Winfried Neuhuber

Functional and chemical anatomy of the afferent vagal system

The results of neural tracing studies suggest that vagal afferent fibers in cervical and thoracic branches innervate the esophagus, lower airways, heart, aorta, and possibly the thymus, and via abdominal branches the entire gastrointestinal tract, liver, portal vein, billiary system, pancreas, but not the spleen. In addition, vagal afferents innervate numerous thoracic and abdominal paraganglia associated with the vagus nerves. Specific terminal structures such as flower basket terminals, intraganglionic laminar endings and intramuscular arrays have been identified in the various organs and organ compartments, suggesting functional specializations. Electrophysiological recording studies have identified mechano- and chemo-receptors, as well as temperature- and osmo-sensors. In the rat and several other species, mostly polymodal units, while in the cat more specialized units have been reported. Few details of the peripheral transduction cascades and the transmitters for signal propagation in the CNS are known. Glutamate and its various receptors are likely to play an important role at the level of primary afferent signaling to the solitary nucleus. The vagal afferent system is thus in an excellent position to detect immune-related events in the periphery and generate appropriate autonomic, endocrine, and behavioral responses via central reflex pathways. There is also good evidence for a role of vagal afferents in nociception, as manifested by affective-emotional responses such as increased blood pressure and tachycardia, typically associated with the perception of pain, and mediated via central reflex pathways involving the amygdala and other parts of the limbic system. The massive central projections are likely to be responsible for the antiepileptic properties of afferent vagal stimulation in humans. Furthermore, these functions are in line with a general defensive character ascribed to the vagal afferent, paraventricular system in lower vertebrates.

Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus

OBJECTIVE To investigate the fate of apoptotic cells in the germinal centers (GCs) of patients with systemic lupus erythematosus (SLE). METHODS Lymph node biopsy specimens obtained from 7 SLE patients with benign follicular hyperplasia, 5 non-SLE patients with benign follicular hyperplasia (non-SLE), 5 patients with malignant follicular lymphoma, and 3 patients with dermatopathic lymphadenitis were stained with monoclonal antibodies against macrophages (CD68) and follicular dendritic cells (CR2/CD21). TUNEL staining and transmission electron microscopy were performed to detect apoptotic cells. Confocal microscopy was used to evaluate the in vivo capacity of tingible body macrophages to remove apoptotic cell material. RESULTS In a subgroup of patients with SLE, apoptotic cells accumulated in the GCs of the lymph nodes. The number of tingible body macrophages, which usually contained engulfed apoptotic nuclei, was significantly reduced in these patients. In contrast to what was observed in all controls, TUNEL-positive apoptotic material from SLE patients was observed to be directly associated with the surfaces of follicular dendritic cells (FDCs). CONCLUSION Our findings suggest that in a sub-group of SLE patients, apoptotic cells are not properly cleared by tingible body macrophages of the GCs. Consequently, nuclear autoantigens bind to FDCs and may thus provide survival signals for autoreactive B cells. This action may override an important control mechanism for B cell development, resulting in the loss of tolerance for nuclear antigens.

PGE(2) selectively blocks inhibitory glycinergic neurotransmission onto rat superficial dorsal horn neurons.

Despite the crucial role that prostaglandins (PGs) have in the sensitization of the central nervous system to pain, their cellular and molecular targets leading to increased pain perception have remained elusive. Here we investigated the effects of PGE2 on fast synaptic transmission onto neurons in the rat spinal cord dorsal horn, the first site of synaptic integration in the pain pathway. We identified the inhibitory (strychnine-sensitive) glycine receptor as a specific target of PGE2. PGE2, but not PGF2α, PGD2 or PGI2, reduced inhibitory glycinergic synaptic transmission in low nanomolar concentrations, whereas GABAA, AMPA and NMDA receptor-mediated transmission remained unaffected. Inhibition of glycine receptors occurred via a postsynaptic mechanism involving the activation of EP2 receptors, cholera-toxin-sensitive G-proteins and cAMP-dependent protein kinase. Via this mechanism, PGE2 may facilitate the transmission of nociceptive input through the spinal cord dorsal horn to higher brain areas where pain becomes conscious.

Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Nav1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Nav1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Nav1.8 knockout (Scn10−/−) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.

Elevated Blood Pressure Linked to Primary Hyperaldosteronism and Impaired Vasodilation in BK Channel–Deficient Mice

Background—Abnormally elevated blood pressure is the most prevalent risk factor for cardiovascular disease. The large-conductance, voltage- and Ca2+-dependent K+ (BK) channel has been proposed as an important effector in the control of vascular tone by linking membrane depolarization and local increases in cytosolic Ca2+ to hyperpolarizing K+ outward currents. However, the BK channel may also affect blood pressure by regulating salt and fluid homeostasis, particularly by adjusting the renin-angiotensin-aldosterone system. Methods and Results—Here we report that deletion of the pore-forming BK channel &agr; subunit leads to a significant blood pressure elevation resulting from hyperaldosteronism accompanied by decreased serum K+ levels as well as increased vascular tone in small arteries. In smooth muscle from small arteries, deletion of the BK channel leads to a depolarized membrane potential, a complete lack of membrane hyperpolarizing spontaneous K+ outward currents, and an attenuated cGMP vasorelaxation associated with a reduced suppression of Ca2+ transients by cGMP. The high level of BK channel expression observed in wild-type adrenal glomerulosa cells, together with unaltered serum renin activities and corticotropin levels in mutant mice, suggests that the hyperaldosteronism results from abnormal adrenal cortical function in BK−/− mice. Conclusions—These results identify previously unknown roles of BK channels in blood pressure regulation and raise the possibility that BK channel dysfunction may underlie specific forms of hyperaldosteronism.

Calcitonin receptor‐like receptor (CLR), receptor activity‐modifying protein 1 (RAMP1), and calcitonin gene‐related peptide (CGRP) immunoreactivity in the rat trigeminovascular system: Differences between peripheral and central CGRP receptor distribution

Calcitonin gene‐related peptide (CGRP) is a key mediator in primary headaches including migraine. Animal models of meningeal nociception demonstrate both peripheral and central CGRP effects; however, the target structures remain unclear. To study the distribution of CGRP receptors in the rat trigeminovascular system we used antibodies recognizing two components of the CGRP receptor, the calcitonin receptor‐like receptor (CLR) and the receptor activity‐modifying protein 1 (RAMP1). In the cranial dura mater, CLR and RAMP1 immunoreactivity (‐ir) was found within arterial blood vessels, mononuclear cells, and Schwann cells, but not sensory axons. In the trigeminal ganglion, besides Schwann and satellite cells, CLR‐ and RAMP1‐ir was found in subpopulations of CGRP‐ir neurons where colocalization of CGRP‐ and RAMP1‐ir was very rare (≈0.6%). CLR‐ and RAMP1‐ir was present on central, but not peripheral, axons. In the spinal trigeminal nucleus, CLR‐ and RAMP1‐ir was localized to “glomerular structures,” partly colocalized with CGRP‐ir. However, CLR‐ and RAMP1‐ir was lacking in central glia and neuronal cell bodies. We conclude that CGRP receptors are associated with structural targets of known CGRP effects (vasodilation, mast cell degranulation) and targets of unknown function (Schwann cells). In the spinal trigeminal nucleus, CGRP receptors are probably located on neuronal processes, including primary afferent endings, suggesting involvement in presynaptic regulation of nociceptive transmission. Thus, in the trigeminovascular system CGRP receptor localization suggests multiple targets for CGRP in the pathogenesis of primary headaches. J. Comp. Neurol. 507:1277–1299, 2008.

Vagal sensors in the rat duodenal mucosa: distribution and structure as revealed by in vivo DiI-tracing

Results from functional studies point to the importance of chemoreceptive endings in the duodenum innervated by vagal afferents in the regulation of gastrointestinal functions such as gastric emptying and acid secretion, as well as in the process of satiation. In order to visualize the vagal sensory innervation of this gut segment, vagal afferents were selectively labeled in vivo by injecting the lipophilic carbocyanine dye DiI into either the left or the right nodose ganglion of young adult rats. Thick cryostat sections or whole-mounted peels of muscularis externa or submucosa of formalinfixed tissue were analyzed with conventional and/or confocal microscopy. In the mucosa, many DiI-labeled vagal afferent fibers were found with terminal arborizations mainly between the crypts and the villous lamina propria. In both areas, vagal terminal branches came in close contact with the basal lamina, but did not appear to penetrate it so as to make direct contact with epithelial cells. Labeled vagal afferent fibers in the villous and cryptic lamina propria were found to be in intimate anatomical contact with fibrocyte-like cells that may belong to the class of interstitial cells of Cajal, and with small granular cells that might be granulocytes or histiocytes. Although our analysis was not quantitative, and considering that labeling was unilateral and not complete, it appears that the overall density of vagal afferent mucosal innervation was variable; many villi showed no evidence for innervation while other areas had quite dense networks of arborizing terminal fibers in several neighboring villi. Analysis of separate whole-mounted muscularis externa and submucosa peels revealed the presence of large bundles of labeled afferent fibers running within the myenteric plexus along the mesenteric attachment primarily in an aboral direction, with individual fibers turning towards the antimesenteric pole, and either penetrating into the submucosa or forming the characteristic intraganglionic laminar endings (IGLEs). Although the possibility of individual fibers issuing collaterals to myenteric IGLEs and at the same time to mucosal terminals was not demonstrated, it cannot be ruled out. These anatomical findings are discussed in the context of absorptive mechanisms for the different macronutrients and the implication of enteroendocrine cells such as CCK-containing cells that may function as intestinal “taste cells”.

Distribution and structure of vagal afferent intraganglionic laminar endings (IGLEs) in the rat gastrointestinal tract.

Abstract Intraganglionic laminar endings (IGLEs) are special terminal structures of vagal afferent fibers and have been demonstrated in the myenteric plexus of esophagus and stomach. In order to quantitatively map their presence and distribution over the entire gastrointestinal tract, including the small and large intestines, vagal afferents were anterogradely labeled in vivo by microinjections of the fluorescent carbocyanine dye DiI into the left or right nodose ganglion of adult male rats. In the most successfully labeled cases the highest density of IGLEs was found in the stomach, with about half to one-third of the myenteric ganglia receiving at least one IGLE. The proportion of myenteric ganglia innervated by IGLEs decreased in the small intestine; however, because of its large surface area this gut segment was estimated to contain the highest total number of IGLEs. Both the cecum and colon also contained significant numbers of IGLEs. In the stomach, this vagal afferent innervation by IGLEs was more or less lateralized, with less than 20% of labeled IGLEs found on the contralateral side with respect to the injection. The left/ventral vagus contributed a larger proportion of IGLEs to the proximal duodenum, while the right/dorsal vagus contributed a larger proportion of IGLEs to the distal duodenum and jejunum. Laser scanning confocal microscopy on select specimens revealed further structural details. The parent axon typically formed two or more branches that flanked the ganglia laterally, and in turn produced numerous highly arborizing laminar terminal branches that covered one or both flat sides of the ganglion in a dome-like fashion. The similar distribution patterns and structural details suggest a uniform function for the IGLEs throughout the gastrointestinal tract, but there is as yet no clear proof for any of the hypothesized roles as specialized mechanosensors or local effector terminals.

Low proliferation and differentiation capacities of adult hippocampal stem cells correlate with memory dysfunction in humans

The hippocampal dentate gyrus maintains its capacity to generate new neurons throughout life. In animal models, hippocampal neurogenesis is increased by cognitive tasks, and experimental ablation of neurogenesis disrupts specific modalities of learning and memory. In humans, the impact of neurogenesis on cognition remains unclear. Here, we assessed the neurogenic potential in the human hippocampal dentate gyrus by isolating adult human neural stem cells from 23 surgical en bloc hippocampus resections. After proliferation of the progenitor cell pool in vitro we identified two distinct patterns. Adult human neural stem cells with a high proliferation capacity were obtained in 11 patients. Most of the cells in the high proliferation capacity cultures were capable of neuronal differentiation (53 ± 13% of in vitro cell population). A low proliferation capacity was observed in 12 specimens, and only few cells differentiated into neurons (4 ± 2%). This was reflected by reduced numbers of proliferating cells in vivo as well as granule cells immunoreactive for doublecortin, brain-derived neurotrophic factor and cyclin-dependent kinase 5 in the low proliferation capacity group. High and low proliferation capacity groups differed dramatically in declarative memory tasks. Patients with high proliferation capacity stem cells had a normal memory performance prior to epilepsy surgery, while patients with low proliferation capacity stem cells showed severe learning and memory impairment. Histopathological examination revealed a highly significant correlation between granule cell loss in the dentate gyrus and the same patients regenerative capacity in vitro (r = 0.813; P < 0.001; linear regression: R²(adjusted) = 0.635), as well as the same patients ability to store and recall new memories (r = 0.966; P = 0.001; linear regression: R²(adjusted) = 0.9). Our results suggest that encoding new memories is related to the regenerative capacity of the hippocampus in the human brain.

Ca2+ -activated K+ channels of the BK-type in the mouse brain.

An antibody against the 442 carboxy-terminal amino acids of the BK channel α-subunit detects high immunoreactivity within the telencephalon in cerebral cortices, olfactory bulb, basal ganglia and hippocampus, while lower levels are found in basal forebrain regions and amygdala. Within the diencephalon, high density was found in nuclei of the ventral and dorsal thalamus and the medial habenular nucleus, and low density in the hypothalamus. The fasciculus retroflexus and its termination in the mesencephalic interpeduncular nucleus are prominently stained. Other mesencephalic expression sites are periaquaeductal gray and raphe nuclei. In the rhombencephalon, BK channels are enriched in the cerebellar cortex and in the locus coeruleus. Strong immunoreactivity is also contained in the vestibular nuclei, but not in cranial nerves and their intramedullary course of their roots. On the cellular level, BK channels show pre- and postsynaptic localizations, i.e., in somata, dendrites, axons and synaptic terminals.