The ontogeny of muscle structure and locomotory function in the long-finned squid Doryteuthis pealeii.

Understanding the extent to which changes in muscle form and function underlie ontogenetic changes in locomotory behaviors and performance is important in understanding the evolution of musculoskeletal systems and also the ecology of different life stages. We explored ontogenetic changes in the structure, myosin heavy chain (MHC) expression and contractile properties of the circular muscles that provide power for jet locomotion in the long-finned squid Doryteuthis pealeii. The circular muscle fibers of newly hatched paralarvae had different sizes, shapes, thick filament lengths, thin:thick filament ratio, myofilament organization and sarcoplasmic reticulum (SR) distribution than those of adults. Viewed in cross section, most circular muscle cells were roughly triangular or ovoid in shape with a core of mitochondria; however, numerous muscle cells with crescent or other unusual cross-sectional shapes and muscle cells with unequal distributions of mitochondria were present in the paralarvae. The frequency of these muscle cells relative to 'normal' circular muscle cells ranged from 1:6 to 1:10 among the 19 paralarvae we surveyed. The thick filaments of the two types of circular fibers, superficial mitochondria-rich (SMR) and central mitochondria-poor (CMP), differed slightly in length among paralarvae with thick filament lengths of 0.83+/-0.15 microm and 0.71+/-0.1 microm for the SMR and CMP fibers, respectively (P 0.05; ANOVA). During ontogeny the thick filament lengths of both the CMP and SMR fibers increased significantly to 1.78+/-0.27 microm and 3.12+/-0.56 microm, respectively, in adults (P<0.0001 for both comparisons; ANOVA with Tukey's highly significant difference post hoc tests). When sectioned parallel to their long axes, the SMR and CMP fibers of both paralarvae and adults exhibited the myofilament arrangements typical of obliquely striated muscle cells but the angle of obliquity of the dense bodies was 22.8+/-2.4 deg. and 4.6+/-0.87 deg. for paralarvae and adults, respectively. There were also differences in the distribution of the anastomosing network of SR. In paralarvae, the outer and central zones of SR were well developed but the intramyoplasmic zone was greatly reduced in some cells or was scattered non-uniformly across the myoplasm. Whereas in adults the intramyoplasmic SR region was composed primarily of flattened tubules, it was composed primarily of rounded vesicles or tubules when present in the paralarvae. The ontogenetic differences in circular muscle structure were correlated with significant differences in their contractile properties. In brief tetanus at 20 degrees C, the mean unloaded shortening velocity of the paralarval circular muscle preparations was 9.1 L(0) s(-1) (where L(0) was the preparation length that generated the peak isometric stress), nearly twice that measured in other studies for the CMP fibers of adults. The mean peak isometric stress was 119+/-15 mN mm(-2) physiological cross section, nearly half that measured for the CMP fibers of adults. Reverse transcriptase-polymerase chain reaction analysis of paralarval and adult mantle samples revealed very similar expression patterns of the two known isoforms of squid MHC. The ontogenetic differences in the structure and physiology of the circular muscles may result in more rapid mantle movements during locomotion. This prediction is consistent with jet pulse durations observed in other studies, with shorter jet pulses providing hydrodynamic advantages for paralarvae.

Evidence of self-correcting spiral flows in swimming boxfishes.

The marine boxfishes have rigid keeled exteriors (carapaces) unlike most fishes, yet exhibit high stability, high maneuverability and relatively low drag given their large cross-sectional area. These characteristics lend themselves well to bioinspired design. Based on previous stereolithographic boxfish model experiments, it was determined that vortical flows develop around the carapace keels, producing self-correcting forces that facilitate swimming in smooth trajectories. To determine if similar self-correcting flows occur in live, actively swimming boxfishes, two species of boxfishes (Ostracion meleagris and Lactophrys triqueter) were induced to swim against currents in a water tunnel, while flows around the fishes were quantified using digital particle image velocimetry. Significant pitch events were rare and short lived in the fishes examined. When these events were observed, spiral flows around the keels qualitatively similar to those observed around models were always present, with greater vortex circulation occurring as pitch angles deviated from 0 degrees . Vortex circulation was higher in live fishes than models presumably because of pectoral fin interaction with the keel-induced flows. The ability of boxfishes to modify their underlying self-correcting system with powered fin control is important for achieving high levels of both stability and maneuverability. Although the challenges of performing stability and maneuverability research on fishes are significant, the results of this study together with future studies employing innovative new approaches promise to provide valuable inspiration for the designers of bioinspired aquatic vehicles.

Aerobic respiratory costs of swimming in the negatively buoyant brief squid Lolliguncula brevis.

Because of the inherent inefficiency of jet propulsion, squid are considered to be at a competitive disadvantage compared with fishes, which generally depend on forms of undulatory/oscillatory locomotion. Some squid, such as the brief squid Lolliguncula brevis, swim at low speeds in shallow-water complex environments, relying heavily on fin activity. Consequently, their swimming costs may be lower than those of the faster, more pelagic squid studied previously and competitive with those of ecologically relevant fishes. To examine aerobic respiratory swimming costs, O(2) consumption rates were measured for L. brevis of various sizes (2-9 cm dorsal mantle length, DML) swimming over a range of speeds (3-30 cm s(-1)) in swim tunnel respirometers, while their behavior was videotaped. Using kinematic data from swimming squid and force data from models, power curves were also generated. Many squid demonstrated partial (J-shaped) or full (U-shaped) parabolic patterns of O(2) consumption rate as a function of swimming speed, with O(2) consumption minima at 0.5-1.5 DML s(-1). Power curves derived from hydrodynamic data plotted as a function of swimming speed were also parabolic, with power minima at 1.2-1.7 DML s(-1). The parabolic relationship between O(2) consumption rate/power and speed, which is also found in aerial flyers such as birds, bats and insects but rarely in aquatic swimmers because of the difficulties associated with low-speed respirometry, is the result of the high cost of generating lift and maintaining stability at low speeds and overcoming drag at high speeds. L. brevis has a lower rate of O(2) consumption than the squid Illex illecebrosus and Loligo opalescens studied in swim tunnel respirometers and is energetically competitive (especially at O(2) consumption minima) with fishes, such as striped bass, mullet and flounder. Therefore, the results of this study indicate that, like aerial flyers, some negatively buoyant nekton have parabolic patterns of O(2) consumption rate/power as a function of speed and that certain shallow-water squid using considerable fin activity have swimming costs that are competitive with those of ecologically relevant fishes.

Swimming mechanics and behavior of the shallow-water brief squid Lolliguncula brevis.

Although squid are among the most versatile swimmers and rely on a unique locomotor system, little is known about the swimming mechanics and behavior of most squid, especially those that swim at low speeds in inshore waters. Shallow-water brief squid Lolliguncula brevis, ranging in size from 1.8 to 8.9 cm in dorsal mantle length (DML), were placed in flumes and videotaped, and the data were analyzed using motion-analysis equipment. Flow visualization and force measurement experiments were also performed in water tunnels. Mean critical swimming speeds (U(crit)) ranged from 15.3 to 22.8 cm s(-1), and mean transition speeds (U(t); the speed above which squid swim exclusively in a tail-first orientation) varied from 9.0 to 15.3 cm s(-1). At low speeds, negatively buoyant brief squid generated lift and/or improved stability by positioning the mantle and arms at high angles of attack, directing high-speed jets downwards (angles >50 degrees ) and using fin activity. To reduce drag at high speeds, the squid decreased angles of attack and swam tail-first. Fin motion, which could not be characterized exclusively as drag- or lift-based propulsion, was used over 50-95 % of the sustained speed range and provided as much as 83.8 % of the vertical and 55.1 % of the horizontal thrust. Small squid (<3.0 cm DML) used different swimming strategies from those of larger squid, possibly to maximize thrust benefits from vortex ring formation. Furthermore, brief squid employed various unsteady behaviors, such as manipulating funnel diameter during jetting, altering arm position and swimming in different orientations, to boost swimming performance. These results demonstrate that locomotion in slow-swimming squid is complex, involving intricate spatial and temporal interactions between the mantle, fins, arms and funnel.

In vivo observation of a non-noradrenergic regulation of arylalkylamine N-acetyltransferase gene expression in the rat pineal complex.

The rodent pineal gland is the end point of several peripheral and central fibers innervating the superficial and deep parts of the gland. Up to now, only the sympathetic transmitter norepinephrine is thought to regulate melatonin synthesis, although numerous biochemical experiments have reported in vitro effects of various transmitters on melatonin synthesis. To find out whether there is non-noradrenergic regulation of in vivo pineal metabolism, the mRNA encoding the enzyme arylalkylamine N-acetyltransferase was studied using the highly sensitive technique of in situ hybridization. The existence of a marked nocturnal increase of arylalkylamine N-acetyltransferase mRNA in the superficial pineal gland was confirmed. Interestingly and for the first time, a similar daily variation was observed in the deep pineal. After removal of superior cervical ganglia, the daily rhythm in arylalkylamine N-acetyltransferase mRNA was abolished in both the superficial and deep pineal indicating that the rhythm is driven by sympathetic input in the entire pineal complex. Interestingly, the remaining arylalkylamine N-acetyltransferase mRNA level in the pineal of day- and night-time ganglionectomized rats was significantly higher than in the pineal of day-time intact animals. These data reveal a sympathetic-dependent day-time inhibition of arylalkylamine N-acetyltransferase gene expression. In addition, the day-time pineal arylalkylamine N-acetyltransferase mRNA expression in ganglionectomized rats persisted after adrenal gland removal but was reduced by 50% after propranolol injection. These results indicate that arylalkylamine N-acetyltransferase mRNA in ganglionectomized rats is not induced by circulating catecholamines and may be caused by both a centrally originated norepinephrine, as already suggested, and other non-adrenergic transmitter(s). In conclusion, this work shows that norepinephrine drives the nocturnal increase of arylalkylamine N-acetyltransferase gene expression both in the superficial and deep pineal and strongly suggests that other neurotransmitters are involved in day-time inhibition and night-time stimulation of pineal metabolism.

Role of aerobic and anaerobic circular mantle muscle fibers in swimming squid: electromyography.

Circular mantle muscle of squids and cuttlefishes consists of distinct zones of aerobic and anaerobic muscle fibers that are thought to have functional roles analogous to red and white muscle in fishes. To test predictions of the functional role of the circular muscle zones during swimming, electromyograms (EMGs) in conjunction with video footage were recorded from brief squid Lolliguncula brevis (5.0-6.8 cm dorsal mantle length, 10.9-18.3 g) swimming in a flume at speeds of 3-27 cm s(-1). In one set of experiments, in which EMGs were recorded from electrodes intersecting both the central anaerobic and peripheral aerobic circular mantle muscles, electrical activity was detected during each mantle contraction at all swimming speeds, and the amplitude and frequency of responses increased with speed. In another set of experiments, in which EMGs were recorded from electrodes placed in the central anaerobic circular muscle fibers alone, electrical activity was not detected during mantle contraction until speeds of about 15 cm s(-1), when EMG activity was sporadic. At speeds greater than 15 cm s(-1), the frequency of central circular muscle activity subsequently increased with swimming speed until maximum speeds of 21-27 cm s(-1), when muscular activity coincided with the majority of mantle contractions. These results indicate that peripheral aerobic circular muscle is used for low, intermediate, and probably high speeds, whereas central anaerobic circular muscle is recruited at intermediate speeds and used progressively more with speed for powerful, unsteady jetting. This is significant because it suggests that there is specialization and efficient use of locomotive muscle in squids.

Lesions of the dorsomedial hypothalamic nucleus do not influence the daily profile of pineal metabolism in rats.

The present study attempted to characterize the effects of electrolytic lesions of the hypothalamic dorsomedial nucleus on the daily profile of pineal metabolism as well as on the inhibition of pineal melatonin synthesis induced by acute light exposure during the night. Adult male Wistar rats (n = 107, 12:12 h light-dark cycle) were left intact (n = 47) or lesioned (n = 60). Lesioned rats and their respective controls were killed at six time points distributed throughout the light-dark cycle. At ZT (zeitgeber time) 18 the animals were killed either in the dark or after 15 min of light stimulation. Pineal glands were assayed using high-performance liquid chromatography with electrochemical detection (HPLC-ED). There was no difference in the amounts of pineal indoles between lesioned and control rats under any of the experimental situations tested. These results suggest that in rats, the hypothalamic dorsomedial nucleus does not participate in either the neural control of daily pineal metabolism or the nocturnal light-induced inhibition of the pineal metabolism.

The role of the retrochiasmatic area in the control of pineal metabolism.

The aim of the present investigation was to study the effect of neurotoxic ibotenic acid lesion of the retrochiasmatic area on the daily profile of pineal N-acetylserotonin and melatonin synthesis and on the pineal metabolic reactivity to nocturnal short-term retinal photostimulation. Groups of rats were killed 6 h after lights off either in the dark of immediately after being photostimulated for 1 or 15 min. Additionally, groups of rats were sacrificed at six different time points throughout the 24-hour light-dark cycle. The results suggested the presence of two functionally distinct territories in the retrochiasmatic area. The basal retrochiasmatic area, an area situated immediately ventral to the third ventricle, behind the suprachiasmatic nuclei and in front of the arcuate nucleus, is implicated in the nocturnal inhibitory process induced by short-term retinal photostimulation. The lateral retrochiasmatic area, which is situated immediately lateral to the anterior periventricular nucleus, below the anterior hypothalamic nucleus and in front of the ventromedial hypothalamic nucleus, is importantly involved in the control of the peak amplitude of the daily production of N-acetylserotonin and melatonin by the pineal gland.

Pinealectomy causes glucose intolerance and decreases adipose cell responsiveness to insulin in rats.

Although the pineal gland influences several physiological systems, only a few studies have investigated its role in the intermediary metabolism. In the present study, male Wistar rats, pinealectomized or sham-operated 6 wk before the experiment, were submitted to both intravenous glucose tolerance tests (IVGTT) and insulin binding as well as glucose transport assays in isolated adipocytes. The insulin receptor tyrosine kinase activity was assessed in liver and muscle. The insulin secretory response during the IVGTT was impaired, particularly in the afternoon, and the glucose transport responsiveness was 33% lower in pinealectomized rats. However, no difference was observed in the insulin receptor number of adipocytes between groups as well as in insulin-stimulated tyrosine kinase activity, indicating that the initial steps in the insulin signaling were well conserved. Conversely, a 40% reduction in adipose tissue GLUT-4 content was detected. In conclusion, pinealectomy is responsible for both impaired insulin secretion and action, emphasizing the influence of the pineal gland on glucose metabolism.

The effect of melatonin chronic treatment upon macrophage and lymphocyte metabolism and function in Walker-256 tumour-bearing rats.

Melatonin is the main hormone involved in the neuroendocrine-immune axis. It also presents antitumour activity. To evaluate the role of melatonin on the progression of Walker-256 tumour in rats we determined the effect of the hormone on some biochemical and functional aspects of macrophage and lymphocytes from cachectic rats. An important finding observed in immune cells from tumour-bearing (TB) rats is the impairment on glutamine and glucose metabolism in such cells. These changes are very similar to those observed in pinealectomized rats (PNX). The increased production of lactate and the flux of glucose through the Krebs cycle and the reduction in glutamine consumption seems to be involved in the immunosuppression presented in the TB and PNX animals. Melatonin treatment restored the changes observed in the metabolism of glucose and glutamine and stimulated the proliferation of lymphocytes from tumour-bearing rats. The results indicate that the effect of melatonin upon tumour growth involves the stimulation of the immune system by the hormone.

The effect of melatonin chronic treatment upon macrophage and lymphocyte metabolism and function in Walker-256 tumour-bearing rats.

Melatonin is the main hormone involved in the neuroendocrine-immune axis. It also presents antitumour activity. To evaluate the role of melatonin on the progression of Walker-256 tumour in rats we determined the effect of the hormone on some biochemical and functional aspects of macrophage and lymphocytes from cachectic rats. An important finding observed in immune cells from tumour-bearing (TB) rats is the impairment on glutamine and glucose metabolism in such cells. These changes are very similar to those observed in pinealectomized rats (PNX). The increased production of lactate and the flux of glucose through the Krebs cycle and the reduction in glutamine consumption seems to be involved in the immunosuppression presented in the TB and PNX animals. Melatonin treatment restored the changes observed in the metabolism of glucose and glutamine and stimulated the proliferation of lymphocytes from tumour-bearing rats. The results indicate that the effect of melatonin upon tumour growth involves the stimulation of the immune system by the hormone.

Pineal metabolic reaction to retinal photostimulation in ganglionectomized rats.

The aim of the present work was to test the pineal gland metabolic reactivity to nocturnal retinal short term photic stimulation in superior cervical ganglionectomized rats. The experimental support for this work is the appearance of a transitory post synaptic hyperactivity in the pineal gland, during the anterograde degenerating process of the conarii sympathetic nerve fibers after surgical removal of the cell body. In this situation the pineal gland is deafferented from the peripheral sympathetic nervous system keeping intact, however, the direct central connections to the deep pineal/lamina intercalaris region (DP). The results show a blockade of the pineal noradrenergic stimulatory process due to the retinal photostimulation. The inactivation of N-acetyltransferase led to a true metabolic shift to the oxidative pathway resulting in a decrease of the amount of N-acetylserotonin and an increase of the amount of serotonin, 5-hydroxyindoleacetic acid and 5-hydroxytryptophan. This inhibitory process brought into action by retinal illumination is dependent on the direct central neural connections to the pineal gland, since rats that were lesioned in the DP, previously to ganglionectomy, did not show any alteration on the indolic content of the pineal gland when subjected to nocturnal retinal photostimulation.

The effects of lesions of the thalamic intergeniculate leaflet on the pineal metabolism.

The aim of the present work was to study, in rats, the effects of lesions of the thalamic intergeniculate leaflet (IGL) and the deep pineal/lamina intercalaris region (DP) on the diurnal profile of N-acetylserotonin (NAS) and on the nocturnal pineal reactivity to acute retinal light stimulation (1 or 15 min). The 24-h experiment shows that there is no phase-shifting on the diurnal NAS curve of groups of rats with bilateral IGL lesion compared to the controls. On the other hand there is a significant reduction on the amplitude of pineal NAS content observed in every nocturnal point of the curve. The pineal glands of IGL-lesioned rats, after 1 min of retinal light stimulation, keep their NAS content equal to the lesioned dark-killed rats. Nonetheless, after 15 min of photostimulation, the pineal NAS content is reduced to nearly zero equally to the control animals. DP lesion does not modify the content of NAS in the pineal gland of rats killed in the dark. However, the pineal photo-inhibition process induced by 1 min of light exposure is impaired. These results suggest that: (1) the intergeniculate leaflet has a role in regulating the amplitude of the diurnal rhythm of pineal NAS production rather than its phase entrainment to light-dark cycle. This effect is not dependent on the direct geniculo-pineal connections. (2) The nocturnal pineal photo-inhibition phenomenon could be decomposed in two processes. One, triggered by short pulses of light and totally dependent on the IGL and partially dependent on the direct monosynaptic pathway between this structure and the pineal gland.(ABSTRACT TRUNCATED AT 250 WORDS)