Central serotonergic neuron deficiency in a mouse model of Zellweger syndrome.

Zellweger syndrome (ZS) is a severe peroxisomal disorder caused by mutations in peroxisome biogenesis, or PEX, genes. A central hallmark of ZS is abnormal neuronal migration and neurodegeneration, which manifests as widespread neurological dysfunction. The molecular basis of ZS neuropathology is not well understood. Here we present findings using a mouse model of ZS neuropathology with conditional brain inactivation of the PEX13 gene. We demonstrate that PEX13 brain mutants display changes that reflect an abnormal serotonergic system - decreased levels of tryptophan hydroxylase-2, the rate-limiting enzyme of serotonin (5-hydroxytryptamine, 5-HT) synthesis, dysmorphic 5-HT-positive neurons, abnormal distribution of 5-HT neurons, and dystrophic serotonergic axons. The raphe nuclei region of PEX13 brain mutants also display increased levels of apoptotic cells and reactive, inflammatory gliosis. Given the role of the serotonergic system in brain development and motor control, dysfunction of this system would account in part for the observed neurological changes of PEX13 brain mutants.

Vascular endothelial growth factor and platelet derived growth factor modulates the glial response to a cortical stab injury.

Traumatic injury to the brain initiates an increase in astrocyte and microglial infiltration as part of an inflammatory response to injury. Increased astrogliosis around the injury impedes regeneration of axons through the injury, while activated microglia release inflammatory mediators. The persistent inflammatory response can lead to local progressive cell death. Modulating the astrocyte and microglial response to traumatic injury therefore has potential therapeutic benefit in brain repair. We examine the modulatory effect of a single bolus of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) in combination on astrocytes and microglia to acute cerebral injury. A combination of VEGF and PDGF (20 pg) was injected into the striatum of adult male Sprague-Dawley rats. The effects of treatment were assessed by quantitative immunofluorescence microscopy analyzing astrocytes and microglia across the stab injury over time. Treatment delayed the onset of astrogliosis in the centre and edge of the stab injury up to day 5; however, increased astrogliosis at areas remote to the stab injury up to day 5 was observed. A persistent astrocytic response was observed in the centre and edge of the stab injury up to day 60. Treatment altered microglia cell morphology and numbers across the stab injury, with a decrease in ramified microglia, but an increase in activated and phagocytic microglia up to day 5 after stab injury. The increased microglial response from 10 until day 60 was comprised of the ramified morphology. Thus, VEGF and PDGF applied at the same time as a stab injury to the brain initially delayed the inflammatory response up to day 5 but evoked a persistent astrogliosis and microglial response up to 60 days.

Survival and engraftment of mouse embryonic stem cell-derived implants in the guinea pig brain.

alpha-Mannosidosis is a lysosomal storage disease resulting from a deficiency of the enzyme alpha-D-mannosidase. A major feature of alpha-mannosidosis is progressive neurological decline, for which there is no safe and effective treatment available. We have a guinea pig model of alpha-mannosidosis that models the human condition. This study investigates the feasibility of implanting differentiated mouse embryonic stem cells in the neonatal guinea pig brain in order to provide a source of alpha-mannosidase to the affected central nervous system. Cells implanted at a low dose (1.5 x 10(3)cells per hemisphere) at 1 week of age were found to survive in very low numbers in some immunosuppressed animals out to 8 weeks. Four weeks post-implantation, cells implanted in high numbers (10(5) cells per hemisphere) formed teratomas in the majority of the animals implanted. Although implanted cells were found to migrate extensively within the brain and differentiate into mature cells of neural (and other) lineages, the safety issue related to uncontrolled cell proliferation precluded the use of this cell type for longer-term implantation studies. We conclude that the pluripotent cell type used in this study is unsuitable for achieving safe engraftment in the guinea pig brain.

Characterization of the intrinsic and extrinsic innervation of the gall bladder epithelium in the Australian Brush-tailed possum (Trichosurus vulpecula).

Intrinsic neurones of the gall bladder modulate its function. Nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) are present in gall bladder neurones and nitric oxide and VIP modulate its epithelial functions. As an extensive extrinsic innervation of the gall bladder is also present, the source of the epithelial innervation is unclear. In this study the source of the gall bladder epithelial innervation is defined. Immunoreactivity for VIP, NOS, substance P (SP), calcitonin gene related peptide (CGRP) and tyrosine hydroxylase (TH) in organotypic cultured and freshly fixed gall bladder were compared. Retrograde tracing in vitro from the epithelium was used to identify putative intrinsic secretomotor neurones, which were then characterized by immunohistochemistry. Abundant spinal afferent and sympathetic innervation of the gall bladder epithelium was demonstrated by CGRP/SP and TH immunohistochemistry, respectively. The intrinsic secretomotor innervation of the epithelium is derived exclusively from neurones of the subepithelial plexus. A majority of these neurones were immunoreactive for NOS. Some of the NOS-immunoreactive neurones of the subepithelial plexus also contained VIP and/or SP. Gall bladder subepithelial plexus neurones, containing NOS and/or VIP/SP, innervate the epithelium, as do extrinsic neurones.

Induction of duodenal motility activates the sphincter of Oddi (SO)-duodenal reflex in the Australian possum in vitro.

1 The aim of this study was to determine if stimulation of duodenal motility by duodenal fluid distension or by administration of carbachol, activates the sphincter of Oddi-duodenal reflex, in an in vitro preparation from the Australian possum. 2 Duodenal distension was achieved by infusion of Krebs solution (0-8 cm H2O). In separate experiments, the sphincter of Oddi (SO) was partitioned from the duodenum and carbachol (10(-7) - 5 x 10(-6) M) added to the duodenal compartment. 3 Fluid distension increased duodenal motility to 120-600% of control activity. These treatments induced increased SO motility (to 120-390% of control) in six preparations, reduced activity (to 60% of control) in one and no response in another. 4 Addition of carbachol to the duodenal compartment resulted in increased duodenal motility. SO motility was increased in seven preparations, reduced in another two and no response were evoked in two others. All SO responses were blocked by tetrodotoxin pretreatment. 5 These data suggest that the SO receives inputs from duodenal mechano and/or stretch receptors resulting in excitatory or inhibitory responses, with the excitatory response dominating. These findings support the role for the SO-duodenal reflex in preventing duodenobiliary/pancreatic reflux during periods of elevated duodenal activity.

Inhibitory motor innervation of the gall bladder musculature by intrinsic neurones containing nitric oxide in the Australian brush-tailed possum (Trichosurus vulpecula).

BACKGROUND: Gall bladder functions are modulated by neurones intrinsic to the organ. Data are available on the neurochemical composition of intrinsic and extrinsic nerves innervating the gall bladder but are lacking on specific functional classes of gall bladder neurones. AIMS: To characterise the intrinsic motor neurones of the gall bladder and identify their roles using pharmacological techniques. METHODS: Retrograde tracing from the possum gall bladder muscle in vitro allowed identification of intrinsic motor neurones. Subsequently, their content of choline acetyltransferase and nitric oxide synthase, markers of acetylcholine and nitric oxide containing neurones, was established using immunohistochemical techniques. Organ bath pharmacology was used to evaluate neurotransmission by acetylcholine and nitric oxide in gall bladder muscle strips. RESULTS: Innervation of the gall bladder musculature by neurones of both the muscular and serosal plexuses was demonstrated. A large proportion (62%) of these motor neurones were immunoreactive for nitric oxide synthase. All gall bladder neurones showed immunoreactivity for choline acetyltransferase. Organ bath pharmacology confirmed the neuroanatomical data, showing acetylcholine and nitric oxide mediating neurotransmission to the gall bladder musculature. CONCLUSIONS: Neurones containing acetylcholine and nitric oxide, located within the muscular and serosal plexuses, provide excitatory and inhibitory motor innervation of the gall bladder, respectively. The large inhibitory innervation suggests active relaxation of the gall bladder during filling, mediated by intrinsic nerves.

Endothelin-1 induces contraction of human and Australian possum gallbladder in vitro.

BACKGROUND: Endothelin-1 (ET-1) stimulates guinea pig gallbladder (GB) muscle strip contractility; however, the role and source of ET-1 in the GB remains to be elucidated. AIMS: To determine the effect of ET-1 on human and possum GB muscle strip contractility and evaluate whether ET-1 is present in GB tissue. METHODS: GB muscle strips were mounted in organ baths to measure isometric tension. ET-1 was added cumulatively with and without pretreatment with the neural blocker tetrodotoxin (TTX) or the ET receptor antagonists BQ-123, BQ-788, and tezosentan. Immunohistochemical localization of ET was performed on freshly fixed and cultured GBs. RESULTS: ET-1 induced concentration-dependent increases in tone in human and possum GB strips (p<0.05). This response was unaffected by BQ-123, BQ-788, and TTX but antagonized by BQ-123+BQ-788 in the human tissue only. Tezosentan (10(-4) mol/l) blocked the ET-1-induced response in human and possum GB strips (p<0.001). Although ET immunoreactivity was absent in freshly fixed possum GB, immunoreactivity was observed in the GB epithelium of freshly fixed human tissue and in both possum and human tissue following 24 h of organ culture. CONCLUSION: ET-1 acts directly on human and possum GB smooth muscle producing contractions, possibly via ET-B receptors. ET may be present under pathophysiological conditions altering GB function.

Distribution of nitric oxide synthase and vasoactive intestinal polypeptide immunoreactivity in the sphincter of Oddi and duodenum of the possum.

The nitrergic innervation of the sphincter of Oddi (SO) and duodenum in the Australian brush-tailed possum and the possible association of this innervation with the neuropeptide vasoactive intestinal polypeptide (VIP) were investigated by using immunohistochemical localisation of nitric oxide synthase (NOS) and VIP, together with the general neuronal marker, protein gene product 9.5 (PGP9.5). Whole-mount preparations of the duodenum and attached SO without the mucosa, submucosa and circular muscle (n=12) were double- and triple-labelled. The density of myenteric nerve cell bodies of the SO in the more distal region (duodenal end) was significantly higher than that in the more proximal region. In the SO, approximately 50% of all cells were NOS-immunoreactive (IR), with 27% of the NOS-IR cells being VIP-IR. Within the duodenal myenteric plexus, NOS immunoreactivity was present in about 25% of all neurons, with 27% of these NOS-IR neurons also being VIP-IR, a similar proportion to that in the SO. Varicose nerve fibres with NOS and VIP immunoreactivity were present within the myenteric and submucous plexuses of the SO and duodenum, and in the circular and longitudinal muscle layers. The NOS-positive cells within both the SO and duodenum were unipolar, displaying a typical Dogiel type I morphology. The myenteric plexuses of the SO and duodenum were in direct continuity, with many interconnecting nerve trunks, some of which showed NOS and VIP immunoreactivity. Thus, the possum possesses an extensive NOS innervation of the SO and duodenum, with a significantly higher proportion of NOS-IR neurons within the SO, a subset of which contains VIP.

Effects of diaspirin cross-linked hemoglobin on motor function of the duodenum and biliary system in the Australian brush-tailed possum in vivo.

Chemically altered hemoglobin-based oxygen carriers have been developed as prototype blood substitutes. Such molecules may affect numerous biological processes, since free hemoglobin scavenges nitric oxide (NO). Diaspirin cross-linked hemoglobin (DCLHb) is a chemically cross-linked molecule, which has a pressor effect on blood pressure, mainly mediated by NO scavenging. However, the effects of DCLHb on the gastrointestinal and biliary motility have not been reported. This study was conducted to investigate the effects of DCLHb on the duodenal and biliary motility and determine if the underlying mechanism involves a NO pathway. Blood pressure, duodenal, sphincter of Oddi and gallbladder motility and trans-sphincteric flow were recorded in anesthetized Australian Brush-tailed possums. The effects of intravenously administered DCLHb (10% solution) or oncotically matched human serum albumin (HSA) solution on these parameters were investigated. To determine the involvement of a NO-mediated pathway in these effects, animals were pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) before DCLHb or HSA was given. DCLHb increased blood pressure and duodenal contraction frequency and slowed trans-sphincteric flow compared with the HSA control. The effects of DCLHb on blood pressure and trans-sphincteric flow were immediate and transient, whereas the effect on duodenal contraction frequency was delayed and long-lived. Pretreatment with L-NAME alone increased blood pressure and duodenal contraction frequency and slowed trans-sphincteric flow. DCLHb-induced changes were not evident in the presence of L-NAME. These findings suggest that DCLHb affects duodenal and trans-sphincteric flow predominantly by NO scavenging.

The projections of 5-hydroxytryptamine-accumulating neurones in the myenteric plexus of the small intestine of the guinea-pig.

Retrograde tracing, combined with immunohistochemistry, was used to study the projections of 5-hydroxytryptamine (5-HT)-accumulating neurones within the ileum of the guinea-pig, with confocal microscopy being used to characterise further their morphology. Two classes of neurones in the myenteric plexus, capable of taking up 5-HT or analogues, were distinguished. One class had Dogiel type I morphology with lamellar dendrites, was located on the edge or in the middle of ganglia and lacked immunoreactivity for somatostatin (SOM). The other class had smooth ovoid cell bodies with multiple filamentous dendrites and a single axon and represented a subset of the SOM-immunoreactive interneurones in the myenteric plexus. Varicosities immunoreactive for 5-HT alone, 5-HT/SOM or SOM alone were present in the myenteric ganglia. Both classes of 5-HT-accumulating neurones had long aboral projections within the myenteric plexus (up to 100 mm long) and to the submucous plexus and probably function as descending interneurones.

Orally projecting interneurones in the guinea-pig small intestine.

1. Orally projecting, cholinergic interneurones are important in mediating ascending excitatory reflexes in the small intestine. We have shown that there is just one major class of orally projecting interneurone, which we have characterized using retrograde labelling in organ culture, combined with immunohistochemistry, intracellular recording and dye filling. 2. Orally projecting interneurones, previously shown to be immunoreactive for choline acetyltransferase, tachykinins, enkephalin, calretinin and neurofilament protein triplet, have axons up to 14 mm long and are the only class of cells with orally directed axons more than 8.5 mm long. 3. They are all small Dogiel type I neurones with short dendrites, usually lamellar in form, and a single axon which sometimes bifurcates. Their axons give rise to short varicose collaterals in myenteric ganglia more than 3 mm oral to their cell bodies. 4. Orally projecting interneurones receive prominent fast excitatory post synaptic potentials (fast EPSPs). A major source of fast EPSPs is other ascending interneurones located further aborally. They also receive fast EPSPs from circumferential pathways. 5. In the stretched preparations used in this study, orally projecting interneurones were highly excitable, firing repeatedly to depolarizing current pulses and had negligible long after-hyperpolarizations following their action potentials. They did not receive measurable non-cholinergic slow excitatory synaptic inputs. 6. Ascending interneurones had a characteristic inflection in their membrane responses to depolarizing current pulses and their first action potential was typically delayed by approximately 30 ms. Under single electrode voltage clamp, ascending interneurones had a transient outward current when depolarized above -70 mV from more hyperpolarized holding potentials. Ascending interneurones also consistently showed marked inward rectification under both current clamp and voltage clamp conditions. 7. This class of cells has consistent morphological, neurochemical and electrophysiological characteristics and are important in mediating orally directed enteric reflexes.