Medium spiny neuron projection from the rat striatum: An intracellular horseradish peroxidase study

The morphological features of striatal projection neurons and the responses of these neurons to electrical stimulation of the substantia nigra were studied in rats through the methods of intracellular recording and intracellular labeling with the enzyme horseradish peroxidase. Under urethane anesthesia, single striatal neurons were first analyzed for responsiveness to nigral stimuli and then filled electrophoretically with the enzyme in order to permit subsequent serial reconstruction.The axon of the medium spiny neuron was found to form an extensive collateral plexus within the striatum before entering the globus pallidus or the internal capsule. These medium spiny projection neurons responded to nigral stimuli with monosynaptic excitation.     

Afferent connections to the lateral hypothalamus: A horseradish peroxidase study in the rat

Horseradish peroxidase, 13% Sigma Type VI, was administered iontophoretically to the mid lateral hypothalamus (LH) of male hooded rats. Animals were perfused intracardially on the following day and brains were removed and sliced in the coronal or sagittal planes into 30–50 μm sections. Sections were processed with DAB and BDH for the brown and blue reaction products and later examined by bright and dark field microscopy for the presence and location of retrogradely labeled neurons. Results indicate that a significant number of afferent connections to the LH originate in the olfactory and accumbens nuclei, pyriform cortex, olfactory tracts, magnocellular and medial preoptic and anterior hypothalamic regions, stria terminalis, stria hypothalamic tract, diagonal tract of Broca, caudate-putamen and globus pallidus, internal capsule, lateral septal nuclei, lateral preoptic area and anterior medial forebrain bundle, the various amygdaloid nuclei, zona incerta, perifornical region, dorsal and ventral medial hypothalamic areas, supraoptic, paraventricular and periventricular nuclei, posterior hypothalamus and medial forebrain bundle, ventral thalamic nuclei, the fields of Forel, arcuate and mammillary nuclei, adjacent to the fasciculus retroflexus, in the ventral tegmental area of Tsai, interpeduncular nucleus, substantia nigra, mesencephalic reticular formation, periaqueductal gray, locus coeruleus and parabrachial region. Results are discussed in terms of previous anatomical and neurophysiological data, probable pathways, and the function of LH neurons.     

Monosynaptic connections between neurons of trigeminal mesencephalic nucleus and jaw-closing motoneurons in the rat: an intracellular horseradish peroxidase labelling study

In order to confirm the monosynaptic connections of muscle spindle-mediated jaw stretch reflexes, 8 neurons of trigeminal mesencephalic nucleus innervating masseteric muscle spindles were identified electrophysiologically and stained intracellularly with horseradish peroxidase. These axon terminals projected to ipsilateral dorsal and dorsolateral divisions of trigeminal motor nucleus and extensive premotor areas. Under electron microscope, labeled terminals made monosynaptic contacts predominantly with dendrites in the jaw-closing motoneuron pools. One labeled and many non-labeled terminals were frequently observed to converge simultaneously on one dendrite in the area. However, it was of particular interest that 28% of the labeled terminals constituted the intermediate component of axo-axodendritic synaptic triads. The present study confirmed, for the first time, monosynaptic connections between jaw-closing muscle spindle afferents and jaw-closing motoneurons. These findings also provided ultrastructural evidence for the monosynaptic excitation of muscle spindle-mediated jaw stretch reflexes which received presynaptic and postsynaptic inhibitions of the premotor neurons from other sources.     

Limbic connections to the lateral preoptic area: a horseradish peroxidase study in the rat

Horseradish peroxidase, 13% Sigma Type VI, was administered iontophoretically to the lateral preoptic area (LPA) of male hooded rats. Animals were perfused intracardially on the following day and brains were removed and sliced in the coronal plane into 50 microns sections. Alternate sections were processed with DAB and BDH for the brown and blue reaction products and later examined by bright and dark field microscopy for the presence and location of retrogradely labeled neurons. Results indicate that there are a significant number of limbic efferent connections to the LPA. Afferents to the LPA originate in the prefrontal corex, nucleus accumbens, diagonal band and olfactory structures, lateral and medial septum, stria hypothalamic tract and stria terminalis, the magnocellular and medial preoptic nuclei, along the extent of the medial forebrain bundle in the LPA and LH, anterior and basolateral amygdala, ventromedial caudate-putamen, stria medullaris and lateral habenula, the stellatocellular-periventricular, ventromedial, arcuate and anterior hypothalamic nuclei, the perifornical area, zona incerta, ventral medial thalamic area, ventral tegmental area of Tsai, interpeduncular nucleus, reticular zone of the substantia nigra, mesencephalic periaqueductal gray and reticular formation, all aspects of the raphe nuclei and the locus coeruleus. Results are discussed in terms of known anatomical and neurophysiological data and the similar limbic inputs observed for lateral hypothalamic neurons which are found along the extent of the medial forebrain bundle.     

Demonstration of caudally directed hippocampal efferents in the rat by intracellular injection of horseradish peroxidase

Axonal projections of rat hippocampal neurons were demonstrated by intracellular injections of horseradish peroxidase. The data indicated a prominent caudally directed projection from pyramidal neurons of hippocampal field CA1, provided evidence that the subiculum is one of its targets, and suggested that the caudally directed efferents from CA1 are more numerous than rostrally directed ones.     

A whole image of the hippocampal pyramidal neuron revealed by intracellular pressure-injection of horseradish peroxidase

The intracellular pressure-injection of HRP was applied to the rat hippocampus and has brought an excellent presentation of the Golgi-like image of the pyramidal neuron. Rats were allowed to survive for 3 days and brain sections were treated with the PAP-immunohistochemical technique to enhance the sensitivity of HRP neurohistochemistry. The pyramidal neuron densely developed axon branches in the ipsilateral hippocampus and sent the commissural axon to the contralateral hippocampus. Moreover, short axon branches diverged from the commissural axon to the bilateral septal nuclei.     

Afferent connections of the subthalamic nucleus: a combined retrograde and anterograde horseradish peroxidase study in the rat

A comprehensive characterization of the afferent connections of the subthalamic nucleus of Luys (STN) is a necessary step in the unraveling of extrapyramidal mechanisms. In the present study, the STN afferents in the rat were systematically investigated with the aid of retrograde and anterograde horseradish peroxidase tracer techniques. The results indicate that, besides a massive input from the dorsal pallidum, the STN receives substantial projections from several districts of the cerebral cortex (the medial division of the prefrontal cortex, the first motor and primary somatosensory areas, and the granular insular territory), the ventral pallidum, the parafascicular nucleus of the thalamus and the pedunculopontine tegmental nucleus, as well as a modest innervation from the dorsal raphe nucleus. In spite of the fact that many additional structures were found to contain retrogradely labeled neurons after tracer injections in the STN, no other projection to the latter nucleus could be effectively established in our anterograde experimental series.     

A quantitative study of synaptic reorganization in red nucleus neurons after lesion of the nucleus interpositus of the cat: an electron microscopic study involving intracellular injection of horseradish peroxidase

A quantitative electron microscopic analysis of the corticorubral projection was performed in the red nucleus (RN) of adult cats to determine morphological correlates of synaptic reorganization that occur following a lesion of the interpositus nucleus (IP).Corticorubral synaptic endings were identified by lesioning the sensorimotor cortex 2–6 days before electrophysiological experiments. Horseradish peroxidase (HRP) was injected into electrophysiologically identified RN neurons. Sagittal sections 100 μm thick were cut and reacted by diaminobenzidine. Sections containing HRP-positive neurons were selected and embedded in Epon.In normal cats, degenerating corticorubral terminals in the RN region frequently made contact with dendritic profiles, having small cross-sections, while a few made contact with somatic profiles. Similar results were obtained when degenerating terminals making contact with HRP-filled dendrites were analyzed.In the experimental animals, the cortical lesion was performed more than 8 weeks after lesion of the IP. In these animals, degenerating corticorubral terminals were frequently found on proximal dendrites and somata in RN region and HRP-positive neurons in contrast to the findings in normal cats.The results indicate that new corticorubral synapses were formed on proximal dendrites and somata of RN neurons as a consequence of IP lesions.     

Electron microscopic observations of axon collateral synaptic endings of cat oculomotor motoneurons stained by intracellular injection of horseradish peroxidase

Motoneurons in the cat oculomotor nucleus have been identified electrophysiologically and stained by intracellular injection of horseradish peroxidase. Axon collateral arborizations with preterminal and terminal boutons identified by light microscopy corresponded to synaptic endings observed by electron microscopy. Despite variations in size and shape, synaptic endings showed similar ultrastructural features and established asymmetrical predominantly axodendritic synaptic contacts usually characterized by the presence of subjunctional dense bodies underlying the postsynaptic membrane densification.     

Morphophysiology of synaptic transmission between type I hair cells and vestibular primary afferents. An intracellular study employing horseradish peroxidase in the lizard, Calotes versicolor.

Intracellular records with glass microelectrodes filled with horseradish peroxidase (HRP) were taken from primary afferents of the horizontal semicircular canal in the lizard, Calotes versicolor. A coefficient of variation (CV) of the interspike intervals of spontaneous action potentials (APs) was calculated and correlated with the terminal morphologies of afferents within the canal crista. Irregular fibers with CV greater than 0.4 always correlated with a nerve chalice or calyx afferent terminal expansion surrounding one or more type I hair cells; more regular fibers with CV less than 0.4 always correlated with a dimorphic or bouton only terminal expansion of afferents. Afferents with a CV greater than 0.4 demonstrated miniature excitatory postsynaptic potentials (mEPSPs) that summated to initiate APs. APs were blocked by tetrodotoxin and mEPSP frequency was modulated by caloric stimulation. Cobalt application reversibly blocked mEPSPs. Electron microscopic examination of physiologically studied afferents with CV greater than 0.4 revealed synaptic profiles consisting of typical synaptic bodies and synaptic vesicles in the type I hair cell presynaptic to the nerve chalice. Examples of the interspike baseline in regular and irregular afferents suggest differential modes of impulse initiation in these two fiber types.     

Morphology of sympathetic preganglionic neurons in the neonatal rat spinal cord: an intracellular horseradish peroxidase study

Understanding the central neural control of autonomic functions requires a knowledge of the morphology of the preganglionic neurons, for the location of the dendritic arborizations of these neurons will indicate which central pathways may have access to them. In the present study, individual sympathetic preganglionic neurons in the neonatal rat spinal cord have been examined by the intracellular injection of horseradish peroxidase (HRP) in an in vitro preparation. Seventeen HRP-labeled preganglionic neurons in thoracic segments T1-T3 were examined in detail; of these, 12 somata were located in the intermediolateral cell column (IML), one in the lateral funiculus (LF), two in the intercalated nucleus (IC), and two at the border between IML and IC. All of the neurons had extensive dendritic arborizations arising from an average of six primary dendrites; the average total dendritic length for these cells was 2,343 microns. The morphology of preganglionic neurons differed depending on the location of their cell bodies. Preganglionic neurons located in the IML were essentially two-dimensional: the cells had some dendrites that coursed rostrocaudally for 300-500 microns within the IML and others that coursed mediolaterally, extending to the lateral surface of the cord and close to the central canal. Axons of these cells coursed ventrally from the cell body and exited from the spinal cord at the first ventral root caudal to the cell body. No intraspinal axon branches were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A horseradish peroxidase study of the cerebellorubral pathway in the rat

The distribution and character of labeled neurons within the cerebellar nuclei of the rat were studied after injection of horseradish peroxidase into the red nucleus. Cells possessing horseradish peroxidase activity were found throughout the contralateral nucleus lateralis (dentate) and interposed complex and were absent from the nucleus medialis (fastigial). Relatively more labeled neurons were present within the anterior part of the interposed complex than in its posterior portion. Peroxidase-stained cells were characteristically large and multipolar, whereas small neurons throughout these centers were devoid of activity. An ipsilateral projection to the red nucleus was not observed.     

Motor and sensory neurons of the rat sural nerve: A horseradish peroxidase study

Neurons of origin of the rat sural nerve were labelled with horseradish peroxidase. Dorsal root ganglionic cells were located in the L4 and L5 ganglia, and occasionally at the L6 level. Most of these sensory neurons measured under 35 μm in diameter. In keeping with previous electrophysiological studies suggesting the presence of motor fibers to plantar muscles in the rat sural nerve, motoneurons were identified at the caudal end of the L5 spinal segment, intermingled in the posterior aspect of the ventral horn with posterior tibial motor cells supplying the foot muscles. A quantitative analysis of HRP-labelled motoneurons revealed no difference between normal (average 67) and deafferented animals (average 70), the values being only marginally lower than counts of motor axons in deafferented sural nerves (average 80).     

Visualization of oligodendrocytes and astrocytes in the intact rat optic nerve by intracellular injection of lucifer yellow and horseradish peroxidase

The morphology of glial cells in the intact rat optic nerve, a central nervous system (CNS) white matter tract, was analysed by filling over 500 macroglial cells intracellularly with horseradish peroxidase (HRP) or Lucifer yellow (LY). Two main cell types were distinguished: fibrous astrocytes and cells presumed to be oligodendrocytes. Intracellularly stained astrocytes were highly complex, with 50-60 long branching processes which passed radially from the cell body and terminated in end-feet at the pial surface or on blood vessels; some processes ended freely in the nerve parenchyma. Astrocytes filled with LY were usually dye-coupled to other astrocytes after the first week of life. Filled oligodendrocytes had a unique appearance that unmistakably distinguished them from astrocytes and were occassionally dye-coupled to nearby oligodendrocytes. These cells had 20-30 longitudinally oriented processes 150-200 microns long, which passed exclusively along the long axis of the nerve parallel to axons; the longitudinal processes were connected to the cell body by thin branches 15-30 microns long. The longitudinal processes probably represent the tongue processes of the internodal myelin sheaths, and thus each oligodendrocyte appears to myelinate 20-30 axons with sheaths that are 150-200 microns in length.     

Central connections of the hepatic branch of the vagus nerve: a horseradish peroxidase histochemical study

The hepatic branch of the vagus nerve has been implicated as an important source of afferent input controlling both physiological and behavioral homeostasis. In addition, it is clear that parasympathetic efferents to the liver can significantly alter hepatic functions. In order to begin physiological studies on the nature of hepatic afferent and efferent relations, it will be necessary to understand the central anatomical organization of the components of this small visceral nerve. By carefully exposing and dissecting the hepatic branch of the vagus and applying crystalline horseradish peroxidase (HRP) to it, we were able to elucidate a predominant pattern of afferent terminations within the left subnucleus gelatinosus, the medial division of the left solitary nucleus and the left lateral edge of the area postrema. Efferent nuclei were concentrated in the left dorsal motor nucleus of the vagus (DMN) with a few scattered neurons located in the right DMN as well as the left anterior nucleus ambiguus.     

Afferent connections of the habenular nuclei in the rat. A horseradish peroxidase study,with a note on the fiber-of-passage problem

The afferent connections of the habenular complex in the rat were examined by injecting horseradish peroxidase (HRP) into discrete portions of the habenular nuclei by microelectrophoresis. 1. HRP deposits confined to the lateral half of the lateral habenular nucleus labeled a multitude of cells in the entopeduncular nucleus. Numerous labeled cells also appeared in such cases in the lateral hypothalamus, indicating that the lateral habenular nucleus is a major convergence point of projections from these otherwise. 2. HRP injected into the medial part of the lateral habenular nucleus labeled cells in the same regions, but more in the diagonal band and fewer in the entopeduncular nucleus than were labeled by more lateral injections. The contrast suggests that the projections from the basal forebrain and entopeduncular nucleus to the lateral habenular nucleus are somewhat topographically organized. 3. Injections of the medial habenular nucleus labeled an abundance of cells in the posterior parts of the supracommissural septum, but also a small number of cells in the diagonal band and mesencephalic raphe. 4. HRP injected into the stria medullaris labeled cells in all of the afore-mentioned areas and, in addition, cells in several olfactory structures, confirming that HRP may be taken up by fibers of passage and label their cells of origin, and suggesting that olfactory structures contribute fibers to the stria medullaris that do not terminate in the habenula.     

Mapping of neuronal contacts with intracellular injection of horseradish peroxidase and Lucifer yellow in combination

A technique for the simultaneous visualization in the light microscope of processes of neurons filled with horseradish peroxidase and Lucifer yellow in combination has been developed. The technique is applied to determine the location, number and distribution of presumptive synaptic sites between neurons in the leech central nervous system.     

Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: a tyrosine hydroxylase immunocytochemical study

In adult rats with a unilateral 6-hydroxydopamine-induced destruction of the nigrostriatal dopamine (DA) pathway, grafts of embryonic substantia nigra can establish a new dopaminergic terminal fiber plexus in the previously denervated neostriatum and compensate for some of the behavioral deficits induced by the nigrostriatal lesion. In the present study the synaptic connections of the ingrowing DA fibers from the graft were analyzed ultrastructurally, using immunocytochemical localization of tyrosine hydroxylase (TH), in animals whose lesion-induced motor asymmetry had been completely compensated for by the nigral grafts. In two of the animals, horseradish peroxidase-wheatgerm agglutinin conjugate was injected into the graft in order to trace possible reciprocal afferent connections to the graft from the host striatum. TH-immunoreactive axons from the graft were seen to make abundant symmetric synapses with neuronal elements in the host neostriatum. Between 85 and 90% of these synapses were on dendritic shafts and spines, and the rest were on neuronal perikarya. Two principal targets were identified: dendrites of spiny neurons, the majority of which are likely to be striatal projection neurons; and the cell bodies of giant neurons, most (or perhaps all) of which are known to be cholinergic interneurons. The synapses made on dendritic spines, which constituted about 40% of all TH-positive synapses formed by the TH-positive neurons in the graft, resembled those seen in normal animals, both in that they made contacts with spine necks and in that they invariably were associated with an asymmetric TH-negative synapse contacting the spine head. The innervation of the giant cell perikarya, which constituted about 6% of all TH-positive synapses found, was strikingly abnormal in that the graft-derived TH-positive fibers formed dense pericellular "baskets" selectively around the giant cell bodies. Such arrangements were never seen in the normal striatum, nor did they occur in the intact contralateral striatum in the grafted animals. It is proposed that this apparent dopaminergic hyperinnervation from the graft could provide a powerful inhibition of the cholinergic interneurons in the reinnervated host striatum, and that such an inhibitory mechanism could assist in the graft-induced functional recovery by potentiating the functional effects of DA synapses terminating on the spiny efferent neurons. This dual innervation may thus help to explain why restoration of only a small proportion of the striatal DA innervation by the graft is sufficient to induce complete compensation of, e.g., motor asymmetry in the lesioned rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Afferent connections to cardiovascular sites in the amygdala: a horseradish peroxidase study in the cat

Either the left or right cervical vagus was electrically stimulated in anesthetized rats before and after selective transection of either the coeliac, gastric and hepatic abdominal branches in order to evaluate the contribution of these branches to vagal controlled insulin secretion. Changes of insulin secretion were estimated on the basis of insulin concentration in venous plasma, sampled by indwelling jugular catheters. Plasma glucose concentration in overnight food-deprived rats was clamped between 130 and 160 mg/dl by means of continuous i.v. glucose infusion, and surgical stress-induced sympathetic activity was blocked by concomitant i.v. infusion of phentolamine and propranolol. Before transection of any abdominal branch, both right and left cervical vagal stimulation induced a 3- to 4-fold increase of plasma insulin concentration and significant increases of plasma glucose concentration, while the heart rate decreased rapidly and significantly. The right cervical vagal stimulation-induced insulin response (integrated incremental area) was significantly decreased by either bilateral coeliac (-37%) or bilateral gastric (-57%), but not by hepatic (-5%) vagotomy. The left cervical vagal stimulation-induced insulin response was significantly decreased (-41%) by hepatic vagotomy. The concomitant rises of plasma glucose concentration may have contributed more than 50% to the vagal stimulation-induced insulin responses. However, calculating the purely neural components revealed that the right cervical vagal stimulation-induced insulin response was still decreased by coeliac (-48%) or gastric (-84%) and not decreased (+24%) by hepatic vagotomy, and the left cervical vagal stimulation-induced insulin response was decreased (-52%) by hepatic branch vagotomy. We conclude that cervical vagal stimulation-induced insulin-secreting activity reaches the pancreas via all 3 abdominal divisions of the vagus nerve, and suggest that pancreatic beta-cells are innervated through all 3 abdominal divisions.     

Intravenous injection of horseradish peroxidase in the rat stimulates corticosterone and adrenocorticotropic hormone release

Summary Wistar rats were given intravenous (i.v.) horseradish peroxidase (HRP) in saline in doses commonly used to study vascular permeability and the blood-brain barrier. Samples of blood were taken from conscious animals via indwelling catheters at intervals up to 6 h after the HRP injection. Plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone were determined and compared with levels in control rats injected with saline alone. Rats injected with saline only presented levels of hormones within the low limits of normal indicating an insignificant influence of stress induced by the experimental procedure. Within 30 min of the i.v. HRP injection, the plasma concentrations of both ACTH and corticosterone increased to very high levels and remained so throughout the period of observation, namely 6 h. The time course of the changes in the concentrations was the same for the two hormones and the actual numerical values were related to the dose of HRP injected. The i. v. injection of HRP in Wistar rats, therefore, induces a marked release of stress hormones which by themselves have profound physiological effects. This phenomenon must be taken into account, in studies on normal vascular permeability using HRP as a tracer and also in similar experiments exploring various pathological conditions of the blood-brain barrier.Dedicated to Prof. F. Seitelberger on the occasion of his seventieth birthdaySupported by Slovak Academy of Sciences and Swedish Medical Research Council project 12X-03020