Brazilian Journal of Medical and Biological Research (2003) 36: 1247-1253 Vascularization of the central nervous system of oblongus 1247 ISSN 0100-879X

Vascular supply of the central nervous system of the (, )

H.G. Nóblega1,2, 1Laboratório de Histofisiologia Comparada, Departamento de Ciências Morfológicas, V. Missaglia1,2, C. Stenert1,2, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, M.C. Faccioni-Heuser1 Porto Alegre, RS, Brasil and M. Achaval1 2Laboratório de Anatomia, Centro de Ciências da Saúde, Universidade do Vale do Rio dos Sinos, São Leopoldo, RS, Brasil

Abstract

Correspondence The vascularization of the central nervous system of the snail Megalo- Key words M. Achaval bulimus oblongus was studied by injection of carmine-gelatin solution • Gastropods Laboratório de Histofisiologia into the arterial system and using a histochemical technique for the • Central nervous system Comparada detection of alkaline phosphatase. The central nervous system of M. • Vascular supply Departamento de Ciências oblongus is irrigated by the anterior aorta, from which a series of small • Trypan blue Morfológicas, ICBS, UFRGS • branches emerge that supply the subesophageal nervous ganglia. In Carmine-gelatin Rua Sarmento Leite, 500 • Alkaline phosphatase turn, these branches give rise to a series of smaller vessels that irrigate 90050-170 Porto Alegre, RS activity Brasil the buccal bulb, the anterior portion of the foot, the cerebral ganglia, Fax: +55-51-3316-3092 the dorsal body gland, and the anterior portion of the reproductive E-mail: [email protected] system. No hemolymph vessels were detected within nervous tissue although such vessels were found in the periganglionic connective Presented at the XV Annual Meeting sheath. This connective sheath contains vascular loops and had a of the Federação de Sociedades de Biologia Experimental, Caxambu, MG, series of overlaps and projections that follow the contour of the Brazil, August 23-26, 2000. nervous ganglia. This arrangement permits a larger area of interaction between the surface of the nervous tissue and the hemolymph and Research partially supported by reduces the distance between the deepest portion of a given ganglion FINEP, CAPES, CNPq and UNISINOS. and the hemolymph vessels.

Received June 14, 2002 Accepted May 27, 2003 Introduction nervous system (CNS). In these ves- sels, sinuses or lacunas are restricted to the The vascular system of mollusks can be periganglionic connective sheath, and no either open, as in the majority of , or vessels are present within the ganglia (3). closed, as in the cephalopods. In gastropods The CNS of Megalobulimus oblongus is this system, in addition to performing trans- similar to that of Helix pomatia and other port functions (1), plays a hydraulic role in species of pulmonates (4). It consists of a the extension of the foot (2). periesophageal ring in which a pair of cere- In the pulmonate gastropods, the hemo- bral ganglia is interconnected by means of lymph is pumped by the heart to the aorta, cerebral-pedal and cerebral-pleural connec- from which the caudal and cephalic branches tives, a subesophageal ring formed of pairs originate. The cephalic branch is responsible of pedal, parietal and pleural ganglia and a for the hemolymph supply to the central single visceral ganglion. Two more distally

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located buccal ganglia are connected to the (Nikon Optiphot II, Tokyo, Japan). periesophageal ring by a pair of cerebral- Eight animals were injected into the pedal buccal connectives (5). musculature with 2% Trypan blue diluted in A detailed description of the circulatory MPS. The total volume (50 µl/g) was system requires a detailed understanding of achieved by 5 injections applied at 15-min the homeostasis and the general behavior of intervals. Animals were anesthetized and the (1). Knowledge of the vascular- sacrificed 2, 3, 4, 5, 6 and 48 h after the last ization of the nervous system is necessary in injection and CNS removal. The CNS was order to ascertain the nature of the existing fixed in 4% paraformaldehyde diluted in PB. neural-hemal interactions and the role played After fixation, horizontal sections were ob- by the cardiovascular system (for a more tained from the cerebral ganglia and the detailed discussion, see Ref. 1). subesophageal ganglia using razor blades The aim of the present study was to deter- and the section surface was examined with a mine which vessels are responsible for the stereomicroscope (STEMI SV11, Zeiss). vascular supply and how this microvascu- Another group of five animals was used larization occurs in the CNS of M. oblongus. to demonstrate alkaline phosphatase activ- ity. Two specimens were previously injected Material and Methods with carmine-gelatin solution as described above. The CNS was fixed in 4% parafor- Adult snails of the spe- maldehyde diluted in PB and cryoprotected cies Megalobulimus oblongus (Müller, 1774; in 15 and 30% sucrose solutions, and 40-µm 60-75 g), from the county of Charqueadas in sections were obtained with a cryostat (Leitz). the State of Rio Grande do Sul, Brazil, were Alkaline phosphatase activity was deter- used. Fifteen animals were anesthetized by mined by incubating the tissue for 2 h at immersion in a menthol-saturated solution 37ºC in a medium containing sodium ß- for 30 to 45 min. After partial removal from glycerophosphate (Sigma, St. Louis, MO, the shell, the mantle and pericardium were USA) as substrate. The material was then sectioned, and the heart cannulated. The vas- immersed in 2% cobalt chloride for 5 min, cular tree was then washed with M. oblongus washed and developed in a 5% ammonium physiological saline solution (MPS) (6) and sulfide solution for 3 min. A control was later injected with 2 to 3 ml of carmine- obtained by omitting the substrate from the gelatin solution (7). The animals were then incubation medium (8). The sections were fixed in Baker’s formalin solution for a mini- mounted on slides and covered with glyc- mum of 24 h and later dissected with the aid erin-gelatin and coverslips. of a stereomicroscope (STEMI SV11, Zeiss, Jena, Germany). Schematic representations Results of the vessel network related to the CNS ganglia of the snail were then prepared. Once The snails injected with carmine-gelatin the anatomical study was completed, the solution had a short common aorta that, after ganglia and vessels were cryoprotected in emerging from the heart, followed a brief both 15 and 30% sucrose solutions, diluted right, lateral-posterior trajectory, after which in 0.1 M phosphate buffer (PB), pH 7.4. it gave rise to the caudal and cephalic aortas. Serial sections (50 µm) were obtained with a The proximal portion of the caudal aorta cryostat (Leitz 1720, Wetzlar, Germany), touched the surface of the hepatopancreas, mounted on slides, covered with glycerin- burying itself within the most distal glandu- gelatin and coverslips and then observed and lar tissue. The cephalic aorta originated from photographed under a light microscope a posterior position in relation to the caudal

Braz J Med Biol Res 36(9) 2003 Vascularization of the central nervous system of Megalobulimus oblongus 1249 branch. The proximal portion of the cephalic the hemolymph to the dorsal body gland, aorta extended in a dorsal-ventral direction where a vast network of capillary vessels is and was enveloped by the hepatopancreas in found. Several vessels filled with carmine- its initial portion. Ventrally, it was located to gelatin solution were observed within the the right of the median plane, from where it connective sheath. Of particular note was took a cephalic trajectory, maintaining a one large vascular space, located at the limit medial position in relation to the reproduc- with the nervous tissue that enveloped the tive system, with which it kept close contact. cerebral ganglia. A similar space was also The distal segment of the anterior aorta, after a short trajectory under the ventral surface of the right visceral and parietal ganglia, in- flected in a dorsal direction, crossing the subesophageal ring, and finally divided into four branches (Figure 1). Under the ventral surface of the same segment of the cephalic CG aorta, four small diameter vessels emerged and supplied the subesophageal ganglia. Of the four branches that arise from the DB CA anterior aorta, two are medial and two are lateral. When the animal retracts itself, the medial anterior branch curves ventrally, fol- lowing the anterior edge of the pedal ganglia RCC and extends towards the foot. The other me- LCC dial branch is the ventral-buccal artery and displays the highest degree of consistency in relation to its point of origin and trajectory. PE This artery is responsible for the vascular- ization of the buccal bulb. The two lateral branches that follow the PL cerebral-pleural and cerebral-pedal connec- tives run in a ventral-dorsal direction when the animal retracts itself. From each lateral branch emerge one or two small diameter PA arteries, the cerebral arteries. These bury themselves in the connective sheath and en- V velop the cerebral ganglia and the dorsal body gland. After the emergence of the cere- bral arteries, the lateral branches divide again, Aa giving rise to the tentacular arteries (Figures 1 and 2A), and the right lateral branch also vBA gives rise to the branches that supply the anterior portion of the reproductive system. Upon penetrating the connective sheath Figure 1. Schematic representation of the periesophageal ring and the vascular supply of that envelops the cerebral ganglia, the cere- the different central nervous ganglia in a posterodorsal view. The anterior aorta in its run bral arteries branch several times, giving rise ventral to the subesophageal ganglia is represented by a discontinuous line. Aa, anterior aorta; CA, cerebral artery; CG, cerebral ganglion; DB, dorsal body gland; LCC, left cervical- to vessels of ever smaller diameter. The pri- cephalic artery; PA, parietal ganglion; PE, pedal ganglion; PL, pleural ganglion; RCC, right mary branches of the cerebral artery supply cervical-cephalic artery; V, visceral ganglion; vBA, ventral-buccal artery.

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observed between the glandular tissue of the large vascular space that bordered the mass dorsal body and the nervous tissue itself of nervous tissue. However, it should be (Figure 2B). noted that the supply to the subesophageal The examination of the subesophageal nerve ring originates from the small branches ring indicated a relation similar to that de- that emerge directly from the cephalic aorta scribed above for the vessels, connective and, after a brief trajectory, penetrate the sheath and nervous tissue. Here, as also ob- connective tissue. served for the cerebral ganglia, there was a All the ganglia that constitute the peri- esophageal ring were penetrated by projec- Figure 2. A, Dorsal view of the cerebral ganglia and buccal bulb. tions of the connective sheath of variable Note the vessels filled with car- thickness. These connective projections were mine-gelatin (arrowheads). Bar = crossed by vessels of various diameters that 2 mm. B, Photomicrograph of a were located close to the edges of the ner- horizontal section of the cere- bral ganglion after carmine-gela- vous tissue. Near the large diameter neurons, tin injection through the circula- the overlaps appeared to be deeper and the tory system. The limit where the vascularization more profuse (Figure 3). vessels are located is identified by a discontinuous line. Bar = The blood vessels present in the perigan- 0.5 mm. B, buccal bulb; CA, ce- glionic connective sheath of buccal ganglia rebral artery; CC, cerebral com- received hemolymph from a pair of arteries missure; CG, cerebral ganglia; DB, dorsal body gland. that emerged from a common branch origi- nating from the middle portion of the ante- rior aorta. These arteries followed the sali- vary glands and their ducts until they reached the buccal ganglia located on the wall behind the buccal bulb, lateral-ventral to the esoph- CG ageal connection. The nervous tissue of M. oblongus only showed alkaline phosphatase activity in the perineuronal regions. In the animals injected with Trypan blue, the dye reached the periganglionic connec- tive sheath and the dorsal body gland, but was not found within the nervous tissue.

Discussion Figure 3. Photomicrograph of the interface region (discontinu- ous line) between the nervous There are a number of similarities be- tissue and the connective tween the vascular network of the CNS of M. sheath (CS). Note the intense alkaline phosphatase activity oblongus and that of other species of Sty- through the nervous tissue (ar- lommatophora gastropod pulmonates. Com- rowheads). Observe the imbri- parison of the vascular arrangement of the cate relationship between the nervous tissue and the connec- CNS of M. oblongus and the vascularization tive sheath where the negative of the central nervous ganglia in Helix hemolymph vessels (arrows) are pomatia reveals a high degree of similarity. housed. Ne, neurons. Bar = 100 In both species the anterior aorta makes con- µm. tact with the subesophageal ring ventrally, between the right visceral and parietal gan-

Braz J Med Biol Res 36(9) 2003 Vascularization of the central nervous system of Megalobulimus oblongus 1251 glia. After reaching the dorsal surface of the work that supplies the central ganglia of subesophageal ganglia it gives rise to a branch Basommatophora such as Lymnaea stagnalis that is responsible for supplying the buccal (3,11) and Biomphalaria glabrata (12) and bulb arteries that go to the foot (sometimes that of M. oblongus. differing in number). Finally, a pair of lateral In Aplysia the abdominal ganglion is sup- arteries (right and left) gives rise to the cere- plied by small branches that originate di- bral arteries that carry the hemolymph sup- rectly from the anterior aorta, which may be ply to the cerebral ganglia. As in M. oblongus, of strategic importance, given that the ma- the subesophageal nerve ring of H. pomatia jority of neurons involved in cardiovascular also receives irrigation from branches that regulation are found within it. The location originate directly from the aorta, though in of these neurons permits the monitoring of this species they approach the ganglia from pressure and chemical indicators of cardio- the dorsal surface (9). vascular and respiratory functions (1). Like The anatomical organization of the ves- Aplysia, both the visceral ganglion and the sels responsible for the irrigation of the CNS subesophageal ganglia of M. oblongus re- in M. oblongus is similar to that found in ceive arterial branches that originate from Strophocheilus lorentzianus (10), a species the anterior aorta. The visceral ganglion per- of the same . In other Stylommatophora forms visceral control functions and thus species such as Deroceras reticulatum, D. similar origins and functions for this ana- caruanae, Limax pseudoflavus, Milax buda- tomical organization may be presumed. pestensis, Arion ater ater and A. hortensis In vertebrates, the vascularization of the (11) there are four branches that originate CNS follows two basic patterns. One con- from the anterior aorta, three of which have sists of a capillary network that originates a range similar to that found in M. oblongus. from isolated vessels that anastomose, and However, the structures supplied by these the other from vessels that penetrate the arteries are not necessarily the same as those nervous tissue and in the distal portion give that were found in this study. A fourth branch, rise to capillary loops. In almost all studied the dorsal-buccal artery, was not found in M. vertebrates, these patterns are exclusive. In- oblongus. Two lateral arteries are respon- vertebrates, for the most part, do not have sible for the irrigation of the cerebral ganglia hemolymph vessels within the nervous tis- and subesophageal nerve ring. Invariably, in sue, though there are some exceptions, such these species, the right lateral artery supplies as the squid that has capillary vessels form- the right cerebral ganglion and the subesoph- ing a reticular pattern and worms that have a ageal nervous ganglionic ring on the same vascular system in the form of capillary loops side, and give rise to the right labial and (13). It appears that there is no correlation tentacular arteries. The left branch, respon- between the phylogenetic position of a de- sible for the left side of the CNS, also gives termined species and the microvasculariza- rise to pedal-penial and ventral-buccal ar- tion pattern of the CNS. Thus, it is difficult to teries. This differs from the distribution ob- establish a primitive or advanced pattern of served in M. oblongus, especially with re- organization of the capillary bed present in spect to the origin of the vessels responsible the CNS. It seems that in the more primitive for the vascular supply to the set of subesoph- organisms, such as invertebrates, the vessels ageal nervous ganglia and in relation to the do not penetrate the nervous tissue. The emergence of the artery of the penis and presence of this primitive organization in M. ventral-buccal artery. oblongus was confirmed by both the ab- There is also some similarity between the sence of areas stained by Trypan blue in the anatomical organization of the arterial net- CNS and the absence of vascular structures

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in the histological sections. the endothelium of the encephalic vessels of The absence of vessels within the nerve vertebrates may reflect the functions of the ganglia raises the following questions. How specific segments of microvascularization. does the exchange between the circulating Ultrastructural studies in mice, rats, cats and fluid and the nerve cells take place? What is humans have demonstrated that in the capil- the importance of the irrigation of the CNS laries the enzymatic activity is located on the to the functioning of the CNS ganglia? The luminal side of the plasma membrane of the most obvious answer to the first question encephalic capillary endothelial cells. This would be interstitial diffusion. The answer would suggest that at these sites there is to the second would depend upon the degree intense dephosphorylation, transphosphory- of interaction and selectivity of the existing lation, and transport of phosphate ions (15). exchanges. In the species studied, we found In M. oblongus, the alkaline phosphatase a series of overlaps and projections of the reaction was limited to the perineuronal re- connective sheath around the nervous gan- gions of the CNS. This confirmed the lack of glia. It was noted that these projections were vessels inside the nervous tissue in the histo- accompanied by hemolymph vessels, sug- logical data. However, it was possible to gesting a pattern of vascular loops, though observe alkaline phosphatase activity in the located outside the nervous tissue. These vascular endothelial cells of the dorsal body connective sheath projections, accompanied gland, as well as in some connective sheath by vessels, allow the distances between the vessels of the nervous ganglia. The absence deepest portions of a certain nervous gan- of alkaline phosphatase activity in some con- glion and the hemolymph vessels to be nective sheath vessels may be related to the equivalent to the distance between the capil- nonexistence or low activity level of the laries and the surface of the same ganglion. enzyme or perhaps the existence of func- Another important property of this arrange- tional differences between the endothelium ment is the possibility of a larger area of present in this invertebrate species and that interaction between the nervous tissue and found in vertebrates. In addition, in verte- the hemolymph. brates, this enzyme is probably one of the In most vertebrates, the endothelial cap- enzymatic components of the blood-brain illary can be characterized by intense alka- barrier (15). At present, some facts point line phosphatase activity, with the exception toward the existence of a hemolymph-neu- of certain pulmonary and sinusoid endothe- ronal barrier in pulmonate gastropods (16), lial capillaries (14). Alkaline phosphatase though it is still not clear which tissue ele- activity is an excellent encephalic microvas- ments might be responsible for such a barrier cularization marker, also for mammals and and what the degree of selectivity of such a birds. However, it has been noted that the barrier might be (17). intensity of the reaction is not uniform in the entire vascular network, being higher in the Acknowledgments endothelium of the precapillary arterioles and capillaries and lower in the small veins. We are grateful to Antonio G. Severino It is believed that these differences between for technical assistance. the levels of alkaline phosphatase reaction in

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References

1. Brownell PH & Ligman SH (1992). Mechanisms of circulatory ho- nervous system of Helix pomatia. Zeitschrift für Zellforschung und meostasis and response in Aplysia. Experientia, 48: 818-827. Mikroskopische Anatomie, 111: 160-178. 2. Voltzow J (1985). Morphology of the pedal circulatory system of the 10. Scott MIH (1939). Estudio anatómico del Borus Strophocheilus marine gastropod Busycon contrarium and its role in locomotion. lorentzianus op (Doer) (Mol. Pul.). Revista del Museo de La Plata, Zoomorphology, 105: 395-400. Sección Zoología, 7: 217-272. 3. Bekius R (1972). The circulatory system of Lymnaea stagnalis (L.). 11. Duval A & Runham NW (1981). The arterial system of six species of Netherlands Journal of Zoology, 22: 1-58. terrestrial slug. Journal of Molluscan Studies, 47: 43-52. 4. Luchtel DL, Martin AW, Deeyrup-Olsen I & Boer HH (1997). Gastro- 12. Basch P (1969). The arterial system of Biomphalaria glabrata (Say). poda: Pulmonata. In: Harrison FW (Editor), Microscopic Anatomy of Malacologia, 7: 169-181. Invertebrates. Vol. 6B ( II). Wiley-Liss, Inc., New York, 459- 13. Sharrer E (1944). The capillary bed of the central nervous system of 718. certain invertebrates. Biological Bulletin, 87: 52-58. 5. Zancan DM, Nóblega HG, Severino AG & Achaval M (1994). Acetyl- 14. Adams CWM (1965) Neurohistochemistry. Elsevier, Amsterdam, cholinesterase distribution in the central nervous system of Mega- The Netherlands. lobulimus oblongus (Gastropoda, Pulmonata). Archives d’Anatomie, 15. Vorbrodt AW (1993). Morphological evidence of the functional polar- d’Histologie, et d’Embryologie Normales et Expérimentales, 75: 75- ization of brain microvascular endothelium. In: Pardridge WM (Edi- 86. tor), The Blood-brain Barrier: Cellular and Molecular Biology. Raven 6. Jaeger CP (1961). Action of acetylcholine on the heart of Press, New York, 137-164. Strophocheilus oblongus. Comparative Biochemistry and Physiolo- 16. Nóblega HG, Rigon F, Faccioni-Heuser MC & Achaval M (2001). gy, 4: 30-32. Does it exist a hemolymph neural barrier in the nervous central 7. Frizzo MES, Campos R, Severino AG & Achaval M (1994). The system of Megalobulimus oblongus (Mollusca, Gastropoda, vasculature of the subfornical organ of the turtle Chrysemys Pulmonata)? Acta Microscopica, 3 (Suppl C): 99-100 (Abstract). dorbigni. Italian Journal of Anatomy and Embryology, 99: 109-121. 17. Reineke M (1976). The glial cells of the cerebral ganglia of Helix 8. Gomori G (1952). Microscopic Histochemistry. Principles and Prac- pomatia L. (Gastropoda, Pulmonata). Cell and Tissue Research, 169: tice. Chicago University Press, Chicago. 361-382. 9. Pentreath VW & Cottrell GA (1970). The blood supply to the central

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