Ontogeny of the Pectoralis muscle in the little brown bat,Myotis lucifugus

The ontogeny of a primary flight muscle, the pectoralis, in the little brown bat (Myotis lucifugus: Vespertilionidae) was studied using histochemical, immunocytochemical, and electrophoretic techniques. In fetal and early neonatal (postnatal age 1–6 days) Myotis, histochemical techniques for myofibrillar ATPase (mATPase) and antibodies for slow and fast myosins demonstrated the presence of two fiber types, here called types I and IIa. These data correlated with multiple transitional myosin heavy chain isoforms and native myosin isoforms demonstrated with SDS-PAGE and 4% pyrophosphate PAGE. There was a decrease in the distribution and number of type I fibers with increasing postnatal age. At postnatal age 8–9 days, the adult phenotype was observed with regard to muscle fiber type (100% type IIa fibers) and myosin isoform profile (single adult MHC and native myosin isoforms). This “adult” fiber type profile and myosin isoform composition preceeded adult function by about 2 weeks. For example, little brown bats were incapable of sustained flight until approximately postnatal day 24, and myofiber size did not achieve adult size until approximately postnatal day 25. Although Myotis pectoralis is unique in being composed of 100% type IIa fibers, transitional fiber types and isoforms were present. These transitional forms had been observed previously in other mammals bearing mixed adult muscle fibers and which undergo transitional stages in muscle ontogeny. However, in Myotis pectoralis, this transition transpires relatively early in development. © 1994 Wiley-Liss, Inc.     

Ontogeny of flight in the little brown bat,Myotis lucifugus: behavior,morphology, and muscle histochemistry

Summary Postnatal changes in wing morphology, flight ability, muscle morphology, and histochemistry were investigated in the little brown bat, Myotis lucifugus. The pectoralis major, acromiodeltoideus, and quadriceps femoris muscles were examined using stains for myofibrillar ATPase, succinate dehydrogenase (SDH), and mitochondrial -glycerophosphate dehydrogenase (-GPDH) enzyme reactions. Bats first exhibited spontaneous, drop-evoked flapping behavior at 10 days, short horizontal flight at 17 days, and sustained flight at 24 days of age. Wing loading decreased and aspect ratio increased during postnatal development, each reaching adult range before the onset of sustained flight. Histochemically, fibers from the three muscles were undifferentiated at birth and had lower oxidative and glycolytic capacities compared to other age groups. Cross-sectional areas of fibers from the pectoralis and acromiodeltoideus muscles increased significantly at an age when dropevoked flapping behavior was first observed, suggesting that the neuromuscular mechanism controlling flapping did not develop until this time. Throughout the postnatal growth period, pectoralis and acromiodeltoideus muscle mass and fiber cross-sectional area increased significantly. By day 17 the pectoralis muscle had become differentiated in glycolytic capacity, as indicated by the mosaic staining pattern for -GPDH. By contrast, the quadriceps fibers were relatively large at birth and slowly increased in size during the postnatal period. Fiber differentiation was evident at the time young bats began to fly, as indicated by a mosaic pattern of staining for myosin ATPase. These results indicate that flight muscles (pectoralis and acromiodeltoideus) are less well developed at birth and undergo rapid development just before the onset of flight. By contrast the quadriceps femoris muscle, which is required for postural control, is more developed at birth than the flight muscles and grows more slowly during subsequent development.     

Functional innervation of the myometrium of Myotis lucifugus, the little brown bat

Myometria of pregnant and nonpregnant Myotis lucifugus were studied in vitro by using electrical field stimulation as well as autonomic agonists and antagonists to determine whether functional responses corresponded with structural evidence showing abundant adrenergic and sparse cholinergic innervation, which uniquely does not disappear during pregnancy. Field stimulation (70 V, 0.6 ms, 5.0-s pulse train, 2.5 - 60 Hz) of myometria from nonpregnant (hibernating) bats produced graded responses consisting of an initial alpha-adrenergic contraction and a subsequent beta-adrenergic relaxation phase. Responses were sensitive to both the nerve poison tetrodotoxin and the adrenergic antagonist guanethidine, demonstrating that they resulted from stimulation of intrinsic adrenergic nerves. Field stimulation responses were unaffected by atropine indicating that there was no functional cholinergic innervation, even though carbachol-induced contraction showed that muscarinic receptors were present. In contrast, functional innervation of cervical tissue was cholinergic and nonadrenergic-non-cholinergic, but not adrenergic. At the beginning of active gestation, some myometrial preparations exhibited little of no response to field stimulation. However, as uterine size increased, the biphasic response to field stimulation was enhanced, particularly the inhibitory (beta-adrenergic) phase. Moreover, the contractile phases, though reduced, was not abolished by alpha-adrenergic antagonists. The residual contractile response was also tetrodotoxin-resistant, suggesting that the myometrium was sensitive to direct electrical stimulation. Near the end of pregnancy, myometrial tissue became nonresponsive to both field stimulation and autonomic agonists, suggesting an absence of available receptor sites on muscle cells.     

Histochemical and biochemical plasticity of muscle fibers in the little brown bat (Myotis lucifugus)

Summary Fiber composition, and glycolytic and oxidative capacities of the pectoralis, gastrocnemius, and cardiac muscles from active and hibernating little brown bats (Myotis lucifugus) was studied. The data were used to test two hypotheses: First, since hibernating bats maintain the capability of flight and make use of leg muscles to maintain a roosting position all winter, the fiber composition of the pectoralis and gastrocnemius muscles should not change with season. Second, we tested the hypothesis of Ianuzzo et al. (in press), who propose that the oxidative potential of mammalian cardiac muscle should increase with increasing heart rate while glycolytic potential should not. Our results indicate that the fiber composition of the pectoralis muscle was uniformly fast-twitch oxidative (FO)_ regardless of the time of year, as predicted. However, the gastrocnemius muscle exhibited a change in FO composition from 83% in active to 61% in hibernating animals. Contrary to the variable change in histochemical properties with metabolic state, a trend of reduced maximal oxidative (CS) and glycolytic (PFK) potential during hibernation in both flight and leg muscles was apparent. The oxidative potential of flight and leg muscles decreased by 15.2% and 56.5%, respectively, while the glycolytic potential of the same muscles decreased by 23.5% and 60.5%, respectively. As predicted, the glycolytic potential of cardiac muscle remained constant between active and hibernating bats, although there was a significant decrease (22.0%) in oxidative potential during hibernation.Abbreviations FO fast-twitch oxidative - FG fast-twitch glycolytic - SO slow-twitch oxidative - Vmax maximal enzyme activity - PFK phosphofructokinase - CS citrate synthase     

Annual skeletal changes in the little brown bat, Myotis lucifugus lucifugus, with particular reference to pregnancy and lactation

Studies of bone from summer-active little brown bats, Myotis lucifugus lucifugus, have demonstrated sex differences in the renewal of skeletal mineral reserves following spring-arousal from hibernation. Patterns of bone remodeling in both sexes of bats indicate that new bone formation does not occur during hibernation: All new bone formation occurs during the summer-active season. Results show that a short period of time elapses after hibernation before the initial demands of a large fetus and rapidly growing neonate are expressed on maternal skeletal reserves. Bone loss in summer-active females was associated with pregnancy and lactation, whereas summer-active males did not show evidence of bone loss but, instead, uninterrupted bone accretion throughout the summer-active season. Osteoclasts and bone-forming osteoblasts, absent during the hibernation period, reappeared on bone surfaces following spring-arousal from hibernation. There was no apparent increase in osteoclast numbers or activity during lactation but resorption cavities were found in deep cortical lamellae distant from bone surfaces. The increase in bone resorption in lactating bats appeared to be by osteocytic osteolysis, suggesting that it might be a significant mechanism of bone/calcium regulation in this hibernating mammal throughout the year.     

A morphometric study of the cochlea of the little brown bat (Myotis lucifugus).

A detailed morphometric study of the basilar membrane was made from serial sections and graphic reconstructions of the cochlea of three little brown bats. Four distinct morphometric changes were observed within the basilar membrane. First, between 0-1.4 mm from the basal end of the cochlea, there is a rapid increase in width and cross-sectional area of the basilar membrane. Secondly, between 1.4-2.5 mm, there is little change in width of the basilar membrane (its cross-sectional area is at its greatest in this region). Thirdly, between 2.7-3.1 mm, there is a sudden decrease in cross-sectional area concomitant with an increase in the width of the basilar membrane. Finally, between 3.1 mm and the apex, there is a gradual decrease in cross-sectional area concomitant with an increase in the width of the basilar membrane. The magnitudes of the cross-sectional areas of the scalae media and vestibuli decrease from base to apex, but this is not true for the scala tympani. The cross-sectional area of the scala tympani appears to decrease from the base to 0.7 mm, then it increases up to 1.4 mm, and then it decreases to the apex. These morphometric changes in the basilar membrane of the little brown bat are compared to those in other echolocating and non-echolocating mammals. The significance of these changes is discussed in relation to the range of hearing in the little brown bat.     

Plasma progesterone levels during pregnancy in the little brown bat Myotis lucifugus (Vespertilionidae)

Plasma progesterone was measured by radioimmunoassay in individual female Myotis lucifugus throughout pregnancy and lactation. Progesterone levels, which averaged 6.7 +/- 0.7 ng/ml in late hibernation, rose to a mean of 18.9 +/- 6.7 ng/ml in unimplanted bats collected in the first two weeks after arrival at a maternity roost. Analysis of progesterone levels in bats in which the developmental stage of the embryo was known revealed two sharp, transient increases in plasma progesterone during the preimplantation period. The first, with values of 30-45 ng/ml, occurred at ovulation. The second, with values of 20-30 ng/ml, coincided with blastocyst formation. Progesterone levels increased exponentially from a mean of 7.4 +/- 1.0 ng/ml during early implantation to peak values of 100-200 ng/ml (means = 136.2 +/- 15.6) in the last two weeks of pregnancy, and showed no evidence of either a midpregnancy or prepartum decline. Despite involution of the corpus luteum at the end of pregnancy, progesterone levels averaged 9.0 +/- 1.0 ng/ml during lactation and did not decline until the end of lactation. In bats undergoing abortion, mean levels of plasma progesterone were already less than 6 ng/ml, equivalent to levels in nonbreeding females. The results indicate that the progesterone profile of pregnant M. lucifugus, though generally resembling those of other bats, exhibits several distinctive features. The sharp rise in plasma progesterone coinciding with blastocyst formation has not been reported in other mammals and suggests a possible role of progesterone in the cavitation process. In addition, peak values of plasma progesterone in late pregnancy were conspicuously higher than levels reported in other verpertilionid bats. The levels did not appear to fall before parturition, although such falls have been reported in other bats.     

Distribution of somatostatinlike immunoreactivity in the brain of the little brown bat (Myotis lucifugus)

The distribution of somatostatinlike immunoreactive (SLI) perikarya, axons, and terminals was mapped in subcortical areas of the brain of the little brown bat, Myotis lucifugus, using light microscopic immunocytochemistry. A preponderance of immunoreactivity was localized in reticular, limbic, and hypothalamic areas including: 1) in the forebrain: the bed nucleus of the stria terminalis; lateral preoptic, dorsal, anterior, lateral and posterior hypothalamic areas; amygdaloid, periventricular, arcuate, supraoptic, suprachiasmatic, ventromedial, dorsomedial, paraventricular, lateral and medial mammillary, and lateral septal nuclei; the nucleus of the diagonal band of Broca and nucleus accumbens septi; 2) in the midbrain: the periaqueductal gray, interpeduncular, dorsal and ventral tegmental, pretectal, and Edinger-Westphal nuclei; and 3) in the hindbrain: the superior central and parabrachial nuclei, nucleus incertus, locus coeruleus, and nucleus reticularis gigantocellularis. Other areas containing SLI included the striatum (caudate nucleus and putamen), zona incerta, infundibulum, supramammillary and premammillary nuclei, medial and dorsal lateral geniculate nuclei, entopeduncular nucleus, lateral habenular nucleus, central medial thalamic nucleus, central tegmental field, linear and dorsal raphe nuclei, nucleus of Darkschewitsch, superior and inferior colliculi, nucleus ruber, substantia nigra, mesencephalic nucleus of V, inferior olivary nucleus, inferior central nucleus, nucleus prepositus, and deep cerebellar nuclei. While these results were similar in some respects to those previously reported in rodents, they also provided interesting contrasts.     

Effects of preservation on wing morphometry of the little brown bat (Myotis lucifugus)

The effects of formalin fixation and subsequent alcohol preservation on various morphometric variables and their derivatives (lifting surface area, wingspan, mass, aspect ratio, wing loading and minimum power speed) and on the results of procedures that estimate lifting surface area of the little brown bat ( Myotis lucifugus ) are identified and quantified. Statisitical analysis demonstrates that the values of all of the examined morphometric variables depends upon the specimen type from which they are obtained (live animal; freshly killed specimen; immediately following formalin fixation; or after 36 weeks in alcohol). Over the short term, the choice of preservation fluid is ot important with respect to determination of the six variables studied. The fixation positin of the wing is an important factor in the determination of all variables except mass. Although originally suggested for study skins and not fluid-preserved specimens, 'intermediate' and 'extended' wing positions are demonstrably better than the conventional 'compressed' position. The estimation procedures of both Pirlot (1977) and Blood & McFarlane (1988) significantly underestimate analogous lifting surface areas determinedby tracing live bats. Smith & Starrett's (1979) procedure was found to yield accurate estimates occasionally: on live animals and preserved specimens with wings fixed in the extended position. Aldridge's (1988) method also yields accurate estimates of lifting surface area, but is limited to those museum specimens where the live or freshly-killed mass is known. Such conclusions permit recommendation of procedures that minimize changes arising through the fixation and preservation process in fluid-preserved museum specimens when compared to the live animal.     

Encoding of sound duration by neurons in the auditory cortex of the little brown bat, Myotis lucifugus

Responses of 117 single- or multi-units in the auditory cortex (AC) of bats (Myotis lucifugus) to tone bursts of different stimulus durations (1– 400 ms) were studied over a wide range of stimulus intensities to determine how stimulus duration is represented in the AC. 36% of AC neurons responded more strongly to short stimulus durations showing short-pass duration response functions, 31% responded equally to all pulse durations (i.e., all-pass), 18% responded preferentially to stimuli having longer durations (i.e., long-pass), and 15% responded to a narrow range of stimulus durations (i.e., band-pass). Neurons showing long-pass and short-pass duration response functions were narrowly distributed within two horizontal slabs of the cortex, over the rostrocaudal extent of the AC. The effects of stimulus level on duration selectivity were evaluated for 17 AC neurons. For 65% of these units, an increase in stimulus intensity resulted in a progressive decrease in the best duration. In light of the unusual intensity-dependent duration responses of AC neurons, we hypothesized that the response selectivities of AC neurons is different from that in the brainstem. This hypothesis was validated by results of study of the duration response characteristics of single neurons in the inferior colliculus. Accepted: 8 November 1996     

Dynamic aspects of the LHRH system associated with ovulation in the little brown bat (Myotis lucifugus)

Adult female bats were collected from natural roosting sites in pre-ovulatory and post-ovulatory conditions. LHRH neurones of these animals were examined using light and electron microscopic immunocytochemistry, and LHRH tissue contents were measured by radioimmunoassay. Comparisons between the two groups of bats revealed that the number of LHRH perikarya detected immunocytochemically, as well as hypothalamic LHRH content, were significantly reduced in post-ovulatory animals. Distributions of immunoreactive perikarya were, however, strikingly similar in both groups. The reduction in immunoreactive cell number observed after ovulation was therefore not restricted to an anatomically defined subset of neurones, but was evident throughout the population. The projection of LHRH-immunoreactive fibres that extend into the pituitary neural lobe in this species also exhibited changes related to endocrine condition. Morphometric indices of fibre density in the neural lobe were significantly reduced in post-ovulatory bats, as was LHRH content of the lower infundibular stalk and neural lobe. Fine structural study of perikarya revealed complex anatomical interactions between LHRH-immunopositive elements, especially in post-ovulatory bats. These interactions included direct apposition of perikarya, as well as more elaborate networks involving various combinations of perikarya and large- and small-caliber processes. These changes in the LHRH system associated with ovulation suggest reduction of stored peptide within perikarya and depletion from terminals within the lower infundibular stem and neural lobe. Parallel reductions in hypothalamic and neural lobe LHRH content during the periovulatory period support the hypothesis that the neural lobe component of the system contributes to control of gonadotrophin secretion in this species. Finally, increased complexity of anatomical contact between components of the LHRH system may be related to activation of this cell population in spring.     

Changes in the fine structure of tanycytes during the annual reproductive cycle of the male little brown bat Myotis lucifugus lucifugus.

The hypothalamic arcuate nucleus in the male little brown bat Myotis lucifugus lucifugus was studied with the electron microscope. Animals were killed by intracardial perfusion at each season throughout the year so that the arcuate nucleus could be examined for seasonal variations in morphology. Striking seasonal changes in the fine structure of ependymal tanycytes lining the arcuate nucleus were observed. Tanycytes in animals collected in the fall and early winter exhibited pale processes characterized by a scant internal framework of microtubules and fine filaments. These processes, which were found throughout the arcuate nucleus, exhibited simple irregular shapes. In animals collected between January and June, tanycyte processes contained dense accumulations of fine filaments intermingled with microtubules, and projected long attenuated extensions that often formed multilamellar sheets around axodendritic terminals or other neuronal elements. Tanycyte processes of animals collected in July and August were densely packed with microtubules and fine filaments. The processes radiated elaborate multilamellar extensions that encapsulated axons, dendrites and even entire neuronal perikarya. Multilamellar sheets consisted of as many as 10 or 12 closely spaced gyres. The seasonal variations in tanycyte structure are suggestive of astrocyte-like behaviour. These changes are discussed with respect to seasonal changes in hypothalamic neuroendocrine activity.