Eberhard Weihe

Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system

Antibodies directed against the C-terminus of the rat vesicular acetylcholine transporter mark expression of this specifically cholinergic protein in perinuclear regions of the soma and on secretory vesicles concentrated within cholinergic nerve terminals. In the central nervous system, the vesicular acetylcholine transporter terminal fields of the major putative cholinergic pathways in cortex, hippocampus, thalamus, amygdala, olfactory cortex and interpeduncular nucleus were examined and characterized. The existence of an intrinsic cholinergic innervation of cerebral cortex was confirmed by both in situ hybridization histochemistry and immunohistochemistry for the rat vesicular acetylcholine transporter and choline acetyltransferase. Cholinergic interneurons of the olfactory tubercle and Islands of Calleja, and the major intrinsic cholinergic innervation of striatum were fully characterized at the light microscopic level with vesicular acetylcholine transporter immunohistochemistry. Cholinergic staining was much more extensive for the vesicular acetylcholine transporter than for choline acetyltransferase in all these regions, due to visualization of cholinergic nerve terminals not easily seen with immunohistochemistry for choline acetyltransferase in paraffin-embedded sections. Cholinergic innervation of the median eminence of the hypothalamus, previously observed with vesicular acetylcholine transporter immunohistochemistry, was confirmed by the presence of vesicular acetylcholine transporter immunoreactivity in extracts of median eminence by western blotting. Cholinergic projections to cerebellum, pineal gland, and to the substantia nigra were documented by vesicular acetylcholine transporter-positive punctate staining in these structures. Additional novel localizations of putative cholinergic terminals to the subependymal zone surrounding the lateral ventricles, and putative cholinergic cell bodies in the sensory mesencephalic trigeminal nucleus, a primary sensory afferent ganglion located in the brainstem, are documented here. The cholinergic phenotype of neurons of the sensory mesencephalic trigeminal nucleus was confirmed by choline acetyltransferase immunohistochemistry. A feature of cholinergic neurons of the central nervous system revealed clearly with vesicular acetylcholine transporter immunohistochemistry in paraffin-embedded sections is the termination of cholinergic neurons on cholinergic cell bodies. These are most prominent on motor neurons of the spinal cord, less prominent but present in some brainstem motor nuclei, and apparently absent from projection neurons of the telencephalon and brainstem, as well as from the preganglionic vesicular acetylcholine transporter-positive sympathetic and parasympathetic neurons visualized in the intermediolateral and intermediomedial columns of the spinal cord. In addition to the large puncta decorating motor neuronal perikarya and dendrites in the ventral horn, vesicular acetylcholine transporter-positive terminal fields are distributed in lamina X surrounding the central canal, where additional small vesicular acetylcholine transporter-positive cell bodies are located, and in the superficial layers of the dorsal horn. Components of the central cholinergic nervous system whose existence has been controversial have been confirmed, and the existence of new components documented, with immunohistochemistry for the vesicular acetylcholine transporter. Quantitative visualization of terminal fields of known cholinergic systems by staining for vesicular acetylcholine transporter will expand the possibilities for documenting changes in synaptic patency accompanying physiological and pathophysiological changes in these systems.

Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis.

The adrenal gland is important for homeostatic responses to metabolic stress: hypoglycemia stimulates the splanchnic nerve, epinephrine is released from adrenomedullary chromaffin cells, and compensatory glucogenesis ensues. Acetylcholine is the primary neurotransmitter mediating catecholamine secretion from the adrenal medulla. Accumulating evidence suggests that a secretin-related neuropeptide also may function as a transmitter at the adrenomedullary synapse. Costaining with highly specific antibodies against the secretin-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and the vesicular acetylcholine transporter (VAChT) revealed that PACAP is found in nerve terminals at all mouse adrenomedullary cholinergic synapses. Mice with a targeted deletion of the PACAP gene had otherwise normal cholinergic innervation and morphology of the adrenal medulla, normal adrenal catecholamine and blood glucose levels, and an intact initial catecholamine secretory response to insulin-induced hypoglycemia. However, insulin-induced hypoglycemia was more profound and longer-lasting in PACAP knock-outs, and was associated with a dose-related lethality absent in wild-type mice. Failure of PACAP-deficient mice to adequately counterregulate plasma glucose levels could be accounted for by impaired long-term secretion of epinephrine, secondary to a lack of induction of tyrosine hydroxylase, normally occurring after insulin hypoglycemia in wild-type mice, and a consequent depletion of adrenomedullary epinephrine stores. Thus, PACAP is needed to couple epinephrine biosynthesis to secretion during metabolic stress. PACAP appears to function as an “emergency response” cotransmitter in the sympathoadrenal axis, where the primary secretory response is controlled by a classical neurotransmitter but sustained under paraphysiological conditions by a neuropeptide.

Localization of vesicular monoamine transporter isoforms (VMAT1 and VMAT2) to endocrine cells and neurons in rat

Polyclonal antipeptide antibodies have been raised against each of the two isoforms of the rat vesicular monoamine transporter, VMAT1 and VMAT2. Antibody specificity was determined by isoform-specific staining of monkey fibroblasts programmed to express either VMAT1 or VMAT2. The expression of VMAT1 and VMAT2 in the diffuse neuroendocrine system of the rat has been examined using these polyclonal antibodies specific for either VMAT1 or VMAT2.VMAT1 is expressed exclusively in endocrine/paracrine cells associated with the intestine, stomach, and sympathetic nervous system. VMAT2 is expressed in neurons of the sympathetic nervous system, and aminergic neurons in the enteric and central nervous systems. VMAT2 is expressed in at least two endocrine cell populations in addition to its expression in neurons. A subpopulation of chromogranin A (CGA)-expressing chromaffin cells of the adrenal medulla also express VMAT2, and the oxyntic mucosa of the stomach contains a prominent population of CGA- and VMAT2-positive endocrine cells.The expression of VMAT2 in neurons, and the mutually exclusive expression of VMAT1 and VMAT2 in endocrine/paracrine cell populations of stomach, intestine, and sympathetic nervous system may provide a marker for, and insight into, the ontogeny and monoamine-secreting capabilities of multiple neuroendocrine sublineages in the diffuse neuroendocrine system.

Molecular anatomy of the neuro-immune connection

Light microscopic immunohistochemistry was employed to elucidate and compare the presence, distribution, and coexistence of various peptides, neuroendocrine markers and enzymes of the catecholamine pathway in nerves supplying lymphoid tissues in a variety of mammalian species. All lymphoid organs and tissues receive innervation by fibers containing dopamine-beta-hydroxylase and/or tyrosine hydroxylase, neural markers like protein gene product 9.5, synaptophysin and neurofilament and a varied spectrum of peptides. The prominent peptides were tachykinins (substance P, neurokinin A), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), and vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI). Opioid innervation was variable. Double immunofluorescence revealed coexistence of tachykinins and CGRP and of tyrosine hydroxylase and NPY. A minor proportion of fibers showed coexistence of NPY and tachykinins and of VIP/PHI and tachykinins. The possible importance of the complex peptidergic innervation of lymphoid tissues in inflammation, allergy, inflammatory pain and psycho-neuro-immuno-endocrine network function is discussed. A special immunomodulatory role of the sensory neurons is suggested.

Effects of Antidepressant Drugs on the Behavioral and Physiological Responses to Lipopolysaccharide (LPS) in Rodents

Antidepressants produce various immunomodulatory effects, as well as an attenuation of the behavioral responses to immune challenges, such as lipopolysaccharide (LPS). To explore further the effects of antidepressants on neuroimmune interactions, rats were treated daily with either fluoxetine (Prozac) or saline for 5 weeks, and various behavioral, neuroendocrine, and immune functions were measured following administration of either LPS or saline. Chronic fluoxetine treatment significantly attenuated the anorexia and body weight loss, as well as the depletion of CRH-41 from the median eminence and the elevation in serum corticosterone levels induced by LPS. Chronic treatment with imipramine also attenuated LPS-induced adrenocortical activation. In rats and in mice, which normally display a biphasic body temperature response to LPS (initial hypothermia followed by hyperthermia), chronic treatment with fluoxetine completely abolished the hypothermic response and facilitated and strengthened the hyperthermic response. The effects of antidepressants on the responsiveness to LPS are probably not mediated by their effects on peripheral proinflammatory cytokine production, because LPS-induced expression of TNFα and IL-1β mRNA in the spleen (assessed by semiquantitative in situ hybridization) was not altered following chronic treatment with either fluoxetine or imipramine. The effects of antidepressants on the acute phase response may have important clinical implications for the psychiatric and neuroendocrine disturbances that are commonly associated with various medical conditions.

Changes in peptidergic innervation in chronic pancreatitis

We sought to identify characteristics of peptidergic innervation that altered in patients with chronic pancreatitis. Pancreatic tissue removed from patients with chronic pancreatitis was analyzed by immunohistochemistry using antisera against neuropeptide Y, tyrosine hydroxylase, vasoactive intestinal polypeptide, peptide histidine isoleucine, calcitonin gene-related peptide, and substance P, respectively. In accordance with recent findings, the number and diameter of intralobular and interlobular nerve bundles were found to be increased as compared with control pancreas from organ donors. The striking change in the peptidergic innervation pattern in chronic pancreatitis concerned these altered nerves. It consisted of an intensification of the immunostaining for calcitonin gene-related peptide and substance P in numerous fibers contained in these nerves. Adjacent sections showed that immunoreactive substance P and immunoreactive calcitonin gene-related peptide coexisted in these fibers. Because both of these peptides are generally regarded as pain transmitter candidates, our findings provide further evidence that changes in pancreatic nerves themselves might be responsible for the long-lasting pain syndrome in chronic pancreatitis.

Homeostatic Scaling of Vesicular Glutamate and GABA Transporter Expression in Rat Neocortical Circuits

Homeostatic control of pyramidal neuron firing rate involves a functional balance of feedforward excitation and feedback inhibition in neocortical circuits. Here, we reveal a dynamic scaling in vesicular excitatory (vesicular glutamate transporters VGLUT1 and VGLUT2) and inhibitory (vesicular inhibitory amino acid transporter VIAAT) transporter mRNA and synaptic protein expression in rat neocortical neuronal cultures, using a well established in vitro protocol to induce homeostatic plasticity. During the second and third week of synaptic differentiation, the predominant vesicular transporters expressed in neocortical neurons, VGLUT1 and VIAAT, are both dramatically upregulated. In mature cultures, VGLUT1 and VIAAT exhibit bidirectional and opposite regulation by prolonged activity changes. Endogenous coregulation during development and homeostatic scaling of the expression of the transporters in functionally differentiated cultures may serve to control vesicular glutamate and GABA filling and adjust functional presynaptic excitatory/inhibitory balance. Unexpectedly, hyperexcitation in differentiated cultures triggers a striking increase in VGLUT2 mRNA and synaptic protein, whereas decreased excitation reduces levels. VGLUT2 mRNA and protein are expressed in subsets of VGLUT1-encoded neocortical neurons that we identify in primary cultures and in neocortex in situ and in vivo. After prolonged hyperexcitation, downregulation of VGLUT1/synaptophysin intensity ratios at most synapses is observed, whereas a subset of VGLUT1-containing boutons selectively increase the expression of VGLUT2. Bidirectional and opposite regulation of VGLUT1 and VGLUT2 by activity may serve as positive or negative feedback regulators for cortical synaptic transmission. Intracortical VGLUT1/VGLUT2 coexpressing neurons have the capacity to independently modulate the level of expression of either transporter at discrete synapses and therefore may serve as a plastic interface between subcortical thalamic input (VGLUT2) and cortical output (VGLUT1) neurons.

Microadenomatosis of the endocrine pancreas in patients with and without the multiple endocrine neoplasia type 1 syndrome.

It has been suggested that microadenomatosis of the endocrine pancreas is a hallmark of the multiple endocrine neoplasia type 1 syndrome (MEN1). This study attempts to elucidate the relationship between pancreatic microadenomatosis and the MEN1 and von Hippel-Lindau (VHL) syndromes. Pancreatic tissue specimens from 37 patients (with either microadenomatosis or the MEN1 syndrome) were analyzed using immunohistochemistry, confocal laser scanning microscopy, and morphometric methods. The MEN1 and the VHL status were assessed on the basis of clinical criteria (all patients) and PCR-based mutational analysis (15 and 5 patients, respectively). Pancreatic microadenomatosis was found in 35 of 37 patients, 28 of whom fulfilled the clinicopathologic criteria and 13 the genetic criteria for MEN1, whereas none of the patients had evidence of a VHL syndrome. Microadenomas were present in 26 of the 28 MEN1 patients, and all these tumors were consistently multihormonal. Five of the 9 patients with microadenomatosis and no clinical evidence for MEN1 or VHL also lacked mutations for the respective genes. Five of these 9 patients suffered from hyperinsulinism and revealed multiple insulin-positive tumors. The other patients were nonsymptomatic and showed multiple glucagon-expressing neoplasms. In microadenomatosis patients with and without the MEN1 syndrome, a subset of morphologically normal-appearing islets showed increased endocrine cell proliferation. In conclusion, endocrine multihormonal microadenomatosis of the pancreas is a feature of MEN1. In addition, a monohormonal type of pancreatic microadenomatosis was identified that consisted of either insulinomas or glucagon-producing tumors and was not associated with MEN1 or VHL.

Chemical coding of the human gastrointestinal nervous system: Cholinergic, VIPergic, and catecholaminergic phenotypes

The aim of this investigation was to identify the proportional neurochemical codes of enteric neurons and to determine the specific terminal fields of chemically defined nerve fibers in all parts of the human gastrointestinal (GI) tract. For this purpose, antibodies against the vesicular monoamine transporters (VMAT1/2), the vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), dopamine β‐hydroxylase (DBH), serotonin (5‐HT), vasoactive intestinal peptide (VIP), and protein gene product 9.5 (PGP 9.5) were used. For in situ hybridization 35S‐labeled VMAT1, VMAT2, and VAChT riboprobes were used. In all regions of the human GI tract, 50–70% of the neurons were cholinergic, as judged by staining for VAChT. The human gut unlike the rodent gut exhibits a cholinergic innervation, which is characterized by an extensive overlap with VIPergic innervation. Neurons containing VMAT2 constituted 14–20% of all intrinsic neurons in the upper GI tract, and there was an equal number of TH‐positive neurons. In contrast, DBH was absent from intrinsic neurons. Cholinergic and monoaminergic phenotypes proved to be completely distinct phenotypes. In conclusion, the chemical coding of human enteric neurons reveals some similarities with that of other mammalian species, but also significant differences. VIP is a cholinergic cotransmitter in the intrinsic innervation of the human gut. The substantial overlap between VMAT2 and TH in enteric neurons indicates that the intrinsic catecholaminergic innervation is a stable component of the human GI tract throughout life. The absence of DBH from intrinsic catecholaminergic neurons indicates that these neurons have a dopaminergic phenotype. J. Comp. Neurol. 459:90–111, 2003.

Chemical neuroanatomy of the vesicular amine transporters

Acetylcholine, catecholamines, serotonin, and histamine are classical neurotransmitters. These small molecules also play important roles in the endocrine and immune/inflammatory systems. Serotonin secreted from enterochromaffin cells of the gut epithelium regulates gut motility; histamine secreted from basophils and mast cells is a major regulator of vascular permeability and skin inflammatory responses; epinephrine is a classical hormone released from the adrenal medulla. Each of these molecules is released from neural, endocrine, or immune/inflammatory cells only in response to specific physiological stimuli. Regulated secretion is possible because amines are stored in secretory vesicles and released via a stimulus‐dependent exocytotic event. Amine storage—at concentrations orders of magnitude higher than in the cytoplasm—is accomplished in turn by specific secretory vesicle transporters that recognize the amines and move them from the cytosol into the vesicle. Immunohistochemical visualization of specific vesicular amine transporters (VATs) in neuronal, endocrine, and inflammatory cells provides important new information about how amine‐handling cell phenotypes arise during development and how vesicular transport is regulated during homeostatic response events. Comparison of the chemical neuroanatomy of VATs and amine biosynthetic enzymes has also revealed cell groups that express vesicular transporters but not enzymes for monoamine synthesis, and vice versa:their function and regulation is a new topic of investigation in mammalian neurobiology. The chemical neuroanatomy of the vesicular amine transporters is reviewed here. These and similar data emerging from the study of the localization of the recently characterized vesicular inhibitory and excitatory amino acid transporters will contribute to understanding chemically coded synaptic circuitry in the brain, and amine‐handling neuroendocrine and immune/inflammatory cell regulation.—Weihe, E., Eiden, L. E. Chemical neuroanatomy of the vesicular amine transporters. The FASEB J. 14, 2435–2449 (2000)