Search

Tuft cells are a rare population of chemosensory cells at the mucosal surface epithelia of hollow organs. Their name-giving morphological feature is an apical tuft of stiff microvilli. Accordingly, the actin-binding protein, villin, was identified as one of the first tuft cell markers in immunohistochemical analysis. Unfortunately, villin expression is not restricted to tuft cells, but is also prominent e.g. in enterocytes, which limits the use of this gene as a marker and as an experimental tool to genetically target tuft cells. Here, we report that the villin-related protein, advillin, is a specific tuft cell marker in the gastro-intestinal and biliary tract epithelia. In situ hybridization and immunohistochemistry revealed that advillin expression, unlike villin, was restricted to solitary cholinergic tuft cells in the mucosal linings of the small and large intestine, and in the gall bladder. In the glandular stomach, villin and advillin mRNA were present in all epithelial cells, while detectable protein levels were confined to solitary tuft cells. Advillin expression was no longer detectable in the mucosa of the intestinal and biliary tract from Pou2f3 deficient mice that lack tuft cells. Finally, crossing Avil-Cre transgenic mice with a double-fluorescent reporter mouse line resulted in specific targeting of gastro-intestinal and biliary tuft cells. Our analysis introduces advillin as a selective marker and tool in histological and functional analysis of the alimentary tract tuft cell system. Electronic supplementary material The online version of this article (doi:10.1007/s10735-020-09893-6) contains supplementary material, which is available to authorized users.

Cholinergic chemosensory cells (CCC) are infrequent epithelial cells with immunosensor function, positioned in mucosal epithelia preferentially near body entry sites in mammals including man. Given their adaptive capacity in response to infection and their role in combatting pathogens, we here addressed the time points of their initial emergence as well as their postnatal development from first exposure to environmental microbiota (i.e., birth) to adulthood in urethra and trachea, utilizing choline acetyltransferase (ChAT)-eGFP reporter mice, mice with genetic deletion of MyD88, toll-like receptor-2 (TLR2), TLR4, TLR2/TLR4, and germ-free mice. Appearance of CCC differs between the investigated organs. CCC of the trachea emerge during embryonic development at E18 and expand further after birth. Urethral CCC show gender diversity and appear first at P6-P10 in male and at P11-P20 in female mice. Urethrae and tracheae of MyD88- and TLR-deficient mice showed significantly fewer CCC in all four investigated deficient strains, with the effect being most prominent in the urethra. In germ-free mice, however, CCC numbers were not reduced, indicating that TLR2/4-MyD88 signaling, but not vita-PAMPs, governs CCC development. Collectively, our data show a marked postnatal expansion of CCC populations with distinct organ-specific features, including the relative impact of TLR2/4-MyD88 signaling. Strong dependency on this pathway (urethra) correlates with absence of CCC at birth and gender-specific initial development and expansion dynamics, whereas moderate dependency (trachea) coincides with presence of first CCC at E18 and sex-independent further development.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Neuronal vacuolization and glial activation are pathologic hallmarks in the superoxide dismutase 1 (SOD1) mouse model of ALS. Previously, we found the neuropeptide calcitonin gene-related peptide (CGRP) associated with vacuolization and astrogliosis in the spinal cord of these mice. We now show that CGRP abundance positively correlated with the severity of astrogliosis, but not vacuolization, in several motor and non-motor areas throughout the brain. SOD1 mice harboring a genetic depletion of the βCGRP isoform showed reduced CGRP immunoreactivity associated with vacuolization, while motor functions, body weight, survival, and astrogliosis were not altered. When CGRP signaling was completely disrupted through genetic depletion of the CGRP receptor component, receptor activity-modifying protein 1 (RAMP1), hind limb muscle denervation, and loss of muscle performance were accelerated, while body weight and survival were not affected. Dampened neuroinflammation, i.e., reduced levels of astrogliosis in the brain stem already in the pre-symptomatic disease stage, and reduced microgliosis and lymphocyte infiltrations during the late disease phase were additional neuropathology features in these mice. On the molecular level, mRNA expression levels of brain-derived neurotrophic factor (BDNF) and those of the anti-inflammatory cytokine interleukin 6 (IL-6) were elevated, while those of several pro-inflammatory cytokines found reduced in the brain stem of RAMP1-deficient SOD1 mice at disease end stage. Our results thus identify an important, possibly dual role of CGRP in ALS pathogenesis.

Each different gas that is used to induce a pneumoperitoneum (PP) exhibits individual effects within the peritoneal cavity. This might include adverse effects such as pain and/or inflammatory reactions. The acute effects of ozonized oxygen (O3/O2), a highly oxidative gas mixture, after being insufflated into the peritoneal cavity are analysed in this study. Using the abdominal constriction response (‘writhing’) assay of chemical nociception in C57BL6/N mice, O3/O2-PP was found not to be associated with visible pain responses and did not alter the c-fos expression in the spinal cord. In addition, mRNA expression levels of the pro-inflammatory cytokines, interleukin (IL)-1β and IL-6, were found unaltered in the spleen 2 h after insufflation. In conclusion, O3/O2-PP is free of adverse pain and does not trigger inflammatory immune responses.

Specialized epithelial cells in the respiratory tract such as solitary chemosensory cells and brush cells sense the luminal content and initiate protective reflexes in response to the detection of potentially harmful substances. The majority of these cells are cholinergic and utilize the canonical taste signal transduction cascade to detect “bitter” substances such as bacterial quorum sensing molecules. Utilizing two different mouse strains reporting expression of choline acetyltransferase (ChAT), the synthesizing enzyme of acetylcholine (ACh), we detected cholinergic cells in the submucosal glands of the murine larynx and trachea. These cells were localized in the ciliated glandular ducts and were neither found in the collecting ducts nor in alveolar or tubular segments of the glands. ChAT expression in tracheal gland ducts was confirmed by in situ hybridization. The cholinergic duct cells expressed the brush cell marker proteins, villin and cytokeratin-18, and were immunoreactive for components of the taste signal transduction cascade (Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel = TRPM5, phospholipase C β2 ), but not for carbonic anhydrase IV. Furthermore, these cells expressed the bitter taste receptor Tas2r131, as demonstrated utilizing an appropriate reporter mouse strain. Our study identified a previously unrecognized presumptive chemosensory cell type in the duct of the airway submucosal glands that likely utilizes ACh for paracrine signaling. We propose that these cells participate in infection-sensing mechanisms and initiate responses assisting bacterial clearance from the lower airways.

Nonmotor neuron-related pathology is a feature of amyotrophic lateral sclerosis (ALS), both in patients and in animal models. There is emerging evidence that sensory systems (olfaction and vision) are affected in humans. Here, we asked whether such sensory neuropathology is recapitulated in the superoxide dismutase 1 (SOD1G93A) mouse model of ALS. Neuronal vacuolization in olfaction and vision pathways was assessed in tissue sections from presymptomatic and symptomatic disease stages, and compared to wild type. In both, the olfactory bulb and retina, vacuolization started around postnatal day 60, and vacuole sizes increased until disease end-stage. Notably, vacuolization was largely restricted to the external plexiform layer of the olfactory bulb and to the inner plexiform layer of the retina. In both layers, hSOD1-immunoreactive vacuoles localized to dendrites of excitatory neurons. Downstream olfaction and vision pathway fiber tracts and relay stations did not display obvious vacuolization. Finally, on a morphological level, there was no evidence for an activation of astrocytes and microglia in the 2 affected areas. Thus, we identified a new pathology hallmark in SOD1G93A ALS mice: a glutamatergic sensory neuron dendropathy restricted to olfactory bulb mitral cells and retinal ganglionic cells.

Expression of Satb2 (Special AT-rich sequence-binding protein-2) elicits expression of the vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) in cultured rat sympathetic neurons exposed to soluble differentiation factors. Here, we determined whether or not Satb2 plays a similar role in cholinergic differentiation in vivo, by comparing the postnatal profile of Satb2 expression in the rodent stellate ganglion to that of VAChT and ChAT. Throughout postnatal development, VAChT and ChAT were found to be co-expressed in a numerically stable subpopulation of rat stellate ganglion neurons. Nerve fibers innervating rat forepaw sweat glands on P1 were VAChT immunoreactive, while ChAT was detectable at this target only after P5. The postnatal abundance of VAChT transcripts in the stellate ganglion was at maximum already on P1, whereas ChAT mRNA levels increased from low levels on P1 to reach maximum levels between P5 and P21. Satb2 mRNA was detected in cholinergic neurons in the stellate ganglion beginning with P8, thus coincident with the onset of unequivocal detection of ChAT immunoreactivity in forepaw sweat gland endings. Satb2 knockout mice exhibited no change in the P1 cholinergic VAChT/ChAT co-phenotype in stellate ganglion neurons. Thus, cholinergic phenotype maturation involves first, early target (sweat-gland)-independent expression and trafficking of VAChT, and later, potentially target- and Satb2-dependent elevation of ChAT mRNA and protein transport into sweat gland endings. In rat sudomotor neurons that, unlike mouse sudomotor neurons, co-express calcitonin gene-related peptide (CGRP), Satb2 may also be related to the establishment of species-specific neuropeptide co-phenotypes during postnatal development.

Chemosensory cells in the mucosal surface of the respiratory tract ("brush cells") use the canonical taste transduction cascade to detect potentially hazardous content and trigger local protective and aversive respiratory reflexes on stimulation. So far, the urogenital tract has been considered to lack this cell type. Here we report the presence of a previously unidentified cholinergic, polymodal chemosensory cell in the mammalian urethra, the potential portal of entry for bacteria and harmful substances into the urogenital system, but not in further centrally located parts of the urinary tract, such as the bladder, ureter, and renal pelvis. Urethral brush cells express bitter and umami taste receptors and downstream components of the taste transduction cascade; respond to stimulation with bitter (denatonium), umami (monosodium glutamate), and uropathogenic Escherichia coli; and release acetylcholine to communicate with other cells. They are approached by sensory nerve fibers expressing nicotinic acetylcholine receptors, and intraurethral application of denatonium reflexively increases activity of the bladder detrusor muscle in anesthetized rats. We propose a concept of urinary bladder control involving a previously unidentified cholinergic chemosensory cell monitoring the chemical composition of the urethral luminal microenvironment for potential hazardous content.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic peptide with autocrine neuroprotective and paracrine anti-inflammatory properties in various models of acute neuronal damage and neurodegenerative diseases. Therefore, we examined a possible beneficial role of endogenous PACAP in the superoxide dismutase 1, SOD1(G93A), mouse model of amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease particularly affecting somatomotor neurons. In wild-type mice, somatomotor and visceromotor neurons in brain stem and spinal cord were found to express the PACAP specific receptor PAC1, but only visceromotor neurons expressed PACAP as a potential autocrine source of regulation of these receptors. In SOD1(G93A) mice, only a small subset of the surviving somatomotor neurons showed induction of PACAP mRNA, and somatomotor neuron degeneration was unchanged in PACAP-deficient SOD1(G93A) mice. Pre-ganglionic sympathetic visceromotor neurons were found to be resistant in SOD1(G93A) mice, while pre-ganglionic parasympathetic neurons degenerated during ALS disease progression in this mouse model. PACAP-deficient SOD1(G93A) mice showed even greater pre-ganglionic parasympathetic neuron loss compared to SOD1(G93A) mice, and additional degeneration of pre-ganglionic sympathetic neurons. Thus, constitutive expression of PACAP and PAC1 may confer neuroprotection to central visceromotor neurons in SOD1(G93A) mice via autocrine pathways. Regarding the progression of neuroinflammation, the switch from amoeboid to hypertrophic microglial phenotype observed in SOD1(G93A) mice was absent in PACAP-deficient SOD1(G93A) mice. Thus, endogenous PACAP may promote microglial cytodestructive functions thought to drive ALS disease progression. This hypothesis was consistent with prolongation of life expectancy and preserved tongue motor function in PACAP-deficient SOD1(G93A) mice, compared to SOD1(G93A) mice. Given the protective role of PACAP expression in visceromotor neurons and the opposing effect on microglial function in SOD1(G93A) mice, both PACAP agonism and antagonism may be promising therapeutic tools for ALS treatment, if stage of disease progression and targeting the specific auto- and paracrine signaling pathways are carefully considered.

The luminal composition of the auditory tube influences its function. The mechanisms involved in the monitoring are currently not known. For the lower respiratory epithelium, such a sentinel role is carried out by cholinergic brush cells. Here, using two different mouse strains expressing eGFP under the control of the promoter of choline acetyltransferase (ChAT), we show the presence of solitary cholinergic villin-positive brush cells also in the mouse auditory tube epithelium. They express the vesicular acetylcholine (ACh) transporter and proteins of the taste transduction pathway such as α-gustducin, phospholipase C beta 2 (PLC(β2)) and transient receptor potential cation channel subfamily M member 5 (TRPM5). Immunoreactivity for TRPM5 and PLCβ2 was found regularly, whereas α-gustducin was absent in approximately 15% of the brush cells. Messenger RNA for the umami taste receptors (TasR), Tas1R1 and 3, and for the bitter receptors, Tas2R105 and Tas2R108, involved in perception of cycloheximide and denatonium were detected in the auditory tube. Using a transgenic mouse that expresses eGFP under the promotor of the nicotinic ACh receptor α3-subunit, we identified cholinoceptive nerve fibers that establish direct contacts to brush cells in the auditory tube. A subpopulation of these fibers displayed also CGRP immunoreactivity. Collectively, we show for the first time the presence of brush cells in the auditory tube. These cells are equipped with all proteins essential for sensing the composition of the luminal microenvironment and for communication of the changes to the CNS via attached sensory nerve fibers.

The balance between excitatory and inhibitory synaptic inputs is critical for the physiological control of motoneurons. The maintenance of a low-intracellular chloride concentration by the potassium chloride cotransporter 2 (KCC2) is essential for the efficacy of fast synaptic inhibition of mature motoneurons in response to the activation of ionotropic γ-aminobutyric acid A and glycine receptors. Altered synaptic balance and excitotoxicity have been proposed as candidate pathophysiological processes in amyotrophic lateral sclerosis (ALS). Therefore, we investigated the expression patterns of KCC2 and its functional opponent, the chloride influx-mediating sodium-potassium chloride cotransporter 1 (NKCC1), in the superoxide dismutase 1 (SOD1-G93A) mouse model of ALS. We detected reduced KCC2 messenger RNA levels and less membrane-bound KCC2 immunoreactivity in ALS-vulnerable motoneurons in lumbar spinal cord and hypoglossal nuclei of SOD1-G93A mice but not in degeneration-resistant oculomotor nuclei. Downregulation of KCC2 started during late presymptomatic stages and accelerated in parallel to hind limb and tongue motor function deficits. In contrast, NKCC1 messenger RNA levels were unaltered in postnatal lumbar spinal cord motoneurons. Our data indicate that reductions in KCC2 gene expression may contribute to selective motor deficits and disease progression in vulnerable motoneurons in a mouse model of ALS.

In our study we investigated the pathology-related expression patterns of the two calcitonin gene-related peptide (CGRP) isoforms in spinal cord motor neurons of SOD1(G93A) mice, an animal model of the human motor neuron disease, amyotrophic lateral sclerosis (ALS). We found that alphaCGRP and betaCGRP gene expression and alphaCGRP immunoreactivity remained unaltered throughout disease, and alphaCGRP gene deficiency had no effect on disease progression. In contrast, betaCGRP immunoreactivity appeared at atypical sites in degenerating motor neuron cell bodies, axons, and dendrites already in the early pre-symptomatic disease phase around postnatal day 40. A close association of betaCGRP-containing dysmorphic dendritic structures with processes of activated astrocytes, in combination with a selective expression of the CGRP receptor by astrocytes, suggests that betaCGRP may function as a motor neuron-derived signaling molecule for astrocyte activation in ALS.

Loss of motor neurons is the key neuropathological feature of amyotrophic lateral sclerosis (ALS). Although these neurons are targeted by many synapses, little is known about the importance of the pre-synaptic excitatory and inhibitory input for motor neuron degeneration. The present study aimed in determining the composition and abundance of neurochemically defined input on lumbar spinal cord motor neurons in the SOD1 mouse model of ALS by employing immunoreactivity (IR) for vesicular neurotransmitter transporter proteins. The first signs of motor neuron degeneration, visualized by vesicular acetylcholine transporter IR, were already evident in the pre-symptomatic phase at day 80 of life. With the beginning of the symptomatic phase at around day 110 of life, surviving motor neurons showed reductions in the abundances of vesicular glutamate transporter 1 and 2 appositions. This loss of excitatory input was paralleled by an essentially unchanged inhibitory input, visualized by vesicular inhibitory amino acid transporter IR. In addition, loss of excitatory and inhibitory fibers and terminals was also evident in non-motor neuron areas of the spinal cord. These data are indicative of an imbalanced synaptic input on spinal cord motor neurons with over-inhibition rather than over-excitation being a neuropathological feature during disease progression that may contribute to motor neuron death.

The expression patterns of Tac2 and NK3 mRNA and of pep2, the neurokinin B (NKB) precursor protein, were compared in rats and mice. Pep2 immunoreactivity was observed in fibers, terminals, and perikarya in the brains of both species, but the number of NKB-immunoreactive cells was generally smaller in mice than in the corresponding nuclei in rats. Congruent distribution patterns of Tac2 mRNA and NKB were found in many nuclei of the thalamus and hypothalamus (habenula, anterodorsal nucleus, preoptic area, arcuate nucleus, paraventricular nucleus). However, mice expressed Tac2 mRNA neither in the hippocampus nor in the nucleus of the lateral olfactory tract, in contrast to rats. Accordingly, mice showed no NKB in the projection areas of these nuclei, such as the olfactory tubercle, whereas a clear NKB signal was present in rat tissues. Surprisingly, we found nearly identical NK3 mRNA expression patterns in both species, despite the species differences in NKB expression. Thus, although the expression patterns of Tac2 and NKB are similar in rats and mice, noteworthy differences exist. Our results have important implications for the interpretation of behavioral results concerning the NKB/NK3 system in these species.

It has long been known that the sympathetic innervation of the sweat glands is cholinergic in most mammalian species, and that during development, rodent sympathetic cholinergic sweat gland innervation transiently expresses noradrenergic traits. We show here that some noradrenergic traits persist in cholinergic sympathetic innervation of the sweat glands in rodents, but that lack of expression of the vesicular monoamine transporter renders these cells functionally non-noradrenergic. Adult human sweat gland innervation, however, is not only cholinergic, but co-expresses all of the proteins required for full noradrenergic function as well, including tyrosine hydroxylase, aromatic amino acid decarboxylase, dopamine ß-hydroxylase, and the vesicular monoamine transporter VMAT2. Thus, cholinergic/noradrenergic co-transmission is apparently a unique feature of the primate autonomic sympathetic nervous system. Furthermore, sympathetic neurons innervating specifically the cutaneous arteriovenous anastomoses (Hoyer Grosser organs) in humans also possess a full cholinergic/noradrenergic co-phenotype. Cholinergic/noradrenergic co-expression is absent from other portions of the human sympathetic nervous system, but is extended in the parasympathetic nervous system to the intrinsic neurons innervating the heart. These observations suggest a mode of autonomic regulation, based on co-release of norepinephrine and acetylcholine at parasympathocardiac, sudomotor, and selected vasomotor neuroeffector junctions, that is unique to the primate peripheral nervous system.

In this study we compared the alpha-calcitonin gene-related peptide (alphaCGRP) and betaCGRP expression patterns in wild-type and knockout mice by using quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. In dorsal root ganglia and spinal cord of wild-type animals, alphaCGRP mRNA was about two times more abundant than betaCGRP mRNA. The betaCGRP mRNA was the only isoform expressed in the intestine. In alphaCGRP knockout mice, we found no change in betaCGRP mRNA levels in dorsal root ganglia and spinal cord compared with wild-type controls, but a twofold decrease in the intestine. CGRP immunoreactivity (IR) was detected in many small and some large neurons in the dorsal root ganglia, was found in sensory fibers and motor neurons in the spinal cord, and labeled neuromuscular junctions in wild-type mice. In the dorsal root ganglia of alphaCGRP knockout mice, punctate betaCGRP-IR again was predominantly found in small neurons. In the spinal cord, betaCGRP-IR fibers were localized to the outermost layer of the dorsal horn. IR was found in the cell bodies of motor neurons, but it was undetectable in neuromuscular junctions. In the intestine, CGRP-IR was localized to neurons of the myenteric plexus and to fibers in the mucosal folds, with similar staining intensity in both wild-type and knockout mice. Finally, CGRP-IR was undetectable in preganglionic fibers and postganglionic sympathetic neurons in mice from both genotypes. Our results indicate that alphaCGRP and betaCGRP are variably coexpressed in different functional aspects of the mouse nervous system. This pattern suggests distinct roles for betaCGRP in pain, neuromuscular, and gastrointestinal systems.

We examined the expression pattern of the vesicular acetylcholine transporter in the mouse nervous system, using rodent-specific riboprobes and antibodies, prior to comparing it with the distribution of vesicular acetylcholine transporter expressed from a human transgene in the mouse, using riboprobes and antibodies specific for human. Endogenous vesicular acetylcholine transporter expression was high in spinal and brainstem somatomotor neurons, vagal visceromotor neurons, and postganglionic parasympathetic neurons, moderate in basal forebrain and brainstem projection neurons and striatal interneurons, and low in intestinal intrinsic neurons. Vesicular acetylcholine transporter expression in intrinsic cortical neurons was restricted to the entorhinal cortex. The sequence of the mouse cholinergic gene locus to 5.1 kb upstream of the start of transcription of the vesicular acetylcholine transporter gene was determined and compared with the corresponding region of the human gene. Cis- regulatory domains implicated previously in human or rat cholinergic gene regulation are highly conserved in mouse, indicating their probable relevance to the regulation of the mammalian cholinergic gene locus in vivo. Mouse lines were established containing a human transgene that included the vesicular acetylcholine transporter gene and sequences spanning 5 kb upstream and 1.8 kb downstream of the vesicular acetylcholine transporter open reading frame. In this transgene, the intact human vesicular acetyl- choline transporter was able to act as its own reporter. This allowed elements within the vesicular acetylcholine transporter open reading frame itself, shown previously to affect transcription in vitro, to be assessed in vivo with antibodies and riboprobes that reliably distinguished between human and mouse vesicular acetylcholine transporters and their messenger RNAs. Expression of the human vesicular acetylcholine transporter was restricted to mouse cholinergic somatomotor neurons in the spinal cord and brainstem, but absent from other central and peripheral cholinergic neurons. The mouse appears to be an appropriate model for the study of the genetic regulation of the cholinergic gene locus, and the physiology and neurochemistry of the mammalian cholinergic nervous system, although differences exist in the distribution of cortical cholinergic neurons between the mouse and other mammals. The somatomotor neuron-specific expression pattern of the transgenic human vesicular acetylcholine transporter suggests a mosaic model for cholinergic gene locus regulation in separate subdivisions of the mammalian cholinergic nervous system. q 2000 IBRO. Published by Elsevier Science Ltd.

The vesicular monoamine transporters VMAT1 and VMAT2 are essential components of monoaminergic neurons and endocrine cells whose expression in development may provide insight into lineage pathways for chemical coding in the diffuse neuroendocrine system. Thus, the brain is a compartment in which only monoaminergic neurons are generated, the gut epithelium generates only endocrine monoamine-containing cells, and the neural crest produces both autonomic monoaminergic neurons and endocrine/paracrine monoaminergic cells. Selection of either the VMAT1 or VMAT2 isoform was examined in these three compartments during development. In the central nervous system VMAT2, but not VMAT1, was expressed in neuroepithelial cells by embryonic day 12 (E12), and all major monoaminergic cell groups by E14. Thalamocortical and hypothalamic neurons that do not express VMAT2 in adulthood were transiently VMAT2-positive from E16 to postnatal day 6 (P6). EC cells of the gut expressed exclusively VMAT1 from E19 on, while histamine-containing enterochromaffin-like (ECL) cells of the stomach expressed only VMAT2 by E19 and throughout postnatal development. VMAT2 and the vesicular acetylcholine transporter VAChT were co-expressed in early development of the primary sympathetic chain as well as in the cranial parasympathetic ganglia. VAChT was progressively restricted to a small population of VMAT2-negative post-ganglionic neurons in the adult sympathetic chain, while VMAT2 expression persisted in sympathetic principal ganglion and SIF cells but was eventually extinguished in cranial parasympathetic ganglia. VMAT1 was co-expressed with VAChT and VMAT2 mRNA in the primary sympathetic chain on E12, but progressively restricted to small intensely fluorescent (SIF) and chromaffin cells thereafter. Thus, expression of the vesicular amine transporters appropriate for chemical coding of brain neurons and gut endocrine cells are pre-determined developmentally. In contrast, the neural crest-derived sympathoadrenal and neural crest-derived parasympathetic cell groups examined here initially co-express two or more vesicular amine transporters, followed by extinction of the inappropriate transporter(s) later in development. Some neural crest-derived neuroendocrine cell populations continue to express both isoforms of VMAT even in adulthood. Lineage distinctions in ontogeny of vesicular amine transporter expression in brain, gut and autonomic nervous system make it likely that the same genes are regulated differently in the autonomic nervous system compared to brain and gut.

In amyotrophic lateral sclerosis (ALS) some motor neurons degenerate while others survive. The molecular mechanisms underlying this selective vulnerability and resistance, respectively, are poorly understood. Since the neuropeptide, calcitonin gene-related peptide (CGRP), is expressed by many but not all motor neurons, we asked if motor neuron CGRP levels predict their vulnerability in the SOD1-G93A mouse model of ALS. In wild type mice three types of somatic motor neurons were distinguished based on their CGRP expression pattern, i.e. highCGRP, lowCGRP, and nonCGRP. Since motor nuclei III, IV and VI contained mostly nonCGRP motor neurons, they defined the oculomotor group. In comparison, the facial group (nuclei V, VII and XII) contained equal numbers of all three types, while the spinomedullary group (ambiguus nucleus and lumbar spinal cord) contained mainly highCGRP motor neurons. Analysis on the transcript level, and of mice lacking the αCGRP isoform, revealed that these group differences in CGRP expression were predominantly based on αCGRP. At disease end-stage in SOD1-G93A mice, group-specific extent of motor neuron loss correlated with CGRP expression as neurons with highCGRP were reduced by 80%, those with lowCGRP by 50%, and nonCGRP motor neurons were not significantly affected in all three groups. Finally, highCGRP motor neuron degeneration preceded lowCGRP motor neuron degeneration during disease progression. Our analysis revealed that the relative abundance of CGRP mRNA and immunoreactivity in motor neurons predicts their vulnerability. CGRP may be an autocrine or paracrine factor promoting motor neuron degeneration in this ALS model.

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.

1. We investigate here for the first time in primate brain the combinatorial expression of the three major functionally relevant proteins for catecholaminergic neurotransmission tyrosine hydroxylase (TH), aromatic acid acid decarboxylase (AADC), and the brain-specific isoform of the vesicular monoamine transporter, VMAT2, using highly specific antibodies and immunofluorescence with confocal microscopy to visualize combinatorial expression of these proteins. 2. In addition to classical TH, AADC, and VMAT2-copositive catecholaminergic neurons, two unique kinds of TH-positive neurons were identified based on co-expression of AADC and VMAT2. 3. TH and AADC co-positive, but VMAT2-negative neurons, are termed "nonexocytotic catecholaminergic TH neurons." These were found in striatum, olfactory bulb, cerebral cortex, area postrema, nucleus tractus solitarius, and in the dorsal motor nucleus of the vagus. 4. TH-positive neurons expressing neither AADC nor VMAT2 are termed "dopaergic TH neurons." We identified these neurons in supraoptic, paraventricular and periventricular hypothalamic nuclei, thalamic paraventicular nucleus, habenula, parabrachial nucleus, cerebral cortex and spinal cord. We were unable to identify any dopaergic (TH-positive, AADC-negative) neurons that expressed VMAT2, suggesting that regulatory mechanisms exist for shutting off VMAT2 expression in neurons that fail to biosynthesize its substrates. 5. In several cases, the corresponding TH phenotypes were identified in the adult rat, suggesting that this rodent is an appropriate experimental model for further investigation of these TH-positive neuronal cell groups in the adult central nervous system. Thus, no examples of TH and VMAT2 co-positive neurons lacking AADC expression were found in rodent adult nervous system. 6. In conclusion, the adult mammalian nervous system contains in addition to classical catecholaminergic neurons, cells that can synthesize dopamine, but cannot transport and store it in synaptic vesicles, and neurons that can synthesize only L-dopa and lack VMAT2 expression. The presence of these additional populations of TH-positive neurons in the adult primate CNS has implications for functional catecholamine neurotransmission, its derangement in disease and drug abuse, and its rescue by gene therapeutic maneuvers in neurodegenerative diseases such as Parkinson's disease.

Prostaglandin E 2 (PGE 2 ) is a crucial mediator of inflammatory pain sensitization. Here, we demonstrate that inhibition of a specific glycine receptor subtype (GlyR α3) by PGE 2 -induced receptor phosphorylation underlies central inflammatory pain sensitization. We show that GlyR α3 is distinctly expressed in superficial layers of the spinal cord dorsal horn. Mice deficient in GlyR α3 not only lack the inhibition of glycinergic neurotransmission by PGE 2 seen in wild-type mice but also show a reduction in pain sensitization induced by spinal PGE 2 injection or peripheral inflammation. Thus, GlyR α3 may provide a previously unrecognized molecular target in pain therapy.

The cholinergic phenotype requires the expression of the vesicular acetylcholine transporter and choline acetyltransferase proteins. Both genes are encoded at one chromosomal location called the cholinergic gene locus. We have identified by in situ hybridization histochemistry distinct patterns of transcription from the cholinergic gene locus in the subdivisions of the rat cholinergic nervous system. The vesicular acetylcholine transporter and choline acetyltransferase are co-expressed in cholinergic neurons at all developmental stages in all major types of cholinergic neurons. The relative levels of vesicular acetylcholine transporter and choline acetyltransferase transcripts, however, change substantially during development in the CNS. They also differ dramatically in distinct subdivisions of the mature cholinergic nervous system, with vesicular acetylcholine transporter mRNA expressed at high levels relative to choline acetyltransferase mRNA in the peripheral nervous system, but at equivalent levels in the CNS. Expression of the R-exon, the presumptive first non-coding exon common to both the vesicular acetylcholine transporter and choline acetyltransferase, was not detectable at any developmental stage in any of the cholinergic neuronal subtypes in the rat nervous system. Thus, in contrast to less complex metazoan organisms, production of the vesicular acetylcholine transporter and choline acetyltransferase via a common differentially spliced transcript does not seem to occur to a significant extent in the rat. We suggest that separate transcriptional start sites within the cholinergic gene locus control vesicular acetylcholine transporter and choline acetyltransferase transcription, while additional elements are responsible for the specific transcriptional control of the entire locus in cholinergic versus non-cholinergic neurons. Independent transcription of the vesicular acetylcholine transporter and choline acetyltransferase genes provides a mechanism for regulating the relative expression of these two proteins to fine-tune acetylcholine quantal size in different types of cholinergic neurons, both centrally and peripherally.

Chemical coding in the sympathetic nervous system involves both noradrenergic and, for a minority of neurons, cholinergic neurotransmission. The expression of the cholinergic phenotype in the developing sympathetic nervous system was examined to determine if coding for cholinergic transmission occurs before or after innervation of peripheral target organs. The vesicular acetylcholine transporter (VAChT) and choline acetyltransferase, the products of the “cholinergic gene locus” determining the cholinergic phenotype, were expressed in principal cells of the paravertebral, but only rarely in prevertebral, sympathetic chains as early as embryonic day 14. A subpopulation of VAChT- and choline acetyltransferase-positive sympathetic ganglion cells persisted throughout development of the stellate and more caudal paravertebral ganglia into anatomically distinct cell groups, and into adulthood. The forepaw eccrine sweat glands, innervated exclusively by the stellate ganglion, received VAChT-positive nerve terminals at least as early as postembryonic day 4, coincident with the development of the sweat glands themselves. These terminals, like the VAChT-positive cell bodies of the developing stellate ganglion, have some noradrenergic traits including expression of tyrosine hydroxylase, but did not express the vesicular monoamine transporter, and are therefore not functionally noradrenergic. Development of the cholinergic phenotype in principal cells of the sympathetic paravertebral ganglia apparently occurs via receipt of instructive cues, or selection, within the sympathetic chain itself or perhaps even during migration of the cells of the neural crest from which the paravertebral ganglia arise.