, by anatomically dissecting their isolated entire respiratory system. Although it is generally similar to that in land-carnivores, we demonstrated that the Arctocephalus australis's respiratory apparatus shows several specific characteristics. Therefore, our results are of great importance for clinical diagnostic and wildlife conservation purposes.

Cortical synapses of the world's smallest mammal: An FIB/SEM study in the Etruscan shrew.

The main aim of the present study was to determine if synapses from the exceptionally small brain of the Etruscan shrew show any peculiarities compared to the much larger human brain. We analyzed the cortical synaptic density and a variety of structural characteristics of 7,239 3D reconstructed synapses, using using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM). We found that some of the general synaptic characteristics are remarkably similar to those found in the human cerebral cortex. However, the cortical volume of the human brain is about 50,000 times larger than the cortical volume of the Etruscan shrew, while the total number of cortical synapses in human is only 20,000 times the number of synapses in the shrew, and synaptic junctions are 35% smaller in the Etruscan shrew. Thus, the differences in the number and size of synapses cannot be attributed to a brain size scaling effect but rather to adaptations of synaptic circuits to particular functions.

An approach to the evaluation of the potency of cannabis resin in Madrid: A health hazard? / Aproximación a la evaluación de la potencia de la resina de cannabis en Madrid: ¿Un riesgo para la salud?

The present study investigates the concentration of Delta (9)-tetrahidrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in 60 samples of cannabis resin acquired on the streets of Madrid region and its potential danger to consumers' health. Additionally, we study the possible correlation between the potency of samples and their organoleptic characteristics. The analysis of cannabinoids was carried out using a high performance liquid chromatography (RP-HPLC-UV). To classify samples, a strength scale based on THC content was established. THC content in 76.7% of the samples was higher than 15%. This potency allows these samples to be classified as Schedule I or drugs with "unacceptable risk" for human health. THC content in 36.7% of the samples was 28.8% on average, which means very high potency. The mean CBD content was 5%, while the correlation between the CBD/THC ratio and potency was negative. The mean content of CBN was 1.74% and the CBN/THC ratio also showed a negative correlation in respect to potency. When investigating the possible correlation between sample potency and organoleptic characteristics, those samples which simultaneously presented sticky texture, high elasticity and light brown colour had very high potency, with an average THC content of 28.7%. Our study shows that the THC content of most of the cannabis that can be purchased in Madrid region is over 15% and poses a health hazard. Additionally, we demonstrate for the first time that only those samples with very high potency can be directly associated with certain organoleptic characteristics.

El presente estudio investiga la concentración de Delta(9)-tetrahidrocannabinol (THC), cannabidiol (CBD) y cannabinol (CBN) en 60 muestras de resina de cannabis adquiridas en las calles de Madrid y su potencial riesgo para la salud del consumidor. Adicionalmente, estudiamos la posible asociación entre la potencia de las muestras y sus características organolépticas. El análisis de cannabinoides se llevó a cabo mediante cromatografía líquida de alta resolución (RP-HPLC-UV). Atendiendo al contenido en THC se estableció una escala de potencia para clasificar las muestras. El 76,7% de las muestras tenía un contenido en THC superior al 15%, esta potencia las cataloga como drogas de Grado I con "riesgo inaceptable" para la salud. El 36,7% de las muestras presentaron un contenido medio en THC del 28,8% (potencia muy alta). El contenido medio en CBD fue del 5% y el de CBN 1,74%; ambas ratios, CBD/THC y CBN/THC, mostraron una correlación negativa con la potencia. Al investigar la posible asociación entra potencia y características organolépticas, se observó que las muestras que presentaban a la vez una textura pegajosa, una elasticidad alta y un color marrón claro, tenían una potencia muy alta, con un contenido medio en THC del 28.7%. Nuestro estudio muestra que el contenido en THC de la mayoría de la resina de cannabis que puede adquirirse en Madrid es superior al 15% y supone un elevado riesgo para la salud. Adicionalmente, demostramos por primera vez que solo aquellas muestras con una potencia muy alta pueden asociarse directamente con ciertas características organolépticas.

The Digestive System of the Arctocephalus australis in Comparison to the Dog as a Land-Carnivore Model.

Marine mammals play a critical ecological role as both predator and prey. They are divided into three groups that share similar adaptations to their aquatic life, but that have very different origins and life patterns: sirenians, pinnipeds, cetaceans. The species object of our interest is the South American fur seal or Arctocephalus australis, a carnivore classified within the group of pinnipeds. The objective of the present study was to evaluate whether the anatomical characteristics of the Arctocephalus australis' digestive system are similar to that of other land-carnivores or if, on the contrary, this species shows anatomical adaptations related to their life in the ocean. The study was carried out on 11 cadavers of the species Arctocephalus australis, made up of two adults and nine juveniles, by means of the anatomical dissection of their entire isolated digestive system. We demonstrate that, with several exceptions, the anatomical characteristics of the digestive system of the Arctocephalus australis are similar to those in other carnivores. Therefore, our data constitute an important contribution for clinical diagnostic and conservation purposes, for both veterinarians and biologists.

Sex Determination in Two Species of Anuran Amphibians by Magnetic Resonance Imaging and Ultrasound Techniques.

The objective of the present study was to evaluate whether gender determination in two amphibian species (Kaloula pulchra and Xenopus laevis) can be reliably carried out by means of magnetic resonance imaging (benchtop magnetic resonance imaging; BT-MRI) or ultrasound (high-resolution ultrasound; HR-US) techniques. Two species of healthy, sexually mature anurans have been used in the present study. Eight Kaloula (blind study) and six Xenopus were used as controls. Magnetic resonance imaging experiments were carried out on a low-field (1 Tesla) benchtop-MRI (BT-MRI) system. HR-US examination was performed with high-resolution equipment. Low-field BT-MRI images provided a clear and quantifiable identification of all the sexual organs present in both genders and species. The HR-US also allowed the identification of testes and ovaries in both species. Results indicate that BT-MRI allowed a very precise sex identification in both anuran species, although its use is limited by the cost of the equipment and the need for anesthesia. HR-US allowed an accurate identification of ovaries of both species whereas a precise identification of testes is limited by the ultrasonographer experience. The main advantages of this technique are the possibility of performing it without anesthesia and the higher availability of equipment in veterinary and zoo institutions.

Estimation of the number of synapses in the hippocampus and brain-wide by volume electron microscopy and genetic labeling.

Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole. Membrane-associated guanylate kinases (MAGUKs) are a family of scaffolding proteins that are expressed in excitatory glutamatergic synapses. We used genetic labeling of two of these proteins (PSD95 and SAP102), and Spinning Disc confocal Microscopy (SDM), to estimate the number of fluorescent puncta in the CA1 area of the hippocampus. We also used FIB-SEM, a three-dimensional electron microscopy technique, to calculate the actual numbers of synapses in the same area. We then estimated the ratio between the three-dimensional densities obtained with FIB-SEM (synapses/µm3) and the bi-dimensional densities obtained with SDM (puncta/100 µm2). Given that it is impractical to use FIB-SEM brain-wide, we used previously available SDM data from other brain regions and we applied this ratio as a conversion factor to estimate the minimum density of synapses in those regions. We found the highest densities of synapses in the isocortex, olfactory areas, hippocampal formation and cortical subplate. Low densities were found in the pallidum, hypothalamus, brainstem and cerebellum. Finally, the striatum and thalamus showed a wide range of synapse densities.

Volume Electron Microscopy Study of the Relationship Between Synapses and Astrocytes in the Developing Rat Somatosensory Cortex.

In recent years, numerous studies have shown that astrocytes play an important role in neuronal processing of information. One of the most interesting findings is the existence of bidirectional interactions between neurons and astrocytes at synapses, which has given rise to the concept of "tripartite synapses" from a functional point of view. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to examine in 3D the relationship of synapses with astrocytes that were previously labeled by intracellular injections in the rat somatosensory cortex. We observed that a large number of synapses (32%) had no contact with astrocytic processes. The remaining synapses (68%) were in contact with astrocytic processes, either at the level of the synaptic cleft (44%) or with the pre- and/or post-synaptic elements (24%). Regarding synaptic morphology, larger synapses with more complex shapes were most frequently found within the population that had the synaptic cleft in contact with astrocytic processes. Furthermore, we observed that although synapses were randomly distributed in space, synapses that were free of astrocytic processes tended to form clusters. Overall, at least in the developing rat neocortex, the concept of tripartite synapse only seems to be applicable to a subset of synapses.

A Fast Method for the Segmentation of Synaptic Junctions and Mitochondria in Serial Electron Microscopic Images of the Brain.

Recent electron microscopy (EM) imaging techniques permit the automatic acquisition of a large number of serial sections from brain samples. Manual segmentation of these images is tedious, time-consuming and requires a high degree of user expertise. Therefore, there is considerable interest in developing automatic segmentation methods. However, currently available methods are computationally demanding in terms of computer time and memory usage, and to work properly many of them require image stacks to be isotropic, that is, voxels must have the same size in the X, Y and Z axes. We present a method that works with anisotropic voxels and that is computationally efficient allowing the segmentation of large image stacks. Our approach involves anisotropy-aware regularization via conditional random field inference and surface smoothing techniques to improve the segmentation and visualization. We have focused on the segmentation of mitochondria and synaptic junctions in EM stacks from the cerebral cortex, and have compared the results to those obtained by other methods. Our method is faster than other methods with similar segmentation results. Our image regularization procedure introduces high-level knowledge about the structure of labels. We have also reduced memory requirements with the introduction of energy optimization in overlapping partitions, which permits the regularization of very large image stacks. Finally, the surface smoothing step improves the appearance of three-dimensional renderings of the segmented volumes.

Facilitation of AMPA receptor synaptic delivery as a molecular mechanism for cognitive enhancement.

Cell adhesion molecules and downstream growth factor-dependent signaling are critical for brain development and synaptic plasticity, and they have been linked to cognitive function in adult animals. We have previously developed a mimetic peptide (FGL) from the neural cell adhesion molecule (NCAM) that enhances spatial learning and memory in rats. We have now investigated the cellular and molecular basis of this cognitive enhancement, using biochemical, morphological, electrophysiological, and behavioral analyses. We have found that FGL triggers a long-lasting enhancement of synaptic transmission in hippocampal CA1 neurons. This effect is mediated by a facilitated synaptic delivery of AMPA receptors, which is accompanied by enhanced NMDA receptor-dependent long-term potentiation (LTP). Both LTP and cognitive enhancement are mediated by an initial PKC activation, which is followed by persistent CaMKII activation. These results provide a mechanistic link between facilitation of AMPA receptor synaptic delivery and improved hippocampal-dependent learning, induced by a pharmacological cognitive enhancer.

A stereological study of synapse number in the epileptic human hippocampus.

Hippocampal sclerosis is the most frequent pathology encountered in resected mesial temporal structures from patients with intractable temporal lobe epilepsy (TLE). Here, we have used stereological methods to compare the overall density of synapses and neurons between non-sclerotic and sclerotic hippocampal tissue obtained by surgical resection from patients with TLE. Specifically, we examined the possible changes in the subiculum and CA1, regions that seem to be critical for the development and/or maintenance of seizures in these patients. We found a remarkable decrease in synaptic and neuronal density in the sclerotic CA1, and while the subiculum from the sclerotic hippocampus did not display changes in synaptic density, the neuronal density was higher. Since the subiculum from the sclerotic hippocampus displays a significant increase in neuronal density, as well as a various other neurochemical changes, we propose that the apparently normal subiculum from the sclerotic hippocampus suffers profound alterations in neuronal circuits at both the molecular and synaptic level that are likely to be critical for the development or maintenance of seizure activity.

Morphological alterations to neurons of the amygdala and impaired fear conditioning in a transgenic mouse model of Alzheimer's disease.

Patients with Alzheimer's disease (AD) suffer from impaired memory and emotional disturbances, the pathogenesis of which is not entirely clear. In APP/PS1 transgenic mice, a model of AD in which amyloid beta (Abeta) accumulates in the brain, we have examined neurons in the lateral nucleus of the amygdala (LA), a brain region crucial to establish cued fear conditioning. We found that although there was no neuronal loss in this region and Abeta plaques only occupy less than 1% of its volume, these mice froze for shorter times after auditory fear conditioning when compared to their non-transgenic littermates. We performed a three-dimensional analysis of projection neurons and of thousands of dendritic spines in the LA. We found changes in dendritic tree morphology and a substantial decrease in the frequency of large spines in plaque-free neurons of APP/PS1 mice. We suggest that these morphological changes in the neurons of the LA may contribute to the impaired auditory fear conditioning seen in this AD model.

Nitric oxide synthase in the external urethral sphincter of the sheep: immunohistochemical and functional study.

PURPOSE: We studied the distribution of neuronal nitric oxide synthase (nNOS) and the effects of nitric oxide (NO) modulating drugs on contractile function of the external urethral sphincter of lambs. Gender differences were evaluated. MATERIALS AND METHODS: Longitudinal and transverse sections of the external urethral sphincter from 10 female and 10 male lambs were studied using reduced nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and nNOS immunocytochemistry. Isometric contractile responses to electrical field stimulation were recorded from external urethral sphincter preparations from 47 female and 45 male lambs and the effects of NO modulating drugs were evaluated. RESULTS: We detected nNOS in the sarcolemma of some but not all striated fibers, where nNOS seems to be concentrated at the neuromuscular junction. In addition, nNOS was present in nerve fibers and intramural ganglia. The density of innervation decreased toward the distal part of the external urethral sphincter and was higher in male preparations. No significant functional effects of the NOS inhibitor NG-nitro-L-arginine (10 mM.) or the NO donors diethylamine and spermine NONOate (Sigma Chemical Co., St. Louis, Missouri) (5 mM. each) on external urethral sphincter isometric contractility were found in either gender. CONCLUSIONS: Despite the evidence for nNOS at the sarcolemma and nerve fibers of the external urethral sphincter the physiological relevance of these immunohistochemical findings remains to be determined.

NADPH-diaphorase distribution in the rabbit superior colliculus and co-localization with calcium-binding proteins.

Nitric oxide (NO) and calcium-binding proteins (CaBP) are important neuromodulators implicated in brain plasticity and brain disease. In addition, the mammalian superior colliculus (SC) has one of the highest concentrations of NO within the brain. The present study was designed to determine the distribution of nitric oxide-synthesizing neurons in the SC of the rabbit by enzyme histochemistry for reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), and its degree of co-localization with CaBP, parvalbumin (PV) and calbindin (CB). NADPH-d-labelled fibres formed dense patches of terminal buttons within the intermediate grey layer and streams of fibres within the deepest layers of SC. Cells expressing NOS constitute a subpopulation of neurons in which practically all cell types are represented. Combined PV/NADPH-d experiments showed a complete lack of co-localization within individual neurons and fibres. On the contrary, double-labelled neurons appeared in CB/NADPH-d-stained sections, only in the superficial layers, and mostly in the SGS and SO. These cells, which were intermingled with other neurons containing either NADPH-d or CB, appear to be a subtype of narrow-field and wide-field vertical cells, and display an anterior-posterior gradient of density. Owing to the involvement of the superficial layers of the SC in the organization and integration of the visual information, it is suggested that these neurons may play a concrete role within the visual circuits. Our data indicate a clear selectivity in the expression of NADPH-d, PV and CB in the SC, and that NO and CB probably serve as co-modulators and/or co-transmitters in the connectivity of the superficial layers of this midbrain structure.