Peripheral T cells from multiple sclerosis patients trigger synaptotoxic alterations in central neurons.

AIMS: The crucial step in the pathogenic events that lead to the development and the progression of multiple sclerosis (MS) is the infiltration of autoreactive T cells in the brain. Data from experimental autoimmune encephalomyelitis (EAE) mice indicate that, together with microglia, T cells are responsible for the enhancement of the glutamatergic transmission in central neurons, contributing to glutamate-mediated excitotoxicity, a pathological hallmark of both EAE and MS brains. Here, we addressed the synaptic role of T cells taken from MS patients. METHODS: A chimeric model of human T cells and murine brain slices was established to record, by Patch Clamp technique, the glutamatergic transmission in the presence of T cells isolated from the peripheral blood of healthy subjects (HS), active (a) and nonactive (na) relapsing remitting MS patients. Intracellular staining and flow cytometry were used to assess tumour necrosis factor (TNF) expression in T cells. RESULTS: Chimeric experiments indicated that, compared to HS and naMS, T cells from aMS induced an increase in glutamatergic kinetic properties of striatal neurons. Such alteration, reminiscent of the those induced by EAE T cells, was blocked by incubation of the slices with etanercept, a TNF receptor antagonist. Of note, T cells from aMS expressed more TNF than naMS patients and HS subjects. CONCLUSION: These data highlight the synaptotoxic potential retained by MS T cells, suggesting that during the inflammatory phase of the disease infiltrating T cells could influence the neuronal activity contributing to the TNF-mediated mechanisms of glutamate excitotoxicity in central neurons.

Epigenetic modifications of Dexras 1 along the nNOS pathway in an animal model of multiple sclerosis.

The development of multiple sclerosis, a major neurodegenerative disease, is due to both genetic and environmental factors that might trigger aberrant epigenetic changes of the genome. In this study, we analysed global DNA methylation in the brain of mice upon induction of experimental autoimmune encephalomyelitis (EAE), and the effect of environmental enrichment (EE). We demonstrate that global DNA methylation decreased in the striatum, but not in the cortex, of EAE mice compared to healthy controls, in particular in neuronal nitric oxide synthase (nNOS)-positive interneurons of this brain area. Also, in the striatum but again not in the cortex, decreased DNA methylation of the nNOS downstream effector, dexamethasone-induced Ras protein 1 (Dexras 1), was observed in EAE mice, and was paralleled by an increase in its mRNA. Interestingly, EE was able to revert EAE effects on mRNA expression and DNA methylation levels of Dexras 1 and reduced gene expression of nNOS and 5-lipoxygenase (Alox5). Conversely, interleukin-1ß (IL-1ß) gene expression was found up-regulated in EAE mice compared to controls and was not affected by EE. Taken together, these data demonstrate an unprecedented epigenetic modulation of nNOS-signaling in the pathogenesis of multiple sclerosis, and show that EE can specifically revert EAE effects on Dexras 1 along this pathway.

FAS-ligand regulates differential activation-induced cell death of human T-helper 1 and 17 cells in healthy donors and multiple sclerosis patients.

Functionally distinct T-helper (Th) subsets orchestrate immune responses. Maintenance of homeostasis through the tight control of inflammatory Th cells is crucial to avoid autoimmune inflammation. Activation-Induced Cell Death (AICD) regulates homeostasis of T cells, and it has never been investigated in human Th cells. We generated stable clones of inflammatory Th subsets involved in autoimmune diseases, such as Th1, Th17 and Th1/17 cells, from healthy donors (HD) and multiple sclerosis (MS) patients and we measured AICD. We find that human Th1 cells are sensitive, whereas Th17 and Th1/17 are resistant, to AICD. In particular, Th1 cells express high level of FAS-ligand (FASL), which interacts with FAS and leads to caspases' cleavage and ultimately to cell death. In contrast, low FASL expression in Th17 and Th1/17 cells blunts caspase 8 activation and thus reduces cell death. Interestingly, Th cells obtained from healthy individuals and MS patients behave similarly, suggesting that this mechanism could explain the persistence of inflammatory IL-17-producing cells in autoimmune diseases, such as MS, where their generation is particularly substantial.

Surgical resection for neuroendocrine tumors of the pancreas: a fourteen years single institutional observation.

OBJECTIVE: Pancreatic neuroendocrine tumors (PNETs) are a rare entity that can present with symptoms of hormone overproduction with surgical resection being the only chance for cure despite the poorly defined tumor behavior. Their management involves a variety of therapies which require a well coordinated multidisciplinary team with the effort to optimize outcomes. PATIENTS AND METHODS: A retrospective analysis of 25 consecutive patients was performed by means of our single institution prospectively maintained database. All patients' files from 1999 to 2013, with histologically proven neuroendocrine tumors of the pancreas, were reviewed for clinical presentation, functional status, treatment, postoperative morbidity and mortality. RESULTS: Of 25 patients a total of 22 patients (11 females, 11 males, average age 49.7 years) underwent surgery with curative intent. We had 3 female patients that underwent palliative surgery because of unresectable disease. Nineteen of the 25 were not functional tumor. For the resected patients the overall morbidity was 38.8%. The 30-day mortality rate was zero. The overall median length of hospital stay was 10.4 days (range 4-23 days). CONCLUSIONS: Surgical resection with regional lymph node dissection is the only potentially curative therapy for patients with localized PNETs with the exceptions of most insulinomas where simple enucleation may be the standard of treatment. The anatomic considerations for determining the resecability are the same as those for pancreatic adenocarcinomas. Careful follow-up after surgery is essential because up to 50% of patients who undergo complete resection develop metachronous liver metastasis. Distant metastatic disease should be resected if possible.

Interleukin-8 is associated with acute and persistent dysfunction after optic neuritis.

BACKGROUND: Acute optic neuritis is often in association with multiple sclerosis (MS). Proinflammatory cytokines trigger neuronal damage in neuroinflammatory disorders but their role in optic neuritis is poorly investigated. OBJECTIVE: The objective of this work is to investigate the associations of intrathecal contents of proinflammatory cytokines with transient and persistent dysfunctions after optic neuritis. METHODS: In 50 MS patients followed for up to six months, cerebrospinal fluid (CSF) levels of IL-1ß, TNF and IL-8 were determined, along with clinical, neurophysiological and morphological measures of optic neuritis severity. RESULTS: Visual impairment, measured by high- and low-contrast visual acuity, and delayed visual-evoked potential (VEP) latencies were significantly correlated to IL-8 levels during optic neuritis. IL-8 at the time of optic neuritis was also associated with persistent demyelination and final axonal loss, inferred by VEP and optical coherence tomography measures, respectively. Contents of IL-8 were correlated to functional visual outcomes, being higher among patients with incomplete recovery. Multivariate analysis confirmed that IL-8 significantly predicted final visual acuity, at equal values of demographics and baseline visual scores. CONCLUSION: Our study points to IL-8 as the main inflammatory cytokine associated with demyelination and secondary neurodegeneration in the optic nerve after optic neuritis.

Interleukin-1ß alters the sensitivity of cannabinoid CB1 receptors controlling glutamate transmission in the striatum.

Proinflammatory cytokines such as tumor necrosis factor-α and interleukin-1ß (IL1ß) regulate both excitatory and inhibitory synaptic transmission in the central nervous system. The interaction between IL1ß and endocannabinoid system (ECS) is also emerging, based on the evidence that IL1ß effects on striatal spontaneous excitatory and inhibitory postsynaptic currents are regulated by transient receptor potential vanilloid 1 (TRPV1) channels, members of the ECS. Furthermore, IL1ß has also been shown to control the sensitivity of cannabinoid CB1 receptors controlling GABA transmission (CB1Rs(GABA)) in the striatum. To better detail the synaptic action of IL1ß, and to clarify its complex interaction with the ECS, here we investigated the possible interplay between IL1ß and CB1Rs controlling glutamate transmission (CB1Rs(glu)), other critical elements of the ECS. Our results show that the sensitivity of CB1Rs(glu) is fully blocked in the presence of IL1ß in corticostriatal brain slices, and that the protein kinase C/TRPV1 pathway is involved in this effect. IL1ß failed to modulate the sensitivity of glutamate synapses to the stimulation of GABAB receptors. We also provided evidence that IL1ß-CB1Rs(GABA) but not IL1ß-CB1Rs(glu) interaction is under the control of the brain-derived neurotrophic factor (BDNF)/trkB signaling and of lipid raft composition, because BDNF gene partial deletion, pharmacological blockade of trkB and membrane cholesterol removal with methyl-ß-cyclodextrin all blocked IL1ß-mediated inhibition of CB1Rs(GABA) but left unaltered the sensitivity of CB1Rs(glu) to this cytokine. Our results provide further evidence that synaptic transmission and the ECS are regulated by IL1ß in the striatum.

Local excision with adjuvant imatinib therapy for anorectal gastrointestinal stromal tumors.

Rectal gastrointestinal stromal tumors (GISTs) are uncommon, and the role of local excision versus a more extensive resection after the advent of effective targeted chemotherapy with imatinib is not known. Our aim is to present two cases of large anorectal GIST treated with local excision through a new anterolateral trans-sphincteric approach followed by adjuvant therapy with imatinib. Two patients (both males, 68 and 63 years old) presented at our institution with anorectal GIST in the period October-November 2010. Their medical records, pathology results, and imaging studies were retrospectively reviewed. Both patients presented with an anterior perianal mass. Imaging studies were characteristic of GIST originating in the lower rectum, circumscribed by a pseudocapsule, and protruding into the ischiorectal fossa. Both patients underwent local excision via an anterolateral trans-sphincteric approach. Both tumors were removed intact, with microscopically negative margins. The maximum tumor diameter was 8 and 9 cm, and the diagnosis of GIST was confirmed by positive CD117 and CD34 staining in both cases. Both tumors had a high (>5/50HPF) mitotic index. The patients had an uneventful postoperative course and were discharged on days 5 and 6. Both patients were started on imatinib 400 mg bid postoperatively. Postoperative magnetic resonance imaging and positron emission tomography computed tomography were carried out at 12 months and did not reveal any signs of recurrence. The patients are currently disease-free at 24 and 23 months of follow-up. In selected cases, complete excision of rectal GIST with negative margins is feasible via a trans-sphincteric approach. With the use of adjuvant therapy, which is currently advocated in all high-risk cases, it is possible that local excision with its reduced morbidity may become a viable alternative, especially in patients who would otherwise require abdominoperineal excision such as the two presented here. Prospective studies with longer follow-up are needed to confirm adequate oncologic results.

Effect of glatiramer acetate on disease reactivation in MS patients discontinuing natalizumab.

BACKGROUND AND PURPOSE: Multiple sclerosis (MS) patients discontinuing natalizumab are at risk of rebound of disease activity. METHODS: In the present multi-center, open-label, non-randomized, prospective, pilot study, we tested whether treatment with glatiramer acetate (GA) is safe and effective after natalizumab in MS patients. The study was performed at academic tertiary medical centers. Forty active relapsing-remitting MS patients who never failed GA therapy and who discontinued natalizumab after 12-18 months of therapy were enrolled. GA was initiated 4 weeks after the last dose of natalizumab. RESULTS: 62.5% of patients were relapse-free 12 months after GA initiation. Annualized relapse rate and time to relapse were significantly lower than before natalizumab. Notably, the frequency of relapses was significantly lower amongst those patients who had experienced ≤2 relapses the year before initiation of natalizumab therapy, compared with patients who had had three or more relapses. No evidence of rebound was observed in magnetic resonance imaging scans. Furthermore, Expanded Disability Status Scale and Multiple Sclerosis Functional Composite were stable in our patients, again suggesting that 12 months of post-natalizumab-GA therapy is not associated with clinical deterioration. CONCLUSIONS: Following discontinuation of natalizumab, 12 months of therapy with GA is safe and well tolerated in MS patients. GA can reduce the risk of early reactivation/rebound of disease activity in this setting.

Risk of acute promyelocytic leukemia in multiple sclerosis: coding variants of DNA repair genes.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in double-strand break repair genes may alter DNA repair capacity and, in turn, confer predisposition to leukemia. We analyzed polymorphic variants of DNA repair and detoxification genes in patients with multiple sclerosis (MS) who developed secondary acute promyelocytic leukemia (sAPL), in most cases after treatment with mitoxantrone (MTZ). METHODS: Using MassARRAY high-throughput DNA analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we genotyped patients with sAPL (n=20) developed after treatment of MS (18 out 20 treated with MTZ) for the presence of 210 SNPs of 22 genes mostly involved in DNA repair and drug detoxification. Patients with MS who did not develop sAPL including 41 treated with MTZ (n=253 and 41, respectively) and healthy blood donors (n=310) were also genotyped as controls. RESULTS: We observed risk allele frequency between MS and sAPL for BRCA2 (rs1801406): 6% and 26%, p=0.007; XRCC5 (rs207906): 2.5% and 15%, p=0.016; CYP3A4 (rs2740574): 4.5% and 25%, p=0.0035. The association of homozygous variants of BRCA2 and XRCC5 yielded higher risk of sAPL (MS vs sAPL: 0.4% and 18%, p=0.001). We also observed a significant association between a SNP in the promoter region (rs2740574) of CYP3A4, an enzyme involved in the metabolism of chemotherapeutic agents and development of sAPL. CONCLUSIONS: Increased susceptibility to develop sAPL in patients with MS receiving MTZ may be linked to genetic variants in DNA repair and drug-metabolizing enzymes that result in impaired detoxification of chemotherapy or inefficient repair of drug-induced genetic damage.

Natalizumab therapy of multiple sclerosis: recommendations of the Multiple Sclerosis Study Group--Italian Neurological Society.

Three years after the introduction of natalizumab (NA) therapy for the second line treatment of relapsing-remitting multiple sclerosis (MS), Italian MS centers critically reviewed the scientific literature and their own clinical experience. Natalizumab was shown to be highly efficacious in the treatment of MS. However, the risk of progressive multifocal leukoencephalopathy was confirmed and defined better. This article summarizes the MS-SIN Study Group recommendations on the use of NA in MS, with particular reference to the appropriate selection and monitoring of patients as well as to the management of adverse events.

Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients.

BACKGROUND: Antibodies against aquaporin-4 (AQP4), a water channel particularly expressed on perivascular astrocytic podocytes, are proposed as a marker for the diagnosis of neuromyelitis optica (NMO). However, a consensus on seroprevalence and optimal detection method has not yet been reached. OBJECTIVES: To investigate the performance of different assays to detect anti-AQP4 antibodies. METHODS: We set up five different assays. Two of them were capable to detect perivascular IgG reactivity on brain tissue by immunofluorescence (NMO-IgG). Other three assays have been set to detect anti-AQP4 antibodies: immunofluorescence and flow cytometry on AQP4-expressing cells, and a radioimmunoprecipitation assay. We assessed sensitivity and specificity of these assays by interrogating sera of 33 NMO patients, 13 patients at high risk to develop NMO (hrNMO), 6 patients affected by acute partial transverse myelitis (APTM), 20 patients with multiple sclerosis (MS), and 67 age- and sex-matched healthy controls. RESULTS: We found that the presence of serum NMO-IgG and anti-AQP4 reactivity is almost exclusively restricted to patients with NMO and hrNMO. Seroprevalence and sensitivity ranged from 30 to 47%, depending on the assay. Specificity ranged from 95 to 100%. Comparing results obtained in the five assays, we noticed lack of concordance in some samples. CONCLUSIONS: Detection of NMO-IgG or anti-AQP4 antibodies may represent a valuable tool to assist neurologists in the differential diagnosis between patients with NMO, hrNMO, APTM, or MS. The current lack of a gold standard to detect anti-AQP4 antibodies implies the necessity to standardize the detection of these antibodies.

Deficits of glutamate transmission in the striatum of experimental hemiballism.

Hemiballism (HB) is a quite rare disorder, generally secondary to stroke, neoplasms or demyelinating plaques, classically considered as almost pathognomonic of a lesion in the subthalamic nucleus (STN). This alteration causes involuntary movements in the chorea-ballism spectrum. One theory is that the output nuclei of the basal ganglia are overinhibited in HB, while little is known about the physiological state of the striatum, the major input structure of the basal ganglia. In the present study, we recorded spontaneous and miniature excitatory and inhibitory postsynaptic currents (sEPSCs, mEPSCs, sIPSCs, mIPSCs) from projection neurons of the striatum of experimental HB. We found a selective reduction of striatal sEPSC and mEPSC frequency following chemical lesion of the STN of the rat, suggesting that reduced synaptic excitation of the input structure of the basal ganglia represents a physiological correlate of HB.

Rapid multiresidue extraction method of organochlorinated pesticides from fish feed.

A rapid multiresidue extraction method for organochlorinated pesticides from fish feed was developed, which is based on the extracted fat treatment by n-hexane, concentrated sulphuric acid and ENVI-carb, a graphitized non-porous carbon material. The final residue, obtained in about 50 min, was dissolved in isooctane and analysed by gas chromatography with an electron capture detector (GC/ECD). The presence of the extracted pesticides was confirmed by gas chromatography-mass spectrometry (GC/MS). Concentration of sulphuric acid and amount of ENVI-carb were optimized in order to improve analytes recovery, accuracy and detection limits. This simple and relatively fast method allowed a high recovery of the HCB, Lindane, HEPO, p,p'-DDE, p,p'-DDD, p,p'-DDT residues, with mean values in the range 68-124% at four fortification levels (12.5, 25.0, 50.0, 100.0 ng/g), and coefficients of variation between 1.9 and 20.2%. Detection limit were equal to 3.0 ng/g, related to fat, for all pesticides, and calibration curves were linear (r > 0.999) in the range of explored concentrations from the detection limit to 100 ng/g. For all pesticides a good repeatability was obtained (CV% values in the range 0.23-4.16%) when a sequence of six injections of the isooctane extraction solution was performed. The usefulness of the proposed method has been tested by the analysis of fish feed samples.

Early ionic and membrane potential changes caused by the pesticide rotenone in striatal cholinergic interneurons.

Mitochondrial metabolism impairment has been implicated in the pathogenesis of several neurodegenerative disorders. In the present work, we combined electrophysiological recordings and microfluorometric measurements from cholinergic interneurons obtained from a rat neostriatal slice preparation. Acute application of the mitochondrial complex I inhibitor rotenone produced an early membrane hyperpolarization coupled to a fall in input resistance, followed by a late depolarizing response. Current-voltage relationship showed a reversal potential of -80 +/- 3 mV, suggesting the involvement of a potassium (K+) current. Simultaneous measurement of intracellular sodium [Na+]i or calcium [Ca2+]i concentrations revealed a striking correlation between [Na+]i elevation and the early membrane hyperpolarization, whereas a significant [Ca2+]i rise matched the depolarizing phase. Interestingly, ion and membrane potential changes were mimicked by ouabain, inhibitor of the Na+-K+ATPase, and were insensitive to tetrodotoxin (TTX) or to a combination of glutamate receptor antagonists. The rotenone effects were partially reduced by blockers of ATP-sensitive K+ channels, glibenclamide and tolbutamide, and largely attenuated by a low Na+-containing solution. Morphological analysis of the rotenone effects on striatal slices showed a significant decrease in the number of choline acetyltransferase (ChAT) immunoreactive cells. These results suggest that rotenone rapidly disrupts the ATP content, leading to a decreased Na+-K+ATPase function and, therefore, to [Na+]i overload. In turn, the hyperpolarizing response might be generated both by the opening of ATP-sensitive K+ channels and by Na+-activated K+ conductances. The increase in [Ca2+]i occurs lately and does not seem to influence the early events.

Activation of beta1-adrenoceptors excites striatal cholinergic interneurons through a cAMP-dependent, protein kinase-independent pathway.

The role of noradrenergic neurotransmission was analyzed in striatal cholinergic interneurons. Conventional intracellular and whole-cell patch-clamp recordings were made of cholinergic interneurons in rat brain slice preparations. Bath-applied noradrenaline (NA) (1-300 microm) dose-dependently induced both an increase in the spontaneous firing activity and a membrane depolarization of the recorded cells. In voltage-clamped neurons, an inward current was induced by NA. This effect was not prevented by alpha-adrenoceptor antagonists, whereas it was mimicked by the beta-adrenoceptor agonist isoproterenol and blocked by the beta1 antagonists propranolol and betaxolol. Interestingly, forskolin, activator of adenylate cyclase, mimicked and occluded the membrane depolarization obtained at saturating doses of both dopamine and NA. Accordingly, SQ22,536, a selective adenylate cyclase inhibitor, reduced the response to NA. Analysis of the reversal potential of the NA-induced current did not provide homogeneous results, indicating the involvement of multiple membrane conductances. Because cAMP is known to modulate Ih, the effects of ZD7288, a selective inhibitor of Ih current, were examined on the NA-induced membrane depolarization/inward current. ZD7288 mostly reduced the response to NA. However, both KT-5720 and H-89, selective protein kinase A (PKA) blockers, failed to prevent the excitatory action of NA. Likewise, calphostin C, antagonist of PKC, genistein, inhibitor of tyrosine kinase, and 8-Bromo-cGMP, blocker of PKG, did not affect the response to NA. Finally, double-labeling experiments combining beta1-adrenoceptor and choline acetyltransferase immunocytochemistry by means of confocal microscopy revealed a strong beta1-adrenoceptor labeling on cholinergic interneurons. We conclude that NA depolarizes striatal cholinergic interneurons via beta1-adrenoceptor activation, through a cAMP-dependent but PKA-independent mechanism.

Adenosine-mediated inhibition of striatal GABAergic synaptic transmission during in vitro ischaemia.

Several reports have shown that energy deprivation, as a result of hypoxia, hypoglycaemia or ischaemia, depresses excitatory synaptic transmission in virtually all brain areas. How this pathological condition affects inhibitory synaptic transmission is still unclear. In the present in vitro study, we coupled whole-cell patch clamp recordings from striatal neurones with focal stimulation of GABAergic nerve terminals in order to characterize the electrophysiological effects of combined oxygen and glucose deprivation (in vitro ischaemia) on inhibitory postsynaptic currents (IPSCs) in this brain area. We found that brief periods (2-5 min) of in vitro ischaemia invariably caused a marked depression of IPSC amplitude. This inhibitory effect was fully reversible on removal of the ischaemic challenge. It was coupled with an increased paired-pulse facilitation, suggesting the involvement of presynaptic mechanisms. Accordingly, the ischaemic inhibition of striatal GABAergic IPSCs was not caused by a shift in the reversal potential of GABA(A)-receptor mediated synaptic currents, and was independ- ent of postsynaptic ATP concentrations. Endogenous adenosine, acting on A1 receptors, appeared responsible for this presynaptic action as the ischaemic depression of IPSCs was prevented by CPT [8-(4-chlorophenylthio) adenosine] and DPCPX, two adenosine A1 receptor antagonists, and mimicked by the application of adenosine in the bathing solution. Conversely, ATP-sensitive potassium channels were not involved in the inhibition of IPSCs by ischaemia, as demonstrated by the fact that tolbutamide and glipizide, two blockers of these channels, were ineffective in preventing this electrophysiological effect. The early depression of GABA-mediated synaptic transmission might play a role in the development of irreversible neuronal injury in the course of brain ischaemia.

An abnormal striatal synaptic plasticity may account for the selective neuronal vulnerability in Huntington's disease.

A marked decrease in the activity of mitochondrial complex II (succinate dehydrogenase, SD) has been found in the brains of Huntington's disease (HD) patients. Here we have examined the possibility that SD inhibitors might produce their toxic action by increasing corticostriatal glutamatergic transmission. We report that SD inhibitors produce a durable augmentation of NMDA-mediated corticostriatal excitation (DANCE) in striatal spiny neurons, but not in striatal cholinergic interneurons. DANCE involves increased intracellular calcium, activation of MAP kinase ERK and is critically dependent upon endogenous dopamine (DA) acting via D2-like receptors. This pathological form of corticostriatal synaptic plasticity might play a key role in the regional and cell-type specific neuronal death observed in HD.

Dopaminergic control of synaptic plasticity in the dorsal striatum.

Cortical glutamatergic and nigral dopaminergic afferents impinge on projection spiny neurons of the striatum, providing the most significant inputs to this structure. Isolated activation of glutamate or dopamine (DA) receptors produces short-term effects on striatal neurons, whereas the combined stimulation of both glutamate and DA receptors is able to induce long-lasting modifications of synaptic excitability. Repetitive stimulation of corticostriatal fibres causes a massive release of both glutamate and DA in the striatum and, depending on the glutamate receptor subtype preferentially activated, produces either long-term depression (LTD) or long-term potentiation (LTP) of excitatory synaptic transmission. D1-like and D2-like DA receptors interact synergistically to allow LTD formation, while they operate in opposition during the induction phase of LTP. Corticostriatal synaptic plasticity is severely impaired after chronic DA denervation and requires the stimulation of DARPP-32, a small protein expressed in dopaminoceptive spiny neurons which acts as a potent inhibitor of protein phosphatase-1. In addition, the formation of LTD and LTP requires the activation of PKG and PKA, respectively, in striatal projection neurons. These kinases appear to be stimulated by the activation of D1-like receptors in distinct neuronal populations.

Dopamine and cAMP-regulated phosphoprotein 32 kDa controls both striatal long-term depression and long-term potentiation, opposing forms of synaptic plasticity.

A complex chain of intracellular signaling events, critically important in motor control, is activated by the stimulation of D1-like dopamine (DA) receptors in striatal neurons. At corticostriatal synapses on medium spiny neurons, we provide evidence that the D1-like receptor-dependent activation of DA and cyclic adenosine 3',5' monophosphate-regulated phosphoprotein 32 kDa is a crucial step for the induction of both long-term depression (LTD) and long-term potentiation (LTP), two opposing forms of synaptic plasticity. In addition, formation of LTD and LTP requires the activation of protein kinase G and protein kinase A, respectively, in striatal projection neurons. These kinases appear to be stimulated by the activation of D1-like receptors in distinct neuronal populations.

Is pharmacological neuroprotection dependent on reduced glutamate release?

BACKGROUND AND PURPOSE: The aim of this study was to determinate the possible role of the ionotropic glutamate receptor in the expression of irreversible electrophysiological changes induced by in vitro ischemia and to test whether the neuroprotective action of various neurotransmitter agonists and drugs of clinical interest is related to a presynaptic inhibitory action at glutamatergic synapses. METHODS: Intracellular and extracellular recordings have been performed in a rat corticostriatal slice preparation. Different pharmacological compounds have been tested on corticostriatal glutamatergic transmission in control conditions and in an in vitro model of ischemia (oxygen and glucose deprivation). RESULTS: In vitro ischemia lasting 10 minutes produced an irreversible loss of the field potential recorded from striatal slices after cortical stimulation. Preincubation of the slices with 3 micromol/L 6-cyano-7-nitroquinoxaline-2,3-dione (an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid [AMPA] receptor antagonist) allowed a significant recovery of the field potential amplitude (P<0.05, n=6), whereas incubation with 30 micromol/L aminophosphonovaleric acid (an N-methyl-D-aspartate receptor antagonist) did not produce a significant recovery after 10 minutes of ischemia (P>0.05, n=7). Bath application of 3 mmol/L glutamate for 5 minutes produced a complete but reversible inhibition of the field potential amplitude. When a similar application was coupled with a brief period of ischemia (5 minutes), which produced, per se, only a transient inhibition of the field potential, it caused an irreversible loss of this parameter. We also tested the possible neuroprotective effect of neurotransmitter agonists reducing the release of glutamate from corticostriatal terminals. Agonists acting on purinergic (adenosine), muscarinic (oxotremorine), and metabotropic glutamate receptors (L-serine o-phosphate [L-SOP]) significantly (P<0.001, n=8 for each agonist) reduced glutamatergic synaptic potentials, with each showing different potencies. The EC(50) was 26.4 micromol/L for adenosine, 0. 08 micromol/L for oxotremorine, and 0.89 micromol/L for L-SOP. Concentrations of these agonists producing the maximal inhibition of the synaptic potential were tested on the ischemia-induced irreversible loss of field potential. Adenosine (P<0.05, n=9) and oxotremorine (P<0.05, n=8) showed significant neuroprotective action, whereas L-SOP was ineffective (P>0.05, n=10). Similarly, putative neuroprotective drugs significantly (P<0.001, n=10 for each drug) reduced the amplitude of corticostriatal potential, with different EC(50) values (phenytoin, 33.5 micromol/L; gabapentin, 96.8 micromol/L; lamotrigine, 26.7 micromol/L; riluzole, 6 micromol/L; and sipatrigine, 2 micromol/L). Concentration of these drugs producing maximal inhibition of the amplitude of corticostriatal potentials showed a differential neuroprotective action on the ischemic electrical damage. Phenytoin (P<0.05, n=10), lamotrigine (P<0.05, n=10), riluzole (P<0.05, n=9), and sipatrigine (P<0.001, n=10) produced a significant neuroprotection, whereas gabapentin (P>0.05, n=11) was ineffective. The neuroprotective action of transmitter agonists and clinical drugs was not related to their ability in decreasing glutamate release, as detected by changes in the paired-pulse facilitation protocol. CONCLUSIONS: Ionotropic glutamate receptors, and particularly AMPA-like receptors, play a role in the irreversible loss of field potential amplitude induced by ischemia in the striatum. Drugs acting by reducing glutamatergic corticostriatal transmission may show a neuroprotective effect. However, their efficacy does not seem to be directly related to their capability to decrease glutamate release from corticostriatal terminals. We suggest that additional modulatory actions on voltage-dependent conductances and on ischemia-induced ion distribution at the postsynaptic site may also exert a crucial role.