Large Cell Bodies Research Articles

Gabapentin reduces CX3CL1 signaling and blocks spinal microglial activation in monoarthritic rats

BackgroundSpinal glia, particularly microglia and astrocytes, are of the utmost importance in the development and maintenance of chronic pain. A recent study from our laboratory revealed that gabapentin, a recommended first-line treatment for multiple neuropathic conditions, could also efficiently antagonize thermal hyperalgesia evoked by complete Freund's adjuvant (CFA)-induced monoarthritis (MA). In the present study, we investigated whether the spinal glia are involved in the anti-hyperalgesic effect of gabapentin and how this event occurs.ResultsUnilateral intra-articular injection of CFA produced a robust activation of microglia and astrocytes. These cells exhibited large cell bodies, thick processes and increases in the ionized calcium binding adapter molecule 1 (Iba-1, a microglial marker) or the glia fibrillary acidic protein (GFAP, an astrocytic marker). These cells also displayed immunoreactive signals, and an upregulation of the voltage-gated calcium channels (VGCCs) α2/δ-1 subunit, CX3CL1 and CX3CR1 expression levels in the spinal cord. These changes were associated with the development of thermal hyperalgesia. Immunofluorescence staining showed that VGCC α2/δ-1 subunit, a proposed gabapentin target of action, was widely distributed in primary afferent fibers terminals and dorsal horn neurons. CX3CL1, a potential trigger to activate microglia, colocalized with VGCC α2/δ-1 subunits in the spinal dorsal horn. However, its receptor CX3CR1 was mainly expressed in the spinal microglia. Multiple intraperitoneal (i.p.) gabapentin injections (100 mg/kg, once daily for 4 days with the first injection 60 min before intra-articular CFA) suppressed the activation of spinal microglia, downregulated spinal VGCC α2/δ-1 subunits decreased CX3CL1 levels and blocked the development of thermal hyperalgesia in MA rats.ConclusionsHere we provide the first evidence that gabapentin diminishes CX3CL1 signaling and spinal microglia activation induced by joint inflammation. We also show that the VGCC α2/δ-1 subunits might be involved in these events.

G-protein-gated inwardly rectifying K+ channel 4 (GIRK4) immunoreactivity in chemically defined neurons of the hypothalamic arcuate nucleus that control body weight

G-protein-gated inwardly rectifying K+ channels (GIRKs; also called Kir3) are a family of K+ channels, which are activated (opened) via a signal transduction cascade starting with ligand-stimulated G-protein-coupled receptors (GPCRs). Four GIRK genes have been identified (GIRK1–4). GIRK4 (Kir3.4) has a role in regulating energy homeostasis, since mice with a targeted mutation in the GIRK4 gene exhibit a predisposition to late-onset obesity. GIRK4 mRNA is expressed in hypothalamic regions that harbor neurons involved in the regulation of food intake and body weight. Using goat and rabbit antisera to the GIRK4 protein, the cellular localization and transmitter content of GIRK4-immunoreactive neurons was determined in the hypothalamic arcuate nucleus, a region that contains neurons which are accessible to circulating hormones and is intimately associated with the control of body weight. GIRK4-immunoreactive large cell bodies were demonstrated in the ventrolateral part of the arcuate nucleus, with smaller neuronal cell bodies in the ventromedial part of the nucleus. Double-labeling showed presence of GIRK4 immunoreactivity in large neurons of the ventrolateral arcuate nucleus containing the peptides α-melanocyte-stimulating hormone (α-MSH), a marker for pro-opiomelanocortin (POMC) neurons, and cocaine- and amphetamine-regulated transcript (CART). GIRK4 immunoreactivity was also seen in neurons of the ventromedial part of the arcuate nucleus containing agouti-regulated peptide (AgRP) and neuropeptide Y (NPY). The results suggest that the GIRK4 channel protein plays a role in regulating membrane excitability in chemically defined neurons of the arcuate nucleus that control body weight.

Molecular Motor Proteins and Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor neurons in the brain, brainstem and spinal cord, which is characterized by motor dysfunction, muscle dystrophy and progressive paralysis. Both inherited and sporadic forms of ALS share common pathological features, however, the initial trigger of neurodegeneration remains unknown. Motor neurons are uniquely targeted by ubiquitously expressed proteins in ALS but the reason for this selectively vulnerability is unclear. However motor neurons have unique characteristics such as very long axons, large cell bodies and high energetic metabolism, therefore placing high demands on cellular transport processes. Defects in cellular trafficking are now widely reported in ALS, including dysfunction to the molecular motors dynein and kinesin. Abnormalities to dynein in particular are linked to ALS, and defects in dynein-mediated axonal transport processes have been reported as one of the earliest pathologies in transgenic SOD1 mice. Furthermore, dynein is very highly expressed in neurons and neurons are particularly sensitive to dynein dysfunction. Hence, unravelling cellular transport processes mediated by molecular motor proteins may help shed light on motor neuron loss in ALS.

Thalamocortical projections to rat auditory cortex from the ventral and dorsal divisions of the medial geniculate nucleus.

The ventral and dorsal medial geniculate (MGV and MGD) constitute the major auditory thalamic subdivisions providing thalamocortical inputs to layer IV and lower layer III of auditory cortex. No quantitative evaluation of this projection is available. Using biotinylated dextran amine (BDA)/biocytin injections, we describe the cortical projection patterns of MGV and MGD cells. In primary auditory cortex the bulk of MGV axon terminals are in layer IV/lower layer III with minor projections to supragranular layers and intermediate levels in infragranular layers. MGD axons project to cortical regions designated posterodorsal (PD) and ventral (VA) showing laminar terminal distributions that are quantitatively similar to the MGV-to-primary cortex terminal distribution. At the electron microscopic level MGV and MGD terminals are non-γ-aminobutyric acid (GABA)ergic with MGD terminals in PD and VA slightly but significantly larger than MGV terminals in primary cortex. MGV/MGD terminals synapse primarily onto non-GABAergic spines/dendrites. A small number synapse on GABAergic structures, contacting large dendrites or cell bodies primarily in the major thalamocortical recipient layers. For MGV projections to primary cortex or MGD projections to PD or VA, the non-GABAergic postsynaptic structures at each site were the same size regardless of whether they were in supragranular, granular, or infragranular layers. However, the population of MGD terminal-recipient structures in VA were significantly larger than the MGD terminal-recipient structures in PD or the MGV terminal-recipient structures in primary cortex. Thus, if terminal and postsynaptic structure size indicate strength of excitation then MGD to VA inputs are strongest, MGD to PD intermediate, and MGV to primary cortex the weakest.

Abstract A22: Social behaviors in medulloblastoma: Functional analysis of tumor-derived, supportive glial cells

Abstract Understanding the interactions between tumor and supportive cells carries great promise of effective and novel treatment methods. However, conventional methods, including mouse cancer models, lack the spatial resolution to tease apart tumor versus supportive cell types for mechanistic studies. To circumvent this problem, our lab makes use of a novel mouse genetic system termed MADM (mosaic analysis with double markers) for cancer modeling. Starting with a mouse heterozygous for a tumor suppressor gene (TSG), MADM can generate sporadic mutant cells that are null for the TSG via Cre-loxP mediated interchromosomal mitotic recombination events. Most importantly, MADM unequivocally labels mutant cells with GFP, enabling us to trace the lineage of mutant cells and easily distinguish them from neighboring normal cells. We used MADM to model medulloblastoma, the most common type of pediatric brain tumor. It is known that such tumors, especially the desmoplastic subtype, are originated from granule neuron precursors (GNPs). Normal GNPs divide exponentially after birth followed by a prompt cease of expansion by postnatal day 21 (P21) in mice and up to 1 year after birth in humans to give rise to mature granule neurons. Medulloblastoma forms when GNPs stop responding to differentiation signals and continue to proliferate beyond the normal developmental time window. To establish our mouse model for medulloblastoma, we used Math1-Cre that expresses specifically in GNPs to generate green mutant cells. The tumor model is successful in two aspects. First, Math1-Cre faithfully induced recombination in GNPs, which gave rise to granule neurons but not other cell types in wild-type cerebellum. Second, medulloblastoma formed in all mice at the age of 2–3 months, and tumors consisted of GFP-labeled mutant cells generated by MADM. Surprisingly, a closer look at the tumor mass led to the observation of a population of green cells with large cell bodies, reminiscent of glial cells rather than GNPs. The fact that the glial cells are GFP-positive suggests that they are derived from mutant GNPs, although normal GNPs are known to be unipotent and should not give rise to any glial cells. We next stained tumor sections with glia-specific markers GFAP and BLBP to further confirm the identity of these cells in the tumor mass, and found 100% co-localization of the glial markers with these cells. Now that MADM-based model revealed an aberrant fate switch in medulloblastoma, the more important question is whether these glial cells have any function in the tumor. To answer this question, we incorporated a TK transgene driven by the GFAP promoter into our mouse model, which can specifically ablate glial cells upon the injection of a drug called GCV. Preliminary data showed that 1 week after the injection of GCV, tumors regressed completely in our mouse model. In summary, these findings suggest that there is a self-organizing social behavior in medulloblastoma: mutant GNPs divert their developmental fate restriction to give rise to glial cells, which in turn provide critical support for the tumor growth. Currently we are focusing on determining the molecular mechanisms by which tumor glial cells are generated from transformed GNPs, and by which they sustain the tumor growth. Dissecting the mode of support provided by tumor-generated glia should shed light on novel targets for more specific therapeutic strategies, circumventing the devastating side effects of current surgical, radio-, and chemotherapies for medulloblastoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A22.

P.1.011 Evidence of hypothalamic mitochondrial dysfunction and neurodegeneration in the anorectic anx/anx mouse model

G-protein-gated inwardly rectifying K+ channels (GIRKs; also called Kir3) are a family of K+ channels, which are activated (opened) via a signal transduction cascade starting with ligand-stimulated G-protein-coupled receptors (GPCRs). Four GIRK genes have been identified (GIRK1–4). GIRK4 (Kir3.4) has a role in regulating energy homeostasis, since mice with a targeted mutation in the GIRK4 gene exhibit a predisposition to late-onset obesity. GIRK4 mRNA is expressed in hypothalamic regions that harbor neurons involved in the regulation of food intake and body weight. Using goat and rabbit antisera to the GIRK4 protein, the cellular localization and transmitter content of GIRK4-immunoreactive neurons was determined in the hypothalamic arcuate nucleus, a region that contains neurons which are accessible to circulating hormones and is intimately associated with the control of body weight. GIRK4-immunoreactive large cell bodies were demonstrated in the ventrolateral part of the arcuate nucleus, with smaller neuronal cell bodies in the ventromedial part of the nucleus. Double-labeling showed presence of GIRK4 immunoreactivity in large neurons of the ventrolateral arcuate nucleus containing the peptides α-melanocyte-stimulating hormone (α-MSH), a marker for pro-opiomelanocortin (POMC) neurons, and cocaine- and amphetamine-regulated transcript (CART). GIRK4 immunoreactivity was also seen in neurons of the ventromedial part of the arcuate nucleus containing agouti-regulated peptide (AgRP) and neuropeptide Y (NPY). The results suggest that the GIRK4 channel protein plays a role in regulating membrane excitability in chemically defined neurons of the arcuate nucleus that control body weight.

Emx1-expressing neural stem cells in the subventricular zone give rise to new interneurons in the ischemic injured striatum

Neural stem cells from different regions within the subventricular zone (SVZ) are able to produce several different subtypes of interneurons in the olfactory bulb throughout life. Previous studies have shown that ischemic stroke induces the production of new neurons in the damaged striatum from the SVZ. However, the origins and genetic profiles of these newborn neurons remain largely unknown as SVZ neural stem cells are heterogeneous. In the present study, using a mouse model of perinatal hypoxic-ischemic (H/I) brain injury combined with BrdU labeling methods, we found that, as in rat brains, virtually all newborn neuroblasts that migrate from the SVZ into the ischemic injured striatum exclusively express the transcription factor Sp8. Furthermore, although newborn neuroblasts are plentiful in the damaged striatum, only a few can differentiate into calretinin-expressing (CR+) interneurons that continuously express Sp8. Genetic fate mapping reveals that newly born CR+ interneurons are generated from Emx1-expressing neural stem cells in the dorsal-lateral SVZ. These results suggest that the fate of the Emx1-expressing lineage in the ischemic damaged striatum is restricted. However, when Sp8 was conditionally inactivated in the Emx1-lineage cells, Pax6 was ectopically expressed by a subpopulation of Emx1-derived CR+ cells in the normal and damaged striatum. Interestingly, these cells possessed large cell bodies and long processes. This work identifies the origin of the newly born CR+ interneurons in the damaged striatum after ischemic brain injury.

Spatial mapping of the mechanical properties of the living retina using scanning force microscopy

The retina is an active soft material, in which mechanosensitive cells are thought to respond to the local mechanical heterogeneity they encounter during development and adult physiological functioning. The retina is also constantly exposed to mechanical stress with shear and traction forces acting at its inner surface. Consequences of these forces depend on the tissue's resistance to deformation, which is characterized by its stiffness. However, currently there is a lack of high-resolution data on retinal mechanical properties. Here, we used scanning force microscopy to determine the apparent elastic modulus K of the retinal inner surface along the length of the eye with sub-millimetre resolution, and compared characteristic K values of the retinal quadrants. We found that the inner retina is a mechanically inhomogeneous tissue. Most elastic moduli were in the range of 940 to 1800 Pa; significant differences were found between areas less than 50 µm apart. To identify the origin of this mechanical inhomogeneity, we investigated the size and distribution of structures comprising the retinal surface: large cell bodies in the ganglion cell layer, nerve fibers, inner limiting membrane, and Muller cell endfeet. Our data suggest that the distribution of compliant nerve fiber bundles and stiff neuronal cell bodies contributes most to the mechanical properties of the inner retina. These data offer a basis for understanding cellular mechanoresistivity and -sensitivity in the retina as a mechanically active tissue, and they may help to understand mechanisms and consequences of a variety of retino-pathological processes and their surgical treatment.

Carbocyanine dye usage in demarcating boundaries of the aged human red nucleus.

BackgroundThough the adult human magnocellular Red nucleus (mNr) is essentially vestigial and its boundaries with neighbouring structures have never been well demarcated, human studies in utero have shown a well developed semilunar mNr wrapping around the caudal parvicellular Red nucleus (pNr), similar to what is seen in quadrupeds. In the present study, we have sought to better delineate the morphological determinants of the adult human Red nucleus (ahRn).Methods and FindingsSerial sections of ahRn show fine myelinated fibers arising from pNr and turning toward the central tegmental tract. DiI was deposited within a well restricted region of ahRn at the fasciculus retroflexus level and the extent of label determined. Nissl-stained serial sections allowed production of a 3-D mNr model, showing rudimentary, vestigial morphology compared with its well developed infant homologue. DiI within this vestigial mNr region at the level of the oculomotor nerve showed labeled giant/large mNr neurons, coarse fiber bundles at the ventral tegmental decussation and lateral lemniscal label.ConclusionsLarge amounts of DiI and a long incubation time have proven useful in aged human brain as a marker of long axons and large cell bodies of projecting neurons such as the rubrospinal projection and for clarifying nuclear boundaries of closed nuclei (e.g., the large human pNr). Our 3D model of adult human mNr appeared shrunken in shape and axially rotated compared with the infant mNr, the rotation being a common feature among mammalian mNr.

NMDA Receptor Contributions to Visual Contrast Coding

In the retina, it is not well understood how visual processing depends on AMPA- and NMDA-type glutamate receptors. Here we investigated how these receptors contribute to contrast coding in identified guinea pig ganglion cell types in vitro. NMDA-mediated responses were negligible in ON alpha cells but substantial in OFF alpha and delta cells. OFF delta cell NMDA receptors were composed of GluN2B subunits. Using a novel deconvolution method, we determined the individual contributions of AMPA, NMDA, and inhibitory currents to light responses of each cell type. OFF alpha and delta cells used NMDA receptors for encoding either the full contrast range (alpha), including near-threshold responses, or only a high range (delta). However, contrast sensitivity depended substantially on NMDA receptors only in OFF alpha cells. NMDA receptors contribute to visual contrast coding in a cell-type-specific manner. Certain cell types generate excitatory responses using primarily AMPA receptors or disinhibition.

Properties of GABAergic Neurons in the Rostral Solitary Tract Nucleus in Mice

The rostral nucleus of the solitary tract (rNST) plays a pivotal role in taste processing. The rNST contains projection neurons and interneurons that differ in morphology and intrinsic membrane properties. Although characteristics of the projection neurons have been detailed, similar information is lacking on the interneurons. We determined the intrinsic properties of the rNST GABAergic interneurons using a transgenic mouse model that expresses enhanced green fluorescent protein under the control of a GAD67 promoter. Glutamic acid decarboxylase-green fluorescent protein (GAD67-GFP) neurons were distributed throughout the rNST but were concentrated in the ventral subdivision with minimal interaction with the terminal field of the afferent input. Furthermore, the density of the GAD67-GFP neurons decreased in more rostral areas of rNST. In whole cell recordings, GAD67-GFP neurons responded with either an initial burst (73%), tonic (18%), or irregular (9%) discharge pattern of action potentials (APs) in response to membrane depolarization. These three groups also differed in passive and AP characteristics. Initial burst neurons had small ovoid or fusiform cell bodies, whereas tonic firing neurons had large multipolar or fusiform cell bodies. Irregular firing neurons had larger spherical soma. Some of the initial burst and tonic firing neurons were also spontaneously active. The GAD67-GFP neurons could also be categorized in subgroups based on colocalization with somatostatin and parvalbumin immunolabeling. Initial burst neurons would transmit the early dynamic portion of the encoded sensory stimuli, whereas tonic firing neurons could respond to both dynamic and static components of the sensory input, suggesting different roles for GAD67-GFP neurons in taste processing.

Immunohistochemical characteristics of submucosal Dogiel type II neurons in rat colon

Secretory and motility reflexes are evoked by physiological stimuli in the isolated rat distal colon, which is therefore expected to contain intrinsic primary afferent (sensory) neurons. Dogiel type II neurons (putative intrinsic primary afferent neurons) exhibit several long processes emerging from large oval or round cell bodies. This study has examined the immunohistochemical characteristics of type II neurons in the submucosal plexus of rat distal colons by using whole-mount preparations. Neuronal cell bodies positive for both substance P (SP) and calretinin have been observed in colchicine-treated rats. Neurofilament 200 immunostaining has confirmed the type II morphology of SP-positive neurons. Moreover, all submucosal type II neurons identified by neurofilament 200 immunoreactivity are positive for calretinin. Calcitonin gene-related peptide (CGRP)-positive neurons in the submucosal plexus are distinct from type II neurons because they are negative for calretinin and have smaller cell bodies than the SP-positive submucosal type II neurons. Most (73%) of the submucosal neurons including type II neurons exhibit immunoreactivity for the neurokinin-1 receptor (NK1R), a receptor for SP, on the surface of cell bodies. Immunoreactivity for the EP3 receptor (EP3R), a receptor for prostaglandin E2, has been detected in 51% of submucosal neurons including type II neurons. Thus, submucosal type II neurons in the rat distal colon are immunopositive for SP/calretinin but immunonegative for CGRP. SP released from submucosal type II neurons probably acts via NK1Rs on type II and non-type II submucosal neurons to mediate intrinsic reflexes. EP3R-positive submucosal type II neurons may be potential targets of prostaglandin E2.

Evidence for a protein tether involved in somatic touch

The gating of ion channels by mechanical force underlies the sense of touch and pain. The mode of gating of mechanosensitive ion channels in vertebrate touch receptors is unknown. Here we show that the presence of a protein link is necessary for the gating of mechanosensitive currents in all low-threshold mechanoreceptors and some nociceptors of the dorsal root ganglia (DRG). Using TEM, we demonstrate that a protein filament with of length ∼100 nm is synthesized by sensory neurons and may link mechanosensitive ion channels in sensory neurons to the extracellular matrix. Brief treatment of sensory neurons with non-specific and site-specific endopeptidases destroys the protein tether and abolishes mechanosensitive currents in sensory neurons without affecting electrical excitability. Protease-sensitive tethers are also required for touch-receptor function in vivo. Thus, unlike the majority of nociceptors, cutaneous mechanoreceptors require a distinct protein tether to transduce mechanical stimuli.

Expression of GLUT 3 in different brain regions of aged rats

To examine the distribution of glucose transport 3 (GLUT 3) in different brain regions of aged rats and to investigate its role in ageing process of the nervous system. The GLUT 3 expression in different brain regions was examined with immunohistochemical method in rats aged 3, 18 and 30 months, respectively. The number of GLUT 3-positive cells varied in the different brain regions in rats of all age groups (P<0.01); the CA1 region contained the greatest number of positive cells,and fewer in the motor cortex and cerebellum. The number of GLUT 3-positive cells was reduced in the brain of aged rats (P<0.01); and the neural cells in 4 different brain regions presented with large cell body and loose alignment. The expression of GLUT 3 decreased in aged rats, which suggests that GLUT 3 may be involved in the ageing process of nervous system.

ENVIRONMENTAL CUE-DEPENDENT DOPAMINERGIC NEURONAL DIFFERENTIATION AND FUNCTIONAL EFFECT OF GRAFTED NEUROEPITHELIAL STEM CELLS IN PARKINSONIAN BRAIN

To physiologically repair damaged neural circuitry using neural transplantation, the donor cells must be able to differentiate into the optimal type and number of neurons for the host brain environment. They must also be capable of functional regulation by the host brain. These features are important to optimize functional outcome and to minimize side effects. In this study, the differentiation of grafted mesencephalic neuroepithelial stem cells in the normal and parkinsonian brain was assessed morphologically and behaviorally to confirm the influence of the host brain environment after neural transplantation regarding functional outcome and side effects of transplants. Green fluorescent protein-positive mesencephalic neuroepithelial stem cells were dissected from early rat embryos and transplanted into normal (n = 20) and Parkinson's disease (PD) rat model striata (n = 30). The differentiation pattern of grafted cells was precisely monitored immunohistochemically, and the functional effects of grafted cells on behavior were assessed using an amphetamine-induced rotation test. The grafted cells survived in both normal and PD rat striata and differentiated into tyrosine hydroxylase-positive cells. In 8-week-old grafts, the number of tyrosine hydroxylase-positive cells was 3-fold higher in parkinsonian brains than in normal brains. The donor-derived tyrosine hydroxylase-positive cells exhibited a mature morphology with long, well-branched cell processes and large cell bodies, especially in parkinsonian brains. Also, the process lengths of these cells in parkinsonian brains were 3.4-fold longer than those in normal brains at 8 weeks after transplantation. The grafted PD rats exhibited a complete recovery from their behavioral defects, and no obvious contralateral rotation to the lesioned and grafted side suggestive of overdose supply from the graft was observed. However, the grafted normal rats did not exhibit any contralateral rotation to the grafted side suggestive of dopamine unbalance. Grafted mesencephalic neuroepithelial stem cells can differentiate into optimal neuron types in response to environmental cues and can affect the behavior of PD rats without any side effects.

Axon Regeneration through Scaffold into Distal Spinal Cord after Transection

We employed Fast Blue (FB) axonal tracing to determine the origin of regenerating axons after thoracic spinal cord transection injury in rats. Schwann cell (SC)-loaded, biodegradable, poly(lactic-co-glycolic acid) (PLGA) scaffolds were implanted after transection. Scaffolds loaded with solubilized basement membrane preparation (without SCs) were used for negative controls, and nontransected cords were positive controls. One or 2 months after injury and scaffold implantation, FB was injected 0–15 mm caudal or about 5 mm rostral to the scaffold. One week later, tissue was harvested and the scaffold and cord sectioned longitudinally (30 μm) on a cryostat. Trans-scaffold labeling of neuron cell bodies was identified with confocal microscopy in all cell-transplanted groups. Large (30–50 μm diameter) neuron cell bodies were predominantly labeled in the ventral horn region. Most labeled neurons were seen 1–10 mm rostral to the scaffold, although some neurons were also labeled in the cervical cord. Axonal growth occurred bidirectionally after cord transection, and axons regenerated up to 14 mm beyond the PLGA scaffolds and into distal cord. The extent of FB labeling was negatively correlated with distance from the injection site to the scaffold. Electron microscopy showed myelinated axons in the transverse sections of the implanted scaffold 2 months after implantation. The pattern of myelination, with extracellular collagen and basal lamina, was characteristic of SC myelination. Our results show that FB labeling is an effective way to measure the origin of regenerating axons.

Insulin-like Growth Factor-I Prevents the Accumulation of Autophagic Vesicles and Cell Death in Purkinje Neurons by Increasing the Rate of Autophagosome-to-lysosome Fusion and Degradation

Continuous macroautophagic activity is critical for the maintenance of neuronal homeostasis; however, unchecked or dysregulated autophagy can lead to cell death. Cultured Purkinje neurons die by an autophagy-associated cell death mechanism when deprived of trophic support. Here, we report that insulin-like growth factor-I (IGF-I) completely blocked the autophagy-associated cell death of Purkinje neurons. To examine the mechanism by which IGF-I influences autophagy, neurons were infected with adeno-RFP-LC3 and subjected to trophic factor withdrawal, and the size and number of autophagosomes were analyzed by live-cell fluorescence imaging. In control neurons, autophagy occurred at a constitutive low level with most autophagosomes measuring less than 0.75 microm. Trophic factor withdrawal increased the number and size of autophagosomes with most autophagosomes ranging between 0.75 and 1.5 microm and some reaching 1.5-2.25 microm. IGF-I added at the time of trophic factor withdrawal prevented the accumulation of the larger autophagosomes; however, it had no effect on the conversion of LC3, an indicator of autophagy induction. Instead, the rate of autophagosome-to-lysosome fusion measured by colocalization of RFP-LC3 and LysoSensor Green was accelerated by IGF-I. Treating the neurons with bafilomycin A(1) in the presence of IGF-I led to the accumulation of autophagosomes even larger than those induced by trophic factor withdrawal alone, indicating that IGF-I regulates autophagic vesicle turnover. Finally, the effect of IGF-I on autophagy was mediated by an Akt/mTOR-de pend ent and an ERK-independent pathway. These data suggest a novel role for IGF-I in protecting Purkinje neurons from autophagy-associated cell death by increasing autophagy efficiency downstream of autophagy induction.

Podocyte polarity signalling

The glomerular filtration barrier is a unique structure characterized by a specialized three-dimensional framework of podocytes. This review is aimed at describing the latest advances made in the understanding of polarity signalling pathways regulating the formation and the maintenance of the complex podocyte architecture. Podocytes are composed of a large cell body that extends primary and secondary processes. An apicobasal polarity axis allows for podocyte orientation between the urinary space and the glomerular basement membrane. Recent studies document that conserved polarity protein complexes such as the partitioning defective 3 (Par3), partitioning defective 6 (Par6) and atypical protein kinase C (aPKC) complex are essential regulators of podocyte morphology. Glomerular development, slit diaphragm targeting and apicobasolateral distribution of molecules seem to be tightly regulated by these polarity signalling pathways. Accumulating evidence indicates that conserved polarity protein complexes are essential for normal podocyte morphology and differentiation. The diseased podocyte, which typically presents with foot process effacement, might require these molecular guideposts when recovering from stress and when restoring normal podocyte morphology.

Characterisation of calcitonin gene-related peptide-immunoreactive neurons in the myenteric plexus of rat colon

A mechanical or chemical stimulus applied to the intestinal mucosa induces motility reflexes in the rat colon. Enteric neurons containing calcitonin gene-related peptide (CGRP) have been suggested as intrinsic primary afferent neurons responsible for mediating such reflexes. In the present study, immunohistochemistry was performed on whole-mount stretch preparations to investigate chemical profiles, morphological characteristics and projections of CGRP-containing neurons in the myenteric plexus of the rat colon. CGRP-positive neuronal cell bodies were detected in preparations incubated with colchicine-containing medium, whereas CGRP-positive nerve fibres were found in colchicine-untreated preparations. These neurons had large oval or round cell bodies that were also immunoreactive for the calcium-binding protein calretinin and neurofilament 200. Myenteric neurons positive for both calretinin and neurofilament 200 had several long processes that emerged from the cell body, consistent with Dogiel type II morphology. Application of the neural tracer DiI to the intestinal mucosa revealed that DiI-labelled myenteric neurons each had an oval or round cell body immunoreactive for calretinin. Thus, CGRP-containing myenteric neurons are Dogiel type II neurons and are immunoreactive for calretinin and neurofilament 200 in the rat colon. These neurons probably project to the intestinal mucosa.

Descending unpaired median neurons with bilaterally symmetrical axons in the suboesophageal ganglion of Manduca sexta larvae

Three large median cell bodies with a diameter between 40 and 70 μm that exhibit octopamine immunoreactivity were identified in the posterior part of the suboesophageal ganglion of the tobacco hawkmoth larva, Manduca sexta. These neurons possess bilaterally symmetrical axons in the posterior neck connectives, and at least one of them extends through the whole ventral nerve cord to the terminal abdominal ganglion. Therefore, these neurons belong to the class of descending ventral unpaired median neurons. From each cell body, a primary neurite ascends anteriorly, which after bending dorsally turns posteriorly and then bifurcates to give rise to two descending axons. From the primary neurite two main dendritic branches ascend anteriorly, and four characteristic branches can be distinguished originating from them: two descending dendritic branches and two ascending dendritic branches. Dense arborizations from all these branches exist in all neuromeres of the suboesophageal ganglion. Intracellular recordings from these neurons show that in contrast to the ventral unpaired median neurons of thoracic and abdominal ganglia, they do not produce overshooting action potentials but exhibit passive soma spikes only. During pharmacologically evoked fictive motor patterns these neurons show coupling to various motor patterns such as crawling, feeding and molting.