Immunoreactive Nerve Terminals Research Articles

Role of the central renin‑angiotensin system in hypertension (Review).

Present in more than one billion adults, hypertension is the most significant modifiable risk factor for mortality resulting from cardiovascular disease. Although its pathogenesis is not yet fully understood, the disruption of the renin-angiotensin system (RAS), consisting of the systemic and brain RAS, has been recognized as one of the primary reasons for several types of hypertension. Therefore, acquiring sound knowledge of the basic science of RAS and the under- lying mechanisms of the signaling pathways associated with RAS may facilitate the discovery of novel therapeutic targets with which to promote the management of patients with cardiovascular and kidney disease. In total, 4 types of angiotensin II receptors have been identified (AT1R-AT4R), of which AT1R plays the most important role in vasoconstriction and has been most extensively studied. It has been found in several regions of the brain, and its distribution is highly associated with that of angiotensin-like immunoreactivity in nerve terminals. The effect of AT1R involves the activation of multiple media and signaling pathways, among which the most important signaling pathways are considered to be AT1R/JAK/STAT and Ras/Raf/MAPK pathways. In addition, the regulation of the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and cyclic AMP response element-binding (CREB) pathways is also closely related to the effect of ATR1. Their mechanisms of action are related to pro-inflammatory and sympathetic excitatory effects. Central AT1R is involved in almost all types of hypertension, including spontaneous hypertension, salt-sensitive hypertension, obesity-induced hypertension, renovascular hypertension, diabetic hypertension, L-NAME-induced hypertension, stress-induced hypertension, angiotensin II-induced hyper- tension and aldosterone-induced hypertension. There are 2 types of central AT1R blockade, acute blockade and chronic blockade. The latter can be achieved by chemical blockade or genetic engineering. The present review article aimed to high- light the prevalence, functions, interactions and modulation means of central AT-1R in an effort to assist in the treatment of several pathological conditions. The identification of angiotensin-derived peptides and the development of AT-2R agonists may provide a wider perspective on RAS, as well as novel therapeutic strategies.

The Effect of Hypoxia and Hyperoxia on Growth and Expression of Hypoxia-Related Genes and Proteins in Spotted Gar Lepisosteus oculatus Larvae and Juveniles.

We studied the molecular responses to different water oxygen levels in gills and swim bladder of spotted gar (Lepisosteus oculatus), a bimodal breather. Fish at swim-up stage were exposed for 71 days to normoxic, hypoxic, and hyperoxic water conditions. Then, all aquaria were switched to normoxic conditions for recovery until the end of the experiment (120 days). Fish were sampled at the beginning of the experiment, and then at 71 days of exposure and at 8 days of recovery. We first cloned three hypoxia-related genes, hypoxia-inducible factor 2α (HIF-2α), Na(+) /H(+) exchanger 1 (NHE-1), and NHE-3, and uploaded their cDNA sequences in the GeneBank database. We then used One Step Taqman® real-time PCR to quantify the mRNA copies of target genes in gills and swim bladder of fish exposed to different water O2 levels. We also determined the protein expression of HIF-2α and neuronal nitric oxide synthase (nNOS) in the swim bladder by using confocal immunofluorescence. Hypoxic stress for 71 days significantly increased the mRNA copies of HIF-2α and NHE-1 in gills and swim bladder, whereas normoxic recovery for 8 days decreased the HIF-2α mRNA copies to control values in both tissues. We did not found significant changes in mRNA copies of the NHE-3 gene in either gills or swim bladder in response to hypoxia and hyperoxia. Unlike in normoxic swim bladder, double immunohistochemical staining in hypoxic and hyperoxic swim bladder using nNOS/HIF-2α showed extensive bundles of HIF-2α-positive nerve fibers in the trabecular musculature associated with a few varicose nNOS immunoreactive nerve terminals.

Dopamine D1 and D2 receptor immunoreactivities in the arcuate-median eminence complex and their link to the tubero-infundibular dopamine neurons.

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and/or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region.

Reduced Number, G Protein Coupling, and Antinociceptive Efficacy of Spinal Mu-Opioid Receptors in Diabetic Rats Are Reversed by Nerve Growth Factor

This study investigated putative mechanisms of impaired spinal opioid antinociception such as a downregulation of mu-opioid receptor (MOR) number, coupling, and efficacy in rats with advanced (12 weeks) streptozotocin (STZ)-induced diabetes. Intravenous injection of STZ (45 mg/kg) in Wistar rats led to selective degeneration of insulin-producing pancreatic ß-cells, elevated blood glucose, and mechanical hyperalgesia. In these animals, dose-dependent and naloxone-reversible intrathecal fentanyl antinociception was significantly impaired and associated with a loss in MOR immunoreactivity of calcitonin gene-related peptide–immunoreactive (CGRP-IR) sensory nerve terminals, membrane-bound MOR binding sites, and MOR-stimulated G protein coupling within the dorsal horn of the spinal cord. Intrathecal delivery of nerve growth factor (NGF) in diabetic animals normalized spinal MOR number and G protein coupling and rescued spinal fentanyl-induced antinociception. These findings identify for the first time a loss in functional MOR on central terminals of sensory neurons as a contributing factor for the impaired spinal opioid responsiveness during advanced STZ-induced diabetes that can be reversed by NGF. Moreover, they support growing evidence of a distinct regulation of opioid responsiveness during various painful states of disease (eg, arthritis, cancer, neuropathy) and may give novel therapeutic incentives. PerspectiveIn diabetic neuropathy a loss in sensory neuron mu-opioid receptor number and coupling contributes to impaired spinal opioid antinociception that can be reversed by NGF. These findings support growing evidence of a distinct regulation of opioid responsiveness during various painful diseases and may give novel therapeutic incentives.

Innervation of papilla vallata in diabetic rats

Introduction: Abnormal sensations such as pain and impairment of taste are symptoms of approximately 10% of patients having diabetes mellitus. Aim: The aim of the study was to investigate and quantify the different neuropeptide containing nerve fibres in the vallate papilla of the diabetic rat. Methods: Immunohistochemical methods were used to study the changes of the number of different neuropeptide containing nerve terminals located in the vallate papillae in diabetic rats. Diabetes was induced in the rats with streptozotocin. Results: Two weeks after streptozotocin treatment the number of the substance P, galanin, vasoactive intestinal polypeptide and neuropeptide Y immunoreactive nerve terminals was significantly increased (p<0.05) in the tunica mucosa of the tongue. The number of the lymphocytes and mast cells was also increased significantly. Some of the immunoreactive nerve terminals were located in the lingual epithelium both intragemmally and extragemmally and were seen to comprise dense bundles in the lamina propria just beneath the epithelium. No taste cells were immunoreactive for any of the investigated peptides. Vasoactive intestinal polypeptide and neuropeptide Y immunoreactive nerve fibres were not detected in the taste buds. For weeks after streptozotocin administration the number of the substance P, calcitonin gene related peptide and galanin immunoreactive nerve terminals was decreased both intragemmally and intergemmally. In case of immediate insulin treatment, the number of the immunoreactive nerve terminals was similar to that of the controls, however, insulin treatment given 1 week later to diabetic rats produced a decreased number of nerve fibers. Morphometry revealed no significant difference in papilla size between the control and diabetic groups, but there were fewer taste buds (per papilla). Conclusions: Increased number of immunoreactive nerve terminals and mast cells 2 weeks after the development of diabetes was the consequence of neurogenic inflammation which might cause vasoconstriction and lesions of the oral mucosa. Taste impairment, which developed 4 weeks after streptozotocin treatment could be caused by neuropathic defects and degeneration or morphological changes in the taste buds and nerve fibres. Orv. Hetil., 2013, 154, 443–448.

Expression of P2X2 and P2X3 receptors in the rat carotid sinus, aortic arch, vena cava, and heart, as well as petrosal and nodose ganglia

With single- and double-labeling immunofluorescence techniques, the distribution patterns and morphological characteristics of P2X(2)- and P2X(3)-immunoreactive nerve fiber terminals and neuronal bodies have been studied in the main circulatory system baroreceptors and the nodose and petrosal ganglia of rats. A high density of P2X(2)- and P2X(3)-immunoreactive nerve fiber terminals was detected in the carotid sinus. P2X(2)- and P2X(3)-immunoreactive nerve fiber terminals were also distributed widely in the aortic arch, atrium, vena cava, and ventricles. Almost all the P2X(2)-immunoreactive nerve fiber terminals were immunoreactive for P2X(3) receptors. P2X(2)- and P2X(3)-immunoreactive neuronal bodies were also detected in the nodose and petrosal ganglia, which are the sources of the P2X(2)- and P2X(3)-immunoreactive nerve terminals. P2X(2) and P2X(3) receptors were expressed in the same ganglionic neurons. These data indicate that extracellular ATP, via the homomeric P2X(2) and P2X(3) receptors, and heteromeric P2X(2/3) receptor in the sensory receptors of carotid sinus, aortic arch, atrium, and vena cava, may be involved in the regulation of systematic circulation blood pressure.

Acute versus long-term effects of 6-hydroxydopamine on oxidative stress and dopamine depletion in the striatum of mice

Oxidative stress is one of the mechanisms which may be important in the pathogenesis of Parkinson's disease. In the current study, the effects of 6-hydroxydopamine (6-OHDA) perfusion on hydroxyl radical formation in the mouse striatum were investigated using the in vivo salicylate trapping microdialysis technique. The latter uses salicylate as a trapping agent for hydroxyl radicals with formation of 2,3-dihydroxybenzoic acid (2,3-DHBA), which is measured by HPLC. Two different approaches of the technique were validated in mice. First, perfusion of the trapping agent salicylate (1 mM) via the probe in combination with 6-OHDA (5 μM) was used to screen for radical scavenging properties of compounds in mice. Alternatively, striatal administration of 6-OHDA in a concentration known to induce nigrostriatal denervation (1 mM), without the trapping agent, allowed to maximally challenge the neuronal microenvironment and as such to investigate both its acute and long-term effects. In the first method, as expected, glutathione (GSH) (1.5 mM) prevented the 6-OHDA-induced increase in 2,3-DHBA levels. In the second method, GSH prevented the hydroxyl radical formation, while depletion of GSH with 2-cyclohexen-1-one (CHO) resulted in significantly higher 2,3-DHBA levels than when 6-OHDA was perfused alone. Three weeks after the local 6-OHDA perfusion, the total striatal dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) content were reduced by 30%, compared to the intact striatum, accompanied by a reduction in striatal tyrosine hydroxylase (TH) immunoreactive (ir) nerve terminals. This suggests that the second method can be used to determine the acute as well as the long-term effects of 6-OHDA in the mouse striatum.

5‐HT1D Receptor Immunoreactivity in the Sphenopalatine Ganglion: Implications for the Efficacy of Triptans in the Treatment of Autonomic Signs Associated With Cluster Headache

To determine if 5-HT(1D) receptors are located in the sphenopalatine ganglion. While the 5-HT(1D) receptor has been described in sensory and sympathetic ganglia in the head, it was not known whether they were also located in parasympathetic ganglia. We used retrograde labeling combined with immunohistochemistry to examine 5-HT(1D) receptor immunoreactivity in rat sphenopalatine ganglion neurons that project to the lacrimal gland, nasal mucosa, cerebral vasculature, and trigeminal ganglion. We found 5-HT(1D) receptor immunoreactivity in nerve terminals around postganglionic cell bodies within the sphenopalatine ganglion. All 5-HT(1D) -immunoreactive terminals were also immunoreactive for calcitonin gene-related peptide but not vesicular acetylcholine transporter, suggesting that they were sensory and not preganglionic parasympathetic fibers. Our retrograde labeling studies showed that approximately 30% of sphenopalatine ganglion neurons innervating the lacrimal gland, 23% innervating the nasal mucosa, and 39% innervating the trigeminal ganglion were in apparent contact with 5-HT(1D) receptor containing nerve terminals. These data suggest that 5-HT(1D) receptors within primary afferent neurons that innervate the sphenopalatine ganglion are in a position to modulate the excitability of postganglionic parasympathetic neurons that innervate the lacrimal gland and nasal mucosa, as well as the trigeminal ganglion. This has implications for triptan (5-HT(1D) receptor agonist) actions on parasympathetic symptoms in cluster headache.

Correlation Between the Distribution of SP and CGRP Immunopositive Neurons in Dorsal Root Ganglia and the Afferent Sensation of Preputial Frenulum

The aim of this study was to explore the distribution of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive nerve terminals in the penis prepuce and the preputial frenulum. The possible correlation between SP- and CGRP-immunopositive neurons in dorsal root ganglia (DRG) and the afferent sensation of the penile preputial frenulum is also discussed. Immunohistochemistry showed SP- and CGRP-positive nerve terminals in the epidermal basal layer of the prepuce and frenulum in adult human males. The majority of the nerve terminals presented as bundles of different lengths and a few as enlarged nodosities. The density of SP- and CGRP-immunopositive nerve terminals in the preputial frenulum was significantly higher than those in the penis prepuce (P<0.01). Fluoro-Gold (FG) retrograde tracing method was used to trace the origin of nerve terminals in Sprague-Dawley rats. SP and CGRP immunofluorescence labeling was employed to detect the distribution of SP- and CGRP-immunoreactive neurons in DRG. FG retro-labeled neurons were localized in L(6) -DRG and S(1) -DRG. All the FG/SP and FG/CGRP double-labeled neurons were medium or small-sized. One-third of the FG-labeled neurons were SP-immunoreactive, and a half of them CGRP-immunoreactive in L(6) -DRG and S(1) -DRG, respectively. The FG/SP/CGRP-labeled neurons accounted for one fifth of the FG retro-labeled neurons. Taken together, these data suggest that the SP- and CGRP-immunopositive nerve fibers may participate in the transmission of afferent sensation in the preputial frenulum.

Induced NG2 expressing microglia in the facial motor nucleus after facial nerve axotomy

Chondroitin sulfate proteoglycan (NG2) expressing cells, ubiquitously distributed in the CNS respond to injured or diseased neurons; however, their behaviors toward injured neurons have remained to be fully explored. In the present study, along with astrocytic and microglial responses, NG2 expressing cells reacted swiftly and robustly in the facial motor nucleus (FMN) subjected to axotomy. With time, hypertrophic NG2 expressing cells gradually adhered to and enwrapped the axotomized motoneurons. Tight encapsulations around axotomized motoneurons were eventually formed at 7, 14, and 28 days after axotomy. NG2 positive processes appeared to interpose between synapsin-1 immunoreactive nerve terminals and surfaces of axotomized motoneurons. Double labeling results showed that NG2 expressing cells encapsulating axotomized facial motoneurons were mainly microglia marked by OX42 and lectin; only a few of them were positive to platelet-derived growth factor-α receptor and none of them positive to ED-1. No Rhodamine particle was detected in the FMN ipsilateral to axotomy after venous injection of the particles. The results suggest that activated microglia in lesioned FMN were induced to express NG2 molecules. It is concluded that axotomized FMN showed two types of NG2 expressing cells namely constitutive NG2 cells and induced-NG2 expressing microglia.

Transient supersensitivity to α‐adrenoceptor agonists, and distinct hyper‐reactivity to vasopressin and angiotensin II after denervation of rat tail artery

Vascular 'denervation' hyper-reactivity has generally been investigated 1-2 weeks after administration of chemicals that temporarily prevent transmitter release, but do not necessarily inactivate the neuronal noradrenaline transporters (NETs). We have investigated the reactivity of rat tail arteries over longer periods after removing the terminals by surgical denervation. Two and 7 weeks after denervation, myography was used to assess contractions of isolated arterial segments to phenylephrine, methoxamine, clonidine, vasopressin and angiotensin II (AII). Denervation was confirmed by lack of tyrosine hydroxylase immunoreactive nerve terminals. The NET inhibitor, desmethylimipramine, increased the pEC(50) for phenylephrine in control, but not denervated arteries after both 2 and 7 weeks. Relative to controls, pEC(50)s for phenylephrine (with desmethylimipramine), methoxamine, clonidine and vasopressin were increased at 2 but not 7 weeks after denervation. The pEC(50) for phenylephrine in the absence of desmethylimipramine was greater than control after both 2 and 7 weeks' denervation. The maximum contraction to vasopressin was larger than in controls at 2 but not 7 weeks after denervation, whereas contractions to AII were markedly enhanced at both time points. Increased vascular reactivity to alpha(1)- and alpha(2)-adrenoceptor agonists, and vasopressin is transient following denervation. After 7 weeks, increased reactivity to phenylephrine can be entirely accounted for by the loss of NETs. Maintained supersensitivity to AII indicates that denervation differentially and selectively affects vascular reactivity to circulating vasoconstrictor agents. This might explain persistent vasoconstriction in denervated skin of humans after nerve injuries.

Presence and distribution of serotonin immunoreactivity in the cyprids of the barnacle Balanus amphitrite

In this work, the presence and distribution of serotonin in the cyprid of the barnacle Balanus amphitrite were investigated by immunohistochemical methods. Serotonin-like immuno-reactive neuronal cell bodies were detected in the central nervous system only. Various clusters of immunoreactive neuronal cell bodies are distributed in the brain (protocerebrum, deutocerebrum, optical lobes), and at least, four pairs of neuronal cell bodies were detected in the centrally positioned neuropil of the posterior ganglion. Rich plexuses of immunoreactive nerve fibers in the neuropil area were also observed. Furthermore, bundles of strongly immunoreactive nerve fibers surrounding the gut wall were localized, and immunoreactive nerve terminals in the antennules and compound eyes were observed. These data demonstrate the presence of a serotonin-like immunoreactive substance in the barnacle cyprids; furthermore, its immunolocalization in the cephalic nerve terminals allows us to postulate the involvement of this bioactive molecule in substrate recognition during the settlement process.

Cholinergic signal transduction in the mouse sphenopalatine ganglion

The sphenopalatine ganglia (SPG) receive their preganglionic innervation from the ventro-lateral reticular formation and nuclei of the caudal pons, and are involved in parasympathetic control of cranial glandular and vascular components including the blood supply to specific brain areas. In 53% of all SPG neurons, a particular member (MOL2.3) of the odorant receptor superfamily is co-expressed with green fluorescent protein (GFP) in MOL2.3 transgenic mouse pups. Choline acetyltransferase and vesicular acetylcholine transporter (VAChT) could be demonstrated in 90% of the GFP-positive, and 60% of the GFP-negative cells, these cells thus representing cholinergic neurons. Some 50% of all SPG neurons were nitrergic at a high rate of VAChT co-expression, the majority of them being GFP-positive. Most SPG neurons received cholinergic innervation as demonstrated by perineuronal VAChT immunoreactive nerve terminals. To characterize cholinergic signal transduction in SPG neurons, calcium imaging experiments were performed in a SPG primary culture system containing GFP-positive and -negative neurons. Ganglionic neurons could repeatedly be activated by cholinergic stimulation in a dose-dependent manner, with calcium entering all cells from the extracellular compartment. Stimulation with specific agonists supported prevalence of nicotinic cholinergic receptors (nAChRs). Inhibition of cholinergically induced intracellular calcium signalling by various ω-conotoxins indicated functional expression of α3β4 and α7 nAChR subtypes in murine SPG cells, which could be supported by RT-PCR analysis of the neonatal mouse SPG. With regard to secondary cholinergic activation, L- but not N-subtype voltage-gated calcium channels might represent a prime target. Nicotinic signal transduction did not prove to be different in GFP-positive as compared to-negative murine SPG neurons.

Influences of surgical decompression on the dorsal horn after chronic constriction injury: Changes in peptidergic and δ-opioid receptor (+) nerve terminals

To understand plastic changes in the dorsal horn related to neuropathic pain, we developed a model of decompression in rats with chronic constriction injury (CCI) and investigated corresponding changes in the dorsal horn. At postoperative week 4 (POW 4) of CCI, rats were divided into a decompression group, in which ligatures were removed, and a CCI group, in which ligatures remained. Spinal cords were immunostained for substance P (SP), the δ-opioid receptor (DOR), and calcitonin gene-related peptide (CGRP). Areas of immunoreactive nerve terminals in the dorsal horn were quantified and expressed as the dorsal horn index (immunoreactive areas of the operated side compared with those of the contralateral side). At POW 4, dorsal horn indexes of all of these molecules were significantly reduced in both groups to similar degrees (0.36∼0.43). At POW 8, neuropathic pain behaviors had completely disappeared in the decompression group with significant reversal of the dorsal horn indexes compared with the CCI group (0.81±0.02 vs. 0.58±0.09, P<0.001 for SP and 0.75±0.04 vs. 0.55±0.03, P<0.001 for DOR). In the CCI group, neuropathic pain behaviors became normalized at POW 12 with corresponding changes in dorsal horn indexes for both SP and DOR similar to those of the decompression group. In contrast, changes in the dorsal horn indexes of CGRP were similar in both the CCI and decompression groups throughout the experimental period. These findings suggest that CCI and decompression cause different patterns in peptidergic and DOR (+) nerve terminals in the dorsal horn.

Sweat gland innervation is pioneered by sympathetic neurons expressing a cholinergic/noradrenergic co-phenotype in the mouse

Classic neurotransmitter phenotypes are generally predetermined and develop as a consequence of target-independent lineage decisions. A unique mode of target-dependent phenotype instruction is the acquisition of the cholinergic phenotype in the peripheral sympathetic nervous system. A body of work suggests that the sweat gland plays an important role to determine the cholinergic phenotype at this target site. A key issue is whether neurons destined to innervate the sweat glands express cholinergic markers before or only after their terminals make target contact. We employed cholinergic-specific over-expression of the vesicular acetylcholine transporter (VAChT) in transgenic mice to overcome sensitivity limits in the detection of initial cholinergic sweat gland innervation. We found that VAChT immunoreactive nerve terminals were present around the sweat gland anlage already from the earliest postnatal stages on, coincident selectively at this sympathetic target with tyrosine hydroxylase–positive fibers. Our results provide a new mechanistic model for sympathetic neuron–target interaction during development, with initial selection by the target of pioneering nerve terminals expressing a cholinergic phenotype, and subsequent stabilization of this phenotype during development.

Role of the amygdaloid cholecystokinin (CCK)/gastrin‐2 receptors and terminal networks in the modulation of anxiety in the rat. Effects of CCK‐4 and CCK‐8S on anxiety‐like behaviour and [3H]GABA release

The amygdala plays a key role in fear and anxiety. The intercalated islands are clusters of glutamate-responsive GABAergic neurons rich in cholecystokinin (CCK)-2 receptors which control the trafficking of nerve impulses from the cerebral cortex to the central nucleus of amygdala. In this study, the nature of the CCK-glutamate-GABA interactions within the rat rostral amygdala, and their relevance for anxiety, were studied. CCK/gastrin-like immunoreactive nerve terminals were found to be mainly restricted to the paracapsular intercalated islands and the rostrolateral part of the main intercalated island. Behaviourally, the bilateral microinjection of CCK-4 (0.043-4.3 pmol/side) or CCK-8S (4.3 pmol/side) into the rostrolateral amygdala reduced the open-arm exploration in the elevated plus-maze without affecting locomotion. In contrast, neither CCK-4 nor CCK-8S (0.043-4.3 pmol/side) had any effects in the shock-probe burying test as compared with their saline-treated controls. Biochemically, CCK-4 (0.3 and 1.5 microm), unlike CCK-8S, enhanced significantly the K(+)-stimulated release of [(3)H]GABA from amygdala slices. These effects were fully prevented by prior superfusion of the slices with either the selective CCK-2 receptor antagonist CR2945 (3 microm), or 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), 10 microm, a glutamatergic (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist. It is suggested that CCK modulates glutamate-GABA mechanisms by acting on CCK-2 receptors via volume transmission occurring at the level of the basolateral amygdaloid nucleus and/or by synaptic or perisynaptic volume transmission in the region of the rostrolateral main and paracapsular intercalated islands, resulting in subsequent disinhibition of the central amygdaloid nucleus and anxiety or panic-like behaviour.

The distribution of dopamine D1 receptor and μ-opioid receptor 1 receptor immunoreactivities in the amygdala and interstitial nucleus of the posterior limb of the anterior commissure: Relationships to tyrosine hydroxylase and opioid peptide terminal systems

Mismatches between dopamine innervation and dopamine D1 receptor (D1) distribution have previously been demonstrated in the intercalated cell masses of the rat amygdala. Here the distribution of enkephalin and β-endorphin immunoreactive (IR) nerve terminals with respect to their μ-opioid receptors is examined in the intercalated cell masses, along with a further immunohistochemical analysis of the dopamine/D1 mismatches. A similar analysis is also made within the extended amygdala.A spatial mismatch in distribution patterns was found between the μ-opioid receptor-1 immunoreactivity and enkephalin IR in the main intercalated island of the amygdala. Discrete cell patches of dopamine D1 receptor and μ-opioid receptor-1 IR were also identified in a distinct region of the extended amygdala, the interstitial nucleus of the posterior limb of the anterior commissure, medial division (IPACM), which displayed sparse tyrosine hydroxylase or enkephalin/β-endorphin IR nerve terminals. Furthermore, distinct regions of the main intercalated island that showed dopamine/D1 receptor matches (the rostral and rostrolateral parts) were associated with strong dopamine and cyclic AMP regulated phosphoprotein, 32 kDa-IR in several D1 IR neuronal cell bodies and dendrites, whereas this was not the case for the dopamine/D1 mismatch areas (the rostromedial and caudal parts) of the main intercalated island.The lack of correlation between the terminal/receptor distribution patterns suggests a role for volume transmission for μ-opioid receptor- and dopamine D1 receptor-mediated transmission in distinct regions of the amygdala and extended amygdala. This may have implications for amygdaloid function, where slow long lasting responses may develop as a result of volume transmission operating in opioid peptide and dopaminergic communication.

Distribution of L1cam mRNA in the adult mouse brain: In situ hybridization and Northern blot analyses

Previous immunohistochemical analysis revealed a wide distribution of L1cam-positive neural and nonneural structures in adult mouse brain. Although there were numerous punctate immunoreactive nerve terminals, only a few immunoreactive neuronal cell somata were present (Munakata et al. [2003] BMC Neurosci. 4:7). To explore the distribution of L1cam mRNA-containing cells, which are interpreted to be L1cam-producing cells, we performed in situ hybridization histochemistry with an antisense L1cam cRNA probe. L1cam mRNA was distributed widely from the olfactory bulb to the upper cervical cord with an uneven localization pattern in adult brain. All positive cell somata with silver grains after emulsion autoradiography were neuronal, and no grains were detected on nonneural cells in the present study. A high density of signals for neuronal L1cam mRNA was found in the thalamus, mammillary body, and hippocampus. In addition, strong hybridization signals were localized in various nuclei: main and accessory olfactory bulb, compact part of the substantia nigra, pontine gray matter, tegmental reticular nucleus, Edinger-Westphal nucleus, trigeminal motor nucleus, locus coeruleus, mesencephalic trigeminal nucleus, raphe nuclei, facial nucleus, ambiguus nucleus, dorsal motor vagal nucleus, and inferior olivary nucleus. Some long projection neurons such as the pyramidal, mitral, principal neurons of several cranial nuclei, and presumably monoaminergic cells containing noradrenalin, dopamine, and serotonin, expressed high levels of L1cam.

Genioglossal hypoglossal muscle motoneurons are contacted by nerve terminals containing delta opioid receptor but not mu opioid receptor-like immunoreactivity in the cat: a dual labeling electron microscopic study

This study has investigated (1) the distribution of delta opioid receptor (DOR) or mu opioid receptor (MOR) containing elements in the hypoglossal nucleus of the adult cat; and (2) the association of these processes with retrogradely labeled genioglossus muscle motoneurons. Cholera toxin B conjugated to horseradish peroxidase (CTB-HRP) was injected into the genioglossus muscle on the right side of four isoflurane-anesthetized cats. Forty-four to 52 h later, the animals were sacrificed. Motoneurons containing HRP were labeled with a histochemical reaction utilizing tetramethylbenzidine (TMB) as the chromogen. The tissues were then processed for immunocytochemistry, using an antiserum raised against DOR or MOR using diaminobenzidine (DAB) as the chromogen. At the light microscopic level, retrogradely labeled cells were observed primarily ipsilaterally in ventral and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were observed immediately caudal to obex. DOR-like immunoreactive processes were apparent at the light microscopic level in the hypoglossal nucleus, but MOR-like immunoreactive processes were not. Both DOR and MOR-like immunoreactive processes were observed in other brainstem areas such as the spinal trigeminal nucleus. At the electron microscopic level, DOR-like immunoreactive nerve terminals formed synaptic contacts with retrogradely labeled genioglossus muscle motoneuronal dendrites and perikarya in the hypoglossal nucleus. Nineteen (19) percent of the DOR terminals contacted retrogradely labeled genioglossus muscle motoneurons. DOR-immunoreactive terminals also synapsed on unlabeled dendrites and somata. Few MOR-like immunoreactive terminals were found at the EM level in the hypoglossal nucleus, and none of these terminals contacted retrogradely labeled neuronal profiles from the GG muscle. These are the first ultrastructural studies demonstrating synaptic interactions between functionally identified hypoglossal motoneurons and DOR terminals, and that enkephalins most likely act presynaptically to modulate the release of other neurotransmitters that affect GG motoneuron activity. These studies demonstrate that hypoglossal motoneurons which innervate the major protruder muscle of the tongue, the genioglossus muscle, are modulated by terminals containing DOR, and that enkephalins acting on DOR but not MOR in the hypoglossal nucleus may play a role in the control of tongue protrusion.

A model of periodontitis in the rat: effect of lipopolysaccharide on bone resorption, osteoclast activity, and local peptidergic innervation.

To establish and characterise a rat model of periodontitis that reiterates the features of human disease. Periodontal inflammation was induced by a single injection of 10 microg liposaccharide (LPS) (Salmonella typhimurium) in 1 microl saline into rat mandibular gingiva at the buccomesial aspect of the second molar. Animals were killed after 3, 7 and 10 days, mandibles dissected and sectioned for histological and immunocytochemical analysis. LPS injection resulted in a significant gingival and periodontal inflammation with inflammatory infiltrate, apical migration of the junctional epithelium, interdental bone loss, and activation of osteoclasts at the site of injection 7 and 10 days after injection. At 10 days post injection, there was a significant trend for bone loss on both sides of the mandible. Periodontal inflammation was associated with alteration in the levels of calcitonin gene-related peptide-like immunoreactivity in nerve terminals innervating the inflamed gingival papilla. Intragingival injection of LPS in the rat provides an easily induced reproducible experimental model of periodontal inflammation that reiterates features of human disease.