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Many neurodegenerative diseases are characterized by the formation of microscopically visible intracellular protein aggregates. α-Synuclein is the key aggregating protein in Parkinson's disease which is characterized by neuronal cytoplasmic Lewy body inclusions. Previous studies have shown relative sparing of neurons in Parkinson's disease and dementia with Lewy bodies that are positive for the vitamin D-dependent calcium-buffering protein, calbindin-D28k, and that α-synuclein aggregates are excluded from calbindin-D28k-positive neurons. Recent cell culture studies have shown that α-synuclein aggregation can be induced by raised intracellular-free Ca(II) and demonstrated that raised intracellular calcium and oxidative stress can act synergistically to promote α-synuclein aggregation. We hypothesized that calcipotriol, a potent vitamin D analogue used pharmaceutically, may be able to suppress calcium-dependent α-synuclein aggregation by inducing calbindin-D28k expression. Immunofluorescence and western blot analysis showed that calcipotriol potently induced calbindin-D28k in a dose-dependent manner in SH-SY5Y human neuroblastoma cells. Calcipotriol significantly decreased the frequency of α-synuclein aggregate positive cells subjected to treatments that cause raised intracellular-free Ca(II) (potassium depolarization, KCl/H 2 O 2 combined treatment, and rotenone) in a dose-dependent manner and increased viability. Suppression of calbindin-D28k expression in calcipotriol-treated cells using calbindin-D28k-specific siRNA showed significantly higher α-synuclein aggregation levels, indicating that calcipotriol-mediated blocking of calcium-dependent α-synuclein aggregation was dependent on the induction of calbindin-D28k expression. These data indicate that targeting raised intraneuronal-free Ca(II) in the brain by promoting the expression of calbindin-D28k at the transcriptional level using calcipotriol could prevent α-synuclein aggregate formation and ameliorate Parkinson's disease pathogenesis., (© 2017 Commonwealth of Australia. Journal of Neurochemistry © 2017 International Society for Neurochemistry.)

Aggregation of the pre-synaptic protein, α-synuclein (α-syn), is the key etiological factor in Parkinson's disease (PD) and other alpha-synucleinopathies, such as multiple system atrophy (MSA) and Dementia with Lewy bodies (DLB). Various triggers for pathological α-syn aggregation have been elucidated, including post-translational modifications, oxidative stress, and binding of metal ions, such as calcium. Raised neuronal calcium levels in PD may occur due to mitochondrial dysfunction and/or may relate to calcium channel dysregulation or the reduced expression of the neuronal calcium buffering protein, calbindin-D28k. Recent results on human tissue and a mouse oxidative stress model show that neuronal calbindin-D28k expression excludes α-syn inclusion bodies. Previously, cell culture model studies have shown that transient increases of intracellular free Ca(II), such as by opening of the voltage-gated plasma calcium channels, could induce cytoplasmic aggregates of α-syn. Raised intracellular free calcium and oxidative stress also act cooperatively to promote α-syn aggregation. The association between raised neuronal calcium, α-syn aggregation, oxidative stress, and neurotoxicity is reviewed in the context of neurodegenerative α-syn disease and potential mechanism-based therapies.

Zellweger syndrome (ZS) is a peroxisome biogenesis disorder that involves significant neuropathology, the molecular basis of which is still poorly understood. Using a mouse model of ZS with brain-restricted deficiency of the peroxisome biogenesis protein PEX13, we demonstrated an expanded and morphologically modified brain mitochondrial population. Cultured fibroblasts from PEX13-deficient mouse embryo displayed similar changes, as well as increased levels of mitochondrial superoxide and membrane depolarization; this phenotype was rescued by antioxidant treatment. Significant oxidative damage to neurons in brain was indicated by products of lipid and DNA oxidation. Similar overall changes were observed for glial cells. In toto, these findings suggest that mitochondrial oxidative stress and aberrant mitochondrial dynamics are associated with the neuropathology arising from PEX13 deficiency., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: The characterisation of dividing brain cells is fundamental for studies ranging from developmental and stem cell biology, to brain cancers. Whilst there is extensive anatomical data on these dividing cells, limited gene transcription data is available due to technical constraints., New Method: We focally isolated dividing cells whilst conserving RNA, from culture, primary neural tissue and xenografted glioma tumours, using a thymidine analogue that enables gene transcription analysis., Results: 5-ethynyl-2-deoxyuridine labels the replicating DNA of dividing cells. Once labelled, cultured cells and tissues were dissociated, fluorescently tagged with a revised click chemistry technique and the dividing cells isolated using fluorescence-assisted cell sorting. RNA was extracted and analysed using real time PCR. Proliferation and maturation related gene expression in neurogenic tissues was demonstrated in acutely and 3 day old labelled cells, respectively. An elevated expression of marker and pathway genes was demonstrated in the dividing cells of xenografted brain tumours, with the non-dividing cells showing relatively low levels of expression., Comparison With Existing Method: BrdU "immune-labelling", the most frequently used protocol for detecting cell proliferation, causes complete denaturation of RNA, precluding gene transcription analysis. This EdU labelling technique, maintained cell integrity during dissociation, minimized copper exposure during labelling and used a cell isolation protocol that avoided cell lysis, thus conserving RNA., Conclusions: The technique conserves RNA, enabling the definition of cell proliferation-related changes in gene transcription of neural and pathological brain cells in cells harvested immediately after division, or following a period of maturation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Intratumor heterogeneity is a primary feature of high-grade gliomas, complicating their therapy. As accumulating evidence suggests that intratumor heterogeneity is a consequence of cellular subsets with different cycling frequencies, we developed a method for transcriptional profiling of gliomas, using a novel technique to dissect the tumors into two fundamental cellular subsets, namely, the proliferating and non-proliferating cell fractions. The tumor fractions were sorted whilst maintaining their molecular integrity, by incorporating the thymidine analog 5-ethynyl-2'-deoxyuridine into actively dividing cells. We sorted the actively dividing versus non-dividing cells from cultured glioma cells, and parental and clonally derived orthotopic tumors, and analyzed them for a number of transcripts. While there was no significant difference in the transcriptional profiles between the two cellular subsets in cultured glioma cells, we demonstrate ∼2-6 fold increase in transcripts of cancer and neuronal stem cell and tumor cell migration/invasion markers, and ∼2-fold decrease in transcripts of markers of hypoxia and their target genes, in the dividing tumor cells of the orthotopic glioma when compared to their non-proliferative counterparts. This suggests the influence of the brain microenvironment in transcriptional regulation and, thereby, the physiology of glioma cells in vivo. When clonal glioma cells were derived from a parental glioma and the resultant orthotopic tumors were compared, their transcriptional profiles were closely correlated to tumor aggression and consequently, survival of the experimental animals. This study demonstrates the resolution of intratumor heterogeneity for profiling studies based on cell proliferation, a defining feature of cancers, with implications for treatment design., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

ABSTRACT Melioidosis is a potentially fatal disease that is endemic to tropical northern Australia and Southeast Asia, with a mortality rate of 14 to 50%. The bacterium Burkholderia pseudomallei is the causative agent which infects numerous parts of the human body, including the brain, which results in the neurological manifestation of melioidosis. The olfactory nerve constitutes a direct conduit from the nasal cavity into the brain, and we have previously reported that B. pseudomallei can colonize this nerve in mice. We have now investigated in detail the mechanism by which the bacteria penetrate the olfactory and trigeminal nerves within the nasal cavity and infect the brain. We found that the olfactory epithelium responded to intranasal B. pseudomallei infection by widespread crenellation followed by disintegration of the neuronal layer to expose the underlying basal layer, which the bacteria then colonized. With the loss of the neuronal cell bodies, olfactory axons also degenerated, and the bacteria then migrated through the now-open conduit of the olfactory nerves. Using immunohistochemistry, we demonstrated that B. pseudomallei migrated through the cribriform plate via the olfactory nerves to enter the outer layer of the olfactory bulb in the brain within 24 h. We also found that the bacteria colonized the thin respiratory epithelium in the nasal cavity and then rapidly migrated along the underlying trigeminal nerve to penetrate the cranial cavity. These results demonstrate that B. pseudomallei invasion of the nerves of the nasal cavity leads to direct infection of the brain and bypasses the blood-brain barrier. IMPORTANCE Melioidosis is a potentially fatal tropical disease that is endemic to northern Australia and Southeast Asia. It is caused by the bacterium Burkholderia pseudomallei, which can infect many organs of the body, including the brain, and results in neurological symptoms. The pathway by which the bacteria can penetrate the brain is unknown, and we have investigated the ability of the bacteria to migrate along nerves that innervate the nasal cavity and enter the frontal region of the brain by using a mouse model of infection. By generating a mutant strain of B. pseudomallei which is unable to survive in the blood, we show that the bacteria rapidly penetrate the cranial cavity using the olfactory (smell) nerve and the trigeminal (sensory) nerve that line the nasal cavity.

Tumor-initiating cells (TICs) often survive therapy and give rise to second-line tumors. We tested the plausibility of sphere cultures as models of TICs. Microarray data and microRNA data analysis confirmed the validity of spheres as models of TICs for breast and prostate cancer as well as mesothelioma cell lines. Microarray data analysis revealed the Trp pathway as the only pathway upregulated significantly in all types of studied TICs, with increased levels of indoleamine-2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme of Trp metabolism along the kynurenine pathway. All types of TICs also expressed higher levels of the Trp uptake system consisting of CD98 and LAT1 with functional consequences. IDO1 expression was regulated via both transcriptional and posttranscriptional mechanisms, depending on the cancer type. Serial transplantation of TICs in mice resulted in gradually increased IDO1. Mitocans, represented by α-tocopheryl succinate and mitochondrially targeted vitamin E succinate (MitoVES), suppressed IDO1 in TICs. MitoVES suppressed IDO1 in TICs with functional mitochondrial complex II, involving transcriptional and posttranscriptional mechanisms. IDO1 increase and its suppression by VE analogues were replicated in TICs from primary human glioblastomas. Our work indicates that IDO1 is increased in TICs and that mitocans suppress the protein., (© 2013 Elsevier Inc. All rights reserved.)

Oligodendroglial inclusion bodies characterize a subset of neurodegenerative diseases. Multiple system atrophy (MSA) is characterized by α-synuclein glial cytoplasmic inclusions and progressive supranuclear palsy (PSP) is associated with glial tau inclusions. The ubiquitin homologue, SUMO-1, has been identified in inclusion bodies in MSA, located in discrete sub-domains in α-synuclein-positive inclusions. We investigated SUMO-1 associated with oligodendroglial inclusion bodies in brain tissue from MSA and PSP and in glial cell models. We examined MSA and PSP cases and compared to age-matched normal controls. Fluorescence immunohistochemistry revealed frequent SUMO-1 sub-domains within and surrounding inclusions bodies in both diseases and showed punctate co-localization of SUMO-1 and the lysosomal marker, cathepsin D, in affected brain regions. Cell counting data revealed that 70-75 % of lysosomes in inclusion body-positive oligodendrocytes were SUMO-1-positive consistently across MSA and PSP cases, compared to 20 % in neighbouring inclusion body negative oligodendrocytes and 10 % in normal brain tissue. Hsp90 co-localized with some SUMO-1 puncta. We examined the SUMO-1 status of lysosomes in 1321N1 human glioma cells over-expressing α-synuclein and in immortalized rat oligodendrocyte cells over-expressing the four repeat form of tau following treatment with the proteasome inhibitor, MG132. We also transfected 1321N1 cells with the inherently aggregation-prone huntingtin exon 1 mutant, HttQ74-GFP. Each cell model showed the association of SUMO-1-positive lysosomes around focal cytoplasmic accumulations of α-synuclein, tau or HttQ74-GFP, respectively. Association of SUMO-1 with lysosomes was also detected in glial cells bearing α-synuclein aggregates in a rotenone-lesioned rat model. SUMO-1 labelling of lysosomes showed a major increase between 24 and 48 h post-incubation of 1321N1 cells with MG132 resulting in an increase in a 90 kDa SUMO-1-positive band that was immunopositive for Hsp90 and immunoprecipitated with an anti-SUMO-1 antibody. That SUMO-1 co-localizes with a subset of lysosomes in neurodegenerative diseases with glial protein aggregates and in glial cell culture models of protein aggregation suggests a role for SUMO-1 in lysosome function.

Trauma to the spinal cord creates an initial physical injury damaging neurons, glia, and blood vessels, which then induces a prolonged inflammatory response, leading to secondary degeneration of spinal cord tissue, and further loss of neurons and glia surrounding the initial site of injury. Angiogenesis is a critical step in tissue repair, but in the injured spinal cord angiogenesis fails; blood vessels formed initially later regress. Stabilizing the angiogenic response is therefore a potential target to improve recovery after spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) can initiate angiogenesis, but cannot sustain blood vessel maturation. Platelet-derived growth factor (PDGF) can promote blood vessel stability and maturation. We therefore investigated a combined application of VEGF and PDGF as treatment for traumatic spinal cord injury, with the aim to reduce secondary degeneration by promotion of angiogenesis. Immediately after hemisection of the spinal cord in the rat we delivered VEGF and PDGF and to the injury site. One and 3 months later the size of the lesion was significantly smaller in the treated group compared to controls, and there was significantly reduced gliosis surrounding the lesion. There was no significant effect of the treatment on blood vessel density, although there was a significant reduction in the numbers of macrophages/microglia surrounding the lesion, and a shift in the distribution of morphological and immunological phenotypes of these inflammatory cells. VEGF and PDGF delivered singly exacerbated secondary degeneration, increasing the size of the lesion cavity. These results demonstrate a novel therapeutic intervention for SCI, and reveal an unanticipated synergy for these growth factors whereby they modulated inflammatory processes and created a microenvironment conducive to axon preservation/sprouting.

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., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in "tagging DNA synthesis" is the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using 'Huisgen's reaction' (1,3-dipolar cycloaddition or 'click chemistry'). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other "unnatural bases". These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.

Bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promise in in vitro neuronal differentiation and in cellular therapy for neurodegenerative disorders, including Parkinson' disease. However, the effects of intracerebral transplantation are not well defined, and studies do not agreed on the optimal neuronal differentiation method. Here, we investigated three growth factor-based neuronal differentiation procedures (using FGF-2/EGF/PDGF/SHH/FGF-8/GDNF), and found all to be capable of eliciting an immature neural phenotype, in terms of cell morphology and gene/protein expression. The neuronal-priming (FGF-2/EGF) method induced neurosphere-like formation and the highest NES and NR4A2 expression by hMSCs. Transplantation of undifferentiated and neuronal-primed hMSCs into the striatum and substantia nigra of 6-OHDA-lesioned hemiparkinsonian rats revealed transient graft survival of 7 days, despite the reported immunosuppressive properties of MSCs and cyclosporine-immunosuppression of rats. Neither differentiation of hMSCs nor induction of host neurogenesis was observed at injection sites, and hMSCs continued producing mesodermal fibronectin. Strategies for improving engraftment and differentiation post-transplantation, such as prior in vitro neuronal-priming, nigral and striatal grafting, and co-transplantation of olfactory ensheathing cells that promote neural regeneration, were unable to provide advantages. Innate inflammatory responses (Iba-1-positive microglia/macrophage and GFAP-positive astrocyte activation and accumulation) were detected around grafts within 7 days. Our findings indicate that growth factor-based methods allow hMSC differentiation toward immature neuronal-like cells, and contrary to previous reports, only transient survival and engraftment of hMSCs occurs following transplantation in immunosuppressed hemiparkinsonian rats. In addition, suppression of host innate inflammatory responses may be a key factor for improving hMSC survival and engraftment.

Rotenone, a mitochondrial complex 1 inhibitor, causes oxidative damage via production of reactive oxygen species. We examined the pathophysiology of neuronal and glial cells of the nigrostriatal pathway following unilateral infusion of varying doses of rotenone into the substantia nigra or medial forebrain bundle of adult male Sprague-Dawley rats, sacrificed 14 and 60 days after infusion. Immunofluorescence techniques were used to qualitatively and quantitatively assay dopaminergic neurons, their projections, glial cells, synapses, and oxidative stress. Rotenone infusion into the substantia nigra at all concentrations caused extensive damage and tissue necrosis, therefore of limited relevance for producing a Parkinson disease model. Infusion of 0.5μg of rotenone targeting the medial forebrain bundle induced oxidative stress in dopaminergic neurons causing ongoing cell stress as defined by an elevation of stress granule and oxidative stress markers. This treatment resulted in the loss of tyrosine hydroxylase immunoreactive cells in the substantia nigra (p≤0.01) and loss of tyrosine hydroxylase immunoreactive nerve fibres and synaptic specialisations in the striatum (p≤0.01). The infusion of 0.5μg of rotenone also caused an increase in astrocytes and microglial cells in the substantia nigra in comparison to control (p≤0.01). We examined the time-dependent reduction of tyrosine hydroxylase-positive nerve fibres and cell bodies in the striatum and substantia nigra respectively, with a progressive reduction evident 60days after infusion (p≤0.01, p≤0.05). Dopaminergic axons exposed to low-dose rotenone undergo oxidative stress, with a resultant ongoing loss of dopaminergic neurons, providing an animal model relevant to Parkinson disease., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Labelling and identifying proliferating cells is central to understanding neurogenesis and neural lineages in vivo and in vitro. We present here a novel thymidine analogue, ethynyl deoxyuridine (EdU) for labelling dividing cells, detected with a fluorescent azide which forms a covalent bond via the "click" chemistry reaction (the Huisgen 1,3-dipolar cycloaddition reaction of an organic azide to a terminal acetylene). Unlike the commonly used BrdU, EdU detection requires no heat or acid treatment. It is quick and easy and compatible with multiple probes for fluorescence immunochemistry, facilitating the characterisation of proliferating cells at high resolution.

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.

The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.

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.

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.

The motor innervation of the duodenum and distal colon remains uncharacterized within the same species. Our aim was to compare the projections and neurochemical properties of duodenal and distal colon circular muscle motor neurons. Circular muscle motor neurons were retrogradely traced by using a neural tracer in vitro, processed for choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) immunoreactivity and then visualized by using indirect immunofluorescence. A mean of 372 +/- 64 and 156 +/- 23 neurons (mean +/- SEM) were tracer-labeled within the duodenum and colon, respectively. The ChAT+/NOS- neurons comprised 57.6 +/- 6.6% and 39.6 +/- 4.4% of all labeled cells in the duodenum and colon, respectively, and projected mainly in the oral direction. Of all labeled cells, the ChAT-/NOS+ neurons comprised 8.5 +/- 2.3% in the duodenum and 46.6 +/- 5.0% in the distal colon and projected mainly in the anal direction. Of the remainder, 20.6 +/- 5.0% and 8.2 +/- 2.4% were ChAT+/NOS+ and 13.2 +/- 0.9% and 5.6 +/- 1.4% were ChAT-/NOS- in the duodenum and distal colon, respectively. Within both regions, the distribution of the ChAT+/NOS- and ChAT-/NOS+ neurons are consistent with the ascending excitatory and descending inhibitory reflexes. The proportion of ChAT-/NOS+ neurons is greater within the colon in comparison with the duodenum. A considerable proportion of duodenal motor neurons were ChAT+/NOS+ and ChAT-/NOS-. These two classes may underlie nonperistaltic motor patterns, which predominate within the duodenum. These findings demonstrate regional differences in the innervation of intestinal circular muscle., (Copyright 2002 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.