Vesicular glutamate transporter DNPI/VGLUT2 mRNA is present in C1 and several other groups of brainstem catecholaminergic neurons

The mouse glutamate vesicular transporter VGLUT2 has recently been characterized. The rat homolog of VGLUT2, differentiation-associated Na(+)/P(i) cotransporter (DNPI), was examined using a digoxigenin-labeled DNPI/VGLUT2 cRNA probe in the present study to determine which, if any, of the various groups of pontine or medullary monoaminergic neurons express DNPI/VGLUT2 mRNA and, thus, are potentially glutamatergic. DNPI/VGLUT2 mRNA was widely distributed within the brainstem and seemed exclusively neuronal. By using a double in situ hybridization method, the presence of the mRNA for DNPI/VGLUT2 and glutamic acid decarboxylase (GAD)-67 was mutually exclusive. By combining DNPI/VGLUT2 mRNA detection and conventional immunohistochemistry, DNPI/VGLUT2 mRNA was undetectable in lower brainstem cholinergic and serotonergic cells, but it was present in several tyrosine hydroxylase-immunoreactive (TH-ir) cell groups. DNPI/VGLUT2 mRNA was detected in most of the adrenergic neurons of the C1, C2, and C3 groups (75-80% of TH-ir neurons), in the A2 noradrenergic group (80%), and in vast numbers of area postrema cells. Within the A1 region, many fewer TH-ir cells contained DNPI/VGLUT2 (16%). Finally, DNPI/VGLUT2 mRNA was undetectable in the pontine noradrenergic cell groups (A5 and A6/locus coeruleus). In conclusion, the general pattern of DNPI/VGLUT2 expression and its exclusion from GABAergic, cholinergic, and serotonergic neurons supports the notion that DNPI/VGLUT2 mRNA identifies a subset of glutamatergic neurons in the lower brainstem. Within this region several catecholaminergic cell groups appear to be glutamatergic, including but not limited to the adrenergic cell groups C1-C3. Based on the present evidence, the noradrenergic cell groups of the pons (A5 and A6) do not contain either known vesicular glutamate transporter and are most likely not glutamatergic.     

Vesicular glutamate transporter DNPI/VGLUT2 is expressed by both C1 adrenergic and nonaminergic presympathetic vasomotor neurons of the rat medulla

The main source of excitatory drive to the sympathetic preganglionic neurons that control blood pressure is from neurons located in the rostral ventrolateral medulla (RVLM). This monosynaptic input includes adrenergic (C1), peptidergic, and noncatecholaminergic neurons. Some of the cells in this pathway are suspected to be glutamatergic, but conclusive evidence is lacking. In the present study we sought to determine whether these presympathetic neurons express the vesicular glutamate transporter BNPI/VGLUT1 or the closely related gene DNPI, the rat homolog of the mouse vesicular glutamate transporter VGLUT2. Both BNPI/VGLUT1 and DNPI/VGLUT2 mRNAs were detected in the medulla oblongata by in situ hybridization, but only DNPI/VGLUT2 mRNA was present in the RVLM. Moreover, BNPI immunoreactivity was absent from the thoracic spinal cord lateral horn. DNPI/VGLUT2 mRNA was present in many medullary cells retrogradely labeled with Fluoro-Gold from the spinal cord (T2; four rats). Within the RVLM, 79% of the bulbospinal C1 cells contained DNPI/VGLUT2 mRNA. Bulbospinal noradrenergic A5 neurons did not contain DNPI/VGLUT2 mRNA. The RVLM of six unanesthetized rats subjected to 2 hours of hydralazine-induced hypotension contained tenfold more c-Fos-ir DNPI/VGLUT2 neurons than that of six saline-treated controls. c-Fos-ir DNPI/VGLUT2 neurons included C1 and non-C1 neurons (3:2 ratio). In seven barbiturate-anesthetized rats, 16 vasomotor presympathetic neurons were filled with biotinamide and analyzed for the presence of tyrosine hydroxylase immunoreactivity and/or DNPI/VGLUT2 mRNA. Biotinamide-labeled neurons included C1 and non-C1 cells. Most non-C1 (9/10) and C1 presympathetic cells (5/6) contained DNPI/VGLUT2 mRNA. In conclusion, DNPI/VGLUT2 is expressed by most blood pressure-regulating presympathetic cells of the RVLM. The data suggest that these neurons may be glutamatergic and that the C1 adrenergic phenotype is one of several secondary phenotypes that are differentially expressed by subgroups of these cells.     

Alpha2A-adrenergic receptors are primarily presynaptic heteroreceptors in the C1 area of the rat rostral ventrolateral medulla

The 2A subtype of the alpha-adrenergic receptor (α_2A-AR) is necessary for the hypotensive effects of clonidine and other sympathoinhibitory adrenergic agonists. This hypotensive response appears to be due to the inhibition of sympathoexcitatory reticulospinal neurons found in the rostral ventrolateral medulla (RVL), including neurons of the C1 adrenergic cell group. The cellular mechanisms underlying this inhibition have not been established. Thus, this study examined the ultrastructural relationships between profiles containing α_2AAR-immunoreactivity (α_2AAR-I) and those containing the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) to determine potential cellular substrates for α_2A-AR inhibition of C1 neuron activity. Consistent with previous light microscopic studies, α_2AAR-I was found in perikarya and large dendrites and the majority of these profiles also contained TH-labeling (70% of 140). However, α_2AAR-I in these cells was primarily found within endosomes and Golgi complexes and in clusters associated with the endoplasmic reticula, probable sites for synthesis and/or trafficking of receptors. In contrast, most of the α_2AAR-I profiles (n=646) in the RVL were axons and axon terminals (68%) which lacked TH immunoreactivity. α_2AAR-labeled axons were small and unmyelinated and labeled terminals usually formed symmetric synapses on the shafts of catecholaminergic or unlabeled dendrites. Most of these α_2AAR-labeled axons were found in close proximity to TH-labeled profiles and approximately one-fifth (17% of 408) of the α_2AAR-labeled axons and axon terminals directly contacted TH-labeled profiles, mostly dendrites. These studies suggest that α_2AARs in the C1 area of the RVL function primarily as heteroreceptors on presynaptic axons and terminals of non-catecholaminergic cells, some of which provide inhibitory synaptic input to C1 neurons. These receptors may be activated by catecholamines released either from the dendrites of C1 neurons or from the terminals of other catecholaminergic neurons via volume transmission.     

An intracellular study of respiratory neurons in the rostral ventrolateral medulla of the rat and their relationship to catecholamine-containing neurons

Intracellular recording and labelling with Lucifer yellow of respiratory neurons in the rostral ventrolateral medulla were carried out in urethane-anaesthetised rats. A combined immunofluorescence and immunoperoxidase technique enabled an assessment of the tyrosine hydroxylase immunoreactivity, as well as an examination of the morphology of inspiratory and expiratory neurons in this part of the medulla oblongata. The results demonstrate: a) that respiratory neurons in the rostral ventrolateral medulla of the rat are intermingled with catecholamine-containing neurons of the C1 cell group, but are not themselves catecholamine-containing; b) that many non-spinally projecting respiratory neurons have axonal arborisations within the ventrolateral medulla in the same region as the C1 cell group, other respiratory neurons, and neurons reported to have a cardiovascular function; and c) that the dendrites of respiratory neurons in this region radiate throughout the ventrolateral medulla and frequently approach the ventral surface.     

Region‐specific deletions of the glutamate transporter GLT1 differentially affect seizure activity and neurodegeneration in mice

Glial glutamate transporter GLT1 plays a key role in the maintenance of extracellular glutamate homeostasis. Recent human genetic studies have suggested that de novo mutations in GLT1 (EAAT2) cause early‐onset epilepsy with multiple seizure types. Consistent with these findings, global GLT1 null mice show lethal spontaneous seizures. The consequences of GLT1 dysfunction vary between different brain regions, suggesting that the role of GLT1 dysfunction in epilepsy may also vary with brain regions. In this study, we generated region‐specific GLT1 knockout mice by crossing floxed‐GLT1 mice with mice that express the Cre recombinase in a particular domain of the ventricular zone. Selective deletion of GLT1 in the diencephalon, brainstem and spinal cord is sufficient to reproduce the phenotypes (excess mortality, decreased body weight, and lethal spontaneous seizure) of the global GLT1 null mice. By contrast, dorsal forebrain‐specific GLT1 knockout mice showed nonlethal complex seizures including myoclonic jerks, hyperkinetic running, spasm and clonic convulsion via the activation of NMDA receptors during a limited period from P12 to P14 and selective neuronal death in cortical layer II/III and the hippocampus. Thus, GLT1 dysfunction in the dorsal forebrain is involved in the pathogenesis of infantile epilepsy and GLT1 in the diencephalon, brainstem and spinal cord may play a critical role in preventing seizure‐induced sudden death.     

Immunohistochemical identification of noradrenaline- and adrenaline- synthesizing neurons in the cat ventrolateral medulla

The distribution and morphology of cell bodies containing the catecholamine biosynthetic enzymes dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the ventrolateral medulla (VLM) of the cat were studied immunohistochemically after intracisternal administration of colchicine. Perikarya immunoreactive to DBH were found throughout the VLM extending from approximately the spinomedullary junction to the level of the superior olivary nucleus. In the caudal VLM DBH neurons were found primarily in the region immediately dorsal to the lateral reticular nucleus (LRN), although a few scattered DBH neurons were also found near the ventral surface of the medulla in and around the parvicellular division of the LRN. These DBH neurons in the caudal VLM were generally fusiform, fusiform-bipolar, or multipolar, with a mean somal area of 422 +/- 32 microns2, and with two to four branching processes. In the rostral VLM DBH neurons formed three distinct groups: one group was found in the nucleus paragigantocellularis lateralis in the region just ventromedial to the retrofacial nucleus (RFN) near the ventrolateral surface of the medulla; the second group was found in the region dorsomedial to the rostral aspects of the nucleus ambiguous and the RFN, and the third group was found in the region along the lateral aspect of the superior olivary nucleus. Perikarya immunoreactive to the adrenaline-synthesizing enzyme PNMT were localized to a more restricted region of the VLM that extended from approximately the rostral aspect of the caudal third of the inferior olivary complex (level of the obex) to the caudal pole of the facial nucleus. These PNMT neurons were fusiform or multipolar with a mean somal area of 273 +/- 21 microns2, and with two to five branching processes. The location, size, shape, and numbers of PNMT-immunoreactive neurons corresponded closely to the rostral groups of DBH neurons, with the exception of the group found along the lateral aspect of the superior olivary nucleus. These data indicate that noradrenaline-synthesizing neurons are primarily found in the caudal VLM and in the region near the superior olivary nucleus, whereas catecholamine neurons in the rostral VLM between these two noradrenergic cell groups synthesize adrenaline. As the VLM has previously been shown to have direct connections with spinal cord, brainstem, and hypothalamic areas implicated in cardiovascular and neuroendocrine regulation, this suggests that DBH- and PNMT-synthesizing neurons are components of neuronal circuits involved in these homeostatic mechanisms.     

Spontaneous activity and barosensitivity of the barosensitive neurons in the rostral ventrolateral medulla of hypertensive rats induced by transection of aortic depressor nerves

In order to determine the role of the rostral ventrolateral medulla (RVLM) in the development of neurogenic hypertension, the aortic depressor nerves of rats were transected (tADN) to produce neurogenic hypertension. The rate and pattern of firing of the barosensitive RVLM neurons of the treated rats were studied. In neurogenic hypertensive rats, the RVLM barosensitive neurons exhibited a faster firing rate and a shorter interspike interval (ISI) than the corresponding values of the control and sham groups, indicating an enhanced spontaneous activity of these neurons in the hypertensive rats. The coefficient of variation (cv) and skewness (sk) of the ISI histogram, parameters reflecting the regularity of neuronal firing, were smaller in neurogenic hypertensive than in the control and sham-operated rats. Following tADN, the responsiveness of these neurons to blood pressure changes was attenuated, suggesting a reduced intrinsic barosensitivity of neurons and/or a reduced baroreceptor input. The increase in spontaneous activity and firing regularity of RVLM barosensitive neurons imply an enhancement in the efficacy of outflow from these neurons. The increased efficacy of the outflow from the RVLM barosensitive neurons and the resetting of the baroreflex may contribute to the genesis of neurogenic hypertension.     

Ultrastructural localization and afferent sources of corticotropin-releasing factor in the rat rostral ventrolateral medulla: Implications for central cardiovascular regulation

We investigated the ultrastructural localization, afferent sources, and arterial pressure effects of corticotropin-releasing factor (CRF) in the nucleus reticularis rostroventrolateralis (RVL), a region of the ventrolateral medulla containing C1 adrenergic neurons and sympatho-excitatory reticulospinal afferents to sympathetic preganglionic neurons. A polyclonal antibody, to CRF was localized in acrolein-fixed sections through the rat RVL by the peroxidase–antiperoxidase (PAP) method. Light microscopy showed that 1–7 perikarya/30 μm section and numerous varicose processes contained CRF-like immunoreactivity (CRF-LI). By electron microscopy, CRF-LI was most intensely localized to large (80–100 nm) dense-core vesicles within numerous terminals and a few perikarya and large dendrites. Approximately half of the terminals containing CRF-LI were in direct contact with unlabeled perikarya or dendrites; the remainder were in apposition to either unlabeled terminals or astrocytes. Most synaptic specializations were asymmetric synapses on small, unlabeled dendrites. To examine potential extrinsic sources of CRF-containing terminals in the C1 area of the RVL, PAP immunocytochemical localization of CRF was combined with retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). In all cases examined, a number of dually labeled neurons were found in the paraventricular nucleus (PVN) of the hypothalamus and a few dually labeled neurons were observed in the nuclei of the solitary tract; these labeled neurons were ipsilateral to the unilateral injection of WGA-HRP into the C1 area. Fewer dually labeled perikarya were detected in the lateral hypothalamic area and the lateral parabrachial nuclei, ipsilateral to the WGA-HRP injection. Additional physiological studies showed that bilateral microinjections of CRF into the C1 area of the RVL of urethane-anesthetized rats elicited a dose-related increase in arterial pressure. The results suggest that within the C1 area of the RVL, CRF released from terminals, arising predominantly from the PVN of the hypothalamus and probably from local neurons as well, may excite sympathoexcitatory reticulospinal neurons. © 1993 Wiley-Liss, Inc.     

Preproenkephalin mRNA is expressed by C1 and non-C1 barosensitive bulbospinal neurons in the rostral ventrolateral medulla of the rat

The autonomic regions of the thoracolumbar spinal cord receive a dense enkephalinergic (ENK) innervation from supraspinal sources, including the rostral ventrolateral medulla (RVLM). In the present study, we sought to determine whether the barosensitive bulbospinal (BSBS) neurons of the RVLM express preproenkephalin (PPE) mRNA. After injection of Fluoro-Gold (FG) into the upper thoracic spinal cord, neurons with PPE mRNA (PPE(+) neurons) were retrogradely labeled throughout the ventrolateral medulla. At the most rostral RVLM level, 29% of bulbospinal PPE+ cells were tyrosine hydroxylase-immunoreactive (TH-ir) and the latter constituted 19.4% of the bulbospinal TH-ir cells. We determined whether the bulbospinal PPE(+) RVLM neurons are barosensitive in two ways. First, we examined Fos production by FG-labeled RVLM neurons after 2 hours of hydralazine-induced hypotension (to 73 +/- 2 mm Hg) in conscious rats. Hydralazine (10 mg/kg i.v.) increased the number of Fos-ir neurons by two- to eightfold at all levels of the ventrolateral medulla examined. In the RVLM, 54% of bulbospinal PPE(+) neurons were Fos-ir, whereas such cells were more rarely found at caudal ventrolateral medullary levels. Second, we recorded individual BSBS RVLM units extracellularly in anesthetized rats and filled them juxtacellularly with biotinamide. Most biotinamide-filled neurons were PPE(+) (10 of 17), and the PPE(+) BSBS cells had a faster axonal conduction velocity than those without PPE mRNA (4.2 vs. 0.67 m/sec). Four of the 10 PPE(+) BSBS RVLM neurons were TH-ir. In summary, PPE mRNA is predominantly expressed by RVLM BSBS neurons with lightly myelinated spinal axons. PPE mRNA is present in most noncatecholaminergic BSBS neurons and also in approximately 20% of the bulbospinal C1 neurons. BSBS RVLM neurons most likely provide a major ENK input to sympathetic preganglionic neurons and PPE mRNA is the first identified positive phenotype of the non-C1 BSBS RVLM neurons.     

A role for KATP-channels in mediating the elevations of cerebral blood flow and arterial pressure by hypoxic stimulation of oxygen-sensitive neurons of rostral ventrolateral medulla

Reticulospinal sympathoexcitatory neurons of rostral ventrolateral medulla (RVL) are selectively excited by hypoxia to elevate arterial pressure (AP) and cerebral blood flow (rCBF), that are elements of the oxygen-conserving (diving) reflex. We investigated whether K_ATP⁺-channels participate in this. Tolbutamide and glibenclamide, K_ATP⁺-channel blockers, microinjected into RVL in anesthetized rats, dose-dependently and site-specifically elevated AP and rCBF and potentiated responses to hypoxemia. K_ATP⁺-channels may mediate hypoxic excitation of oxygen-sensing RVL neurons.     

A role for KATP+-channels in mediating the elevations of cerebral blood flow and arterial pressure by hypoxic stimulation of oxygen-sensitive neurons of rostral ventrolateral medulla.

Reticulospinal sympathoexcitatory neurons of rostral ventrolateral medulla (RVL) are selectively excited by hypoxia to elevate arterial pressure (AP) and cerebral blood flow (rCBF), that are elements of the oxygen-conserving (diving) reflex. We investigated whether KATP+-channels participate in this. Tolbutamide and glibenclamide, KATP+-channel blockers, microinjected into RVL in anesthetized rats, dose-dependently and site-specifically elevated AP and rCBF and potentiated responses to hypoxemia. KATP+-channels may mediate hypoxic excitation of oxygen-sensing RVL neurons.     

Expression and transport function of the glutamate transporter EAAC1 in Xenopus oocytes is regulated by syntaxin 1A

The function of several membrane proteins is regulated by interaction with the SNARE protein syntaxin 1A; this includes regulation of GAT1, the transporter for the dominating inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Here we demonstrate that also EAAC1, the transporter for the dominating excitatory neurotransmitter, is down-regulated by interaction with syntaxin 1A. This is shown by coexpression of EAAC1 and syntaxin 1A in Xenopus oocytes. Total EAAC1 expression is not significantly affected by the coexpression of syntaxin 1A, but more proteins become targeted to the membrane as demonstrated by biotinylation. Colocalization by coimmunoprecipitation suggests direct interaction between the two proteins. In contrast to the number of transporters, the glutamate transport activity becomes reduced, and even stronger inhibition is observed for the EAAC1-mediated conductance uncoupled from glutamate translocation. We conclude that the interaction of syntaxin 1A with EAAC1 particularly disrupts the structure of the conductance pathway of EAAC1.     

Differential regulation of 5' splice variants of the glutamate transporter EAAT2 in an in vivo model of chemical hypoxia induced by 3-nitropropionic acid

Defective glutamate uptake has been implicated as a pathogenic event of neuronal damage related to cerebral ischemia and hypoxia. In several models of ischemia-hypoxia, a reduced immunoreactivity and altered RNA expression of excitatory amino acid transporter 2 (EAAT2), the major excitatory amino acid transporter, have been reported. However, the gene regulation of EAAT2 under these conditions is incompletely understood. In this study, we investigated alternative splicing of EAAT2 in an in vivo mouse model of chemical hypoxia as induced by 3-nitropropionic acid (3-NP). The neurotoxin 3-NP is an inhibitor of mitochondrial energy production. Furthermore, it is known to inhibit glutamate reuptake directly, representing at least one of the mechanisms responsible for 3-NP-induced neurodegeneration. Here we report an expression analysis of five known (mEAAT2/5UT1-5) and two novel (mEAAT2/5UT6, -7) 5' splice variants of EAAT2 using semiquantitative PCR. The RNA expression was studied at 2, 12, 24, 48, and 72 hr and 7 days after 3-NP administration. mEAAT2/5UT4 and mEAAT2/5UT5 were up-regulated in the frontal cortex and down-regulated in the hippocampus 12-72 hr after chemical hypoxia. In the cerebellum, there was an increased expression of mEAAT2/5UT4 and a down-regulation of mEAAT2/5UT5. mEAAT2/5UT3 show a different regional expression pattern, being regulated in the cerebellum only. mEAAT2/5UT1-7 encoded distinct 5' regulatory sequences, including conserved elements of translational control. It is easily conceivable that expression alterations of 5' splice variants of EAAT2 are related to glutamate transporter malfunction after chemical hypoxia. Our findings contribute to the hypothesis that RNA splicing events can serve as a molecular mechanism of posthypoxic gene regulation.     

Deletion of the CCK2 receptor gene reduces mechanical sensitivity and abolishes the development of hyperalgesia in mononeuropathic mice

Previous studies suggest that cholecystokinin (CCK) is implicated in the modulation of pain sensitivity and the development of neuropathic pain. We used CCK(2) receptor deficient (CCK(2) (-/-)) mice and assessed their mechanical sensitivity using Von Frey filaments, as well as the development and time course of mechanical hyperalgesia in a model of neuropathic pain. We found that CCK(2) (-/-) mice displayed mechanical hyposensitivity, which was reversed to the level of wild-type animals after administration of naloxone (0.1-10 mg/kg). On the other hand, injection of L-365260 (0.01-1 mg/kg), an antagonist of CCK(2) receptors, decreased dose-dependently, mechanical sensitivity in wild-type mice. The mechanism of reduced mechanical sensitivity in CCK(2) (-/-) mice may be explained by changes in interactions between CCK and opioid systems. Indeed, CCK(2) (-/-) mice natively expressed higher levels of lumbar CCK(1), opioid delta and kappa receptors. Next, we found that CCK(2) (-/-) mice did not develop mechanical hyperalgesia in the Bennett's neuropathic pain model. Induction of neuropathy resulted in decrease of lumbar pro-opiomelanocortin (POMC) gene expression in wild-type mice, but increase of POMC expression in CCK(2) (-/-) mice. In addition, induction of neuropathy resulted in further increase of opioid delta receptor in CCK(2) (-/-) mice. Gene expression results indicate up-regulation of opioid system in CCK(2) (-/-) mice, which apparently result in decreased neuropathy score. Our study suggests that not only pain sensitivity, but also mechanical sensitivity and the development of neuropathic pain are regulated by antagonistic interactions between CCK and opioid systems.     

Rescue of abnormal phenotypes in δ2 glutamate receptor-deficient mice by the extracellular N-terminal and intracellular C-terminal domains of the δ2 glutamate receptor

The δ2 glutamate receptor (GluRδ2) is expressed predominantly in cerebellar Purkinje cells. GluRδ2 knock-out mice show impaired synaptogenesis and loss of long-term depression (LTD) at parallel fiber/Purkinje cell synapses, and persistent multiple climbing fiber (CF) innervation of Purkinje cells, resulting in severe ataxia. To identify domains critical for GluRδ2 function, we produced various GluRδ2 deletion constructs. Using lentiviral vectors, those constructs were expressed in Purkinje cells of GluRδ2-deficient mice at postnatal day (P) 6 or 7, and rescue of abnormal phenotypes was examined beyond P30. Most constructs failed to rescue the defects of GluRδ2-deficient mice, mainly because they were not efficiently transferred to the postsynaptic sites. However, a construct carrying only the extracellular N-terminal domain (NTD) and the intracellular C-terminal domain (CTD) linked with the fourth transmembrane domain of GluRδ2 (NTD-TM4-CTD) caused incomplete, but significant rescue of ataxia, consistent with relatively better transport of the construct to the synapses. Notably, the expression of NTD-TM4-CTD in GluRδ2-deficient Purkinje cells restored abrogated LTD, and aberrant CF territory in the molecular layer. Although the expression of NTD-TM4-CTD failed to rescue persistent multiple CF innervation of GluRδ2-deficient Purkinje cells, a similar construct in which only TM4 was replaced with a transmembrane domain of CD4 successfully rescued the multiple CF innervation, probably due to more efficient transport of the protein to postsynaptic sites. These results suggest that NTD and CTD are critical domains of GluRδ2, which functions substantially without conventional ligand binding and ion channel structures.     

Differential regulation of the dopamine D1, D2 and D3 receptor gene expression and changes in the phenotype of the striatal neurons in mice lacking the dopamine transporter

Mice with a genetic disruption of the dopamine transporter (DAT-/-) exhibit locomotor hyperactivity and profound alterations in the homeostasis of the nigrostriatal system, e.g. a dramatic increase in the extracellular dopamine level. Here, we investigated the adaptive changes in dopamine D1, D2 and D3 receptor gene expression in the caudate putamen and nucleus accumbens of DAT-/- mice. We used quantitative in situ hybridization and found that the constitutive hyperdopaminergia results in opposite regulations in the gene expression for the dopamine receptors. In DAT-/- mice, we observed increased mRNA levels encoding the D3 receptor (caudate putamen, +60-85%; nucleus accumbens, +40-107%), and decreased mRNA levels for both D1 (caudate putamen, -34%; nucleus accumbens, -45%) and D2 receptors (caudate putamen, -36%; nucleus accumbens, -33%). Furthermore, we assessed the phenotypical organization of the striatal efferent neurons by using double in situ hybridization. Our results show that in DAT+/+ mice, D1 and D2 receptor mRNAs are segregated in two different main populations corresponding to substance P and preproenkephalin A mRNA-containing neurons, respectively. The phenotype of D1 or D2 mRNA-containing neurons was unchanged in both the caudate putamen and nucleus accumbens of DAT-/- mice. Interestingly, we found an increased density of preproenkephalin A-negative neurons that express the D3 receptor mRNA in the nucleus accumbens (core, +35%; shell, +46%) of DAT-/- mice. Our data further support the critical role for the D3 receptor in the regulation of D1-D2 interactions, an action being restricted to neurons coexpressing D1 and D3 receptors in the nucleus accumbens.     

NMDA glutamate receptor stimulation is required for the expression of D2 dopamine mediated responses in apomorphine primed 6-hydroxydopamine lesioned rats

Three priming injections with the D1/D2 dopamine agonist apomorphine permits a challenge with the D2 agonist quinpirole to elicit robust contralateral rotation and ipsilateral striatal Fos expression in 6-hydroxydopamine lesioned rats. Pretreatment with NMDA glutamate antagonists MK-801 or CPP dose-dependently attenuates these quinpirole-mediated responses. These findings suggest that concomitant NMDA receptor stimulation is required for the expression of D2-mediated responses in apomorphine primed dopamine-depleted rats.     

ATP binding cassette transporter ABC1 is required for the release of interleukin-1beta by P2X7-stimulated and lipopolysaccharide-primed mouse Schwann cells

Schwann cells are best known as myelinating glial cells of the peripheral nervous system, but they also participate actively in the sphere of immunity by producing pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta). In a previous study, we demonstrated that posttranslational processing of IL-1beta by immune-challenged Schwann cells required the P2X7 receptor. Remarkably, the release of IL-1beta was not associated with cell death, indicating the involvement of an active mechanism. ATP binding cassette (ABC) transporters are known to transport leaderless secretory proteins, such as IL-1beta; therefore, we investigated whether such transporters were at work in Schwann cells. Mouse Schwann cells expressed ABC1 transporter mRNA and displayed the functional protein. Glybenclamide and diisothiocyanato-stilbene-disulfonic acid (DIDS), two blockers of chloride fluxes that drive the export activity of ABC1 transporters, inhibited IL-1beta release without altering its intracellular processing. Enhancing chloride efflux potentiated the release of IL-1beta, while decreasing it led to a strong reduction in its release. Because the stimulation of the P2X7 receptor also activates a chloride conductance, we investigated the possibility of a sole anionic pathway mobilized by the P2X7 receptor and ABC1. Glybenclamide and DIDS had no significant effects on the P2X7-activated chloride current suggesting therefore the existence of two different pathways. In summary, ABC1 transporters are required for the release of IL-1beta by mouse Schwann cells. Being associated together with chloride conductance, P2X7 receptors and ABC1 transporters delineate a subtle and complex regulation of IL-1beta production in mammalian Schwann cells. Furthermore, ABC1 transporters could be a target of therapeutic interest for regulating IL-1beta activity in neuroinflammation disorders.