The epidermal growth factor receptor mediates allergic airway remodelling in the rat.

The chronicity of bronchial asthma is attributed to persistent airway inflammation and to a variety of structural changes, or remodelling, that includes smooth muscle and goblet cell hyperplasia. To investigate the mechanisms of airway remodelling, the current authors used an established allergen (ovalbumin; OVA)-driven rodent model (the Brown Norway rat). Brown Norway rats were sensitised to OVA and challenged three times at 5-day intervals to evoke airway remodelling. The effects of an epidermal growth factor (EGF) receptor inhibitor, AG1478, and a cysteinyl leukotriene-1 receptor antagonist, montelukast, on epithelial and airway smooth muscle (ASM) cell proliferation in vivo in response to repeated OVA challenge were tested. Three challenges with leukotriene (LT)D(4) were given, to examine their effects on remodelling with and without AG1478 pretreatment. OVA challenges caused ASM hyperplasia, with an increase in mass, epithelial cell proliferation and goblet cell proliferation. AG1478 prevented the changes, as did montelukast. Multiple OVA challenges increased heparin-binding EGF-like growth factor but not EGF expression by airway epithelium. LTD(4) reproduced the changes in remodelling induced by OVA and this was blocked by AG1478. Allergen-induced airway epithelial and airway smooth muscle remodelling is mediated by cysteinyl leukotrienes via the cysteinyl leukotriene-1 receptor with downstream effects on the epidermal growth factor receptor axis.

Modulation of smooth muscle phenotype in vitro by homologous cell substrate.

We have developed a novel cell culture system that supports the shortening of smooth muscle cells. Primary rat airway smooth muscle cells were plated on an ethanol-fixed, confluent monolayer of homologous smooth muscle cells (homologous cell substrate, HCS). Cells grown on HCS exhibited morphological and functional characteristics consistent with a differentiated phenotype. Cells on HCS were spindle shaped with a well-defined long axis, whereas cells grown on glass were larger and irregularly shaped. Smooth muscle-specific alpha-actin immunostained diffusely in cells on HCS, whereas it appeared as stress fibers in cells on glass. Agonists recruited a greater fraction of HCS cells to contract, resulting in greater changes in cell area or length on average, but the maximal capacity of shortening of individual cells was similar between the groups. Unlike cells on glass, cells on HCS shortened to methacholine. HCS was reversible and persisted over several passages. Agonists stimulated intracellular Ca(2+) oscillations in cells on HCS, whereas they elicited biphasic peak and plateau transients in cells on glass. HCS modulates smooth muscle cell phenotype in vitro.

Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats.

Inbred Fischer 344 rats display airway hyperresponsiveness (AHR) in vivo compared with the normoresponsive Lewis strain. Fischer AHR has been linked with increased airway smooth muscle (ASM) contraction ex vivo and enhanced ASM cell intracellular Ca(2+) mobilization in response to serotonin compared with Lewis. To determine the generality of this association, we tested whether bradykinin (BK) also stimulates greater contraction of Fischer airways and greater Ca(2+) mobilization in Fischer ASM cells. Explants of Fischer intraparenchymal airways constricted faster and to a greater degree in response to BK than Lewis airways. BK also evoked higher Ca(2+) transients in Fischer than in Lewis ASM cells. ASM cell B(2) receptor expression was similar between the two strains. BK activated both phosphatidylinositide-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific PLC to mobilize Ca(2+) in Fischer and Lewis ASM cells. PI-PLC activity, as measured by inositol polyphosphate accumulation, was similar in the two strains. PKC inhibition with GF109203X, Go6973, or Go6983 attenuated BK-mediated Ca(2+) transients in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 did not affect Ca(2+) transients in Lewis cells. Enhanced Ca(2+) mobilization in ASM cells can arise from variations in PKC and may be an important component of nonspecific, innate AHR.

Calcium and growth responses of hyperresponsive airway smooth muscle to different isoforms of platelet-derived growth factor (PDGF).

Airway smooth muscle (ASM) mass is likely to be an important determinant of airway responsiveness. Highly inbred Fisher rats model innate hyperresponsiveness, and also have more ASM in vivo than control Lewis rats. Platelet derived growth factor (PDGF) is an important endogenous growth factor for ASM, and partially purified PDGF-AB causes enhanced growth of Fisher rat ASM cells, compared to Lewis cells. The aim of the present study was to determine the mitogenic effects of all three recombinant PDGF isoforms on ASM cells, and investigate the mechanisms of enhanced Fisher ASM growth responses. The potential mechanisms assessed include PDGF receptor expression and activation (tyrosine phosphorylation), and intracellular calcium (Ca2+) responses to PDGF isoforms. Fisher ASM cells had a greater mitogenic response to PDGF-AB and -AA, and a greater Ca2+ response to -BB than Lewis ASM cells. A Ca2+ response was not necessary for a mitogenic response, and the effects of PDGF isoforms on Ca2+ were not associated with their effects on growth. Therefore, we suggest that enhanced Fisher mitogenic response to PDGF-AA and -AB is not mediated by differences in Ca2+ signalling. Western analysis of the PDGF receptor indicated a similar expression of beta-PDGF receptor in ASM cells from the two rat strains, but a greater expression of alpha-PDGF receptor in Fisher cells; however, phosphorylation of the PDGF receptor following growth stimulation did not differ between strains. This suggests a role for post-receptor signals, in addition to enhanced receptor expression, in the enhanced growth response of Fisher ASM cells to PDGF-AA and -AB.

Inositol (1,4,5)trisphosphate metabolism and enhanced calcium mobilization in airway smooth muscle of hyperresponsive rats.

Airway hyperresponsiveness (AHR) is a phenotype of asthma and can be modeled by the inbred Fisher strain of rat, which is hyperresponsive in vivo relative to the Lewis strain. Enhanced airway smooth muscle (ASM) contractility and Ca(2+) mobilization are associated with the AHR observed in Fisher rats. In this study, we investigated whether the interstrain differences in Ca(2+) mobilization to serotonin (5HT) result from differences in inositol (1,4,5)trisphosphate (IP(3)) metabolism and/or IP(3) receptor (IP(3)R) sensitivity. Ca(2+) mobilization by 5HT in cultured ASM cells from both rat strains was phospholipase C (PLC) dependent. Inositol polyphosphate accumulation, and hence PLC activity, was similar in both rat strains, but a specific IP(3) transient was detectable only in Fisher myocytes in response to 5HT. These findings suggested that IP(3) degradation rather than production differed between the two strains. The Vmax and Michaelis constant (K(m)) of IP(3)-specific 5-phosphatase activity were higher in the particulate fraction of Lewis than in Fisher ASM cell homogenates and appeared to be related to a greater expression of two isoforms of 5-phosphatase (type I and type II) in Lewis cells as shown by Western blot analysis. The sensitivity of the IP(3)R to IP(3) was similar between Fisher and Lewis ASM cells, indicating that the interstrain intracellular Ca(2+) differences were unrelated to IP(3)R function. We propose that interstrain variations in 5-phosphatase activity and expression may give rise to the interstrain differences in IP(3)-mediated Ca(2+) release in ASM and may be a determinant of AHR.

Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells.

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.

Enhanced Ca(2+) mobilization in airway smooth muscle contributes to airway hyperresponsiveness in an inbred strain of rat.

The mechanisms underlying airway hyperresponsiveness are still unknown but increased contractility of airway smooth muscle may play a role. This study sought to demonstrate a relationship between in vivo airway responsiveness and a number of measures of airway smooth muscle responsiveness ex vivo, including intracellular Ca(2+) signaling, by comparing three inbred strains of rat with different degrees of airways responsiveness to methacholine. Lewis, ACI, and Fisher strains of rat were characterized for their pulmonary responses to 5-hydroxytryptamine (5HT) in vivo and Fisher rats were found to be hyperresponsive to 5HT compared with ACI and Lewis rats. The responsiveness of the airways from these strains of rat ex vivo revealed that intraparenchymal airways from Fisher rats significantly narrowed to a greater degree and at a faster rate to 5HT than Lewis rat airways, consistent with their differences in vivo. Intraparenchymal ACI airways, however, narrowed to the same degree as Fisher airways but took longer to do so at a high concentration of 5HT. 5HT caused concentration-dependent increases in intracellular Ca(2+) in airway smooth muscle cells from all three strains of rat, but Fisher and ACI displayed higher responses than Lewis airway smooth muscle. Our results demonstrate that the degree of intracellular Ca(2+) mobilization by 5HT in airway smooth muscle parallels the rate and degree of intraparenchymal airway narrowing and suggest that the degree of intracellular Ca(2+) mobilization plays a role in determining airway smooth muscle contractility.

Nitric oxide synthesis by tracheal smooth muscle cells by a nitric oxide synthase-independent pathway.

Nitric oxide (NO) is known to be synthesized from L-arginine in a reaction catalyzed by NO synthase. Liver cytochrome P-450 enzymes also catalyze the oxidative cleavage of C==N bonds of compounds containing a -C(NH2)==NOH function, producing NO in vitro. The present study was designed to investigate whether there was evidence of a similar pathway for the production of NO in tracheal smooth muscle cells. Formamidoxime (10(-2) to 10(-4) M), a compound containing -C(NH2)==NOH, relaxed carbachol-contracted tracheal rings and increased intracellular cGMP in cultured tracheal smooth muscle cells, whereas L-arginine had no such effect. NO was detectable in the medium containing cultured tracheal smooth muscle cells when incubated with formamidoxime. Ethoxyresorufin (10(-7) to 10(-4) M), an alternate cytochrome P-450 substrate, inhibited formamidoxime-induced cGMP accumulation as well as tracheal ring relaxation in cultured tracheal smooth muscle cells. The NO synthase inhibitors Nomega-nitro-L-arginine (10(-3) M) and NG-monomethyl-L-arginine (10(-3) M) had no effect on formamidoxime-induced cGMP accumulation. These results suggest that NO can be synthesized from formamidoxime in tracheal smooth muscle cells, presumably by a reaction catalyzed by cytochrome P-450.

Effects of cAMP on serotonin evoked calcium transients in cultured rat airway smooth muscle cells.

Agents increasing intracellular adenosine 3',5'-cyclic monophosphate (cAMP) cause relaxation of airway smooth muscle. However, the mechanisms of their action are not fully understood. We investigated the role of cAMP in the modulation of intracellular Ca2+ concentration ([Ca2+]i) transients evoked by serotonin (5-HT) in cultured rat tracheal smooth muscle (TSM) cells. Forskolin (10(-7) M) caused a significant elevation of intracellular cAMP and a 60% relaxation of tracheal rings contracted with 5-HT but did not affect [Ca2+]i in TSM cells. Forskolin (10(-5) M) completely relaxed tracheal rings and significantly decreased [Ca2+]i during the sustained phase of the 5-HT response. Forskolin-induced relaxation was attenuated by the cAMP-dependent protein kinase A (PKA) inhibitor Rp diastereomer of cAMP (Rp-cAMPS; 10(-4) M) and by the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor [Rp isomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, 10(-4) M]. The effects of forskolin on [Ca2+]i were not altered by the PKA inhibitor but were abolished by the PKG inhibitor and thapsigargin. These results indicate that, in rat TSM, the relaxant effects of high concentrations of cAMP may be mediated, at least in part, by facilitating the sequestration of Ca2+ into intracellular stores by a mechanism involving PKG.

Effects of purine nucleotides and nucleoside on cytosolic calcium levels in rat tracheal smooth muscle cells.

Extracellular adenosine triphosphate (ATP) has a range of effects on a wide variety of cells through the activation of specific purinoceptors. The aim of this study was to establish whether P2 purinoceptors are present on airway smooth muscle cells. Experiments were conducted on cultured rat tracheal smooth-muscle cells (first through third passage). Intracellular Ca2+ ([Ca2+]i) was measured using Fura-2 and dual-excitation wavelength microfluorometry. The effects of ATP, adenosine diphosphate (ADP), uridine triphosphate (UTP), and adenosine (ADO) were measured in concentrations from 10(-6) to 10(-3) M. At a concentration of 10(-4) M, the peak [Ca2+]i was 502 +/- 92 nM for ATP and 543 +/- 76 nM for UTP (mean +/- standard error of the mean). ADO had no significant effect on Ca2+ release. Peak [Ca2+]i induced by ATP was not dependent on extracellular Ca2+ but was blocked by U-73122, an inhibitor of phospholipase C. Pretreatment with adenosine deaminase and desensitization with alphabeta-MeATP had no effect on ATP-induced Ca2+ release. The effects of ATP (10(-4) M) on peak [Ca2+]i were potentiated by the presence of ADO 10(-5) M (969 +/- 257 nM; P < 0.05). The presence of XAC, a blocker of A1 and A2 ADO receptors did not prevent this effect. In the presence of XAC, ADO 10(-6) M potentiated the effects of ATP (peak [Ca2+]i: 1,300 +/- 229 nM). The addition of 1433U83, a blocker of A3 ADO receptors, blocked the synergistic effect of ADO 10(-6) M on ATP. These data show that P2 purinoceptors, most likely of the P2U subtype, are present on airway smooth muscle cells and that the newly discovered A3 ADO receptor appears to be also present.

Modulation of Ca2+ transients in neonatal and adult rat cardiomyocytes by angiotensin II and endothelin-1.

Vasoactive peptides may exert inotropic and chronotropic effects in cardiac muscle by modulating intracellular calcium. This study assesses effects of angiotensin II (ANG II) and endothelin-1 (ET-1) on intracellular free calcium concentration ([Ca2+]i) in cultured cardiomyocytes from neonatal and adult rats. [Ca2+]i was measured microphotometrically and by digital imaging using fura 2 methodology. Receptor subtypes through which these agonists induce responses were determined pharmacologically and by radioligand binding studies. ANG II and ET-1 increased neonatal atrial and ventricular cell [Ca2+]i transients in a dose-dependent manner. ANG II (10(-11) to 10(-7) M) failed to elicit [Ca2+]i responses in adult cardiomyocytes, whereas ET-1 increased [Ca2+]i in a dose-dependent manner. The ETA receptor antagonist BQ-123 significantly reduced (P < 0.05) ET-1 induced responses, and the ETB receptor agonist IRL-1620 (10(-7) to 10(-5) M) significantly increased (P < 0.05) [Ca2+]i in neonatal and adult cardiomyocytes. ET-1 binding studies demonstrated 85% displacement by BQ-123 and approximately 15% by the ETB receptor agonist sarafotoxin S6c, suggesting a predominance of ETA receptors. Competition binding studies for ANG II failed to demonstrate significant binding on adult ventricular myocytes, indicating the absence or presence of very few ANG II receptors. These data demonstrate that ANG II and ET-1 have stimulatory [Ca2+]i effects on neonatal cardiomyocytes, whereas in adult cardiomyocytes, ANG II-induced effects are insignificant, and only ET-1-induced responses, which are mediated predominantly via ETA receptors, are preserved. Cardiomyocyte responses to vasoactive peptides may thus vary with cardiac development.

Serotonin-evoked calcium transients in airway smooth muscle cells.

Serotonin (5-HT) is an important mediator of allergic airway narrowing in several animal species. The present study was designed to characterize the receptor subtypes that mediate 5-HT-induced airway responses in the rat. To do this, we measured Ca2+ transients and adenosine 3',5'-cyclic monophosphate (cAMP) production evoked by 5-HT in cultured rat tracheal smooth muscle cells as well as 5-HT-induced contractions of isolated tracheal rings. 5-HT (10(-6) to 10(-4) M) triggered a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a second phase of sustained, elevated, and sometimes oscillating levels of [Ca2+]i. Sustained but not peak [Ca2+]i levels were dependent on Ca2+ influx but were not attenuated by nifedipine (10(-5) M). Oscillations were observed in cells in Ca2+ -free medium, suggesting Ca2+ -induced Ca2+ release independent of Ca2+ influx. The effects of 5-HT were inhibited by thapsigargin (10(-6) M) and ketanserin (10(-7) M). In cells incubated with LY-278,584 (5-HT3 antagonist) and (-)pindolol (5-HT1 antagonist), 5-HT-evoked responses were not significantly different from the control values. 5-methyltryptamine (5-MT), a ligand with higher affinity for 5-HT2C receptors than "classical" 5-HT2 receptors, elicited higher responses than dipropyl-5-carboxamidotryptamine (DP-5-CT), which possesses lower affinity for 5-HT2C receptors than 5-MT, but an affinity for the classical 5-HT2 receptor similar to 5-MT, but an affinity for the classical 5-HT2 receptor similar to 5-MT.(ABSTRACT TRUNCATED AT 250 WORDS)

Blunted attenuation of angiotensin II-mediated Ca2+ transients by insulin in cultured unpassaged vascular smooth muscle cells from spontaneously hypertensive rats.

Insulin attenuates vasoconstrictor-stimulated intracellular calcium ([Ca2+]i) responses in cells from normotensive rats. To determine whether these effects may be altered in hypertension, this study assesses the effects of physiologic concentrations of insulin on angiotensin II-stimulated [Ca2+]i in primary unpassaged cultured vascular smooth muscle cells (VSMC) from mesenteric arteries from spontaneously hypertensive rats (SHR), Wistar Kyoto (WKY), and Wistar rats. [Ca2+]i was measured microphotometrically in cells from 3-, 9-, and 17-week-old rats by the Fura 2 methodology. Basal, angiotensin II-stimulated (1 nmol/L), and insulin-attenuated (70 microU/mL) angiotensin II-induced [Ca2+]i did not differ in VSMC obtained from 3-week-old WKY, Wistar, and SHR. Basal and angiotensin II-stimulated [Ca2+]i in VSMC from 9- and 17-week-old SHR was significantly greater (P < .01) than that in cells from age-matched WKY and Wistar rats. Insulin decreased angiotensin II-stimulated [Ca2+]i responses in all groups, but the effect was significantly blunted in cells from 9- and 17-week-old SHR. The magnitude of inhibition of angiotensin II-stimulated [Ca2+]i responses induced by insulin was 63 +/- 12 nmol/l (WKY), 60 +/- 10 nmol/L (Wistar), and 28 +/- 8 nmol/L (SHR), (P < .01 v normotensive) in cells from 9-week-old rats and 70 +/- 15 nmol/L (WKY), 67 +/- 12 nmol/L (Wistar), and 25 +/- 10 nmol/L (SHR) (P < .01 v normotensive) in cells from 17-week-old rats. Insulin increased angiotensin II-stimulated [Ca2+]i recovery rate to basal values in cells from WKY rats, but had no effect on the rate of recovery in VSMC from SHR. (ABSTRACT TRUNCATED AT 250 WORDS)

Mesenteric vascular smooth muscle cells from spontaneously hypertensive rats display increased calcium responses to angiotensin II but not to endothelin-1.

OBJECTIVE: To determine the differential calcium responses to two vasoconstrictor peptides, angiotensin II (Ang II) and endothelin-1, in vascular smooth muscle cells derived from mesenteric arteries from young and adult normotensive and hypertensive rats. METHODS: Effects of Ang II and endothelin-1 on cytosolic free calcium concentration in primary cultured unpassaged single vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto (WKY), Wistar and spontaneously hypertensive rats (SHR) aged 3, 9 and 17 weeks were examined microphotometrically using fura-2 methodology. RESULTS: Basal cytosolic free calcium concentration was significantly increased in cells from SHR aged 9 and 17 weeks compared with cells from age-matched WKY and Wistar rats. Ang II and endothelin-1 significantly increased cell cytosolic free calcium in all rat groups at all ages. Responses to low concentrations of Ang II (1 nmol/l) were significantly higher in cells from SHR aged 9 and 17 weeks than in age-matched controls. This was confirmed in cells from rats aged 17 weeks with full concentration-response curves, which also showed that the pD2 for Ang II for WKY rats was significantly different from that of SHR. In cells from SHR at all ages Ang II-stimulated cytosolic free calcium remained persistently high, whereas in cells from WKY and Wistar rats basal levels were reached within 100 s after the maximal response. Low concentrations of endothelin-1 elicited significantly lower cytosolic free calcium responses in cells from SHR aged 17 weeks compared with age-matched controls. The time course of cytosolic free calcium responses to endothelin-1 were similar in the groups. CONCLUSIONS: In primary cultured unpassaged mesenteric vascular smooth muscle cells from adult SHR, cytosolic free calcium concentration responses to Ang II are enhanced, whereas responses to low concentrations of endothelin-1 are slightly reduced. The differential effects of these two vasoconstrictor peptides may contribute to their relative roles in modulating vascular smooth muscle cell cytosolic free calcium in SHR.

Insulin attenuates agonist-evoked calcium transients in vascular smooth muscle cells.

Insulin may decrease the contractile response of vascular smooth muscle to vasoactive agents. This could be due to interactions of insulin with the effects of vasoactive agonists on intracellular free calcium transients in vascular smooth muscle cells. This study assesses the effects of physiological doses of insulin (70 microU/mL) on calcium responses in cultured vascular smooth muscle cells (primary unpassaged and passaged) to angiotensin II (1 nmol/L), arginine vasopressin (10 nmol/L), and norepinephrine (10 mumol/L). Intracellular free Ca2+ concentrations in single cells were measured microphotometrically using fura 2-AM. Insulin, angiotensin II, arginine vasopressin, and norepinephrine significantly increased calcium (to 115 +/- 7, 183 +/- 20, 184 +/- 15, and 168 +/- 12 nmol/L, respectively, from basal calcium of 90 +/- 10 nmol/L). Insulin significantly attenuated the agonist-induced calcium responses. The effects of insulin were almost completely inhibited by diltiazem, staurosporine, calphostin C, and thapsigargin. In conclusion, insulin stimulates calcium transients but blunts agonist-mediated calcium rises in vascular smooth muscle cells. These responses are related to regulatory effects of insulin on cellular calcium homeostasis and may explain how insulin modulates vascular smooth muscle contraction.

Tracing neural pathways in snail olfaction: from the tip of the tentacles to the brain and beyond.

The anatomical organization of the olfactory system of terrestrial snails and slugs is described in this paper, primarily on the basis of experiments using the African snail Achatina fulica. Behavioral studies demonstrate the functional competence of olfaction in mediating food finding, conspecific attraction, and homing. The neural substrate for olfaction is characterized by an extraordinarily large number of neurons relative to the rest of the nervous system, and by the fact that many of them are unusually small. There exist multiple serial and parallel pathways connecting the olfactory organ, located at the tip of the tentacle, with integrative centers in the central nervous system. Our methods of studying these pathways have relied on the selective neural labels horseradish peroxidase and hexamminecobaltous chloride. One afferent pathway contains synaptic glomeruli whose ultrastructure is similar to that of the glomeruli seen in the mammalian olfactory bulb and the insect olfactory lobe. All of the olfactory neuropils, but especially the tentacle ganglion, contain large numbers of morphologically symmetrical chemical synapses. The procerebrum is a unique region of the snail brain that possesses further features analogous with olfactory areas in other animal groups. Olfactory axons from the tentacle terminate in the procerebrum, but the intrinsic neurons do not project outside of it. An output pathway from the procerebrum to the pedal ganglion has been identified and found to consist of inter-ganglionic dendrites. The major challenge for future studies is to elucidate the pattern of connectivity within, rather than between, the various olfactory neuropils.

Synaptic innervation of the giant cerebral neuron in sated and hungry snails.

The giant cerebral neuron (GCN) is a serotoninergic cell that facilitates feeding behaviour in gastropod molluscs. We have examined the morphology of its cerebral arborizations after labelling them by intracellular injection of hexamminecobalt. Observations in the light microscope reveal extensive arborizations, with similar overall distributions, in the terrestrial snails Achatina fulica and Rumina decollata. All the major peripheral nerves terminate within the zone covered by the GCN arbor. In a sample of 370 synapses in which the GCN participated, the GCN was identified as the postsynaptic element in every case, thereby establishing the dendritic nature of the cerebral arborizations. A total of approximately 1,000 synapses contacts the GCN, with no evident regional differences in innervation density. The synaptic membrane of the presynaptic profile is characterized by an agglomeration of small clear vesicles and small dense vesicles. At nonsynaptic membranes there are agglomerations of larger dense and dense core vesicles, suggestive of nonsynaptic release. The dendrites of the GCN also contain vesicles. Starvation for five days (Rumina decollata) caused a significant increase in the proportion of curved synapses relative to flat synapses. This might be a plastic change allowing for a greater efficiency of transmission between sensory afferents and the GCN.

Interganglionic dendrites constitute an output pathway from the procerebrum of the snail Achatina fulica.

The procerebrum is an olfactory processing region that occupies approximately one-third of the total brain area in pulmonate gastropod molluscs. It has many unusual features, including a development separate from the rest of the brain and the absence of axons belonging to its intrinsic neurons. We have investigated the input and output pathways of the procerebrum in the terrestrial snail Achatina fulica by using hexamminecobalt chloride as a selective label. Both the tentacle nerve and the cerebropedal connective nerve contribute to a fine neural plexus that is distributed throughout the neuropile region of the procerebrum. The fibers from the tentacle nerve are predominantly presynaptic, whereas those from the cerebropedal connective are predominantly postsynaptic. The postsynaptic fibers (dendrites) were traced to two groups of nerve cells (total number, 20-25) near the ventral surface of the ipsilateral pedal ganglion. No evidence was obtained for any other numerically significant output pathway from the procerebrum. Since locomotion is known to be controlled by the pedal ganglion, these results provide an anatomical substrate for the strong influence of olfaction on locomotor behavior in snails. The pathway is unusual in that the dendrites are interganglionic and can be as long as 5 mm.

Evidence for differential DNA endoreplication during the development of a molluscan brain.

The DNA content of neurons in the cerebral ganglion of Achatina fulica was determined by the two-wavelength method of microspectrophotometry using Feulgen-stained sections. DNA measurements of mouse hepatocytes were used as a control of the methods. All sampled neurons with a nuclear diameter greater than 7 microns were polyploid. The shape of the frequency histogram of DNA contents is not compatible with an interpretation that assumes one or more simple duplications of the genome. It is suggested, instead, that the results are due to either the underreplication of some DNA sequences or the selective amplification of genes. Additional experiments using [3H]-thymidine autoradiography showed that the incidence of neuronal DNA synthesis is highest during the period of the animal's greatest growth and then declines rapidly near the onset of sexual maturity. The mesocerebrum incorporated greater amounts of [3H]-thymidine than the rest of the brain, whereas the procerebrum remained diploid throughout the course of the study.