Injections Of Horseradish Peroxidase Research Articles

The pathways connecting the hippocampal formation, the thalamic reuniens nucleus and the thalamic reticular nucleus in the rat

Most dorsal thalamic nuclei send axons to specific areas of the neocortex and to specific sectors of the thalamic reticular nucleus; the neocortex then sends reciprocal connections back to the same thalamic nucleus, directly as well indirectly through a relay in the thalamic reticular nucleus. This can be regarded as a 'canonical' circuit of the sensory thalamus. For the pathways that link the thalamus and the hippocampal formation, only a few comparable connections have been described. The reuniens nucleus of the thalamus sends some of its major cortical efferents to the hippocampal formation. The present study shows that cells of the hippocampal formation as well as cells in the reuniens nucleus are retrogradely labelled following injections of horseradish peroxidase or fluoro-gold into the rostral part of the thalamic reticular nucleus in the rat. Within the hippocampal formation, labelled neurons were localized in the subiculum, predominantly on the ipsilateral side, with fewer neurons labelled contralaterally. Labelled neurons were seen in the hippocampal formation and nucleus reuniens only after injections made in the rostral thalamic reticular nucleus (1.6-1.8 mm caudal to bregma). In addition, the present study confirmed the presence of afferent connections to the rostral thalamic reticular nucleus from cortical (cingulate, orbital and infralimbic, retrosplenial and frontal), midline thalamic (paraventricular, anteromedial, centromedial and mediodorsal thalamic nuclei) and brainstem structures (substantia nigra pars reticularis, ventral tegmental area, periaqueductal grey, superior vestibular and pontine reticular nuclei). These results demonstrate a potential for the thalamo-hippocampal circuitry to influence the functional roles of the thalamic reticular nucleus, and show that thalamo-hippocampal connections resemble the circuitry that links the sensory thalamus and neocortex.

The Milk-Ejection Reflex in the Sheep: An Anatomical Study on the Afferent Pathway

The present study is an attempt to reveal the spinal and supraspinal organization of the ascending branch of the milk-ejection reflex in the ewe by means of a tract-tracing technique. For this purpose, injections of horseradish peroxidase (HRP) were performed into the lateral cervical nucleus (LCN) and into the hypothalamic paraventricular nucleus (PVN). Peroxidase injections into the LCN revealed retrogradely labelled neurons in the medial part of laminae I-III of the ipsilateral L3 and L4 spinal segments, while injections of HRP into the PVN revealed retrogradely labelled cells in the contralateral LCN and the medial cuneate nucleus. Taking into account the results obtained, it is concluded that the transmission of the afferent input from the nipples to the PVN is accomplished by at least two pathways: one employing a single relay station located in the medial cuneate nucleus, and another possessing two relay stations located in the medial part of laminae I-III of the dorsal horn of L3 and L4 spinal segments and in the LCN.

GABA- and glycine-like immunoreactivity in axonal endings presynaptic to the vibrissa afferents in the cat trigeminal interpolar nucleus

The goal of this study was to analyze the synaptic interaction of primary afferents with GABA- and/or glycine-immunopositive presynaptic endings in the cat trigeminal interpolar nucleus (Vi). Fast adapting vibrissa afferents were labeled by intra-axonal injections of horseradish peroxidase. Postembedding immunogold labeling on serially cut ultrathin sections and quantitative ultrastructural analysis of the labeled boutons and their presynaptic endings (p-endings) in the Vi were performed. The majority of p-endings presynaptic to labeled boutons (83%) were immunopositive for both GABA and glycine and 8% were immunopositive for glycine alone. A small fraction of p-endings were immunopositive for GABA alone (4%) or immunonegative for both GABA and glycine (4%). Ultrastructural parameters related to synaptic release, i.e. bouton volume, mitochondrial volume, and active zone area, were significantly larger in the labeled boutons of primary afferents than in the p-endings. The volume of labeled boutons was positively correlated with the number of the postsynaptic dendrites and p-endings. In addition, fairly large-sized labeled boutons and p-endings were frequently observed in the Vi. These results reveal that large majority of vibrissa afferents in the Vi are presynaptically modulated by interneurons immunopositive for both GABA and glycine, and suggest that the Vi plays a distinct role in the processing of orofacial sensory information, different from that of other trigeminal sensory nuclei.

Matrix Metalloproteinase-9 Is Involved in the Development and Plasticity of Retinotectal Projections in Rats

Objective: During postnatal development, retinotectal projections undergo a process of misplaced axon elimination, leading to a topographical matching between the retinal surface and the superior colliculus. Matrix metalloproteinases (MMPs) have been implicated in the development and plasticity of the nervous system. We studied the expression and role of MMPs during normal development of retinotectal projections and after monocular enucleation-induced plasticity. Material and Methods: Lister hooded rats at different postnatal ages received subpial ethylene vinyl acetate 40W implants to deliver an MMP inhibitor or vehicle to the superior colliculus. Animals received intraocular injections of horseradish peroxidase for anterograde tracing of ipsilateral projections. For immunoblotting and zymography, colliculi were removed without fixation. Results: We observed the highest MMP activity in the first postnatal week, with decreasing activity thereafter. Monocular enucleation at postnatal day 10 yielded a rapid increase in MMP activity, 24 h following denervation of the contralateral colliculus. Importantly, inhibition of MMP activity in vivo induced a marked delay of axonal clustering along the medial aspect of colliculus. Conclusions: Our data indicate that MMPs are crucial in retinotectal development concurring to the fine tuning of topographical order and synaptic specificity of these connections.

Vascular Endothelial Growth Factor and Nitric Oxide Production in Response to Hypoxia in the Choroid Plexus in Neonatal Brain

Damage to the choroid plexus in 1-day-old Wistar rats subjected to hypoxia was investigated. The mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), endothelial, neuronal, inducible nitric oxide synthase (eNOS, nNOS, iNOS), and vascular endothelial growth factor (VEGF) along with nitric oxide (NO) production and VEGF concentration was up-regulated significantly in hypoxic rats. Ultrastructurally, the choroid plexus epithelial cells showed massive accumulation of glycogen. A striking feature was the extrusion of cytoplasmic fragments from the apical cell surfaces into the ventricular lumen following the hypoxic insult. Intraventricular macrophages showed increased expression of complement type 3 receptors, major histocompatibility complex class I and II antigens, and ED1 antigens. Following an intravenous injection of horseradish peroxidase (HRP), a large number of intraventricular macrophages were labeled suggesting enhanced leakage of the tracer from the blood vessels in the choroid plexus connective tissue stroma into the ventricular lumen. We suggest that increased production of NO in hypoxia is linked to the structural alteration of the choroid plexus, and along with VEGF, may lead to increased vascular permeability. Melatonin treatment reduced VEGF and NO levels as well as leakage of HRP suggesting its potential value in ameliorating damage in choroid plexus pathologies.

Tectorotundal connections in turtles: An electron microscopic tracing and GABA-immunocytochemical study

The nucleus rotundus of the turtles Emys orbicularis and Testudo horsfieldi was analysed by axonal tracing methods and post-embedding GABA immunocytochemistry. After injections of horseradish peroxidase or biotinylated dextran amine into the optic tectum, electron microscopic observations showed that the vast majority of ipsilateral tectorotundal axon terminals were small in size, had smooth contours and contained small, round, densely packed synaptic vesicles. These terminals were GABA-immunonegative, often gathered in clusters, and established asymmetrical synaptic contacts with either small- or medium-sized GABA-negative dendritic profiles and with GABA-immunoreactive (GABA-ir) dendrites, which did not contain synaptic vesicles. Occasional GABA-ir-labelled axon terminals were observed; these may arise from the rare GABAergic neurons in the central tectal layer, or from neurons in the ventral pretectal nucleus, which projects both to the optic tectum and nucleus rotundus. In addition to tracer-labelled axon terminals, we observed both GABA-negative and GABA-ir cell bodies and dendrites also labelled by the tracer. No GABA-ir presynaptic dendritic profiles containing synaptic vesicles were observed. The existence in reptiles of reciprocal connections between the nucleus rotundus and the optic tectum as a phylogenetically ancient feedback system is discussed.

Blood–retinal barrier disruption and ultrastructural changes in the hypoxic retina in adult rats: the beneficial effect of melatonin administration

Reactive changes in astrocytes and Müller cells in the retina of adult rats subjected to hypoxia were investigated. Along with this, the integrity of the blood-retinal barrier (BRB) was assessed using fluorescent and electron-dense tracers. In hypoxic rats, mRNA and protein expression of glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQ4) were significantly increased. AQ4 immunoreactive cells were identified as astrocytes and Müller cells by double immunofluorescence labelling. Another alteration in the hypoxic retina was marked reduction in melatonin content compared to controls. In this connection, administration of exogenous melatonin reduced the tissue concentration of vascular endothelial growth factor (VEGF) and nitric oxide (NO); both were elevated in hypoxic rats. A major structural change in the hypoxic retina was swelling of astrocyte and Müller cell processes but this was noticeably attenuated after melatonin administration. Following an intraperitoneal or intravenous injection of rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP), leakage of both tracers was observed in the retina in hypoxic rats but not in the controls, indicating that the functional integrity of the BRB is compromised in hypoxia/reoxygenation. It is suggested that enhanced tissue concentration of VEGF and NO production in the hypoxic retina contribute to increased permeability of the retinal blood vessels. The concurrent up-regulation of AQ4, a water-transporting protein, in astrocytes and Müller cells in hypoxia suggests its involvement in oedema formation. Since melatonin effectively reduced the vascular permeability in the retina of hypoxic rats, as evidenced by reduced leakage of RhIC, we suggest that its administration may be of potential benefit in the management of retinal oedema associated with retinal hypoxia.

The somatotopic organization of trigeminal premotoneurons in the cat brainstem

This study was performed to complement the results of prior intracellular recording and labeling studies by investigating the general distribution pattern of trigeminal premotoneurons in the cat brainstem using the retrograde tracing methods. The results of the present study reconfirmed the presence of premotoneurons in the trigeminal principal and oral nuclei following horseradish peroxidase injections into the jaw-opening (JO) or jaw-closing (JC) nucleus. Furthermore, we found that labeled cells from the JO nucleus and JC nucleus located in the reticular regions surrounding the trigeminal motor nucleus (Vmo; Vmo shell region) were arranged in a topographic fashion, while those in the parabrachial nucleus, supratrigeminal nucleus, lateral reticular formation caudal to the shell region and raphe nuclei were intermingled with each other. The labeling in the individual nuclei was bilateral with an ipsilateral predominance to each injection site, with the exception of the mesencephalic trigeminal nucleus, where the labeling was ipsilateral to the injection site in the JC nucleus. These results, combined with the data of the previous intracellular tracing studies, indicate that based on the presence of somatotopic organization, premotoneurons can be largely divided into two groups; those projecting to either the JO or the JC nucleus and those projecting to the two nuclei, and we offer the suggestion that roles of premotoneurons for jaw movements differ among the individual nuclei.

Macromolecular transport in heart valves. I. Studies of rat valves with horseradish peroxidase

The present study aims to experimentally elucidate subtle structural features of the rat valve leaflet and the related nature of macromolecular transport across its endothelium and in its subendothelial space, information necessary to construct a rational theoretical model that can explain observation. After intravenous injection of horseradish peroxidase (HRP), we perfusion-fixed the aortic valve of normal Sprague-Dawley rats and found under light microscopy that HRP leaked through the leaflet's endothelium at very few localized brown spots, rather than uniformly. These spots grew nearly as rapidly with HRP circulation time before euthanasia as aortic spots, particularly when the time axis only included the time the valve was closed. These results suggest that macromolecular transport in heart valves depends not only on the direction normal to, but also parallel to, the endothelial surface and that convection, as well as molecular diffusion, plays an important role in macromolecular transport in heart valves. Transmission electron microscopy of traverse leaflet sections after 4-min HRP circulation showed a very thin ( approximately 150 nm), sparse layer immediately beneath the endothelium where the HRP concentration was much higher than that in the matrix below it. Nievelstein-Post et al.'s (Nievelstein-Post P, Mottino G, Fogelman A, Frank J. Arterioscler Thromb 14: 1151-1161, 1994) ultrarapid freezing/rotary shadow etching of the normal rabbit valve's subendothelial space supports the existence of this very thin, very sparse "valvular subendothelial intima," in analogy to the vascular subendothelial intima.

Breathing and calling: Neuronal networks in the Xenopus laevis hindbrain

Xenopus laevis is an aquatic anuran with a complex vocal repertoire. Unlike terrestrial frogs, vocalizations are independent of respiration, and a single muscle group--the laryngeal dilators--produces underwater calls. We sought to identify the premotor neural network that underlies vocal behaviors. Vocal patterns generated by premotor networks control laryngeal motor neurons in cranial nucleus (n.) IX-X. Glottal motor neurons, active during respiration, are also present in n.IX-X. We used horseradish peroxidase (HRP), Lucifer yellow, and fluorescently conjugated dextrans to characterize the organization of n.IX-X and to trace premotor neuron projections. Premotor nuclei include the inferior reticular formation (Ri) adjacent to n.IX-X and the pretrigeminal nucleus of the dorsal tegmental area of the medulla (DTAM), the primary descending input to n.IX-X. Intramuscular HRP injections revealed a spatially segregated pattern, with glottal motor neurons in anterior n.IX-X and laryngeal motor neurons in the caudal portion of the nucleus. Dextran injections identified commissural n.IX-X neurons that project to the contralateral motor nucleus and DTAM-projecting n.IX-X neurons. Both neuronal types are clustered in anteromedial n.IX-X, closely associated with glottal motor neurons. Ri neurons project to ipsilateral and contralateral DTAM. Projections from DTAM target n.IX-X bilaterally, and all four identified subtypes receive DTAM input. In contrast, Ri neurons receive little input from DTAM. We hypothesize that connectivity between neurons in n.IX-X, Ri and DTAM may provide mechanisms to generate laryngeal and glottal activity patterns and that DTAM may coordinate vocal and respiratory motor pools, perhaps acting to switch between these two mutually exclusive behaviors.

Functional connection from Mauthner cell to homologous reticulospinal neurons in the goldfish hindbrain

Crossed vestibular inhibition first described in mammals, also occurs in many species ranging from reptiles to mammals, but was nct observed in frog and toadfish. During a study of the inhibitory pathways of the Mauthner cell (M-cell) of a teleost, two classes of interneurons were individualized. Their identification was made possible by intracellular injection of horseradish peroxidase (HRP) after they had been physiologicaIly identified by the presence of an intracellularly recorded passive hyperpolarizing potential induced by the firing of their adjacent M-cell. Interneurons of the first group are part of the recurrent collateral network of the M-cell; they have some similarities with the Renshaw cells. The second group is composed of commissural vestibule-vestibular neurons. In a recent study of the neurons inhibiting the M-cell of teleosts, it was demonstrated that each bouton established on the M-cell by the commissural and recurrent collateral interneurons functions as an independent all-or-none unit. This result has prompted the investigation of the ultrastructure of these endings to determine if they were morphologically different from those found at other central chemically transmitting synapses.This chapter deals with deals with the relation that was found between the morphology and the function of a set of neurons that are known to inhibit the M-cell, in this case second order vestibulo-vestibular interneurons.

Spatial distribution patterns of excitatory and inhibitory synapses in the dendritic tree differ between jaw-closing and -opening motoneurons

This paper reviews recent data on the spatial distribution of inhibitory and excitatory synapses on the dendritic tree of jaw-closing (JC) and -opening (JO) motoneurons in the cat, in which a combination of techniques employing intracellular injections of horseradish peroxidase and postembedding immunogold labelling was used. The dendritic tree is divided into three segments: primary and distal dendrites and intermediate dendrites between the two segments. The proportion of inhibitory boutons (immunoreactive for GABA and/or glycine) is slightly higher than proportion of excitatory boutons (immunoreactive for glutamate) in JC motoneurons, but this trend is reversed in JO motoneurons. In the two kinds of motoneuron, boutons immunoreactive to glycine alone are more numerous than boutons double-labelled to GABA and glycine, which, in turn, occur more frequently than boutons immunoreactive to GABA alone. In JC motoneurons, the packing density (number of boutons per 100 μm 2) of the inhibitory boutons decreases somatofugally, but this trend is not applicable to the excitatory boutons. In contrast, the packing density of the inhibitory and excitatory boutons in JO motoneurons does not significantly differ among the three dendritic compartments, though it is slightly higher for the excitatory than the inhibitory ones on each dendritic segment. These differences have important implications for synaptic integration in JC and JO motoneurons.

The spinal cord connections of the myofascial trigger spots

Background Recent electrophysiological studies revealed that endplate noise (EPN) could be specifically recorded from a myofascial trigger point (MTrP) region. EPN has been considered as the focal graded potentials due to excessive acetylcholine release in neuromuscular junction. A recent histological study has demonstrated a free nerve ending at the vicinity of the site, from where EPN could be recorded in an MTrP region. However, the sensory (afferent) and the motor (efferent) connections between an MTrP and the spinal cord still has never been fully studied. Aims The aim of this study was to delineate both motor and sensory connections between an MTrP and the spinal cord by applying the stain with horseradish peroxidase (HRP). Methods Nine Wistar rats were studied. When the rat was anesthetized, its biceps femoris muscles were exposed for localizing the myofascial trigger spot (MTrS, equivalent to MTrP in human). In one side, a monopolar Teflon-coated, hollow-needle electrode was used for searching EPN in an MTrS region, and then HRP was injected via this hollow-needle electrode into the site where EPN was recorded. HRP was also injected into a normal (non-taut band, non-MTrS) site in the contralateral side to obtain the control data. Two days after HRP injection, the rats were sacrificed and their spinal cords and dorsal root ganglia (DRG) were sectioned for the identification of the sites where neurons were labeled with HRP. Results The HRP-labeled neurons were found in the ventral horn of the spinal cord and in the DRG over L3, L4, and L5, while most were found in the L5 level. The mean numbers of HRP-labeled neurons in the EPN side looked smaller than that in the control side, but the difference did not reach statistically significant level ( P > 0.05). The mean values of the diameters of the HRP-labeled neurons in the DRG were not significantly different between the EPN side and the control side ( P > 0.05). However, HRP-neurons in the ventral horn of the spinal cord in the EPN side showed mild tendency to be smaller than that in the control side. Conclusions The spinal cord connections of an MTrS are basically similar to that for a normal tissue region. The motor neurons related to MTrS tended to be smaller in their diameters. The findings in this study further supported the previously proposed hypotheses for the pathogenesis of an MTrP.

Premotor circuits controlling eyelid movements in conjunction with vertical saccades in the cat: II. Interstitial nucleus of Cajal

Vertical saccadic eye movements are accompanied by concurrent eyelid movements in the same direction. The interstitial nucleus of Cajal (InC) controls eye position for vertical eye movements and may also control saccade-related lid position as well. This study investigates whether the InC serves as a premotor center for eyelid saccades, by employing dual-tracer methods in cats to label both the projections of the InC and the motoneurons supplying the levator palpebrae superioris (LPS) muscle, which lie in the caudal central subdivision (CCS) of the oculomotor complex. Injections of biotinylated dextran amine (BDA) into the InC anterogradely labeled axons that terminated bilaterally throughout the CCS and in the oculomotor nuclei proper. Labeled terminals lay in close association with labeled LPS motoneurons, which were retrogradely labeled following injections of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the muscle. Ultrastructural investigation revealed that most terminals contained spherical vesicles and formed asymmetric synaptic contacts with the labeled motoneurons. These results strongly suggest that the InC monosynaptically controls lid movements in conjunction with vertical eye movements, including saccades. To identify the neurons of origin for this pathway, WGA-HRP injections were centered in the CCS. These experiments indicate that lid and eye motoneurons may share a common source of bilateral InC input. Thus, a common vertical position signal may be employed to maintain the lid and eye at appropriate elevations during fixation, such that the lid sits just above the pupil, allowing unobstructed vision, but at the ready to protect the cornea.

Ultrastructural synaptic features differ between α‐ and γ‐motoneurons innervating the tibialis anterior muscle in the rat

We investigated the synaptology of retrogradely labeled spinal motoneurons after injection of horseradish peroxidase into the tibialis anterior (TA) muscle of adult rat. In total, 32 TA motoneurons were investigated in the electron microscope and demonstrated a bimodal size distribution with cell diameter peaks at 40 microm and 20 microm, likely representing alpha- and gamma-motoneurons, respectively. Both alpha- and gamma-motoneurons were apposed by S- and F-type synaptic boutons, whereas only alpha-motoneurons demonstrated inputs by the large M- and C-type boutons. The proportion of cell body membrane covered by synaptic inputs was surprisingly indistinguishable between alpha-motoneurons (72.2%) and gamma-motoneurons (63.5%). The ratio between the number of F- and S-type boutons in apposition with the motoneuron cell body (F/S ratio) and the ratio between the soma membrane coverage provided by F- and S-type boutons were both significantly higher in alpha- than in gamma-motoneurons. When comparing our data with previous findings in other species, we conclude that rat TA alpha-motoneurons are similar to cat and primate alpha-motoneurons with regard to synaptic terminal morphology, frequency, and distribution. However, rat gamma-motoneurons show a markedly higher total synaptic coverage and frequency than cat gamma-motoneurons, although both species exhibit appositions made by the same synaptic types and similar ratios between inhibitory and excitatory inputs.

Ultrastructure of synaptic input to medial olivocochlear neurons

Medial olivocochlear (MOC) neurons project from the brain to the cochlea to form the efferent limb of the MOC reflex. To study synaptic inputs to MOC neurons, we retrogradely labeled these neurons using horseradish peroxidase injections into the cochlea. Labeled neurons were identified in the ventral nucleus of the trapezoid body and documented with the light microscope before being studied with serial-section electron microscopy. MOC somata and dendrites were innervated by three different types of synapses, distinguished as either having: 1) large, round synaptic vesicles and forming asymmetric contacts; 2) small, round vesicles plus a few dense core vesicles and forming asymmetric contacts; or 3) pleomorphic vesicles and forming symmetric contacts. The first two types were the most frequent on somata. Acetylcholinesterase-stained material confirmed that the type containing large, round vesicles is most common on dendrites. We kept track of the synaptic terminals in serial sections and compiled them into three-dimensional swellings. Swellings with large, round vesicles formed up to seven synapses per swelling, were largest in size, and sometimes formed complex arrangements engulfing spines of MOC neurons. Swellings with small, round vesicles formed up to four synapses per swelling. The morphology of this type of synapse, and the moderate sizes of the swellings forming it, suggests that it originates from posteroventral cochlear nucleus stellate/multipolar neurons. This input may thus provide the sound-evoked input to MOC neurons that causes their reflexive response to sound.

Hypoxia‐induced astrocytic reaction and increased vascular permeability in the rat cerebellum

Hypoxia is an important factor linked to induction of vascular leakage and formation of brain edema. In this connection, astrocytes associated closely with the blood vessels are deemed to be involved. This study investigated the response of astrocytes to hypoxia in the adult rat cerebellum, and along with this, the integrity of the blood-brain barrier (BBB) was assessed using fluorescent and electron dense tracers. In rats subjected to hypoxia, mRNA and protein expression of hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), glial fibrillary acidic protein (GFAP), and aquaporin-4 (AQ4) was significantly increased. VEGF and AQ4 immunoreactive cells were identified as astrocytes by double immunofluorescence labeling. Increased VEGF tissue concentration and astrocytic swelling as observed in hypoxic rats were reduced after melatonin administration. Following intraperitoneal or intravenous injection of rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP), leakage of both tracers was observed in hypoxic rats but not in the controls indicating that functional integrity of BBB is compromised in hypoxia/reoxygenation. Enhanced gene and protein expression of VEGF may contribute to increased permeability of blood vessels. AQ4, a water transporting protein, is upregulated in astrocytes in hypoxia suggesting the cells are involved in edema formation. To this end, melatonin may be beneficial in reducing edema as it reduced VEGF concentration and, hence, vascular permeability.

Connections of the zona incerta to the reticular nucleus of the thalamus in the rat.

This study demonstrated that there is a pathway from the zona incerta to the thalamic reticular nucleus. Injections of horseradish peroxidase or Fluorogold were made, using stereotaxic coordinates, into the rostral, intermediate or caudal regions of the thalamic reticular nucleus of adult Sprague-Dawley rats. The results show that the different regions of the thalamic reticular nucleus have distinct patterns of connections with the sectors of the zona incerta. In terms of the relative strength of the connections, injections made into the rostral regions of the thalamic reticular nucleus showed the highest number of labelled cells within the rostral and ventral sectors of the zona incerta; injections made into the intermediate regions of the thalamic reticular nucleus showed labelled cells in the dorsal and ventral sectors; while injections to the caudal regions of the thalamic reticular nucleus showed only a few labelled cells in the caudal sector of the zona incerta. Previous studies have shown that the zona incerta projects to the higher order thalamic nuclei but not first order thalamic nuclei. The labelling observed in the present study may represent collaterals of zona incerta to higher order thalamic nuclei projections.

Differential effects of hydroxyacetophenone analogues on the transcytotic vesicular pathway in rat liver

Insertion of transporter proteins into the apical canalicular membrane via vesicular transport is one of several choleretic mechanisms. Based on different choleretic activities of hydroxyacetophenone analogues including 4-mono; 2,6-di and 2,4,6-trihydroxy-acetophenone (MHA, DHA and THA), the present study aims to determine if these compounds stimulated vesicular transport in hepatocytes. Hydroxyacetophenone was continuously infused into the duodenum of the bile fistula rat. Bile flow rate was allowed to stabilize and then followed by an intraportal injection of horseradish peroxidase, a marker of the transcytotic vesicle pathway. MHA which stimulates bile acid independent flow, showed a dose-dependent increase in both the early (paracellular) and late (transcellular) peak of horseradish peroxidase excretion in bile. THA, which stimulates both bile acid dependent flow and bile acid independent flow, did not alter the pattern of horseradish peroxidase excretion into bile. However, DHA, which is more hydrophobic and increases only bile acid dependent flow, decreased the late peak. The stimulating effects of MHA on bile flow and horseradish peroxidase excretion were markedly inhibited by colchicine, suggesting that its choleretic action involves stimulation of exocytosis, as well as increase in paracellular permeability. In contrast, the lack of a stimulatory effect of THA and DHA on biliary horseradish peroxidase excretion suggested that their choleretic action is not associated with vesicular exocytosis. These results demonstrate a variable effect of hydroxyacetophenones on the transcytotic vesicular pathway reflecting different choleretic mechanisms and therapeutic potential.

Synaptic plasticity in the adult spinal dorsal horn: The appearance of new functional connections following peripheral nerve regeneration

Peripherally regenerated fibers were impaled in the dorsal columns. Each impaled fiber's adequate stimulus was determined and the fiber was activated by passing brief (200 ms) current pulses through the microelectrode. Cord dorsum potentials (CDPs) elicited by fiber stimulation were recorded at 8 sites, and then the fiber was injected with Neurobiotin (NB). In the same preparations, dorsal horn cells were impaled and their receptive fields (RFs) mapped; areas of skin from which the most vigorous responses were elicited were noted. Needle electrodes inserted into these cutaneous “hot spots” were used to electrically activate minimal numbers of peripherally regenerated fibers while simultaneously recording the resulting CDPs and any intracellular EPSPs. This allowed determination of connectivity between regenerated fibers and dorsal horn cells with overlapping RFs. In agreement with findings in intact animals, NB revealed long-ranging collaterals which were not seen using intraaxonally injected horseradish peroxidase (HRP). Although there was no qualitative difference in their morphology compared to those seen in controls, the correlation between spatial distribution of boutons and amplitudes of the monosynaptic CDPs of peripherally regenerated fibers revealed significant shifts in the functional efficacy of many central connections. Transcutaneous electrical stimulation revealed a significantly higher incidence of connectivity between regenerated fibers and cells with overlapping RFs at 9–12 months (86%) than at 5–6 months (34%). Although there was no obvious anatomical reorganization of afferent projections in the dorsal horn, the observed functional changes with time following transection show the formation of new functional central connections.