Terminal Plexus Research Articles

Ultrastructure of substance P-immunoreactive terminals and their relation to vascular smooth muscle cells of rat small mesenteric arteries

Mesenteric arteries of the rat are surrounded by a plexus of primary afferent nerve terminals which contain both substance P (SP) and calcitonin gene-related peptide (CGRP). The ultrastructural arrangement of the innervation was studied in second-order branches of the rat mesenteric artery using immunohistochemical labelling with antibodies against SP. The structure and distribution of SP-immunoreactive (SP+) and SP-negative (SP-, i.e., virtually all noradrenergic) axons and their terminals within the adventitia of the artery have been determined. Sixteen percent of axons and 22% of varicosities in the perivascular plexus were SP+. Most of the SP+ varicosities lay between 0.4 and 2 microm from the smooth muscle cells, whereas most SP- varicosities lay much closer to the vessel (i.e., <1 microm). SP+ varicosities typically contained the same number and size of small synaptic vesicles and mitochondria as SP- varicosities, but there were more large dense-cored vesicles in the SP+ varicosities. Unlike SP- varicosities, the peptidergic varicosities did not show clustering of synaptic vesicles toward one part of the axon membrane, and none of them formed junctions with the smooth muscle cells. Close relationships between SP+ and SP- varicosities lacked any detectable structural specialization. The arrangement of SP+ (primary afferent) terminals and their association with vascular smooth muscle cells indicates that peptide released from afferent terminals must diffuse further than noradrenaline from sympathetic terminals to reach the vascular smooth muscle.

Distribution of P2X receptors in the rat adrenal gland.

The distribution of each of the seven subtypes of ATP-gated P2X receptors was investigated in the adrenal gland of rat utilizing immunohistochemical techniques with specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. A small number of chromaffin cells showed positive immunoreaction for P2X5 and P2X7, with the relative occurrence of P2X7-immunoreactive chromaffin cells exceeding that of P2X5. The preganglionic nerve fibres that form terminal plexuses around some chromaffin cells showed P2X1 immunoreactivity. Intrinsic adrenal neurones were observed to be positively stained for P2X2 and P2X3 receptors. P2X2 immunoreactivity occurred in several neurones found singly or in groups in the medulla, while only a small number of neurones were immunoreactive for P2X3. Adrenal cortical cells were positively immunostained for P2X4-7. Immunoreactivity for P2X4 was confined to the cells of the zona reticularis, while P2X5-7 immunoreactivities occurred in cells of the zona fasciculata. The relative occurrence of immunoreactive cortical cells of the zona fasciculata was highest for P2X6, followed by P2X7 and then P2X5. The smooth muscle of some capsular and subcapsular blood vessels showed P2X2 immunoreactivity. The specific and widespread distribution of P2X receptor subtypes in the adrenal gland suggests a significant role for purine signalling in the physiology of the rat adrenal gland.

Organization of projections from the anterior pole of the nucleus reticularis thalami (NRT) to subdivisions of the motor thalamus: Light and electron microscopic studies in the Rhesus monkey

Projections to the motor-related thalamic nuclei from the anterior pole of the reticular thalamic nucleus (NRT) were studied after injections of biotinylated dextran amine and wheat germ agglutinin conjugated horseradish peroxidase at light and electron microscopic levels, respectively. Each injection resulted in anterograde labeling in the three subdivisions of the ventral anterior nucleus (pars parvicellularis, VApc; pars densicellularis, VAdc; and pars magnocellularis, VAmc) and in the ventral lateral nucleus (VL). NRT fibers had beaded shapes and coursed in a posterior direction giving rise to relatively diffuse terminal plexuses. The average size of the beads (0.7μm2) and their density per 100μm of fiber length (23.7–25.7) were similar between the nuclei studied. At the electron microscopic level, anterogradely labeled boutons displayed positive immunoreactivity for γ-aminobutyric acid (GABA), contained pleomorphic synaptic vesicles, and formed relatively long (∼0.4μm) symmetric synaptic contacts. Usually, a single terminal formed synapses on more than one postsynaptic structure. Synaptic contacts were on projection and local circuit neurons and targeted mainly their distal dendrites. In the VAmc, synapses on local circuit neurons composed 48% of the total sample, in the VAdc/VApc and in the VL the proportion was higher, 65% and 62%, respectively. The results suggest that the input from the anterior pole of the monkey reticular nucleus to the motor-related thalamic nuclei is organized differently from what is known on the organization of connections of NRT with sensory thalamic nuclei in other species in that the terminal fields of individual fibers are diffuse rather than focal and that at least 50% of synapses are established on GABAergic local circuit neurons. J. Comp. Neurol. 409:369–384, 1999. © 1999 Wiley-Liss, Inc.

Organization of projections from the anterior pole of the nucleus reticularis thalami (NRT) to subdivisions of the motor thalamus: Light and electron microscopic studies in the Rhesus monkey

Projections to the motor-related thalamic nuclei from the anterior pole of the reticular thalamic nucleus (NRT) were studied after injections of biotinylated dextran amine and wheat germ agglutinin conjugated horseradish peroxidase at light and electron microscopic levels, respectively. Each injection resulted in anterograde labeling in the three subdivisions of the ventral anterior nucleus (pars parvicellularis, VApc; pars densicellularis, VAdc; and pars magnocellularis, VAmc) and in the ventral lateral nucleus (VL). NRT fibers had beaded shapes and coursed in a posterior direction giving rise to relatively diffuse terminal plexuses. The average size of the beads (0.7 microm2) and their density per 100 microm of fiber length (23.7-25.7) were similar between the nuclei studied. At the electron microscopic level, anterogradely labeled boutons displayed positive immunoreactivity for gamma-aminobutyric acid (GABA), contained pleomorphic synaptic vesicles, and formed relatively long (approximately 0.4 microm) symmetric synaptic contacts. Usually, a single terminal formed synapses on more than one postsynaptic structure. Synaptic contacts were on projection and local circuit neurons and targeted mainly their distal dendrites. In the VAmc, synapses on local circuit neurons composed 48% of the total sample, in the VAdc/VApc and in the VL the proportion was higher, 65% and 62%, respectively. The results suggest that the input from the anterior pole of the monkey reticular nucleus to the motor-related thalamic nuclei is organized differently from what is known on the organization of connections of NRT with sensory thalamic nuclei in other species in that the terminal fields of individual fibers are diffuse rather than focal and that at least 50% of synapses are established on GABAergic local circuit neurons.

Hormonal Regulation of Glutamate Receptor Gene Expression in the Anteroventral Periventricular Nucleus of the Hypothalamus

Glutamate plays an important role in mediating the positive feedback effects of ovarian steroids on gonadotropin secretion, and the preoptic region of the hypothalamus is a likely site of action of glutamate. The anteroventral periventricular nucleus (AVPV) of the preoptic region is an essential part of neural pathways mediating hormonal feedback on gonadotropin secretion, and it appears to provide direct inputs to gonadotropin releasing hormone (GnRH)-containing neurons. Immunohistochemistry and in situ hybridization were used in this study to define the distribution and hormonal regulation of glutamate receptor subtypes in the AVPV of juvenile female rats. Neurons that express the NMDAR1 receptor subtype are abundant in the AVPV, as are cells that express AMPA receptor subtypes (GluR1, GluR2, and GluR3 but not GluR4), and the AVPV appears to contain a dense plexus of NMDAR1-immunoreactive presynaptic terminals. However, AVPV neurons do not seem to express detectable levels of kainate receptor (GluR5, GluR6, and GluR7) or metabotropic receptor (mGluR1-6) subtypes. Treatment of ovariectomized juvenile rats with estradiol induced expression of GluR1 mRNA but did not alter levels of GluR2 or GluR3 mRNA. Treatment of estrogen-primed ovariectomized juvenile rats with progesterone caused an initial increase in GluR1 mRNA expression, followed by a small decrease 24 hr after treatment. In contrast, estrogen appears to suppress levels of NMDAR1 mRNA in the AVPV, which remained unchanged after progesterone treatment. Thus, one mechanism whereby ovarian steroids may provide positive feedback to GnRH neurons is by altering the sensitivity of AVPV neurons to glutamatergic activation.

The ascending serotonergic system in the hamster: comparison with projections of the dorsal and median raphe nuclei

The ascending serotonergic projections are derived largely from the midbrain median and dorsal raphe nuclei, and contribute to the regulation of many behavioral and physiological systems. Serotonergic innervation of the hamster circadian system has been shown to be substantially different from earlier results obtained with other methods and species. The present study was conducted to determine whether similar differences are observed in other brain regions. Ascending projections from the hamster dorsal or median raphe were identified using an anterograde tracer, Phaseolus vulgaris leucoagglutinin, injected by iontophoresis into each nucleus. Brains were processed for tracer immunoreactivity, and drawings were made of the median raphe and dorsal raphe efferent projection patterns. The efferents were also compared to the distribution of normal serotonergic innervation of the hamster midbrain and forebrain. The results show widespread, overlapping projection patterns from both the median and dorsal raphe, with innervation generally greater from the dorsal raphe. In several brain regions, including parts of the pretectum, lateral geniculate and basal forebrain, nuclei are innervated by the dorsal, but not the median, raphe. The hypothalamic suprachiasmatic nucleus is the only site innervated exclusively by the median and not by the dorsal raphe. The pattern of normal serotonin fiber and terminal distribution is generally more robust than would be inferred from the anterograde tracer material. However, there is good qualitative similarity between the two sets of data. The oculomotor nucleus and the medial habenula are unusual to the extent that each has a moderately dense serotonin terminal plexus, although neither receives innervation from the median or dorsal raphe. In contrast, the centrolateral thalamic nucleus and lateral habenula have little serotonergic innervation, but receive substantial other neural input from the raphe nuclei. The normal serotonergic innervation of the hamster brain is similar to that in the rat, although there are exceptions. The anterograde tracing of ascending median or dorsal raphe projections reveals a high, but imperfect, degree of correspondence with the serotonin innervation data, and with data from rats derived from immunohistochemical and autoradiographic tract-tracing techniques.

Regenerative effect of human recombinant NGF on capsaicin-lesioned sensory neurons in the adult rat

Nerve growth factor (NGF) has the ability to increase the content of peptide transmitter in intact primary sensory afferents of the adult rat. We have previously shown that NGF can also induce a refill of peptide transmitters in capsaicin-depleted peptidergic nerve terminals of the rat paw skin upon intraplantar injection. The present study was aimed at investigating the neurochemical, immunohistochemical and functional recovery of peripheral and central terminals of capsaicin-lesioned afferents following administration of recombinant human NGF-β (rhNGF-β). The systemic capsaicin treatment in adult rats by 50 mg/kg s.c. (day 0) was followed by intraplantar rhNGF-β injections (4 μg each) into one hind paw on days 1, 2, 3, 5, 6 and by the analysis on day 8. The content of the marker peptide calcitonin gene-related peptide (CGRP) showed a 100% NGF-induced recovery in the peripheral (sciatic nerve) and central axons (lumbar dorsal roots) on the side of the NGF treatment and also in the contralateral sciatic nerve and lumbar dorsal roots. In the terminals of the hind paw skin, the recovery of the CGRP content, as measured by radioimmunoassay, was 100% in the plantar and 80% in the dorsal skin ipsilaterally, and 55% in the dorsal and plantar hind paw skin contralaterally. In the lumbar dorsal spinal cord, CGRP content recovered by 85% bilaterally. The morphological appearance of the sensory nerve terminals was visualized by CGRP-immunohistochemistry. In the paw skin, the CGRP-immunoreactive (CGRP-IR) nerve endings were restricted to a fragmentary subepidermal plexus after the capsaicin treatment, whereas the subsequent NGF treatment caused a bilateral recovery of the subepidermal plexus and an intact reinnervation of the epidermis and blood vessels with free nerve terminals. The capsaicin-induced fragmentation of the CGRP terminal plexus in laminae I and II of the lumbar spinal dorsal horn was also markedly repaired on both sides by the intraplantar NGF injections. The NGF treatment caused the CGRP nerve terminals in the spinal cord to regain their ability of releasing transmitter upon capsaicin stimulation as shown in tissue slice superfusion experiments. These results show that within one week, rhNGF-β can induce a complete reinnervation of skin and spinal cord with intact CGRP-IR nerve terminals after an acute capsaicin lesion.

Experimentally induced lysosomal dysfunction disrupts processing of hypothalamic releasing factors

Previous studies have shown that experimentally induced lysosomal dysfunction elicits various features of aging in the cortical telencephalon. The present study used cultured slices to test if: (1) it causes similar changes in the hypothalamus, and/or (2) modifies the processing of two releasing factors important to aging. A 2-day exposure to N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), a selective inhibitor of cathepsins B and L, triggered a pronounced increase in the numbers of lysosomes in the ventromedial and dorsomedial nuclei, and in lateral hypothalamus. Continued incubation with the inhibitor for 3-12 days resulted in the spread of endosomes-lysosomes into dendrites and, in the lateral hypothalamus, the formation of massive, lysosome-filled expansions of neuronal processes (meganeurites). These effects did not occur in the arcuate nucleus, making it the first region so far examined in which lysosomal proliferation is not initiated by hydrolase inhibitors. Despite this, a dense plexus of axons and terminals in the median eminence was partially depleted of growth hormone releasing hormone (GHRH) within 48 hours after addition of ZPAD. Moreover, the inhibitor caused axonal GHRH to become collected into large puncta, an effect highly suggestive of a partial failure in axonal transport. GHRH mRNA levels were not greatly affected by 6 days of ZPAD exposure, indicating that reduced expression did not play a major role in the peptide changes seen at 48 hours. Similar but less pronounced immunocytochemical changes were recorded for the somatostatin system in the arcuate and periventricular nucleus. It is concluded that lysosome dysfunction: (1) has different consequences for the arcuate nucleus than other brain regions, and (2) disrupts transport of hypothalamic releasing factors. The potential significance of the results to endocrine senescence is discussed.

Experimentally induced lysosomal dysfunction disrupts processing of hypothalamic releasing factors

Previous studies have shown that experimentally induced lysosomal dysfunction elicits various features of aging in the cortical telencephalon. The present study used cultured slices to test if: (1) it causes similar changes in the hypothalamus, and/ or (2) modifies the processing of two releasing factors important to aging. A 2-day exposure to N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), a selective inhibitor of cathepsins B and L, triggered a pronounced increase in the numbers of lysosomes in the ventromedial and dorsomedial nuclei, and in lateral hypothalamus. Continued incubation with the inhibitor for 3–12 days resulted in the spread of endosomes-lysosomes into dendrites and, in the lateral hypothalamus, the formation of massive, lysosome-filled expansions of neuronal processes (meganeurites). These effects did not occur in the arcuate nucleus, making it the first region so far examined in which lysosomal proliferation is not initiated by hydrolase inhibitors. Despite this, a dense plexus of axons and terminals in the median eminence was partially depleted of growth hormone releasing hormone (GHRH) within 48 hours after addition of ZPAD. Moreover, the inhibitor caused axonal GHRH to become collected into large puncta, an effect highly suggestive of a partial failure in axonal transport. GHRH mRNA levels were not greatly affected by 6 days of ZPAD exposure, indicating that reduced expression did not play a major role in the peptide changes seen at 48 hours. Similar but less pronounced immunocytochemical changes were recorded for the somatostatin system in the arcuate and periventricular nucleus. It is concluded that lysosome dysfunction: (1) has different consequences for the arcuate nucleus than other brain regions, and (2) disrupts transport of hypothalamic releasing factors. The potential significance of the results to endocrine senescence is discussed. J. Comp. Neurol. 401:382–394, 1998. © 1998 Wiley-Liss, Inc.

Golgi cells in the superficial granule cell domain overlying the ventral cochlear nucleus: Morphology and electrophysiology in slices

Golgi cells are poised to integrate multimodal influences by participating in circuits involving granule cells in the cochlear nuclei. To understand their physiological role, intracellular recordings were made from anatomically identified Golgi cells in slices of the cochlear nuclei from mice. Cell bodies, dendrites, and terminals for all seven labeled cells were restricted to the narrow plane of the superficial granule cell domain over the ventral cochlear nucleus. The axonal arborization was the most striking feature of all Golgi cells; a dense plexus of terminals covered an area 200-400 microm in diameter in the vicinity of the cell body and dendrites. Axonal beads often surrounded granule cell bodies, indicating that granule cells are probable targets. Cells had input resistances up to 130 M omega and fired regular, overshooting action potentials. Golgi cells probably receive auditory nerve input, because shocks to the cut end of the auditory nerve excited Golgi cells with excitatory postsynaptic potentials (EPSPs). The latency of EPSPs shortened to a minimum and the amplitude of EPSPs grew in several steps as the strength of shocks was increased. The minimum latency of EPSPs in Golgi cells was on average 1.3 milliseconds, 0.6 milliseconds longer than the minimum latencies of EPSPs in nearby octopus and T stellate cells. The long latency raises the possibility that Golgi cells receive input from slowly conducting, unmyelinated auditory nerve fibers. Golgi cells are also excited by interneurons with N-methyl-D-aspartate receptors, probably granule cells, because repetitive shocks and single shocks in the absence of extracellular Mg2+ evoked late EPSPs that were reversibly blocked by DL-2-amino-5-phosphono-valeric acid.

3-D characterization of ganglion cells of the terminal nerve plexus of rat atrioventricular junction

Three-dimensional (3-D) morphology of neurons of the terminal nerve plexus of the atrioventricular junction was examined in a scanning electron microscope. Distributions of different cell types encountered as well as their relations to different structures of the atrioventricular specialized tissue were also studied. Most neurons were found disseminated in a thin connective tissue layer separating different segments of the atrioventricular conductive tissue from the interventricular septum. Sometimes, they formed small pluricellular ganglia (up to 5 neurons) but, frequently, they occurred isolated in the terminal ramifications of the intramural nerve plexus of specialized tissue. Some intranodal neurons could also be identified. According to their 3-D morphology, nerve cells of the perinodal ganglionated plexus could be divided into three categories: (1) Large unipolar neurons were scattered throughout the atrioventricular junction. Their long and thin axonal projections were often directed towards the interventricular septum. (2) Large pseudounipolar or bipolar neurons were located at a few specific loci, namely all along the bundle of His and its bifurcation into the right and left bundle branches. Frequently, they occurred solitary and immersed amongst strands of surrounding muscle cells. Only occasional synaptic impacts could be identified on the surface of neuronal bodies of these bipolar neurons. On the other hand, their dendritic varicosities were richly innervated. Due to their irregular shape, intimate association with muscular elements and their topographical superposition with occasional spindle-like structures, these nerve cells recall prospective sensory neurons involved in integration of mechanical and neural stimuli to the heart. (3) Small multipolar interneurons could be identified in the retronodal ganglion and within right and left bundle branches. The present description of morphological heterogeneity of intramural nerve cells agrees with recent morphological and functional classifications of autonomic neurons and supports the idea that, at the level of the atrioventricular junction, a self-governed neuronal network may be operating.

Nigral innervation of cholinergic and glutamatergic cells in the rat mesopontine tegmentum: Light and electron microscopic anterograde tracing and immunohistochemical studies

The substantia nigra (SN) has long been known as an important source of afferents to the pedunculopontine tegmental nucleus (PPN). However, it has not been established which of the chemospecific cell populations receive this synaptic input. We sought to address this issue by a correlative light and electron microscopic approach that combines anterograde tracing of nigral efferents with pre-embedding choline acetyltransferase (ChAT) and/or glutamate (Glu) immunohistochemistry. Following large bilateral injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) in the SN, the labeled nigrotegmental fibers were concentrated in a small area of the mesopontine tegmentum which contained very few ChAT-immunoreactive (ChAT-ir) cell bodies. However, strands of fine varicose fibers penetrated to adjacent regions of the PPN which harbored numerous cholinergic perikarya. The anterogradely labeled boutons were often seen in the proximity of ChAT-ir perikarya and dendrites, but the majority (82-93%) established symmetric synaptic junctions with noncholinergic profiles. In the pars dissipata of the PPN (PPNd), one-third of the labeled terminals synapsed onto noncholinergic perikarya and primary dendrites, while in the pars compacta of the PPN (PPNc) axosomatic synapses were rare. The possibility that the perikarya receiving a rich synaptic input from the SN are glutamatergic was tested in experiments combining anterograde transport of biotinylated tracers biocytin and dextran-amine (BDA) with glutamate immunohistochemistry. In double-labeled sections, Glu-ir perikarya within the terminal plexus of nigrotegmental fibers were surrounded by synaptic terminals. The PPNd also contained retrogradely BDA-labeled neurons which were contacted by anterogradely labeled terminals. These results indicate that although a small subpopulation of cholinergic neurons in the mesopontine tegmentum receive direct synaptic input from the SN, the primary target of nigrotegmental fibers are glutamatergic cells in the PPNd. Our results also provide ultrastructural evidence that some nigrotegmental fibers innervate pedunculonigral neurons.

Arborisation and termination of single motor thalamocortical axons in the rat.

The aim of this study was to examine the arborisations and terminations of individual thalamocortical axons in the motor system of the rat. Small, extracellular injections of an anterograde tracer (dextran-biotin) were made into the ventrolateral (VL) or ventral posterolateral (VPL) thalamic nuclei to label thalamocortical projections. Eleven motor axons and one somatosensory axon were reconstructed through serial sections just rostral from the injection site to their terminations in sensorimotor cortex. The smallest arbor arising from a single motor axon extended approximately 0.9 mm rostrocaudal and 0.9 mm mediolateral, the largest extended 3.9 mm rostrocaudal and 1.0 mm mediolateral. In some cases, two distinct plexuses of terminals were formed by an axon. In addition, motor axons formed terminals in cortical layer V only or in layers I, III, and V. By contrast (and in keeping with previous reports), the somatosensory axon formed a single plexus of terminals in layer IV of the cortex that extended approximately 0.3 mm rostrocaudal and 0.4 mm mediolateral. It is concluded that individual motor thalamocortical neurones are in a position to influence much more widespread cortical regions than somatosensory thalamocortical neurones.

The catecholaminergic innervation of the rat amygdala.

The present study is the first to demonstrate conclusively and to analyze systematically synaptic contacts of all three types of catecholaminergic afferent fibers in different nuclei of the rat amygdala and to relate the catecholaminergic innervation to neurochemically identified target neurons. 4.1.1 Central Nucleus: The central nucleus is the amygdaloid nucleus receiving the most dense catecholaminergic innervation. In the medial central nucleus, dopaminergic, noradrenergic and adrenergic terminal plexus overlap, in the central lateral central nucleus mainly dopaminergic plexus are found. The lateral capsular central nucleus is generally scarcely innervated, but individual neurons of this subnucleus possess a dense dopaminergic innervation. Colocalization of neurotensin in dopaminergic afferents is rare, the majority of the dense neurotensin-ir terminal plexus consist of non-dopaminergic fibers. The catecholaminergic innervation of the medial central nucleus is directed preferentially at peripheral neuronal structures, and has thus presumably modulatory functions. Dopaminergic terminals form predominantly symmetric, noradrenergic and adrenergic terminals from preferentially asymmetric synapses. A characteristic feature of the dopaminergic innervation is the dense perisomatic innervation of selected neurons. Adrenergic and the majority of noradrenergic afferent fibers to the medial central nucleus originate from cell groups in the medulla oblongata and contain high levels of NPY. GAD mRNA-detection suggests that most target neurons of catecholaminergic afferent fibers are capable of synthesizing GABA in the medial central nucleus. In its dorsal part, GABA is possibly colocalized with somatostatin, and many neurons express the dopamine-1-receptor subtype mRNA. In the posteroventral medial central nucleus, on the other hand, enkephalin mRNA-r and dopamine-2-receptor subtype mRNA-reactive neurons show a similar distribution as the GAD mRNA-reactive ones. Contacts could be shown between dopaminergic, noradrenergic and adrenergic axons and NPY- and somatostatin-immunoreactive neurons which are supposedly among the brainstem projection neurons of the medial central nucleus. The dopaminergic innervation of the central lateral central nucleus resembles that of the neighboring striatum in many respects. The synaptic density is high. As in the medial subnucleus, distal neuronal elements are the preferential target structures, indicating a modulatory function possibly regulating the selectivity of the target neurons for stimuli transmitted by other afferent fibers. Besides, individual neurons possess a dense perisomatic, presumably non-selective dopaminergic innervation. The innervation does not appear to be targeted at one specific neurochemical type of neuron in the central lateral central nucleus, but rather contacts somatostatin- and neurotensin-immunoreactive neurons (which are possibly also GABAergic), in addition to GABA/enkephalin-synthesizing and other (e.g., CHAT-immunoreactive) neurons. Individual neurons of the central lateral central nucleus express the dopamine-2-receptor subtype mRNA. The dopaminergic fiber baskets of the lateral capsular central nucleus are found surrounding enkephalin mRNA-reactive neurons. Codistribution studies suggest that they express the dopamine-2-receptor subtype mRNA. 4.1.2 Basal Complex: The basal complex receives dopaminergic and noradrenergic innervation, the latter mainly originating in the locus coeruleus. Some of the dopaminergic afferents contain neurotensin, and in contrast to the central nucleus, all neurotensin-immunoreactive afferent fibers are dopaminergic. In the noradrenergic afferent fibers NPY is not detectable. These results and the innervation pattern displaying mostly peripheral neuronal target structures resemble dopaminergic and noradrenergic innervation patterns documented in cortical areas. (ABSTRACT TRUNCATED)

Localization of the adenomatous polyposis coli tumour suppressor protein in the mouse central nervous system

The adenomatous polyposis coli gene is mutated in familial adenomatous polyposis and in sporadic colorectal tumours. The adenomatous polyposis coli gene product is a 300 000 mol. wt cytoplasmic protein that binds to at least three other proteins; β-catenin, a cytoplasmic E-cadherin-associated protein; hDLG, a human homologue of the Drosophila discs large tumour suppressor protein and glycogen synthase kinase 3 β, a mammalian homologue of the Drosophila ZESTE WHITE 3 protein. The adenomatous polyposis coli gene is highly expressed in the brain, suggesting that it may be involved in nerve function. Here we show that adenomatous polyposis coli is localized in the pericapillary astrocytic endfeet throughout the mouse central nervous system. Adenomatous polyposis coli is also localized in the astrocytic processes in the cerebellar granular layer, and displays concentrated expression in the terminal plexuses of the basket cell fibres around Purkinje cells. Adenomatous polyposis coli is further expressed in neuronal cell bodies and/or nerve fibres in the olfactory bulb, hippocampus, brain stem, spinal cord and dorsal root ganglia. Adenomatous polyposis coli is demonstrated to be co-localized with β-catenin and/or hDLG in neurons and nerve fibres, but not in astrocytes. From these results, adenomatous polyposis coli is suggested to participate in a signal transduction pathway in astrocytes which is independent of β-catenin and hDLG, and also in regulation of neuronal functions in association with β-catenin and hDLG.

Zinc, nervous system and aging.

required for quick inactivation of hormones or neurotransmitters justify its involvement also in actual performance of the nervous system. Thus, zinc-dependent dopamine-beta-hydrolase and phenylethanolamine-N-methyl transferase are required to convert dopamine to norepinephrine and this later into epinephrine respectively; glutamate dehydrogenase is involved in the metabolism of glutamic acid. Zinc acts as a cofactor in the metalloenzyme enkephalinase, thus contributing to the degradation of enkephalin and, possibly, also of cholecystokinin-octapeptide. Zinc may interfere with the binding to receptor of gamma-aminobutyric acid, of acetylcholine and of benzodiazepines. Finally, many of the so-called metaboreceptors are also zinc-dependent. The ionic zinc, which is the fraction of tissue zinc that can be demonstrated histochemically, appears to be intimately associated with the secretory-signaling functions of certain neurones. The highest levels of the ion occur in the hippocampal mossy fiber (MF) pathway. This system forms a dense plexus of terminals on apical dendrites of CA3 pyramidal neurons and is involved in the modulation of processes related to spatial learning and memory. The synaptic terminals of the mossy fibers are uniquely rich in Zn 2+ , which is actively taken up and stored in a pool of synaptic vesicles. Stimulation of the MF can induce Zn release, suggesting that the ion may affect neurotransmission. It has been observed that a reduction of the hippocampal Zn levels, obtained with chelating agents, can induce in adult animals spatial memory deficits and reduced learning ability. In humans some evidence exists of impaired concentration, jitteriness, lethargy, depression and mood lability, as well as disturbed taste and smell perception in individuals suffering from suspected zinc deficiency. From a clinical point of view many are the neuropathologies associated with alterations of zinc status. Zinc has been found reduced in schizophrenia, mental depression, dyslexia, multiple sclerosis, Guillain

Neuropeptide Y and enkephalin immunoreactivity in retinorecipient nuclei of the hamster pretectum and thalamus.

This investigation was stimulated by the historical confusion concerning the identity of certain pretectal nuclei and by large differences reported between species with respect to which nuclei receive retinal innervation. Subcortical visual nuclei were studied using immunohistochemistry to identify retinal projections labeled following intraocular injection of cholera toxin, b fragment. In addition, neuropeptide Y (NPY) or enkephalin (ENK) immunoreactive cells and fibers were also evaluated in the retinorecipient pretectal and thalamic areas. The results confirm the established view that the retina directly innervates the nucleus of the optic tract (NOT), posterior (PPT), and olivary pretectal (OPT) nuclei. However, the retina also innervates the hamster medial (MPT) and anterior (APT; dorsal division) pretectal nuclei, results not previously reported in rodents. A commissural pretectal area (CPT) sparsely innervated by retina is also described. The data show for the first time that the posterior limitans nucleus (PLi) receives a moderately dense, direct retinal input. The PLi does not project to the cortex and appears to be a pretectal, rather than thalamic, nucleus. All retinal projections are bilateral, although predominantly contralateral. The PLi contains a moderately dense plexus of NPY- and ENK-IR fibers and terminals. However, peptidergic fibers also traverse the ATP and connect with the dorsomedial pretectium. The OPT contains ENK- and NPY-IR neurons and fibers, but is specifically identifiable by a moderately dense plexus of ENK-IR terminals. Numerous ENK-IR neurons are found in the NOT and PPT. The latter also has moderate numbers of ENK-IR fibers and terminals, but few NPY-IR neurons or fibers. The MPT contains modest numbers of ENK-IR fibers. The APT has no NPY-IR neurons or terminals, but an occasional ENK-IR neuron is seen and there is sparse ENK-IR innervation. Peptidergic innervation of the visual nuclei does not appear to be derived from the retina. The results show a set of retinally innervated, contiguous nuclei extending from the thalamic ventrolateral geniculate nucleus dorsomedially to the midbrain CPT. These nuclei plus the superior colliculus comprise a dorsal "visual shell" embracing a central core of caudal thalamus and rostral midbrain.

Arborization pattern of sympathetic preganglionic axons in the rat superior cervical and stellate ganglia

Anterograde labeling technique with Phaseolus Vulgaris leucoagglutinin (PHA-L) was employed to observe how a single preganglionic axon arborizes in the superior cervical ganglion (SCG) and stellate ganglion (STG) of rats. PHA-L was injected into the intermediolateral nucleus of the spinal cord at the middle point between segments T1 and T2, and labeled axons were detected immunohistochemically in serial sections. We traced and drew three preganglionic axons over their full length in the SCG and STG. In SCG, the labeled axons bifurcated repeatedly and extended to a length of 600–700 μm in the rostrocaudal direction, and about 200 μm in the transverse direction. These three preganglionic axons made 11, 14 and 11 dense terminal plexus regions along their trajectory. The pattern of the most dense terminal plexus corresponded to the pericellular type dendritic plexus, one of the plexus patterns of dendritic collaterals of SCG neurons. In the STG, the extent of axonal arborization was more variable than that in the SCG, ranging from 400 to 800 μm in the rostrocaudal direction and about 400 μm in the transverse direction. The three analyzed axons made 21, 19 and 20 dense terminal plexus regions along their trajectory, with a similar pattern to those in SCG. These results indicated that there might be a columnar or ellipsoidal organization of postganglionic neurons which are innervated by single preganglionic axons.

Dynamic imaging of functional nerve terminals and Schwann cells in presynaptic 'nerve plates' isolated from the skate electric organ

The cholinergic innervation and its glial support were isolated in a functional state from the electric organ of the skate (Raja species) using a combined enzymatic and mechanical dissociation technique. Examination using light and electron microscopy showed that this 'nerve plate' is a disc-shaped structure several hundred micrometres in diameter consisting of a dense plexus of nerve terminals attached to branching nerve fibrils with numerous associated myelinating and perisynaptic Schwann cells. In unfixed nerve plates, depolarisation and Ca2+-dependent staining of the nerve terminals was seen with RH-414, a fluorescent marker for functional motor terminals. The components of the nerve plate could be loaded with the Ca2+-sensitive dyes Fluo-3 and Fura-2. Depolarisation of nerve plates loaded with either dye leads to an immediate increase of intracellular Ca2+ levels ([Ca2+]i) in the nerve terminals. This response was blocked by certain Ca2+ channel antagonists from the -conotoxin family. Glial cell responses to depolarisation were in general minimal. However, increases of [Ca2+]i were seen in these cells, but not in nerve terminals, during applications of ATP and acetylcholine. These results show that both nerve terminal and glial elements in this 'nerve plate' preparation are functional. The ease of this preparation and its functional reliability give it considerable potential as a useful model for elucidating presynaptic mechanisms.

Chapter 16 Intrinsic properties and environmental factors in the regeneration of adult cerebellar axons

The success of axon regeneration in the adult mammalian brain depends on the presence of growth-permissive environmental conditions as well as on specific properties of the affected neurons. To investigate the relative contribution of extrinsic cues and intrinsic determinants to reparative processes we have investigated the regenerative properties of olivocerebellar and Purkinje cell axons. When these axon populations are severed in the cerebellar white matter and confronted with embryonic neural grafts of cerebellar or extracerebellar origin, the former vigorously regenerate into the transplant, whereas the latter invariably fail to do so (Rossi et al., 1995). The same response occurs when dissociated Schwann cells are implanted in the lesion site: Purkinje cell axons fail to regrow, whereas olivocerebellar fibres regenerate for considerable distances. Within the graft, regenerating fibres follow tortuous courses along Schwann cell bundles and sometimes end with poorly developed terminal plexuses. Some of them, however, succeed in crossing the graft and grow further into the host cortex, where they break into fine terminal branches confined to the granular layer. The remarkable regenerative response of olivocerebellar axons revealed by these experiments might be an intrinsic reaction of the affected neurons to axon injury or it might be elicited by growth promoting cues derived from the grafts. To elucidate this point we have undertaken the investigation of cellular changes occurring in adult inferior olivary neurons following the transection of the inferior cerebellar peduncle. Our results show that axotomy induces a series of cellular changes, or reparative and regressive character, which ultimately lead to cell death. Interestingly, however, these modifications are not uniformly distributed throughout the whole inferior olive. (i) Neuronal atrophy and degeneration progress more rapidly in the PO and DAO than in the MAO. (ii) A subpopulation of inferior olivary neurons become reactive for NADPH-diaphorase histochemistry, and their preferential localisation in the MAO suggests that this modification is related to the longer survival of these cells after axotomy. (iii) The developmentally regulated calcitonin gene-related peptide (CGRP) is reexpressed by a subset of neurons in the caudal nuclear compartments. These results further emphasise the conclusion that the dissimilar regenerative response of Purkinje cell and olivocerebellar axons confronted with permissive environmental conditions is due to different intrinsic properties of these neuronal populations. The reexpression of developmentally regulated substances by axotomised inferior olivary neurons suggests that their reparative reaction is triggered by axon injury. However, the pattern of growth of regenerating olivocerebellar axons is strongly conditioned by environmental constraints, which, in the present experimental conditions, do not allow them to reattain denervated Purkinje cells.