Wheat Germ Agglutinin-conjugated Horseradish Peroxidase Research Articles

Neural Tracing Protein-Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons.

Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.

Overall patterns of eye-specific retino-geniculo-cortical projections to layers III, IV, and VI in primary visual cortex of the greater galago (Otolemur crassicudatus), and correlation with cytochrome oxidase blobs.

Studies in the greater galago have not provided a comprehensive description of the organization of eye-specific retino-geniculate-cortical projections to the recipient layers in V1. Here we demonstrate the overall patterns of ocular dominance domains in layers III, IV, and VI revealed following a monocular injection of the transneuronal tracer wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP). We also correlate these patterns with the array of cytochrome oxidase (CO) blobs in tangential sections through the unfolded and flattened cortex. In layer IV, we observed for the first time that eye-specific domains form an interconnected pattern of bands 200-250μm wide arranged such that they do not show orientation bias and do not meet the V1 border at right angles, as is the case in macaques. We also observed distinct WGA-HRP labeled patches in layers III and VI. The patches in layer III, likely corresponding to patches of K lateral geniculate nucleus (LGN) input, align with layer IV ocular dominance columns (ODCs) of the same eye dominance and overlap partially with virtually all CO blobs in both hemispheres, implying that CO blobs receive K LGN input from both eyes. We further found that CO blobs straddle the border between layer IV ODCs, such that the distribution of CO staining is approximately equal over ipsilateral and contralateral ODCs. These results, together with studies showing that a high percentage of cells in CO blobs are monocular, suggest that CO blobs consist of ipsilateral and contralateral subregions that are in register with underlying layer IV ODCs of the same eye dominance. In macaques and humans, CO blobs are centered on ODCs in layer IV. Our finding that CO blobs in galago straddle the border of neighboring layer IV ODCs suggests that this novel feature may represent an alternative way by which visual information is processed by eye-specific modular architecture in mammalian V1.

A New Neural Pathway from the Ventral Striatum to the Nucleus Basalis of Meynert with Functional Implication to Learning and Memory

The cholinergic neurons in the nucleus basalis of Meynert (NBM) are among the first group of neurons known to become degenerated in Alzheimer’s disease, and thus the NBM is proposed to be involved in learning and memory. The marginal division (MrD) of the striatum is a newly discovered subdivision at the ventromedial border of the mammalian striatum and is considered to be one part of the ventral striatum involved in learning and memory. The present study provided evidence to support the hypothesis that the MrD and the NBM were structurally connected at cellular and subcellular levels with functional implications in learning and memory. First, when wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was stereotaxically injected into the NBM, fusiform neurons in the MrD were retrogradely labeled with WGA-HRP gray-blue particles and some of them were double stained in brown color by AchE staining method. Thus, cholinergic neurons of the MrD were shown to project to the neurons in the NBM. Second, in anterograde tract-tracing experiments where WGA-HRP was injected to the MrD, the labeled WGA-HRP was found to be anterogradely transported in axons from the MrD to the synaptic terminals with dendrites, axons, and perikaryons of the cholinergic neurons in the NBM when observed under an electronic microscope, indicating reciprocal structural connections between the MrD and the NBM. Third, when bilateral lesions of the MrD were injured with kainic acid in rats, degenerative terminals were observed in synapses of the NBM by an electronic microscope and severe learning and memory deficiency was found in these rats by the Y-maze behavioral test. Our results suggest reciprocal cholinergic connections between the MrD of the ventral striatum and the NBM, and implicate a role of the MrD-NBM pathway in learning and memory. The efferent fibers of cholinergic neurons in the NBM mainly project to the cortex, and severe reduction of the cholinergic innervation in the cortex is the common feature of Alzheimer’s patients. The newly discovered cholinergic neural pathway between the MrD of the ventral striatum and the NBM is supposed involved in the memory circuitries of the brain and probably might play a role in the pathogenesis of the Alzheimer’s disease.

Diaphragmatic recovery in rats with cervical spinal cord injury induced by a theophylline nanoconjugate: Challenges for clinical use

Context: Following a spinal cord hemisection at the second cervical segment the ipsilateral hemidiaphragm is paralyzed due to the disruption of the rostral ventral respiratory group (rVRG) axons descending to the ipsilateral phrenic motoneurons (PN). Systemically administered theophylline activates a functionally latent crossed phrenic pathway (CPP) which decussates caudal to the hemisection and activates phrenic motoneurons ipsilateral to the hemisection. The result is return of function to the paralyzed hemidiaphragm. Unfortunately, in humans, systemically administered theophylline at a therapeutic dose produces many unwanted side effects.Design and setting: A tripartite nanoconjugate was synthesized in which theophylline was coupled to a neuronal tracer, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), using gold nanoparticles as the coupler. Following intradiaphragmatic injection of the nanoconjugate, WGA-HRP selectively targets the theophylline-bound nanoconjugate to phrenic motoneurons initially, followed by neurons in the rVRG by retrograde transsynaptic transport.Participants: (N/A)Interventions: (N/A)Outcome Measures: Immunostaining, Electromyography (EMG).Results: Delivery of the theophylline-coupled nanoconjugate to the nuclei involved in respiration induces a return of respiratory activity as detected by EMG of the diaphragm and a modest return of phrenic nerve activity.Conclusion: In addition to the modest return of phrenic nerve activity, there were many difficulties using the theophylline nanoconjugate because of its chemical instability, which suggests that the theophylline nanoconjugate should not be developed for clinical use as explained herein.

Mediolateral and dorsoventral projection patterns of cutaneous afferents within transverse planes of the mouse spinal dorsal horn.

The central projection patterns of cutaneous afferents from the forelimb and shoulder of mice were studied in the spinal dorsal horn after intracutaneous injection of AlexaFluor 488-conjugated and/or 594-conjugated cholera toxin subunit B (CTB). Based on their dermatomes, the following eight skin regions are thought to be innervated by spinal nerves from the sixth to eighth cervical spinal nerve roots: the dorsal surface of the shoulder, brachium, proximal forearm, distal forearm, hand, palmar surface of the second and third digits, and palm. The termination areas of afferents from the dorsal surface of the shoulder and forearm were narrow, distributed in a dorsoventral direction, and aligned in order from lateral to medial within the sixth to eighth cervical dorsal horns. By contrast, the termination areas of the palmar surface of the second and third digits largely overlapped. We also injected CTB into the dorsal surface of the hindlimb and pelvic regions. Skin regions there are thought to be innervated by nerves from the third to fifth lumbar spinal nerve roots. The observed projection patterns in the lumbar dorsal horn were similar to the cervical patterns. Injection of a mixture of CTB and wheat-germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), which are thought to label Aβ and Aδ/C fibers, respectively, showed segregated termination areas of CTB- and WGA-HRP-labeled afferents. Moreover, alignment of the termination areas was in the dorsoventral direction. These results suggest there is fine somatotopic (mediolateral axis) and modality-specific (dorsoventral axis) organization within the spinal dorsal horn.

Thalamo-insular pathway conveying orofacial muscle proprioception in the rat

Little is known about how proprioceptive signals arising from muscles reach to higher brain regions such as the cerebral cortex. We have recently shown that a particular thalamic region, the caudo-ventromedial edge (VPMcvm) of ventral posteromedial thalamic nucleus (VPM), receives the proprioceptive signals from jaw-closing muscle spindles (JCMSs) in rats. In this study, we further addressed how the orofacial thalamic inputs from the JCMSs were transmitted from the thalamus (VPMcvm) to the cerebral cortex in rats. Injections of a retrograde and anterograde neuronal tracer, wheat-germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), into the VPMcvm demonstrated that the thalamic pathway terminated mainly in a rostrocaudally narrow area in the dorsal part of granular insular cortex rostroventrally adjacent to the rostralmost part of the secondary somatosensory cortex (dGIrvs2). We also electrophysiologically confirmed that the dGIrvs2 received the proprioceptive inputs from JCMSs. To support the anatomical evidence of the VPMcvm–dGIrvs2 pathway, injections of a retrograde neuronal tracer Fluorogold into the dGIrvs2 demonstrated that the thalamic neurons projecting to the dGIrvs2 were confined in the VPMcvm and the parvicellular part of ventral posterior nucleus. In contrast, WGA-HRP injections into the lingual nerve area of core VPM demonstrated that axon terminals were mainly labeled in the core regions of the primary and secondary somatosensory cortices, which were far from the dGIrvs2. These results suggest that the dGIrvs2 is a specialized cortical region receiving the orofacial proprioceptive inputs. Functional contribution of the revealed JCMSs–VPMcvm–dGIrvs2 pathway to Tourette syndrome is also discussed.

Chemiluminescence-based quantification of the colonization rates of Lotus japonicus roots by arbuscular mycorrhizal fungi

ABSTRACTSimple evaluation of the levels of root colonization by arbuscular mycorrhizal (AM) fungi is challenging for conventional histochemical staining because most roots show an endogenous background signal. Herein, we report a chemiluminescence-based novel method for the quantification of the relative colonization rates of AM fungi. Lotus japonicus seedlings were grown in two types of soils with different inoculum potential [i.e., the former vegetation was maize (host) or buckwheat (non-host)]. Indigenous AM fungi colonizing roots were labelled with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), which specifically targets the N-acetylglucosamine polymers of fungal cell walls. After the roots were removed from the soil and rinsed with water, all procedures were conducted in 24-well plates. AM fungi were detected by chemiluminescence in the presence of enhanced chemiluminescence (ECL) reagents, and the signal strengths were measured by image analyses. Post-staining of AM fungal structures in the presence of 3,3ʹ-diaminobenzidine (DAB) and H2O2 (DAB staining) revealed that even small colonization events (e.g., hyphopodia) could be detected, whereas the background signals observed in DAB staining were eliminated in the chemiluminescence method. Chemiluminescence thus provides both high sensitivity and high specificity. The chemiluminescence-based colonization levels of roots grown in maize soil were significantly higher than those of roots grown in buckwheat soil, demonstrating the feasibility for high-throughput quantification of the relative colonization rates of L. japonicus seedlings by AM fungi.

Central pupillary light reflex circuits in the cat: I. The olivary pretectal nucleus.

The central pathways subserving the feline pupillary light reflex were examined by defining retinal input to the olivary pretectal nucleus (OPt), the midbrain projections of this nucleus, and the premotor neurons within it. Unilateral intravitreal wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) injections revealed differences in the pattern of retinal OPt termination on the two sides. Injections of WGA-HRP into OPt labeled terminals bilaterally in the anteromedian nucleus, and to a lesser extent in the supraoculomotor area, centrally projecting Edinger-Westphal nucleus, and nucleus of the posterior commissure. Labeled terminals, as well as retrogradely labeled multipolar cells, were present in the contralateral OPt, indicating a commissural pathway. Injections of WGA-HRP into the anteromedian nucleus labeled fusiform premotor neurons within the OPt, as well as multipolar cells in the nucleus of the posterior commissure. Connections between retinal terminals and the pretectal premotor neurons were characterized by combining vitreous chamber and anteromedian nucleus injections of WGA-HRP in the same animal. Fusiform-shaped, retrogradely labeled cells fell within the anterogradely labeled retinal terminal field in the OPt. Ultrastructural analysis revealed labeled retinal terminals containing clear spherical vesicles. They contacted labeled pretectal premotor neurons via asymmetric synaptic densities. These results provide an anatomical substrate for the pupillary light reflex in the cat. Pretectal premotor neurons receive direct retinal input via synapses suggestive of an excitatory drive, and project directly to nuclei containing preganglionic motoneurons. These projections are concentrated in the anteromedian nucleus, indicating its involvement in the pupillary light reflex.

Regeneration of optic nerve fibers with unoprostone, a prostaglandin-related antiglaucoma drug, in adult cats

We investigated the effects of unoprostone on neurite extension of cultured retinal pieces and axonal regeneration of retinal ganglion cells in the crushed optic nerve of adult cats. The retinal pieces were cultured with unoprostone or its primary metabolite, M1, dissolved in DMSO or polysorbate for 14 days, and the number and length of Tau-1-positive neurites and glial processes labeled with anti-glial fibrillary acidic protein antibodies were examined. After the optic nerve was crushed, unoprostone was injected into the vitreous body and the crushed site. On day 12, wheat germ agglutinin-conjugated horseradish peroxidase was injected into the vitreous body to anterogradely label the regenerated axons. On day 14, the optic nerve was excised and longitudinally sectioned. After peroxidase reaction, the number of axons regenerating beyond the crush site was examined. The greatest number of neurites protruded from the cultured retinal pieces in 3 μM unoprostone and 3 μM M1. The neurite length was also the longest at 3 μM unoprostone and 3 μM M1, in which no glial processes were detected. After injections of 3 μM unoprostone, the final concentration in the vitreous humor, into the vitreous body and the crush site, the optic nerve fibers regenerated and extended beyond the crush site. In contrast, almost no fibers extended beyond the crush site after injection of phosphate-buffered saline. The results indicate that intravitreal injection of unoprostone promotes regeneration of crushed optic nerve fibers in adult cats.

Synaptic Connections between Endomorphin 2-Immunoreactive Terminals and μ-Opioid Receptor-Expressing Neurons in the Sacral Parasympathetic Nucleus of the Rat

The urinary bladder is innervated by parasympathetic preganglionic neurons (PPNs) that express μ-opioid receptors (MOR) in the sacral parasympathetic nucleus (SPN) at lumbosacral segments L6-S1. The SPN also contains endomorphin 2 (EM2)-immunoreactive (IR) fibers and terminals. EM2 is the endogenous ligand of MOR. In the present study, retrograde tract-tracing with cholera toxin subunit b (CTb) or wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) via the pelvic nerve combined with immunohistochemical staining for EM2 and MOR to identify PPNs within the SPN as well as synaptic connections between the EM2-IR terminals and MOR-expressing PPNs in the SPN of the rat. After CTb was injected into the pelvic nerve, CTb retrogradely labeled neurons were almost exclusively located in the lateral part of the intermediolateral gray matter at L6-S1 of the lumbosacral spinal cord. All of the them also expressed MOR. EM2-IR terminals formed symmetric synapses with MOR-IR, WGA-HRP-labeled and WGA-HRP/MOR double-labeled neuronal cell bodies and dendrites within the SPN. These results provided morphological evidence that EM2-containing axon terminals formed symmetric synapses with MOR-expressing PPNs in the SPN. The present results also show that EM2 and MOR might be involved in both the homeostatic control and information transmission of micturition.

Projections of the superior colliculus to the pulvinar in prosimian galagos (Otolemur garnettii) and VGLUT2 staining of the visual pulvinar

An understanding of the organization of the pulvinar complex in prosimian primates has been somewhat elusive due to the lack of clear architectonic divisions. In the current study we reveal features of the organization of the pulvinar complex in galagos by examining superior colliculus (SC) projections to this structure and comparing them with staining patterns of the vesicular glutamate transporter, VGLUT2. Cholera toxin subunit β (CTB), Fluoro-ruby (FR), and wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) were placed in topographically different locations within the SC. Our results showed multiple topographically organized patterns of projections from the SC to several divisions of the pulvinar complex. At least two topographically distributed projections were found within the lateral region of the pulvinar complex, and two less obvious topographical projection patterns were found within the caudomedial region, in zones that stain darkly for VGLUT2. The results, considered in relation to recent observations in tree shrews and squirrels, suggest that parts of the organizational scheme of the pulvinar complex in primates are present in rodents and other mammals.

Cerebral origins of the auditory projection to the superior colliculus of the cat

The superior colliculus (SC) is critical for directing accurate head and eye movements to visual and acoustic targets. In visual cortex, areas involved in orienting of the head and eyes to a visual stimulus have direct projections to the SC. In auditory cortex of the cat, four areas have been identified to be critical for the accurate orienting of the head and body to an acoustic stimulus. These areas include primary auditory cortex (A1), the posterior auditory field (PAF), the dorsal zone of auditory cortex (DZ), and the auditory field of the anterior ectosylvian sulcus (fAES). Therefore, we hypothesized that these four regions of auditory cortex would have direct projections to the SC. To test this hypothesis, deposits of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) were made into the superficial and deep layers of the SC to label, by means of retrograde transport, the auditory cortical origins of the corticotectal pathway. Bilateral examination of auditory cortex revealed that the vast majority of the labeled cells were located in the hemisphere ipsilateral to the SC injection. In ipsilateral auditory cortex, nearly all the labeled neurons were found in the infragranular layers, predominately in layer V. The largest population of labeled cells was located in the fAES. Few labeled neurons were identified in A1, PAF, or DZ. Thus, in contrast to the visual system, only one of the auditory cortical areas involved in orienting to an acoustic stimulus has a strong direct projection to the SC. Sound localization signals processed in primary (A1) and other non-primary (PAF and DZ) auditory cortices may be transmitted to the SC via a multi-synaptic corticotectal network.

Secretion-related structures of hypothalamo-hypophysial terminals in the rat posterior pituitary

Hypothalamic terminals were investigated in the rat posterior pituitary (PP). Injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) and co-injection of WGA-HRP with Rab3A-siRNA were made into the hypothalamus, respectively. Additional injection of WGA-HRP was made into the hypothalamus in the animals exposed to ethanol. These injections resulted in heavy labeling of fibers exclusively confined to the PP. Ultrastructural observations showed terminals, fibers, pituicytes, capillaries and vascular spaces in the PP. Although the majority of terminals were observed to contain large dense core vesicles (LDCVs) and HRP-reaction products (HRP-RPs), exocytosis of LDCVs in close proximity to cell membrane was not found. Interestingly, a few terminals showed alteration of cell membrane called "apocrine-like structure" containing LDCV and RP. The narrow neck portion of the structure gave the appearance that it may have been in some stage of separating from terminals. Other remarkable feature was that terminals occasionally reveal the structure of "leakage" of RP discharged into vascular spaces crossing cell membrane. Such hormone-releasing mechanism might be involved in one of "diacrine-like secretion". In the present study secretion-related structures of hypothalamic terminals in the PP are quite different from normal vesicular exocytosis.

A Neurobehavioral Analysis of the Prevention of Visual Impairment in the DBA/2J Mouse Model of Glaucoma

Timoptic-XE treatment was used to examine the relationship between age-related changes in intraocular pressure (IOP), retinal cell loss, visual ability, and neuronal labeling in the superior colliculus in the DBA/2J mouse model of pigmentary glaucoma. Mice were administered Timoptic-XE (0.0%, 0.25%, or 0.50%) daily from 9 weeks to 12 months of age. Visual ability and IOP were evaluated at 3, 6, 9, and 12 months of age. Mice from each group were then given intraocular injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), and estimates of the number of cells in the ganglion cell layer of the retina, WGA-HRP transneural labeling of cells, cell count, and cross-sectional area of Nissl-stained cells in the superior colliculus were obtained. Mice treated with 0.50% and 0.25% Timoptic-XE maintained a high level of performance in behavioral vision tasks, while 12-month-old untreated mice (0.0% Timoptic-XE) exhibited impaired visual performance. Timoptic-XE therapy reduced IOP and cell loss in the ganglion cell layer of the retina and prevented somal shrinkage and the decrease in WGA-HRP transneural labeling in the superior colliculus that occurred in untreated mice at 12 months of age. This study provides a comprehensive assessment of the efficacy of Timoptic-XE in DBA/2J mice by correlating age-related visual system changes in the retina and brain with changes in IOP and visual ability. These results showed that reducing IOP not only rescued retinal ganglion cell atrophy but also restored visual function and altered patterns of neurodegeneration that occur with blindness.

A Perioculomotor Nitridergic Population in the Macaque and Cat

We determined the distribution of cells containing synthetic enzymes for the unconventional neurotransmitter, nitric oxide, with respect to the known populations within the oculomotor complex. The oculomotor complex was investigated in monkeys and cats by use of histochemistry to demonstrate nicotinamide adenine dinucleotide phosphate diaphorase positive (NADPHd(+)) cells and antibodies to localize neuronal nitric oxide synthase positive (NOS(+)) cells. In some cases, wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) was injected into extraocular muscles to allow comparison of retrogradely labeled and NADPHd(+) cell distributions. The distribution of the NADPHd(+) and NOS(+) neurons did not coincide with that of preganglionic and extraocular motoneurons in the oculomotor complex. However, labeled perioculomotor neurons were observed. Specifically, in monkeys, they lay in an arc that extended from between the oculomotor nuclei into the supraoculomotor area (SOA). Comparison of WGA-HRP-labeled medial and superior rectus motoneurons with NADPHd staining confirmed that the distributions overlapped, but showed that the C- and S-group cells were not NADPHd(+). This suggested that NADPHd(+) cells are part of the centrally projecting Edinger-Westphal population (EWcp). Examination of the NADPHd(+) cell distribution in the cat showed that these cells were indeed found primarily within its well-defined EWcp. Based on their similar distributions, it appears that the peptidergic EWcp neurons, which project widely in the brain, also may be nitridergic. While the preganglionic and C- and S-group motoneuron populations do not use this nonsynaptic neurotransmitter, nitric oxide produced by surrounding NADPHd(+) cells may modulate the activity of these motoneurons.

Morphological Evidence of an Altered Process of Synaptic Transcytosis in Adult Rats Exposed to Ethanol

The effects of ethanol exposure on synaptic structure were investigated in the nucleus of solitary tract (NST) in rats, using the horse-radish peroxidase (HRP) method. Eight-week-old experimental rats were allowed free access to a liquid diet containing ethanol for 3 weeks, while controls were given an isocaloric diet. Some of the control and experimental animals were given an injection of wheat germ agglutinin conjugated with HRP (WGA-HRP) into the vagus nerve toward the end of the treatment period. After the treatment, the neuropil region of the NST was examined under an electron microscope. We observed that a few terminals were characterized by deep indentation of axodendritic membranes into the post-synaptic neurons. This appeared to be similar to that commonly seen in exocrine glands. Interestingly, the indented portion often contained various sizes of vacuoles and flattened cisternae. HRP-reaction product (RP) transported to terminals was recognized easily as an electron-dense lysosomal substance when lead citrate staining was omitted. Terminals containing HRP-RP also revealed quite a similar structure with indentation of axodendritic membranes as described earlier. The results are considered to confirm that terminals forming 'apocrine-like structures' observed in the ethanol-fed animals with no injection of WGA-HRP originate from afferent fibers of the vagus nerve. The present study suggests the possibility that the alteration of the synaptic structure induced by ethanol exposure can lead to the neuronal transcytosis of materials including proteins which is different from the normal vesicular exocytosis involved in chemical synaptic transmission.

Increased transvascular transport of WGA-peroxidase after chronic perinatal stress in the hippocampal microvasculature of the rat

Brain endothelial ultrastructural properties contribute to maintain proper blood–brain barrier (BBB) function. Several physiological and pathological conditions have been shown to alter BBB permeability to blood-borne molecules, acute and chronic stress among them. In the rat, early life stress increased transvascular transport of Evans blue, however, the route of tracer extravasation is not fully known; therefore the aim of the present experiment was to describe the ultrastructural changes in endothelial cells subsequent to chronic perinatal stress in order to ascertain the route for transvascular transport of an electrodense tracer. Pregnant Wistar rats and their litters were used. Four pregnant rats were subjected to forced swimming between gestational days 10 to 20. After delivery, half of the control litters underwent 180 min maternal separation from postnatal day 2 to 20. Controls were kept free of any stress manipulation. At sacrifice between postnatal days 1 to 30 subjects were given intracardially the lectin wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). WGA-HRP stained hippocampi were processed for ultrastructural analysis, transmission electron micrographs were obtained and endothelial ultrastructural parameters quantified using the ImageJ software. Both stress procedures accelerated gross microvessel development by decreasing capillary wall thickness and endothelial microvilli. However, early-life stress also neutralized endothelial glycocalyx, increased vesicle-mediated transport and tended to promote the formation of secondary lysosomes containing endocytosed WGA-HRP vesicles, all parameters of altered endothelial cell function. Tight junction development in both stress groups was similar to the control pups.

RA-GEF-1 (Rapgef2) is essential for proper development of the midline commissures

The cerebral hemispheres are directly connected by three major interhemispheric fibers: the corpus callosum, the anterior commissure, and the hippocampal commissure. RA-GEF-1 (also termed Rapgef2) is a guanine nucleotide exchange factor responsible for sustained activation of Rap1. We previously reported anatomical defects of the major forebrain commissures in the adult dorsal telencephalon-specific RA-GEF-1 conditional knockout (cKO) mice. In this study, we use neuroanatomical tracing and immunohistochemistry to study the formation of the commissural fibers during early postnatal development. DiI anterograde tracing reveals the inability of the callosal axons to cross the midline in cKO mice, thereby forming Probst bundles on the ipsilateral side, which is associated with the absence of the indusium griseum glia and the glial sling at the cortical midline. Wheat germ agglutinin-conjugated horseradish peroxidase retrograde tracing verifies the agenesis of the anterior commissure in cKO mice, and DiI anterograde tracing confirms the deviation of the fibers from their original tract. As for the hippocampal commissure, agenesis and hypoplasia are observed in its dorsal and ventral parts, respectively. These results indicate the essential role of RA-GEF-1 in the proper formation of the cerebral midline commissures.

A possible new relay for tongue thermal sense in the dorsal margin of the trigeminal principal nucleus in rats

Neurons responding to innocuous thermal stimulation of the anterior tongue were newly identified in the dorsal margin of the trigeminal principal nucleus (PV). Connections of the dorsal margin of the PV were neuroanatomically identified using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) with electrophysiological techniques. Injection of WGA-HRP into the dorsal margin of the PV demonstrated anterograde labels distributed from the most dorsomedial portion of the posteromedial ventral nucleus (VPM) to the dorsolateral portion of the parvicellular part of the VPM and retrograde labels in the superficial layers of the caudal subnucleus of the spinal trigeminal nucleus (SpVc). Injection of WGA-HRP in the most dorsomedial portion of the VPM demonstrated retrogradely labeled cells in the dorsal margin of the PV as well as in the superficial layers of the SpVc. Tracer injection into the superficial layers of the SpVc resulted in anterograde labels in the dorsal margin of the PV as well as in the dorsomedial portion of the VPM. These findings show that neurons in the PV respond to innocuous thermal stimulation of the trigeminal field and suggest that the dorsal margin of the PV is a possible new relay for the tongue thermal sense, receiving thermal information from the superficial layers of the SpVc, the primary thermal relay for the trigeminal field, and passing the information to the thalamic relay.

Alteration of the Expression Levels of Rab3A Affects the Extent of Transcytosis of HRP-Labeled Marker Proteins in Rat CNS Neurons

It has been hypothesized that Rab3A, a small GTPase, may be closely involved in the process of dense core vesicle exocytosis in various cell types. This possibility was investigated by disrupting the expression levels of Rab3A-mRNA using a small interfering RNA of the Rab3A GTPase (Rab3A-siRNA) and examining the effect of this on transcytosis of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP). Rab3A-siRNA and WGA-HRP were injected into the right vagus nerves of adult rats which were killed 12, 24 or 48 hours later. In some animals, portions of the brain stem containing the nucleus of solitary tract (NST) were prepared for electron microscopy. In other animals, the nodose ganglion of the vagus nerve was used to determine the levels of expression of Rab3A-mRNA using RT-PCR techniques. It was found that the expression of Rab3A-mRNA was markedly depressed in animals at 12 h after the Rab3A-siRNA injection. In the NST, there was an accumulation of HRP-reaction product (RP), recognized as electron dense lysosomal-like structures, in both axons and terminals in the NST 12 h after injection. Some HRP-RP was found in membrane bound vesicles in close proximity to cell membranes and appeared to be in the process of transcytosis. This neuronal transcytosis of HRP-RP appeared to occur at random locations over the axodendritic membranes. These findings indicate that inhibiting the expression of Rab3A-mRNA using Rab3A-siRNA can modulate the level of transcytosis of proteins across neuronal membranes confirming the potentially important role of this GTPase in the process of transcytosis.