Wheat Germ Agglutinin-horseradish Peroxidase Research Articles

Activated Oxytocin Neurons in the PVN-DVC Pathway in Asthmatic Rats.

Asthma is a heterogeneous disease, and the central nervous system (CNS) also participates in the pathogenesis of asthma. We previously reported the amygdala might regulate asthmatic attacks via projecting to the paraventricular hypothalamic nucleus (PVN). The dorsal vagal complex (DVC) is a crucial region that modulates respiratory. This study aimed to observe the activity in both PVN and DVC and the connection between PVN and DVC in asthmatic rats. Immunohistochemistry was conducted to observe the changes in Fos and oxytocin (OT) expression. Retrograde tracing using wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and double immunohistochemistry for OT and Fos was used to observe the HRP/OT/Fos positive neurons distribution in the PVN. The results showed that during an asthma attack, the Fos positive neurons increased in both PVN and DVC over time. The expression of OT positive neurons in PVN showed a similar trend in parallel to the c-Fos positive neurons in PVN. The HRP retrograde-labeled neurons were densely distributed in the medial and lateral subnucleus in the PVN. OT+/HRP+ and Fos+/OT+/HRP+ accounted for 18.14%, and 2.37% of HRP-labeled neurons, respectively. Our study showed PVN and DVC were activated and the expression of OT positive neurons in PVN were increased over time during an asthma attack. The existence of connection between PVN and DVC suggested the OT neurons in PVN might project to DVC which might be involved in the pathogenesis of asthma.

The amygdala via the paraventricular nucleus regulates asthma attack in rats

AimsThis study aimed to investigate the functions of the amygdala in rat asthma model.Main methodsWheat germ agglutinin‐horseradish peroxidase (WGA‐HRP) was used for tracing from the paraventricular nucleus (PVN) to the amygdala, and nuclear lesions were performed to observe changes in respiratory function and airway inflammation.ResultsThis study showed that the extracellular neuronal discharged in the medial amygdala (MeA) and central amygdala (CeA), and the expression of Fos significantly increased in asthmatic rat compared to control group. The distribution of Fos‐ and oxytocin (OT)‐positive neurons and Fos/OT dual‐positive neurons evidently increased in the PVN. WGA‐HRP was injected into the PVN for tracing, and Fos/HRP‐dual‐positive neurons were observed to be distributed in the MeA. By using kainic acid (KA) to injure the MeA and CeA in asthmatic rats, expiratory and inspiratory times (TE/TI) and airway resistance (Raw) decreased, and minute ventilation volume (MVV) and dynamic pulmonary compliance (Cdyn) increased accordingly. In the bronchoalveolar lavage fluid (BALF), the number of eosinophils and the concentration of IL‐4 were lower than those of the control group, and the ratio of Th1/Th2 cells was higher than that of the control group. In the PVN, the distribution of Fos‐, OT‐positive cells and Fos/OT double‐positive cells decreased compared with those of the control group. The activities of the MeA and CeA and of OT neurons in the PVN of the rats were correlated with the occurrence of asthma.ConclusionsAsthma attack could induce neural activities in the MeA and CeA, and OT neurons in the PVN may be involved in the process of asthma attack.

Vagal baro- and chemoreceptors in middle internal carotid artery and carotid body in rat.

The glossopharyngeal nerve, via the carotid sinus nerve (CSN), presents baroreceptors from the internal carotid artery (ICA) and chemoreceptors from the carotid body. Although neurons in the nodose ganglion were labelled after injecting tracer into the carotid body, the vagal pathway to these baro- and chemoreceptors has not been identified. Neither has the glossopharyngeal intracranial afferent/sensory pathway that connects to the brainstem been defined. We investigated both of these issues in male Sprague-Dawley rats (n=40) by injecting neural tracer wheat germ agglutinin-horseradish peroxidase into: (i) the peripheral glossopharyngeal or vagal nerve trunk with or without the intracranial glossopharyngeal rootlet being rhizotomized; or (ii) the nucleus of the solitary tract right after dorsal and ventral intracranial glossopharyngeal rootlets were dissected. By examining whole-mount tissues and brainstem sections, we verified that only the most rostral rootlet connects to the glossopharyngeal nerve and usually four caudal rootlets connect to the vagus nerve. Furthermore, vagal branches may: (i) join the CSN originating from the pharyngeal nerve base, caudal nodose ganglion, and rostral or caudal superior laryngeal nerve; or (ii) connect directly to nerve endings in the middle segment of the ICA or to chemoreceptors in the carotid body. The aortic depressor nerve always presents and bifurcates from either the rostral or the caudal part of the superior laryngeal nerve. The vagus nerve seemingly provides redundant carotid baro- and chemoreceptors to work with the glossopharyngeal nerve. These innervations confer more extensive roles on the vagus nerve in regulating body energy that is supplied by the cardiovascular, pulmonary and digestive systems.

Superior Cervical Ganglion: Axonal Passage and Inputs

Axons of the rostral middle (MCG) and inferior (ICG) cervical ganglion were reported to bypass the superior cervical ganglion (SCG). However, the distribution of these neurons has not been determined. Meanwhile, it has been inconsistent with the distribution of the preganglionic neurons of the SCG. To examine the above issues, we recently injected wheat germ agglutinin- horseradish peroxidase (WGA-HRP) into the entire or partial (rostral or caudal) SCG of the rat (Anatomical Record, 301:1906-1916, 2018). By preparing complete whole-mounts of the sympathetic tissues and sections of the brainstem and spinal cord, we labeled neurons that are distributed evenly in the MCG (left: 258 ＞ right: 121) ICG (left: 848, right: 681) and CST (up to 770). It is found specifically a novel cervical ganglion and termed it as the ＂pre-middle cervical ganglion＂ (pMCG) that is located rostral to the MSG and sends axons (left: 120 ＞ right: 82) to bypass the SCG. In addition to the preganglionic neurons in the cervical (C1-C4) and thoracic (T1-T4) segments, we also labeled neurons (233) that extend dendrites into the vestibular nuclear complex and locate in the intermediate reticular nuclei (96%) and perifacial zone (4%) of the brainstem ranging from Bregma -10.0 to -11.0 mm, mainly in -10.80 mm. With tracer injection into the rostral or caudal SCG, we labeled neurons correspondingly in the brainstem, C1-C2 or T1-T2. These data indicated that many neurons in pMCG, MCG and ICG run rostrally within the CST to project to their peripheral targets rather than segmentally via the closest rami. For their close anatomical location, neurons in pMCG and MCG may have similar or complementary function. Numerous neurons in the CST and caudal cervical ganglia provide bypass axons via SCG may imply special function that is useful for electroceuticals. The newly identified preganglionic neurons located in the rostral brainstem may be involved in the vestibulo-autonomic interaction for their extending dendrites into the vestibular nuclear complex that has been known to form an interaction pathway with the vagal complex.

Axons of Passage and Inputs to Superior Cervical Ganglion in Rat.

Wheat germ agglutinin-horseradish peroxidase was injected into the entire (0.8 μL) or partial (rostral or caudal, 0.1-0.3 μL) superior cervical ganglion (SCG) of the rat (male Sprague-Dawley, N = 35) to examine the distribution of neurons in the middle (MCG) and inferior (ICG) cervical ganglion that send axons bypass the SCG. Whole-mounts of the SCG, cervical sympathetic trunk (CST), MCG, ICG, and sections of the brainstem and spinal cord were prepared. With entire SCG tracer injection, neurons were labeled evenly in the MCG (left: 258, right: 121), ICG (left: 848, right: 681), and CST (up to 770). Some neurons grouped in a single bulge just rostral to the MCG, which we termed as the "premiddle cervical ganglion" (pMCG). The left pMCG (120) is larger and has more neurons than the right pMCG (82). Centrally, neurons were labeled in lamina IX of cervical segments (C1: 18%, C2: 46%, C3: 33%, C4: 3%), intermediate zone of thoracic segments (T1: 31%, T2: 35%, T3: 27%, T4: 7%), and intermediate reticular nuclei (96%) and perifacial zone (4%) of brainstem. The rostral and caudal SCG injection selectively labeled neurons mainly in brainstem, C1-C2 and in T1-T2, respectively. Before projecting to their peripheral targets, many neurons in pMCG, MCG and ICG run rostrally within the CST rather than segmentally through the closest rami, from the level of SCG or above. Neurons in pMCG and MCG may have similar or complementary function and those in brainstem may be involved in the vestibulo-autonomic interaction. Anat Rec, 301:1906-1916, 2018. © 2018 Wiley Periodicals, Inc.

Cerebellar projections to the ventral lateral geniculate nucleus and the thalamic reticular nucleus in the cat.

The ventral lateral geniculate nucleus (LGNv) is a retinorecipient part of the ventral thalamus and in cats, it consists of medial (M), medial intermediate (IM), lateral intermediate (IL), lateral (L), and dorsal (D) subdivisions. These subdivisions can be differentiated not only by their cytoarchitecture, but also by their connectivity and putative functions. The LGNv may play a role in visuomotor gating, in that there is evidence of cerebellar afferent projections to the intermediate subdivisions. The cerebellar posterior interpositus (IP) and lateral (LC) nuclei are known to project to IM and IL, but the specifics of these projections are unclear. We hypothesized that the IP and LC project differentially to IM and IL. To evaluate LGNv innervation by the deep cerebellar nuclei, we injected the tract-tracer wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into several different regions of the LGNv and cerebellar nuclei of adult cats in either sex. Small injections into the middle and posterior LGNv retrogradely labeled cells in the ventral part of the IP. However, injections in the anterior regions of the LGNv, with or without diffusion into the thalamic reticular nucleus (Re), retrogradely labeled cells in the ventral part of both the IP and the LC. Confirmatory injections into the IP and LC produced terminal-like labeling distributed in IM, IL, and Re; injections mostly localized to the LC resulted in labeling mainly in IM and Re. We concluded that the IP projects to IL whereas the LC projects to IM and Re.

Central projections and connections of lumbar primary afferent fibers in adult rats: effectively revealed using Texas red-dextran amine tracing

Signals from lumbar primary afferent fibers are important for modulating locomotion of the hind-limbs. However, silver impregnation techniques, autoradiography, wheat germ agglutinin-horseradish peroxidase and cholera toxin B subunit-horseradish peroxidase cannot image the central projections and connections of the dorsal root in detail. Thus, we injected 3-kDa Texas red-dextran amine into the proximal trunks of L4 dorsal roots in adult rats. Confocal microscopy results revealed that numerous labeled arborizations and varicosities extended to the dorsal horn from T12-S4, to Clarke's column from T10-L2, and to the ventral horn from L1-5. The labeled varicosities at the L4 cord level were very dense, particularly in laminae I-III, and the density decreased gradually in more rostral and caudal segments. In addition, they were predominately distributed in laminae I-IV, moderately in laminae V-VII and sparsely in laminae VIII-X. Furthermore, direct contacts of lumbar afferent fibers with propriospinal neurons were widespread in gray matter. In conclusion, the projection and connection patterns of L4 afferents were illustrated in detail by Texas red-dextran amine-dorsal root tracing.

Vagal nerve endings in visceral pleura and triangular ligaments of the rat lung.

The inner thoracic cavity is lined by the parietal pleura, and the lung lobes are covered by the visceral pleura. The parietal and visceral plurae form the pleural cavity that has negative pressure within to enable normal respiration. The lung tissues are bilaterally innervated by vagal and spinal nerves, including sensory and motor components. This complicated innervation pattern has made it difficult to discern the vagal vs. spinal processes in the pulmonary visceral pleura. With and without vagotomy, we identified vagal nerve fibres and endings distributed extensively in the visceral pleura ('P'-type nerve endings) and triangular ligaments ('L'-type nerve endings) by injecting wheat germ agglutinin-horseradish peroxidase as a tracer into the nucleus of solitary tract or nodose ganglion of male Sprague-Dawley rats. We found the hilar and non-hilar vagal pulmonary pleural innervation pathways. In the hilar pathway, vagal sub-branches enter the hilum and follow the pleural sheet to give off the terminal arborizations. In the non-hilar pathway, vagal sub-branches run caudally along the oesophagus and either directly enter the ventral-middle-mediastinal left lobe or follow the triangular ligaments to enter the left and inferior lobe. Both vagi innervate: (i) the superior, middle and accessory lobes on the ventral surfaces that face the heart; (ii) the dorsal-rostral superior lobe; (iii) the dorsal-caudal left lobe; and (iv) the left triangular ligament. Innervated only by the left vagus is: (i) the ventral-rostral and dorsal-rostral left lobe via the hilar pathway; (ii) the ventral-middle-mediastinal left lobe and the dorsal accessory lobe that face the left lobe via the non-hilar pathway; and (iii) the ventral-rostral inferior lobe that faces the heart. Innervated only by the right vagus, via the non-hilar pathway, is: (i) the inferior (ventral and dorsal) and left (ventral only) lobe in the area near the triangular ligament; (ii) the dorsal-middle-mediastinal left lobe; and (iii) the right triangular ligament. Other regions innervated with unknown vagal pathways include: (i) the middle lobe that faces the superior and inferior lobe; (ii) the rostral-mediastinal inferior lobe that faces the middle lobe; and (iii) the ventral accessory lobe that faces the diaphragm. Our study demonstrated that most areas that face the dorsal thoracic cavity have no vagal innervation, whereas the interlobar and heart-facing areas are bilaterally or unilaterally innervated with a left-rostral vs. right-caudal lateralized innervation pattern. This innervation pattern may account for the fact that the respiratory regulation in rats has a lateralized right-side dominant pattern.

Transporter protein and drug-conjugated gold nanoparticles capable of bypassing the blood-brain barrier.

Drug delivery to the central nervous system (CNS) is challenging due to the inability of many drugs to cross the blood-brain barrier (BBB). Here, we show that wheat germ agglutinin horse radish peroxidase (WGA-HRP) chemically conjugated to gold nanoparticles (AuNPs) can be transported to the spinal cord and brainstem following intramuscular injection into the diaphragm of rats. We synthesized and determined the size and chemical composition of a three-part nanoconjugate consisting of WGA-HRP, AuNPs, and drugs for the treatment of diaphragm paralysis associated with high cervical spinal cord injury (SCI). Upon injection into the diaphragm muscle of rats, we show that the nanoconjugate is capable of delivering the drug at a much lower dose than the unconjugated drug injected systemically to effectively induce respiratory recovery in rats following SCI. This study not only demonstrates a promising strategy to deliver drugs to the CNS bypassing the BBB but also contributes a potential nanotherapy for the treatment of respiratory muscle paralysis resulted from cervical SCI.

Characterization of glutamatergic neurons in the rat atrial intrinsic cardiac ganglia that project to the cardiac ventricular wall

The intrinsic cardiac nervous system modulates cardiac function by acting as an integration site for regulating autonomic efferent cardiac output. This intrinsic system is proposed to be composed of a short cardio-cardiac feedback control loop within the cardiac innervation hierarchy. For example, electrophysiological studies have postulated the presence of sensory neurons in intrinsic cardiac ganglia (ICG) for regional cardiac control. There is still a knowledge gap, however, about the anatomical location and neurochemical phenotype of sensory neurons inside ICG. In the present study, rat ICG neurons were characterized neurochemically with immunohistochemistry using glutamatergic markers: vesicular glutamate transporters 1 and 2 (VGLUT1; VGLUT2), and glutaminase (GLS), the enzyme essential for glutamate production. Glutamatergic neurons (VGLUT1/VGLUT2/GLS) in the ICG that have axons to the ventricles were identified by retrograde tracing of wheat germ agglutinin–horseradish peroxidase (WGA–HRP) injected in the ventricular wall. Co-labeling of VGLUT1, VGLUT2, and GLS with the vesicular acetylcholine transporter (VAChT) was used to evaluate the relationship between post-ganglionic autonomic neurons and glutamatergic neurons. Sequential labeling of VGLUT1 and VGLUT2 in adjacent tissue sections was used to evaluate the co-localization of VGLUT1 and VGLUT2 in ICG neurons. Our studies yielded the following results: (1) ICG contain glutamatergic neurons with GLS for glutamate production and VGLUT1 and 2 for transport of glutamate into synaptic vesicles; (2) atrial ICG contain neurons that project to ventricle walls and these neurons are glutamatergic; (3) many glutamatergic ICG neurons also were cholinergic, expressing VAChT; (4) VGLUT1 and VGLUT2 co-localization occurred in ICG neurons with variation of their protein expression level. Investigation of both glutamatergic and cholinergic ICG neurons could help in better understanding the function of the intrinsic cardiac nervous system.

Reconstruction of atonic bladder innervation after spinal cord injury: A bladder reflex arc with afferent and efferent pathways

BackgroundEstablishing bladder reflex arcs only with the efferent pathway to induce micturition after spinal cord injury (SCI) has been successful. However, the absence of sensory function and micturition desires can lead to serious complications.ObjectivesTo reconstruct a bladder reflex arc with both afferent and efferent pathways to achieve atonic bladder innervation after SCI.MethodsA reflex arc was established by microanastomosis of the S2 dorsal root to the peripheral process of the L5 dorsal ganglion and the L5 ventral root to the S2 ventral root. The functions of the reflex arc were evaluated using electrophysiology, wheat germ agglutinin–horseradish peroxidase (WGA–HRP) tracing, and calcitonin gene-related peptide (CGRP) immunocytochemistry analysis. Hind-paw motion was evaluated by CatWalk gait.ResultsCompound action potentials and compound muscle action potentials were recorded at the right L5 dorsal root following electrical stimulation of right S2 dorsal root. Similar to the control side, these were not significantly different before or after the spinal cord destruction between L6 and S4. WGA–HRP tracing and CGRP immunocytochemistry showed that construction of the afferent and efferent pathways of the bladder reflex arc encouraged axonal regeneration of motor and sensory nerves, which then made contact with the anterior and posterior horns of the spinal cord, ultimately reestablishing axoplasmic transportation. Gait analysis showed that at 3 months following the operation, only the regularity index was significantly different as compared with 1 day before the operation, other parameters showing no difference.ConclusionBladder reflex arc with the afferent and efferent pathways reconstructs the micturition function without great influence on the motion of leg.

Topographic distribution of cortical projection cells in the rat subiculum

The topographic distribution of subicular pyramidal cells, which give rise to projections to the entorhinal cortex, presubiculum, parasubiculum, and the retrosplenial granular cortex, was investigated in the rat using retrograde labeling with wheat germ agglutinin-horseradish peroxidase. Using two-dimensional unfolded maps of the entire hippocampal and parahippocampal fields, we found that the cells originating the projections to the above cortical areas were consistently observed throughout the entire septotemporal extent of the subiculum. In the transverse plane, most of the cortical projection cells were vertically located in the middle region of the subicular pyramidal cell layer. The cells giving rise to the projections to the lateral entorhinal cortex were predominantly located in the most proximal (near CA1), superficial region. Few cortical projection cells were located in the deepest (adjacent to the angular bundle) region. The distribution of cortical projection cells showed an oblique tri-laminar pattern, which was similar to the previously reported laminal pattern of subcortical projection cells in the subiculum. These results suggest that cortical projection cells in middle and superficial regions of the subiculum may correspond to layer V of the isocortex and cells in the deepest region corresponding to layer VI.

Metabotropic glutamate receptor 5 shows different patterns of localization within the parallel visual pathways in macaque and squirrel monkeys.

Glutamate is used as an excitatory neurotransmitter by the koniocellular (K), magnocellular (M), and parvocellular (P) pathways to transfer signals from the primate lateral geniculate nucleus (LGN) to primary visual cortex (V1). Glutamate acts through both fast ionotropic receptors, which appear to carry the main sensory message, and slower, modulatory metabotropic receptors (mGluRs). In this study, we asked whether mGluR5 relates in distinct ways to the K, M, and P LGN axons in V1. To answer this question, we used light microscopic immunocytochemistry and preembedding electron microscopic immunogold labeling to determine the localization of mGluR5 within the layers of V1 in relation to the K, M, and P pathways in macaque and squirrel monkeys. These pathways were labeled separately via wheat germ agglutinin–horseradish peroxidase (WGA–HRP) injections targeting the LGN layers. mGluR5 is of interest because it: 1) has been shown to be expressed in the thalamic input layers; 2) appears to be responsible for some types of oscillatory firing, which could be important in the binding of visual features; and 3) has been associated with a number of sensory-motor gating-related pathologies, including schizophrenia and autism. Our results demonstrated the presence of mGluR5 in the neuropil of all V1 layers. This protein was lowest in IVCα (M input) and the infragranular layers. In layer IVC, mGluR5 also was found postsynaptic to about 30% of labeled axons, but the distribution was uneven, such that postsynaptic mGluR5 label tended to occur opposite smaller (presumed P), and not larger (presumed M) axon terminals. Only in the K pathway in layer IIIB, however, was mGluR5 always found in the axon terminals themselves. The presence of mGluR5 in K axons and not in M and P axons, and the presence of mGluR5 postsynaptic mainly to smaller P and not larger M axons suggest that the response to the release of glutamate is modulated in distinct ways within and between the parallel visual pathways of primates.

Somatotopic Organization of Trigeminal Ganglion

Clearing the somatotopic organization of trigeminal ganglion can help us to improve the precision of treatment for trigeminal neuralgia. The distribution of primary afferent perikarya of 3 branches of trigeminal nerve in the trigeminal ganglion was investigated in the rabbit, and 3D model was reconstructed then. After application of wheat germ agglutinin-horseradish peroxidase and DiI to the cut endings of the 3 branches of trigeminal nerve, ophthalmic cells were found in the anteromedial part of the trigeminal ganglion, mandibular cells in the posterolateral part, and maxillary cells in the middle part. The results suggest that the somatotopic organization of the ganglion in rabbits is a mediolateral direction reflecting the mediolateral order of the ophthalmic, maxillary, and mandibular nerves.

Cytoarchitecture of the olfactory bulb in the laggard mutant mouse

The laggard (lag) mutant mouse, characterized by hypomyelination and cerebellar ataxia, is a spontaneously occurring mutant mouse caused by mutation in the Kif14 gene. In this mutant mouse, the laminated structures such as the cerebral and cerebellar cortices and the dentate gyrus are cytoarchitecturally abnormal. Macroscopically, the olfactory bulb of the lag mutant mouse is smaller in size and more transparent than the normal counterpart. Hematoxylin–eosin staining reveals that the mutant olfactory bulb has normal lamination in general, but detailed analysis has demonstrated that olfactory periglomerular cells and granule cells are reduced in number. In the mutant, olfactory glomeruli are cytoarchitecturally disorganized and mitral cells are arranged in multiple cell layers instead of being arranged in a single layer. The rostral migratory stream in the mutant becomes gradually thinner or obliterated during early postnatal days. Some of mitral cells and periglomerular cells are multinucleated, suggesting that Kif14 mutation leads to an abnormal cell division. In the mutant, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the subventricular zone of the lateral ventricle are increased in number, especially at perinatal age, suggesting that the decreased population of granule cells in the lag mutant mouse is caused by the increased apoptotic cell death. The olfactory input appears to be intact, as indicated by anterograde labeling of olfactory nerves with an injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the olfactory mucosa. In conclusion, the olfactory bulb of the lag mutant mouse is cytoarchitecturally affected, suggesting that the causal gene for lag mutation, i.e., Kif14, has multiple effects on the development of laminated structures in the central nervous system in addition to the myelin formation.

Vesicular glutamate transporter 1‐immunoreactive sensory neurons in the rat intrinsic cardiac ganglia

Sensory neurons that reside in intrinsic cardiac ganglia (ICG) have an important role in monitoring the cardiac milieu for regional cardiac dynamic control. We recently described sensory neurons in ICG that project to the cardiac ventricular wall and currently are evaluating their neurochemical phenotype. Since many primary sensory neurons are glutamatergic, we hypothesized that ICG sensory neurons are glutamatergic, utilizing vesicular glutamate transporter 1 (VGLUT1) to package glutamate into synaptic vesicles. In the present study, the rat ventricle wall was injected (retro‐diaphragmatic, 8μl) with the retrograde tracer wheat germ agglutinin‐horseradish peroxidase (WGA‐HRP). After 72h, rats were euthanized and transcardially perfused with fixative. ICG were processed for immunohistochemistry studies. Our results demonstrated that: (1) VGLUT1‐immunoreactive (ir) neurons were present in ICG; (2) Some VGLUT1‐ir neurons were labeled from the ventricles with WGA‐HRP. Our previous studies indicated that sensory ICG neurons contain glutaminase, the synthetic enzyme for glutamate. These and the current results indicate that ICG sensory neurons are glutamatergic in neurochemical phenotype. Further investigation of VGLUTs during cardiac physiological and pathological conditions will help in deciphering the function of glutamate as a neurotransmitter in the ICG. Supported by NIH AR47410 (KEM).Grant Funding Source: NIH AR47410

The brainstem localization of gastric preganglionic parasympathetic neurons in the agouti (Dasyprocta leporina).

This study was designed to determine qualitatively, the source of gastric vagal nerve fibres in the Agouti. A total of 18 male and female adult agoutis were used for the present investigation. Following anaesthesia, laparotomy was performed and the stomach exteriorized. Multiple intramuscular injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were then made into different areas of the stomach in the experimental animals. The control animals were divided into four groups of two animals each. The first group had intraperitoneal injection of the tracer, the second had intramuscular injection of normal saline, the third group had injection of tracer into the hepatic portal vein and the last group had injection of the tracer into the gastric walls followed immediately by bilateral vagotomy. Following a survival period of five to seven days, the animals were sacrificed by transcardial perfusion, first with normal saline followed by fixative and finally with 20% buffered sucrose. Following perfusion, the brainstem was extracted from the brain, immersed in 20% buffered sucrose and kept refrigerated overnight for cryoprotection. The brainstems were subsequently sectioned serially, processed for WGA-HRP neurohistochemistry and then analysed under light and dark-field illuminations. The analysis of the sections taken from the experimental animals revealed bilateral presence of WGA-HRP labelled neurons in the dorsal motor nucleus of the vagus nerve (DMNV) and the nucleus ambiguus (nA) of the medulla oblongata. No labelled neurons were seen in any of the sections taken from the control animals. The implications of the findings are discussed.

An experimental study on effect of bladder reflex arc with sensory afferent pathway in the treatment of atonic bladder after spinal cord injury

Objective To establish bladder reflex arc with sensory afferent pathway using SD rats,and evaluate its effect in the treatment of atonic bladder after spinal cord injury.Methods Twenty-four male SD rats were used in the study.For each rat,the right side was the experimental side,and the left side was the control side.In the right side,the L5 ventral root (VR) was anastomosed to S2 VR,and the distal end of S2 dorsal root (DR) was anastomosed to the proximal end of L5 peripheral process of dorsal ganglion.In the left side,no treatment was done.In order to evaluate the validity of the bladder reflex arc,general observation,neuro-electrophysiological test and wheat germ agglutinin horseradish peroxidase (WGA-HRP) method were used before and after the spinal cord destruction between L6 and S2 level at 3 months postoperatively.Results Twenty-one rats survived 3 months after the operation,and anastomotic nerves were separated successfully only in seven rats.Compound action potentials (CAPs) of plexus vesica and compound muscle action potentials (CMAPs) of bladder smooth muscle were found by electrical stimulation in distal end of the anastomotic stoma of the right S2 DR.There was no statistically significant difference in action potential before and after paraplegia.No action potential was detected in control sides after paraplegia.The curves of CAPs and CMAPs in the right side were similar to those in the control side,and the mean maximum amplitude reached respectively 71.9％ and 82.4％ of those in the left side before paraplegia.In addition,WGAHRP labeled cells were observed in L5 anterior horn and posterior horn in the experimental side after WGAHRP injection.Conclusion Reconstruction of bladder reflex arc with sensory afferent pathway can promote axonal regeneration of motor and sensory nerves,and then the regenerated axon could contact with cells in anterior and posterior horn of spinal cord through parasympathetic nerves,ultimately the capability of axoplasmic transportation could be reestablished.Therefore,this method can be used for treating atonic bladder. Key words: Spinal cord injuries; Urinary bladder; Afferent pathways

Abdominal vagal afferent pathways and their distributions of intraganglionic laminar endings in the rat duodenum

We examined abdominal vagal afferents (n = 33) and the distributions of their intraganglionic laminar endings (IGLEs) in the duodenum. Rats (male, Sprague-Dawley) received a partial subdiaphragmatic vagotomy that spared a single branch. Wheat germ agglutinin-horseradish peroxidase (0.5-1.0 μl) was injected into the nodose ganglion ipsilateral to the vagotomized side. We observed that the hepatic branch does not project to the stomach, that the accessory celiac and celiac branches course along the celiac artery and innervate the intestines, and that the left nodose afferents innervate predominantly the duodenum. The hepatic branch innervates the duodenum via the "hepatoduodenal" subbranch and has the densest IGLE distribution in both the dorsoventral and the rostrocaudal extensions of the first 4-cm segment. Both gastric branches have two subbranches that innervate the duodenum; the "lesser curvature" subbranches follow the lesser curvature artery and may join the "hepatoduodenal" subbranch, whereas the "pyloric" subbranches run through the antrum and pylorus to reach the proximal duodenum. Moreover, the subbranches of ventral and dorsal gastric branches innervate more in the ventral and dorsal parts of the duodenum, respectively, and have more IGLEs in the rostral region than in the caudal. A posteriori comparisons indicate that, in the first-centimeter segment, the ventral gastric branch has significantly more IGLEs, whereas, in the third- and fourth-centimeter segments, the hepatic branch has more IGLEs. The finding that three different vagal branches innervate the duodenum with different densities of afferent endings might indicate a viscerotopic receptive field that coordinates digestive functions in feeding.

Another Component of the Pelvic Plexus That Innervates the Penis in the Rat

To determine whether the small plexus located at the base of the penis, consisting of nerves arising from the major pelvic ganglion, connecting with the sensory branch of the pudendal nerve, contains postganglionic neurons innervating the erectile tissue and urethra of the penis. A total of 22 adult male Wistar rats 3-4 months of age (weight 250-350 g) were used. The features, distribution, and topography of the small plexus were determined by gross anatomy and histologic studies (Masson's trichrome and Nissl stain). Postganglionic neurons were traced with horseradish peroxidase-wheat germ agglutinin injected in the cavernosum, spongiosum corpora, and penile urethra. The small plexus located at the base of the penis has ganglia containing cells with large nuclei. The cells were stained with the Nissl technique confirming they are neurons. The neurons were positively labeled with horseradish peroxidase-wheat germ agglutinin after the tracer was injected in the penile structures. In the male rat, the small plexus located at the base of the penis contains postganglionic neurons innervating the penile structures. These neurons can contribute to the regulation of the sexual and urinary function of the genitourinary tract. Because the rat is the main laboratory animal used in urologic studies, it is important to consider, when designing experiments or discussing results, that in addition to the major pelvic ganglion, other postganglionic neurons of the penis are provided by the small plexus. The small plexus could be named the minor pelvic ganglia and considered a component of the pelvic plexus.