Effects of +Gz Loads on Structural Organization of Central Autonomic Nuclei.

Structural alterations in the central autonomic nuclei (dorsal vagal complex and intermediolateral nucleus) of the centrifuged random-bred male rats subjected to +Gz loads were examined. Acute exposure to gravitational loads predominantly produced the reactive changes in these nuclei, while persistently repeated regular loads resulted in cumulation of the destructive alterations. The structural perturbations in the central autonomic nuclei can disturb the autonomic regulation of physiological functions. The character of such disturbances is partially determined by the peculiarities in structural organization of these nuclei.

The delayed phase of cisplatin-induced emesis is mediated by the area postrema and not the abdominal visceral innervation in the ferret.

The anti-cancer chemotherapeutic agent cisplatin induces an acute (approximately 24 h) and delayed (approximately 24-72 h+) emetic response in humans; whereas the mechanism mediating the acute phase has been characterised, the delayed phase is relatively poorly understood. We have used nerve lesions (abdominal vagus, VX; greater splanchnic nerve, GSNX) and area postrema ablation (APX) in the ferret model of cisplatin (5 mg/kg, i.p.) delayed emesis and demonstrated that VX and VX+GSNX did not significantly modify the delayed emetic response (24-72 h), which consisted of 276.0+/-62.8 retches+vomits (R+V) in sham-operated ferrets and 167.2+/-34.0R+V and 214.8+/-40.2R+V, in the VX and VX+GSNX groups, respectively. APX virtually abolished the delayed phase of emesis and sham-operated ferrets had 93.0+/-22.9R+V whilst only 6.0+/-3.6R+V (p=0.009) were observed in APX animals. These data suggest that, in contrast to the acute emetic response triggered by cisplatin, the delayed phase does not rely on abdominal visceral afferents but is mediated via the area postrema.

A diffusion barrier between the area postrema and nucleus tractus solitarius.

The blood-brain barrier (BBB) is a structural and functional barrier that prevents free exchange of circulating substances with the brain, where the endothelial cells of microvessels are joined by tight junctions. The circumventricular organs (CVOs), by contrast, lack tight junctions and exhibit more direct communication with the circulating blood and cerebrospinal fluid. Despite many outstanding morphological studies at the electron microscopic level, there remain misconceptions that the CVOs provide direct passage of blood-borne substances to the rest of the brain. This study will show the structure of the anatomical borders of the dorsal vagal complex in the brainstem. A distinct diffusion barrier between the area postrema (AP, a CVO) and the nucleus tractus solitarius (NTS) was illustrated by immunohistochemistry at both the light and electron microscopic levels. The border zone between the AP and NTS was underlined by a continuous monolayer of columnar cells that were immunopositive for both the tight junction protein zona occludin-1 and the astrocyte marker glial fibrillary acidic protein. This observation of a diffusion barrier between the AP and NTS resolves a long-standing dispute about whether the NTS is a structural extension of the AP with a leaky BBB.

A size selective vascular barrier in the rat area postrema formed by perivascular macrophages and the extracellular matrix.

The fenestrated microvasculature of the area postrema shows a less restrictive blood-brain barrier than is found in other areas of the CNS. We have studied the expression and relationship of vascular endothelial tight junctional proteins, astrocytes, macrophages, and the extracellular matrix with the extravasation of fluorescently tagged dextrans and sodium fluorescein in the rat area postrema. Glial fibrillary acidic protein (GFAP) -positive astrocytes were present within the area postrema which was surrounded by a dense zone of highly GFAP-reactive astrocytes. Expression of the tight junction proteins claudin-5, -12 and occludin was absent, although diffuse cytoplasmic claudin-1 immunoreactivity was present. The extracellular matrix of the endothelium showed two non-fused thickened layers of laminin immunoreactivity. CD163 and CD169 immunoreactive perivascular macrophages were located within lacunae between these two laminin layers. Fluorescently tagged dextrans (10-70 kDa) passed from the vasculature but were retained between the inner and outer laminin walls and rapidly sequestered by the perivascular CD163 and CD169 macrophages. Three-kilodalton dextran diffused into the parenchyma, but was retained within the boundary of the area postrema at the interface with the highly reactive GFAP-astrocytes, while sodium fluorescein (0.3 kDa) passed from the area postrema into surrounding CNS areas. Our observations suggest that despite the absence of a tight blood-brain barrier, a size selective barrier restricting the movement of blood solutes into the parenchyma is present in the area postrema. We suggest that the rapid uptake by CD163 and CD169 macrophages together with the non-fused laminin immunoreactive layers of the extracellular matrix plays a size selective role in restricting movement of serum proteins and other blood borne macromolecules over 10 kDa in to the area postrema.

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.

Neural-immune interface in the rat area postrema.

The area postrema functions as one interface between the immune system and the brain. Immune cells within the area postrema express immunoreactivity for the pro-inflammatory cytokine, interleukin-1beta following challenge with immune stimulants, including lipopolysaccharide (from bacterial cell walls). As a circumventricular organ, the area postrema accesses circulating immune-derived mediators, but also receives direct primary viscerosensory signals via the vagus nerve. Neurons in the area postrema contribute to central autonomic network neurocircuitry implicated in brain-mediated host defense responses. These experiments were directed toward clarifying relationships between immune cells and neurons in the area postrema, with a view toward potential mechanisms by which they may communicate. We used antisera directed toward markers indicating microglia (CR3/CD11b; OX-42), resident macrophages (CD163; ED-2), or dendritic cell-like phenotypes (major histocompability complex class II; OX-6), in area postrema sections from lipopolysaccharide-treated rats processed for light, laser scanning confocal, and electron microscopy. Lipopolysaccharide treatment induced interleukin-1beta-like immunoreactivity in immune cells that either associated with the vasculature (perivascular cells, a subtype of macrophage) or associated with neuronal elements (dendritic-like, and unknown phenotype). Electron microscopic analysis revealed that some immune cells, including interleukin-1beta-positive cells, evinced membrane apposition with neuronal elements, including dendrites and terminals, that could derive from inputs to the area postrema such as vagal sensory fibers, or intrinsic area postrema neurons. This arrangement provides an anatomical substrate by which immune cells could directly and specifically influence individual neurons in the area postrema, that may support the induction and/or maintenance of brain responses to inflammation.

Characterization of orexin A immunoreactivity in the rat area postrema.

The distribution of orexin A immunoreactivity and the synaptic relationships of orexin A-positive neurons in the rat area postrema were studied using both light and electron microscopy techniques. At the light microscope level, numerous orexin A-like immunoreactive fibers were found within the area postrema. Using electron microscopy, immunoreactivity within fibers was confined primarily to the axon terminals, most of which contained dense-cored vesicles. Both axo-somatic and axo-dendritic synapses made by orexin A-like immunoreactive axon terminals were found, with these synapses being both symmetric and asymmetric in form. Orexin A-like immunoreactive axon terminals could be found presynaptic to two different immunonegative profiles including the perikarya and dendrites. Occasionally, some orexin A-like immunoreactive profiles, most likely to be dendrites, could be seen receiving synaptic inputs from immunonegative or immunopositive axon terminals. The present results suggest that the physiological function of orexin A in the area postrema depends on synaptic relationships with other immunopositive and immunonegative neurons, with the action of orexin A mediated via a self-modulation feedback mechanism.

Ultrastructural localization of serotonin 2A and N-methyl-D-aspartate receptors in somata and dendrites of single neurons within rat dorsal motor nucleus of the vagus.

Both glutamate and serotonin are potent modulators of autonomic functions involving the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMNV) at the level of the area postrema. Moreover, many of the dendrites in this NTS region express both N-methyl-D-aspartate (NMDA) and serotonin (5HT) 2A receptors, and some of these dendrites may arise from the adjacent DMNV. Thus, single neurons in DMNV may also express both receptors. To test this hypothesis, we used electron microscopic immunocytochemistry for dual localization of the essential R1 subunit of the NMDA receptor (NR1) and the 5HT2A receptor in rat intermediate DMNV, a region serving mainly gastrointestinal functions. Gold particles representing NR1 and peroxidase reaction product for 5HT2A receptors were seen in the cytoplasm, as well as on distinct segments of the plasma membrane of many dendrites. Of the NR1-labeled dendrites, 31% (254/814) also contained 5HT2A immunoreactivity; among the 5HT2A-labeled dendrites, 52% (254/485) expressed NR1. The 5HT2A labeling was also present in numerous small unmyelinated axons, axon terminals, and glial processes. These profiles were largely without NR1 immunoreactivity, although NR1 was detected in some of the dendrites postsynaptic to 5HT2A-labeled terminals. Our results suggest that calcium entry through NMDA channels and 5HT2A receptor activation may dramatically affect postsynaptic excitability of single neurons in the DMNV. In addition, the findings also indicate that the 5HT2A receptor is strategically positioned for involvement in modulation of the presynaptic release of neurotransmitters affecting the postsynaptic activity of DMNV neurons responsive to NMDA activation.

Alpha-2A-adrenergic receptors are present on neurons in the central nucleus of the amygdala that project to the dorsal vagal complex in the rat.

The descending pathway between the central nucleus of the amygdala (CeA) and the dorsal vagal complex (DVC) is an important substrate for autonomic functions associated with emotion. Activity in this circuit is crucially modulated by catecholamines and agonists of the alpha-2A-adrenergic receptor (alpha(2A)-AR), which relieve cardiovascular and gastrointestinal symptoms associated with experience of aversive stimuli. The subcellular distribution of alpha(2A)-AR within the CeA, however, has not been characterized. It is also not known if any alpha(2A)-AR-expressing neurons in the CeA project to the dorsal vagal complex. In order to address these questions, we examined the immunocytochemical labeling of alpha(2A)-AR in the CeA of rats receiving microinjection of the retrograde tracer fluorogold (FG) into the dorsal vagal complex at the level of the area postrema, an area involved in cardiorespiratory and gastrointestinal functions. Of all alpha(2A)-AR-labeled profiles in the CeA, the majority were either dendrites (42%) or somata (24%). alpha(2A)-AR labeling was often present on the plasmalemma in dendrites and was mainly found in endosome-like organelles in somata. Of all alpha(2A)-AR immunoreactive somata, 62% also contained immunolabeling for FG and 23% of all dendrites also showed labeling for the retrograde tracer. The intracellular distribution of alpha(2A)-AR did not differ in somata or dendrites with or without detectable FG. The remaining singly labeled alpha(2A)-AR profiles consisted of axons (11%), axon terminals (12%), and glial processes (13%). In numerous instances, alpha(2A)-AR-labeled glia or axon terminals were apposed to DVC projecting neurons. Together, this evidence suggests that the principal site for alpha(2A)-AR activation is at extrasynaptic sites on dendrites of CeA neurons, many of which project to the DVC and also show endosomal receptor labeling. In addition, these results indicate that activation of alpha(2A)-AR in the CeA may influence the activity of DVC projecting neurons through indirect mechanisms, including changes in presynaptic transmitter release or glial function. These results suggest that alpha(2A)-AR agonists in the CeA may modulate numerous processes including stress-evoked autonomic reactions and feeding behavior.