Innervation of the gallbladder: structure, neurochemical coding, and physiological properties of guinea pig gallbladder ganglia.

The muscle and epithelial tissues of the gallbladder are regulated by a ganglionated plexus that lies within the wall of the organ. Although these ganglia are derived from the same set of precursor neural crest cells that colonize the gut, they exhibit structural, neurochemical and physiological characteristics that are distinct from the myenteric and submucous plexuses of the enteric nervous system. Structurally, the ganglionated plexus of the guinea pig gallbladder is comprised of small clusters of neurons that are located in the outer wall of the organ, between the serosa and underlying smooth muscle. The ganglia are encapsulated by a shell of fibroblasts and a basal lamina, and are devoid of collagen. Gallbladder neurons are rather simple in structure, consisting of a soma, a few short dendritic processes and one or two long axons. Results reported here indicate that all gallbladder neurons are probably cholinergic since they all express immunoreactivity for choline acetyltransferase. The majority of these neurons also express substance P, neuropeptide Y, and somatostatin, and a small remaining population of neurons express vasoactive intestinal peptide (VIP) immunoreactivity and NADPH-diaphorase enzymatic activity. We report here that NADPH-diaphorase activity, nitric oxide synthase immunoreactivity, and VIP immunoreactivity are expressed by the same neurons in the gallbladder. Physiological studies indicate that the ganglia of the gallbladder are the site of action of the following neurohumoral inputs: 1) all neurons receive nicotinic input from vagal preganglionic fibers; 2) norepinephrine released from sympathetic postganglionic fibers acts presynaptically on vagal terminals within gallbladder ganglia to decrease the release of acetylcholine from vagal terminals; 3) substance P and calcitonin gene-related peptide, which are co-expressed in sensory fibers, cause prolonged depolarizations of gallbladder neurons that resemble slow EPSPs; and 4) cholecystokinin (CCK) acts presynaptically within gallbladder ganglia to increase the release of acetylcholine from vagal terminals. Results reported here indicate that hormonal CCK can readily access gallbladder ganglia, since there is no evidence for a blood-ganglionic barrier in the gallbladder. Taken together, these results indicate that gallbladder ganglia are not simple relay stations, but rather sites of complex modulatory interactions that ultimately influence the functions of muscle and epithelial cells in the organ.

Development of differential preganglionic projections to pre- and paravertebral sympathetic ganglia.

Sympathetic preganglionic axons project to spatially distinct targets in the periphery. A precise topographic pattern exists within the thoracic preganglionic cell column relative to the direction of axonal projections within the sympathetic chain. In this study, the time course and pattern of axonal outgrowth from different populations of preganglionic neurons in the chicken embryo is examined in detail to clarify the origin of the topography in this system. Projections to prevertebral targets are established by development of the splanchnic nerves by stage 25, well after the earliest somatic motor projections at stage 19 but at least two stages before the reported onset of paravertebral projections. Further, preganglionic axons that project rostrally into the sympathetic chain may do so earlier than those that project caudally in the chain. The separation of preganglionic axons into prevertebral, rostral paravertebral or caudal paravertebral directions occurs at a common site in the ventral mesenchyme, established by the initial ventromedial projection of the splanchnic nerves. Analysis of the axonal trajectories of rostrally and caudally projecting cells reveals that preganglionic axons are not selectively fasciculated before their point of separation at the sympathetic chain. The patterning of the preganglionic cell column is specified before the establishment of functional connections within the chain, indicating that target contact is not a determinant of the segmental pattern. We suggest that the differential outgrowth of preganglionic axons to peripheral targets is determined by the unique identities of underlying subpopulations of preganglionic axons.

Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx.

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K+]o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca2+]i) caused by Ca2+ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca2+ sequestration. In medium containing a normal concentration of extracellular Ca2+ ([Ca2+]o), thapsigargin caused a sustained rise of intracellular Ca2+ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca2+]o in the presence of thapsigargin further increased [Ca2+]i, suggesting that the sustained rise of [Ca2+]i was caused by a thapsigargin-induced Ca2+ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca2+ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca2+]i and enhanced survival caused by depolarization with elevated [K+]o, suggesting that these effects are mediated by Ca2+ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca2+]i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca2+ through L-type channels. These results provide additional evidence that increased [Ca2+]i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca2+]i for investigation of this and other Ca(2+)-dependent phenomena.

Structure of neurons and ganglia of the guinea pig gallbladder: light and electron microscopic studies.

This study was undertaken to examine the morphological features of cells within ganglia of the guinea pig gallbladder, and to examine the ultrastructure of the ganglionated plexus. Gallbladder neurons are large, with a relatively simple form, having only one or two major processes. Neurobiotin often filled axons to their varicose arbors on smooth muscle in close proximity to the interganglionic connectives. With the exception of connective tissue clefts that sometimes penetrated into them, ganglia were devoid of intercellular spaces, capillaries, or connective tissue elements such as collagen and basal laminae. However, ganglia were surrounded by a single, continuous basal lamina that was enclosed within a fibroblast and collagen capsule. Within ganglia, neurons were insulated by the processes of cells that resembled the astrocyte-like glia of enteric ganglia. Although few classical synapses were observed, numerous sites of direct apposition were identified between vesicle-rich profiles and processes of gallbladder neurons. Direct appositions between vesicle-rich profiles and the ganglion-limiting basal laminae were also observed. Vesiculated profiles contained small clear vesicles and large dense-core vesicles. Within interganglionic connectives, axons were unmyelinated and were isolated from one another by processes of glia that resembled Schwann cells. As was seen in the ganglia, direct appositions between vesicle-rich profiles and the connective-limiting basal laminae were observed. The results of this study demonstrate that gallbladder ganglia are similar, ultrastructurally, to enteric ganglia in the CNS-like composition of the neuropil. However, the greater degree of glial investment, lesser degree of innervation, and simpler neurons indicated differences from the enteric nervous system that may be functionally significant.

Transmitter diversity in ganglion cells of the guinea pig gallbladder: an immunohistochemical study.

Several neurotransmitters have been reported to exist in the ganglionated plexus of the guinea pig gallbladder. These include substance P, neuropeptide Y (NPY), calcitonin gene-related peptide, vasoactive intestinal peptide (VIP), acetylcholine, norepinephrine, serotonin, and dopamine. To determine which neuropeptides are intrinsic to gallbladder ganglia, we performed immunohistochemistry on colchicine-treated preparations. In separate, single-labeled preparations, a majority of neurons contained substance P-, NPY-, or somatostatin-like immunoreactivity. In double-labeled preparations, a large majority of the neurons that contained substance P-like immunoreactivity also contained NPY-like immunoreactivity and somatostatin-like immunoreactivity. Immunoreactivity for VIP was present in a small percentage of the gallbladder neurons which did not contain substance P-like immunoreactivity. Additional experiments were done to test for the presence of other compounds, known to exist in the neurons of the gut. Although immunoreactivity was found in control preparations of small intestine, the ganglionated plexus of the gallbladder lacked immunoreactivity for galanin, dynorphin, enkephalin, gastrin-releasing peptide, or gamma-aminobutyric acid. We conclude that ganglia of the guinea pig gallbladder contain at least two populations of neurons, based on transmitter phenotype. One of these populations appears to contain substance P, NPY, and somatostatin. Another population, which represents a small contingent of the total population of neurons, contains VIP.

Sexually dimorphic distribution of a galanin-like peptide in the central nervous system of the teleost fish Poecilia latipinna.

Immunohistochemical techniques were used to visualize areas of the brain and spinal cord containing a galanin-like peptide in the teleost fish, the sailfin molly. Galanin-like immunoreactivity (GAL-LI) in both males and females was identified in neurons in the nucleus preopticus periventricularis, nucleus lateralis tuberis, and nucleus commissuralis. GAL-LI fibers had a comparable distribution in the forebrain, preoptic, hypothalamic, and visceral sensory areas of both sexes. In striking contrast to these areas, the optic tectum, torus semicircularis, brainstem tegmentum, and spinal cord of the male contained much higher levels of GAL-LI than the female. GAL-LI in these dimorphic areas in the female was limited to single fiber bundles in the ventromedial tegmentum and in the trigeminal system. Additionally, a population of neurons in the preoptic nucleus was found to contain GAL-LI in the male only. Sexual dimorphism was especially prominent in the spinal cord, where extensive GAL-LI fibers were found in the male only. These fibers were oriented in the longitudinal plane and confined largely to the gray matter. Comparative studies were performed on the goldfish spinal cord, in which GAL-LI was localized solely in the dorsal horn and exhibited no sexual dimorphism. Further, examination of spinal cord material from neonatal mollies revealed a lack of spinal GAL-LI at this developmental stage. As the extent of GAL-LI in the male molly spinal cord differs from both the goldfish and from that reported for the mammalian spinal cord, and a prominent sexual dimorphism in GAL-LI extends from the diencephalon to the caudal spinal cord, it is suggested that a galanin-like peptide may play a unique, sex-specific role in this species.

Source of sexually dimorphic galanin-like immunoreactive projections in the teleost fish Poecilia latipinna.

A galanin-like peptide has a sexually dimorphic distribution in the teleost fish, the sailfin molly. An extensive system of galanin-like immunoreactive (GAL-LI) fibers has been described in the brainstem and spinal cord of the male molly, which is absent in the female (Cornbrooks and Parsons, companion paper). As GAL-LI in the mammalian spinal cord has been localized to neurons of origin in the dorsal root ganglia and dorsal and ventral horns, the present study was undertaken to determine whether the sexually dimorphic GAL-LI in the male molly may originate in part from corresponding sources in this species. Colchicine treatments of the spinal cord and dorsal root ganglia did not result in GAL-LI staining in neuronal somata in these regions. Following complete transection of the spinal cord and at any level of the spinal cord, there was a complete absence of GAL-LI caudal to the lesion site. In fish that received unilateral spinal transection, there was a loss of GAL-LI ipsilateral and caudal to the lesion. Finally, in fish that received lesions in the rostral hypothalamus, there was a complete loss of GAL-LI in the sexually dimorphic fiber system in the brainstem and spinal cord, but not in non-dimorphic GAL-LI regions of the brainstem. Thus the sexually dimorphic fiber system in the male molly may originate in neurons of the preoptic nucleus that are sexually dimorphic for a GAL-LI peptide. This preoptico-spinal pathway may mediate sex-specific behaviors in this species.