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Fast Blue (FB) and Cholera Toxin-B (CTB) are two retrograde tracers extensively used to label alpha-motoneurons (α-MNs). The overall goals of the present study were to (1) assess the effectiveness of different FB and CTB protocols in labeling α-MNs, (2) compare the labeling quality of these tracers at standard concentrations reported in the literature (FB 2% and CTB 0.1%) versus lower concentrations to overcome tracer leakage, and (3) determine an optimal protocol for labeling α-MNs in young B6SJL and aged C57Bl/J mice (when axonal transport is disrupted by aging). Hindlimb muscles of young B6SJL and aged C57Bl/J mice were intramuscularly injected with different FB or CTB concentrations and then euthanized at either 3 or 5 days after injection. Measurements were performed to assess labeling quality via seven different parameters. Our results show that tracer protocols of lower concentration and shorter labeling durations were generally better in labeling young α-MNs, whereas tracer protocols of higher tracer concentration and longer labeling durations were generally better in labeling aged α-MNs. A 0.2%, 3-day FB protocol provided optimal labeling of young α-MNs without tracer leakage, whereas a 2%, 5-day FB protocol or 0.1% CTB protocol provided optimal labeling of aged α-MNs. These results inform future studies on the selection of optimal FB and CTB protocols for α-MNs labeling in normal, aging, and neurodegenerative disease conditions. [ABSTRACT FROM AUTHOR]

Cannabis sativa is the drug of abuse most cultivated, trafficked, and consumed worldwide. One of several techniques used to detect cannabinoids is based on the thin‐layer chromatography (TLC). However, the designation of the colors observed can be inaccurate and not reproducible. The designation of colors goes beyond physical and physiological aspects, because what is conventionally called color is a socio‐cultural construction. Thus, the objective of this paper was to evaluate the different TLC methods to detection of cannabinoids, and apply standardization method in naming of colors. TLC analysis performed using silica gel 60 F254 as a stationary phase. Three mobile phase compositions [hexane:chloroform (8:2 v:v), hexane:ethyl ether (8:2 v:v), and chloroform:hexane (8:2 v:v)], as well as, two different solutions of Fast Blue B salt (FBBS, Azoic Diazo No. 48) and Fast Blue RR (FBRR, Azoic Diazo No. 24) were evaluated. Determination of colors names was realized through the Sci‐Chromus® software. The best resolution was obtained using hexane:ethyl ether (8:2 v:v) as a mobile phase. It was observed that although the cannabidiol (CBD), delta‐9‐tetrahydrocannabinol (Δ9‐THC), cannabinol (CBN), and cannabigerol (CBG) were detect using both the FBBS‐ and FBRR‐acidified solutions, the best visualization was achieved using the latter reagent. To the best of our knowledge, this is the first study that applied and demonstrated a method for standardization and denomination of colors in the TLC analysis of cannabinoids. This method was able to reduce the subjectivity in naming the colors observed and presented several application possibilities. [ABSTRACT FROM AUTHOR]

The autonomic nervous system is a sophisticated and independent structure composed of two antagonistic (opposing) divisions (sympathetic and parasympathetic) that control many vital functions including: homeostasis maintenance, heart rate, blood circulation, secretion, etc. Thymus is one of the most important primary lymphoid organs playing a role in the developing of a juvenile's immune system mainly by maturation, development, and migration of T-cells (T lymphocytes). In the last decades, several studies identifying sources of the thymic autonomic supply have been undertaken in humans and several laboratory rodents but not in higher mammals such as the pig. Therefore, in the present work, retrograde tracing technique of Fast Blue and DiI was used to investigate the sources of sympathetic efferent supply to the porcine thymus. After Fast Blue injection into the right lobe of the thymus, the presence of Fast Bluepositive neurons was found in the unilateral cranial cervical ganglion (82.8 ± 3.0% of total Fast Blue-positive neurons) as well as in the middle cervical ganglion (17.2 ± 3.0%). Injection of DiI resulted in the presence of retrograde tracer in neurons of the cranial cervical ganglion (80.4 ± 2.3% of total amount of DiI-labelled neurons), the middle cervical ganglion (18.4 ± 1.9%), and the cervicothoracic ganglion (1.2 ± 0.8%). The present report provides the first data describing in details the localization of primary efferent sympathetic neurons innervating the porcine thymus. [ABSTRACT FROM AUTHOR]

[Display omitted] • Mg x Zn 1-x O (x = 0, 0.25, 0.5, 0.75 and 1) NPs were synthesized by Chonemorpha fragrans leaf extract assisted solution combustion method. • The photocatalytic degradation of Fast Blue (FB) and Malachite Green (MG) dyes were tested for all the samples. • MG dyes has higher (90%) degradation compared with FB (87% degradation) dye due to rise in the hydroxyl ions. • The low charge transfer resistance and high capacitance value of Mg x Zn 1-x O electrode were studied for all the samples. • The Nyquist plot is in the order MgO ˃ Zn 0.25 Mg 0.75 O ˃ Zn 0.50 Mg 0.50 O ˃ Zn 0.75 Mg 0.25 O ˃ ZnO. Chonemorpha fragrans leaf extract assisted solution combustion method was carried out for synthesizing Mg x Zn 1−x O (x = 0, 0.25, 0.5, 0.75 and 1) nanoparticles. The obtained samples were characterized using powder X-ray diffraction, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, and diffuse reflection spectroscopy techniques. The powder X-ray diffraction patterns of Mg x Zn 1−x O (x = 0 and 1) show pure hexagonal and cubic phases respectively and x = 0.25, 0.5, and 0.75 shows mixed phases. The Rietveld refinement study was done to refine structural parameters of × = 0, 0.5, and 1. A scanning electron microscope shows the high agglomeration with increasing 'x' content. Based on the Williamson-Hall plot, the average size of crystallites was found to be between 25 and 40 nm. Transmission electron microscope results also revealed the formation of nanoparticles. The energy bandgap was calculated from diffuse reflectance spectra using the Kubelka Munk relation and found to vary from 3.2 to 5.28 eV. The photocatalytic degradation of Fast Blue and Malachite Green dyes for all the Mg x Zn 1−x O nanoparticles catalysts was tested under the irradiation of UV light. Mg x Zn 1−x O nanoparticles showed enhanced degradation with increasing 'x' contents for Fast Blue (87%) and Malachite Green (90%) dyes under UV light. This enhanced degradation is due to variation in the bandgap of Mg x Zn 1−x O. Mg x Zn 1−x O electrodes exhibit a reversible electrode reaction based on their low charge transfer resistance and high capacitance value. Based on our results, the synthesized nanoparticles are promising candidates for wastewater treatment and lead sensor applications. [ABSTRACT FROM AUTHOR]

The study was aimed to establish the contribution of the dorsal branch of the accessory nerve ( DBXI) to the innervation of the porcine trapezius muscle ( TRAP). Combined retrograde tracing using fluorescent tracer Fast blue ( FB) and surgical denervation procedure (excision of DBXI segment) were applied . FB+ neurons supplying the cervical (c- TRAP) and thoracic part (th- TRAP) of TRAP were localized in following nerve centres: the ipsilateral ventral horn of the grey matter of cervical neuromers, ipsilateral spinal ganglia and bilateral sympathetic chain ganglia. After the excision of DBXI segment, no FB+ motoneurons supplying c- TRAP were found while the mean number of those supplying th- TRAP was significantly decreased. A slight decrease in average numbers of sensory and autonomic neurons implemented in the innervation of both parts of TRAP was also observed. This study has revealed that in the pig DBXI is the only motor pathway to c- TRAP while the vast majority of motoneurons supplying porcine th- TRAP send their axons via DBXI. [ABSTRACT FROM AUTHOR]

Background IGLEs represent the only low-threshold vagal mechanosensory terminals in the tunica muscularis of the esophagus. Previously, close relationships of vesicular glutamate transporter 2 ( VGLUT2) immunopositive IGLEs and cholinergic varicosities suggestive for direct contacts were described in almost all mouse esophageal myenteric ganglia. Possible cholinergic influence on IGLEs requires specific acetylcholine receptors. In particular, the occurrence and location of neuronal muscarinic acetylcholine receptors (mAChR) in the esophagus were not yet characterized. Methods This study aimed at specifying relationships of VGLUT2 immunopositive IGLEs and vesicular acetylcholine transporter ( VAChT)-immunopositive varicosities using pre-embedding electron microscopy and the location of mAChR1-3 (M1-3) within esophagus and nodose ganglia using multilabel immunofluorescence and retrograde tracing. Key Results Electron microscopy confirmed synaptic contacts between cholinergic varicosities and IGLEs. M1- and M2-immunoreactivities (-iry; -iries) were colocalized with VGLUT2-iry in subpopulations of IGLEs. Retrograde Fast Blue tracing from the esophagus showed nodose ganglion neurons colocalizing tracer and M2-iry. M1-3-iries were detected in about 80% of myenteric ganglia and in about 67% of myenteric neurons. M1- and M2-iry were present in many fibers and varicosities within myenteric ganglia. Presynaptic M2-iry was detected in all, presynaptic M3-iry in one-fifth of motor endplates of striated esophageal muscles. M1-iry could not be detected in motor endplates of the esophagus, but in sternomastoid muscle. Conclusions & Inferences Acetylcholine probably released from varicosities of both extrinsic and intrinsic origin may influence a subpopulation of esophageal IGLEs via M2 and M1-receptors. [ABSTRACT FROM AUTHOR]

A method is described for studying the morphological features of extensive axonal projections within the central nervous system of the gerbil, Meriones anguiculatus. Potentially long descending axonal projections between the auditory thalamus and lower brainstem were used as a model. The inferior colliculus (IC) in the tectum was injected in vivo with a fluorescent retrograde tracer, Fluoro-Gold, to label cells in the medial geniculate body (MGB) that had descending projections to the IC, and cells in the superior olivary complex (SOC) that had ascending projections to the IC. Another fluorescent retrograde tracer, fast blue, was injected into the cochlea to label olivocochlear (OC) cells in the SOC. Inferomedially curved parasagittal slices containing ipsilateral auditory cell groups from the thalamus to the brainstem were cut and descending axons of the pre-labeled MGB cells were traced anterogradely with Biocytin. After visualizing histologically the injected Biocytin, discretely labeled IC-projecting axons of the MGB cells were traced including their collaterals that extended further into the SOC. In the SOC, these axons terminated on pre-labeled cells including OC cells. The combination of anterograde and retrograde tracing in the slice preparations described here demonstrated extensive descending axonal projections from the thalamus to their targets in the lower brainstem that had known ascending/descending projections within the auditory system. [ABSTRACT FROM AUTHOR]

Abstract: The amygdala is activated by fear and plays an important role in the emotional response to life-threatening situations. When rats feel threatened, they respond by biting fiercely. Bite strength is regulated by the trigeminal motor nucleus and the mesencephalic trigeminal nucleus (Me5). The Me5 relays proprioceptive signals from the masticatory muscles and the periodontal ligaments to the trigeminal motor and premotor nuclei. The amygdala projects to the trigeminal motor nucleus and the premotor reticular formation. However, it is unknown whether the amygdala projects directly to the Me5. In the present study, neurons of the central amygdaloid nucleus (ACe) were labeled following injection of a retrograde tracer, Fast Blue, into the caudal Me5, and fibers and terminal buttons from the ACe to the Me5 were examined after injections of an anterograde neuronal tracer, biotinylated dextran amine into the ACe. Furthermore, wheat germ agglutinin-conjugated to horseradish peroxidase was injected into the ACe, and labeled fibers and terminal buttons in the Me5 were examined by electron microscopy. Labeled terminal buttons on Me5 somata were more abundant in the caudal than the rostral Me5. Electron microscopic observation revealed that a part of these terminal buttons formed axo-somatic synapses. These results indicate that the ACe sends direct projections to the Me5, and suggest that the amygdala regulates bite strength by modifying neuronal activity in the Me5. [Copyright &y& Elsevier]

Background: Whole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round. Results: We show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated. Conclusion: Since POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms. [ABSTRACT FROM AUTHOR]

Abstract: Despite the contribution of nodose ganglia neurons to the innervation of the heart being the subject of several studies, specific neuronal subpopulations innervating the four different chambers of the heart have not been distinguished. In our study, the application of Fast Blue-loaded patch to the epicardial surface of different chambers of the rat heart (the right or left atrium or the right or left ventricle) resulted in labeling of discrete populations of immunohistochemically diverse neurons. About one half (55%) of these neurons showed immunoreactivity for the 200-kDa neurofilament protein (marker of myelinated neurons), with a higher proportion of positive staining among neurons projecting to the left than to the right ventricle. Isolectin B4 immunoreactivity (characteristic for a subset of nonmyelinated non-peptidergic neurons) was more abundant among neurons projecting to the right side of the heart (right atria and right ventricles) compared to the left side (23% vs. 16%). Calretinin immunoreactivity (possible marker of mechanosensitive neurons) was significantly higher among neurons projecting to the ventricles than among those projecting to atria (36% vs. 11%). These findings reveal that chambers of the rat heart are innervated with immunohistochemically different subpopulations of neurons from the nodose ganglia. [Copyright &y& Elsevier]

Preconditioning sciatic nerve injury enhances axonal regeneration of ascending sensory neurons after spinal cord injury. A key question is whether direct injury of sensory nerves is necessary for the enhanced regeneration. The lumbar 5 ventral root transection (L5 VRT) model, a model of selective motor nerve injury, provides a useful tool to address this question. Here we examined the effects of a preconditioning L5 VRT on the regeneration after a subsequent dorsal column transection (DCT) in adult Sprague–Dawley rats. We found that L5 VRT 1 week before DCT increased the number of Fast Blue (FB)-labeled neurons in the L5 dorsal root ganglia (DRG) and promoted sprouting/regenerating axons to grow into the glial scar. L5 VRT also induced a dramatic upregulation of expression of brain-derived neurotrophic factor (BDNF) in the preconditioned DRG and in the injured spinal cord. Moreover, almost all of the FB-labeled sprouting/regenerating neurons expressed BDNF, and approximately 55% of these neurons were surrounded by p75 neurotrophin receptor-positive glial cells. This combined injury led to an increase in the number of BDNF- and TrkB-immunoreactive nerve fibers in the dorsal column caudal to the lesion site. Taken together, these findings demonstrate that L5 VRT promotes sprouting/regeneration of ascending sensory neurons, indicating that sensory axotomy may not be essential for the plasticity of injured dorsal column axons. Thus, the sensory neurons could be preprimed in the regenerative milieu of Wallerian degeneration and neuroinflammation, which might alter the expression of neurotrophic factors and their receptors, facilitating sprouting/regeneration of ascending sensory neurons. [ABSTRACT FROM AUTHOR]

[Display omitted] • Activation of 5-HT7Rs improved behavioral impairments. • CA1 pyramidal excitability enhanced and synaptic capacity decreased. • Activation of 5-HT7Rs restored LTP, but their inhibition produced LTP impairment. • Blockade of 5-HT7Rs did not prevent resistance to the LTD induction. • Retrograde fast blue-labeled neurons in raphe nuclei were reduced. Autism spectrum disorder (ASD) is a severe life-long neuropsychiatric disorder. Alterations and imbalance of several neurochemical systems may be involved in ASD pathophysiology, of them, serotonergic neurotransmission dysfunction and deficiency may underlie behavioral abnormalities associated with ASD. However, the functional importance of serotonergic receptors, particularly 5HT7 receptors in ASD pathology remains poorly defined. Serotonin receptor subtype 7 (5-HT7R) plays a direct regulatory role in the development and also for the mature function of the brain, therefore, further studies are necessary to elucidate the role of these receptors in the etiology of autism. To address this issue, we combined here behavioral, electrophysiological methods to further characterize the contribution of 5-HT7Rs in the prenatal valproic acid (VPA) exposure-induced impairment in synaptic plasticity and their impact on the associated behavioral changes. This may help to unravel the underlying cellular mechanisms involved in ASD and can lead to new treatment and/or prevention therapies based on the role of the serotonergic system for autism. Findings revealed that compared to control, autistic-like offspring showed increased anxiety-like behavior, reduced social interaction, decreased locomotor activity, and impaired identification of the novel object. However, administration of 5-HT7Rs agonist, LP-211, for 7 consecutive days before testing from postnatal day 21 to 27 reversed all behavioral deficits induced by prenatal exposure to VPA in offspring. Also, both short-term depression and long-term potentiation were impaired in the autistic-like pups, but activation of 5-HT7Rs rescued the LTP impairment in the autistic-like group so that there was no significant difference between the two groups. Blockade of 5-HT7Rs caused LTP impairment following HFS in the autistic-like group. Besides, there was a significant difference in LTD induction following SB-269970 application between the control and the autistic-like groups measured at first 10 min following TPS. Moreover, both the number and the size of retrograde fast blue-labelled neurons in the raphe nuclei were reduced. Overall, these results provide for the first time, as far as we know, functional evidence for the restorative role of 5-HT7Rs activation against prenatal VPA exposure induced behavioral deficits and hippocampal synaptic plasticity impairment. Therefore, these receptors could be a potential and promising pharmacotherapy target for the treatment of autism. [ABSTRACT FROM AUTHOR]

Abstract: It is morphologically demonstrated that the subfornical organ (SFO) projects to the paraventricular hypothalamic nucleus (PVN) and also projects to the nucleus preopticus medianus (POMe), a relay nucleus of indirect projections from the SFO to PVN. However, it remains unknown, whether or not SFO neurons project collaterally to the POMe and PVN. To confirm this, a double retrograde labeling method was performed on rats using two fluorescent tracers. One tracer (red-colored FluoSpheres: FSR) was injected into the POMe and the other (Fast Blue: FB) was injected into the unilateral PVN at the same time. As a result, many retrogradely labeled neurons were found in the entire SFO. Of these, some neurons showed both FSR and FB fluorescence. Double-labeled neurons were found in about 8.7% of FSR-labeled neurons and 15.5% of FB-labeled neurons. The existence of double-labeled neurons indicates that single neurons in the SFO project simultaneously to the POMe and PVN via collateral axon branches. The data suggest that there are complicated neuronal pathways originating from the SFO in regulating cardiovascular and body fluid homeostasis. [Copyright &y& Elsevier]

Abstract: A common method for identifying collateral projections is to inject different retrograde tracers into two targets and examine labeled cells for the presence of both tracers. Double-labeled cells are considered to have collateral projections to the two injection sites. This method is widely considered to underestimate the extent of collaterals. To test the efficiency of double-labeling, we mixed equal volumes of two tracers, injected them into one site in a guinea-pig brain, and counted the resulting labeled cells. Ideally, the tracers would have precisely overlapping injection sites and all labeled cells would contain both tracers. We tested several combinations of tracers: 1) Fast Blue and fluorescein dextran; 2) fluorescein dextran and FluoroGold; 3) fluorescein dextran and FluoroRuby; 4) FluoroGold and green beads; 5) FluoroGold and red beads; 6) FluoroRuby and green beads; and, 7) green beads and red beads. For each combination, a mixture was injected into the left inferior colliculus. After 1 week to allow for transport, labeled cells were counted in the right inferior colliculus and the left temporal cortex. For each mixture, the results were similar for the two areas. The percentage of cells that were double-labeled varied from 0% to 100%, depending on tracer combination. The highest efficiencies (>96%) were observed with red beads and green beads or with FluoroRuby and fluorescein dextran. The limited efficiency of other mixtures could be accounted for only in part by incomplete overlap of the two tracers at the injection site. The results indicate that the specific combination of tracers used to search for collateral projections can greatly affect the findings. [Copyright &y& Elsevier]

The topography and the main characteristics of the vestibular nuclear complex (VNC) in cattle have been studied in serially transversally cut Nissl and Gles-stained sections. By using computerized image analysis software, the cell size, the maximum and minimum diameter of the neurons of each vestibular nucleus were obtained. These parameters were statistically analyzed by comparing the cell population from different nuclei and different parts of each nucleus. Furthermore, in order to investigate the lumbo-sacral projections, the fluorescent tracer Fast Blue was injected into the L6-S1 spinal cord of three calves. Among the vestibular nuclei, the superior was the least extensive rostro-caudally, the medial was the most extensive and contained the smallest cells, the lateral showed the largest neurons, and the descending nucleus contained cells of intermediate size which decreased in a rostrocaudal direction. Concerning the lumbo-sacral projections of the bovine VNC, the present study showed that only the fibers coming from the lateral vestibular nucleus reached the L6-S1 spinal cord. The labelled neurons were most heavily concentrated in the dorsal portion of this nucleus, but scattered neurons were also observed throughout the entire extension of the nucleus. The differences between the descriptions of cattle and other species were described. [ABSTRACT FROM AUTHOR]

We examined transient receptor potential (TRP) V1 and TRPV2 expression in calcitonin gene-related peptide (CGRP) positive (+) primary sensory neurons projecting to the skin and skeletal muscles of the rat dorsum. Among the dorsal root ganglia at the levels from C2 to Th1, 34.9% of neurons projecting to the skin were positive for CGRP, and 32.6% or 21.6% of neurons projecting to the trapezius muscle or the longissimus muscle were positive for CGRP. Of the small CGRP+ neurons projecting to the skin, 53.5% were positive for TRPV1, 11.6% were positive for TRPV2. Of the small CGRP+ neurons projecting to the trapezius or the longissimus, 53.1 or 53.2% were positive for TRPV1, 8.8 or 8.3% were positive for TRPV2, respectively. In the periphery, 29.3% of CGRP+ nerve fibers were positive for TRPV1 in the skin, whereas 65.0 or 59.8% were positive in the trapezius or the longissimus. Therefore, the present study showed that the percentage of CGRP+ neurons projecting to the trapezius is higher than that to the longissimus, and that the co-localization percentage of CGRP and TRPV1 on the sensory nerves was also higher in the trapezius than in the longissimus and the skin. [ABSTRACT FROM AUTHOR]

Previous publications have provided different descriptions of the topographical organization of the facial nucleus of the pig. Since swine is used in biomedical research due to its embryological, anatomical and physiological similarities to human, we have reinvestigated the anatomical organization of the facial nucleus with application of fluorescent retrograde tracer Fast Blue, antibody to choline acetyltransferase and acetylcholinesterase histochemistry. Our findings demonstrate that in the porcine medulla facial motoneurons constitute a large cellular group occupying the ventro-lateral medulla. The neuronal group is interposed rostro-caudally between the superior and inferior olive, and located ventro-medially to the spinal nucleus of the trigeminal nerve. The present results clarify the anatomical description of this important brain stem nucleus in the pig. [ABSTRACT FROM AUTHOR]

Abstract: This study assesses the effects of ATP and GTP on the kinetic properties of voltage-gated K+ currents in anatomically identified postganglionic sympathetic neurons innervating the submandibular gland and the masseter muscle in rats. Three types of K+ currents were isolated: the I Af steady-state inactivating at more hyperpolarized potentials, I As steady-state inactivating at less hyperpolarized potentials than I Af and the I K current independent of membrane potential. The kinetic properties of these currents were tested in neurons with ATP (4 mM) and GTP (0.5 mM) or without ATP and GTP in the intracellular solution.In glandular and muscular neurons in the absence of ATP and GTP in the intracellular solution, the current density of I Af was significantly larger (142 pA/pF and 166 pA/pF, respectively) comparing to cells with ATP and GTP (96 pA/pF and 100 pA/pF, respectively). The I As was larger only in glandular neurons (52 pA/pF vs. 37 pA/pF).Conversely, I K current density was smaller in glandular and muscular neurons without ATP and GTP (17 pA/pF and 31 pA/pF, respectively) comparing to cells with ATP and GTP (57 pA/pF and 58 pA/pF, respectively). In glandular (15.5 nA/ms vs. 6.9 nA/ms) and muscular (10.9 nA/ms vs. 7.5 nA/ms) neurons, the I Af activated faster in the absence of ATP and GTP. Half inactivation voltage of I Af in glandular (−110.0 mV vs. −119.7 mV) and muscular (−108.4 vs. −117.3 mV) neurons was shifted towards depolarization in the absence of ATP and GTP. We suggest that the kinetic properties of K+ currents in glandular and muscular sympathetic neurons change markedly in the absence of ATP and GTP in the cytoplasm. Effectiveness of steady-state inactivated currents (I Af and IAS) increased, while effectiveness of steady-state noninactivated currents decreased in the absence of ATP and GTP. The effects were more pronounced in glandular than in muscular neurons. [Copyright &y& Elsevier]

Voltage-gated Na+ currents were recorded from anatomically identified postganglionic muscular sympathetic neurons without and with ATP and GTP in the intracellular solution. The main findings of the study were that cells without ATP and GTP in the intracellular solution express a higher amplitude and greater density of voltage-gated Na+ current, and their Na+ current activates faster and also inactivates faster time dependently. The current is also steady-state inactivated to a lesser degree and recovers from inactivation more slowly in cells without added ATP and GTP. These findings suggest that the presence of ATP and GTP, substrates for channel phosphorylation, changes the kinetic properties of Na+ currents. [Copyright &y& Elsevier]

The expression and functional responses of P2X receptors in bladder and cutaneous sensory neurons of adult rats and mice have been studied using immunohistochemistry and patch clamp techniques. Cell bodies of bladder pelvic afferents were identified in L6 and S1 dorsal root ganglia (DRG), following Fast Blue injection into the muscle wall of the urinary bladder. Similarly, cutaneous sensory neurons were identified in L3 and L4 DRG, following Fast Blue injection into the saphenous nerve innervating the skin. Bladder sensory neurons contained only weak to moderate P2X3-immunoreactivity (IR), in contrast to strong P2X3-IR observed in a sub-population of cutaneous afferents. Whole-cell patch-clamp recordings revealed that approximately 90% of bladder afferent neurons responded to αβ-methylene ATP (αβmeATP) and ATP (30 μM) with persistent currents, which were inhibited by 2′,3′-O-trinitrophenyl-ATP (TNP-ATP) (0.3 μM) to 6.4±1.9% and 8.0±2.6% of control, respectively (n=8). The remaining bladder sensory neurons demonstrated biphasic, transient or no response to P2X agonists. In contrast, only 24% of cutaneous afferent neurons gave persistent currents to αβmeATP (30 μM), with 66% of cells giving transient or biphasic currents and the remaining 10% being non-responsive. Our results suggest that, in contrast to DRG neurons in general, bladder sensory neurons projecting via pelvic nerves express predominantly P2X2/3 heteromeric receptors, which are likely to mediate the important roles of ATP as a signaling molecule of urinary bladder filling and nociception. [Copyright &y& Elsevier]

Whereas basic features of post-axotomy muscle reinnervation have been extensively studied in rats, little is known about axonal regrowth and pathfinding in cats. To address the question, adult cats were subjected to facial-facial anastomosis (FFA). First group served to establish optimal parameters for labeling of the zygomatic and buccal facial branches with 1,1' dioctadecyl - 3,3,3,'3' - tetramethylindo-carbocyanine perchlorate (DiI) and Fast Blue (FB) placed onto respective transected nerves. The second group of animals underwent identical bilateral labeling 3 months after transection and suture of the right facial nerve. This group served to establish the number of motoneurons, which had branched after surgery and projected into both facial branches. On control side. DiI application onto zygomotico-orbital branch labeled 3883 ± 598 (mean ± S.D.) perikarya were confined to the dorsal and intermediate facial subnuclei, meanwhile an application of FB onto the buccal branch labeled 1617 ± 552 perikarya in the lateral and ventrolateral subnuclei. There were no doubled-labeled cells. Three months after FFA all retrogradely labeled motoneurons were scattered throughout the entire facial nucleus. To establish the proportion of perikarya, that er-grew multiple axonal branches into both nerves, double-labeled (FB + DiI) mononeurons were counted from digital images. The zygomatico-orbital nerve contained 3311 ± 430 DiI-labeled whereas the buccal nerve 1500 ± 442 FB-labeled motoneurons. The occurrence of 311 ± 103 double-labeled perikaya (DiI+FB) suggested that approximately 6% of all retrogradely labeled motoneurons branched axons into both nerves. I conclude that malfunctioning axonal pathfinding rather than deviant reinnervation contributed to poor recovery of function after FFa in the cat. [ABSTRACT FROM AUTHOR]

We have assessed the expression and kinetics of voltage-gated K+ currents in cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of a fluorescent tracer into the pericardial sack. Ninety-nine neurons were labelled: 24% small (diameter<30 μm), 66% medium-sized (diameter 30 μm>.48 μm) and 10% large (>48 μm) neurons. Current recordings were performed in small and medium-sized neurons. The kinetic and pharmacological properties of K+ currents recorded in these two groups of neurons were identical and the results obtained from these neurons were pooled. Three types of K+ currents were identified:a) IAs, slowly activating and slowly time-dependently inactivating current, with V1/2 of activation −18 mV and current density at +30 mV equal to 164 pA/pF, V1/2 of inactivation at −84 mV. b) IAf current, fast activating and fast time-dependently inactivating current, with V1/2 of activation at two mV and current density at +30 mV equal to 180 pA/pF, V1/2 of inactivation at −26 mV. At resting membrane potential IAs was inactivated, whilst IAf, available for activation. The IAs currents recovered faster from inactivation than IAf current. 4-Aminopiridyne (4-AP) (10 mM) and tetraethylammonium (TEA) (100 mM) produced 98% and 92% reductions of IAf current, respectively and 27% and 66% of IAs current, respectively. c) The IK current that did not inactivate over time. Its V1/2 of activation was −11 mV and its current density equaled 67 pA/pF. This current was inhibited by 95% (100 mM) TEA, whilst 4-AP (10 mM) produced its 23% reduction. All three K+ current components (IAs, IAf and IK) were present in every small and medium-sized cardiac DRG neuron.We suggest that at hyperpolarized membrane potentials the fast reactivating IAs current limits the action potential firing rate of cardiac DRG neurons. At depolarised membrane potentials the IAf K+ current, the reactivation of which is very slow, does not oppose the firing rate of cardiac DRG neurons. [Copyright &y& Elsevier]

The dental pulp is a favorable model for studies of interactions between nociceptive sensory neurons and their peripheral target tissues. In the present study, we retrogradely labeled pulpal afferent neurons with an improved method that permits monitoring of changes in neuronal phenotype in response to controlled tooth injuries. The capacity of retrograde neuronal tracers to diffuse through dentinal tubules was exploited, thereby avoiding the severe injury to the pulp associated with previous tracer application methods. The strategy was to apply the durable fluorescent tracer, Fluoro-gold (FG), to exposed dentin in the floor of shallow cavities in molars, in order to pre-label pulpal neurons in trigeminal ganglia of young adult Sprague–Dawley rats. A high percentage of pupal afferent neurons were retrogradely labeled by application of FG to exposed dentin and the FG fluorescent signal persisted in most labeled neurons for at least 8 weeks. Following tracer application to dentin, the pulp tissue appeared normal histologically, with the exception that a layer of reactive dentin was deposited at the pulp-dentin border beneath the shallow cavities. Assessment of expression of calcitonin gene-related peptide (CGRP) and brain derived neurotrophic factor (BDNF) indicated that pulpal neurons remained in a quiescent, baseline condition cytochemically following application of tracer to cavities in dentin and upregulation of these markers could be detected in neurons that projected to teeth that received a test injury subsequent to tracer application. Thus, labeling of trigeminal neurons via dentinal tubules provides the basis for a useful model for precisely assessing properties of pulpal afferents in both quiescent and activated states. [Copyright &y& Elsevier]

This study presents the kinetic and pharmacological properties of voltage-gated Ca2+ currents in anatomically defined cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of fluorescent tracer Fast Blue into the pericardial sack. There were three distinct groups of neurons with respect to cell size: small (27% of total; cell capacitance <30 pF), medium (65% of total; capacitance 30–80 pF) and large neurons (8% of total; capacitance >80 pF). The properties of Ca2+ currents were tested in small and medium-sized neurons. In large neurons currents could not be adequately controlled and were not analysed. Ca2+ currents did not completely inactivate during 100 ms depolarising voltage steps. The activation thresholds in small (−36.9±1.3 mV) and medium (−39.0±1.3 mV) size neurons were similar. Current densities were 105.8 pA/pF in small and 97.4 pA/pF in large neurons and also did not differ. The kinetic properties of activation and inactivation did not differ between small and medium-sized cardiac DRG neurons. At membrane potentials between −50 and −60 mV (the expected resting membrane potential in these neurons) 55 to 70% of Ca2+ currents in small and medium-sized neurons were available for activation. Both, small and medium-sized neurons expressed similar proportions of L (7.5%), N (25%) and P/Q (36%) type Ca2+ currents. We conclude that small and medium-sized cardiac DRG neurons are homogeneous with respect to the expression and properties of voltage-gated Ca2+ currents. Voltage-gated Ca2+ currents probably play an important role in action potential generation in cardiac DRG neurons due to their availability for activation at resting membrane potential, their high density and voltage threshold close to the threshold for voltage-gated Na+ currents. [Copyright &y& Elsevier]

The expression and properties of voltage-gated Na+ currents in cardiac dorsal root ganglion (DRG) neurons were assessed in this study. Cardiac DRG neurons were labelled by injecting the Fast Blue fluorescent tracer into the pericardium. Recordings were performed from 138 cells. Voltage-dependent Na+ currents were found in 115 neurons. There were 109 neurons in which both tetrodotoxin-sensitive (TTX-S, blocked by 1 μM of TTX) and tetrodotoxin-resistant (TTX-R, insensitive to 1 μM of TTX) Na+ currents were present. Five cells expressed TTX-R current only and one cell only the TTX-S current. The kinetic properties of Na+ currents and action potential waveform parameters were measured in neurons with cell membrane capacitance ranging from 15 to 75 pF. The densities of TTX-R (110.0 pA/pF) and TTX-S (126.1 pA/pF) currents were not significantly different. Current threshold was significantly higher for TTX-R (−34 mV) than for TTX-S (−40.4 mV) currents. V1/2 of activation for TTX-S current (−19.6 mV) was significantly more negative than for TTX-R current (−9.2 mV), but k factors did not differ significantly. V1/2 and the k constant for inactivation for TTX-S currents were −35.6 and −5.7 mV, respectively. These values were significantly lower than those recorded for TTX-R current for which V1/2 and k were −62.3 and −7.7 mV, respectively. The action potential threshold was lower, the 10–90% rise time and potential width were shorter before than after the application of TTX. Based on this we drew the conclusion that action potential recorded before adding tetrodotoxin was mainly TTX-S current dependent, while the action potential recorded after the application of toxin was TTX-R current dependent. We also found 23 cells with mean membrane capacitance ranging from 12 to 35 pF (the smallest labelled DRG cells found in this study) that did not express the Na+ current. The function of these cells is unclear. We conclude that the overwhelming majority of cardiac dorsal root ganglion neurons in which voltage-dependent Na+ currents were present, exhibited both TTX-S and TTX-R Na+ currents with remarkably similar expression and kinetic properties. [Copyright &y& Elsevier]

Abstract Insulin and glucose play a key role in the control of body energy homeostasis. However, the anatomical organization of the network of central insulin and glucose sensitive areas is still unclear. In the present study, we used a multiple-labelling technique combining retrograde tracing and Fos-like immunohistochemistry, to analyse the anatomical projections from hypothalamic neurones activated by the combined stimulus of insulin and glucose. After intraperitoneal injections of a bolus of insulin plus glucose, Fos-like immunoreactive neurones were observed in the paraventricular nucleus (PVN), ventromedial and arcuate nuclei, as well as the lateral hypothalamic area. In addition, neurones projecting to the autonomic preganglionic levels in the brainstem and spinal cord potentially involved in the control of glucose metabolism were identified by injections of fluorochrome tracers. Thus, Fluorogold was injected into the intermediolateral cell column of the lower spinal cord and Fast Blue was injected into the dorsal motor nucleus of the vagus. Perikarya of descending neurones were detected chiefly in the dorsal, medial and lateral parvocellular subnuclei and also in the posterior magnocellular subnucleus of the PVN. In contrast, insulin-glucose activated neurones in the PVN were observed mainly in the medial parvocellular and posterior magnocellular subnuclei. Fluorogold/Fos double-labelled neurones were only observed in the ventral zone of the medial parvocellular subnucleus. These data indicate that, within the PVN, there could be neurones responding to insulin-glucose administration, which are involved in the sympathetic control of the classical regulatory structures of body energy homeostasis, such as the liver and pancreas, and which could play a role in the output of the neuronal circuitry controlling food intake. [ABSTRACT FROM AUTHOR]

Abstract This paper presents the kinetic and pharmacological properties of voltage-gated K+ currents in anatomically identified glandular postganglionic sympathetic neurons isolated from the superior cervical ganglia in rats. The neurons were labelled by injecting the fluorescent tracer Fast Blue into the submandibular gland. The first group of neurons remained intact, i.e. innervated by the preganglionic axons until the day of current recordings (control neurons). The second group of neurons was denervated by severing the superior cervical trunk 4–6 weeks prior to current recordings (decentralized neurons). In every control and decentralized neuron three categories of voltage-dependent K+ currents were found. (i) The I Af K+ current, steady state, inactivated at hyperpolarized membrane potentials. This current was fast activated and fast time-dependently inactivated, insensitive to TEA and partially depressed by 4-AP. (ii) The I As K+ current, which was steady-state inactivated at less hyperpolarized membrane potentials than I Af . The current activation and time-dependent inactivation kinetics were slower than those of I Af . I As was blocked by TEA and partially inhibited by 4-AP. (iii) The I K K+ current did not undergo steady-state inactivation. In decentralized compared to control neurons the maximum I Af K+ current density (at +50 mV) increased from 116.9 ± 8.2 to 189.0 ± 11.5 pA/pF, the 10–90% current rise time decreased from 2.3 to 0.7 ms and the recovery from inactivation was faster. Similarly, in decentralized compared to control neurons the maximum I As K+ current density (at +50 mV) increased from 49.9 ± 3.5 to 74.3 ± 5.0 pA/pF, the 10–90% current rise time shortened from 29 to 16 ms and the recovery from inactivation of the current was also... [ABSTRACT FROM AUTHOR]