PAC1 deficiency reduces chondrogenesis in atherosclerotic lesions of hypercholesterolemic ApoE-deficient mice.

BACKGROUND: Induction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide (PACAP) aggravates atherosclerosis in ApoE deficient (ApoE-/-) mice. PACAP signaling regulates chondrogenesis and osteogenesis during cartilage and bone development. Therefore, this study aimed to decipher whether PACAP signaling is related to atherogenesis-related chondrogenesis in the ApoE-/- mouse model of atherosclerosis and under the influence of a high-fat diet. METHODS: For this purpose, PACAP-/-/ApoE-/-, PAC1-/-/ApoE-/-, and ApoE-/- mice, as well as wildtype (WT) mice, were studied under standard chow (SC) or cholesterol-enriched diet (CED) for 20 weeks. The amount of cartilage matrix in atherosclerotic lesions of the brachiocephalic trunk (BT) with maximal lumen stenosis was monitored by alcian blue and collagen II staining on deparaffinized cross sections. The chondrogenic RUNX family transcription factor 2 (RUNX2), macrophages [(MΦ), Iba1+], and smooth muscle cells (SMC, sm-α-actin) were immunohistochemically analyzed and quantified. RESULTS: ApoE-/- mice fed either SC or CED revealed an increase of alcian blue-positive areas within the media compared to WT mice. PAC1-/-/ApoE-/- mice under CED showed a reduction in the alcian blue-positive plaque area in the BT compared to ApoE-/- mice. In contrast, PACAP deficiency in ApoE-/- mice did not affect the chondrogenic signature under either diet. CONCLUSIONS: Our data show that PAC1 deficiency reduces chondrogenesis in atherosclerotic plaques exclusively under conditions of CED-induced hypercholesterolemia. We conclude that CED-related chondrogenesis occurs in atherosclerotic plaques via transdifferentiation of SMCs and MΦ, partly depending on PACAP signaling through PAC1. Thus, PAC1 antagonists or PACAP agonists may offer therapeutic potential against pathological chondrogenesis in atherosclerotic lesions generated under hypercholesterolemic conditions, especially in familial hypercholesterolemia. This discovery opens therapeutic perspectives to be used in the treatment against the progression of atherosclerosis.

Species-specific vesicular monoamine transporter 2 (VMAT2) expression in mammalian pancreatic beta cells: implications for optimising radioligand-based human beta cell mass (BCM) imaging in animal models.

AIMS/HYPOTHESIS: Imaging of beta cell mass (BCM) is a major challenge in diabetes research. The vesicular monoamine transporter 2 (VMAT2) is abundantly expressed in human beta cells. Radiolabelled analogues of tetrabenazine (TBZ; a low-molecular-weight, cell-permeant VMAT2-selective ligand) have been employed for pancreatic islet imaging in humans. Since reports on TBZ-based VMAT2 imaging in rodent pancreas have been fraught with confusion, we compared VMAT2 gene expression patterns in the mouse, rat, pig and human pancreas, to identify appropriate animal models with which to further validate and optimise TBZ imaging in humans. METHODS: We used a panel of highly sensitive VMAT2 antibodies developed against equivalently antigenic regions of the transporter from each species in combination with immunostaining for insulin and species-specific in situ hybridisation probes. Individual pancreatic islets were obtained by laser-capture microdissection and subjected to analysis of mRNA expression of VMAT2. RESULTS: The VMAT2 protein was not expressed in beta cells in the adult pancreas of common mouse or rat laboratory strains, in contrast to its expression in beta cells (but not other pancreatic endocrine cell types) in the pancreas of pigs and humans. VMAT2- and tyrosine hydroxylase co-positive (catecholaminergic) innervation was less abundant in humans than in rodents. VMAT2-positive mast cells were identified in the pancreas of all species. CONCLUSIONS/INTERPRETATION: Primates and pigs are suitable models for TBZ imaging of beta cells. Rodents, because of a complete lack of VMAT2 expression in the endocrine pancreas, are a 'null' model for assessing interference with BCM measurements by VMAT2-positive mast cells and sympathetic innervation in the pancreas.

Cholinergic chemosensory cells in the auditory tube.

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(ß2)) and transient receptor potential cation channel subfamily M member 5 (TRPM5). Immunoreactivity for TRPM5 and PLCß2 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.

Distribution and kinetics of superantigen-induced cytokine gene expression in mouse spleen.

The polyclonal stimulation of T cells by bacterial superantigens is involved in the pathogenesis of the toxic shock syndrome in certain staphylococcal and streptococcal infections. Here we describe the onset and kinetics of superantigen-induced cytokine production in situ in spleens of normal BALB/c mice monitored at the level of cytokine mRNA expression by in situ hybridization. Messenger RNAs for interleukin 2 (IL-2), interferon gamma, and tumor necrosis factors (TNF) alpha and beta were not expressed at detectable levels in spleens of unstimulated animals but became visible already 30 min after intraperitoneal application of 50 micrograms staphylococcal enterotoxin B. All mRNA levels showed peak expression approximately 3 h after injection and a slow decrease up to 24 h after injection. Expression of the mRNAs was restricted to the T cell-dependent area of the periarteriolar lymphatic sheets of the spleen. Interestingly, TNF-alpha mRNA showed a biphasic response, the early appearing mRNA had the same localization as the other mRNAs, whereas after 3 h TNF-alpha mRNA showed a broader distribution indicating a second cell population producing TNF-alpha. The expression of IL-2 and TNF proteins in the serum increased in parallel to the observed mRNA changes with a slight delay. The presence of macrophages was not required for the expression of the cytokine mRNAs in the spleen as the expression was unchanged in macrophage-depleted mice. Only the second phase of TNF-alpha mRNA expression was abrogated in such animals. The expression of all mRNAs was completely suppressed by prior administration of cyclosporin A. These data show that nonphagocytic cells are the essential superantigen-presenting cells in vivo and indicate that at least part of the pathogenetic TNF-alpha is T cell derived.

Sweat gland innervation is pioneered by sympathetic neurons expressing a cholinergic/noradrenergic co-phenotype in the mouse.

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.

Role of virus-induced neuropeptides in the brain in the pathogenesis of rabies.

Rabies virus (RABV) infection is characterized by the rapid neuronal spread of RABV into the CNS before a protective immune response is raised. Therefore, a typical feature of RABV infection is the paucity of inflammatory reactions in the brain. Here we examined whether the induction of immunosuppressive neuropeptides, in particular CGRP, may contribute to the ability of RABV to evade immune responses. RABV infection of mice caused a strong induction of calcitonin gene-related peptide (CGRP) in neurons and fibres in the neocortex as well as in the dentate gyrus and CA1 region of the hippocampus although RABV did not infect neurons in which CGRP expression was upregulated. Neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP) expressing neurons also were not infected by RABV. In contrast, somatostatin neurons were infected by RABV. There was evidence for an RABV-induced increase of VIP and somatostatin but not of NPY. To test how CGRP expression is related to TNFalpha-induced enhancement of CNS innate and adaptive immunity during RABV infection, we used recombinant RABVs that contained either an active (SPBN-TNFalpha(+)) or an inactive (SPBN-TNFalpha(-)) TNFalpha gene. As compared to SPBN-TNFalpha(-), infection with SPBN-TNFalpha(+) attenuated the induction of CGRP but simultaneously enhanced induction of the invariant chain of MHC II, microglial activation and T cell infiltration. In conclusion, distinct neuropeptidergic neurons in the brain are remarkably spared from RABV infection suggesting a pivotal role of neuropeptides during CNS virus infection. Given the inhibitory effect of CGRP on antigen presentation, we propose that the strong RABV-induced upregulation of CGRP in the brain may contribute to the mechanism by which RABV escapes immune detection. Targeting the expression of neuropeptides, in particular CGRP, that are induced during RABV infection may open a new avenue for therapeutic intervention in human rabies.

The phenotype of the RABV glycoprotein determines cellular and global virus load in the brain and is decisive for the pace of the disease.

The Rabies lyssavirus glycoprotein (RABV-G) is largely responsible for the neuroinvasiveness of the virus and the induction of antiviral immune responses. To study the effects of RABV-G we compared the G of the attenuated RABV variant SPBN with that of the pathogenic DOG4 strain. Infection via the olfactory route caused 100% mortality in mice with both virus variants. Of note, with the attenuated SPBN, progression of the disease was accelerated, microglia response less pronounced and IL-6 expression higher than in the presence of RABV-G from the pathogenic DOG4. However, while virus spread was less extensive, viral gene expression in individual neurons was actually higher in SPBN-infected brains without causing apoptosis of infected neurons. These differences between the two variants were not observed in infected neuronal cultures indicating that the effects of RABV-G on virus spread and viral gene expression depend on factors only present in the intact brain.

Adaptive plasticity in tachykinin and tachykinin receptor expression after focal cerebral ischemia is differentially linked to gabaergic and glutamatergic cerebrocortical circuits and cerebrovenular endothelium.

To test the hypothesis of an involvement of tachykinins in destabilization and hyperexcitation of neuronal circuits, gliosis, and neuroinflammation during cerebral ischemia, we investigated cell-specific expressional changes of the genes encoding substance P (SP), neurokinin B (NKB), and the tachykinin/neurokinin receptors (NK1, NK2, and NK3) after middle cerebral artery occlusion (MCAO) in the rat. Our analysis by quantitative in situ hybridization, immunohistochemistry, and confocal microscopy was concentrated on cerebrocortical areas that survive primary infarction but undergo secondary damage. Here, SP-encoding preprotachykinin-A and NK1 mRNA levels and SP-like immunoreactivity were transiently increased in GABAergic interneurons at 2 d after MCAO. Coincidently, MCAO caused a marked expression of SP and NK1 in a subpopulation of glutamatergic pyramidal cells, and in some neurons SP and NK1 mRNAs were coinduced. Elevated levels of the NKB-encoding preprotachykinin-B mRNA and of NKB-like immunoreactivity at 2 and 7 d after MCAO were confined to GABAergic interneurons. In parallel, the expression of NK3 was markedly downregulated in pyramidal neurons. MCAO caused transient NK1 expression in activated cerebrovenular endothelium within and adjacent to the infarct. NK1 expression was absent from activated astroglia or microglia. The differential ischemia-induced plasticity of the tachykinin system in distinct inhibitory and excitatory cerebrocortical circuits suggests that it may be involved in the balance of endogenous neuroprotection and neurotoxicity by enhancing GABAergic inhibitory circuits or by facilitating glutamate-mediated hyperexcitability. The transient induction of NK1 in cerebrovenular endothelium may contribute to ischemia-induced edema and leukocyte diapedesis. Brain tachykinin receptors are proposed as potential drug targets in stroke.

Striatal tyrosine hydroxylase-positive neurons are associated with L-DOPA-induced dyskinesia in hemiparkinsonian mice.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is the therapeutic gold standard in Parkinson's disease. However, long-term treatment is complicated by the induction of debilitating abnormal involuntary movements termed L-DOPA-induced dyskinesias (LIDs). Until today the underlying mechanisms of LID pathogenesis are not fully understood. The aim of this study was to reveal new factors, which may be involved in the induction of LID. We have focused on the expression of striatal tyrosine hydroxylase-positive (TH+) neurons, which are capable of producing either L-DOPA or dopamine (DA) in target areas of ventral midbrain DAergic neurons. To address this issue, a daily L-DOPA dose was administered over the course of 15 days to mice with unilateral 6-hydroxydopamine-induced lesions of the medial forebrain bundle and LIDs were evaluated. Remarkably, the number of striatal TH+ neurons strongly correlated with both induction and severity of LID as well as ΔFosB expression as an established molecular marker for LID. Furthermore, dyskinetic mice showed a marked augmentation of serotonergic fiber innervation in the striatum, enabling the decarboxylation of L-DOPA to DA. Axial, limb and orolingual dyskinesias were predominantly associated with TH+ neurons in the lateral striatum, whereas medially located TH+ neurons triggered locomotive rotations. In contrast, identified accumbal and cortical TH+ cells did not contribute to the generation of LID. Thus, striatal TH+ cells and serotonergic terminals may cooperatively synthesize DA and subsequently contribute to supraphysiological synaptic DA concentrations, an accepted cause in LID pathogenesis.

Identification of cholinergic chemosensory cells in mouse tracheal and laryngeal glandular ducts.

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(ß2)), 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.

Fine structural analysis of the synaptic junction of Merkel cell-axon-complexes.

The ultrastructure of synaptic contact areas in Merkel cell-axon-complexes from sinus hair follicles and touch domes of various mammals (nude mice, rats, cats, rabbits, opossums and monkeys) was investigated by electron microscopy of ultrathin sections from perfusion fixed tissue. Synapses between Merkel cells and axons were a common feature in all analyzed species. Special staining with digallic acid and goniometric tilting facilitated the resolution of the membranous and paramembranous synaptic elements. The synaptic contact revealed the typical characteristics of a chemical synapse, except for presynaptic clear vesicles: a postsynaptic membrane thickening and dense projections at the presynaptic membrane (i.e., the Merkel cell membrane). The cleft material was resolved as a fuzzy coating of the outer leaflets of the synaptic membranes with occasional bridges across the synaptic cleft. The presence of a synapse in the Merkel cell-axon-complexes emphasizes the receptor function of the Merkel cell besides other possible functions of this cell.

Chromogranin A in the mammalian Merkel cell: cellular and subcellular distribution.

Chromogranin-A (CGA), which accounts for more than half the soluble matrix protein in secretory granules of various neuroendocrine cells, has a wide spectrum of potential biological roles and is considered an important marker of the diffuse neuroendocrine system (DNES). Light and electron microscopic immunohistochemistry of mammalian skin revealed that Merkel cells are exclusively CGA-immunoreactive (ir) and that the immunoreaction is localized in the secretory granules. This finding supports the classification of the Merkel cell as a member of the DNES. The CGA immunoreactivity was restricted to Merkel cells of pigs and humans. In human embryonic skin, CGA was expressed in Merkel cells as early as week 11 of gestation. The antisera differed in their ability to stain Merkel cells in different species and developmental stages, reflecting a variable chemical coding for CGA. CGA probably represents a precursor for smaller regulatory peptides or acts as a messenger on its own on various target tissues, suggesting a neurosecretory function of the Merkel cell.

Immunohistochemical localization of vasoactive intestinal polypeptide (VIP) in Merkel cells of various mammals: evidence for a neuromodulator function of the Merkel cell.

Since met-enkephalin-like substance has been demonstrated only in Merkel cells of some rodents but not of cat, dog, pig, and humans, Merkel cells of these species were analyzed by immunohistochemistry using a variety of different antisera for the occurrence of neuropeptides different from met-enkephalin. In various locations of all species investigated Merkel cells were found to be immunoreactive exclusively to vasoactive intestinal polypeptide (VIP) but not to any of the other antisera used. Thus, in mammalian Merkel cells, neuropeptides occur that are different from met-enkephalin. It is suggested that the Merkel cell-axon complex represents a complex regulatory system involving a presumptive receptor or modulator function whereby the Merkel cell may influence the threshold of the sensory nerve ending via release of a neuropeptide (VIP- or met-enkephalin-like material).

Cytopathologic and neurochemical correlates of progression to motor/cognitive impairment in SIV-infected rhesus monkeys.

Neurochemical, pathologic, virologic, and histochemical correlates of simian immunodeficiency virus (SIV)-associated central nervous system (CNS) dysfunction were assessed serially or at necropsy in rhesus monkeys that exhibited motor and cognitive deficits after SIV infection. Some infected monkeys presented with signs of acquired immunodeficiency disease (AIDS) at the time of sacrifice. Seven of eight animals exhibited motor skill impairment which was associated with elevated quinolinic acid in cerebrospinal fluid (CSF). Examination of the brains revealed diffuse increases in glial fibrillary acidic protein immunoreactivity in cerebral cortex in all animals, regardless of evidence of immunodeficiency disease. Reactive astrogliosis preceded or was coincident with the onset of neuropsychological impairments. Virus rescue from CSF of six of eight infected animals showed that one of three animals with AIDS and none of three animals without AIDS at necropsy had virus rescue-positive CSF. Multinucleated giant cells were seen in the brain of only one animal with end-stage AIDS and high systemic virus burden at death. Neither systemic nor CNS virus burden was associated with the onset of CNS dysfunction. SIV-associated motor/cognitive impairment is associated with subtle, widespread changes in CNS cytology and neurochemistry, rather than with large increases in brain virus burden or widespread virus-associated brain lesions.

Prodynorphin gene expression in the rat intermediate pituitary lobe: gender differences and postpartum regulation.

The distribution of prodynorphin (proDyn) messenger RNA (mRNA) was examined in the rat pituitary using Northern and in situ hybridization analysis. Anterior pituitary gonadotrophs are known to express ProDyn, but the present study demonstrated that proDyn mRNA was also expressed in the intermediate lobe melanotrophs and was colocalized with POMC mRNA. The 2.6-kilobase proDyn transcript observed in the intermediate lobe was shown to be translatable by polysome analysis. Immunohistochemical studies showed dynorphin (Dyn)-like immunoreactivity in all intermediate lobe melanotrophs. Intermediate lobe proDyn gene expression was not regulated by dopamine, in contrast to intermediate lobe POMC mRNA levels, which were increased with haloperidol and decreased with bromocriptine treatment, as expected. A gender difference in ProDyn gene expression was noted, since intermediate lobes of male rats had nearly 2-fold higher proDyn mRNA levels than intermediate lobes of female rats. In contrast, no gender difference of intermediate lobe POMC mRNA levels were detected. ProDyn mRNA levels were up-regulated by 3- to 4-fold in the intermediate lobes of postpartum females as compared to pregnant or nonpregnant female rats, whereas POMC mRNA levels were unchanged, suggesting a role for intermediate lobe ProDyn in the postpartum period of the female rat. Although our results demonstrate proDyn and POMC coexpression in the pituitary intermediate lobe melanotrophs and show a differential regulational control for each gene in this tissue, the present data also strengthen the notion that proDyn is a precursor that has a role to play in reproductive functions.

Neuronal expression of fractalkine in the presence and absence of inflammation.

Fractalkine is the only as yet known member of a novel class of chemokines. Besides its novel Cys-X-X-X-Cys motif, fractalkine exhibits features which have not been described for any other member of the chemokine family, including its unusual size (397 amino acids human, 395 mouse) and the possession of a transmembrane anchor, from which a soluble form may be released by extracellular cleavage. This report demonstrates the abundant mRNA and fractalkine protein expression in neuronal cells. The neuronal expression of fractalkine mRNA is unaffected by experimentally induced inflammation of central nervous tissue.

Regional specificities in the distribution, chemical phenotypes, and coexistence patterns of neuropeptide containing nerve fibres in the human anal canal.

Despite the pivotal clinical significance of the human anal canal, little is known about its total and specific innervation. This study assessed the comparative distribution and histotopology of nerve fibres immunoreactive for neural markers and a variety of regulatory active neuropeptides in the human anal canal by light microscopic immunohistochemistry. Depending on the epithelial zone and region of the anal canal, the neural elements were differentially immunoreactive for the pan-neural marker protein gene product 9.5, the catecholamine marker tyrosine hydroxylase, the neuroendocrine marker chromogranin A, and various neuropeptides. Protein gene product 9.5-immunoreactive nerve fibres were ubiquitously abundant in the anal canal. In the anal transitional zone, ectopic epithelial types were supplied by the same pattern of peptidergic nerves as the respective type of epithelium in normotopic location. In the dermis of the squamous zone and in the perianal epidermis, unusual distribution patterns of nerve fibres, referred to as areas of high nerve fibre density, were encountered. Double immunohistochemistry revealed region-specific coexistence patterns of neuropeptidergic nerve fibres, and novel peptide coexistence patterns were detected in anal nerve fibres. Subsets of nerve fibres formed close spatial relationships with chromogranin A-positive neuroendocrine cells, most frequently in the anal transitional zone. Chromogranin-A positive cells were shown to be present in the epithelium of perianal eccrine sweat glands. The differential distribution, peptide phenotypes and coexistence patterns of different nerve fibre populations in the human anal canal may reflect topospecific regulatory functions of neurally released neuropeptides in health and disease.

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 TNFalpha and IL-1beta 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.

Regeneration of implanted splenic tissue in the rat: re-innervation is host age-dependent and necessary for tissue development.

The loss of spleen may lead to fatal bacterial infections. To prevent this, splenic autotransplantation has been performed in humans and experimental animals. However, there is still controversy about the protective function of this procedure. Since innervation plays an important role in splenic function, we investigated whether splenic regenerates are re-innervated, and whether this depends on the donor and host age. Splenic tissue (30 mg) was implanted into the greater omentum of either young (2 days) or old (12 months) rats, from either young or old syngeneic animals. After 3 months of regeneration, the weight of the regenerates was determined, PGP+ nerve fibers were revealed by immunohistology, and subdivided into nerve fibers of sympathetic (TH+, NPY+) or sensory (SP+, CGRP+) origin. In addition, proliferating (Ki-67 proliferation antigen+) and apoptotic cells (TUNEL technique+) were likewise investigated. No innervation of splenic regenerates was observed after implantation into old hosts, correlating with poorly developed splenic compartments. In contrast, almost normal re-innervation occurred in young hosts after implantation of both young and old splenic tissue. These regenerates showed well-developed splenic compartments and a normal number and tissue distribution of proliferating and apoptotic cells. However, after the implantation of young tissue, the final size of splenic regenerates was three times larger (140 +/- 30 vs. 40 +/- 10 mg). Thus, re-innervation of splenic implants is necessary for their subsequent development. It is determined by host age, whereas the final size of the splenic regenerates is regulated by donor age-dependent factors. This model is useful for studying both the process leading to initial innervation and the consequences of this innervation.

Independent patterns of transcription for the products of the rat cholinergic gene locus.

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.