Sustained hypertension increases the density of AMPA receptor subunit, GluR1, in baroreceptive regions of the nucleus tractus solitarii of the rat.

AMPA-type glutamate receptors in the nucleus tractus solitarii (NTS) are necessary for the baroreceptor reflex, a primary mechanism for homeostatic regulation of blood pressure. Within NTS, the GluR1 subunit of the AMPA receptor is found primarily in dendritic spines. We previously showed that both GluR1 and dendritic spine density are increased in NTS of spontaneously hypertensive rats (SHRs). We hypothesize that both receptor and synaptic plasticity are induced by a sustained elevation in arterial pressure. To test the general nature of this hypothesis, we examined whether similar changes in GluR1 density are found in a renovascular model of hypertension, the DOCA-salt rat, and if these changes are preventable by normalizing blood pressure with hydralazine, a peripherally acting vasodilator. Using immunoperoxidase detection, GluR1 appears as small puncta at the light microscopic level, and is found in dendritic spines at the ultrastructural level. Following the development of hypertension, GluR1 spine and puncta counts were significantly greater in DOCA-salt rats than controls. Hydralazine treatment (4-5 weeks) prevented the development of hypertension in DOCA-salt rats and reduced blood pressure of SHRs to normotensive levels. The density of GluR1 puncta in the NTS was significantly reduced by hydralazine treatment in the SHR model. These results show that hypertension alters dendritic spines containing AMPA-type glutamate receptors within NTS, suggesting that adjustments in GluR1 expression within NTS are part of the synaptic adaptations to the hypertensive state.

Cranial visceral afferent pathways through the nucleus of the solitary tract to caudal ventrolateral medulla or paraventricular hypothalamus: target-specific synaptic reliability and convergence patterns.

Cranial visceral afferents activate central pathways that mediate systemic homeostatic processes. Afferent information arrives in the brainstem nucleus of the solitary tract (NTS) and is relayed to other CNS sites for integration into autonomic responses and complex behaviors. Little is known about the organization or nature of processing within NTS. We injected fluorescent retrograde tracers into two nuclei to identify neurons that project to sites involved in autonomic regulation: the caudal ventrolateral medulla (CVLM) or paraventricular nucleus of the hypothalamus (PVN). We found distinct differences in synaptic connections and performance in the afferent path through NTS to these neurons. Anatomical studies using confocal and electron microscopy found prominent, primary afferent synapses directly on somata and dendrites of CVLM-projecting NTS neurons identifying them as second-order neurons. In brainstem slices, afferent activation evoked large, constant latency EPSCs in CVLM-projecting NTS neurons that were consistent with the precise timing and rare failures of monosynaptic contacts on second-order neurons. In contrast, most PVN-projecting NTS neurons lacked direct afferent input and responded to afferent stimuli with highly variable, intermittently failing synaptic responses, indicating polysynaptic pathways to higher-order neurons. The afferent-evoked EPSCs in most PVN-projecting NTS neurons were smaller and unreliable but also often included multiple, convergent polysynaptic responses not observed in CVLM-projecting neurons. A few PVN-projecting NTS neurons had monosynaptic EPSC characteristics. Together, we found that cranial visceral afferent pathways are structured distinctly within NTS depending on the projection target. Such, intra-NTS pathway architecture will substantially impact performance of autonomic or neuroendocrine reflex arcs.

Ultrastructure of primary afferent terminals and synapses in the rat nucleus of the solitary tract: comparison among the greater superficial petrosal, chorda tympani, and glossopharyngeal nerves.

The greater superficial petrosal (GSP), chorda tympani (CT), and glossopharyngeal (IX) nerves terminate in overlapping patterns in the brainstem in the rat nucleus of the solitary tract (NTS). There is one region, in particular, that receives overlapping inputs from all three nerves and is especially plastic during normal and experimentally altered development. To provide the requisite data necessary ultimately to delineate the circuitry in this region, we characterized the morphology of the synaptic inputs provided by the GSP, CT, and IX nerves through transmission electron microscopy. Although all three nerves had features characteristic of excitatory nerve terminals, ultrastructural analysis revealed dimorphic morphologies differentiating IX terminals from GSP and CT terminals. IX terminals had a larger area than GSP and CT terminals, and more synapses were associated with IX terminals compared with GSP and CT terminals. Additionally, IX terminals formed synapses most often with spines, as opposed to GSP and CT terminals, which formed synapses more often with dendrites. IX terminals also exhibited morphological features often associated with synaptic plasticity more often than was seen for GSP and CT terminals. These normative data form the basis for future studies of developmentally and environmentally induced plasticity in the rodent brainstem.

Ultrastructural evidence for selective noradrenergic innervation of CNS vagal projections to the fundus of the rat.

We reported pharmacological data suggesting that stimulation of the vago-vagal reflex activates noradrenergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the fundus, but not to the antrum [Ferreira Jr., M., Sahibzada, N., Shi, M., Panico, W., Neidringhaus, M., Wasserman, A., Kellar, K.J., Verbalis, J., Gillis, R.A., 2002. CNS site of action and brainstem circuitry responsible for the intravenous effects of nicotine on gastric tone. J. Neurosci. 22, 2764-2779.]. The purpose of this study was to use an ultrastructural approach to test the hypothesis that noradrenergic terminals form synapses with DMV fundus-projecting neurons, but not with DMV antrum-projecting neurons. A retrograde tracer, CTbeta-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTbeta-HRP, and immunocytochemically for either DbetaH or PNMT by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 482 synapses on 238 neurons that projected to the fundus revealed that 17.4+/-2.7% (n=4) of synaptic contacts were with DbetaH-IR terminals. Of 165 fundus-projecting neurons, 4.4+/-1.5% (n=4) formed synaptic contacts with PNMT-IR terminals. In contrast, the examination of 384 synapses on 223 antrum-projecting neurons revealed no synaptic contact with DbetaH-IR terminals. These data provide proof that norepinephrine containing nerve terminals synapse with DMV fundus-projecting neurons but not with DMV antrum-projecting neurons. These data also suggest that brainstem circuitry controlling the fundus differs from circuitry controlling the antrum.

Pattern differentiation of excitatory and inhibitory synaptic inputs on distinct neuronal types in the rat caudal nucleus of the tractus solitarius.

Region- and size-specific neuronal organizations of the caudal nucleus of the tractus solitarius (cNTS) were investigated, followed by analyses of excitatory and inhibitory synaptic input patterns onto specific cell types by patch clamp recordings and immunoelectron microscopy. Cell size distribution and numerical density of cNTS neurons were examined in subregions at levels of the area postrema. In the subpostremal and dorsomedial subnuclei, characterized by the presence of dense glutamatergic and sparse GABAergic somata, small calbindin neurons constituted 42% of the total cells. The medial subnucleus contained large numbers of glutamatergic, GABAergic, and catecholaminergic somata and large tyrosine hydroxylase-containing cells constituted 13% in this region. In total, small neurons (<150 microm2) represented about 80% of the cell population in the cNTS. Predominant excitatory postsynaptic currents were observed in the adult small neurons, while inhibitory postsynaptic currents were more evident in larger neurons, irrespective of subnuclear location. This distinct differentiation of postsynaptic current patterns was not evident in neonates. GABAergic synapses were more frequently associated with dendrites of large catecholaminergic cells (73%) than with those of small calbindin-containing cells (10%) in adults. These results indicate that differential synaptic input patterns were developmentally established in distinct small and large neurons.

Morphological evidence for GABA/glycine-cocontaining terminals in synaptic contact with neurokinin-1 receptor-expressing neurons in the sacral dorsal commissural nucleus of the rat.

Previous studies have shown that neurons in the sacral dorsal commissural nucleus (SDCN) express neurokinin-1 receptor (NK1R) and can be modulated by the co-release of GABA and glycine (Gly) from single presynaptic terminal. These results raise the possibility that GABA/Gly-cocontaining terminals might make synaptic contacts with NK1R-expressing neurons in the SDCN. In order to provide morphological evidence for this hypothesis, the triple-immunohistochemical studies were performed in the SDCN. Triple-immunofluorescence histochemical study showed that some axon terminals in close association with NK1R-immunopositive (NK1R-ip) neurons in the SDCN were immunopositive for both glutamic acid decarboxylase (GAD) and glycine transporter 2 (GlyT2). In electron microscopic dual- and triple-immunohistochemistry for GAD/GlyT2, GAD/NK1R, GlyT2/NK1R, or GAD/GlyT2/NK1R also revealed dually labeled (GAD/GlyT2-ip) synaptic terminals upon SDCN neurons, as well as GAD- and/or GlyT2-ip axon terminals in synaptic contact with NK1R-ip SDCN neurons. These results suggested that some synaptic terminals upon NK1R-expressing SDCN neurons co-released both GABA and Gly.

Subcellular localization of neuronal nitric oxide synthase in the rat nucleus of the solitary tract in relation to vagal afferent inputs.

In the nucleus of the solitary tract (NTS), nitric oxide (NO) modulates neuronal circuits controlling autonomic functions. A proposed source of this NO is via nitric oxide synthase (NOS) present in vagal afferent fibre terminals, which convey visceral afferent information to the NTS. Here, we first determined with electron microscopy that neuronal NOS (nNOS) is present in both presynaptic and postsynaptic structures in the NTS. To examine the relationship of nNOS to vagal afferent fibres the anterograde tracer biotinylated dextran amine was injected into the nodose ganglion and detected in brainstem sections using peroxidase-based methods. nNOS was subsequently visualised using a pre-embedding immunogold procedure. Ultrastructural examination revealed nNOS immunoreactivity in dendrites receiving vagal afferent input. However, although nNOS-immunoreactive terminals were frequently evident in the NTS, none were vagal afferent in origin. Dual immunofluorescence also confirmed lack of co-localisation. Nevertheless, nNOS immunoreactivity was observed in vagal afferent neurone cell bodies of the nodose ganglion. To determine if these labelled cells in the nodose ganglion were indeed vagal afferent neurones nodose ganglion sections were immunostained following application of cholera toxin B subunit to the heart. Whilst some cardiac-innervating neurones were also nNOS immunoreactive, nNOS was never detected in the central terminals of these neurones. These data show that nNOS is present in the NTS in both pre- and postsynaptic structures. However, these presynaptic structures are unlikely to be of vagal afferent origin. The lack of nNOS in vagal afferent terminals in the NTS, yet the presence in some vagal afferent cell bodies, suggests it is selectively targeted to specific regions of the same neurones.

Vanilloid receptor 1 expression in the rat urinary tract.

Previous findings have shown that the capsaicin sensitivity of sensory fibres is due to the expression of a specific membrane protein, the vanilloid receptor type 1 (VR1). In the present work we studied the distribution, morphology and the neurochemical content of nerve fibres expressing this receptor in the rat urinary tract. Immunolabelling was performed against the VR1 and the positive fibres were examined by light and electron microscopy. Colocalisation of VR1 and substance P or calcitonin gene-related peptide immunoreactivities, and isolectin B4 binding, was evaluated under the confocal microscope. In addition, the effect of intravesical administration of resiniferatoxin, an ultra-potent vanilloid receptor agonist, in the receptor expression in the bladder was also studied. Numerous VR1-immunoreactive fibres were found in the mucosa and muscular layer of the entire urinary tract except the kidney. In the bladder, most fibres were also substance P- or calcitonin gene-related peptide-immunoreactive but did not bind isolectin B4. Under the electron microscope VR1 immunoreactivity was confined to unmyelinated axons and varicosities containing small clear and large dense-core synaptic vesicles. They occurred beneath or among epithelial cells or closely apposed to smooth muscle cells. Intravesical resiniferatoxin decreased VR1 immunoreactivity transiently. These data indicate that primary sensory fibres expressing VR1 are extremely abundant in the rat urinary tract and that, in contrast to the skin, they belong almost exclusively to the peptide-containing sub-population of primary afferents. As capsaicin-sensitive bladder afferents are involved in nociception and reflex micturition control, the numerous free terminal nerve endings expressing VR1 in the mucosa seem more adequate to accomplish the former function. However, the close apposition between VR1-expressing fibres and smooth muscle cells suggests that they may also encode the tonus of the muscular layer.

Association of extracellular acetylcholinesterase with gustatory nerve terminal fibers in the nucleus of the solitary tract.

Acetylcholinesterase (AChE) staining is associated with terminal fields of the glossopharyngeal and chorda tympani nerves in the nucleus of the solitary tract (NST). To address AChE function at these sites, the location of the staining was examined at the fine structural level in combination with the labeling of chorda tympani nerve fibers with biotinylated dextran in golden Syrian hamsters. AChE staining was located in the endoplasmic reticulum of geniculate ganglion neuronal somata, and extracellularly, surrounding labeled chorda tympani terminal fibers and boutons in the NST. Neuronal profiles adjacent to these labeled fibers were stained less intensely, whereas most non-adjacent profiles were unstained. The location of staining is consistent with the secretion of AChE into the extracellular space by primary afferent chorda tympani fibers. AChE staining was reduced in the dextran-labeled chorda tympani fibers and terminals as well as adjacent non-labeled profiles 2 weeks following nerve transection and dextran application. The distribution of staining outside synapses and the loss of staining following denervation is suggestive of a non-cholinergic role for AChE in the intact gustatory system.

Synaptic connections between trigemino-parabrachial projection neurons and gamma-aminobutyric acid- and glycine-immunoreactive terminals in the rat.

The synaptic connections between gamma-aminobutyric acid (GABA)- and glycine-immunoreactive terminals and neurons projecting to the lateral parabrachial region were examined by a combination of retrograde tracing and immunohistochemical staining in the rat medullary dorsal horn. After injection of horseradish peroxidase (HRP) into the right lateral parabrachial region, HRP retrogradely labeled neurons were observed bilaterally in laminae I, II and III of the medullary dorsal horn with an ipsilateral predominance. GABA- and glycine-like immunoreactive terminals were found in laminae I, II and III. Some of these GABA- and glycine-like immunoreactive terminals were observed chiefly to make symmetric synapses with HRP-labeled neuronal cell bodies and dendritic processes. The present results indicate that neurons in the medullary dorsal horn projecting to the lateral parabrachial region might be modulated by GABAergic and glycinergic inhibitory intrinsic neurons, which might be significantly involved in the regulation of the noxious information transmission.

Binding of the isolectin I-B4 from Griffonia simplicifolia to the general visceral afferents in the vagus nerve: a light- and electron-microscope study in the rat.

Prominent binding to the isolectin I-B4 from Griffonia simplicifolia (I-B4) was observed not only in the terminal area of general somatic afferents (lamina II of the medullary and the spinal dorsal horns), but also in the terminal area of the general visceral afferents (nucleus of the solitary tract, NST). The vast majority of neuronal cell bodies in the nodose ganglion (NG), including those with substance P-like immunoreactivity, also showed the I-B4-binding. After ganglionectomy of the NG, the I-B4-binding in the solitary tract and NST was highly reduced throughout the solitary tract and the NST on the side ipsilateral to the operation. The I-B4-binding was electron microscopically observed in many axon terminals within the NST.

Subcellular distributions of adenosine A1 and A2A receptors in the rat dorsomedial nucleus of the solitary tract at the level of the area postrema.

Adenosine A1 and A2A receptors mediate distinct cardiovascular components of defense reactions that are ascribed, in part, to opposing actions within the nucleus tractus solitarius. To assess the cellular sites of relevance to these actions, we examined the light and electron microscopic immunolabeling of adenosine A1 and A2A receptors in the rat dorsomedial nucleus of the solitary tract at the level of the area postrema (dmNTS-AP), a region crucial for cardiovascular regulation involving vagal baroreceptor afferents. Immunoreactivity for each receptor was independently localized to distinct segments of plasma membranes and endomembranes in somatodendritic, axonal, and glial profiles. The dendritic labeling for each receptor also was detected within and near asymmetric, excitatory-type synapses. Of all peroxidase labeled profiles exclusive of somata, approximately 58% were A1- and 39% were A2A-labeled dendrites. Dendrites and astrocytic glia were the profiles that most often expressed both subtypes of adenosine receptors. The axonal labeling for A2A receptors was seen mainly in unmyelinated axons, whereas the A1 receptors were prominently localized within axon terminals. These terminals often formed single or multisynaptic excitatory-type junctions or single symmetric synapses on dendrites, a few of which expressed A1 and A2A receptors. These results provide the first ultrastructural evidence that A1 and A2A receptors have distributions conductive to their dual involvement in modulating the output of single neurons and glial function in the dmNTS-AP, where the predominate presynaptic effects of adenosine are mediated through A1 receptors.

How many kinds of visceral afferents?

Most afferent signals from the viscera do not give rise to conscious experience and yet they participate in the complex neural control of visceral functions. Surprisingly little information is available on the origin, morphology, and receptor functional characteristics of the nerve endings of most primary afferent neurones to the digestive tract. This review deals with the morphological nature of the afferent neurones that supply the gastrointestinal tract specifically.

Receptor targeting in medullary nuclei mediating baroreceptor reflexes.

The sympathoinhibitory component of the baroreceptor reflex prominently involves glutamatergic visceral afferents terminating in the nuclei of the solitary tract (NTS) and C1 adrenergic neurons of the rostral ventrolateral medulla (RVLM). As reviewed, we have used electron microscopic immunocytochemical dual labeling in these regions to precisely analyze (1) the cellular sites for synergistic or opposing responses attributed to activation of different receptor subtypes on single neurons and (2) interactions involving monoaminergic neurons identified by their content of neurotransmitter synthesizing enzymes, vesicular monoamine transporter, and frequent coexpression of endogenous opioid peptides. The summarized results provide important cellular substrates for N-methyl-D-aspartate (NMDA)-mediated glutamatergic transmission and activation of either serotoninergic (5-HT2A), adrenergic (alpha 2A), or mu- or delta opioid receptors within the baroreceptor reflex circuit.

Serotonin transporters (SERTs) within the rat nucleus of the solitary tract: subcellular distribution and relation to 5HT2A receptors.

Serotonergic transmission is terminated by serotonin transporter (SERT)-mediated uptake following activation of serotonin receptors, several subtypes of which are present in the medial nucleus of the solitary tract (mNTS) at the area postrema level. In this region, serotonin (5HT) is a major modulator of the baroreceptor reflex and also affects gastric motility. This serotonin is derived from multiple sources including local neurons and inputs from raphe and visceral vagal afferents. To determine the relevant functional sites for serotonin uptake in the mNTS, we examined the electron microscopic localization of SERTs using both immunoperoxidase and immunogold labeling in rat brain. In addition, we combined these methods for dual labeling of SERTs and 5HT2A receptors to detect whether the SERT in this region was located near or at a distance from the sites of activation of these G-protein coupled receptors. Intensive SERT immunolabeling was seen on plasma membranes of axons and morphologically heterogeneous axon terminals that formed symmetric or asymmetric synapses on dendrites without detectable 5HT2A immunoreactivity (IR). 5HT2A-IR was, however, located in other nearby neuronal and glial profiles, some of which apposed intensively SERT-labeled terminals or terminals containing lower intensity of SERT immunolabeling. In somatodendritic profiles, co-expression of SERT and 5HT2A receptor immunolabeling was seen near synapses and Golgi lamellae. Our results suggest that in the mNTS 5HT activates 5HT2A receptors at a distance from SERT-mediated uptake sites in diverse cell types including some that express both 5HT2A receptors and SERTs.

Alpha(2A)-adrenergic receptors are present in mu-opioid receptor containing neurons in rat medial nucleus tractus solitarius.

Agonists of the alpha-2A-adrenergic- (alpha(2A)-AR) and the mu-opioid-receptor (muOR) jointly affect autonomic functions that are also disregulated in animals undergoing withdrawal from chronic administration of the muOR agonist morphine. Cardiovascular and gastrointestinal reflexes are mediated, in part, by the medial nucleus of the solitary tract (mNTS) at caudal (cNTS) and intermediate (iNTS) subregions. Together, this evidence suggests that alpha(2A)-AR and muOR may be colocalized within many of the same neuronal profiles in both the intermediate and caudal mNTS. In order to examine whether alpha(2A)-AR and muOR are present within common somata, dendrites, or axon terminals in the mNTS, we used electron microscopic immunocytochemistry for the detection of antisera against each receptor at intermediate and caudal levels of this brain region. Most of the dually labeled profiles were somata and dendrites. Of all dual-labeled profiles in the iNTS 49% were somata and were 47% dendrites, whereas in the cNTS 61% were somata and 32% were dendrites. Within dual-labeled profiles, the intracellular distribution of alpha(2A)-AR and muOR differed. MuOR was more frequently associated with the plasmalemma, whereas alpha(2A)-AR was often affiliated with vesicular organelles. Few axon terminals, and even fewer glia, contained both markers. We also frequently observed single-labeled alpha(2A)-AR glia that apposed exclusively muOR-containing dendrites or axon terminals. These findings indicate that somata and dendrites contain functional sites for convergent muOR and alpha(2A)-AR activation. In addition, each receptor is positioned for involvement in intercellular signaling between apposed neurons and glia. Activation of alpha(2A)-AR on muOR-containing somata or dendrites, or on glia apposed to muOR-containing neurons, may help to account for the efficacy of alpha(2A)-AR agonists in relieving some of the autonomic symptoms of opiate withdrawal.