GABA-like immunoreactivity in the suboesophageal ganglion of the locust Schistocerca gregaria

Summary Neurones in the suboesophageal ganglion of the locust Schistocerca gregaria were stained with an antiserum raised against gamma amino butyric acid (GABA). This ganglion consists of the fused mandibular, maxillary and labial neuromeres. Immunoreactive cell bodies of similar size and distribution occur in the lateral, ventral and middorsal regions of all three neuromeres. Approximately 200 cell bodies stain in both the mandibular and maxillary neuromeres and 270 in the labial neuromere. A few distinctly larger cells occur in the ventral groups and one large pair occurs in the lateral group of the maxillary neuromere. Dorsal commissures DCIV and DCV are composed mainly of stained fibres, while DCI–DCIII are largely unstained. A ventral commissure also stains in the maxillary neuromere. All longitudinal tracts contain both stained and unstained fibres. Many processes within the neuropil are also immunoreactive. A stained axon is found in the posterior tritocerebral commissure which enters the anterior dorsal region of the mandibular neuromere. The salivary branch of the 7th nerve contains one stained axon and two axons stain in nerve 8 which innervates neck muscles.     

C. elegans dystroglycan coordinates responsiveness of follower axons to dorsal/ventral and anterior/posterior guidance cues

Neural development in metazoans is characterized by the establishment of initial process tracts by pioneer axons and the subsequent extension of follower axons along these pioneer processes. Mechanisms governing the fidelity of follower extension along pioneered routes are largely unknown. In C. elegans, formation of the right angle‐shaped lumbar commissure connecting the lumbar and preanal ganglia is an example of pioneer/follower dynamics. We find that the dystroglycan ortholog DGN‐1 mediates the fidelity of follower lumbar commissure axon extension along the pioneer axon route. In dgn‐1 mutants, the axon of the pioneer PVQ neuron faithfully establishes the lumbar commissure, but axons of follower lumbar neurons, such as PVC, frequently bypass the lumbar commissure and extend along an oblique trajectory directly toward the preanal ganglion. In contrast, disruption of the UNC‐6/netrin guidance pathway principally perturbs PVQ ventral guidance to pioneer the lumbar commissure. Loss of DGN‐1 in unc‐6 mutants has a quantitatively similar effect on follower axon guidance regardless of PVQ axon route, indicating that DGN‐1 does not mediate follower/pioneer adhesion. Instead, DGN‐1 appears to block premature responsiveness of follower axons to a preanal ganglion‐directed guidance cue, which mediates ventral‐to‐anterior reorientation of lumbar commissure axons. Deletion analysis shows that only the most N‐terminal DGN‐1 domain is required for these activities. These studies suggest that dystroglycan modulation of growth cone responsiveness to conflicting guidance cues is important for restricting follower axon extension to the tracts laid down by pioneers. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Changes in molluscan neurosecretory cells during reproductive cessation: cause or effect?

The pond snail Lymnaea stagnalis has a maximum life span of about 22 months. At the age of about 250 days animals start to decrease egg laying activity and at about 500 days most animals ceased egg laying activity. At the age of cessation of egg laying the neurosecretory caudodorsal cells (CDCs) which control egg laying in Lymnaea exhibit reduced branching patterns. At this stage the cells still exhibit their physiological properties. CDCs still contain biologically active peptides and in the isolated CNS they still exhibit an afterdischarge upon electrical stimulation. Probably in the intact animal cessation of egg laying occurs because the CDCs are not activated anymore by natural egg laying inducing stimuli. In very old animals CDCs exhibit signs of degeneration indicating that cell death occur. After an extended period of no egg laying of Lymnaea physiological changes occur in the CDCs. CDCs from animals after an extended period of no egg laying failed to exhibit an afterdischarge. In such CDCs chemical and electrical coupling among the CDCs are reduced. Morphologically reduced CDCs predominantly fail to exhibit an afterdischarge. However, there are minimally branched CDCs that still could give an afterdischarge. Probably morphological reduction is not the only factor that defines afterdischarge failure. At present we suggest the following sequence of changes. 1. Morphological reduction of CDC branching patterns. 2. Cessation of egg laying. 3. Physiological changes in the CDCs resulting in afterdischarge failure. 4. Further morphological and physiological deterioration of CDCs.     

Terminal degeneration in the mediodorsal cerebral cortex of the lizard Agama agama: Light and electron microscopy

The degeneration of axon terminals in the small-celled part of the mediodorsal cortex (sMDC) of the lizard Agama agama has been studied after lesions in the dorsal cortex at various survival periods. The Fink-Heimer stain was used to map and demonstrate terminal degeneration with the light and electron microscope. Electron microscopy was used to identify and describe degenerating boutons ultrastructurally. One sham-operated and three unoperated animals served as controls. Between 6 and 21 days postsurgically, degenerating terminals can be seen through 80% of the superficial plexiform layer, the zone adjacent to the cellular layer remaining free of degeneration. Swelling of dendrites in the outer part of the superficial plexiform layer and increased numbers of vacuolar invaginations, both present at short (24 hr–6 days; peak at 48–54 hr) survival periods, can be regarded as reaction to the surgical trauma. Degeneration of axon terminals takes three forms, all of the electron-dense type: gray boutons, degenerating bouton-dendritic spine complexes surrounded or engulfed by glia, and degeneration debris inside glial processes. Several forms of terminal degeneration occur concomitantly at any short (3–12 days) survival time. At longer survival times (15–21 days) only debris is present. From 6 days on, considerable numbers of degenerating structures are present, but the majority of degenerating boutons and debris are associated with reactive glia rather than with dendrites. From these observations it is concluded that in this lizard application of the combined degeneration-Golgi-EM technique would probably lead to little success. Electron microscopy of Fink-Heimer-stained sections suggests that degenerating bouton-dendritic spine complexes and degeneration debris accumulate silver particles, whereas gray boutons do not.     

Structure of allotransplanted ganglia and regenerated neuromuscular connections in crayfish

In adult crayfish, Procambarus clarkii, motoneurons to a denervated abdominal superficial flexor muscle regenerate long-lasting and highly specific synaptic connections as seen from recordings of excitatory postsynaptic potentials, even when they arise from the ganglion of another crayfish. To confirm the morphological origins of these physiological connections we examined the fine structure of the allotransplanted tissue that consisted of the third abdominal ganglion and the nerve to the superficial flexor muscle (the fourth ganglion and the connecting ventral nerve cord were also included). Although there is considerable degeneration, the allotransplanted ganglia display intact areas of axon tracts, neuropil, and somata. Thus in both short (6–8 weeks) and long (24–30 weeks) term transplants approximately 20 healthy somata are present and this is more than the five axons regenerated to the host muscle. The principal neurite and dendrites of these somata receive both excitatory and inhibitory synaptic inputs, and these types of synaptic contacts also occur among the dendritic profiles of the neuropil. Axon tracts in the allotransplanted ganglia and ventral nerve cord consist largely of small diameter axons; most of the large axons including the medial and lateral giant axons are lost. The transplanted ganglia have many blood vessels and blood lacunae ensuring long-term survival. The transplanted superficial flexor nerve regenerates from the ventral to the dorsal surface of the muscle where it has five axons, each consisting of many profiles rather than a single profile. This indicates sprouting of the individual axons and accounts for the enlarged size of the regenerated nerve. The regenerated axons give rise to normal-looking synaptic terminals with well-defined synaptic contacts and presynaptic dense bars or active zones. Some of these synaptic terminals lie in close proximity to degenerating terminals, suggesting that they may inhabit old sites and in this way ensure target specificity. The presence of intact somata, neuropil, and axon tracts are factors that would contribute to the spontaneous firing of the transplanted motoneurons. © 1996 John Wiley & Sons, Inc.     

Patterns and projections of crustacean cardioactive-peptide-immunoreactice neurones of the terminal ganglion of crayfish

Three distinct clusters of crustacean cardioactive-peptide-immunoreactive neurones occur in the terminal abdominal ganglion of the crayfish species Orconectes limosus, Astacus leptodactylus, Astacus astacus and Procambarus clarkii, as revealed by immunocytochemistry of whole-mount preparations and sections. They exhibit similar topology and projection patterns in all four studied species. An anterior ventral lateral and a posterior lateral cluster contain one small, strongly stained perikaryon and two large, less intensely stained perikarya, each showing contralateral projections. A posterior medial lateral cluster of up to six cells also contains these two types of perikarya. Whereas the small type perikarya belong to putative interneurones, the large type perikarya give rise to extensive neurohaemal plexuses in perineural sheaths of the third roots of the fifth abdominal ganglia, the connectives, the dorsal telson nerves, the ganglion itself, its roots and arteriolar supply. Thin fibres from these plexuses reach newly discovered putative neurohaemal areas around the hindgut and anus via the intestinal and the anal nerves, and directly innervate the phasic telson musculature. A comparison with earlier investigations of motoneurones and segmentation indicates that these three cell groups containing putative neurosecretory neurones may be members of at least three neuromeres in this ganglion. Crustacean cardioactive peptide released from these neurones may participate in the neurohumoral and modulatory control of different neuronal and muscle targets, thereby exceeding its previously established hindgut and heart excitatory effects.Abbreviations AG abdominal ganglion - adpl arteria dorsalis pleica - Ala arreria lateralis abdominalis - Asub arteria subneuralis - CCAP crustacean cardioactive peptide - CNS central nervous system - IR immunoreactive - LG lateral giant axon - LTr lateral tract - MDT medial dorsal tract - MG medial giant axon - M Tr medial tract - mcan musculus compressor ani - mfltp museulus flexor telsonos posterior - nan nervus ani (AG6 N5) - nant nervus anterior (AG6 N1, N2) - nia nervus intestinal anterior - nin nervus intestinalis (AG6 N7) - nip nervus intestinalis posterior - nteld nervus telsonos dorsalis (AG6 N6) - nielv nervus telsonos ventralis (AG6 N4) - nur nervus uropedalis (AG6 N3) - nven nervus ventralis (AG5 N3) - PIR peri-intestinal ring - PTF posterior telson flexor - VLT ventral lateral tract - VMT ventral medial tract - VNC ventral nerve cord - VIF ventral telson flexor - AVLC, PLC, PMLC anterior ventral lateral, posterior lateral, posterior medial lateral CCAP-immunoreactive cell cluster - A6AVC, A7AVC anterior ventral commissures - A7DCI dorsal commissure I - A7PVC posterior ventral commissure - A7SCII sensory commissure II - A7VCII, A7VCIII ventral commissures II and III of the sixth (A6) and seventh (A7) abdominal neuromer     

Intragranular colocalization of arginine vasopressin and methionine-enkephalin-octapeptide in CRF-axons in the rat median eminence

Summary Ultrastructural appearances of axonal terminals containing corticoliberin (CRF) were examined in the rat median eminence prepared by a freeze-drying procedure. Immunolabeling was performed by using 5-, 8-, or 15-nm gold-antibody complexes for CRF, arginine vasopressin (VP) and methionine-enkephalin-octapeptide (Enk-8), singly or in combination. In intact animals, the CRF-containing secretory granules were only slightly labeled with goldanti-VP or -Enk-8. In adrenalectomized rats, granules within single axons appeared to be labeled with all the immunogold complexes. This intragranular colocalization of the three antigens was confirmed by using three neighboring sections of the same axon terminals which were stained separately with each one of the antibodies and visualized with the avidin-biotin-peroxidase complex method. The granules labeled for CRF had decreased 9 days after adrenalectomy but had increased again by day 21, while those labeled for VP steadily increased after adrenalectomy. However, this did not correspond with the appearances of cell bodies in the paraventricular nucleus; the cell bodies labeled for both CRF and VP steadily increased in number and in stainability. By contrast, Enk-8 immunoreactivity in the axonal terminals and cell bodies was not affected by adrenalectomy. These findings suggest that although the three peptides could be released simultaneously from the axonal terminals, VP may play some special role in the expression of CRF activity.     

Histology and lectin-binding patterns in the skin of the terrestrial horned frog Ceratophrys ornata

The terrestrial horned frog, Ceratophrys ornata, lives on a wet substratum and absorbs water through the ventral epidermis; water is lost by evaporation from the dorsal skin. Thus, this species may be useful as a model for determining whether or not skin histology and lectin-binding patterns, indicative of glycoconjugates, are related to skin functions such as osmoregulation and water balance. With this in mind, a histological and lectin-histochemical study was carried out on dorsal and ventral skin of aquatic tadpoles and of a young terrestrial frog of C. ornata. Sections of skin were stained with various dyes to demonstrate general histological features and with two horseradish peroxidase (HRP)-conjugated lectins, Ulex europaeus agglutinin (UEA 1) and soybean agglutinin (SBA) which bind to specific terminal sugar residues of glycoconjugates, namely L-fucose and N-acetyl-D-galactosamine or D-galactose, respectively. In early stage tadpoles both lectin-binding patterns were similar in the bilaminar epidermis of dorsal and ventral skin (i.e., each lectin stained the apical cell layer). However, metamorphic changes resulted in a young frog with typical adult-type skin composed of a stratified squamous epidermis and three distinct types of glands containing glycoconjugates in their secretions. Strikingly different lectin-binding patterns were evident in the epidermis from dorsal and ventral regions of the body. The epidermis from the dorsal region was stained by both lectins; in contrast, that from the ventral region although stained strongly by HRP-SBA, did not react with HRP-UEA 1 indicating that few, if any, fucose residues were present in the ventral epidermis. These findings, as suggested in the discussion, indicate that different glycoconjugate patterns in dorsal and ventral skin may be associated with the regulation of water balance in the frog.     

Axon transport of steroid hormones via spinal root fibers in old rats

Labelled steroid hormones,³H-hydrocortisone and¹⁴C-testosterone, being injected in the gray matter of theL ₅−L ₆ spinal cord segments were shown to be transported via ventral and dorsal root fibers (antero- and retrograde directions, respectively) of old (25 to 28 months) rats with a lower velocity than in adult young (6 to 11 months) animals. The averaged maximum velocities of axon transport (AT) through the ventral and dorsal roots were: for³H-hydrocortisone, 756±63 and 738±46 mm per day, and for¹⁴H-testosterone, 624±54 and 608±80 mm per day, respectively. Therefore, in old rats the AT velocities for³H-hydrocortisone and¹⁴C-testosterone were about four and seven-eight times lower than those in adult rats. In the course of anterograde, AT through the ventral roots in old rats the inclusion of³H-hydrocortisone is sharply suppressed (by more than an order of magnitude), as compared with than in adult animals. The doses of non-labelled steroid hormones within a 10⁻⁷ – 10⁻⁶ range, injected into the lumbar spinal segments, resulted in hyperpolarization of muscle fibers of themm. gastrocnemius anddeltoideus, but this phenomenon developed in old rats much later than in adult rats. It is obvious that AT of steroid hormones can be considered one of the mechanisms of their effects on the tissue of an organism, and this mechanism undergoes extremely intensive modifications with aging.     

Survival of egg-laying controlling neuroendocrine cells during reproductive senescence of a mollusc

During brain aging neuronal degradation occurs. In some neurons this may result in degeneration and cell death, still other neurons may survive and maintain their basic properties. The present study deals with survival of the egg-laying controlling neuroendocrine caudodorsal cells (CDCs) during reproductive senescence of the pond snail Lymnaea stagnalis. In senescent animals CDCs exhibited reduced branching patterns but still maintained their electrophysiological characteristics. In the isolated CNS the cells could still respond with an afterdischarge upon electrical stimulation. After an extended period of no egg laying of Lymnaea CDCs failed to exhibit an afterdischarge. In senescent CDCs that failed an afterdischarge, discharge activity could be restored by exposure to peptides released by CDCs from reproductive animals. Moreover, raising the intracellular cAMP level could induce discharge activity in CDCs with afterdischarge failure. Discharge activity also occurred during depolarization of senescent CDCs by exposure of the cells to saline with a high potassium concentration. These results indicate that in senescent CDCs the pacemaking mechanism of the afterdischarge is still intact but that the initial activation fails. Chemical (auto)transmission of CDCs in such animals was indeed reduced as indicated by the small amplitude of the depolarizing afterpotential (DAP) induced by electrical stimulation. Interestingly, CDCs of senescent animals contained a relative large amount of a particular small peptide. The artificially synthesized peptide appeared to suppress DAP induction in CDCs. Possibly, release of the peptide contributes to the prevention of afterdischarge induction in senescent CDCs. The results so far indicate that in senescent Lymnaea neurons electrophysiological functions persist even after long periods of inactivity and severe morphological reduction.     

Ultrastructural changes in the superficial layers of the superior colliculus in Galago crassicaudatus (primates) after eye enucleation

Summary Several types of terminals were found in the three superficial collicular layers of Galago. At least two axon terminals with round vesicles (R1 and R2) could be distinguished on the basis of vesicle packing and electron density of the cytoplasmic and mitochondrial matrices. R1 axon terminals were characterized by aggregations of vesicles in an electron lucent cytoplasm and mitochondria with a relatively dark matrix, while in R2 axon terminals the vesicles were more evenly distributed in an electron dense cytoplasm and the mitochondrial matrix was pale. R2 endings occurred in clusters in the stratum griseum superficiale; they were absent in the stratum zonale. R1 endings were found in all three superficial collicular layers. Both types of R terminals made asymmetrical contacts with small dendrites, dendritic spines and F profiles. Profiles containing flattened vesicles and establishing symmetrical contacts were numerous, and many could be identified as dendrites by accepting as criteria for dendrites evenly spaced microtubules, clusters of ribosomes and the fact that these F profiles were postsynaptic to other terminals. F terminals were presynaptic to other F profiles, dendrites and somata; they were postsynaptic to R terminals and took part in serial synapses. Dendrodendritic contacts were frequent, somatodendritic contacts rare. After eye enucleation most R2 axon terminals underwent the electron dense degenerative reaction. The degeneration process was a lengthy one; many degenerating boutons were found 30 days after axotomy and some persisted up to 180 days postoperatively. There was strong indication that the superior colliculus received more crossed than uncrossed retinofugal fibers. The crossed and uncrossed retinocollicular axons terminated in two different substrata of the stratum griseum superficiale.This study was supported by N.I.H. Grant RR-00165 to Yerkes Regional Primate Research Center and N.I.H Grant EY 00638-03 to J. Tigges. — The opportunity to use the electron microscopic facilities of the Fernbank Science Center for the initial stage of this work is gratefully acknowledged.     

Ultrastructural relation between cortical efferents and terminals containing enkephalin-like immunoreactivity in rat neostriatum

The interrelationships between cortical efferents and terminals containing enkephalin-like immunoreactivity (ELI) were examined by combining anterograde degeneration with electron microscopic immunocytochemistry in the adult rat neostriatum. Two days following unilateral removal of the cerebral cortex, the brains were fixed by aortic arch perfusion, then sectioned and processed for the immunocytochemical localization of an antiserum directed against methionine (Met5)-enkephalin. The observed relationships between the degenerating cortical efferents and immunocytochemically labeled terminals were of two types. In the first, the degenerating and ELI containing terminals converged on the same unlabeled dendrite or dendritic spine. In the second, terminal and preterminal axons of the ELI containing neurons had one surface directly apposed to the plasma membrane of a degenerating axon terminal. These findings support the concept that neurons containing opioid peptides and cortical efferents modulate the output of common recipient neurons and may also directly interact with each other through presynaptic axonal mechanisms in the rat neostriatum.     

The temporo-spatial course of degeneration after cutting cortico-cortical connections in adult rats

Summary Adult albino rats received callosotomies or lesions in the paracingular cortex. Between 12 h and 3 months after injury the structure and topography of the degeneration products were studied by light- and electron-microscopy. The degeneration process was quantified by television-image analysis applied to sections prepared according to a new technique that stains reliably degenerating terminals and lysosomes (Gallyas et al. 1980). All types of cortico-cortical connections show a multiphasic degeneration process: During a precursor stage a small number of dense bodies and mitochondrial granules are stained. These and the few early degenerating axon terminals are much more diffusely distributed than the large number of terminals that degenerate during the following period. The terminal degeneration shows a biphasic time course. One maximum appears at 2–7 days post operation, which corresponds to the well known direct consequence of axotomy. The second peak at 10–20 days post operation could be caused by transneuronal reorganization of the cortical connectivity. Terminal degeneration always begins along the borders between cortical regions and areas, but it may change its laminar and columnar distribution pattern during the second phase. The degeneration products that are phagocytosed by astrocytes seem to be removed by intracellular transport to their perivascular endfeet. The degeneration process ends with fiber degeneration which, especially in laminae I and VI, may form a separate peak after 20 days or more.On leave from: Department of Neurosurgery, University of Göttingen, Federal Republic of Germany;On leave from: First Department of Anatomy, Semmelweis University, Medical School, Budapest, Hungary     

The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio

We have examined neuronal differentiation and the formation of axon tracts in the embryonic forebrain and midbrain of the zebrafish, between 1 and 2 days postfertilisation. Axons were visualised with three techniques; immunocytochemistry (using HNK-1 and antiacetylated tubulin antibodies) and horseradish peroxidase (HRP) labelling in whole-mounted brains, and transmission electron microscopy. Differentiation was monitored by histochemical staining for acetylcholinesterase (AChE). These independent methods demonstrated that a simple grid of tracts and commissures forms the initial axon scaffold of the brain. At 1 day, the olfactory nerve, four commissures, their associated tracts and three other non-commissural tracts are present. By 2 days, these tracts and commissures have all greatly enlarged and, in addition, the optic nerve and tract, and three new commissures and their associated tracts have been added. Small applications of HRP at various sites revealed the origins and projections of some of these earliest axons. Retrogradely labelled cell bodies originated from regions that were also positive for AChE activity. At 1 day, HRP-labelled axons were traced: (1) from the olfactory placode through the olfactory nerve to the dorsal telencephalon; (2) from the telencephalon into the tract of the anterior commissure and also to the postoptic region of the diencephalon; (3) from the hindbrain through the ventral midbrain and diencephalon to the postoptic commissure; (4) from the dorsal diencephalon (in or near the epiphysis) to the tract of the postoptic commissure; (5) from ventral and rostral midbrain through the posterior commissure. Three new projections were demonstrated at 2 days: (1) from the retina through the tract of the postoptic commissure to the tectum; (2) from the telencephalon to the contralateral diencephalon; and (3) from the telencephalon to the ventral flexure. These results show that at 1 day, the zebrafish brain is impressively simple, with a few small, well-separated tracts but by 2 days the brain is already considerably more complex. Most of the additional axons added onto pre-existent tracts rather than pioneered new ones supporting the notion that other axons play a crucial role in the guidance of early central nervous system (CNS) axons.     

SALMFamide2 and serotonin immunoreactivity in the nervous system of some acoels (Xenacoelomorpha)

Acoel worms are simple, often microscopic animals with direct development, a multiciliated epidermis, a statocyst, and a digestive parenchyma instead of a gut epithelium. Morphological characters of acoels have been notoriously difficult to interpret due to their relative scarcity. The nervous system is one of the most accessible and widely used comparative features in acoels, which have a so‐called commissural brain without capsule and several major longitudinal neurite bundles. Here, we use the selective binding properties of a neuropeptide antibody raised in echinoderms (SALMFamide2, or S2), and a commercial antibody against serotonin (5‐HT) to provide additional characters of the acoel nervous system. We have prepared whole‐mount immunofluorescent stainings of three acoel species: Symsagittifera psammophila (Convolutidae), Aphanostoma pisae, and the model acoel Isodiametra pulchra (both Isodiametridae). The commissural brain of all three acoels is delimited anteriorly by the ventral anterior commissure, and posteriorly by the dorsal posterior commissure. The dorsal anterior commissure is situated between the ventral anterior commissure and the dorsal posterior commissure, while the statocyst lies between dorsal anterior and dorsal posterior commissure. S2 and serotonin do not co‐localise, and they follow similar patterns to each other within an animal. In particular, S2, but not 5‐HT, stains a prominent commissure posterior to the main (dorsal) posterior commissure. We have for the first time observed a closed posterior loop of the main neurite bundles in S. psammophila for both the amidergic and the serotonergic nervous system. In I. pulchra, the lateral neurite bundles also form a posterior loop in our serotonergic nervous system stainings.     

Effect of aging on the response of biochemical markers in the rabbit subjected to short-term partial bladder obstruction

Purpose Partial bladder outlet obstruction (PBOO) results in marked biochemical alterations in the bladder. In this study, we focused on comparison of thapsigargin sensitive sarco/endoplasmic reticulum Ca²⁺ ATPase activity (SERCA) and Citrate Synthase after short term PBOO in young versus old rabbits. Materials and methods A total of 20 young and 20 mature male rabbits were divided into 4 sub-groups of 5 rabbits each (4 obstructed and 1 sham-control rabbit). The rabbits in the groups were evaluated after 1, 3, 7, and 14 days of obstruction, respectively. The activities of SERCA and citrate synthase were examined as markers for sarcoplasmic reticular calcium storage and release and mitochondrial function, respectively. Results The SERCA activity of bladder body smooth muscle in the young animals increased at 7 and 14 days. For the old rabbits, the SERCA activity decreased significantly by 1 day and remained this level throughout the course of obstruction, and was significantly lower than young at all time periods. The citrate synthase activity in the young animals decreased over the 1–7 days, and then returned toward control level by 14 days following obstruction. In the old animals, citrate synthase activity of bladder body smooth muscle progressively decreased over the course of the study, and was significantly lower in the old than the young animals after 14 days obstructed. Conclusion The urinary bladders of the young rabbits have a considerable greater ability to adapt to PBOO than do those of the old rabbits. The deterioration of mitochondrial and SR function may be important mechanisms underlying geriatric voiding dysfunction.     

Immuno-electron microscopy of sorting and release of neuropeptides in Lymnaea stagnalis

Summary The cerebral caudodorsal cells of the pulmonate snail Lymnaea stagnalis control egg laying and egglaying behavior by releasing various peptides derived from two precursors. The biosynthesis, storage, intracellular breakdown and release of three caudodorsal cell peptides were studied by means of immuno-electron microscopy using antisera raised to fragments of these peptides: (1) Caudodorsal Cell Hormone-I (CDCH-I; derived from precursor I), (2) Caudodorsal Cell Hormone-II (CDCH-II; from precursor II), and (3) -Caudodorsal Cell Peptide ( CDCP; from both precursors). After affinity purification of the antisera, the specificity of the sera was confirmed with dotting immunobinding assays. From the ultrastructural immunocytochemical data it has been concluded that the precursor molecules are cleaved at the level of the Golgi apparatus after which the C-terminal parts (containing CDCP) and N-terminal parts (containing CDCH-I or CDCH-II) are sorted and preferentially packaged into large electron-dense granules (MD 150 nm), respectively. Very probably, the content of the large electron-dense granules is degraded within the cell body. The immunoreactivity of the secretory granules increases during discharge from the Golgi apparatus, indicating further processing. At least a portion of the secretory granules contains all three peptides, as shown by double and triple immunopositive stainings whereas other granules appear to contain only one or two of these peptides. The caudodorsal cells release multiple peptides via exocytosis from neurohemal axon terminals into the hemolymph and from blindly ending axon collaterals into the intercellular space of the cerebral commissure (nonsynaptic release).     

Age-regulated cycling metabolites are relevant for behavior

Circadian cycles of sleep:wake and gene expression change with age in all organisms examined. Metabolism is also under robust circadian regulation, but little is known about how metabolic cycles change with age and whether these contribute to the regulation of behavioral cycles. To address this gap, we compared cycling of metabolites in young and old Drosophila and found major age-related variations. A significant model separated the young metabolic profiles by circadian timepoint, but could not be defined for the old metabolic profiles due to the greater variation in this dataset. Of the 159 metabolites measured in fly heads, we found 17 that cycle by JTK analysis in young flies and 17 in aged. Only four metabolites overlapped in the two groups, suggesting that cycling metabolites are distinct in young and old animals. Among our top cyclers exclusive to young flies were components of the pentose phosphate pathway (PPP). As the PPP is important for buffering reactive oxygen species, and overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key component of the PPP, was previously shown to extend lifespan in Drosophila, we asked if this manipulation also affects sleep:wake cycles. We found that overexpression in circadian clock neurons decreases sleep in association with an increase in cellular calcium and mitochondrial oxidation, suggesting that altering PPP activity affects neuronal activity. Our findings elucidate the importance of metabolic regulation in maintaining patterns of neural activity, and thereby sleep:wake cycles.     

The tritocerebral commissure ''dwarf'' (TCD): a major GABA-immunoreactive descending interneuron in the locust

Summary The minor branch of the tritocerebral commissure of the locust,Locusta migratoria, contains only two axons which are from interneurons in the brain descending to the ventral cord ganglia. The smaller of these two neurons, the tritocerebral commissure dwarf (TCD), is immunoreactive to GABA, suggesting that it may be an inhibitory interneuron. We have exploited the accessibility of its axon in the commissure, first, to fill it with cobalt to define its morphology, and second, to record its input characteristics. It has a cell body and arborization of fine branches in the deutocerebrum of the brain, its axon passes contralateral through the tritocerebral commissure and it forms bilateral arborizations in the suboesophageal and three thoracic ganglia. It receives mechanosensory input from many regions of the ipsilateral body and head, and it is sensitive to illumination levels, generally showing greater spontaneous activity in the dark.It is one of the largest GABA-immunoreactive descending interneurons in the locust, suggesting it plays a prominent role in behaviour. Since it is easily accessible for physiological recording, its roles in circuits for particular components of behaviour should be amenable to investigation.     

In vivo recordings of neuroendocrine cells (caudo-dorsal cells) in the pond snail

Summary Ovulation and egg-laying behavior in the pond snailLymnaea stagnalis are controlled by the neuroendocrine caudodorsal cells (CDCs), constituting two clusters — one in each cerebral ganglion — totaling about 100 cells. In vitro studies have shown that the CDCs release their products, including the ovulation hormone, during a burst of spiking activity lasting for about 30 min (CDC discharge). This burst can be initiated by repeated intracellular stimulation with depolarizing current pulses, in which case the firing pattern is termed afterdischarge.Using cuff electrodes we recorded extracellularly from the intercerebral commissure, (the neurohaemal area of the CDCs) to study the activity of these cells during spontaneous egg-laying of freely behaving snails.The cuff-implanted snails showed long-lasting spiking activity prior to every bout of egg-laying. These spontaneous long-lasting discharges had several features in common with the intracellularly recorded activity of the CDCs in vitro: the time courses of spike broadening and of firing rates in the cuff-implanted animals were very similar to the characteristic patterns found in the isolated brain. Firing rates were higher and durations were longer in the cuff-implanted animals, however. In vitro, the duration of the discharge could be prolonged appreciably by recording in blood instead of normal saline, indicating that the bathing fluid normally used causes shortening of the CDC discharge. The way in which CDC discharges are triggered is discussed as a possible explanation for the differences in firing rates.The pattern of locomotion during spontaneous egg-laying was largely similar in cuff-implanted and unoperated animals. The level of locomotion was lower in the experimental animals. In addition, the rate of locomotion only partially returned to pre-oviposition levels. This is ascribed to the effect of the operation.It is concluded that the afterdischarge is the natural firing pattern of the caudodorsal cells ofLymnaea, and that this firing pattern constitutes the central event in the egg-laying behavior of this animal.Abbreviations CDC caudodorsal cells - CDCH caudodorsal cell hormone - CDCA caudodorsal cell autotransmitter