Manuel Lisardo Sánchez

Mapping of somatostatin-28 (1-12) in the alpaca diencephalon.

Using an immunocytochemical technique, we report for the first time the distribution of immunoreactive cell bodies and fibers containing somatostatin-28 (1-12) in the alpaca diencephalon. Somatostatin-28 (1-12)-immunoreactive cell bodies were only observed in the hypothalamus (lateral hypothalamic area, arcuate nucleus and ventromedial hypothalamic nucleus). However, immunoreactive fibers were widely distributed throughout the thalamus and hypothalamus. A high density of such fibers was observed in the central medial thalamic nucleus, laterodorsal thalamic nucleus, lateral habenular nucleus, mediodorsal thalamic nucleus, paraventricular thalamic nucleus, reuniens thalamic nucleus, rhomboid thalamic nucleus, subparafascicular thalamic nucleus, anterior hypothalamic area, arcuate nucleus, dorsal hypothalamic area, around the fornix, lateral hypothalamic area, lateral mammilary nucleus, posterior hypothalamic nucleus, paraventricular hypothalamic nucleus, suprachiasmatic nucleus, supraoptic hypothalamic nucleus, and in the ventromedial hypothalamic nucleus. The widespread distribution of somatostatin-28 (1-12) in the thalamus and hypothalamus of the alpaca suggests that the neuropeptide could be involved in many physiological actions.

Mapping of CGRP in the alpaca diencephalon.

We report the distribution of immunoreactive cell bodies and fibers containing calcitonin gene-related peptide in the alpaca diencephalon. This study was carried out in alpacas that lived from birth to death at 0 m above sea level. Immunoreactive fibers were widely distributed throughout the thalamus and hypothalamus. A moderate density of these fibers was found in the zona incerta, the central medial, subparafascicular, reuniens and rhomboid thalamic nuclei, in the preoptic, anterior, lateral and dorsal hypothalamic areas, around the fornix, in the posterior, ventromedial and paraventricular hypothalamic nuclei and in the lateral mammillary nucleus. Cell bodies were only found in the hypothalamus: a high density in the paraventricular and supraoptic hypothalamic nuclei and a low density in the anterior, lateral and dorsal hypothalamic areas, around the fornix, and in the posterior and ventromedial hypothalamic nuclei. The widespread distribution of calcitonin gene-related peptide in the alpaca diencephalon suggests that it is involved in many physiological actions that must be investigated in-depth in the future, since alpacas lives from 0 m above sea level to altitudes of up to 5000 m altitude and hence the involvement of neuropeptides in special and unique regulatory physiological mechanisms could be suggested.

Distribution of methionine-enkephalin in the minipig brainstem

We have studied the distribution of immunoreactive cell bodies and axons are containing methionine-enkephalin in the minipig brainstem. Immunoreactive axons were widely distributed, whereas the distribution of perikarya was less widespread. A high or moderate density of axons containing methionine-enkephalin were found from rostral to caudal levels in the substantia nigra, nucleus interpeduncularis, nucleus reticularis tegmenti pontis, nucleus dorsalis raphae, nucleus centralis raphae, nuclei dorsalis and ventralis tegmenti of Gudden, locus ceruleus, nucleus sensorius principalis nervi trigemini, nucleus cuneatus externalis, nucleus tractus solitarius, nuclei vestibularis inferior and medialis, nucleus ambiguus, nucleus olivaris inferior and in the nucleus tractus spinalis nervi trigemini. Immunoreactive perikarya were observed in the nuclei centralis and dorsalis raphae, nucleus motorius nervi trigemini, nucleus centralis superior, nucleus nervi facialis, nuclei parabrachialis medialis and lateralis, nucleus ventralis raphae, nucleus reticularis lateralis and in the formatio reticularis. We have also described the presence of perikarya containing methionine-enkephalin in the nuclei nervi abducens, ruber, nervi oculomotorius and nervi trochlearis. These results suggest that in the minipig the pentapeptide may be involved in many physiological functions (for example, proprioceptive and nociceptive information; motor, respiratory and cardiovascular mechanisms).

Immunohistochemical mapping of pro-opiomelanocortin- and pro-dynorphin-derived peptides in the alpaca (Lama pacos) diencephalon

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibres containing non-opioid peptides (adrenocorticotropin hormone (ACTH), alpha-melanocyte-stimulating hormone) and opioid peptides (beta-endorphin (1-27), alpha-neo-endorphin, leucine-enkephalin) in the alpaca diencephalon. No immunoreactive cell bodies containing ACTH were found. Perikarya containing the other four peptides were observed exclusively in the hypothalamus and their distribution was restricted. Perikarya containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin showed a more widespread distribution than those containing leucine-enkephalin or beta-endorphin (1-27). Cell bodies containing pro-opiomelanocortin-derived peptides were observed in the arcuate nucleus, anterior and lateral hypothalamic areas and in the ventromedial and supraoptic hypothalamic nuclei, whereas perikarya containing alpha-neo-endorphin (a pro-dynorphin-derived peptide) were found in the arcuate nucleus, dorsal and lateral hypothalamic areas, and in the paraventricular, ventromedial and supraoptic hypothalamic nuclei. Immunoreactive cell bodies containing leucine-enkephalin were found in the lateral hypothalamic area and in the paraventricular hypothalamic nucleus. Immunoreactive fibres expressing pro-opiomelanocortin-derived peptides were more numerous than those expressing pro-dynorphin-derived peptides. A close anatomical relationship was observed: in all the diencephalic nuclei in which beta-endorphin (1-27)-immunoreactive fibres were found, fibres containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were also observed. Fibres containing beta-endorphin (1-27), alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were widely distributed throughout the diencephalon, but fibres containing ACTH or leucine-enkephalin showed a moderate distribution. The distribution of the five peptides studied here is also compared with that reported previously in other mammalian species. The widespread distribution observed indicates that both the pro-dynorphin and the pro-opiomelanocortin systems are involved in multiple physiological actions (e.g., food intake, thermoregulation, neuroendocrine and reproductive mechanisms) in the alpaca diencephalon.

Mapping of methionine-enkephalin-arg6-gly7-leu8 in the human diencephalon

Using an immunohistochemical technique, we mapped the immunoreactive structures containing methionine-enkephalin-Arg6-Gly7-Leu8 (Met-8) (a marker for the pro-enkephalin system) in the human diencephalon. Compared with previous studies, we observed a more widespread distribution of Met-8 in the human diencephalon. Met-8-immunoreactive cell bodies and fibers exhibited a more widespread distribution in the hypothalamus than in the thalamus. We observed six populations of Met-8-immunoreactive cell bodies. These perikarya were observed in the paratenial thalamic nucleus, ventromedial and dorsomedial hypothalamic nuclei, lateral hypothalamic area, pallidohypothalamic nucleus and in the paraventricular hypothalamic nucleus (posterior part). In the thalamus, Met-8-immunoreactive fibers were primarily observed in the midline region, whereas in the hypothalamus, these fibers were widely distributed. In general, a moderate/low density of Met-8-immunoreactive fibers was observed in the diencephalic nuclei. A moderate density was observed in the paraventricular thalamic nucleus, reuniens thalamic nucleus, lateral and medial geniculate nuclei, dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus (posterior part) and ventromedial hypothalamic nucleus. The present study is the first to demonstrate the presence of clusters of Met-8-immunoreactive cell bodies in the human thalamus and hypothalamus, the distribution of fibers containing neuropeptides in the hypothalamus and the presence of these fibers in several thalamic nuclei. This neuroanatomical study will serve to elucidate the physiological roles of Met-8 in future studies of the human diencephalon.

Distribution of Neurotensin and Somatostatin-28 (1-12) in the Minipig Brainstem.

Using an indirect immunoperoxidase technique, an in depth study has been carried out for the first time on the distribution of fibres and cell bodies containing neurotensin and somatostatin‐28 (1–12) (SOM) in the minipig brainstem. The animals used were not treated with colchicine. The distribution of neurotensin‐ and SOM‐immunoreactive fibres was seen to be quite similar and was moderate in the minipig brainstem: a close anatomical relationship between both neuropeptides was observed. The distribution of cell bodies containing neurotensin or SOM was quite different and restricted. Cell bodies containing neurotensin were found in four brainstem nuclei: nucleus centralis raphae, nucleus dorsalis raphae, in the pars centralis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis raphae. Cell bodies containing SOM were found in six nuclei/regions of the brainstem: nucleus ambiguus, nucleus dorsalis motorius nervi vagus, formatio reticularis, nucleus parabrachialis medialis, nucleus reticularis lateralis and nucleus ventralis raphae. According to the observed anatomical distribution of the immunoreactive structures containing neurotensin or SOM, the peptides could be involved in sleep‐waking, nociceptive, gustatory, motor, respiratory and autonomic mechanisms.

Mapping of somatostatin-28 (1-12) in the alpaca (Lama pacos) brainstem.

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibers containing somatostatin‐28 (1‐12) in the alpaca brainstem. Immunoreactive fibers were widely distributed throughout the whole brainstem: 34 brainstem nuclei/regions showed a high or a moderate density of these fibers. Perikarya containing the peptide were widely distributed throughout the mesencephalon, pons and medulla oblongata. Cell bodies containing somatostatin‐28 (1‐12) were observed in the lateral and medial divisions of the marginal nucleus of the brachium conjunctivum, reticular formation (mesencephalon, pons and medulla oblongata), inferior colliculus, periaqueductal gray, superior colliculus, pericentral division of the dorsal tegmental nucleus, interpeduncular nucleus, nucleus of the trapezoid body, vestibular nucleus, motor dorsal nucleus of the vagus, nucleus of the solitary tract, nucleus praepositus hypoglossi, and in the substantia nigra. This widespread distribution indicates that somatostatin‐28 (1‐12) is involved in multiple physiological actions in the alpaca brainstem. Microsc. Res. Tech. 78:363–374, 2015.

Distribution of CGRP in the Minipig Brainstem

For the first time, an in‐depth study has been made of the distribution of fibers and cell bodies containing calcitonin gene‐related peptide (CGRP) in the minipig brainstem using an indirect immunoperoxidase technique. The animals studied were not treated with colchicine. Cell bodies containing CGRP were found in 20 nuclei/regions of the brainstem. These perikarya were located in somatomotor, brachiomotor and raphae nuclei, nucleus ambiguus, substantia nigra, nucleus reticularis tegmenti pontis, nucleus prepositus hypoglossi, nuclei olivaris inferior and superior, nuclei pontis, formatio reticularis, nucleus dorsalis tegmenti of Gudden, and in the nucleus reticularis lateralis. Fourteen of the 20 brainstem nuclei showed a high density of immunoreactive cell bodies. In comparison with other species, the minipig, together with the rat, show the most widespread distribution of cell bodies containing CGRP in the mammalian brainstem. Immunoreactive fibers were also observed in the brainstem. However, in the minipig brainstem the density of these fibers is low, as in many brainstem nuclei only single immunoreactive fibers were observed. A high density of immunoreactive fibers was only observed in the pars caudalis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis tegmenti of Gudden. According to the observed anatomical distribution of the immunoreactive structures containing CGRP, the peptide could be involved in motor, somatosensory, gustative, and autonomic mechanisms. Microsc. Res. Tech. 77:374–384, 2014.

Immunohistochemical mapping of neurotensin in the alpaca diencephalon

INTRODUCTION The distribution of the immunoreactive cell bodies and fibers containing neurotensin in the alpaca diencephalon was determined by an immunohistochemical technique. MATERIAL AND METHODS The study was carried out in four male alpacas that lived at sea level. Brains of deeply anesthetized animals were fixed by perfusion with 4% paraformaldehyde. Cryostat sections were stained by a standard immunohistochemical method. RESULTS Cell bodies containing neurotensin were observed in the zona incerta and hypothalamus. A low/moderate density of these cell bodies was observed in the lateral hypothalamic area, anterior and dorsal hypothalamic areas, suprachiasmatic nucleus, periventricular region of the hypothalamus and in the ventromedial hypothalamic nucleus. In both thalamus and hypothalamus, immunoreactive fibers showed a widespread distribution. In the thalamus, a high density of these fibers was mainly found in the midline nuclei, whereas in the hypothalamus a high density was in general observed in the whole structure. CONCLUSIONS In comparison with other mammals, the thalamus of the alpaca showed the most widespread distribution of neurotensin-immunoreactive fibers. The widespread distribution of neurotensin through the alpaca diencephalon suggests that the peptide can be involved in many physiological actions.

Generation of specific antisera directed against D-amino acids: focus on the neuroanatomical distribution of D-glutamate and other D-amino acids

This review updates the findings about the anatomical distribution (using immunohistochemical techniques) and possible functions of D-glutamate in the central nervous system of mammals, as well as compares the distribution of D-glutamate with the distribution of the most studied D-amino acids: D-serine and D-aspartate. The protocol used to obtain highly specific antisera directed against D-amino acids is also reported. Immunoreactivity for D-glutamate was found in dendrites and cell bodies, but not in nerve fibers. Perikarya containing D-glutamate were found in the mesencephalon and thalamus. The highest density of cell bodies was found in the dorsal raphe nucleus, the mesencephalic central grey matter, the superior colliculus, and in the subparafascicular thalamic nucleus. In comparison with the distribution of immunoreactive cell bodies containing D-serine or D-aspartate, the distribution of D-glutamate-immunoreactive perikarya is less widespread. Currently, the physiological actions mediated by D-glutamate in the brain are unknown but the restricted neuroanatomical distribution of this D-amino acid suggests that D-glutamate could be involved in very specific physiological mechanisms. In this sense, the possible functional roles of D-glutamate are discussed.