The group 2 metabotropic glutamate receptor agonist LY379268 rescues neuronal, neurochemical and motor abnormalities in R6/2 Huntington's disease mice

Excitotoxic injury to striatum by dysfunctional cortical input or aberrant glutamate uptake may contribute to Huntington's disease (HD) pathogenesis. Since corticostriatal terminals possess mGluR2/3 autoreceptors, whose activation dampens glutamate release, we tested the ability of the mGluR2/3 agonist LY379268 to improve the phenotype in R6/2 HD mice with 120-125 CAG repeats. Daily subcutaneous injection of a maximum tolerated dose (MTD) of LY379268 (20mg/kg) had no evident adverse effects in WT mice, and diverse benefits in R6/2 mice, both in a cohort of mice tested behaviorally until the end of R6/2 lifespan and in a cohort sacrificed at 10weeks of age for blinded histological analysis. MTD LY379268 yielded a significant 11% increase in R6/2 survival, an improvement on rotarod, normalization and/or improvement in locomotor parameters measured in open field (activity, speed, acceleration, endurance, and gait), a rescue of a 15-20% cortical and striatal neuron loss, normalization of SP striatal neuron neurochemistry, and to a lesser extent enkephalinergic striatal neuron neurochemistry. Deficits were greater in male than female R6/2 mice, and drug benefit tended to be greater in males. The improvements in SP striatal neurons, which facilitate movement, are consistent with the improved movement in LY379268-treated R6/2 mice. Our data indicate that mGluR2/3 agonists may be particularly useful for ameliorating the morphological, neurochemical and motor defects observed in HD.     

Glutamate receptor mGlu2 and mGlu3 knockout striata are dopamine supersensitive,with elevated D2High receptors and marked supersensitivity to the dopamine agonist (+)PHNO

The finding that the mGlu2/3 metabotropic glutamate receptor agonist, LY404039, improves clinical symptoms in schizophrenia warrants a search for a possible interaction between mGlu2/3 receptors and dopamine D2 receptors. Here, this topic is examined in striatal tissue of mice lacking either mGlu2 or mGlu3 receptor. Such mice are known to be behaviorally supersensitive to dopamine receptor agonists. Therefore, to determine the basis of this dopamine supersensitivity, the proportion of dopamine D2^High receptors was measured in the striata of mGlu2 and mGlu3 receptor knockout mice. The proportion of D2^High receptors was found to be elevated by 220% in the striata of both knockouts. To measure the functional dopamine supersensitivity, the D2 agonist (+)PHNO was used to stimulate the incorporation of GTP‐γ‐S in the striatal homogenates in the presence of drugs that blocked the dopamine D1, D3, and D5 receptors. Compared with control striata, the mGlu2 receptor knockout tissues were 67‐fold more sensitive to (+)PHNO, while the mGlu3 receptor knockout tissues were 17‐fold more sensitive. These data suggest that group II mGlu receptors—mGlu2 receptors in particular—may normally regulate D2 receptors by reducing the proportion of high‐affinity D2 receptors in membranes. Such regulation may contribute to the antipsychotic action of mGlu2/3 receptor agonists. Synapse 63:247–251, 2009. © 2008 Wiley‐Liss, Inc.     

Blockade of M2‐like muscarinic receptors enhances long‐term potentiation at corticostriatal synapses

Acetylcholine (ACh) exerts a crucial role in learning and memory. The striatum contains the highest concentration of this transmitter in the brain. This structure expresses two different forms of synaptic plasticity, long-term depression (LTD) and long-term potentiation (LTP), which might contribute to the storage of motor skills and some cognitive processes. We have investigated the role of M2-like muscarinic receptors in striatal LTP by utilizing intracellular recordings in vitro from a rat corticostriatal slice preparation. Methoctramine (250 nm ), an antagonist of M2-like muscarinic receptors, enhanced striatal LTP induced in the absence of external magnesium (Mg²⁺) by high-frequency stimulation (HFS) of corticostriatal fibres. Methoctramine did not affect the amplitude of excitatory postsynaptic potentials (EPSPs) when bath applied either before or after the conditioning tetanus suggesting that a critical increase of ACh concentrations is produced only during HFS. Methoctramine per se failed to enhance the NMDA-mediated EPSPs recorded in the absence of external Mg²⁺ and in the presence of 10 μm CNQX. Methoctramine antagonized the presynaptic inhibitory action of neostigmine, an inhibitor of ACh-esterase, and oxotremorine, an agonist of M2-like muscarinic receptors. These data indicate that the activation of M2-like muscarinic receptors exerts a negative influence on striatal LTP, probably by reducing the release of glutamate from corticostriatal fibres and they suggest a complex modulatory effect of ACh in striatal synaptic plasticity.     

Effect of metabotropic glutamate receptor activation on receptor-mediated cyclic AMP responses in primary cultures of rat striatal neurones

Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. -glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA), with EC₅₀ values of 3.41±0.39 and 5.69±1.64 μM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 μM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, -glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC₅₀ values to those observed for the augmentation of NECA cyclic AMP responses (5.19±1.18 and 3.78±1.42 μM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu₅ receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.     

Perinatal exposure to low-dose of bisphenol A causes anxiety-like alteration in adrenal axis regulation and behaviors of rat offspring: A potential role for metabotropic glutamate 2/3 receptors

AimsThe present study focuses on detecting anxiety-like behavior and associated neurochemical alterations in adolescent rats exposed perinatally to bisphenol A (BPA), an estrogen-mimicking endocrine disrupter and investigating the possible involvement of metabotropic glutamate 2/3 receptors (mGlu2/3 receptors) in BPA-induced anxiogenic effects.Methods and resultsWhen female breeders were administered orally with BPA (40 μg/kg/d) during pregnancy and lactation, their pups (here named ‘BPA-exposed offspring’) developed an anxiety-like phenotype, characterized by the hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis, impaired glucocorticoid receptor (GR)-mediated negative feedback regulation of the HPA axis, altered hippocampal synaptic plasticity and increased anxiety-like behaviors. BPA-exposed offspring also showed a reduced expression of mGlu2/3 receptors in the hippocampus. BPA-exposed offspring further subjected to systemic administration of mGlu2/3 receptor agonist (LY379268, 0.5 mg/kg, i.p.) or antagonist (LY341495, 1.5 mg/kg, i.p.) twice per day for 6 days. The results indicated that chronic LY379268 treatment corrected the anxiety-like behaviors and associated neurochemical and endocrinological alterations in BPA-exposed offspring.ConclusionOur data demonstrate for the first time that the perinatal BPA exposure induces an anxiety-like phenotype in behaviors and -related neuroendocrinology, and suggest that the changes in mGlu2/3 receptor might lie at the core of the pathological reprogramming triggered by early-life adversity. mGlu2/3 receptor may serve as a novel biomarker and potential therapeutic target for anxiety disorders associated with adverse early-life agents including perinatal BPA exposure.     

Comparison of the effect of glutamate receptor modulators in the 6 Hz and maximal electroshock seizure models

Glutamatergic ionotropic and metabotropic receptor modulators have been shown to produce anticonvulsant activity in a number of animal seizure models, e.g. maximal electroshock (MES) and DBA/2 sensory-induced seizures. The 6 Hz model of partial seizures is an alternative low frequency, long duration stimulation paradigm resulting in a seizure characterized by jaw and forelimb clonus, immobility, and an elevated tail (Straub-tail). A unique aspect of this model is that it is the only acute electrically-induced seizure model in which levetiracetam has displayed anticonvulsant activity, suggesting that the 6 Hz seizure model may be useful in identifying compounds with unique anticonvulsant profiles. The purpose of the present study was to examine the role of glutamate receptors in the MES and 6 Hz seizure models using a number of NMDA, AMPA/KA, and mGlu receptor modulators. The pharmacological profile of the 6 Hz seizure model was compared to that of the MES model using eight ionotropic glutamate receptor antagonists and eight mGlu receptor modulators. The ionotropic receptor antagonists MK-801, LY235959, NBQX, LY293558, GYKI 52466, LY300168, and LY377770 produced complete protection from tonic extension in the MES model. Furthermore, the noncompetitive mGlu1 (LY456236) and mGlu5 (MPEP) metabotropic receptor antagonists and the mGlu8 metabotropic receptor agonist (PPG) were also effective in the MES model whereas the competitive mGlu1 (LY367385) receptor antagonist, the mGlu2/3 (LY379268 and LY389795) and Group III (L-AP4) metabotropic receptor agonists were ineffective. In contrast, all of the compounds tested, produced dose-dependent protection in the 6 Hz model with an increase in potency as compared to the MES model. The largest protective indices (P.I.=TD50/ED50) observed were associated with the iGlu5 antagonist LY382884 and the mGlu2/3 receptor agonists LY379268 and LY389795 (P.I.=>14, 14, and 4.9, respectively) in the 6 Hz model. The results from the present study support the continued search for glutamate receptor modulators as potential antiepileptic agents. Furthermore these results illustrate the importance of using several different animal seizure models in the search for novel AEDs and the potential utility of the 6 Hz seizure model in identifying novel AEDs.     

Octopamine is the synaptic transmitter between identified neurons in the buccal feeding network of the pond snail Lymnaea stagnalis

We report the pharmacological properties of synaptic connections from the three octopamine-containing OC interneurons to identified buccal feeding neurons in the pond snail, Lymnaea stagnalis. Intracellular stimulation of an OC interneuron evokes inhibitory postsynaptic potentials in the B3 motoneurons and N2 (d) interneurons, while the synapse between OC and N3 (phasic) interneurons has two components: an initial electrical excitation followed by chemical inhibition. All synaptic responses persist in a saline with elevated calcium and magnesium suggesting that the connections are monosynaptic. Local perfusion of 10⁻⁴ M octopamine produces the same inhibitory membrane responses from these buccal neurons as OC stimulation. These responses also persist in high Mg²⁺/Ca²⁺ saline indicating direct membrane effects. The similarities in reversal potentials for the synaptic hyperpolarization evoked on B3 neurons after OC stimulation (−89.0 mV, S.E.M.=14.1, n=10) and the octopamine response of the B3 neurons (−84.7 mV, S.E.M.=6.6, n=6) indicate that increased K⁺-conductance underlies both responses. Bath application of the octopaminergic drugs phentolamine (10⁻⁶ M), epinastine (10⁻⁶ M) or DCDM (10⁻⁴ M) blocks the inhibitory synapse onto B3 or N2 neurons and the chemical component of the N3 response. They also block the octopamine-evoked inhibition of B3, N2 and N3 neurons. NC-7 (2×10⁻⁵ M) has a hyperpolarizing agonist effect (like octopamine) on these neurons and also blocks their chemical synaptic input from the OC interneurons. These results provide pharmacological evidence that the neurotransmitter between the octopamine-immunopositive OC interneurons and its followers is octopamine. This is the first example of identified octopaminergic synaptic connections within the snail CNS.     

PINK1 heterozygous mutations induce subtle alterations in dopamine‐dependent synaptic plasticity

Homozygous or compound heterozygous mutations in the phosphatase and tensin homolog‐induced putative kinase 1 (PINK1) gene are causative of autosomal recessive, early onset Parkinson's disease. Single heterozygous mutations have been detected repeatedly both in a subset of patients and in unaffected individuals, and the significance of these mutations has long been debated. Several neurophysiological studies from non‐manifesting PINK1 heterozygotes have demonstrated the existence of neural plasticity abnormalities, indicating the presence of specific endophenotypic traits in the heterozygous state. We performed a functional analysis of corticostriatal synaptic plasticity in heterozygous PINK1 knockout (PINK1^+/?) mice using a multidisciplinary approach and observed that, despite normal motor behavior, repetitive activation of cortical inputs to striatal neurons failed to induce long‐term potentiation (LTP), whereas long‐term depression was normal. Although nigral dopaminergic neurons exhibited normal morphological and electrophysiological properties with normal responses to dopamine receptor activation, a significantly lower dopamine release was measured in the striatum of PINK1^+/? mice compared with control mice, suggesting that a decrease in stimulus‐evoked dopamine overflow acts as a major determinant for the LTP deficit. Accordingly, pharmacological agents capable of increasing the availability of dopamine in the synaptic cleft restored normal LTP in heterozygous mice. Moreover, monoamine oxidase B inhibitors rescued physiological LTP and normal dopamine release. Our results provide novel evidence for striatal plasticity abnormalities, even in the heterozygous disease state. These alterations might be considered an endophenotype to this monogenic form of Parkinson's disease and a valid tool with which to characterize early disease stage and design possible disease‐modifying therapies. © 2013 International Parkinson and Movement Disorder Society     

Dopamine and 5-HT turnover are increased by the mGlu2/3 receptor agonist LY379268 in rat medial prefrontal cortex, nucleus accumbens and striatum

We have shown, using in vivo microdialysis sampling, that systemic administration of the selective group II metabotropic (mGlu) receptor agonist LY379268, like the atypical antipsychotic clozapine, increased extracellular levels of dopamine, dopamine metabolites DOPAC and HVA, and the major 5-HT metabolite 5-HIAA, in rat medial prefrontal cortex (mPFC). Here, we have compared the effects of LY379268 with clozapine as well as risperidone on ex vivo tissue levels of dopamine, DOPAC, HVA, 5-HT and 5-HIAA in multiple brain regions. One to two hours following administration of LY379268, mPFC tissue levels of DOPAC, HVA and 5-HIAA were increased in a dose-dependent manner. Increases evoked by LY379268 (10 mg/kg s.c.) at the 2 h point were 189, 245 and 139% of basal levels, respectively. These effects were reversed within 4 h of administration. Clozapine (10 mg/kg s.c.) and risperidone (1 mg/kg s. c.) also increased levels of the dopamine metabolites to a similar extent but were without significant effect on tissue levels of 5-HIAA. LY379268 (10 mg/kg s.c.) also increased tissue levels of DOPAC, HVA and 5-HIAA by 169, 221 and 134% of basal levels in nucleus accumbens, respectively, and by 131, 179 and 132% of basal levels in striatum, respectively. These data show that activation of mGlu2/3 receptors can increase the turnover of dopamine and 5-HT in the areas of the brain implicated in the actions of atypical antipsychotics.     

Excitation of rat subthalamic nucleus neurones in vitro by activation of a group I metabotropic glutamate receptor

The subthalamic nucleus (SThN) provides a glutamate mediated excitatory drive to several other component nuclei of the basal ganglia, thereby significantly influencing locomotion and control of voluntary movement. We have characterised functionally the metabotropic glutamate (mGlu) receptors in the SThN using extracellular single unit recording from rat midbrain slices. SThN neurones fired action potentials spontaneously at a rate of 10 Hz which was increased by the group I/II mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD; 1–30 μM) and the group I selective agonist (S,R)-dihydroxyphenylglycine (DHPG; 1–30 μM). However, both the group II selective agonist (1S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; 1 μM) and the group III selective agonist (S)-2-amino-4-phosphonobutanoic acid (L-AP4; 10 μM) were without effect, indicating that the excitation was mediated by a group I mGlu receptor. The excitation caused by DHPG (3 μM) was reversed by co-application of the mGlu receptor antagonist (+)-α-methyl-4-carboxyphenylglycine (MCPG; 500 μM). Thus a group I mGlu receptor mediates excitation of SThN neurones, and suggests a use for group I mGlu receptor ligands for treatment of both hypo- and hyperkinetic disorders of basal ganglia origin, such as Parkinson's disease and Huntington's disease.     

Clinical features and [<Superscript>11</Superscript>C]-CFT PET analysis of PARK2, PARK6, PARK7-linked autosomal recessive early onset Parkinsonism

Mutations in the Parkin, PINK1, and DJ-1 genes can cause autosomal recessive early onset Parkinsonism. We studied three families with the mutations of the Parkin, PINK1 and DJ-1 genes, respectively, with a dopamine transporter ligand [¹¹C]-CFT positron emission tomography. A marked bilaterally and dissymmetrically decrement of [¹¹C]-CFT uptake was found in all these patients, and putamen as well as caudate nucleus was affected. We also found asymptomatic Parkin and PINK1 heterozygotes showed a mild but significant decrement in [¹¹C]-CFT uptake, but this phenomenon was not found in the DJ-1-heterozygotes. Our results suggested the three autosomal recessive forms of early onset are similar to each other on pathophysiological grounds, a sub-clinical disease process in Parkin and PINK1-heterozygotes, but not in DJ-1-heterozygotes.     

Higher free D-aspartate and N-methyl-D-aspartate levels prevent striatal depotentiation and anticipate L-DOPA-induced dyskinesia

In Parkinson's disease (PD) progressive alteration of striatal N-methyl-d-aspartate receptors (NMDARs) signaling has emerged as a considerable factor for the onset of the adverse motor effects of long-term levodopa (l-DOPA) treatment. In this regard, the NMDAR channel blocker amantadine is so far the only drug available for clinical use that attenuates l-DOPA-induced dyskinesia (LID). In this study, we examined the influence of a basal corticostriatal hyper-glutamatergic transmission in the appearance of dyskinesia, using a genetic mouse model lacking d-Aspartate Oxidase (DDO) enzyme (Ddo^−/− mice). We found that, in Ddo^−/− mice, non-physiological, high levels of the endogenous free d-amino acids d-aspartate (d-Asp) and NMDA, known to stimulate NMDAR transmission, resulted in the loss of corticostriatal synaptic depotentiation and precocious expression of LID. Interestingly, the block of depotentiation precedes any change in dopaminergic transmission associated to 6-OHDA lesion and l-DOPA treatment. Indeed, lesioned mutant mice display physiological l-DOPA-dependent enhancement of striatal D1 receptor/PKA/protein phosphatase-1 and ERK signaling. Moreover, in line with synaptic rearrangements of NMDAR subunits occurring in dyskinetic animal models, a short l-DOPA treatment produces a dramatic and selective reduction of the NR2B subunit in the striatal post-synaptic fraction of Ddo^−/− lesioned mutants but not in controls. These data indicate that a preexisting hyper-glutamatergic tone at NMDARs in Ddo^−/− mice produce abnormal striatal synaptic changes that, in turn, facilitate the onset of LID.     

Evidence forN-methyl-d-aspartic acid receptor-mediated modulation of the commissural input to central vestibular neurons of the frog

We have investigated the role ofN-methyl-d-asparte (NMDA) receptors in the excitatory synaptic transmission to central vestibular neurons in the isolated superfused brainstem of the frog. In superfusate containing 1 mM Mg²⁺ field potentials in the vestibular nuclei evoked by electrical stimulation of either the ipsi- or the contralateral VIIIth nerve were not affected by bath-appliedd-2-amino-5-phosphonovaleric acid (D-APV, 25–50 μM), a selective NMDA antagonist. In a low Mg²⁺ solution postsynaptic field potential components were larger than control but still unaffected by D-APV. Ipsi- and contralaterally evoked excitatory postsynaptic potentials (EPSPs) differed in their shape parameters as well as their pharmacological sensitivity. Ipsilaterally evoked EPSPs were not affected by D-APV and had a rise time that was faster than that of contralaterally evoked EPSPs. The peak amplitude of the latter was reduced by D-APV (25–50 μM) to about 65% of the control value in the presence of 1 mM Mg²⁺. During bath application of NMDA (100 μM) an increased input resistance and repetitive de- and hyperpolarizing membrane potential shifts were observed. Similar events were observed during a reduction of the Mg²⁺ concentration. Bath application of NMDA (0.1–1 μM) resulted in an enhanced size of the recorded EPSPs. Dendritic and somatic EPSPs were stimulated on a computer with the assumption of a constant NMDA receptor activation and a pulse-like non-NMDA receptor activation. The results of these stimulations are consistent with the hypothesis that the efficacy of non-NMDA-mediated vestibular commissural synaptic transmission is modulated through tonically activated NMDA receptors.     

The formation of cerebrospinal fluid in two amphibians,Rana catesbeiana andRana pipiens

The cerebrospinal fluid (CSF) system of two amphibians,Rana catesbeiana andRana pipiens has been perfused from the lateral ventricle to a site in the subarachnoid space close to the roof of the fourth ventricle. The CSF production rate (V_f) was measured by the dilution method using a perfusion fluid containing [¹⁴C]dextran in artificial CSF. ForRana catesbeiana V_f was 1.43 μl min⁻¹ or 0.2 μl min⁻¹ mg choroid plexus⁻¹ and forRana pipiens V_f was 0.2 μl min⁻¹ or 0.1 μl min⁻¹ mg chorotid plexus⁻¹. These control values declined by 35% and 14% respectively in the second half of the experimental period. Ouabain (5 × 10^−5M) in the perfusion fluid for the second half of the experiment resulted in a much greater reduction in V_f, viz. 65% forRana catesbeiana and 71% forRana pipiens. Acetazolamide (1 × 10⁻⁴M) in the perfusion fluid for the second half of the experiment gave no change in V_f values when compared with control experiments. Mean ventricular volume measured inRana catesbeiana was74.5 ± 4.8 μl.     

Localization of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor subtypes, and vasopressin in the mouse hypothalamus

The hypothalamic angiotensin II (Ang II) system plays an important role in pituitary hormone release. Little is known about this system in the mouse brain. We studied the distribution of angiotensin-converting-enzyme (ACE), Ang II, Ang II receptor subtypes, and vasopressin in the hypothalamus of adult male mice. Autoradiography of binding of the ACE inhibitor [¹²⁵I]351A revealed low levels of ACE throughout the hypothalamus. Ang II- and vasopressin-immunoreactive neurons and fibers were detected in the paraventricular, accessory magnocellulary, and supraoptic nuclei, in the retrochiasmatic part of the supraoptic nucleus and in the median eminence. Autoradiography of Ang II receptors was performed using [¹²⁵I]Sar¹–Ang II binding. Ang II receptors were present in the paraventricular, suprachiasmatic, arcuate and dorsomedial nuclei, and in the median eminence. In all areas [¹²⁵I]Sar¹–Ang II binding was displaced by the AT₁ receptor antagonist losartan, indicating the presence of AT₁ receptors. In the paraventricular nucleus [¹²⁵I]Sar¹–Ang II binding was displaced by Ang II (K_i=7.6×10⁻⁹) and losartan (K_i=1.4×10⁻⁷) but also by the AT₂ receptor ligand PD 123319 (K_i=5.0×10⁻⁷). In addition, a low amount of AT₂ receptor binding was detected in the paraventricular nucleus using [¹²⁵I]CGP 42112 as radioligand, and the binding was displaced by Ang II (K_i=2.4×10⁻⁹), CGP 42112 (K_i=7.9×10⁻¹⁰), and PD 123319 (K_i=2.2×10⁻⁷). ACE, Ang II, and AT₁ as well as AT₂ receptor subtypes are present in the mouse hypothalamus. Our data are the basis for further studies on the mouse brain Ang II system.     

Effect of glutamate receptor antagonists on excitatory postsynaptic potentials in striatum

Glutamate, as the main transmitter of corticostriatal pathway, has a crucial role in the regulation of the activity of striatal cells as well as in pathogenesis of some diseases characterized by striatal malfunction caused by overexcitation of neurons. In the present study, the role of ionotropic excitatory amino acid receptors was investigated in the striatal synaptic transmission. Using conventional intracellular electrophysiological methods in brain slices, we have investigated the effects of the N-methyl-D-aspartate (NMDA) antagonist (±) 2-amino-5-phosphono-valerate (APV) and the α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) antagonist (±) 1-(4-aminophenyl)-3-methyl-carbamoyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 53655) on the excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of corpus callosum. The AMPA antagonist significantly decreased electrically evoked responses and a weak inhibition was also observed after APV application. The results were compared to similar data obtained in a cortical slice study.     

Potentiation of excitatory postsynaptic potentials by a metabotropic glutamate receptor agonist (1S,3R-ACPD) in frog spinal motoneurons

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg²⁺, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 μM ACPD and in 7 of 12 cells with 0.5 μM ACPD. The EPSP amplitudes with 5 and 0.5 μM ACPD were 142±10% (mean±S.E.M., n=19) and 130±13% (n=12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 μM ACPD were not significantly different in cells perfused with the low Ca²⁺-high Mg²⁺ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence ofd-(−)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg²⁺. Five μM ACPD potentiated monosynaptic EPSPs in 4 of 6 cells with 1 mM Mg²⁺ in the solution and in 2 of 17 cells with 4 mM Mg²⁺, and the EPSP amplitude was 123±9% (n=6) and 98±3% (n=17) of those before application of ACPD, respectively. These results suggest that activation of metabotropic glutamate receptors potentiates LC-EPSPs via mechanisms sensitive to Mg²⁺ and may work as a positive feedback mechanism at the excitatory amino acid-mediated synapses between the descending fibers and lumbar spinal motoneurons.     

Changes in rat serum corticosterone after treatment with metabotropic glutamate receptor agonists or antagonists

From previous work, it appears that glutamate can activate the hypothalamic-pituitary-adrenocortical (HPA) axis by an interaction at either ionotopic or metabotropic (G-protein coupled) receptors. For example, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD), a metabotropic glutamate (mGlu) receptor agonist, has been shown to increase the levels of serum corticosterone in rats. The present study was undertaken to further characterize which of the mGlu receptors are substantially involved in control of the HPA axis. The group I mGlu receptor agonists, 3,5-dihydroxyphenylglycine (DHPG), 1S,3R-ACPD, and 2-chloro-5-hydroxyphenylglycine (CHPG) but not the inactive isomer 1R,3S-ACPD were found to dose-dependently increase serum corticosterone 1 h after intracerebroventricular (i.c.v.) injection in male rats. The relative potency, DHPG (EC50 = 520 nmol) > 1S,3R-ACPD (1.4 micromol) = CHPG (2.7 micromol) > 1R,3S-ACPD (> 3 micromol) is consistent with activation of group I (mGlu1/5) receptors. The effects of DHPG were long lasting with substantial elevations in corticosterone remaining for at least 3 h. In a similar manner, the group III mGlu receptor agonists, L-AP4 (4-phosphono-2-aminobutyric acid) and L-SOP (serine-O-phosphate), were found to increase serum corticosterone levels at 1 h. In contrast, the mGlu group II selective agonists LY354740 (10 mg/kg, i.p.) and subtype-selective doses of the group II antagonist LY341495 (1 mg/kg, i.p.) did not significantly elevate serum corticosterone. Given the group I agonists results, it was surprising to find that group I selective and mGlu1 selective antagonists given alone also increased serum corticosterone. As with the agonists, the rise in serum corticosterone with LY393675 (an mGlu1/5 antagonist, EC50 = 20 nmol, i.c.v.) and LY367385 (an mGlu1 antagonist, 325 nmol, i.c.v.) were dose-dependent and consistent with their relative affinity for the group I mGlu receptors. The selective mGlu5 antagonist MPEP [2-methyl-6-(phenylethylnyl)pyridine] increased serum corticosterone but only at high doses (> 30 mg/kg, i.p.). A model involving the high glutamatergic tone on GABAergic interneurons in the paraventricular nucleus of the hypothalamus is discussed as a possible explanation for these results.     

Endogenous ACh enhances striatal NMDA‐responses via M1‐like muscarinic receptors and PKC activation

Cortical glutamatergic fibres and cholinergic inputs arising from large aspiny interneurons converge on striatal spiny neurons and play a major role in the control of motor activity. We have investigated the interaction between excitatory amino acids and acetylcholine (ACh) on striatal spiny neurons by utilizing intracellular recordings, both in current- and in voltage-clamp mode in rat brain slices. Muscarine (0.3–10 μm ) produced a reversible and dose-dependent increase in the membrane depolarizations/inward currents induced by brief applications of N-methyl-d -aspartate (NMDA), while it did not affect the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-induced responses. These concentrations of muscarine did not alter the membrane potential and the current-voltage relationship of the recorded cells. Neostigmine (0.3–10 μm ), an ACh-esterase inhibitor, mimicked this facilitatory effect. The facilitatory effects of muscarine and neostigmine were antagonized either by scopolamine (3 μm ) or by pirenzepine (10–100 nm ), an antagonist of M1-like muscarinic receptors, but not by methoctramine (300 nm ), an antagonist of M2-like muscarinic receptor. Accordingly, these facilitatory effects were mimicked by McN-A-343 (1–10 μm ), an agonist of M1-like muscarinic receptors, but not by oxotremorine (300 nm ), an agonist of M2-like receptors. Tetrodotoxin (TTX) did not block the facilitatory effect produced by the activation of muscarinic receptors suggesting that this effect is postsynaptically mediated. The action of neostigmine was prevented either by the intracellular calcium (Ca²⁺) chelator BAPTA (200 mm ) or by preincubating the slices with inhibitors of protein kinase C (PKC) (staurosporine 100 nm or calphostin C 1 μm ). McN-A-343 did not alter the excitatory post synaptic potentials (EPSPs) evoked by corticostriatal stimulation in the presence of physiological concentration of magnesium (Mg²⁺ 1.2 mm ), while it enhanced the duration of these EPSPs recorded in the absence of external magnesium. Our data show that endogenous striatal ACh exerts a positive modulatory action on NMDA responses via M1-like muscarinic receptors and PKC activation.