Tetramethylbenzidine Reaction Research Articles

Surface-carboxylated cobaltous oxide with oxidase-like activity for conveniently assessing total antioxidant capacity of Cordyceps militaris at neutral pH

Reassessment of total antioxidant capacity (TAC) in Cordyceps militaris is imperative for providing dietary guidance and facilitating health monitoring. Herein, surface-carboxylated cobaltous oxide (COOH-Co3O4) nanozyme was achieved via a dual-stage procedure encompassing reduction preparation and surface modification. COOH-Co3O4 exhibited exceptional oxidase-like activity under neutral pH conditions, facilitating conversion of dissolved oxygen into superoxide anion free radicals and subsequently catalyzing oxidation of colorless 3, 3′, 5, 5′-tetramethylbenzidine (TMB) to yield blue oxidized TMB (TMBox) at neutral pH. Additionally, antioxidants can eliminate O2·− produced in the above reaction system. Moreover, the absorbance at 652 nm of the COOH-Co3O4 + TMB reaction system gradually diminishes with an increase in ascorbic acid concentration. Based on it, a colorimetric evaluation system (using ascorbic acid as a reference) for TAC of C. militaris was established. This innovative approach offers an impressively low limitation of detection (LOD) calculated as 0.78 μM. Furthermore, it was employed to evaluate TAC of real C. militaris samples, yielding a satisfactory recovery (96.1 %–102.3 %), thereby demonstrating its exceptional accuracy. The TAC of C. militaris extracted using different solvents was determined, indicating that the highest TAC is observed in C. militaris extracted by deionized water. This study not only offers a rapid, cost-effective, and convenient analytical tool for evaluating TAC of C. militaris but also expands the potential applications of nanozymes.

Flexible microfluidic colorimetric detection chip integrated with ABTS·+ and Co@MnO2 nanozyme catalyzed TMB reaction systems for bio-enzyme free detection of sweat uric acid

BackgroundThe development of wearable detection devices that can achieve noninvasive, on-site and real-time monitoring of sweat metabolites is of great demand and practical significance for point-of-care testing and healthcare monitoring. Monitoring uric acid (UA) content in sweat provides a simple and promising way to reduce the risk of gout and hyperuricemia. Traditional bioenzyme based UA assays suffer from high cost, poor stability, inconvenience for storage and easy deactivation of bioenzymes. Wearable microfluidic colorimetric detection device for sweat UA detection has not been reported. The development of novel wearable microfluidic colorimetric detection chip with no requirement of bioenzymes for sweat UA detection is of great importance for health care monitoring. ResultsFirstly, Co@MnO2 nanozyme with high oxidase-like activity was synthesized and characterized. Co@MnO2 can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) directly to generate blue-green colored ox-TMB. Green colored 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) radical (ABTS·+) was produced by the oxidation of ABTS by potassium persulfate. UA exhibits distinct quenching effect on Co@MnO2 catalyzed TMB colorimetric reaction system and ABTS·+ based colorimetric system, leading to obvious color fading of the two colorimetric systems. Then, a flexible microfluidic colorimetric detection chip for UA detection was fabricated by assembling Co@MnO2/TMB modified paper chips and ABTS·+ modified paper chips into a polydimethylsiloxane (PDMS) microfluidic chip. The fabricated microfluidic colorimetric detection chip exhibits good linear relationship for sweat UA detection. The linear range is from 20 to 200 μmol/L with detection limit as low as 6.6 μmol/L. Good results were obtained for the detection of UA in actual sweat from three volunteers. SignificanceThis work provides two bio-enzyme free colorimetric detection systems for UA detection. Furthermore, a simple, low-cost and selective flexible wearable microfluidic colorimetric detection chip was fabricated for noninvasive and on-site detection of sweat UA, which holds great application potential for personal health monitoring and point-of-care testing.

Pt/NiCo2O4 nanocomposite with extremely low Pt loading as an oxidase-like nanozyme for colorimetric detection of acetaminophen in natural water

BackgroundIn the last COVID-19-three years, acetaminophen (AP) consumption has increased significantly, potentially causing water contamination. Due to its ease of use and great performance, colorimetric detection of AP based on Pt-based nanozymes is gaining popularity. A significant difficulty is how to reduce Pt loading content while retaining a high catalytic activity. MethodsControlling the deamination of Co-NH3 complexes, including Ni(OH)2, led to the formation of small NiCo2O4 nanoparticles, which were afterwards decorated with a 0.37wt% Pt loading. Significant findingsWith a low Michaelis-Menten constant of 0.0112 mM, the 3,3′,5,5′-tetramethylbenzidine (TMB) was oxidized with O2 utilizing the Pt(0.37wt%)/NiCo2O4 nanocomposite. The detection settings were tuned for nanozyme and TMB concentrations, pH, temperature, and reaction duration. Utilizing AP's inhibitory impact on the oxidase-like activity at pH = 1, an AP colorimetric sensor was created. It was possible to establish a linear response with a detection limit (LOD) of 2.4 μM and a range of 10 μM–0.25 mM. In natural water, such as spring, lake, and river water, this colorimetric sensor was effectively employed to detect AP with good selectivity over interfering substances.

SERS-based error calibration of a TMB-H2O2 colorimetric system.

3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 is widely used as an effective colorimetric system, in which the color reaction is implemented with peroxidase-catalyzed TMB oxidation by H2O2 that usually measured UV-vis absorption spectra or Raman spectra. However, its low accuracy significantly limits its application. Blue charge transfer complex (CTC), which is the product of TMB and H2O2 reaction and is used as the basis for partial colorimetric methods, usually causes colorimetric error owing to changes in the UV-vis absorption and Raman spectra during TMB oxidation under various environmental conditions (catalyst type, temperature, H2O2 concentration). Herein, we propose a surface-enhanced Raman spectrum (SERS)-based error calibration method to improve the accuracy of the TMB-H2O2 colorimetric system. It is found that under 633 nm laser excitation, TMB has three Raman peaks at 1189, 1335 and 1609 cm-1 in the single-electron oxidation phase, and these peaks disappear completely in the two-electron oxidation phase. By comparing these Raman peaks, we can conveniently obtain the actual process information during TMB oxidation. Using the proposed method, the accuracy of the TMB-H2O2 colorimetric system improved by more than 15%. Importantly, this SERS-based TMB-H2O2 error calibration method will open a new horizon for enzyme-linked immunosorbent assay (ELISA) and other biomedical applications.

Highly sensitive colorimetric detection of arsenite based on reassembly-induced oxidase-mimicking activity inhibition of dithiothreitol-capped Pd nanozyme

Given that hypertoxic arsenite (As3+) poses a serious threat to the environment and global health, developing high-performance, low-cost and easy-to-use approaches to specifically monitor As3+ in environmental matrices turns to be of great importance. In the present work, we reported a novel colorimetric assay of As3+ based on the reassembly-induced oxidase-like activity inhibition of dithiothreitol-capped Pd nanoparticles (Pd-DTT). The Pd-DTT nanozyme exhibits good oxidase-like catalytic activity to trigger the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of O2, forming a blue product TMBox. When As3+ exists, it can chelate the sulfydryl groups in DTT more strongly, leading to a reassembly of Pd-DTT. Compared with the original nanozyme, the reassembled one has much less oxidase-like activity, resulting in the significantly suppressed TMB reaction. Based on this principle, highly sensitive detection of As3+ was achieved, providing a wide linear range from 33 ng L−1 to 333.3 μg L−1 and an ultralow detection limit of 35 ng L−1. With the help of EDTA as a chelating agent, the method could also offer excellent selectivity for the determination of As3+ against other metal ions. Besides, our colorimetric assay was employed to accurately measure the analyte in spiked drinking and river water, revealing its great promise in environmental analysis application.

Localization of intracellular lipid hydroperoxides using the tetramethylbenzidine reaction for transmission electron microscopy.

Histochemical reactions for lipid hydroperoxides (LHP) using indophenol, benzidine, or phenylendiamine as the electron donor have been described previously for auto-oxidized adipose () and neuronal () tissue. More recently, tetramethylbenzidine (TMB) has been proposed as another chromagen (). The usefulness of the TMB reaction for ultrastructural studies of lipid peroxidation was demonstrated in retina where LPH was generated by incubation with exogenous lipoxygenase. The glutaraldehyde fixed tissue, which was reacted with TMB and then postfixed in osmium tetroxide, showed an electron-dense product (). This technique allows intra- and extracellular localization, as well as a comparison of relative intensity amoung various cell types and subcellular organelles (). In light-induced lipid peroxidation, discs of the outer segments, which are rich in oxidizable long-chain polyunsaturated fatty acids, stain strongly and appear as bubble-like structures (). These are however, quite similar to fingerprint profiles seen acutely in outer segments and chronically in neurons, which are visualized without TMB following exogenous exposure to LHP (,). A possible caveat to the reported method is that peroxidized protein and carbohydrates many also react and so the TMB method has not been proven to be specific for LHP only.

Ultrastructural evidence for synaptic inputs of enkephalinergic nerve terminals to target neurons in the rat arcuate nucleus

The morphological support of interactions between enkephalins and three systems—β-endorphin (β-END), tyrosine hydroxylase (TH), or neuropeptide Y (NPY)—well represented in the arcuate nucleus, was examined by using an electron microscopic double immunostaining combining two sensitive chromogens, diaminobenzidine (DAB) and tetramethylbenzidine (TMB). The first step consisted of visualizing Met-enkephalinergic terminals with DAB reaction product, and the second one involved detecting the antigens TH, β-END, and NPY in their respective neurons with TMB reaction product. Ultrastructural analysis revealed enkephalinergic terminals presynaptic to TH-immunopositive cells and dendrites, principally in the dorsal portion of the arcuate nucleus. Enkephalinergic nerve terminals also contacted synaptically ventrolaterally located β-END-immunoreactive cells. In the ventromedial arcuate nucleus, few synaptic contacts were observed between enkephalinergic boutons and NPY neurons, which were principally in close apposition with glial processes. Enkephalin-immunoreactive synapses were more frequently seen on TH-immunopositive neurons. This TH neuronal group is known to correspond to the dopaminergic tuberoinfundibular neurons implicated in the control of reproductive functions. The pattern of distribution of the different synapses within the arcuate nucleus (TH dorsally, β-END ventrolaterally; NPY ventromedially) suggests that enkephalins may play a role in the neuroendocrine regulation of gonadotropin and prolactin secretion. The results provide evidence that enkephalins, in the arcuate nucleus, exert a postsynaptic action on the β-END cells in addition to the presynaptic regulation previously demonstrated in the mediobasal hypothalamus, related to β-END release. Moreover, the arcuate nucleus is a site of intercellular relationships between enkephalins and dopamine and between enkephalins and other peptides such as NPY.

The tungstate-stabilized tetramethylbenzidine reaction for light and electron microscopic immunocytochemistry and for revealing biocytin-filled neurons

A peroxidase reaction product that can be easily distinguished from standard diaminobenzidine (DAB) reaction products is needed for pre-embedding electron microscopic double-antibody labelling studies. Benzidine dihydrochloride (BDHC) and gold-substituted silver peroxidase reactions are unsatisfactory for double labelling because they lack sensitivity and reliability and/or compromise ultrastructure. We show here that light and electron microscopic immunocytochemistry can be done with a modification of the tungstate-stabilized tetramethylbenzidine (TMB) reaction (Weinberg and Van Eyck 1991) which yields a crystalline reaction product. With this method, we have obtained excellent immunolabelling for a variety of antigens, including tyrosine hydroxylase, enkephalin, serotonin, Fos protein and retrogradely transported cholera toxin B subunit (CTB). The TMB-tungstate reaction is useful for ultrastructural double labelling because the crystals contrast well with the amorphous product of diaminobenzidine reactions. The TMB-tungstate reaction is more sensitive and reliable for immunocytochemistry than the benzidine dihydrochloride reaction and gives better ultrastructure than the gold-substituted silver peroxidase reaction. We also show that neurons filled with biocytin by intracellular injection can be visualized with TMB-tungstate for either light (LM) or electron (EM) microscopy.

Transcallosal non‐pyramidal cell projections from visual cortex in the cat

Non-pyramidal cells with transcallosal projections were identified in the area 17/18 border region of the cat by retrograde transport of horseradish peroxidase injected into border region of the opposite hemisphere. From several hundred neurons filled with a Golgi-like diaminobenzidine (DAB) reaction product, seven cells were identified by their radially oriented smooth dendrites as possible non-pyramidal cells. Following thin-sectioning and examination with the electron microscope, four of the neurons proved to be layer IV spiny stellate cells with incompletely filled dendritic spines, and two proved to be layer III pyramidal cells with an incompletely labelled apical dendrite and dendritic spines. The remaining neuron was a non-pyramidal cell whose essentially smooth dendrites were covered with synapses, and whose cell body formed both symmetric and asymmetric synapses with presynaptic terminals. To better assess how many non-pyramidal cells might be labelled, thin sections of the area 17/18 border were surveyed using material processed with tetramethylbenzidine (TMB), and another five labelled non-pyramidal cells with transcallosal projections were identified by the needle-like crystals of TMB reaction product they contained. During the study it became evident that both the DAB and TMB reaction products in the lightly labelled neurons tended to be associated with granules that are 0.5 microns or larger in diameter and that had the characteristics of lysosomes. These granules are also visible in the light microscope as dark puncta. The numbers of puncta in profiles of pyramidal and of non-pyramidal cells in layers II/III and IVa of the area 17/18 border region and in the control acallosal region of area 17 were counted and compared. These comparisons revealed that labelled transcallosally projecting non-pyramidal cells may constitute 10-32% of the non-pyramidal cell population at the area 17/18 border region. Similar values were also obtained for pyramidal cells in this region. Consequently, it is concluded that significant numbers of non-pyramidal cells have axons that project through the corpus callosum to the contralateral hemisphere.

A double-label pre-embedding immunoperoxidase technique for electron microscopy using diaminobenzidine and tetramethylbenzidine as markers.

Techniques for correlative double-label immunocytochemistry (ICC) at light and electron microscopic (EM) level are useful for determining the neurotransmitter phenotype of inputs onto immunocytochemically identified neurons. Tetramethylbenzidine (TMB) has been used as a chromogen at the EM level for horseradish peroxidase tract tracing. We have found that TMB, in combination with diaminobenzidine (DAB), can be used in a double-label immunocytochemical protocol to examine neuropeptide Y inputs onto luteinizing hormone-releasing hormone cells in the sheep preoptic area. At both light and EM levels, TMB reaction product is visibly distinct from DAB reaction product. The ultrastructural preservation we have been able to obtain with our technique is better than that obtained with techniques that use TMB at a lower pH. Furthermore, this technique allows the demonstration of synaptic contacts between neurochemically identified terminals and cells with different neurotransmitter phenotypes.

Improvement of the tetramethyl benzidine reaction with ammonium molybdate as a stabilizer for light and electron microscopic ligand-HRP neurohistochemistry, immunocytochemistry and double-labelling

Ammonium heptamolybdate (AHM) was used as a stabilizing agent in the tetramethyl benzidine (TMB) reaction of choleragen subunit B conjugated horseradish peroxidase (CB-HRP) neurohistochemistry (TMB-AHM method). In comparison with Mesulam's TMB method employing sodium nitroprusside as a stabilizing agent (TMB-SNP method), the TMB-AHM procedure offfers a similar sensitivity with regard to the visualization of CB-HRP labelled neurons and their extranuclear Golgi-phobic dendrites. However, it is less sensitive for the demonstration of anterogradely transported CB-HRP in axon terminals. At the nearly physiological pH value of the reaction medium (pH 6–8), it demonstrates better preservation of tissue and cell structures in the reacted sections. Under the electron microscope, the specific reaction product can be clearly distinguished and little damage of cellular and subcellular structures occurred. Preliminary application of TMB-AHM method to choleragen subunit B (CB) immunocytochemistry and double labelling technique which paired the neuronal tracing methods of HRP neurohistochemistry and CB immunocytochemistry, was also carried out with small modification.

Stabilization of tetramethylbenzidine (TMB) reaction product at the electron microscopic level by ammonium molybdate

The ability to use the tetramethylbenzidine (TMB) method for studying neuronal connections at the electron microscopic level is often difficult because the conditions of osmification and dehydration used in processing the tissue may result in significant loss and/or decreased electron density of the reaction product. In the present study, we report that stabilization of TMB reaction product with 5% ammonium molybdate (AM) prior to osmificating the tissue results in the formation of TMB-AM crystals that are many times more electron dense and resistant to ethanol extraction than non-stabilized TMB crystals. The nature of the chemical interaction that underlies the stabilization of TMB by AM is uncertain, but it may involve the formation of an insoluble salt between molybdic ions and the TMB polymer. The use of this simple procedure increases the sensitivity of the TMB procedure at the electron microscopic level and may be used to label neuronal pathways in the peripheral and central nervous systems with equal success.

Brainstem projections to lumbar motoneurons in rat—II. An ultrastructural study by means of the anterograde transport of wheat germ agglutinin coupled to horseradish peroxidase and using the tetramethyl benzidine reaction

The descending projections from the ventrolateral medial reticular formation to the lumbar motoneuronal cell groups were studied in rat at the ultrastructural level using the anterograde transport of wheat germ agglutinin coupled to horseradish peroxidase in combination with the chromogen tetramethyl benzidine. The tissue containing the horseradish peroxidase reaction products was processed for electron microscopy using the method of chemical dehydration. In the ultrathin sections of the lumbar motoneuronal cell groups reaction products were easily recognized by their electron-dense crystal-like structure. These crystals were mostly found in terminals (66%) and to a lesser extent in axons (10%) and dendrites (10%). In the lumbar motoneuronal cell groups six different types of terminals were distinguished. It was found that F-type terminal profiles most frequently contained crystals, whereas fewer S- and G-types did so. The various findings are basically the same as those of a similar ultrastructural autoradiographic study with [ 3H]leucine as a tracer, except for the labelling of dendrites, which is discussed. It is concluded that the horseradish peroxidase technique employed in the present study can be reliably used for anterograde tracing at the ultrastructural level. Its advantages and disadvantages in comparison with the electron microscopical autoradiographic technique are discussed.

Origin and distribution of cerebral vascular innervation from superior cervical, trigeminal and spinal ganglia investigated with retrograde and anterograde WGA-HRP tracing in the rat

Peripheral sources of cerebral vascular innervation have been investigated with retrograde and anterograde neuronal tracing of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) in the rat. For retrograde identification of sources of innervation, WGA-HRP was applied to the exposed basilar artery through a fine slit in the overlying meninges, and sections of brain and peripheral ganglia were reacted with tetramethylbenzidine for detection of the tracer. A high density of tetramethylbenzidine reaction product was observed around the basilar artery and in the surrounding pial tissue, but the application sites were not completely selective since some tracer always had spread into the ventral brain stem. Retrogradely labelled cell bodies were identified in the superior cervical, stellate, first and second spinal, and trigeminal ganglia, i.e. these ganglia may represent origins of basilar artery innervation. In a second series of experiments, microinjections of WGA-HRP were placed into the indicated ganglia to obtain anterograde labelling of nerve fibres on whole-mounts of the cerebral vessels. Injections into trigeminal ganglia labelled nerve fibres on the ipsilateral half of the circle of Willis, as well as the contralateral anterior cerebral artery and the rostral part of the basilar artery. The first and second spinal ganglia projected to the vertebrobasilar arteries, while the ipsilateral part of the internal carotid (outside the circle of Willis) received fibres from the second spinal ganglion. Nerve fibres originating in trigeminal and spinal ganglia were organised in bundles, and between these a sparse plexus of thin single fibres appeared. Injection of WGA-HRP into superior cervical ganglion labelled a plexus of nerve fibres on the ipsilateral circle of Willis and the (rostral) basilar artery. These experiments demonstrated the origin and distribution of sympathetic and sensory innervation to major cerebral arteries in the rat.

Somatotopic organization of cutaneous afferent terminals and dorsal horn neuronal receptive fields in the superficial and deep laminae of the rat lumbar spinal cord

The somatotopic organization of A- and C-afferent fibre terminals in the dorsal horn of the rat lumbar spinal cord was compared with the spatial location of second-order dorsal horn neuronal mechanoreceptive fields. The central terminal fields of the sural, saphenous, and tibial nerve were mapped by labelling the nerves with horseradish peroxidase (HRP). A previous study used the transganglionic transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) to produce a somatotopic map of high-threshold C-fibre terminal fields in lamina II (Swett and Woolf: J. Comp. Neurol. 231:66-77, '85). In the present study the terminal fields of low-threshold A beta afferents that terminate in laminae III and IV were mapped by using unconjugated HRP at prolonged survival times (72 hours). Unfixed tissue was used to increase the sensitivity of the tetramethylbenzidine reaction, thus allowing these afferent terminals to be clearly seen. The general spatial arrangement of the terminal fields in laminae III/IV closely resembled that found in lamina II in the mediolateral and rostrocaudal planes but because of a dorsoventral obliquity of the afferent terminals, the superficial and deeper fields are not in strict vertical register. The input to laminae II-IV of the dorsal horn may therefore be viewed as two horizontally arranged sheets of afferent terminals both accurately representing the skin surface, the more superficial sheet representing the high-threshold C-afferents and the deeper sheet, low-threshold A-beta afferents. The spatial organization of high-threshold A-delta afferents in laminae I and V appears to be quite different, with a transverse rather than a longitudinal orientation. To study dorsal horn cell receptive field organization two single units with mechanoreceptive fields were recorded extracellularly in each of 87 vertical tracks in the lumbar spinal cord, one unit in the superficial dorsal horn and the second in the deep dorsal horn. In general the somatotopic organization of the receptive fields of both sets of units followed that of the afferent terminal fields but there were cells with receptive fields that were anomalous relative to the recording site. No evidence of any vertical relation or columnar arrangement in receptive field size, threshold, or location on the body surface was found when comparing the two units in a pair. Furthermore, no laminar functional specialization was found, the majority of neurones having both low- and high-threshold inputs.(ABSTRACT TRUNCATED AT 400 WORDS)

Tyrosine hydroxylase in the rat parabrachial region: ultrastructural localization and extrinsic sources of immunoreactivity

We sought to determine the ultrastructural localization and the extrinsic sources of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), in the lateral parabrachial region (PBR) of adult male rats. In the first portion of the study, a rabbit antiserum to TH was immunocytochemically localized in coronal sections through the lateral PBR from acrolein-fixed brains using the peroxidase-antiperoxidase method. Electron-microscopic analysis revealed that perikarya and dendrites with peroxidase immunoreactivity for TH constituted only 17% of the total labeled profiles. Afferents to the TH-labeled perikarya and dendrites usually failed to exhibit immunoreactivity and were thus considered noncatecholaminergic. Somatic synapses were most commonly detected on small immunoreactive perikarya in the central lateral nucleus of the PBR. Other labeled perikarya located in the dorsal lateral or ventral lateral nuclei received few somatic synapses and were morphologically distinct in terms of their larger size, infolded nuclear membrane, and abundance of cytoplasmic organelles. Axons and axon terminals with peroxidase immunoreactivity constituted the remaining labeled profiles in the lateral PBR. These terminals primarily formed symmetric synapses with unlabeled and a few labeled dendrites. The labeled axon terminals were categorized into 2 types: Type I was small (0.3-0.6 micron), contained many small clear vesicles, and exhibited few well-defined synaptic densities. The second type was large (0.8-1.4 micron), contained both small clear and large dense core vesicles, and exhibited well-defined synaptic densities. The 2 types of terminals were morphologically similar to dopaminergic terminals. The location of catecholaminergic neurons contributing to the TH-labeled terminals was determined by combining peroxidase-antiperoxidase immunocytochemistry for TH with retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). The tracer was unilaterally injected into the PBR of anesthetized adult rats. Immunocytochemical labeling for TH was seen as a brown reaction product within neurons in known catecholaminergic cell groups. A black granular reaction product formed by a cobalt-intensified and diaminobenzidine-stabilized tetramethyl benzidine reaction for WGA-HRP was evident within many TH-labeled and unlabeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Topographical distribution of dorsal and median raphe neurons projecting to motor, sensorimotor, and visual cortical areas in the rat.

The present study was conducted to examine the spatial organization of dorsal (DR) and median (MR) raphe neurons that project to rostrocaudally aligned areas of the rat cerebral cortex. An additional goal was to determine if individual DR cells that send efferents to forelimb sensorimotor or visual regions of the neocortex also send axon collaterals to forelimb (crus II) or visual (paraflocculus) areas of the cerebellum. Long-Evans hooded rats received unilateral pressure injections of horseradish peroxidase (HRP) in either motor (n = 4) or sensorimotor (n = 5) or visual (n = 4) cortex to determine the intranuclear location of DR and MR neurons that project to specific neocortical regions. Coronal sections (40-100 microns) through the pons and midbrain were examined by light microscopy after the tetramethyl benzidine reaction and neutral red counterstaining were carried out. The locations of retrogradely labeled cells were recorded relative to a three-dimensional biological coordinate system maintained by a computer linked to the light microscope. For double labeling studies, unilateral injections of fast blue and nuclear yellow were made in paired motor (sensorimotor cortex and crus II of the lateral cerebellum) or visual (cortical area 17 and paraflocculus) areas of the CNS. Coronal tissue sections (35 microns) were collected on coverslips and examined on a Leitz fluorescence microscope (wavelength = 365 nm). DR neurons labeled from cerebrocortical injections of HRP were concentrated in the rostral two-thirds of the nucleus. HRP-filled neurons were distributed such that individual groups of neurons projecting to motor, sensorimotor, or visual cortex were aligned in a partially overlapping, rostral to caudal array. In the dorsoventral dimension, retrogradely labeled cells were clustered in three distinct groupings such that neurons projecting to the motor, sensorimotor, and visual areas were concentrated in dorsal, intermediate, and ventral portions of the DR nucleus, respectively. For all cases, the majority of HRP-filled cells were positioned along the midline or displaced to the side of the nucleus that was ipsilateral to the cortical injection site. A small number of retrogradely labeled neurons were observed in the MR following injections in the motor cortex. Computer-assisted reconstruction of the neuroanatomical data facilitated the visualization of spatial relationships between groups of DR neocortical projection neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural localization and afferent sources of substance P in the rat parabrachial region

The ultrastructural morphology and afferent sources of terminals containing substance P-like immunoreactivity were examined in the rat parabrachial region. In the first portion of the study, a polyclonal antiserum to substance P was localized in the ventrolateral parabrachial region using the peroxidase-antiperoxidase labeling technique combined with electron microscopy. The antiserum was tested for cross-reaction with substance P, physalaemin, substance K and neuromedins B, C and K. Cross-reactivity was most intense with substance P. However, substance K, neuromedin K and physalaemin also exhibited limited cross-reactions with the antiserum. In the ventrolateral parabrachial region of untreated adult animals, substance P-like immunoreactivity was localized in axon terminals containing numerous small (40–60 nm) clear vesicle and 1–3 large (90–120 nm) dense-core vesicles. At least 54% of the labeled terminals formed asymmetric synapses with unlabeled dendrites; and at least 30% of the recipient dendrites received more than one labeled axon terminal. In addition, the labeled terminals were associated less frequently with other unlabeled soma, axon terminals and blood vessels. In the second part of the study, we examined whether or not perikarya in various extrinsic regions contributed to the substance P-like immunoreactivity in axon terminals in the parabrachial region. Wheat-germ agglutinin conjugated horseradish peroxidase was injected unilaterally into the parabrachial region of adult rats two days prior to being killed and one day prior to intraventricular injection of colchicine (100 μg in 7.5μ1 saline) which enhanced the detection of immunoreactivity in perikarya. Sections were first processed by a tetramethylbenzidine reaction stabilized with cobalt-diaminobenzidine for demonstration of the transported peroxidase then were immunocytochemically labeled for substance P. Perikarya containing both the black granular retrograde labeling and brown peroxidase-immunoreactivity were found in the nuclei of the solitary tracts, the caudal ventrolateral reticular formation, the lateral dorsal tegmental nucleus and the paraventricular, dorsomedial and lateral hypothalamic nuclei. The projections were largely, but not exclusively, from perikarya located on the same side as the parabrachial injection. We conclude that substance P, or a closely related tachykinin, is a putative transmitter or modulator within a number of pathways to the parabrachial region and that these afferents act primarily through axodendritic synapses with intrinsic neurons.

Stabilization of TMB Reaction Product for electron microscopic retrograde and anterograde fiber tracing

Use of the highly sensitive tetramethylbenzidine (TMB) method of horseradish peroxidase histochemistry for electron microscopy has been limited by the solubility of the reaction product in aqueous and alcoholic solutions. We have found that following the TMB reaction with a diaminobenzidine-cobalt (DAB-Co) step causes the TMB crystals to become coated with DAB-Co. The resultant reaction complex is insoluble, and easily localized using electron microscopy. By systematically varying the pH at which the TMB reaction is run, the size and shape of the reaction complex can be controlled. The pH 4.0 reaction complex was the most suitable for electron microscopic identification of labeled structures less than 1.0 μm in diameter (e.g., axon terminals).

Evidence for a ‘Paravascular’ fluid circulation in the mammalian central nervous system, provided by the rapid distribution of tracer protein throughout the brain from the subarachnoid space

The protein tracer, horseradish peroxidase (HRP), was infused into the lateral cerebral ventricles or subrachnoid space of anesthetized cats and dogs after insertion of a cisternal cannula to permit drainage of cerebrospinal fluid (CSF) and tre tracer solution. The intracerebral distribution of the tracer was then determined by light microscopy of serial brain sections after postinfusion intervals of 4 min-2 h. For the localization of HRP, sections were incubated with diaminobenzidine (DAB) or the much more sensitive chromogen, tetramethylbenzidine (TMB). The TMB reaction showed a consistent ‘paravascular’ distribution of tracer reaction product, within the perivascular spaces (PVS) around large penetrating vessels and in the basal laminae around capillaries, far beyond the termination of the PVS. After infusion of HRP over 4 min, arterioles were surrounded by the tracer, but capillaries and venules were usually less densely demarcated; by 6 min, however, the intraparenchymal microvasculature was outlined in toto throughout the forebrain and brainstem. Electron microscopy of sections incubated in DAB after 10 or 20 min HRP circulation confirmed the paravascular location of the reaction product, which was also dispersed throughout the extracellular spaces (ECS) of the adjacent parenchyma. Our results demonstrate that solutes in the CSF have access to the ECS throughout the neuraxis within minutes via fluid pathways paralleling the intraparenchymal vasculature. The rapid paravascular influx of HRP could be prevented by stopping or diminishing the pulsations of the cerebral arteries by aortic occlusion or by partial ligation of the brachiocephalic artery. The exchange of solutes between the CSF and the cerebral ECS has generally been attributed to diffusion, however, HRP enters the neuraxis along the intraparenchymal microvasculature far more rapidly than can be explained on this basis. This apparent convective tracer influx may be facilitated by transmission of the pulsations of the cerebral arteries to the microvasculature. We postulate that a fluid circulation through the CNS occurs via paravascular pathways.