Denise Maria Zancan

The terrestrial Gastropoda Megalobulimus abbreviatus as a useful model for nociceptive experiments: effects of morphine and naloxone on thermal avoidance behavior

We describe the behavior of the snail Megalobulimus abbreviatus upon receiving thermal stimuli and the effects of pretreatment with morphine and naloxone on behavior after a thermal stimulus, in order to establish a useful model for nociceptive experiments. Snails submitted to non-functional (22 degrees C) and non-thermal hot-plate stress (30 degrees C) only displayed exploratory behavior. However, the animals submitted to a thermal stimulus (50 degrees C) displayed biphasic avoidance behavior. Latency was measured from the time the animal was placed on the hot plate to the time when the animal lifted the head-foot complex 1 cm from the substrate, indicating aversive thermal behavior. Other animals were pretreated with morphine (5, 10, 20 mg/kg) or naloxone (2.5, 5.0, 7.5 mg/kg) 15 min prior to receiving a thermal stimulus (50 degrees C; N = 9 in each group). The results (means +/- SD) showed an extremely significant difference in response latency between the group treated with 20 mg/kg morphine (63.18 +/- 14.47 s) and the other experimental groups (P < 0.001). With 2.5 mg/kg (16.26 +/- 3.19 s), 5.0 mg/kg (11.53 +/- 1.64 s) and 7.5 mg/kg naloxone (7.38 +/- 1.6 s), there was a significant, not dose-dependent decrease in latency compared to the control (33.44 +/- 8.53 s) and saline groups (29.1 +/- 9.91 s). No statistically significant difference was found between the naloxone-treated groups. With naloxone plus morphine, there was a significant decrease in latency when compared to all other groups (minimum 64% in the saline group and maximum 83.2% decrease in the morphine group). These results provide evidence of the involvement of endogenous opioid peptides in the control of thermal withdrawal behavior in this snail, and reveal a stereotyped and reproducible avoidance behavior for this snail species, which could be studied in other pharmacological and neurophysiological studies.

Seasonal variation of the carbohydrate and lipid metabolism in a land pulmonate gastropod, Megalobulimus oblongus

Abstract Seasonal variations of carbohydrate and lipid metabolism in Megalobulimus oblongus were investigated. Haemolymph glucose levels were higher in summer than in other seasons. The glycogen content of the hepatopancreas, muscle and mantle was constant throughout the year, except in winter when lower values were found. Hepatopancreas total lipid increased in spring, whereas gonad total lipid increased in winter and spring. Glycogen metabolism seems to constitute the source of energy substrate during winter while lipid metabolism may be related to the annual reproductive cycle.

Seasonal variations in the biochemical composition and reproductive cycle of the ghost crab Ocypode quadrata (Fabricius, 1787) in Southern Brazil.

The ghost crab, Ocypode quadrata, is found on sandy beaches from the United States to Southern Brazil. Because there is still little information about the metabolism and reproduction of O. quadrata on the southern coast of Brazil, the objectives of this study were to (i) evaluate the effect of seasonal variations on the carbohydrate, lipid, and protein metabolism of O. quadrata at Rondinha Beach, a beach with high anthropogenic activity, and to compare it with data from Siriú Beach, which has less human activity; and (ii) describe the effect of seasonal variations on the histological characteristics of male and female gonads, in order to assess the reproductive capacity of the crabs. The gonads of male crabs showed no significant variations in the gonadosomatic index (GSI) and glycogen levels; however, histological analysis of the testes revealed that they are mature in the summer. In females, the GSI and glycogen values are higher in summer, concomitantly with the presence of mature oocytes. These results suggest that the reproductive peak of O. quadrata occurs in summer. The seasonal analysis of the biochemical parameters, as well as comparison with Siriú Beach, demonstrated that the ghost crabs of Rondinha Beach have a different pattern of metabolism than those of Siriú. This difference may be a consequence of differences in the environmental conditions as well as in the anthropogenic pressures, such as vehicle traffic and the increase in human population at the beach in summer.

MONOAMINE-CONTAINING NEURONS IN THE CENTRAL NERVOUS SYSTEM OF MEGALOBULIMUS OBLONGUS (GASTROPODA, PULMONATA)

Abstract The central nervous system (CNS) of the pulmonate gastropod Megalobulimus oblongus was subjected to histofluorescence by the glyoxylic acid method and to 5,7-dihydroxytryptamine (5,7-DHT) treatment to reveal the presence of monoaminergic neurons. Glyoxylic acid histochemistry labeled catecholamine cell bodies in the buccal, cerebral and pedal ganglia and monoaminergic fiber tracts distributed throughout the CNS of Megalobulimus. Serotonergic cell bodies were detected by the glyoxylic acid method and 5,7-DHT administration (until 16 weeks of treatment) in the cerebral, right parietal, visceral and pedal ganglia. The pedal ganglion was the region that showed the greatest number of serotonergic neurons. Mapping of monoaminergic neurons in M. oblongus established comparisons with the CNS of other gastropods.

Control of carbohydrate metabolism in an anoxia‐tolerant nervous system

Anoxia-tolerant animal models are crucial to understand protective mechanisms during low oxygen excursions. As glycogen is the main fermentable fuel supporting energy production during oxygen tension reduction, understanding glycogen metabolism can provide important insights about processes involved in anoxia survival. In this report we studied carbohydrate metabolism regulation in the central nervous system (CNS) of an anoxia-tolerant land snail during experimental anoxia exposure and subsequent reoxygenation. Glucose uptake, glycogen synthesis from glucose, and the key enzymes of glycogen metabolism, glycogen synthase (GS) and glycogen phosphorylase (GP), were analyzed. When exposed to anoxia, the nervous ganglia of the snail achieved a sustained glucose uptake and glycogen synthesis levels, which seems important to maintain neural homeostasis. However, the activities of GS and GP were reduced, indicating a possible metabolic depression in the CNS. During the aerobic recovery period, the enzyme activities returned to basal values. The possible strategies used by Megalobulimus abbreviatus CNS to survive anoxia are discussed.

Experimental model of tympanic colic (acute abdomen) in chinchillas (Chinchilla lanigera)

Digestive disorders caused by sudden changes in diet or inappropriate diet are among the most common disorders of the digestive system. Cecal or intestinal tympany, one consequence of inappropriate diet, is characterized by the accumulation of gases, marked distension of the cecum and colon and the induction of inflammatory processes. To know the effects of intestinal tympany on the enteric plexuses, we developed a method of experimental tympanic colic (TC) in the Chinchilla lanigera. This species was used in view of its susceptibility to TC. TC was induced with a diet rich in alfalfa associated with grain overload for two weeks. Physical and clinical examination including the von Frey test confirmed the diagnosis. The chinchillas with acute abdomen were treated with 1% ketoprofen and resumption of a balanced diet. Necropsy and histopathological analysis showed tympany-induced alterations mainly in the cecum and colon. After treatment, the control conditions were restored. The TC protocol is proposed as an experimental approach designed to aid the study of the effects of acute intestinal inflammation and obstruction caused by an inappropriate diet.

Enteric glial reactivity to systemic LPS administration: Changes in GFAP and S100B protein

Lipopolysaccharide (LPS) is used to induce inflammation and promotes nervous system activation. Different regions of the brain present heterogeneous glial responses; thus, in order to verify whether systemic LPS-induced inflammation affects the enteric glia differently across the intestinal segments, we evaluated the expressions of two glial activity markers, GFAP and S100B protein, in different intestine segments, at 1h, 24h and 7days after acute systemic LPS administration (0.25 or 2.5mgkg-1) in rats. Histological inflammatory analysis indicated that the cecum was most affected when compared to the duodenum and proximal colon at the highest doses of LPS. LPS induced an increased S100B content after 24h in all three regions, which decreased at 7days after the highest dose in all regions. Moreover, at 24h, this dose of LPS increased ex-vivo S100B secretion only in the cecum. The highest dose of LPS also increased GFAP in all regions at 24h, but earlier in the cecum, where LPS-induced enteric S100B and GFAP alterations were dependent on dose, time and intestine region. No associated changes in serum S100B were observed. Our results indicate heterogeneous enteric glial responses to inflammatory insult, as observed in distinct brain areas.

General Morphology and Innervation of the Midgut and Hindgut of Megalobulimus abbreviatus (Gastropoda, Pulmonata)

We describe the morphology and innervation of the midgut and hindgut of the giant land snail Megalobulimus abbreviatus for the first time. The midgut (stomach and intestine) and hindgut (rectum and anus) are innervated by the subesophageal ganglia, through the gastrointestinal branch (originated from the visceral nerve) and the rectum-anal nerve, respectively. Backfilling through these nerves revealed neuronal bodies, mainly in the right parietal and visceral ganglia. The enteric plexuses of the midgut and hindgut are formed by extensive axonal networks and several neuronal somata arranged in clusters or as isolated cells. The gastrointestinal branch and the rectum-anal nerve directly innervate the enteric plexuses of the intestine and the hindgut, respectively. However, the outer wall of the stomach has a stomatogastric nervous system, which consists of four ganglia: stomatogastric, gastric, cardic, and pyloric. Fibers of the gastrointestinal branch project to these ganglia. Anterograde tracing from stomatogastric system ganglia revealed that the enteric plexus of the stomach is innervated only by these peripheral ganglia. Anterograde tracing of the gastrointestinal branch did not result in labeling in the enteric plexus of the stomach. Therefore, the midgut and hindgut of M. abbreviatus is controlled by an intrinsic innervation, constituted by the submucous and myenteric plexuses, which are innervated directly by neurons from the subesophageal ganglia or indirectly via the stomatogastric nervous system (for the stomach).

The Stomatogastric and Enteric Nervous System of the Pulmonate Snail Megalobulimus abbreviatus: A Neurochemical Analysis

Chemical coding of stomatogastric nervous system (STNS) and enteric nervous system (ENS) of midgut and hindgut in the snail Megalobulimus abbreviatus was investigated using histochemistry, histofluorescence, and immunohistochemistry. The gastrointestinal plexuses, constituted by intrinsic neurons and fibers originating from the subesophageal ganglia and/or STNS, showed intense acetylcholinesterase (AChE) and nicotinamide adenine dinucleotide diaphorase (NADPHd) activity. The enteric neurons and fibers with AChE activity are scattered in the submucosa and between both muscular layers of gastrointestinal tract, whereas NADPHd neurons and fibers are more abundant between muscular layers than in the submucosa. Catecholaminergic nerve fibers and varicosities are found mainly within the submucosa across the mid- and hindgut. Serotoninand FMRFamide-immunoreactive neurons and fibers originating from the STNS are distributed in the submucosa of the intestine and rectum. FMRFamide-immunoreactive neurons and fibers are present in the mucosa, submucosa, and muscular layers of mid- and hindgut. The neuron-like intraepithelial cells exhibited AChE activity, a few NADPHd activity, and immunoreactivity for serotonin and FMRFamide. Intense glial fibrillary acidic protein (GFAP) immunoreaction is found throughout the intestine plexuses and in the STNS ganglia. The GFAP immunoreaction in intramural plexuses suggests the presence of glial cells as an important component of ENS in this pulmonate snail.

Comparative Anatomy of the Peripheral Nerves

Abstract This chapter provides a comparative review of the topography, origin, and distribution of the spinal nerves that constitute the brachial and lumbosacral plexuses between humans and mammal species recognized as experimental models in the study of plexopathies and peripheral nerve injuries. A brief description of brachial and lumbosacral plexuses in lower tetrapods (birds, reptiles and amphibians) is included. The description of the brachial and lumbosacral plexuses in nonhuman primates is based on three genera of Hominidae, on the genus Hylobates, and some primate species of experimental interest. The most widely used experimental models in plexopathies (rat, guinea pig, dog, cat, and pig) are described and the significant anatomical differences in relation to humans are indicated.