Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor. 2005 Elsevier Ltd. All rights reserved.
Glutamate; Metabotropic; LTP; LTD; PKC; Plasticity
Recent reports have shown that the selective dopamine D1-like agonist SKF 83822 [which stimulates adenylate cyclase, but not phospholipase C] induces prominent behavioral seizures in mice, whereas its benzazepine congener SKF 83959 [which stimulates phospholipase C, but not adenylate cyclase] does not. To investigate the relative involvement of D1 vs D5 receptors in mediating seizures, ethological behavioral topography and cortical EEGs were recorded in D1, D5 and DARPP-32 knockout mice in response to a convulsant dose of SKF 83822. SKF 83822-induced behavioral and EEG seizures were gene dose-dependently abolished in D1 knockouts. In both heterozygous and homozygous D5 knockouts, the latency to first seizure was significantly increased and total EEG seizures were reduced relative to wild-types. The majority (60%) of homozygous DARPP-32 knockouts did not have seizures; of those having seizures (40%), the latency to first seizure was significantly increased and the number of high amplitude, high frequency polyspike EEG events was reduced. In addition, immunoblotting was performed to investigate downstream intracellular signalling mechanisms at D1-like receptors following challenge with SKF 83822 and SKF 83959. In wild-types administered SKF 83822, levels of ERK1/2 and GluR1 AMPA receptor phosphorylation increased two-fold in both the striatum and hippocampus; in striatal slices DARPP-32 phosphorylation at Thr34 increased five-fold relative to vehicle-treated controls. These findings indicate that D1, and to a lesser extent D5, receptor coupling to DARPP-32, ERK1/2 and glutamatergic signalling is involved in mediating the convulsant effects of SKF 83822.
Dopamine D1-like receptors; Seizures; Adenylate cyclase; DARPP-32; ERK1/2; Knockout
Mu-opioid and CB1-cannabinoid agonists produce analgesia; however, adverse effects limit use of drugs in both classes. Additive or synergistic effects resulting from concurrent administration of low doses of mu- and CB1-agonists may produce analgesia with fewer side effects. Synergism potentially results from interaction between mu-opioid receptors (MORs) and CB1 receptors (CB1Rs). AM-251 and rimonabant are CB1R antagonist/inverse agonists employed to validate opioid-cannabinoid interactions, presumed to act selectively at CB1Rs. Therefore, the potential for direct action of these antagonists at MORs is rarely considered. This study determined if AM-251 and/or rimonabant directly bind and modulate the function of MORs. Surprisingly, AM-251 and rimonabant, but not a third CB1R inverse agonist AM-281, bind with mid-nanomolar affinity to human MORs with a rank order of affinity (Ki) of AM-251 (251 nM) > rimonabant (652 nM) > AM281 (2135 nM). AM-251 and rimonabant, but not AM-281, also competitively antagonize morphine induced G-protein activation in CHO-hMOR cell homogenates (Kb = 719 or 1310 nM, respectively). AM-251 and rimonabant block morphine inhibition of cAMP production, while only AM-251 elicits cAMP rebound in CHO-hMOR cells chronically exposed to morphine. AM-251 and rimonabant (10 mg/kg) attenuate morphine analgesia, whereas the same dose of AM-281 produces little effect. Therefore, in addition to high CB1R affinity, AM-251 and rimonabant bind to MORs with mid-nanomolar affinity and at higher doses may affect morphine analgesia via direct antagonism at MORs. Such CB1-independent actions of these antagonists may contribute to reported inconsistencies when CB1/MOR interactions are examined via pharmacological methods in CB1-knockout versus wild-type mice.
Morphine; Marijuana; CB1 Receptors; G-protein; Adenylyl Cyclase; Analgesia
Currently there is no treatment for juvenile Batten disease, a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. The Cln3-knockout (Cln3Δex1-6) mouse model recapitulates several features of the human disorder. Cln3Δex1-6 mice, similarly to juvenile Batten disease patients, have a motor coordination deficit detectable as early as postnatal day 14. Previous studies demonstrated that acute attenuation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor activity by the non-competitive AMPA antagonist, EGIS-8332, in both 1- and 6–7-month-old Cln3Δex1-6 mice results in improvement in motor coordination. Here we show that acute inhibition of N-methyl-D-aspartate (NMDA)-type glutamate receptors by memantine (1 and 5 mg/kg i.p.) had no effect on the impaired motor coordination of one-month-old Cln3Δex1-6 mice. At a later stage of the disease, in 6–7-month-old Cln3Δex1-6 mice, memantine induced a delayed but extended (8 days) improvement of motor skills similarly to that observed previously with EGIS-8332 treatment. An age-dependent therapeutic effect of memantine implies that the pathomechanism in juvenile Batten disease changes during disease progression. In contrast to acute treatment, repeated administration of memantine or EGIS-8332 (1 mg/kg, once a week for 4 weeks) to 6-month-old Cln3Δex1-6 mice had no beneficial effect on motor coordination. Moreover, repeated treatments did not impact microglial activation or the survival of vulnerable neuron populations. Memantine did not affect astrocytosis in the cortex. EGIS-8332, however, decreased astrocytic activation in the somatosensory barrelfield cortex.
Acute inhibition of NMDA receptors can induce a prolonged therapeutic effect, identifying NMDA receptors as a new therapeutic target for juvenile Batten disease.
juvenile Batten disease; neuronal ceroid lipofuscinoses; Cln3; NMDA receptor; AMPA receptor; rotarod
Palonosetron is a potent 5-HT3 receptor antagonist with a unique structure and some unusual properties. Here we explore the properties of palonosetron at heterologously expressed 5-HT3A and 5-HT3AB receptors. We used receptors expressed in HEK293 cells, and functionally analysed them using a membrane potential sensitive dye in a Flexstation, which revealed IC50s of 0.24 nM and 0.18 nM for 5-HT3A and 5-HT3AB receptors respectively. Radioligand binding studies with [3H]palonosetron revealed similar Kds: 0.34 nM for 5-HT3A and 0.15 nM for 5-HT3AB receptors. Kinetic studies showed palonosetron association and dissociation rates were slightly faster in 5-HT3AB than 5-HT3A receptors, and for both subtypes dissociation rates were ligand-dependent, with antagonists causing more rapid dissociation than agonists. Similar ligand effects were not observed for [3H]granisetron dissociation studies. These data support previous studies which show palonosetron has actions distinct to other 5-HT3 receptor antagonists, and the slow rates observed for agonist induced dissociation (t1/2 > 10 h) could at least partly explain the long duration of palonosetron effects in vivo.
•Palonosetron IC50s and Kds for 5-HT3A and 5-HT3AB receptors in HEK293 cells are similar.•Palonosetron association and dissociation rates are slower at 5-HT3A than 5-HT3AB receptors.•Agonist-induced palonosetron dissociation rates are slower than those for antagonists in both 5-HT3A and 5-HT3AB receptors.•Agonist- and antagonist-induced granisetron dissociation rates are similar in both 5-HT3A and 5-HT3AB receptors.•Palonosetron and granisetron have distinct actions.
Serotonin receptor; Allosteric binding site; Site-directed mutagenesis; Radioligand binding; FlexStation assays; 5-HT, 5-hydroxytryptamine (serotonin); HEK, human embryonic kidney; IC50, concentration of antagonist required for half-maximal inhibition; Kd, affinity constant
Essential tremor is a common disorder that lacks molecular targets for therapeutic development. T-type calcium channel activation has been postulated to underlie rhythmicity in the olivo-cerebellar system that is implicated in essential tremor. We therefore tested whether compounds that antagonize T-type calcium channel currents suppress tremor in two mouse models that possess an essential tremor-like pharmacological response profile. Tremor was measured using digitized spectral motion power analysis with harmaline-induced tremor and in the GABAA receptor α1 subunit-null model. Mice were given ethosuximide, zonisamide, the neuroactive steroid (3β,5α,17β)-17-hydroxyestrane-3-carbonitrile (ECN), the 3,4-dihydroquinazoline derivative KYS05064, the mibefradil derivative NNC 55-0396, or vehicle. In non-sedating doses, each compound reduced harmaline-induced tremor by at least 50% (range of maximal suppression: 53–81%), and in the GABAA α1-null model by at least 70% (range 70–93%). Because the T-type calcium channel Cav3.1 is the dominant subtype expressed in the inferior olive, we assessed the tremor response of Cav3.1-deficient mice to harmaline, and found that null and heterozygote mice exhibit as much tremor as wild-type mice. In addition, ECN and NNC 55-0396 suppressed harmaline tremor as well in Cav3.1-null mice as in wild-type mice. The finding that five T-type calcium antagonists suppress tremor in two animal tremor models suggests that T-type calcium channels may be an appropriate target for essential tremor therapy development. It is uncertain whether medications developed to block only the Cav3.1 subtype would exhibit efficacy.
Essential tremor; Harmaline; Ethosuximide; Zonisamide; Calcium channel; Cerebellum; Inferior olive
Absence epilepsy is generated by the cortico-thalamo-cortical network, which undergoes a finely tuned regulation by metabotropic glutamate (mGlu) receptors. We have shown previously that potentiation of mGlu1 receptors reduces spontaneous occurring spike and wave discharges (SWDs) in the WAG/Rij rat model of absence epilepsy, whereas activation of mGlu2/3 and mGlu4 receptors produces the opposite effect. Here, we have extended the study to mGlu5 receptors, which are known to be highly expressed within the cortico-thalamo-cortical network. We used presymptomatic and symptomatic WAG/Rij rats and aged-matched ACI rats. WAG/Rij rats showed a reduction in the mGlu5 receptor protein levels and in the mGlu5-receptor mediated stimulation of polyphosphoinositide hydrolysis in the ventrobasal thalamus, whereas the expression of mGlu5 receptors was increased in the somatosensory cortex. Interestingly, these changes preceded the onset of the epileptic phenotype, being already visible in pre-symptomatic WAG/Rij rats. SWDs in symptomatic WAG/Rij rats were not influenced by pharmacological blockade of mGlu5 receptors with MTEP (10 or 30 mg/kg, i.p.), but were significantly decreased by mGlu5 receptor potentiation with the novel enhancer, VU0360172 (3 or 10 mg/kg, s.c.), without affecting motor behaviour. The effect of VU0360172 was prevented by co-treatment with MTEP. These findings suggest that changes in mGlu5 receptors might lie at the core of the absence-seizure prone phenotype of WAG/Rij rats, and that mGlu5 receptor enhancers are potential candidates to the treatment of absence epilepsy.
Absence epilepsy; WAG/Rij rats; mGlu5 receptor; VU0360172; MTEP
Metabotropic glutamate (mGlu) receptors were discovered in the mid 1980s and originally described as glutamate receptors coupled to polyphosphoinositide hydrolysis. Almost 6500 articles have been published since then, and subtype-selective mGlu receptor ligands are now under clinical development for the treatment of a variety of disorders such as Fragile-X syndrome, schizophrenia, Parkinson’s disease and L-DOPA-induced dyskinesias, generalized anxiety disorder, chronic pain, and gastroesophageal reflux disorder. Prof. Erminio Costa was linked to the early times of the mGlu receptor history, when a few research groups challenged the general belief that glutamate could only activate ionotropic receptors and all metabolic responses to glutamate were secondary to calcium entry. This review moves from those nostalgic times to the most recent advances in the physiology and pharmacology of mGlu receptors, and highlights the role of individual mGlu receptor subtypes in the pathophysiology of human disorders. This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’.
Metabotropic glutamate receptors; Receptor structure; mGlu receptor ligands; Clinical studies
Tetrabenazine (TBZ), a benzoquinolizine derivative, binds with high affinity to the vesicular monoamine transporter-2 (VMAT2), inhibiting uptake of cytosolic monoamines. The current study aimed to provide preclinical evidence supporting the potential use of TBZ as a treatment for methamphetamine abuse. Effects of TBZ on function of the dopamine transporter (DAT) and serotonin transporter (SERT) in striatal and hippocampal synaptosomes, respectively, and on VMAT2 function in isolated striatal synaptic vesicles were determined. Effect of TBZ (acute, 0.1 - 3.0 mg/kg, s.c.; repeated, 1.0 mg/kg for 7 days) on locomotor activity in methamphetamine-sensitized rats was assessed. Ability of TBZ (0.1 -3.0 mg/kg; s.c.) or vehicle to decrease the discriminative effect of methamphetamine also was determined. Ability of TBZ (acute, 0.1 - 1.0 mg/kg, s.c.; repeated, 0.1 or 1.0 mg/kg for 7 days) to specifically decrease methamphetamine self-administration was determined; for comparison, a separate group of rats was assessed for effects of TBZ on food-maintained responding. Results show that TBZ was 11-fold more potent inhibiting DAT than SERT, and 2.5-fold more potent inhibiting VMAT2 than DAT. Results from behavioral studies showed that the lowest dose of TBZ transiently increased methamphetamine self-administration, whereas higher TBZ doses decreased methamphetamine self-administration. Also, TBZ at high doses decreased methamphetamine locomotor sensitization and discriminative stimulus effects, as well as food-maintained responding. Thus, despite acting as a potent VMAT2 inhibitor, these preclinical results indicate that TBZ lacks behavioral specificity as an inhibitor of methamphetamine-induced reinforcement, diminishing its viability as a suitable treatment for methamphetamine abuse.
methamphetamine; tetrabenazine; vesicular monoamine transporter 2; self-administration; locomotor activity; discriminative stimulus
Butorphanol is hypothesized to induce analgesia via opioid pathways, although the precise mechanisms for its effects remain unknown. In this study, we investigated the role of the μ-opioid receptor (MOP) in thermal, mechanical, and visceral chemical antinociception induced by butorphanol using MOP knockout (KO) mice. Butorphanol-induced thermal antinociception, assessed by the hot-plate and tail-flick tests, was significantly reduced in heterozygous and abolished in homozygous MOP-KO mice compared with wildtype mice. The results obtained from our butorphanol-induced mechanical antinociception experiments, assessed by the Randall-Selitto test, were similar to the results obtained from the thermal antinociception experiments in these mice. Interestingly, however, butorphanol retained its ability to induce significant visceral chemical antinociception, assessed by the writhing test, in homozygous MOP-KO mice. The butorphanol-induced visceral chemical antinociception that was retained in homozygous MOP-KO mice was completely blocked by pretreatment with nor-binaltorphimine, a κ-opioid receptor (KOP) antagonist. In vitro binding and cyclic adenosine monophosphate assays also showed that butorphanol possessed higher affinity for KOPs and MOPs than for δ-opioid receptors. These results molecular pharmacologically confirmed previous studies implicating MOPs, and partially KOPs, in mediating butorphanol-induced analgesia.
Opioid receptor; Knockout mice; Butorphanol; Antinociception
Cajal-Retzius cells are thought to play an important role for cortical development, and receive primarily spontaneous GABAergic input mediated by GABAA receptors. However, neither the effects of synaptically-released GABA on their excitability nor the cellular source(s) of spontaneous GABAergic currents have been yet determined. By directly recording electrophysiological responses from identified Cajal-Retzius cells of the CXCR4-EGFP mouse, we show that GABAergic input can trigger supra-threshold responses, and that the pharmacological activation of mGlu1α receptors with the group I agonist DHPG powerfully increases the frequency of spontaneous GABAergic currents. These effects appeared mediated by a network mechanism, because responses to DHPG were completely prevented both by surgical disconnection of layer I from lower layers and by exposure of slices to TTX.
We propose that the cellular source underlying the observed effect of DHPG are layer I-targeting Martinotti-like interneurons, which we show express functional group I mGluRs and respond to DHPG with supra-threshold depolarization already at early developmental stages.
In conclusion, our work suggests that conditions of enhanced glutamate release may be critical at early developmental stages for the recruitment of an mGlu1α-dependent micro-circuit, which then leads to the activation of Cajal-Retzius cells.
Trifluoroacetic acid is a metabolite of the inhaled anesthetics halothane, desflurane and isoflurane as well as a major contaminant in HPLC-purified peptides. Ligand-gated ion channels, including cys-loop receptors such as the glycine receptor, have been the targets of peptide-based drug design and are considered to be likely candidates for mediating the effects of anesthetics in vivo, but the possible secondary contributions of contaminants and metabolites to these effects have not been studied. We used two-electrode voltage-clamp electrophysiology to test glycine, GABAA and 5-HT3 receptors expressed in Xenopus oocytes for their sensitivities to sodium trifluoroacetate. Trifluoroacetate (100 μM–3 mM) enhanced the currents elicited by low concentrations of glycine applied to α1 homomeric and α1β heteromeric glycine receptors, but it had no effects when co-applied with a maximally-effective glycine concentration. Trifluoroacetate had no effects on α1β2γ2S GABAA or 5-HT3A receptors at any GABA or serotonin concentration tested. The results demonstrate that trifluoroacetate acts as an allosteric modulator at the glycine receptor with greater specificity than other known modulators. These results have important implications for both the secondary effects of volatile anesthetics and the presence of contaminating trifluoroacetate in HPLC-purified peptides, which is potentially an important source of experimental variability or error that requires control.
Anesthetic metabolism; Allosteric modulation; Glycine receptor; GABAA receptor; 5-HT3 receptor; Trifluoroacetate
Habenulo-interpeduncular nicotinic receptors, particularly those containing α3, β4 and α5 subunits, have recently been implicated in the reinforcing effects of nicotine. Our laboratory has shown that injection of α3β4 nicotinic receptor antagonists into the medial habenula (MHb) decreases self-administration of multiple abused drugs, including nicotine (Glick et al., 2006; 2008; 2011). However, it is unclear whether blockade of MHb nicotinic receptors has a direct effect on mesolimbic dopamine. Here, we performed in vivo microdialysis in female rats. Microdialysis probes were implanted into the nucleus accumbens (NAcc) and α3β4 nicotinic receptor antagonists (18-methoxycoronaridine; 18-MC or α-conotoxin AuIB; AuIB), were injected into the ipsilateral MHb, just prior to systemic nicotine (0.4 mg/kg, s.c.). Dialysate samples were collected before and after drug administration and levels of extracellular dopamine and its metabolites were measured using HPLC. Acute nicotine administration increased levels of extracellular dopamine and its metabolites in the NAcc. Pre-treatment with intra-habenular AuIB or 18-MC prevented nicotine-induced increases in accumbal dopamine. Neither drug had an effect on nicotine-induced increases in dopamine metabolites, suggesting that α3β4 receptors do not play a role in dopamine metabolism. The effect of intra-habenular blockade of α3β4 receptors on NAcc dopamine was selective for acute nicotine: neither AuIB nor 18-MC prevented increases in NAcc dopamine stimulated by acute d-amphetamine or morphine. These results suggest the mesolimbic response to acute nicotine, but not to acute administration of other drugs of abuse, is directly modulated by α3β4 nicotinic receptors in the MHb, and emphasize a critical role for habenular nicotinic receptors in nicotine’s reinforcing effects.
Recent studies have demonstrated that exposure to environmental enrichment (EE) during withdrawal periods reduces the risks of relapse to drug-seeking behavior. In this study, we investigated whether EE could prevent the development of time-dependent increases in cocaine-seeking behavior (incubation of craving). In addition, we investigated whether EE could eliminate already developed incubation and whether the effects of EE would last when enrichment is discontinued. For this, we allowed rats to self-administer cocaine for 10 daily 6h sessions and measured cocaine seeking 1, 30 and 60 days after the last self-administration session. In between these tests, rats were kept in forced abstinence and housed either in EE or standard environments (SE). Between day 30 and 60 of withdrawal, half of the rats in each group were maintained in their original environmental condition and the other half was switched to the other environmental condition. We found that exposure to EE prevents development of incubation of cocaine craving and eliminates already developed incubation. In addition, contrary to our expectations, when EE was discontinued, its positive effects on incubation of craving disappeared. These results indicate that EE can reduce cocaine seeking but only temporarily and questions the hypothesis that EE can permanently eliminate the neural consequences of exposure to drugs of abuse. Therefore, stimulating environments could have positive effects on the treatment of cocaine addiction only if they are maintained for long periods of abstinence that encompass the time-frame during which addicts are most vulnerable to relapse.
addiction; treatment; relapse; neuroadaptation; stress; withdrawal
Patients suffering from amphetamine---induced psychosis display repetitive behaviors, partially alleviated by antipsychotics, which are reminiscent of rodent stereotypies. Due to recent evidence implicating endocannabinoid involvement in brain disorders, including psychosis, we studied the effects of endocannabinoid signaling on neuronal oscillations of rats exhibiting methamphetamine stereotypy. Neuronal network oscillations were recorded with multiple single electrode arrays aimed at the nucleus accumbens of freely moving rats. During the experiments, animals were dosed intravenously with the CB1 receptor antagonist rimonabant (0.3 mg/kg) or vehicle followed by an ascending dose regimen of methamphetamine (0.01, 0.1, 1, and 3 mg/kg; cumulative dosing). The effects of drug administration on stereotypy and local gamma oscillations were evaluated. Methamphetamine treatment significantly increased high frequency gamma oscillations (~ 80 Hz). Entrainment of a subpopulation of nucleus accumbens neurons to high frequency gamma was associated with stereotypy encoding in putative fast-spiking interneurons, but not in putative medium spiny neurons. The observed ability of methamphetamine to induce both stereotypy and high frequency gamma power was potently disrupted following CB1 receptor blockade. The present data suggest that CB1 receptor-dependent mechanisms are recruited by methamphetamine to modify striatal interneuron oscillations that accompany changes in psychomotor state, further supporting the link between endocannabinoids and schizophrenia spectrum disorders.
The role of CB2 in the central nervous system, particularly in neurons, has generated much controversy. Fueling the controversy are imperfect tools, which have made conclusive identification of CB2-expressing neurons problematic. Imprecise localization of CB2 has made it difficult to determine its function in neurons. Here we avoid the localization controversy and directly address the question if CB2 can modulate neurotransmission. CB2 was expressed in excitatory hippocampal autaptic neurons obtained from CB1 null mice. Whole-cell patch clamp recordings were made from these neurons to determine the effects of CB2 on short-term synaptic plasticity. CB2 expression restored depolarization induced suppression of excitation to these neurons, which was lost following genetic ablation of CB1. The endocannabinoid 2-arachidonylglycerol (2-AG) mimicked the effects of depolarization in CB2 expressing neurons. Interestingly, ongoing basal production of 2-AG resulted in constitutive activation of CB2, causing a tonic inhibition of neurotransmission that was relieved by the CB2 antagonist AM630 or the diacylglycerol lipase inhibitor RHC80267. Through immunocytochemistry and analysis of spontaneous EPSCs, paired pulse ratios and coefficients of variation we determined that CB2 exerts its function at a presynaptic site of action, likely through inhibition of voltage gated calcium channels. Therefore CB2 expressed in neurons effectively mimics the actions of CB1. Thus, neuronal CB2 is well suited to integrate into conventional neuronal endocannabinoid signaling processes, with its specific role determined by its unique and highly inducible expression profile.
CB2; cannabinoid receptor; autaptic neurons; DSE; endocannabinoid2
Vascular endothelial growth factor (VEGF) is a hypoxia-induced angiogenic protein that exhibits a broad range of neurotrophic and neuroprotective effects in the central nervous system. Given that neurogenesis occurs in close proximity to blood vessels, increasing evidence has suggested that VEGF may constitute an important link between neurogenesis and angiogenesis. Although it is known that VEGF can directly stimulate the proliferation of neuronal progenitors, the underlying signaling pathways responsible in this process are not fully understood. Thus, in the present study, we set out to examine the requirement of two downstream targets of the VEGF/Flk-1 signaling network, the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways, in producing the mitogenic effects of VEGF. Both in vivo and in vitro experiments showed that a single treatment of VEGF activated Erk1/2 and Akt signaling pathways in the adult rat hippocampus and in cultured hippocampal neuronal progenitor cells. This effect was blocked with the VEGF/Flk-1 inhibitor SU5416. Importantly, microinfusion of VEGF into the rat brain also induced pCREB expression in the dentate gyrus and increased the number of BrdU-labeled cells in the dentate subgranular zone. Double immunofluorescence labeling revealed that a large proportion of BrdU-labeled cells expressed activated forms of Flk-1, Erk1/2, and Akt. Interestingly, treatment with the SSRI fluoxetine, which is well known to stimulate neurogenesis and VEGF-signaling, also produced a similar expression pattern of Erk1/2 and Akt in proliferating cells. Finally, pharmacological experiments showed that administration of inhibitors of either MAPK/ERK (U0126) or PI3K (LY294002) blocked VEGF-stimulation of hippocampal cell proliferation in vivo and in vitro. Taken together, our findings demonstrate that the proliferative actions of VEGF require activation of both ERK and Akt signaling cascades and that these intracellular pathways are stimulated almost exclusively in actively proliferating neuronal progenitor cells of the adult hippocampus.
VEGF; neurotrophic factors; MEK/ERK; PI3K/AKT; fluoxetine; neurogenesis; hippocampus
Systemic administration of NMDA receptor antagonists elevates extracellular glutamate within prefrontal cortex. The cognitive and behavioral effects of NMDA receptor blockade have direct relevance to symptoms of schizophrenia, and recent studies demonstrate an important role for nitric oxide and GABAB receptors in mediating the effects of NMDA receptor blockade on these behaviors. We sought to extend those observations by directly measuring the effects of nitric oxide and GABAB receptor mechanisms on MK-801-induced glutamate release in the prefrontal cortex. Systemic MK-801 injection (0.3 mg/kg) to male Sprague-Dawley rats significantly increased extracellular glutamate levels in prefrontal cortex, as determined by microdialysis. This effect was blocked by pretreatment with the nitric oxide synthase inhibitor L-NAME (60 mg/kg). Reverse dialysis of the nitric oxide donor SNAP (0.5 – 5 mM) directly into prefrontal cortex mimicked the effect of systemic MK-801, dose-dependently elevating cortical extracellular glutamate. The effect of MK-801 was also blocked by systemic treatment with the GABAB receptor agonist baclofen (5 mg/kg). In combination, these data suggest increased nitric oxide formation is necessary for NMDA antagonist-induced elevations of extracellular glutamate in the prefrontal cortex. Additionally, the data suggest GABAB receptor activation can modulate the NMDA antagonist-induced increase in cortical glutamate release.
glutamate; nitric oxide; MK-801; GABA; NMDA receptor; schizophrenia
Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3β4 binding sites and other subunits, including β4, β2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 β4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and β4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and β4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3β4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for β4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations.
Smoking cessation; drug screening; ganglionic receptors; partial agonists
Cannabinoids suppress nocifensive behaviors in rodents. We presently investigated peripheral endocannabinoid modulation of itch- and pain-related behaviors elicited from facial vs. spinally-innervated skin of rats. Intradermal (id) injection of the pruritogen serotonin (5-HT) elicited significantly more hindlimb scratch bouts, and longer cumulative time scratching, when injected in the rostral back compared to the cheek. Pretreatment of skin with inhibitors of degrading enzymes for the endocannabinoids anandamide (URB597) or 2-arachidonoylglycerol (JZL184) significantly reduced scratching elicited by 5-HT in the rostral back. These effects were prevented by co-treatment with antagonists of the CB1 (AM251) or CB2 receptor (AM630), implicating both receptor subtypes in endocannabinoid suppression of scratching in spinally-innervated skin. Conversely, pretreatment with either enzyme inhibitor, or with AM630 alone, increased the number of scratch bouts elicited by id 5-HT injection in the cheek. Moreover, pretreatment with JZL184 also significantly increased pain-related forelimb wipes directed to the cheek following id injection of the algogen, allyl isothiocyanate (AITC; mustard oil). Thus, peripheral endocannabinoids have opposite effects on itch-related scratching behaviors in trigeminally- vs. spinally-innervated skin. These results suggest that increasing peripheral endocannabinoid levels represents a promising therapeutic approach to treat itch arising from the lower body, but caution that such treatment may not relieve, and may even exacerbate, itch and pain arising from trigeminally-innervated skin of the face or scalp.
endocannabinoid; itch; scratch; C-fiber; cannabinoid receptor; CB1; CB2; G-protein-coupled receptor; serotonin
The selective 5-HT1 receptor agonist sumatriptan is an effective therapeutic for migraine pain yet the antimigraine mechanisms of action remain controversial. Pain-responsive fibres containing calcitonin gene-related peptide (CGRP) densely innervating the cranial dura mater are widely believed to be an essential anatomical substrate for the development of migraine pain. 5HT1 receptors in the dura colocalize with CGRP fibres in high density and thus provide a possible peripheral site of action for sumatriptan. In the present study, we used high-resolution optical imaging selectively within individual mouse dural CGRP nociceptive fibre terminations and found that application of sumatriptan caused a rapid, reversible dose-dependent inhibition in the amplitude of single action potential evoked Ca2+ transients. Pre-application of the 5-HT1 antagonist GR127935 or the selective 5-HT1D antagonist BRL 15572 prevented inhibition while the selective 5-HT1B antagonist SB 224289 did not, suggesting this effect was mediated selectively through the 5-HT1D receptor subtype. Sumatriptan inhibition of the action potential evoked Ca2+ signaling was mediated selectively through N-type Ca2+ channels. Although the T-type Ca2+ channel accounted for a greater proportion of the Ca2+ signal it did not mediate any of the sumatriptan inhibition. Our findings support a peripheral site of action for sumatriptan in inhibiting the activity of dural pain fibres selectively through a single Ca2+ channel subtype. This finding adds to our understanding of the mechanisms that underlie the clinical effectiveness of 5HT1 receptor agonists such as sumatriptan and may provide insight for the development of novel peripherally targeted therapeutics for mitigating the pain of migraine.
migraine; Ca2+ signaling; pain; imaging; 5-HT1; dura
Pregabalin, an anticonvulsant and anxiolytic compound that binds to α2-δ auxiliary subunit Types 1 and 2 of voltage-gated calcium channels, has been shown to reduce excitatory neurotransmission partially through modulation of glutamatergic signaling. Prepulse inhibition (PPI) of startle is an operational measure of sensorimotor gating impacted by disruption of the glutamatergic system and is reduced in schizophrenia patients. Dysregulation of the glutamatergic system has also been implicated in the pathophysiology of schizophrenia. Here we tested the hypothesis that pregabalin may ameliorate PPI in a model of deficient gating in humans and mice. In study 1, 14 healthy human subjects participated in a within subjects, cross-over study with placebo, 50 mg or 200 mg pregabalin treatment prior to undergoing a PPI task. In study 2, 24 C57BL/6 mice underwent a similar procedure with vehicle, 30 and 100 mg/kg dose treatments. In both studies, subjects were assigned to a “Low” or “High” gating group using a median split procedure based on their PPI performance during placebo/vehicle. Drug effects were then examined across these groups. In humans, pregabalin treatment significantly increased PPI performance in the “low gating” group. In mice, pregabalin treatment significantly increased PPI in the low gating group but reduced PPI in the high gating group. Across species, pregabalin treatment improves PPI in subjects with low gating. These data support further exploration of pregabalin as a potential treatment for disorders characterized by sensorimotor gating deficits and glutamatergic hypersignaling, such as schizophrenia.
Schizophrenia; Pre-pulse inhibition; Glutamate; Pregabalin; Startle; Sensorimotor gating
Recent evidence suggests that GABAA receptor ligands may regulate ethanol intake via effects at both synaptic and extrasynaptic receptors. For example, the endogenous neurosteroid, allopregnanolone (ALLO) has a similar pharmacological profile as ethanol, and it alters ethanol intake in rodent models. Additionally, recent evidence suggests that δ-subunit containing extrasynaptic GABAA receptors may confer high sensitivity to both ethanol and neurosteroids. The purpose of the present study was to determine the effects of ganaxolone (GAN; an ALLO analogue) and gaboxadol (THIP; a GABAA receptor agonist with selectivity for the extrasynaptic δ-subunit) on ethanol intake, drinking patterns, and bout characteristics in operant and limited access self-administration procedures. In separate studies, the effects of GAN (0 – 10 mg/kg) and THIP (2 – 16 mg/kg) were tested in C57BL/6J male mice provided with two-hour access to a two-bottle choice of water or 10% ethanol or trained to respond for 30 minutes of access to 10% ethanol. GAN had no overall significant effect on operant ethanol self-administration, but tended to decrease the latency to consume the first bout. In the limited-access procedure, GAN dose-dependently decreased ethanol intake. THIP dose-dependently decreased ethanol intake in both paradigms, altering both the consummatory and appetitive processes of operant self-administration as well as shifting the drinking patterns in both procedures. These results add to literature suggesting time-dependent effects of neurosteroids to promote the onset, and to subsequently decrease, ethanol drinking behavior, and they support a role for extrasynaptic GABAA receptor activation in ethanol reinforcement.
L-Dopa-induced dyskinesias are a serious side effect that develops in most Parkinson’s disease patients on dopamine replacement therapy. Few treatment options are available to manage dyskinesias; however, recent studies show that nicotine reduces these abnormal involuntary movements (AIMs) in parkinsonian animals by acting at nicotinic acetylcholine receptors (nAChRs). Identification of the nAChR subtypes that mediate this reduction in AIMs is important as it will help in the development of nAChR subtype selective drugs for their treatment. Here we investigate the role of α6β2* nAChRs, a subtype selectively present in the nigrostriatal pathway, using α6 nAChR subunit null mutant (α6(-/-)) mice. Wildtype and α6(-/-) mice were lesioned by unilateral injection of 6-hydroxydopamine (3 μg/μl) into the medial forebrain bundle. They were then given L-dopa (3 mg/kg) plus benserazide (15 mg/kg) 2-3 wk later. L-dopa-induced AIMs developed to a similar extent in α6(-/-) and wildtype mice. However, AIMs in α6(-/-) mice declined to ~50% of that in wildtype mice with continued L-dopa treatment. Nicotine treatment also decreased AIMs by ~50% in wildtype mice, although not in α6(-/-) mice. There were no effects on parkinsonism under any experimental condition. To conclude, the similar declines in L-dopa-induced AIMs in nicotine-treated wildtype mice and in α6(-/-) mice treated with and without nicotine indicate an essential role for α6β2* nAChRs in the maintenance of L-dopa-induced AIMs. These findings suggest that α6β2* nAChR drugs have potential for reducing L-dopa-induced dyskinesias in Parkinson’s disease.
alpha6; dyskinesia; L-dopa; nicotine; 6-hydroxydopamine; Parkinson’s disease
Thalamocortical (TC) neurons provide the major sensory input to the mammalian somatosensory cortex. Decreased activity of these cells may be pivotal in the ability of general anesthetics to induce loss of consciousness and promote sleep (hypnosis). T-type voltage-gated calcium currents (T-currents) have a key function regulating the cellular excitability of TC neurons and previous studies have indicated that volatile general anesthetics may alter the excitability of these neurons.
Using a patch-clamp technique, we investigated the mechanisms whereby isoflurane, a common volatile anesthetic, modulates isolated T-currents and T-current-dependent excitability of native TC neurons in acute brain slices of the rat.
In voltage-clamp experiments, we found that isoflurane strongly inhibited peak amplitude of T-current, yielding an IC50 of 1.1% at physiological membrane potentials. Ensuing biophysical studies demonstrated that inhibition was more prominent at depolarized membrane potentials as evidenced by hyperpolarizing shifts in channel availability curves. In current-clamp experiments we found that isoflurane decreased the rate of depolarization of low-threshold-calcium spikes (LTCSs) and consequently increased the latency of rebound spike firing at the same concentrations that inhibited isolated T-currents. This effect was mimicked by a novel selective T-channel blocker 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2). In contrast, isoflurane and TTA-P2 had minimal effect on resting membrane potential and cell input resistance. We propose that depression of thalamic T-currents may contribute to some clinical properties of isoflurane.
T-type calcium current; rodent thalamocortical neurons; isoflurane; anesthetics; burst firing; low-voltage-activated