Transport Of Horseradish Peroxidase Research Articles

Stepwise Optimization of Inducible Expression System for the Functional Secretion of Horseradish Peroxidase in Saccharomyces cerevisiae.

Horseradish peroxidase (HRP) is a plant-derived glycoprotein that can be developed as a food additive to cross-link proteins or biopolymers. Although Saccharomyces cerevisiae has advantages in the production of food-grade HRP, the low expressional level and inefficient secretion hindered its application values. After comparing the effects of constitutive and inducible expression on cell growth, the strength of HRP expression was roughly tuned by replacing core regions of the promoter in the GAL80-knockout strain and further finely tuned by terminator screening. Additionally, the most suitable signal peptide was selected, and the pre-peptide with pro-peptides was modified to balance the transport of HRP in the endoplasmic reticulum. The extracellular HRP activity of the best strain reached 13 506 U/L at the fermenter level, 330-fold higher than the previous result of 41 U/L in S. cerevisiae. The strategy can be applied to alleviate the inhibition of cell growth caused by the expression of toxic proteins and improve their secretion.

Choice-selective sequences dominate in cortical relative to thalamic inputs to NAc to support reinforcement learning.

SUMMARYHow are actions linked with subsequent outcomes to guide choices? The nucleus accumbens, which is implicated in this process, receives glutamatergic inputs from the prelimbic cortex and midline regions of the thalamus. However, little is known about whether and how representations differ across these input pathways. By comparing these inputs during a reinforcement learning task in mice, we discovered that prelimbic cortical inputs preferentially represent actions and choices, whereas midline thalamic inputs preferentially represent cues. Choice-selective activity in the prelimbic cortical inputs is organized in sequences that persist beyond the outcome. Through computational modeling, we demonstrate that these sequences can support the neural implementation of reinforcement-learning algorithms, in both a circuit model based on synaptic plasticity and one based on neural dynamics. Finally, we test and confirm a prediction of our circuit models by direct manipulation of nucleus accumbens input neurons.

Spatial Organization of the Projections of the Dorsal Raphe Nucleus to Functionally Diverse Areas of the Basal Ganglia in Dogs

Objective. To study the projections of individual subregions of the dorsal raphe nucleus (DRN) to functionally diverse areas of the basal ganglia in the dog brain. Materials and methods. A method based on the retrograde axonal transport of horseradish peroxidase (HRP) was used in 43 mongrel dogs given injections into functionally diverse areas of the basal ganglia to study the spatial organization of the projections from different parts of the DRN to these structures. At 48 h after perfusion of the brain, tetramethylbenzidine was used for the histochemical detection of HRP in DRN neurons in frontal serial sections of the brain and the numbers of labeled neurons in each area, as identified on serial frontal celloidin brain sections stained with toluidine, were counted under the microscope. Results. The spatial organization of projections to functionally diverse areas of the basal ganglia from individual functionally diverse parts of the DRN was studied in dogs using a method based on the retrograde axonal transport of HRP. The topographic characteristics of projection connections were identified, pointing to the possible segregated conduction of information from the dorsal part of the DRN, which has connections with limbic structures, i.e., limbic subareas of the corpus striatum. The overlapping of the terminal fields of neurons from all parts of the DRN receiving and sending functionally diverse information to the basal ganglia and structures connected to them seen in the entopeduncular nucleus, the ventral pallidum, the deep mesencephalic nucleus, and the medial part of the pedunculopontine nucleus of the tegmentum is evidence that this information may be integrated in these nuclei. The structural basis for information processing in the morphofunctional system of the basal ganglia was analyzed. Conclusions. Data are presented on the topographical organization of projections directed to functionally diverse areas of the basal ganglia from different parts of the DRN, providing evidence of the possible influences of different parts of the DRN on a wide spectrum of behavioral and physiological processes involving the basal ganglia. The connection system identified here is involved in conducting information and integrating it in the morphofunctional system of the basal ganglia and provides the structural basis for understanding the mechanisms of their functioning in health and disease.

Organization of the Connections of the Rostromedial Tegmental Nucleus with Structures of the Morphofunctional Basal Ganglia System in the Dog Brain

The spatial organization of the projections of the rostromedial tegmental nucleus (RMTg) to functionally diverse segments of the structures of the basal ganglia in dogs (n = 25) were studied by retrograde axonal transport of horseradish peroxidase. Connections between the limbic segments of the RMTg and the limbic segments of the zona incerta and the pallidum constituted evidence pointing to the segregated conduction of limbic information. In addition, these studies showed that the projections of the limbic medial and motor lateral segments of the RMTg overlap, within the pedunculopontine and entopeduncular nuclei, with fibers arriving in these nuclei from neurons in functionally diverse nuclei. The presence of labeled sparsely branched, long-axon “reticular” RMTg neurons projecting to the structures of interest is also evidence that this nucleus has an integrative function. Thus, the morphological substrate for information processing in the morphofunctional system of the basal ganglia underlying the mechanisms of their functioning was analyzed.

Efferent projections of dorsal root afferents in the spinal cord of the lamprey Lampetra fluviatilis

Arborization of dorsal root afferents was studied in the lamprey spinal cord by the method of horseradish peroxidase transport. Direct evidence was obtained for the presence of efferent fibers in dorsal roots, representing collaterals that depart from ascending and descending intraspinal branches of sensory axons and travel towards the periphery through the adjacent roots.

Organization of the Projections of the Lateral Midbrain Tegmental Nuclei to the Basal Ganglia in the Dog Brain

The organization of the projections of the lateral nuclei of the midbrain tegmentum (the peripeduncular and perilemniscal nuclei, the nucleus of the brachium of the posterior colliculus) to the functionally diverse nuclei of the basal ganglia system of the brain was studied in dogs (n = 34) using a method based on the retrograde axonal transport of horseradish peroxidase. The study nuclei of the midbrain were found to be involved in functionally diverse circuits whose components include the basal ganglia. These nuclei innervate the area of the putamen, globus pallidus, cuneate nucleus, and subcuneate nucleus, which on the basis of their predominant connections with the motor or limbic nuclei of the brain are classified as motor or limbic nuclei, as well as the area of the caudate and accumbens nuclei and the compact zone of the pedunculopontine nucleus, which receive projections from functionally diverse structures. Analysis of Nissl-stained frontal sections allowed the topographical anatomy of the individual lateral nuclei of the midbrain tegmentum to be refined. Histochemical reactions for NADPH diaphorase was used to determine the cholinergic nature of their neurons.

Organization of the Pallidal Projections of the Rostromedial Tegmental Nucleus of the Dog Brain

The organization of the projections of the rostromedial tegmental nucleus (RMTN) to functionally diverse nuclei of the pallidum in the dog brain (n = 13) was studied by retrograde and anterograde transport of horseradish peroxidase. These studies showed that fibers formed by neurons in the limbic medial segments of the rostral and caudal divisions of the RMTN are directed to the limbic parts of the pallidum – the ventral zone of the globus pallidus and the ventral pallidum. Reciprocal connections were observed between the ventral pallidum and the medial part of the rostral RMTN. These data identify the potential for segregated conduction of limbic information via these projection systems. However, most pallidal structures showed convergence of projection fibers arising from neurons in functionally diverse parts of the RMTN. Thus, projection fibers from neurons in the motor lateral and limbic medial parts of the RMTN are directed to the limbic ventral segment of the globus pallidus and the entopeduncular nucleus, which are innervated by fibers formed by neurons in functionally diverse structures. The possible pathways for the conduction of functionally different information and its integration in the projection systems studied here are discussed.

Protein transport across membranes: Comparison between lysine and guanidinium-rich carriers

The mechanism(s) by which certain small peptides and peptide mimics carry large cargoes across membranes through exclusively non-covalent interactions has been difficult to resolve. Here, we use the droplet-interface bilayer as a platform to characterize distinct mechanistic differences between two such carriers: Pep-1 and a guanidinium-rich peptide mimic we call D9. While both Pep-1 and D9 can carry an enzyme, horseradish peroxidase (HRP) across a lipid bilayer, we found that they do so by different mechanisms. Specifically, Pep-1 requires voltage or membrane asymmetry while D9 does not. In addition, D9 can facilitate HRP transport without pre-forming a complex with HRP. By contrast, complex formation is required by Pep-1. Both carriers are capable of forming pores in membranes but our data hints that these pores are not responsible for cargo transport. Overall, D9 appears to be a more potent and versatile transporter when compared with Pep-1 because D9 does not require an applied voltage or other forces to drive transport. Thus, D9 might be used to deliver cargo across membranes under conditions where Pep-1 would be ineffective.

Neuron Populations Giving Rise to Corticothalamic Projections to the Partially Deafferented Ventrolateral Nucleus of the Thalamus

The characteristics of the distribution and morphological composition of neuron populations in the motor cortex projecting to the cerebellar recipient site, the ventrolateral nucleus of the thalamus, after its partial deafferentation were studied in adult cats. Partial deafferentation of the ventrolateral nucleus was produced by prior (three months) electrolytic lesioning of the contralateral intermediate nucleus of the cerebellum. A retrograde axonal horseradish peroxidase transport method with local administration of marker into the ventrolateral nucleus of the thalamus was used. All labeled neurons were members of the populations of nonpyramidal and pyramidal neurons, and most were of small or intermediate size and were located in the deep cortical layers – the lower subdivision of layer V and, mainly, layer VI. Labeled neurons were seen in cortical fi elds 4γ and 6αβ. These data demonstrate the absence of any structural reorganization of cortical projections to the deafferented ventrolateral nucleus of the thalamus. It is suggested that this is due to the high level of specialization of this movement program-triggering system. Neuroplastic changes were apparent in anomalous transformations of the proximal parts of dendrites and the presence of larger numbers of “paired” pyramidal neurons than in intact animals.

Organization of Projections of the Rostromedial Tegmental Nucleus to the Striatum in the Dog Brain

The organization of the projections of the rostromedial tegmental nucleus (RMTg) to functionally diverse areas of striatal structures in dogs (n = 26) was studied by a method based on retrograde axonal transport of horseradish peroxidase and fluorochromes. Projection fibers from neurons in the limbic medial part of the RMTg were found to be directed only to the limbic segments of the caudate nucleus, putamen, and nucleus accumbens. However, most segments of striatal structures showed convergence of projection fibers running from neurons in functionally diverse parts of the RMTg. Thus, the motor dorsolateral segment of the caudate nucleus, as well as striatal segments innervated by fibers from neurons in functionally diverse structures (the ventrolateral segment of the caudate nucleus and the lateral segment of the nucleus accumbens) received fibers from neurons in the motor lateral part of the RMTg and limbic medial part of the RMTg. The possible pathways conducting functionally diverse information and integrating it in the projection systems studied here are assessed.

Cortico-Cortical, Cortico-Striatal, and Cortico-Thalamic White Matter Fiber Tracts Generated in the Macaque Brain via Dynamic Programming

Probabilistic methods have the potential to generate multiple and complex white matter fiber tracts in diffusion tensor imaging (DTI). Here, a method based on dynamic programming (DP) is introduced to reconstruct fibers pathways whose complex anatomical structures cannot be resolved beyond the resolution of standard DTI data. DP is based on optimizing a sequentially additive cost function derived from a Gaussian diffusion model whose covariance is defined by the diffusion tensor. DP is used to determine the optimal path between initial and terminal nodes by efficiently searching over all paths, connecting the nodes, and choosing the path in which the total probability is maximized. An ex vivo high-resolution scan of a macaque hemi-brain is used to demonstrate the advantages and limitations of DP. DP can generate fiber bundles between distant cortical areas (superior longitudinal fasciculi, arcuate fasciculus, uncinate fasciculus, and fronto-occipital fasciculus), neighboring cortical areas (dorsal and ventral banks of the principal sulcus), as well as cortical projections to the hippocampal formation (cingulum bundle), neostriatum (motor cortical projections to the putamen), thalamus (subcortical bundle), and hippocampal formation projections to the mammillary bodies via the fornix. Validation is established either by comparison with in vivo intracellular transport of horseradish peroxidase in another macaque monkey or by comparison with atlases. DP is able to generate known pathways, including crossing and kissing tracts. Thus, DP has the potential to enhance neuroimaging studies of cortical connectivity.

Organization of the Projections of the Substructures of the Deep Mesencephalic Nucleus to the Striatum in the Dog Brain

Method of retrograde axonal transport of horseradish peroxidase was used to study the organization of the projections of the individual substructures of the deep mesencephalic nucleus complex to the functionally diverse regions of striatal structures of the dog brain (n=20). It was shown that the projection fibers of the neurons of the nucleus profundus mesencephali were directed to the ventro-lateral segment of the nucleus caudatus and the ventral segment of the putamen. The dorsal segment of the putamen obtains the fibers from the neurons of the nucleus cuneiformis, while the lateral segment of the nucleus accumbens received them from the neurons of the nucleus subcuneiformis. The possible pathways for conduction of the functionally diverse information and its integration in the investigated projection systems are discussed.

Interaction of the Pallidum, Pedunculopontine Nucleus, Zona Incerta, and Deep Mesencephalic Nucleus – Structures of the Morphofunctional Basal Ganglia System

The organization of the projections of substructures of the zona incerta (ZI), pedunculopontine nucleus (PPN), and the deep mesencephalic nucleus (NPM) complex and functionally diverse pallidal structures of the dog brain (n = 20) were studied by a method based on anterograde and posterograde axonal transport of horseradish peroxidase. The globus pallidus and entopeduncular nucleus were found to be connected by reciprocal connections with the dorsal, ventral, and caudal sectors of the ZI and with the NPM, and the lateral segment of the diffuse zone and the compact zone of the PPN. These same pallidal nuclei were connected to the rostral sector of the ZI, the cuneate and subcuneate nuclei of the NPM, the medial segment of the diffuse zone of the PPN via ipsilateral projections. Of all the structures studied, reciprocal connections with the ventral pallidum were seen only with the lateral segment of the diffuse zone and the compact zone of the PPN. The possible pathways for conduction of functionally diverse information and its integration in the projection systems studied here are discussed.

Striopallidal Projections of the Zona Incerta of the Dog Diencephalon

The projections of individual sectors of the zona incerta (ZI) in dog diencephalon to the functionally different striatal segments were studied by the method of the antero- and retrograde axonal transport of horseradish peroxidase. The data on the interconnections of ZI with all striopallidal structures, obtained by the author before and in the present work were summarized. Among all the striatal nuclei, the the rostral, ventral and caudal ZI sectors contained the neurons that projected their fibers only to the dorsal segments of the putamen. Not a single striatal structure was found to form projections to ZI. ZI and the dorsal pallidal nuclei (globus pallidus, nucleus entopeduncularis) are reciprocally connected with almost all ZI sectors. The connections of the ventral pallidum with the ZI were not detected.

A novel path for rapid transverse communication of vestibular signals in turtle cerebellum

Voltage-sensitive dye activity within the thin, unfoliated turtle cerebellar cortex (Cb) was recorded in vitro during eighth cranial nerve (nVIII) stimulation. Short latency responses were localized to the middle of the lateral edges of both ipsilateral and contralateral Cb [vestibulocerebellum (vCb)]. Even with a severed contralateral Cb peduncle, stimulation of the nVIII ipsilateral to the intact peduncle evoked contralateral vCb responses with a mean latency of only 0.25 ms after the ipsilateral responses, even though the distance between them was ∼ 5 mm. We investigated whether a rapidly conducting commissure exists between each vCb by stimulating one of them directly. Responses in both vCb spread sagittally, but, surprisingly, there was no sequential activation along a transverse Cb beam between them. In contrast, stimulation medial to either vCb evoked transverse beams that required ∼ 20 ms to cross the Cb. Therefore, the rapid commissural connection between each vCb is not mediated by slowly conducting parallel fibers. Also, the vCb was not strongly activated by climbing fiber stimulation, suggesting that inputs to vCb involve distinct cerebellar circuits. Responses between the two vCb remained following knife cuts through the rostral and caudal Cb along the midline, through both peduncles, and even shallow midline cuts to the middle Cb through its white matter and granule cell layer. Commissural responses were still observed only with a narrow transverse bridge between each vCb or in thick transverse Cb slices. Horseradish peroxidase transport from one vCb labeled transverse axons traveling within the Purkinje cell layer that were larger than parallel fibers and lacked varicosities. In sagittal sections, cross-section profiles of myelinated axons were observed around Purkinje cells midway between the rostral and caudal Cb. This novel pathway for transverse communication between lateral edges of turtle Cb suggests that afferents may directly conduct vestibular information rapidly across the Cb to coordinate vestibulomotor reflex behaviors.

Direct projections of omnipause neurons to reticulospinal neurons: A double‐labeling light microscopic study in the cat

Omnipause neurons (OPNs) are inhibitory neurons located in the midline region of the caudal pons. Their role in gating the discharges of saccade-related burst neurons is well known, but there is no agreement concerning their influence on brainstem neurons that control other muscle groups participating in rapid gaze shifts. In the present study, we inquired whether OPNs project directly to pontobulbar reticulospinal neurons (RSNs) in the cat. Retrograde transport of horseradish peroxidase from the cervical spinal cord was used to label RSNs and an anterograde tracer (biocytin) was iontophoresed at sites of extracellular recording of the OPN activity. Somadendritic characteristics of biocytin-labeled OPNs were largely similar to those obtained previously with intracellular labeling. Three-dimensional reconstruction of axonal trajectories and collaterals revealed that projections of OPNs, regarded as a population, are bilateral. Their terminals were restricted to the reticular formation and midline structures throughout the rostral bulbar and pontine tegmentum. Appositions of synaptic boutons originating from five fully stained OPNs were detected on 38 retrogradely labeled RSNs, each of the OPNs contacting 3-13 cells. The numbers of boutons (1-46; mean 11.8) on the RSN somata and proximal dendrites indicate that the anatomical strength of paired OPN-RSN connections is comparable to that of other similarly studied inhibitory neurons in the cat. The existence of connections with RSNs supports the hypothesis of a generalized influence of OPNs on several effectors participating in orienting gaze shifts as opposed to the idea of their strict specialization for the control of eye saccades.

Connections between the Zona Incerta of the Dog Diencephalon and the Substantia Nigra, Ventral Tegmental Field, and Pedunculopontine Tegmental Nucleus

Studies using technique based on retrograde and anterograde transport of horseradish peroxidase were performed to address the organization of the connections of different sectors of the zone incerta (ZI) of the diencephalon with substructures of the substantia nigra, ventral tegmental field, and pedunculopontine nucleus of the midbrain in dogs. These structures were found to be interconnected. The organization of these connections included elements demonstrating that segregated conduction of functionally diverse information can be transmitted via these pathways. In addition, the convergence of projections fibers from neurons in substructures of the midbrain nucleus described in all sectors of the ZI and to these neurons from neurons in all ZI sectors is evidence that functionally diverse information can be integrated both at the level of the ZI and at the level of the midbrain structures studied here.

Receptor-Mediated and Fluid-Phase Transcytosis of Horseradish Peroxidase across Rat Hepatocytes

Horseradish peroxidase (HRP) is often used as a fluid-phase marker to characterize endocytic and transcytotic processes. Likewise, it has been applied to investigate the mechanisms of biliary secretion of fluid in rat liver hepatocytes. However, HRP contains mannose residues and thus binds to mannose receptors (MRs) on liver cells, including hepatocytes. To study the role of MR-mediated endocytosis of HRP transport in hepatocytes, we determined the influence of the oligosaccharid mannan on HRP biliary secretion in the isolated perfused rat liver. A 1-minute pulse of HRP was applied followed by marker-free perfusion. HRP appeared in bile with biphasic kinetics: a first peak at 7 minutes and a second peak at 15 minutes after labeling. Perfusion with 0.8 mg/mL HRP in the presence of a twofold excess of mannan reduced the first peak by 41% without effect on the second one. Together with recently published data on MR expression in rat hepatocytes this demonstrates two different mechanisms for HRP transcytosis: a rapid, receptor-mediated transport and a slower fluid-phase transport.

Organization of Projections of the Zona Incerta of the Diencephalon to Pallidal Structures in the Dog Brain

Detailed studies of the organization of individual sectors in the zona incerta of the diencephalon to functionally diverse pallidal structures in the dog brain were performed by antero- and retrograde axon transport of horseradish peroxidase. The results showed that neurons in the caudal sector of the zona incerta innervate the globus pallidus and entopeduncular nucleus, which receive more extensive innervation from motor structures. The same pallidal structures receive projections from occasional neurons in the dorsal and causal sectors of the zona incerta. No connections of the zona incerta with the limbic ventral pallidum were found.

Location of Neurons in the Central Nucleus of the Amygdaloid Body Projecting to the Paraventricular Nucleus of the Hypothalamus

Experiments were performed on rats using retrograde axonal transport of horseradish peroxidase with the aim of identifying cells in the individual structures of the central nucleus of the amygdaloid body (CNAB) innervating the paraventricular nucleus of the hypothalamus. Labeled cells were seen in the ipsilateral CNAB throughout its rostrocaudal extent. The largest number of labeled neurons was seen in the middle third of the nucleus in the area corresponding to the intermediate subnucleus of the CNAB. Occasional cells were located in the medial and lateral subnuclei of the CNAB. Labeled neurons in the intermediate subnucleus were oval cells or cells of indeterminate shape.