Synaptology of three peptidergic neuron types in the central nucleus of the rat amygdala

The synaptology of neurotensin (NT)-, somatostatin (SS)- and vasoactive intestinal polypeptide (VIP)-immunoreactive neurons was studied in the central nucleus of the rat amygdala (CNA). Three types of axon terminals formed synaptic contacts with peptide-immunoreactive neurons in the CNA: Type A terminals containing many round or oval vesicles; Type B terminals containing many pleomorphic vesicles; and Type C terminals containing fewer, pleomorphic vesicles. Peptide-immunoreactive terminals were type A. All three types of terminals formed symmetrical axosomatic and asymmetrical axodendritic contacts. However, type B and peptide-immunoreactive terminals frequently formed symmetrical axodendritic synaptic contacts. VIP-immunoreactive terminals also formed asymmetrical axodendritic contacts. SS- and NT-immunoreactive terminals commonly formed symmetrical contacts on SS- and NT-immunoreactive cell bodies, respectively. VIP-immunoreactive axon terminals were postsynaptic to nonreactive terminals. Type B terminals appeared more frequently on VIP neurons than on NT or SS neurons.     

Chemically defined collateral projections from the pons to the central nucleus of the amygdala and hypothalamic paraventricular nucleus in the rat

Triple fluorescence labelling was employed to reveal the distribution of chemically identified neurons within the pontine laterodorsal tegmental nucleus and dorsal raphe nucleus which supply branching collateral input to the central nucleus of the amygdala and hypothalamic paraventricular nucleus. The chemical identity of neurons in the laterodorsal tegmental nucleus was revealed by immunocytochemical detection of choline-acetyltransferase or substance P; in the dorsal raphe nucleus, the chemical content of the neurons was revealed with antibody recognizing serotonin. The projections were defined by injections of two retrograde tracers, rhodamine-and fluorescein-labelled latex microspheres, in the central nucleus of the amygdala and paraventricular nucleus, respectively. Neurons projecting to both the central nucleus of the amygdala and the paraventricular nucleus were distributed primarily within the caudal extensions of the laterodorsal tegmental nucleus and dorsal raphe nucleus. Approximately 11% and 7% of the labelled cells in the laterodorsal tegmental nucleus and dorsal raphe nucleus projected via branching collaterals to the paraventricular nucleus and central nucleus of the amygdala. About half of these neurons in the laterodorsal tegmental nucleus were cholinergic, and one-third were substance-P-ergic; in the dorsal raphe nucleus, approximately half of the neurons containing both retrograde tracers were serotonergic. These results indicate that pontine neurons may simultaneously transmit signals to the central nucleus of the amygdala and paraventricular nucleus and that several different neuroactive substances are found in the neurons participating in these pathways. This coordinated signalling may lead to synchronized responses of the central nucleus of the amygdala and paraventricular nucleus for the maintenance of homeostasis. Interactions between different neuroactive substances at the target site may serve to modulate the responses of individual neurons.     

Involvement of amygdala central nucleus in the regulation of cardiovascular functions

Effects of high frequency stimulation of the amygdala central nucleus involved a selective decrease in the activity: an increase in the activity of the inferior cardiac nerve and a simultaneous decrease in the activity of the vertebral nerve, as well as an obvious BP increase. Bilateral electrolytic lesions of the same amygdala structure accompanied with an overload of the higher nervous activity induced no hypertensions. The role of the amygdala central nucleus in control of cardiovascular functions is discussed.     

Interstitial Cajal-like cells in rat mesentery: an ultrastructural and immunohistochemical approach

Interstitial Cajal-like Cells (ICLC) were recently recognized in a plethora of non-digestive organs. Here, we describe a cell type of rat mesentery sharing ultrastructural and immunohistochemical features with ICLC. Mesenteric ICLC were demonstrated by transmission electron microscopy (TEM) and further tested by light microscope immunohistochemistry. The cell described here fulfils the TEM diagnostic criteria accepted for ICLC: location in the connective interstitium; close vicinity to nerves, capillaries and other interstitial cells; characteristic long, moniliform cell processes; specialized cell-to-cell junctions; caveolae; mitochondria at 5-10% of cytoplasmic volume; rough endoplasmic reticulum at about 1-2%; intermediate and thin filaments, microtubules; undetectable thick filaments. The processes of this mesenteric ICLC were particularly long, with a mean length of 24.91 microm (10.27-50.83 micorm), and a convolution index of 2.32 (1.37-3.63) was calculated in order to measure their potential length. Mean distances versus main target cells of ICLC-nerve bundles, vessels, adipocytes and macrophages-were 110.69, 115.80, 205.07 and 34.65 nm, respectively. We also tested the expression of CD117/c-kit, CD34, vimentin, alpha-smooth muscle actin, nestin, NK-1, tryptase and chymase and the antigenic profile of the mesenteric ICLC was comparable if not identical with that recently observed in ICLC from other extra-digestive tissues. Due to the peculiar aspect of the mesenteric ICLC processes it can be hypothesized that these cells form a three-dimensional network within the mesentery that is at the same time resistant and deformable following stretches consequent to intestine movements, mainly avoiding blood vessels closure or controlling blood vessels rheology. It remains, however, to be established if and how such cells are connected with the archetypal enteric ICC.     

Neurotensin concentrations in rat striatum and nucleus accumbens: Further studies of their regulation

The possible influence of cholinergic and dopaminergic mechanisms on neurotensin-containing neurones was examined at two different levels; nucleus accumbens and striatum in the rat brain. The acute treatment with the anticholinergic drugs atropine and scopolamine increased neurotensin concentrations in the striatum and, in the former case, also in the nucleus accumbens. Subchronic administration of atropine resulted in tolerance to its neurotensin-elevating action within the accumbens, but not within the striatum. Combined treatment with submaximal doses of haloperidol and atropine resulted in increases in neurotensin content which were greater than those seen with either agent alone. This was true regardless of whether the drugs were administered acutely or subchronically. This observation demonstrated that the tolerance phenomena occurring after subchronic elozapine and fluperlapine were not attributable to their anticholinergic activity. The control of striatal and accumbal neurotensin content by antidopaminergic and anticholinergic drugs seemed to be quite specific: drugs with actions on noradrenergic, serotoninergic, GABA-ergic and opiate systems did not influence the neurotensin content in these two structures. Preliminary studies on the effects of haloperidol on neurotensin release from striatal slices in vitro and that of cycloheximide on haloperidol's effect in vivo, suggest a possible inhibitory action of dopamine receptor blockade on neurotensin release.     

神经降压素对迷走运动背核神经元兴奋作用的离体的研究

Intracellular recordings were made from neurons of the dorsal motor nucleus of the vagus (DMV) in rat brain slices. Most of DMV neurons (88%) were depolarized by droplets or perfusion of neurotensin (NT) in a dose-dependent manner. The depolarization, accompanied by an increase in membrane resistance, depended on extracellular K+ concentration and reversed polarity at about -82 mV. Perfusion with Ca(2+)-free/high-Mg2+ solution or with 1 mumol/L TTX solution, which blocked synaptic activities, did not eliminate NT-induced depolarization. The results indicate that NT excites DMV neurons through postsynaptic mechanism and this exciting depolarization may result from a decrease in K+ conductance.     

The central nucleus of the amygdala: cytoarchitechtonics, neuronal organization, and histophysiology

Neurologists' attention is attracted by amygdala's central nucleus which takes part in formation of stress response and adaptive behaviour in animals. The data on cytoarchitectonics, neuronal organization and histophysiology of this important centre are based on current knowledge of its subnuclear organization.     

Peptide-containing pathways from the parabrachial complex to the central nucleus of the amygdala

The present investigation was undertaken to examine the organization of peptidergic projections that exist between the parabrachial nuclear complex (PB) and the central nucleus of the amygdala (CNA). The retrograde tracer True Blue was injected into the CNA of adult rats. The brain tissue was then reacted immunocytochemically to localize neurotensin (NT), substance P (SP), methionine enkephalin (ENK), vasoactive intestinal polypeptide (VIP), somatostatin (SS), and cholecystokinin octapeptide (CCK). Following microinjection of True Blue in the CNA, retrogradely-labeled neurons were located primarily in the external lateral subnucleus, abutting the brachium conjunctivum. In animals that received colchicine pretreatment, two populations of neurons, containing either SP or NT, were found to project to the CNA. In addition, cells containing CCK, ENK, VIP, or SS were not found to be a part of this projection system. These data suggest that neurons in the PB project to the CNA and are, in part peptide-containing.     

Somatostatinergic projections from the central nucleus of the amygdala to the vagal nuclei

In the rat, somatostatin immunoreactivity was identified in neurons of the central nucleus of the amygdala that were retrogradely labeled by injection of fluorescent dyes into the nucleus tractus solitarius and dorsal motor nucleus of the vagus nerve. The double-labeled neurons are located in the medial subdivision of the central nucleus and appear to comprise less than one fifth of the descending pathway. These results suggest that somatostatin may act as a neurotransmitter in a pathway which mediates cardiovascular and other autonomic responses to fear-producing and other emotional stimuli.     

Somatostatin-like immunoreactivity in the amygdala of the pig

The distribution and morphology of neurons containing somatostatin (SOM) was investigated in the amygdala (CA) of the pig. The SOM-immunoreactive (SOM-IR) cell bodies and fibres were present in all subdivisions of the porcine CA, however, their number and density varied depending on the nucleus studied. The highest density of SOM-positive somata was observed in the layer III of the cortical nuclei, in the anterior (magnocellular) part of the basomedial nucleus and in the caudal (large-celled) part of the lateral nucleus. Moderate to high numbers of SOM-IR cells were also observed in the medial and basolateral nuclei. Many labeled neurons were also consistently observed in the lateral part of the central nucleus. In the remaining CA regions, the density of SOM-positive cell bodies varied from moderate to low. In any CA region studied SOM-IR neurons formed heterogeneous population consisting of small, rounded or slightly elongated cell bodies, with a few poorly branched smooth dendrites. In general, morphological features of these cells clearly resembled the non-pyramidal Golgi type II interneurons. The routine double-labeling studies with antisera directed against SOM and neuropeptide Y (NPY) demonstrated that a large number of SOM-IR cell bodies and fibers in all studied CA areas contained simultaneously NPY. In contrast, co-localization of SOM and cholecystokinin (CCK) or SOM and vasoactive intestinal polypeptide (VIP) was never seen in cell bodies and fibres in any of nuclei studied. In conclusion, SOM-IR neurons of the porcine amygdala form large and heterogeneous subpopulation of, most probably, interneurons that often contain additionally NPY. On the other hand, CCK- and/or VIP-IR neurons belonged to another, discrete subpopulations of porcine CA neurons.     

Peptide YY-like immunoreactivity in the central nervous system of the rat

The concentration of peptide YY (PYY)-like immunoreactivity in rat brain and spinal cord was determined by radioimmunoassay. The highest concentrations were found in the cervical spinal cord (18.1 +/- 1.3 ng/g, mean +/- S.E.M.) and in the medulla oblongata (16.3 +/- 1.5 ng/g). Lower amounts were found in the pons and in the hypothalamus. Chromatographic analysis of the PYY-like immunoreactivity from various regions of the brain revealed 95% of the immunoreactive material to be indistinguishable from synthetic porcine PYY. PYY-immunoreactive nerve cell bodies could be demonstrated by immunocytochemistry in the medulla oblongata of colchicine-treated rats, the largest group of cells being found in the midline area between and partly in the raphe pontis and obscurus nuclei. Another large group of immunoreactive cells was detected more laterally in the medial parts of the gigantocellular reticular nucleus. A few cells, finally, were seen in the dorsal parts of the medulla, including the nucleus of the solitary tract. Varicose nerve fibers displaying PYY immunoreactivity were observed in many parts of the hypothalamus, pons, medulla and spinal cord.     

The morphology of somatostatin-immunoreactive neurons in the lateral nucleus of the rat amygdala

Three types of somatostatin-immunoreactive neurons are described in the lateral nucleus of the rat amygdala. These three types closely correspond to neurons previously reported in Golgi preparations of the lateral nucleus. Class I somatostatin neurons have triangular- or piriform-shaped somata with large primary dendrites and spiny secondary dendrites. Class II somatostatin neurons have small to medium-sized oval perikarya and are fusiform or multipolar in shape. Class III somatostatin neurons have small spheroid somata with small thinner relatively aspinous dendrites. Class I somatostatin neurons give rise to axons which project outside the lateral nucleus whereas class II and III neurons innervate other somatostatin-positive and non-somatostatin neurons within the lateral nucleus. Somatostatin neurons within the lateral nucleus are hypothesized to function as part of a network of somatostatin neurons extending from cortical regions through the amygdala to basal telencephalic and lower brain stem regions.     

Alcohol enhances characteristic releases of dopamine and serotonin in the central nucleus of the amygdala

The amygdaloid complex (AMY) is implicated in emotional and motivational aspects of behavior, including the formation of positive reinforcement association. AMY may also associated with brain rewarding circuitry. In the present study, the effect of ethanol (EtOH) on the release of dopamine (DA) and serotonin (5-HT) was studied in the central amygdaloid nucleus (CeAMY), and projecting excitatory afferents to the ventral tegmental area (VTA), of freely moving Wistar rats by brain microdialysis. Within 20 min of i.p. injection of EtOH (2 g/kg), the levels of DA and 5-HT in the CeAMY dialysate increased over the baseline value by 270 and 160% (N = 6-7), respectively. Addition of EtOH (25, 50 and 100 mM) to the microdialysis perfusion medium for 1 h caused a 115-150% dose-related increase in the extracellular level of DA in the CeAMY. 100 mM EtOH-induced CeAMY DA release continued to increase for 1 h after the perfusion medium was returned to normal perfusion medium. In contrast, the CeAMY 5-HT level was increased only by the addition of 100 mM EtOH for 1 h to 130% for 80 min. The stimulation of the CeAMY by EtOH through the microdialysis membrane showed delayed responses of DA and 5-HT compared with the i.p. injection of EtOH. Overall, the present findings are not sufficient to conclude whether EtOH acts directly or indirectly on the major monoamine nerve cells in the CeAMY, but the degree of acute EtOH action affected the differences in time at the peak response on EtOH-induced DA and 5-HT releases in the CeAMY via VTA.     

Quantitative distribution of galanin-like immunoreactivity in the rat central nervous system

Using an antiserum directed against synthetic galanin (GAL) a sensitive radioimmunoassay was developed. The antiserum interaction with GAL was characterized by displacement curve characteristics and high performance liquid chromatography. Besides the main GAL-immunoreactive peak several small peaks with GAL-like immunoreactivity were observed. No cross-reactivity of the GAL-antiserum with several other peptides was observed. GAL-like immunoreactivity was measured in 37 microdissected areas of the rat central nervous system. High concentrations (greater than 2000 fmol/mg protein) were observed in the amygdaloid nuclei, the septum, globus pallidus, bed nuclei of the stria terminalis, all hypothalamic nuclei, the superior colliculus, locus coeruleus, the nucleus of the solitary tract and the neurointermediate lobe of the pituitary. Moderate concentrations (1000-2000 fmol/mg protein) were observed in the hippocampus, the nucleus accumbens and nucleus of the diagonal tract, the caudate-putamen, the central gray, the nucleus, tract and substantia gelatinosa of the spinal trigeminal nerve. The results generally correlate with those previously published by immunocytochemistry. The widespread distribution of GAL-like immunoreactivity in the rat central nervous system suggests an involvement of GAL in a variety of brain functions.     

Daily changes of neuropeptide Y-like immunoreactivity in the suprachiasmatic nucleus of the rat

Most of the biochemical, physiological and behavioural events in living organisms show diurnal fluctuations, normally synchronized with 24-h environmental rhythms, such as the light-dark cycle. The suprachiasmatic nucleus (SCN) of the hypothalamus is considered to be a pacemaker of the circadian rhythms in several mammals. The light-dark cycle is the primary synchronizing agent for many of the circadian rhythms which are regulated by the SCN. The photic information reaches the SCN also through a neuropeptide Y(NPY)-like immunoreactive pathway from the ventro-lateral geniculate nucleus. We found that in 12-h-dark and 12-h-light housed rats the NPY-like immunoreactive innervation of the ventro-lateral part of the SCN shows a 24 h rhythm with values rising gradually during the light phase and falling during the dark phase. Besides this rhythm, we found two peaks corresponding to the switching on and switching off of the light. The average level of NPY-like immunoreactivity, as assessed by means of semiquantitative immunocytochemistry and expressed in 'arbitrary units', is reduced in rats housed in total darkness for 2 weeks. These results confirm the physiological role of NPY in the timing of the circadian activity of the SCN.     

Neurotensin receptors in the rat bronchi

The isolated, circular preparations of the left and right bronchi of the rat were examined for mechanical responses to neurotensin (NT) and other vasoactive peptides. NT caused concentration-dependent increases in the isometric tension of the unstimulated preparations, with an apparent affinity higher than for the cholinergic agonists but with considerably lower intrinsic activity. Pronounced tachyphylaxis to NT was observed. NT potentiated the atropine-sensitive increase in tension resulting from electrical field stimulation. Neither atropine nor methysergide abolished the response to NT in the unstimulated preparations. Bradykinin and to a lesser extent angiotensin II contracted the unstimulated preparations and both systemic peptides enhanced the cholinergic output in response to field stimulation. Substance P and VIP on the other hand were without effects in the stimulated and unstimulated bronchi. The results are consistent with the presence of receptors for NT on the presynaptic cholinergic terminals as well as on the post-synaptic smooth muscles of the rat bronchi.     

Neurotensin receptor 1 immunoreactivity in the peripheral ganglia and carotid body

In the present study we investigated, through immunohistochemistry, the presence and location of neurotensin receptor 1 (NTR1) in the peripheral ganglia and carotid body of 16 humans and 5 rats. In both humans and rats, NTR1 immunostained ganglion cells were found in superior cervical ganglia (57.4±11.6% and 72.4±11.4%, respectively, p<0.05), enteric ganglia (51.9±10.4% and 64.6±6.1%, p<0.05), sensory ganglia (69.2±10.7% and 73.0±13.1%, p>0.05) and parasympathetic ganglia (52.1±14.1% and 59.4±14.0%, p>0.05), supporting a modulatory role for NT in these ganglia. Positivity was also detected in 45.6±9.2% and 50.8±6.8% of human and rat type I glomic cells, respectively, whereas type II cells were negative. Our findings suggest that NT produced by type I cells acts in an autocrine or paracrine way on the same cell type, playing a modulatory role on chemoception.Key words: neurotensin receptor 1, carotid body, autonomic ganglia, sensory ganglia, immunohistochemistry.Neurotensin (NT) is a tridecapeptide which was first isolated from bovine hypothalamus (Carraway and Leeman, 1973) and is widely distributed in the nervous system and intestine. In the nervous system, neurotensin acts as a neurotransmitter and neuromodulator (Dobner, 2006); in the periphery, as a paracrine or endocrine factor (Mazzocchi et al., 1997; Malendowicz, 1998). It also acts as a growth factor on various cell types (Malendowicz, 1993; Markowska et al., 1994a, 1994b; Evers, 2006).Three different NT receptors, termed NTR1, NTR2 and NTR3, have been identified and cloned to date. NTR1 and NTR2 are, respectively, high- and low-affinity seven trans-membrane domain G protein-coupled receptors. NTR3 is a high-affinity single trans-membrane domain type 1 receptor, with 100% homology with the sorting protein, gp95/sortilin (Kitabgi, 2006; Mazella et al., 1998). NTR3 can also form heterodimers with NTR1 in the plasma membrane (Martin et al., 2002). Nuclear internalization of the NTR1 has been reported and has been suggested to play a role in the production of long-term genomic effects (Feldberg et al., 1998; Laduron, 1992). It has also been reported that NTR2, but not NTR1, returns to the plasma membrane after NT-induced sequestration (Mazella and Vincent, 2006).In the peripheral nervous system, pregangliar fibers containing NT have been found in sympathetic, parasympathetic and enteric ganglia, and functional studies also suggest the expression of NTRs in ganglion cells. However, direct evidence of NTR1 protein expression in the different cell types of the ganglia has not yet been provided for human and rat. Only in rat dorsal root ganglia has evidence of NTR1 expression been given through hybridization in situ (Zhang et al., 1995), but there are no data on protein location or internalization.The carotid body is an arterial chemoreceptor, sensitive to reductions in partial blood oxygen pressure and pH and to increases in partial CO2 pressure, the stimulation of which induces increases in ventilatory frequency and volume.The carotid body is situated at the carotid bifurcation, and is composed of parenchymal lobules separated by connective tissue, in which afferent fibers of the glossopharyngeal nerve, arising from the petrosal ganglion, occur (Porzionato et al., 2005).Two different cell populations are present in the carotid body: type I cells, in turn separated into light, dark and pyknotic, and type II cells, at the edges of the clusters. Post-ganglionic sympathetic nerve fibers from the superior cervical ganglion are present, innervating blood vessels and type I cells, and preganglionic parasympathetic and sympathetic fibers reaching ganglion cells near the glomic cells. NT has been detected in glomic cells (Heath et al., 1988; Heym and Kummer, 1989; Smith et al., 1990) but the presence of the corresponding receptors in the various glomic cell types has not yet been investigated.Thus, the aim of the present study was to investigate, through immunohistochemistry, the presence and location of NTR1 in the peripheral ganglia and carotid body of both human and rat, with particular reference to the different cell types.