KCNQ1 is an essential mediator of the sex-dependent perception of moderate cold temperatures.

Low temperatures and cooling agents like menthol induce cold sensation by activating the peripheral cold receptors TRPM8 and TRPA1, cation channels belonging to the TRP channel family, while the reduction of potassium currents provides an additional and/or synergistic mechanism of cold sensation. Despite extensive studies over the past decades to identify the molecular receptors that mediate thermosensation, cold sensation is still not fully understood and many cold-sensitive peripheral neurons do not express the well-established cold sensor TRPM8. We found that the voltage-gated potassium channel KCNQ1 (Kv7.1), which is defective in cardiac LQT1 syndrome, is, in addition to its known function in the heart, a highly relevant and sex-specific sensor of moderately cold temperatures. We found that KCNQ1 is expressed in skin and dorsal root ganglion neurons, is sensitive to menthol and cooling agents, and is highly sensitive to moderately cold temperatures, in a temperature range at which TRPM8 is not thermosensitive. C-fiber recordings from KCNQ1-/- mice displayed altered action potential firing properties. Strikingly, only male KCNQ1-/- mice showed substantial deficits in cold avoidance at moderately cold temperatures, with a strength of the phenotype similar to that observed in TRPM8-/- animals. While sex-dependent differences in thermal sensitivity have been well documented in humans and mice, KCNQ1 is the first gene reported to play a role in sex-specific temperature sensation. Moreover, we propose that KCNQ1, together with TRPM8, is a key instrumentalist that orchestrates the range and intensity of cold sensation.

Neurochemical Monitoring of Traumatic Brain Injury by the Combined Analysis of Plasma Beta-Synuclein, NfL, and GFAP in Polytraumatized Patients.

Traumatic brain injury (TBI) represents a major determining factor of outcome in severely injured patients. However, reliable brain-damage-monitoring markers are still missing. We therefore assessed brain-specific beta-synuclein as a novel blood biomarker of synaptic damage and measured the benchmarks neurofilament light chain (NfL), as a neuroaxonal injury marker, and glial fibrillary acidic protein (GFAP), as an astroglial injury marker, in patients after polytrauma with and without TBI. Compared to healthy volunteers, plasma NfL, beta-synuclein, and GFAP were significantly increased after polytrauma. The markers demonstrated highly distinct time courses, with beta-synuclein and GFAP peaking early and NfL concentrations gradually elevating during the 10-day observation period. Correlation analyses revealed a distinct influence of the extent of extracranial hemorrhage and the severity of head injury on biomarker concentrations. A combined analysis of beta-synuclein and GFAP effectively discriminated between polytrauma patients with and without TBI, despite the comparable severity of injury. Furthermore, we found a good predictive performance for fatal outcome by employing the initial plasma concentrations of NfL, beta-synuclein, and GFAP. Our findings suggest a high diagnostic value of neuronal injury markers reflecting distinct aspects of neuronal injury for the diagnosis of TBI in the complex setting of polytrauma, especially in clinical surroundings with limited imaging opportunities.

The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels encode neuronal and cardiac pacemaker currents. The composition of pacemaker channel complexes in different tissues is poorly understood, and the presence of additional HCN modulating subunits was speculated. Here we show that vesicle-associated membrane protein-associated protein B (VAPB), previously associated with a familial form of amyotrophic lateral sclerosis 8, is an essential HCN1 and HCN2 modulator. VAPB significantly increases HCN2 currents and surface expression and has a major influence on the dendritic neuronal distribution of HCN2. Severe cardiac bradycardias in VAPB-deficient zebrafish and VAPB-/- mice highlight that VAPB physiologically serves to increase cardiac pacemaker currents. An altered T-wave morphology observed in the ECGs of VAPB-/- mice supports the recently proposed role of HCN channels for ventricular repolarization. The critical function of VAPB in native pacemaker channel complexes will be relevant for our understanding of cardiac arrhythmias and epilepsies, and provides an unexpected link between these diseases and amyotrophic lateral sclerosis.-Silbernagel, N., Walecki, M., Schäfer, M.-K. H., Kessler, M., Zobeiri, M., Rinné, S., Kiper, A. K., Komadowski, M. A., Vowinkel, K. S., Wemhöner, K., Fortmüller, L., Schewe, M., Dolga, A. M., Scekic-Zahirovic, J., Matschke, L. A., Culmsee, C., Baukrowitz, T., Monassier, L., Ullrich, N. D., Dupuis, L., Just, S., Budde, T., Fabritz, L., Decher, N. The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function.

Satb2-independent acquisition of the cholinergic sudomotor phenotype in rodents.

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.

The sympathetic nervous system modulates CD4(+)Foxp3(+) regulatory T cells via noradrenaline-dependent apoptosis in a murine model of lymphoproliferative disease.

The sympathetic nervous system (SNS) plays a crucial role in the course and development of autoimmune disease in Fas-deficient lpr/lpr mice. As regulatory T cells (Tregs) are considered important modulators of autoimmune processes, we analyzed the interaction between the SNS and Tregs in this murine model of lymphoproliferative disease. We found that the percentage of Tregs among CD4(+) T cells is increased in the spleen, lymph nodes, and thymus of lpr/lpr mice as compared to age-matched C57Bl/6J (B6) mice. Furthermore, noradrenaline (NA), the main sympathetic neurotransmitter, induced apoptosis in B6- and lpr/lpr-derived Tregs. NA also reduced the frequency of Foxp3(+) cells and Foxp3 mRNA expression via ß2-adrenoceptor (ß2-AR)-mediated mechanisms in a concentration and time-dependent manner. Destruction of peripheral sympathetic nerves by 6-hydroxydopamine significantly increased the percentage of Tregs in B6 control mice to an extent comparable to aged-matched lpr/lpr mice. The concentration of splenic NA negatively correlated with the frequency of CD4(+)Foxp3(+) Tregs. Additionally, 60days after sympathectomy, a partial recovery of NA concentrations led to Treg percentages comparable to those of intact, vehicle-treated controls. Immunohistochemical analysis of the spleen revealed localization of single Foxp3(+) Tregs in proximity to NA-producing nerve fibers, providing an interface between Tregs and the SNS. Taken together, our data suggest a relation between the degree of splenic sympathetic innervation and the size of the Treg compartment. While there are few examples of endogenous substances capable of affecting Tregs, our results provide a possible explanation of how the magnitude of the Treg compartment in the spleen can be regulated by the SNS.

Popeye domain containing proteins modulate the voltage-gated cardiac sodium channel Nav1.5.

Popeye domain containing (POPDC) proteins are predominantly expressed in the heart and skeletal muscle, modulating the K2P potassium channel TREK-1 in a cAMP-dependent manner. POPDC1 and POPDC2 variants cause cardiac conduction disorders with or without muscular dystrophy. Searching for POPDC2-modulated ion channels using a functional co-expression screen in Xenopus oocytes, we found POPDC proteins to modulate the cardiac sodium channel Nav1.5. POPDC proteins downregulate Nav1.5 currents in a cAMP-dependent manner by reducing the surface expression of the channel. POPDC2 and Nav1.5 are both expressed in different regions of the murine heart and consistently POPDC2 co-immunoprecipitates with Nav1.5 from native cardiac tissue. Strikingly, the knock-down of popdc2 in embryonic zebrafish caused an increased upstroke velocity and overshoot of cardiac action potentials. The POPDC modulation of Nav1.5 provides a new mechanism to regulate cardiac sodium channel densities under sympathetic stimulation, which is likely to have a functional impact on cardiac physiology and inherited arrhythmias.

PACAP signaling exerts opposing effects on neuroprotection and neuroinflammation during disease progression in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

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.

Contingent Social Interaction Does Not Prevent Habituation towards Playback of Pro-Social 50-kHz Calls: Behavioral Responses and Brain Activation Patterns.

Rats, which are highly social animals, are known to communicate using ultrasonic vocalizations (USV) in different frequency ranges. Calls around 50 kHz are related to positive affective states and promote social interactions. Our previous work has shown that the playback of natural 50-kHz USV leads to a strong social approach response toward the sound source, which is related to activation in the nucleus accumbens. In male Wistar rats, the behavioral response habituates, that is, becomes weaker or is even absent, when such playback is repeated several days later, an outcome found to be memory-dependent. Here, we asked whether such habituation is due to the lack of a contingent social consequence after playback in the initial test and whether activation of the nucleus accumbens, as measured by c-fos immunohistochemistry, can still be observed in a retest. To this end, groups of young male Wistar rats underwent an initial 50-kHz USV playback test, immediately after which they were either (1) kept temporarily alone, (2) exposed to a same-sex juvenile, or (3) to their own housing group. One week later, they underwent a retest with playback; this time not followed by social consequences but by brain removal for c-fos immunohistochemistry. Consistent with previous reports, behavioral changes evoked by the initial exposure to 50-kHz USV playback included a strong approach response. In the retest, no such response was found, irrespective of whether rats had experienced a contingent social consequence after the initial test or not. At the neural level, no substantial c-fos activation was found in the nucleus accumbens, but unexpected strong activation was detected in the anterior cingulate cortex, with some of it in GABAergic cells. The c-fos patterns did not differ between groups but cell numbers were individually correlated with behavior, i.e., rats that still approached in response to playback in the retest showed more activation. Together, these data do not provide substantial evidence that the lack of a contingent social consequence after 50-kHz USV playback accounts for approach habituation in the retest. Additionally, there is apparently no substantial activation of the nucleus accumbens in the retest, whereas the exploratory findings in the anterior cingulate cortex indicate that this brain area might be involved when individual rats still approach 50-kHz USV playback.

Protein Expression of the Microglial Marker Tmem119 Decreases in Association With Morphological Changes and Location in a Mouse Model of Traumatic Brain Injury.

The activation of microglia and the infiltration of macrophages are hallmarks of neuroinflammation after acute brain injuries, including traumatic brain injury (TBI). The two myeloid populations share many features in the post-injury inflammatory response, thus, being antigenically indistinguishable. Recently Tmem119, a type I transmembrane protein specifically expressed by microglia under physiological conditions, was proposed as a tool to differentiate resident microglia from blood-borne macrophages, not expressing it. However, the validity of Tmem119 as a specific marker of resident microglia in the context of acute brain injury, where microglia are activated and macrophages are recruited, needs validation. Our purpose was to investigate Tmem119 expression and distribution in relation to the morphology of brain myeloid cells present in the injured area after TBI. Mice underwent sham surgery or TBI by controlled cortical impact (CCI). Brains from sham-operated, or TBI mice, were analyzed by in situ hybridization to identify the cells expressing Tmem119, and by Western blot and quantitative immunofluorescence to measure Tmem119 protein levels in the entire brain regions and single cells. The morphology of Iba1+ myeloid cells was analyzed at different times (4 and 7 days after TBI) and several distances from the contused edge in order to associate Tmem119 expression with morphological evolution of active microglia. In situ hybridization indicated an increased Tmem119 RNA along with increased microglial complement C1q activation in the contused area and surrounding regions. On the contrary, the biochemical evaluation showed a drop in Tmem119 protein levels in the same areas. The Tmem119 immunoreactivity decreased in Iba1+ myeloid cells found in the contused cortex at both time points, with the cells showing the hypertrophic ameboid morphology having no Tmem119 expression. The Tmem119 was present on ramifications of resident microglia and its presence was decreased as a consequence of microglial activation in cortical areas close to contusion. Based on the data, we conclude that the decrease of Tmem119 in reactive microglia may depend on the process of microglial activation, which involves the retracting of their branchings to acquire an ameboid shape. The Tmem119 immunoreactivity decreases in reactive microglia to similar levels than the blood-borne macrophages, thus, failing to discriminate the two myeloid populations after TBI.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP)-Glutamate Co-transmission Drives Circadian Phase-Advancing Responses to Intrinsically Photosensitive Retinal Ganglion Cell Projections by Suprachiasmatic Nucleus.

Results from a variety of sources indicate a role for pituitary adenylate cyclase-activating polypeptide (PACAP) in light/glutamate-induced phase resetting of the circadian clock mediated by the retinohypothalamic tract (RHT). Attempts to block or remove PACAP's contribution to clock-resetting have generated phenotypes that differ in their responses to light or glutamate. For example, previous studies of circadian behaviors found that period-maintenance and early-night phase delays are intact in PACAP-null mice, yet there is a consistent deficit in behavioral phase-resetting to light stimulation in the late night. Here we report rodent stimulus-response characteristics of PACAP release from the RHT, and map these to responses of the suprachiasmatic nucleus (SCN) in intact and PACAP-deficient mouse hypothalamus with regard to phase-resetting. SCN of PACAP-null mice exhibit normal circadian rhythms in neuronal activity, but are "blind" to glutamate stimulating phase-advance responses in late night, although not in early night, consistent with previously reported selective lack of late-night light behavioral responsiveness of these mice. Induction of CREB phosphorylation, a hallmark of the light/glutamate response of the SCN, also is absent in SCN-containing ex vivo slices from PACAP-deficient mouse hypothalamus. PACAP replacement to the SCN of PACAP-null mice restored wild-type phase-shifting of firing-rate patterns in response to glutamate applied to the SCN in late night. Likewise, ex vivo SCN of wild-type mice post-orbital enucleation are unresponsive to glutamate unless PACAP also is restored. Furthermore, we demonstrate that the period of efficacy of PACAP at SCN nerve terminals corresponds to waxing of PACAP mRNA expression in ipRGCs during the night, and waning during the day. These results validate the use of PACAP-deficient mice in defining the role and specificity of PACAP as a co-transmitter with glutamate in ipRGC-RHT projections to SCN in phase advancing the SCN circadian rhythm in late night.

Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1.

To examine the role of the vanilloid receptor TRPV1 in neuropathic pain, we assessed the effects of the receptor antagonist thioxo-BCTC and antisense oligonucleotides against the TRPV1 mRNA in a rat model of spinal nerve ligation. In order to identify accessible sites on the mRNA of TRPV1, the RNase H assay was used, leading to the successful identification of binding sites for antisense oligonucleotides. Cotransfection studies using Cos-7 cells were employed to identify the most effective antisense oligonucleotide efficiently inhibiting the expression of a fusion protein consisting of TRPV1 and the green fluorescent protein in a specific and concentration-dependent manner. In an in vivo rat model of spinal nerve ligation, intravenous application of the TRPV1 antagonist thioxo-BCTC reduced mechanical hypersensitivity yielding an ED(50) value of 10.6mg/kg. Intrathecal administration of the antisense oligonucleotide against TRPV1, but not the mismatch oligonucleotide or a vehicle control, reduced mechanical hypersensitivity in rats with spinal nerve ligation in a similar manner. Immunohistochemical analysis revealed neuropathy- and antisense-associated regulation of TRPV1 protein expression in spinal cord and dorsal root ganglia. Our data demonstrate comparative analgesic effects of a TRPV1 anatagonist and a rationally designed TRPV1 antisense oligonucleotide in a spinal nerve ligation model of neuropathic pain and thus, lend support to the validation of TRPV1 as a promising target for the treatment of neuropathic pain.

Preclinical evaluation of PAC1 targeting with radiolabeled Maxadilan.

There is an ongoing search for new tracers to optimize imaging of beta cell-derived tumors (insulinomas). The PAC1 receptor, expressed by insulinomas, can be used for targeting of these tumors. Here, we investigated whether radiolabeled maxadilan could be used for insulinoma imaging. Maxadilan was C- or N-terminally conjugated with DTPA (termed maxadilan-DPTA or DTPA-maxadilan respectively). BALB/c nude mice bearing subcutaneous INS-1 tumors were injected with either In-111-labeled maxadilan-DTPA or In-111-DTPA-maxadilan. Biodistribution studies were carried out at 1, 2 and 4 hours after injection and SPECT/CT imaging 1 and 4 hours after injection of maxadilan-DTPA-111In. Radiolabeling of maxadilan-DTPA (680 MBq/nmol) was more efficient than of DTPA-maxadilan (55 MBq/nmol). Conjugation with DTPA slightly reduced receptor binding affinity in vitro: IC50 values were 3.2, 21.0 and 21.0 nM for maxadilan, natIn-DTPA-maxadilan and maxadilan-DTPA-natIn respectively. Upon i.v. injection maxadilan-DTPA-111In accumulated specifically in INS-1 tumors (7.30 ± 1.87%ID/g) and in the pancreas (3.82 ± 0.22%ID/g). INS-1 tumors were clearly visualized by small animal SPECT/CT. In conclusion, this study showed that the high affinity of maxadilan to the PAC1 receptor was maintained after DTPA conjugation. Furthermore, radiolabeled maxadilan-DTPA accumulated specifically in INS-1 tumors and, therefore, may qualify as a useful tracer to image insulinomas.

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.

Fractalkine expression in the rhesus monkey brain during lentivirus infection and its control by 6-chloro-2',3'-dideoxyguanosine.

Existing data concerning the role of the delta-chemokine fractalkine (CX3CL1) and its receptor (CX3CR1) in lentivirus-induced encephalitis are limited and controversial. We explored, by quantitative in situ hybridization and immunohistochemistry, the cell-specific changes of CX3CL1 and CX3CR1 in rhesus macaque brain during simian immunodeficiency virus (SIV) infection and antiretroviral treatment. Neuronal expression of CX3CL1 was significantly reduced in cortex and striatum of AIDS-diseased monkeys as compared with uninfected and asymptomatic SIV-infected monkeys. CX3CL1 mRNA was increased in some endothelial cells and newly induced in astrocytes and macrophages focally in areas of SIV burden and inflammatory infiltrates. In most CX3CL1-positive astrocytes and macrophages, the transcription factor NF-kappaB was translocated to the nucleus. CX3CR1 was upregulated in scattered, nodule, and giant cell-forming microglia/macrophages and mononuclear infiltrates close to CX3CL1-induced cells in the brain. Treatment of AIDS monkeys with the central nervous system-permeant 6-chloro-2',3'-dideoxyguanosine fully reversed SIV burden, productive inflammation, nuclear NF-kappaB translocation as well as focal induction of CX3CL1 in astrocytes and macrophages and downregulation in neurons. In contrast, diffuse CX3CR1-positive microgliosis and GFAP-positive astrogliosis were partially reversed by 6-chloro-2',3'-dideoxyguanosine. Thus, focally induced CX3CL1 may be a target for therapeutic intervention to limit ongoing inflammatory infiltration into brain in lentivirus infection.

Vesicular monoamine transporter 2 (VMAT2) expression in hematopoietic cells and in patients with systemic mastocytosis.

Uptake of monoamines into secretory granules is mediated by the vesicular monoamine transporters VMAT1 and VMAT2. In this study, we analyzed their expression in inflammatory and hematopoietic cells and in patients suffering from systemic mastocytosis (SM) and chronic myelogenous leukemia (CML). Normal human and monkey tissue specimens and tissues from patients suffering from SM and CML were analyzed by means of immunohistochemistry, radioactive in situ hybridization, real time RT-PCR, double fluorescence confocal laser scanning microscopy, and immunoelectron microscopy. In normal tissue specimens, VMAT2, but not VMAT1, was expressed in mast cells, megakaryocytes, thrombocytes, basophil granulocytes, and cutaneous Langerhans cells. Further hematopoietic and lymphoid cells showed no expression of VMATs. VMAT2 was expressed in all types of SM, as indicated by coexpression with the mast cell marker tryptase. In CML, VMAT2 expression was retained in neoplastic megakaryocytes and basophil granulocytes. In conclusion, the identification of VMAT2 in mast cells, megakaryocytes, thrombocytes, basophil granulocytes, and cutaneous Langerhans cells provides evidence that these cells possess molecular mechanisms for monoamine storage and handling. VMAT2 identifies normal and neoplastic mast cells, megakaryocytes, and basophil granulocytes and may therefore become a valuable tool for the diagnosis of mastocytosis and malignant systemic diseases involving megakaryocytes and basophil granulocytes.

Activity-dependent regulation of vesicular glutamate and GABA transporters: a means to scale quantal size.

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.

l-DOPA-induced dyskinesia is associated with a deficient numerical downregulation of striatal tyrosine hydroxylase mRNA-expressing neurons.

l-3,4-Dihydroxyphenylalanine (l-DOPA) is the therapeutic gold standard in Parkinson's disease. However, most patients develop debilitating abnormal involuntary movements termed l-DOPA-induced dyskinesia (LID) as therapy-complicating side effects. The underlying mechanisms of LID pathogenesis are still not fully understood. Recent evidence suggests an involvement of striatal tyrosine hydroxylase (TH) protein-expressing neurons, as they are capable of endogenously producing l-DOPA and possibly dopamine. The aim of this study was to elucidate changes of TH transcription in the striatum and nucleus accumbens that occur under experimental conditions of LID. Mice with a unilateral 6-hydroxydopamine-induced lesion of the medial forebrain bundle were treated daily with l-DOPA for 15days to provoke dyskinesia. In situ hybridization analysis revealed a significant numerical decrease of TH mRNA-positive neurons in the striatum and nucleus accumbens of mice not exhibiting LID, whereas dyskinetic animals failed to show this reduction of TH transcription. Interestingly, similar changes were observed in intact non-deafferentiated striata, demonstrating an l-DOPA-responsive transcriptional TH regulation independently from nigrostriatal lesion severity. Consolidation with our previous study on TH protein level (Keber et al., 2015) impressively highlights that LID is associated with both a deficient downregulation of TH transcription and an excessive translation of TH protein in intrastriatal neurons. As TH protein levels in comparison to mRNA levels showed a stronger correlation with development and severity of LID, antidyskinetic treatment strategies should focus on translational and posttranslational modulations of TH as a promising target.

Cell-specific expression and lipopolysaccharide-induced regulation of tumor necrosis factor alpha (TNFalpha) and TNF receptors in rat dorsal root ganglion.

The proinflammatory and lipopolysaccharide (LPS)-inducible cytokine tumor necrosis factor alpha (TNFalpha) has been shown to enhance primary sensory nociceptive signaling. However, the precise cellular sites of TNFalpha and TNF receptor synthesis are still a matter of controversy. Therefore, we differentiated the neuronal and non-neuronal sites of TNFalpha, TNFR1, and TNFR2 mRNA synthesis in dorsal root ganglion (DRG) of control rats and evaluated how their expression is altered under systemic challenge with LPS. In situ hybridization (ISH), RT-PCR analysis of laser-microdissected cells, and immunocytochemistry revealed absence of TNFalpha from DRG neurons and LPS-induced expression of TNFalpha exclusively in a subpopulation of non-neuronal DRG cells. Using RT-PCR and Northern blotting TNFR1 and TNFR2 mRNAs were found to be constitutively expressed and increased after LPS. TNFR1 mRNA was expressed in virtually all neurons and in non-neuronal cells with increased levels after LPS in both. TNFR2 was exclusively expressed and regulated in non-neuronal cells. RT-PCR analysis of microdissected DRG neurons and of the sensory neuronal cell line F11 confirmed the neuronal expression of TNFR1 and excluded that of TNFR2. Double ISH revealed varying levels of TNFR1 mRNA in virtually all DRG neurons including putative nociceptive neurons coding for calcitonin gene-related peptide, substance P, or vanilloid receptor 1. Taken together, we provide evidence that non-neuronally synthesized TNFalpha may directly act on primary afferent neurons via TNFR1 but not TNFR2. This is likely to be relevant under conditions of inflammatory pain and infections accompanied by widespread TNFalpha synthesis and release and may drive sickness behavior.

Identification of the differentiation-associated Na+/PI transporter as a novel vesicular glutamate transporter expressed in a distinct set of glutamatergic synapses.

Glutamate transport into synaptic vesicles is a prerequisite for its regulated neurosecretion. Here we functionally identify a second isoform of the vesicular glutamate transporter (VGLUT2) that was previously identified as a plasma membrane Na+-dependent inorganic phosphate transporter (differentiation-associated Na+/P(I) transporter). Studies using intracellular vesicles from transiently transfected PC12 cells indicate that uptake by VGLUT2 is highly selective for glutamate, is H+ dependent, and requires Cl- ion. Both the vesicular membrane potential (Deltapsi) and the proton gradient (DeltapH) are important driving forces for vesicular glutamate accumulation under physiological Cl- concentrations. Using an antibody specific for VGLUT2, we also find that this protein is enriched on synaptic vesicles and selective for a distinct class of glutamatergic nerve terminals. The pathway-specific, complementary expression of two different vesicular glutamate transporters suggests functional diversity in the regulation of vesicular release at excitatory synapses. Together, the two isoforms may account for the uptake of glutamate by synaptic vesicles from all central glutamatergic neurons.

Excitatory stimulation of neurons in the arcuate nucleus initiates central CRF-dependent stimulation of colonic propulsion in rats.

It is well established that autonomic control of digestive function is modulated by central autonomic neurotransmission. In this context it has been shown that digestive function can be modulated by exogenous neuropeptides microinjected into specific brain sides. Furthermore, there is considerable evidence suggesting that neurons projecting from the arcuate nucleus (ARC) to the PVN may be the source of endogenous neuropeptide release in the PVN. Neuronal projections from the ARC have been proposed to target corticotropin-releasing factor (CRF)-positive neurons in the PVN. Exogenous CRF in the PVN has been shown to modulate digestive function like gastric acid secretion and GI motility. Recently we have demonstrated that activation of ARC neurons inhibits gastric acid secretion via central CRF receptor dependent mechanisms. This poses the question whether neuronal activation of the ARC alters digestive function beside gastric acid secretion. In the present study we investigated whether CRF pathways in the ARC-PVN axis are involved in the modulation of colonic motility. First we examined the effect of an excitatory amino acid, kainate, microinjected into the ARC on colonic motility in anesthetized rats. Colonic motility was measured with a non-absorbable radioactive marker using the geometric center method. Kainate (120 pmol/rat) bilaterally microinjected into the ARC induced a significant stimulation of colonic propulsion. To assess the contribution of hypothalamic CRF to the effects of neuronal stimulation in the ARC on colonic motility we performed consecutive bilateral microinjections of an antagonist to CRF receptors into the PVN and the excitatory amino acid kainate into the ARC. Microinjection of the non-selective CRF receptor antagonist, astressin (100 ng), into the PVN abolished the stimulatory effect of neuronal activation in the ARC by kainate on colonic motor function. The data indicate that activation of neurons in the ARC stimulates colonic motility via CRF-receptor-mediated mechanism in the PVN and underlines the important role of the ARC-PVN circuit for the integrative CNS regulation of GI function.