Partial nicotinic receptor blockade unmasks a modulatory role of nitric oxide on urethral striated neuromuscular transmission.

The objective of this study was to investigate the possible modulatory role of endogenous nitric oxide (NO) production on the urethral striated muscle (USM) function in the sheep urethra. Significant NO synthase (NOS) activity was measured in both the particulate and cytosolic fractions of USM homogenates. NOS activity was calcium-dependent and showed greater inhibition by NOS inhibitors selective of the neural NOS isoform (nNOS). nNOS immunoreactivity was present in intramural nerves as well as in the sarcolemma of some striated fibers, being denser at the neuromuscular junction (NMJ). Double immunolabeling showed co-localization of nNOS with both alpha-bungarotoxin and choline acetyltransferase, at the USM endplates. For the first time, functional data support a role of NO on the USM contractility "in vitro," which became evident following partial nicotinic receptor inactivation with low concentrations of D-tubocurarine. Only under D-tubocurarine (0.25 microM) treatment, different NOS inhibitors, specially N(G)-propyl-L-arginine, as well as the guanylate cyclase inhibitor ODQ, all showed a significant enhancing effect on contractions induced by electrical field stimulation of intrinsic somatic nerves. These data suggest that local production of NO at the urethral NMJ may modulate release and/or action of acetylcholine on motor endplates by cyclic GMP-mediated effects. This modulatory action could be especially relevant when neuromuscular transmission at the USM is impaired.

Stereological study of the external urethral sphincter in the female urethra of the lamb: a new model for studies on urinary continence.

Some of the morphometrical features and functional properties of the lamb external urethral sphincter (EUS) were determined by means of ATPase histochemistry. In this study, eight female urethras of 2- to 3-month-old lambs were used. The samples were studied by using an unbiased stereological design and were statistically evaluated with a Kolmogorov-Smirnov test. Additionally, the mechanical activity of the different fibre types was analysed by measuring the isometric changes in tension induced by electrical field stimulation (EFS) of intrinsic nerves. The lamb EUS has an average fibre density of 1142.37 fibres/mm(2) (SD = 279.33). Our data showed the predominance of fast-twitch fibres or type II fibres, with a proportion of 30% (type I) and 70% (type II) in all cases. These percentages remained the same along the length of the EUS. However, a clear tendency to increase the number of both fibre types in the mid-EUS was observed. Interestingly, type I and II fibres are intermingled throughout the whole lamb EUS. Dynamic parameters of the contraction induced in lamb EUS preparations by EFS of intramural nerves, were intermediate between fast- and slow-twitch muscles. Furthermore, contractile activity showed no decrease in amplitude upon repetitive stimulation, suggesting the presence of a mixture of fibre types, in which fast but fatigue-resistant fibres (probably type IIA) seem to predominate. Our results indicate that lambs could be suggested as a new biological model to study those pathological processes, which involve the sphincteric regulation of the EUS.

Neurochemical heterogeneity of the thalamic reticular and perireticular nuclei in developing rabbits: patterns of calbindin expression.

The thalamic reticular nucleus (TRN) forms an essential part of the circuits that link the thalamus to the cortex, whereas the perireticular thalamic nucleus (PRN) consists of scattered neurons that are located in the internal capsule, in close relation to the TRN. A common feature of these nuclei in different species is the immunoreactivity for some calcium binding proteins with a developmental pattern of expression. In the present study, sections from rabbits at different ages were examined to determine the calbindin (CB) expression in the developing TRN and PRN at the first stages of development. These CB-expressing cells constitute an important subpopulation of neurons in the caudal half of the developing TRN. In the adult, there are still positive CB somata in the middle and caudal halves of the nucleus. In the PRN, where the developmental pattern of CB expression has not been described before, the number of CB perireticular cells decreases progressively. Our results, together with previous data in the rabbit suggest the existence of remarkable neurochemical heterogeneity in the TRN and PRN of the rabbit.

The thalamic reticular and perireticular nuclei in developing rabbits: patterns of parvalbumin expression.

Due to its strategic position, the thalamic reticular nucleus (TRN) plays an important role within the thalamo-cortical circuits. The perireticular thalamic nucleus (PRN) is a smaller group of cells, which is associated with the TRN and lies among the fibres of the internal capsule (IC). Studies of nuclei in rodents and carnivores have been conducted employing a number of different tools. The use of calcium-binding proteins is one example. It needs to be noted that rabbits have been regarded as intermediate between rodents and carnivores in relation to local GABAergic circuits. In the present study, sections from rabbits at different ages (prenatal, postnatal and adult) were examined to determine the parvalbumin (PV) expression in the developing TRN and PRN. In the TRN, there is one wave of PV expression during development, from caudal parts of the nucleus towards the rostral pole. At E22 there is already an incipient PV expression. In the adult stage, the TRN is completely positive to PV. The present study clearly indicates the presence of the PRN in the developing rabbit. The first PV positive cells were visible at E24, meanwhile the immunoreactivity was at its maximum at early postnatal stages (P0-P8). Two different types of perireticular cells in the IC were identified and the changes concerning neuronal morphology and orientation were described. The comparison between these results and previous data obtained in rats, ferrets or cats suggest that rabbits could represent an intermediate stage in the evolution of thalamic circuits and could be considered as useful neurobiological model.

Age-related changes in the ventricular system of the dog brain.

The cerebral ventricles represent cavities of the brain which are used for diagnostic purposes. Although a wide variety of age related changes have been described in the Central Nervous System (CNS) of many species, few studies about the effect of ageing on the canine brain have been published until now. However, to date there is no previous data concerning the ventricular system of dogs. The present study deals with the morphometry of the various parts of the cerebral cavities of the German Shepherd dog by means of Batson's casts and the possible changes which take place in the ventricular system with age and/or sex. In this study, two age groups were considered: young (2-5 years) and old (10-12 years). A total number of forty seven dogs (12 young males, 13 old males, 13 young females and 9 old females), weighing 34-42 kg, were used for experimentation. Our results describe the enlargement which takes place in the ventricular system with age, which is probably related to a general age-related atrophy of neural tissue. On the contrary, there are no remarkable changes related to sex. These age-related changes are similar to the best known changes which occur in the human species. Our data could corroborate the usefulness of the dog as a natural animal model for the study of normal ageing and neurodegenerative diseases.

Calbindin D28k and parvalbumin immunoreactivity in the rabbit superior colliculus: an anatomical study.

The expression pattern of two calcium binding proteins (CaBP), calbindin D28k (CB) and parvalbumin (PV), in the superior colliculus (SC) of the adult rabbit, as well as the morphology of the immunoreactive cells were examined. The study was performed on 12 rabbits. Coronal sections from postmortem SC were analyzed by light microscopy, and drawings of CaBP-labeled cells were obtained using a drawing tube. No previous information is available on either the CB/PV expression or the morphology of CB/PV positive cells in the SC of the adult rabbit. Therefore, in this study we show that CB neurons and neuropil form three main tiers: the first located within the stratum zonale (SZ) and the upper part of the stratum griseum superficiale (SGS), the second located within the stratum griseum intermedium (SGI), and the third, located within the medial and central areas of the stratum griseum profundum (SGP). In contrast to this layer labeling, almost no CB-positivity is found within the other collicular layers. On the other hand, the densest concentration of PV labeled cells and terminals is found within a single dense tier that spanned the ventral part of the startum griseum superficiale (SGS) and the dorsal part of the stratum opticum (SO). Anti-PV neurons are also scattered through the deeper layers below the dense tier. In contrast, almost no anti-PV labeled neurons or neuropil are found within the stratum zonale (SZ) and upper SGS. This distribution represents a new pattern of sublamination in the SC of this species. All the previously described cell types in other mammals are observed in the rabbit SC: marginal cells, horizontal cells, pyriform cells, narrow-field vertical cells, wide-field vertical cells, and stellate/multipolar cells. Detailed drawings of all these cellular types are represented to show their complete morphology. The results of this study indicate that both CB and PV are present in a variety of neurons, which present a number of homologies between mammals, but have a different location and/or distribution, according to the different species. These findings are thus relevant to better understand the organisation of the SC in mammals.

Serotonergic connections to the ventral oral pontine reticular nucleus: implication in paradoxical sleep modulation.

Cholinergic microstimulation of the ventral part of the oral pontine reticular nucleus (vRPO) in cats generates and maintains paradoxical sleep. The implication of rostral raphe nuclei in modulating the sleep-wakefulness cycle has been based on their serotonergic projections to the pontine structures responsible for the induction of paradoxical sleep. However, serotonergic neurons have also been described in brainstem structures other than the raphe nuclei. The aim of the present work is to trace the origin of the serotonergic afferents to the vRPO and to the locus coeruleus alpha and perilocus coeruleu alpha nuclei, closely related with different paradoxical sleep events. Anterograde and retrograde horseradish peroxidase conjugated with wheat germ agglutinin tracer injections in these nuclei in cats were combined with serotonin antiserum immunohistochemistry. Our results demonstrate that reciprocal connections linking the rostral raphe nuclei to those oral pontine nuclei are scarce. The percentage of double-labeled neurons after injections in the vRPO averaged 18% in rostral raphe nuclei, while a level of 82% was estimated in mesopontine tegmentum structures other than the raphe nuclei. These results showed that the main source of serotonin to the vRPO, implicated in generation and maintenance of paradoxical sleep, arises from these mesopontine tegmentum structures. This indicates that the serotonin modulation of paradoxical sleep could be the result of activation in non-raphe mesopontine tegmentum structures. The existence of a complicated network in the vRPO, which maintains a balance between different neurotransmitters responsible for the generation and alternance of paradoxical sleep episodes, is discussed.

A quantitative study of ganglion cells in the goat retina.

As in a number of mammals, the most prominent feature of the ganglion-cell layer in the retina of the murciano-granadina goat is an increase in the density of ganglion cells in the central area, as well as a concentration along a ridge extending horizontally across the retina, below the optic disc, and in the upper temporal retina. Thus, there is an area of maximum density and two streaks that are known as the 'horizontal' and 'vertical' streak. The isodensity lines of ganglion-cell distribution is toughly concentric, with their values varying from 304 cells/mm2 in the periphery to 3592 cells/mm2 in the central area, with the cells densely packed. There were some individual differences amongst the animal studied, although all of them were purebred animals.

Spinal cord central canal of the German shepherd dog: morphological, histological, and ultrastructural considerations.

This study deals with some macroscopical, microscopical, and ultrastructural aspects of the spinal cord central canal of the German shepherd dog. The caudal end of the spinal cord is constituted by the conus medullaris, which may extend to the first sacral vertebra, the terminal ventricle, and the filum terminale. The latter structure is considered as internum (second to third sacral vertebrae) or externum (fifth caudal vertebra), according to its relation to the dura mater. Occasionally, there is a second anchorage which is close to the level of the sixth caudal vertebra. The central canal is surrounded by a ciliated ependymal epithelium, which differs depending upon the levels. The most caudal part of the filum terminale bears a columnar ciliated ependymal epithelium surrounded by two layers of glia and pia mater, which separate the central canal from the subarachnoid space. Microfil injections show a communication between the cavity and the subarachnoid space, as the plastic is able to pass through the ependymal epithelium. At the level of the terminal ventricle there are real separations of the ependymal epithelium, which seem to connect the lumen of the spinal canal with the subarachnoid space. These structures probably constitute one of the drainage pathways of the cerebrospinal fluid. The diameter of the central canal is related to the age of the animal. However, even in very old animals the spinal cord central canal reaches the tip of the filum terminale and remains patent until death. At the ultrastructural level the ependymal cells present villi, located on cytoplasmic projections, cilia, dense mitochondria, and oval nuclei.

A quantitative study of ganglion cells in the German shepherd dog retina.

As in the number of mammals, the most prominent feature of the ganglion-cell layer in the retina of the German shepherd dog is the sharp increase in the density of ganglion cells in the central area. There is an area of maximum density and also a 'cat-like' visual streak, located dorsal to the optic disc. The isodensity lines of ganglion-cell distribution is roughly concentric. Their values vary from 5300-13,000 cells/mm2 in the central area, with the cells densely packed, to 1000 cells/mm2 or less in the periphery, where the cells are sparsely distributed. There were some individual differences amongst the animals studied, although all of them were pure-bred dogs. This suggests that the configuration of the retina in the canine species is not only dependent on the breed itself but also on some other parameters such as phylogenetic heritage, environment, aptitude, lifestyle, or even training.

Location and anatomical connections of a paradoxical sleep induction site in the cat ventral pontine tegmentum.

The brainstem mechanisms for the generation of paradoxical sleep are under considerable debate. Previous experiments in cats have demonstrated that injections of the cholinergic agonist carbachol into the oral pontine tegmentum elicit paradoxical sleep behaviour and its polygraphic correlates. The different results on the pontine structures that mediate this effect do not agree. We report here that limited microinjections of a carbachol solution into the ventral part of the oral pontine reticular nucleus in the cat induce, with a short latency, a dramatic, long-lasting increase in paradoxical sleep. Moreover, neuronal tracing experiments show that this pontine site is connected with brain structures responsible for the different bioelectric events of paradoxical sleep. These two facts suggest that the ventral part of the oral pontine reticular nucleus is a nodal link in the neuronal network underlying paradoxical sleep mechanisms.

GABA-synthesizing neurons in the medulla: their relationship to serotonin-containing and spinally projecting neurons in the rat.

GABA-synthesizing neurons were identified in the medulla of the rat by peroxidase-antiperoxidase (PAP) immunohistochemistry for glutamic acid decarboxylase (GAD). Using diaminobenzidine (DAB) either alone or intensified with silver, a relatively large number of GAD-immunoreactive neurons were evident within the reticular formation, raphe nuclei and vestibular nuclei. In all these areas, profuse GAD-immunoreactive varicosities appeared to contact the soma and dendrites of both non-GABA and GABA neurons. These observations suggest that GABA neurons may act as interneurons or local projection neurons within the medulla and accordingly exert a potent inhibitory and/or disinhibitory control on bulbar projection neurons. Within the ventral reticular formation (pars alpha and ventralis of the gigantocellular reticular field) and raphe magnus, large numbers of prominent GAD-immunoreactive neurons resembled in size and morphology and overlapped in distribution the serotonin-immunoreactive neurons of the same regions. However, by sequential double immunostaining utilizing DAB as a chromogen for serotonin (5-HT) and benzidine dihydrochloride (BDHC) for GAD, it was found that GAD-containing neurons were distinct from 5-HT-containing neurons. Following injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the upper cervical spinal cord and combined processing for WGA-HRP (using tetramethylbenzidine [TMB] with cobalt) and immunohistochemistry (with DAB), a contingent of spinally projecting neurons were found to contain GAD. The GAD-immunoreactive reticulo- and raphe-spinal neurons were most frequent within the pars alpha and ventralis of the gigantocellular reticular fields and the raphe magnus, where they were approximately equal in number to the coexistent, but distinct 5-HT spinally projecting neurons. GABA neurons of the medulla may thus contribute directly to the bulbar inhibitory influence upon spinal sensory and motor systems.