The roles of mesoporous silica and carbon nanoparticles in antigen stability and intensity of immune response against recombinant subunit B of cholera toxin in a rabbit animal model.

Vaccines are the front line in the fight against diseases. However, setbacks with existing cholera vaccines have ignited a considerable effort to develop more suitable vaccine formulations. In this study, we aim to investigate the effect of antigen stability and controlled release in inducing an immune response. Therefore, two types of silica and carbon mesoporous nanoparticles of the same size and shape but different pore architectures were synthesized and loaded with recombinant cholera toxin subunit B to serve as a model for antigen stability and controlled release of antigenic CTB. In order to evaluate immune response efficacy for these model formulations, IgG and IgA responses and fluid accumulation (FA) index were measured in immunized rabbits, which were challenged with wild-type Vibrio cholerae. Our result suggests that mesoporous silica nanoparticles have greater efficacy in inducing mucosal immune responses, and it proved more proficiency in overall immune responses in challenge experiments and FA index (p < 0.05). These findings indicate that mesoporous nanoparticles and, in particular, mesoporous silica nanoparticles, could be used in oral vaccine formulation against cholera.

Second-order spinal cord pathway contributes to cortical responses after long recoveries from dorsal column injury in squirrel monkeys.

Months after the occurrence of spinal cord dorsal column lesions (DCLs) at the cervical level, neural responses in the hand representation of somatosensory area 3b hand cortex recover, along with hand use. To examine whether the second-order spinal cord pathway contributes to this functional recovery, we injected cholera toxin subunit B (CTB) into the hand representation in the cuneate nucleus (Cu) to label the spinal cord neurons, and related results to cortical reactivation in four squirrel monkeys (Saimiri boliviensis) at least 7 months after DCL. In two monkeys with complete DCLs, few CTB-labeled neurons were present below the lesion, and few neurons in the affected hand region in area 3b responded to touch on the hand. In two other cases with large but incomplete DCLs, CTB-labeled neurons were abundant below the lesion, and the area 3b hand cortex responded well to tactile stimulation in a roughly somatotopic organization. The proportions of labeled neurons in the spinal cord hand region reflected the extent of cortical reactivation to the hand. Comparing monkeys with short and long recovery times suggests that the numbers of labeled neurons below the lesion increase with time following incomplete DCLs (<95%) but decrease with time after nearly complete DCLs (≥95%). Taken together, these results suggest that the second-order spinal cord pathway facilitates cortical reactivation, likely through the potentiation of persisting tactile inputs from the hand to the Cu over months of postlesion recovery.

Reinforcing Mucus Barrier Properties with Low Molar Mass Chitosans.

The mucus gel covers the wet epithelia that forms the inner lining of the body. It constitutes our first line of defense protecting the body from infections and other deleterious molecules. Failure of the mucus barrier can lead to the inflammation of the mucosa such as in inflammatory bowel diseases. Unfortunately, there are no effective strategies that reinforce the mucus barrier properties to recover or enhance its ability to protect the epithelium. Herein, we describe a mucus engineering approach that addresses this issue where we physically cross-link the mucus gel with low molar mass chitosan variants to reinforce its barrier functions. We tested the effect of these chitosans on mucus using in-lab purified porcine gastric mucins, which mimic the native properties of mucus, and on mucus-secreting HT29-MTX epithelial cell cultures. We found that the lowest molar mass chitosan variant (degree of polymerization of 8) diffuses deep into the mucus gels while physically cross-linking the mucin polymers, whereas the higher molar mass chitosan variants (degree of polymerization of 52 and 100) interact only superficially. The complexation resulted in a tighter mucin polymer mesh that slowed the diffusion of dextran polymers and of the cholera toxin B subunit protein through the mucus gels. These results uncover a new use for low molar mass mucoadhesive polymers such as chitosans as noncytotoxic mucosal barrier enhancers that could be valuable in the prevention and treatment of mucosal diseases.

Cholera Toxin Subunit B Enabled Multifunctional Glioma-Targeted Drug Delivery.

Glioma is among the most formidable brain cancers due to location in the brain. Cholera toxin subunit B (CTB) is investigated to facilitate multifunctional glioma-targeted drug delivery by targeting the glycosphingolipid GM1 expressed in the blood-brain barrier (BBB), neovasulature, and glioma cells. When modified on the surface of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (CTB-NPs), CTB fully retains its bioactivity after 24 h incubation in the fresh mouse plasma. The formed protein corona (PC) of CTB-NP and plain PLGA nanoparticles (NP) after incubation in plasma is analyzed using liquid chromatography tandem massspectrometry (nano-LC-MS/MS). CTB modification does not alter the protein components of the formed PC, macrophage phagocytosis, or pharmacokinetic profiles. CTB-NP can efficiently penetrate the in vitro BBB model and target glioma cells and human umbilical vascular endothelial cells. Paclitaxel is loaded in NP (NP/PTX) and CTB-NP (CTB-NP/PTX), and their antiglioma effects are assessed in nude mice bearing intracranial glioma. CTB-NP/PTX can efficiently induce apoptosis of intracranial glioma cells and ablate neovasulature in vivo, resulting in significant prolongation of survival of nude mice bearing intracranial glioma (34 d) in comparison to those treated with NP/PTX (29 d), Taxol (24 d), and saline (21 d). The present study suggests a potential multifunctional glioma-targeted drug delivery system enabled by cholera toxin subunit B.

Adaptive reorganization of retinogeniculate axon terminals in dorsal lateral geniculate nucleus following experimental mild traumatic brain injury.

The pathologic process in traumatic brain injury marked by delayed axonal loss, known as diffuse axonal injury (DAI), leads to partial deafferentation of neurons downstream of injured axons. This process is linked to persistent visual dysfunction following mild traumatic brain injury (mTBI), however, examination of deafferentation in humans is impossible with current technology. To investigate potential reorganization in the visual system following mTBI, we utilized the central fluid percussion injury (cFPI) mouse model of mTBI. We report that in the optic nerve of adult male C57BL/6J mice, axonal projections of retinal ganglion cells (RGCs) to their downstream thalamic target, dorsal lateral geniculate nucleus (dLGN), undergo DAI followed by scattered, widespread axon terminals loss within the dLGN at 4days post-injury. However, at 10days post-injury, significant reorganization of RGC axon terminals was found, suggestive of an adaptive neuroplastic response. While these changes persisted at 20days post-injury, the RGC axon terminal distribution did not recovery fully to sham-injury levels. Our studies also revealed that following DAI, the segregation of axon terminals from ipsilateral and contralateral eye projections remained consistent with normal adult mouse distribution. Lastly, our examination of the shell and core of dLGN suggested that different RGC subpopulations may vary in their susceptibility to injury or in their contribution to reorganization following injury. Collectively, these findings support the premise that subcortical axon terminal reorganization may contribute to recovery following mTBI, and that different neural phenotypes may vary in their contribution to this reorganization despite exposure to the same injury.

Genetic inactivation of glutamate neurons in the rat sublaterodorsal tegmental nucleus recapitulates REM sleep behaviour disorder.

SEE SCHENCK AND MAHOWALD DOI101093/AWW329 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Idiopathic REM sleep behaviour disorder is characterized by the enactment of violent dreams during paradoxical (REM) sleep in the absence of normal muscle atonia. Accumulating clinical and experimental data suggest that REM sleep behaviour disorder might be due to the neurodegeneration of glutamate neurons involved in paradoxical sleep and located within the pontine sublaterodorsal tegmental nucleus. The purpose of the present work was thus to functionally determine first, the role of glutamate sublaterodorsal tegmental nucleus neurons in paradoxical sleep and second, whether their genetic inactivation is sufficient for recapitulating REM sleep behaviour disorder in rats. For this goal, we first injected two retrograde tracers in the intralaminar thalamus and ventral medulla to disentangle neuronal circuits in which sublaterodorsal tegmental nucleus is involved; second we infused bilaterally in sublaterodorsal tegmental nucleus adeno-associated viruses carrying short hairpin RNAs targeting Slc17a6 mRNA [which encodes vesicular glutamate transporter 2 (vGluT2)] to chronically impair glutamate synaptic transmission in sublaterodorsal tegmental nucleus neurons. At the neuroanatomical level, sublaterodorsal tegmental nucleus neurons specifically activated during paradoxical sleep hypersomnia send descending efferents to glycine/GABA neurons within the ventral medulla, but not ascending projections to the intralaminar thalamus. These data suggest a crucial role of sublaterodorsal tegmental nucleus neurons rather in muscle atonia than in paradoxical sleep generation. In line with this hypothesis, 30 days after adeno-associated virus injections into sublaterodorsal tegmental nucleus rats display a decrease of 30% of paradoxical sleep daily quantities, and a significant increase of muscle tone during paradoxical sleep concomitant to a tremendous increase of abnormal motor dream-enacting behaviours. These animals display symptoms and behaviours during paradoxical sleep that closely mimic human REM sleep behaviour disorder. Altogether, our data demonstrate that glutamate sublaterodorsal tegmental nucleus neurons generate muscle atonia during paradoxical sleep likely through descending projections to glycine/GABA premotor neurons in the ventral medulla. Although playing a role in paradoxical sleep regulation, they are, however, not necessary for inducing the state itself. The present work further validates a potent new preclinical REM sleep behaviour disorder model that opens avenues for studying and treating this disabling sleep disorder, and advances potential regions implicated in prodromal stages of synucleinopathies such as Parkinson's disease.

Acute hypoxia activates hypothalamic paraventricular nucleus-projecting catecholaminergic neurons in the C1 region.

Catecholaminergic C1 cells reside in the rostral and intermediate portions of the ventrolateral medulla (RVLM) and can be activated by hypoxia. These neurons regulate the hypothalamic pituitary axis via direct projections to the hypothalamic paraventricular nucleus (PVH) and regulate the autonomic nervous system via projections to sympathetic and parasympathetic preganglionic neurons. Based on the various effects attributed to the C1 cells and what is currently known of their synaptic inputs, our hypothesis is that acute hypoxia (AH) activates RVLM projecting catecholaminergic neurons to PVH. Anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L) was unilaterally injected into the RVLM and a retrograde tracer Cholera toxin b (CTb) was unilaterally injected into the PVH region. After ten days, male Wistar rats that received CTb injection into the PVH were subjected to AH (8% O2, balanced with N2) or normoxia (21% O2) for 3h. Acute hypoxia significantly increased Fos immunoreactivity in the C1 region (68±2 neurons), and half of the RVLM cells activated are catecholaminergic (35±2 neurons). We observed that 23±4% of the RVLM projecting PVH cells that were activated by AH were also C1 cells. The presence of varicosities containing PHA-L in PVH region was also observed. The present results suggest that catecholaminergic C1-PVH projection is hypoxia-sensitive and the pathway between these two important brain areas can be one more piece in the complex puzzle of neural control of autonomic regulation during hypoxia.

ZOT-derived peptide and chitosan functionalized nanocarrier for oral delivery of protein drug.

In this study, we developed a dual ligand functionalized pluronic-based nanocarrier (NC) for oral delivery of insulin. Chitosan and zonula occludins toxin (ZOT)-derived, tight junction opening peptide were conjugated to NC to increase the permeability of loaded insulin across the small intestine through the paracellular pathway. Surface functionalized NC, either by chitosan or peptide, could modulate the tight junction (TJ) integrity in contrast to no effect of unmodified NC, as evidenced by the change in transepithelial electrical resistance (TEER) and immunostaining of Claudin-4, a tight junction marker, in Caco-2 cell monolayer. On the other hand, dual ligand (chitosan and peptide) functionalized NC significantly further increased the permeation of insulin across Caco-2 cell monolayer. More importantly, insulin loaded, dual ligand functionalized NC could increase the plasma insulin level and efficiently regulate the glycemic response for a prolonged period of time (∼1 day) upon oral administration to diabetic rats, whereas delivery of insulin by single ligand functionalized NCs, either by chitosan or peptide, as well as by unmodified NC and free insulin, could not induce the effective regulation of the blood glucose level. The use of fluorescence dye labeled insulin (FITC-insulin) and Cy5.5 labeled NC revealed that both insulin and dual ligand functionalized NC were adequately penetrated across the whole intestine villi in contrast to limited adsorption of insulin and NC mainly onto the epithelial surface of the intestine for single ligand functionalized NCs. These results suggest that dual conjugation of ZOT-derived peptide and chitosan is a promising approach to functionalize the surface of nanocarrier for oral delivery of protein drugs.

Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes.

We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.

Bilateral neuroinflammatory processes in visual pathways induced by unilateral ocular hypertension in the rat.

BACKGROUND: Glaucoma is one of the leading causes of irreversible blindness in the world. The major risk factor is elevated intraocular pressure (IOP) leading to progressive retinal ganglion cell (RGC) death from the optic nerve (ON) to visual pathways in the brain. Glaucoma has been reported to share mechanisms with neurodegenerative disorders. We therefore hypothesize that neuroinflammatory mechanisms in central visual pathways may contribute to the spread of glaucoma disease. The aim of the present study was to analyze the neuroinflammation processes that occur from the pathological retina to the superior colliculi (SCs) in a rat model of unilateral ocular hypertension induced by episcleral vein cauterization (EVC). RESULTS: Six weeks after unilateral (right eye) EVC in male Long-Evans rats, we evaluated both the neurodegenerative process and the neuroinflammatory state in visual pathway tissues. RGCs immunolabeled (Brn3a(+)) in ipsilateral whole flat-mounted retina demonstrated peripheral RGC loss associated with tissue macrophage/microglia activation (CD68(+)). Gene expression analysis of hypertensive and normotensive retinas revealed a significant increase of pro-inflammatory genes such as CCL2, IL-1ß, and Nox2 mRNA expression compared to naïve eyes. Importantly, we found an upregulation of pro-inflammatory markers such as IL-1ß and TNFα and astrocyte and tissue macrophage/microglia activation in hypertensive and normotensive RGC projection sites in the SCs compared to a naïve SC. To understand how neuroinflammation in the hypertensive retina is sufficient to damage both right and left SCs and the normotensive retina, we used an inflammatory model consisting in an unilateral stereotaxic injection of TNFα (25 ng/µl) in the right SC of naïve rats. Two weeks after TNFα injection, using an optomotor test, we observed that rats had visual deficiency in both eyes. Furthermore, both SCs showed an upregulation of genes and proteins for astrocytes, microglia, and pro-inflammatory cytokines, notably IL-1ß. In addition, both retinas exhibited a significant increase of inflammatory markers compared to a naïve retina. CONCLUSIONS: All these data evidence the complex role played by the SCs in the propagation of neuroinflammatory events induced by unilateral ocular hypertension and provide a new insight into the spread of neurodegenerative diseases such as glaucoma.

Cholera Toxin B Subunit Shows Transneuronal Tracing after Injection in an Injured Sciatic Nerve.

Cholera toxin B subunit (CTB) has been extensively used in the past for monosynaptic mapping. For decades, it was thought to lack the ability of transneuronal tracing. In order to investigate whether biotin conjugates of CTB (b-CTB) would pass through transneurons in the rat spinal cord, it was injected into the crushed left sciatic nerve. For experimental control, the first order afferent neuronal projections were defined by retrograde transport of fluorogold (FG, a non-transneuronal labeling marker as an experimental control) injected into the crushed right sciatic nerve in the same rat. Neurons containing b-CTB or FG were observed in the dorsal root ganglia (DRG) at the L4-L6 levels ipsilateral to the tracer injection. In the spinal cord, b-CTB labeled neurons were distributed in all laminae ipsilaterally between C7 and S1 segments, but labeling of neurons at the cervical segment was abolished when the T10 segment was transected completely. The interneurons, distributed in the intermediate gray matter and identified as gamma-aminobutyric acid-ergic (GABAergic), were labeled by b-CTB. In contrast, FG labeling was confined to the ventral horn neurons at L4-L6 spinal segments ipsilateral to the injection. b-CTB immunoreactivity remained to be restricted to the soma of neurons and often appeared as irregular patches detected by light and electron microscopy. Detection of monosialoganglioside (GM1) in b-CTB labeled neurons suggests that GM1 ganglioside may specifically enhance the uptake and transneuronal passage of b-CTB, thus supporting the notion that it may be used as a novel transneuronal tracer.

Role of the Suprachiasmatic and Arcuate Nuclei in Diurnal Temperature Regulation in the Rat.

In mammals, daily changes in body temperature (Tb) depend on the integrity of the suprachiasmatic nucleus (SCN). Fasting influences the Tb in the resting period and the presence of the SCN is essential for this process. However, the origin of this circadian/metabolic influence is unknown. We hypothesized that, not only the SCN but also the arcuate nucleus (ARC), are involved in the Tb setting through afferents to the thermoregulatory median preoptic nucleus (MnPO). Therefore, we investigated by neuronal tracing and microdialysis experiments the possible targeting of the MnPO by the SCN and the ARC in male Wistar rats. We observed that vasopressin release from the SCN decreases the temperature just before light onset, whereas α-melanocyte stimulating hormone release, especially at the end of the dark period, maintains high temperature. Both peptides have opposite effects on the brown adipose tissue activity through thermoregulatory nuclei such as the dorsomedial nucleus of the hypothalamus and the dorsal raphe nucleus. The present study indicates that the coordination between circadian and metabolic signaling within the hypothalamus is essential for an adequate temperature control. SIGNIFICANCE STATEMENT: When circadian and metabolic systems are not well synchronized, individuals may develop metabolic diseases. The underlying mechanisms are unknown. Here, we demonstrate that the balance between the releases of neuropeptides derived from the biological clock and from a metabolic sensory organ as the arcuate nucleus, are essential for an adequate temperature control. These observations show that brain areas involved in circadian and metabolic functions of the body need to interact to produce a coherent arrangement of physiological processes associated with temperature control.

A novel fluorescent retrograde neural tracer: cholera toxin B conjugated carbon dots.

The retrograde neuroanatomical tracing method is a key technique to study the complex interconnections of the nervous system. Traditional tracers have several drawbacks, including time-consuming immunohistochemical or immunofluorescent staining procedures, rapid fluorescence quenching and low fluorescence intensity. Carbon dots (CDs) have been widely used as a fluorescent bio-probe due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility and low toxicity. Herein, we develop a novel fluorescent neural tracer: cholera toxin B-carbon dot conjugates (CTB-CDs). It can be taken up and retrogradely transported by neurons in the peripheral nervous system of rats. Our results show that CTB-CDs possess high photoluminescence intensity, good optical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a direct and more economical way of performing retrograde labelling experiments. Therefore, CTB-CDs are reliable fluorescent retrograde tracers.

Role of cerebrospinal fluid-contacting nucleus in sodium sensing and sodium appetite.

The brainstem plays an important role in controlling sodium and water homeostasis. It is a major regulatory site for autonomic and motor functions. Moreover, it integrates cerebrospinal fluid (CSF) signals with neuronal and hormonal signals. Evidence suggests that the CSF-contacting nucleus (CSF-CN) transmits and integrates CSF signals, but, the definitive role of CSF-CN in sodium homeostasis is poorly understood. In this study, we used c-Fos as a marker of neuronal activity and causing colocalization of Nax channel and 5-HT. This proved that CSF-CN played a role in sensing the increase of CSF sodium level. Then, we determined the role of the CSF-contacting nucleus in increasing the sodium appetite of rats. So, we performed targeted lesion of the CSF-contacting nucleus in the brainstem using the cholera toxin subunit B-saporin (CB-SAP), a cytotoxin coupled to cholera toxin subunit B. The lesion of the CSF-CN showed decreased and degenerative neurons, while sodium appetite have increased and Fos immunocytochemistry detected neuronal activity in the lateral parabrachial nucleus (LPBN), but not in the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT). These results indicate that the CSF-CN plays an important role in sensing CSF sodium level and satiating sodium appetite by influencing the LPBN but not SFO and OVLT. The Nax channel and 5-HT might be the molecular mechanisms through which contribute to sodium homeostasis.

Sensory Cortical Control of a Visually Induced Arrest Behavior via Corticotectal Projections.

Innate defense behaviors (IDBs) evoked by threatening sensory stimuli are essential for animal survival. Although subcortical circuits are implicated in IDBs, it remains largely unclear whether sensory cortex modulates IDBs and what the underlying neural pathways are. Here, we show that optogenetic silencing of corticotectal projections from layer 5 (L5) of the mouse primary visual cortex (V1) to the superior colliculus (SC) significantly reduces an SC-dependent innate behavior (i.e., temporary suspension of locomotion upon a sudden flash of light as short as milliseconds). Surprisingly, optogenetic activation of SC-projecting neurons in V1 or their axon terminals in SC sufficiently elicits the behavior, in contrast to other major L5 corticofugal projections. Thus, via the same corticofugal projection, visual cortex not only modulates the light-induced arrest behavior, but also can directly drive the behavior. Our results suggest that sensory cortex may play a previously unrecognized role in the top-down initiation of sensory-motor behaviors.

Expression of cholera toxin B-lumbrokinase fusion protein in Pichia pastoris--the use of transmucosal carriers in the delivery of therapeutic proteins to protect rats against thrombosis.

Cholera toxin B-subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. However, whether CTB can be used as a transmucosal carrier in the delivery of not only vaccines but also therapeutic proteins has not been widely studied. Thus, we investigate here the concept of receptor-mediated oral delivery of lumbrokinase (LK) proteins which is an important fibrinolytic enzyme derived from earthworm. CTB and LK, separated by a furin cleavage site, was expressed via Pichia pastoris. The activity and proper folding of recombinant protein in yeast were confirmed by Western blot analysis, fibrin plate assays, and G(M1)-ganglioside ELISA. Following oral administration of recombinant protein, the thrombosis model of rats and mice revealed that the oral treatment of rCTB-LK has a more significant anti-thrombotic effect on animals compared with rLK. It is possible to conclude that CTB can successfully enhance its fusion protein LK to be absorbed. The use of CTB as a transmucosal carrier in the delivery of not only vaccines but also therapeutic proteins was supported.

Jaw muscle spindle afferents coordinate multiple orofacial motoneurons via common premotor neurons in rats: an electrophysiological and anatomical study.

Jaw muscle spindle afferents (JMSA) in the mesencephalic trigeminal nucleus (Vme) project to the parvocellular reticular nucleus (PCRt) and dorsomedial spinal trigeminal nucleus (dm-Vsp). A number of premotor neurons that project to the trigeminal motor nucleus (Vmo), facial nucleus (VII) and hypoglossal nucleus (XII) are also located in the PCRt and dm-Vsp. In this study, we examined whether these premotor neurons serve as common relay pool for relaying JMSA to multiple orofacial motoneurons. JMSA inputs to the PCRt and dm-Vsp neurons were verified by recording extracellular responses to electrical stimulation of the caudal Vme or masseter nerve, mechanical stimulation of jaw muscles and jaw opening. After recording, biocytin in recording electrode was inotophorized into recording sites. Biocytin-Iabeled fibers traveled to the Vmo, VII, XII, and the nucleus ambiguus (Amb). Labeled boutons were seen in close apposition with Nissl-stained motoneurons in the Vmo, VII, XII and Amb. In addition, an anterograde tracer (biotinylated dextran amine) was iontophorized into the caudal Vme, and a retrograde tracer (Cholera toxin B subunit) was delivered into either the VII or Xll to identify VII and XII premotor neurons that receive JMSA input. Contacts between labeled Vme neuronal boutons and premotor neurons were observed in the PCRt and adjacent dm-Vsp. Confocal microscopic observations confirmed close contacts between Vme boutons and VII and XII premotor neurons. This study provides evidence that JMSA may coordinate activities of multiple orofacial motor nuclei, including Vmo, VII, XII and Amb in the brainstem via a common premotor neuron pool.

Thalamocortical projections of the anterodorsal thalamic nucleus in the rabbit.

The anterior thalamic nuclei consist of the anterodorsal (AD), anteroventral, and anteromedial nuclei, each of which are highly differentiated and may contribute to different aspects of various cognitive and memory functions. In particular, the AD is unique in that it is implicated in learning at the earliest stage of discriminative avoidance conditioning in the rabbit. To better understand the functional roles played by the AD in memory and learning processes, we analyzed the organization of thalamocortical projections of the AD in the rabbit, using the anterograde tracer biotinylated dextran amine and the retrograde tracer cholera toxin subunit B. The data show that the AD provides strong projections to layers I and IV of area 30 and to layers I, III, IV, and VI of area 29 in the retrosplenial cortex, and to layers I and III-VI of the presubiculum. The projections to the retrosplenial cortex are organized such that the rostral and caudal AD, respectively, project to the caudal and rostral retrosplenial cortex. In contrast, the projections to the presubiculum are not organized topographically. Other minor projections were also observed in the parasubiculum and part of the medial entorhinal area. These results indicate that the AD provides strong projections to the retrosplenial cortex and presubiculum, suggesting that these projections constitute essential pathways to these cortical regions for transmitting mnemonic information, such as a novel conditioning stimulus during the initial stage of avoidance learning.

Arf6-dependent intracellular trafficking of Pasteurella multocida toxin and pH-dependent translocation from late endosomes.

The potent mitogenic toxin from Pasteurella multocida (PMT) is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH(4)Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn) and cholera toxin (CT), the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity.

Is there any sense in the Palisade endings of eye muscles?

Palisade endings (PEs), which are unique to the eye muscles, are associated with multiply innervated muscle fibers. They lie at the myotendinous junctions and form a cap around the muscle fiber tip. They are found in all animals investigated so far, but their function is not known. Recently, we demonstrated that cell bodies of PEs and tendon organs lie around the periphery of the oculomotor nucleus in the C- and S-groups. A morphological analysis of these peripheral neurons revealed the existence of different populations within the C-group. We propose that a small group of round or spindle-shaped cells gives rise to PEs, and another group of multipolar neurons provide the multiple motor endings. If PEs have a sensory function, then their cell body location close to motor neurons would be in an ideal location to control tension in extraocular muscles; in the case of the C-group, its proximity to the preganglionic neurons of the Edinger-Westphal nucleus would permit its participation in the near response. Despite their unusual properties, PEs may have a sensory function.