James N.C. Kew

A novel mechanism of activity‐dependent NMDA receptor antagonism describes the effect of ifenprodil in rat cultured cortical neurones.

1. Ifenprodil is a selective, atypical non‐competitive antagonist of NMDA receptors that contain the NR2B subunit with an undefined mechanism of action. Ifenprodil is neuroprotective in in vivo models of cerebral ischaemia but lacks many of the undesirable side‐effects associated with NMDA antagonist. 2. Using whole‐cell voltage‐clamp recordings, we have studied the mechanism of inhibition of NMDA‐evoked currents by ifenprodil in rat cultured cortical neurones in the presence of saturating concentrations of glycine. 3. Ifenprodil antagonized NMDA receptors in an activity‐dependent manner, whilst also increasing the receptor affinity for glutamate recognition‐site agonists. Ifenprodil inhibition curves against 10 and 100 microM NMDA‐evoked currents yielded IC50 values of 0.88 and 0.17 microM, respectively. Thus, the apparent affinity of ifenprodil for the NMDA receptor is increased in an NMDA concentration‐dependent manner. 4. Currents evoked by 0.3 and 1 microM NMDA were potentiated to approximately 200% of control levels in the presence of 3 microM ifenprodil. Thus, with increasing concentration of NMDA the effect of ifenprodil on NMDA‐evoked currents changed from one of potentiation to one of increasing inhibition. 5. These results are predicted by a reaction scheme in which ifenprodil exhibits a 39‐ and 50‐fold higher affinity for the agonist‐bound activated and desensitized states of the NMDA receptor, respectively, relative to the resting, agonist‐unbound state. Furthermore, ifenprodil binding to the NMDA receptor results in a 6‐fold higher affinity for glutamate site agonists. 6. This represents a novel mechanism of NMDA receptor antagonism that, together with the subunit selectivity, probably contributes to the attractive neuropharmacological profile of this and related compounds.

Pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent

Atrophy of the medial temporal lobes, including the glutamatergic cortical-hippocampal circuitry, is an early event in Alzheimers disease (AD) and probably contributes to the characteristic short-term mnemonic decline. Pharmacological strategies directly targeted to ameliorating this functional decline may represent a novel approach for the symptomatic treatment of AD. Presynaptic group II metabotropic glutamate receptors (i.e. mGlu2 and mGlu3) exert a powerful modulatory influence on the function of these pathways, in particular the perforant pathway. Using a combination of mGlu2 receptor knockout mice and the group II agonist LY354740, we show that activation of mGlu2 receptors produces a cognitive impairment, i.e. a delay-dependent deficit in delayed matching and non-matching to position, and impaired spatial learning in a Morris water maze. Conversely, a group II antagonist, LY341495, improved acquisition of spatial learning. LY354740 potently reduced field excitatory postsynaptic potentials in hippocampal slices from wild type but not mGlu2 receptor knockout mice. Taken together, these results suggest that activation of mGlu2 receptors evokes a powerful inhibitory effect on hippocampal synaptic transmission and mGlu2 agonists produce a cognitive deficit consistent with this change. Conversely, mGlu2 receptor antagonists may improve certain aspects of cognition and thus represent a novel approach for the symptomatic treatment of AD.

Prepulse inhibition deficits of the startle reflex in neonatal ventral hippocampal–lesioned rats: reversal by glycine and a glycine transporter inhibitor

BACKGROUNDnNeonatal ventral hippocampal (NVH) lesions in rats induce behavioral abnormalities at adulthood thought to simulate some aspects of the positive, negative, and cognitive deficits classically observed in schizophrenic patients. Such lesions induce a postpubertal emergence of prepulse inhibition (PPI) deficits of the startle reflex reminiscent of the sensorimotor gating deficits observed in a majority of schizophrenic patients. To study the potential involvement of the glycinergic neurotransmission in such deficits, we investigated the capacity of glycine (an obligatory N-methyl-D-aspartate [NMDA] receptor co-agonist) and ORG 24598 (a selective glycine transporter 1 inhibitor) to reverse NVH lesion-induced PPI deficits in rats.nnnMETHODSnIbotenic acid was injected bilaterally into the ventral hippocampus of 7-day-old pups. Prepulse inhibition of the startle reflex was measured at adulthood.nnnRESULTSnGlycine (.8 and 1.6 g/kg IP) and ORG 24598 (10 mg/kg IP) fully and partially reversed lesion-induced PPI deficits, respectively.nnnCONCLUSIONSnThese findings confirm that an impaired glutamatergic neurotransmission may be responsible for PPI deficits exhibited by NVH-lesioned rats and support the hypoglutamatergic hypothesis of schizophrenia. They also suggest that drugs acting either directly at the NMDA receptor glycine site or indirectly on the glycine transporter 1 could offer promising targets for the development of novel therapies for schizophrenia.

Activity-dependent presynaptic autoinhibition by group II metabotropic glutamate receptors at the perforant path inputs to the dentate gyrus and CA1.

Pharmacological activation of metabotropic glutamate receptors (mGluRs) can inhibit synaptic transmission; however, relatively little evidence exists regarding the physiological conditions under which such autoreceptors are activated by synaptically released glutamate. Bath application of selective group II mGluR agonists profoundly inhibited field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the perforant path inputs to both the mid-molecular layer of the dentate gyrus and the stratum lacunosum moleculare of the CA1. Application of the group II selective mGluR antagonist LY341495 resulted in an increase in the relative amplitude of a test fEPSP evoked 200 ms after a conditioning burst, but not after a single conditioning stimulus, in both pathways. Antagonist application also resulted in a marked increase in the relative amplitude of test population spikes evoked in the dentate gyrus following a conditioning burst. These observations are consistent with a presynaptic autoinhibitory action of group II metabotropic receptors that is revealed following burst stimulation of the pathway, consistent with their localisation in the preterminal zone. Activation of group II mGluRs during theta-gamma pattern discharge of projection neurones in the entorhinal cortex is likely to play an important role in the regulation of synaptic transmission and plasticity in the perforant pathway.

An allosteric interaction between the NMDA receptor polyamine and ifenprodil sites in rat cultured cortical neurones

1 The atypical NR2B subunit‐selective NMDA receptor antagonist ifenprodil was originally believed to act as a competitive antagonist at the polyamine binding site of the NMDA receptor. However, a number of studies have suggested that ifenprodil might bind to a distinct site. 2 Using whole‐cell voltage clamp recordings, we have studied the interaction of spermine with both ifenprodil and the related NR2B selective antagonist Ro 8–4304 at the NMDA receptor in rat cultured cortical neurones in the presence of saturating concentrations of glycine. 3 Ifenprodil and Ro 8‐4304 inhibited steady‐state currents evoked by 100 μm NMDA in the absence of spermine with IC50 values of 0.3 and 0.6 μm, respectively. In the presence of 1 and 3 mm spermine, IC50 values for ifenprodil were 1.4 and 1.8 μm and for Ro 8‐4304 they were 3.0 and 7.5 μm, respectively. 4 In the presence of spermine, the on‐time constant of receptor blockade by both antagonists was significantly slower than control and the off‐time constant of recovery from receptor blockade following removal of Ro 8‐4304 was significantly faster. 5 Fast application of spermine during an NMDA steady‐state current in the continuous presence of a subsaturating concentration of either antagonist resulted in a biphasic increase in the current, consistent with a fast increase upon spermine binding and a slow increase resultant from dissociation of antagonist due to spermine binding‐induced allosteric reduction in receptor antagonist affinity. In agreement with this, at higher, saturating concentrations of antagonist, the slow increase in current amplitude was markedly reduced or absent. 6 These observations are consistent with a non‐competitive, allosteric interaction between spermine and the antagonists, such that spermine binding to the NMDA receptor results in a reduction in receptor affinity for the antagonists and vice versa. 7 The effects of Mg2+ on the NMDA‐evoked currents and its interaction with ifenprodil were similar to those of spermine, supporting the suggestion that Mg2+ might be the physiological ligand acting at the spermine site mediating glycine‐independent stimulation.

A combined pharmacological and genetic approach to investigate the role of orphanin FQ in learning and memory

Using a combination of the selective opioid receptor‐like1 (ORL1) receptor agonist, Rou200364‐6198, and orphanin FQ/nociceptin (OFQ/N) peptide knockout (KO) mice, the influence of OFQ/N on cognition has been studied in the rodent. In wild type, C57BL/6J mice, Rou200364‐6198 (0.3–1u2003mg/kg i.p.) impaired the acquisition of spatial learning in the Morris water maze, although a mild neurological impairment was evident which complicated precise interpretation. In Lister hooded rats, Rou200364‐6198 (6u2003mg/kg i.p.) produced delay dependent impairments in rats performing either a delayed matching or a delayed nonmatching to position task with only a modest (<u200320%) effect on omissions – an effect consistent with a short‐term memory impairment. Electrophysiological studies demonstrated an inhibitory effect of OFQ/N on LTP recorded from the CA1 region of wild type mice, but not in ORL1 receptor knockout mice. In contrast to the ORL1 agonist, mice deficient in the OFQ/N peptide showed some evidence of improved spatial learning, fear conditioning and passive avoidance retention. However, CA1 LTP was similar between OFQ/N peptide KO mice and wild type controls. Subsequent receptor radioautography studies demonstrated the presence of ORL1 receptors within various regions of the medial temporal lobe system: i.e. CA1, dentate gyrus molecular layer, subiculum, perirhinal cortex. Taken together, these results suggest a bi‐directional effect of OFQ/N containing systems on aspects of cognitive behaviour, particularly those elements associated with hippocampal function. This is consistent with a likely modulatory role of OFQ/N on hippocampal and associated cortical circuitry.

Differential regulation of synaptic transmission by mGlu2 and mGlu3 at the perforant path inputs to the dentate gyrus and CA1 revealed in mGlu2 -/- mice.

Group II metabotropic glutamate (mGlu) receptors can act as presynaptic autoinhibitory receptors at perforant path inputs to the hippocampus under conditions of high frequency synaptic activation. We have used mGlu2 -/- mice to examine the relative roles of mGlu2 and mGlu3 in the regulation of perforant path synaptic transmission mediated by both the selective group II receptor agonist, DCG-IV, and by synaptically released glutamate. Field excitatory postsynaptic potentials evoked by stimulation of either the perforant path inputs to the dentate gyrus mid-moleculare or the CA1 stratum lacunosum moleculare were inhibited by DCG-IV with IC(50) values and maximum percentage inhibition of: 169 nM (60%) and 41 nM (72%) in wild-type mice and 273 nM (19%) and 116 nM (49%) in mGlu2 -/- mice, respectively. Activation of presynaptic group II mGlu autoreceptors by synaptically released glutamate, as revealed by a LY341495-mediated increase in the relative amplitude of a test fEPSP evoked after a conditioning burst, was observed in both the dentate gyrus and the stratum lacunosum of wild-type, but not mGlu2 -/- mice. These observations demonstrate that activation of mGlu3 receptors can regulate synaptic transmission at perforant path synapses but suggest that mGlu2 is the major presynaptic group II autoreceptor activated by synaptically released glutamate.

State‐dependent NMDA receptor antagonism by Ro 8‐4304, a novel NR2B selective, non‐competitive, voltage‐independent antagonist

Subunit‐selective blockade of N‐methyl‐D‐aspartate (NMDA) receptors provides a potentially attractive strategy for neuroprotection in the absence of undesirable side effects. Here, we describe a novel NR2B‐selective NMDA antagonist, 4‐{3‐[4‐(4‐fluoro‐phenyl)‐3,6‐dihydro‐2H‐pyridin‐1‐yl]‐2‐hydroxy‐propoxy}‐benzamide (Ro 8‐4304), which exhibits >100 fold higher affinity for recombinant NR1001/NR2B than NR1001/NR2A receptors. Ro 8‐4304 is a voltage‐independent, non‐competitive antagonist of NMDA receptors in rat cultured cortical neurones and exhibits a state‐dependent mode of action similar to that described for ifenprodil. The apparent affinity of Ro 8‐4304 for the NMDA receptor increased in an NMDA concentration‐dependent manner so that Ro 8‐4304 inhibited 10 and 100u2003μM NMDA responses with IC50s of 2.3 and 0.36u2003μM, respectively. Currents elicited by 1u2003μM NMDA were slightly potentiated in the presence of 10u2003μM Ro 8‐4304, and Ro 8‐4304 binding slowed the rate of glutamate dissociation from NMDA receptors. These results were predicted by a reaction scheme in which Ro 8‐4304 exhibits a 14 and 23 fold higher affinity for the activated and desensitized states of the NMDA receptor, respectively, relative to the agonist‐unbound resting state. Additionally, Ro 8‐4304 binding resulted in a 3–4 fold increase in receptor affinity for glutamate site agonists. Surprisingly, whilst exhibiting a similar affinity for NR2B‐containing NMDA receptors as ifenprodil, Ro 8‐4304 exhibited markedly faster kinetics of binding and unbinding to the NMDA receptor. This spectrum of kinetic behaviour reveals a further important feature of this emerging class of NR2B‐selective compounds.

Long-term transgene expression can be mediated in the brain by adenoviral vectors when powerful neuron-specific promoters are used

Adenoviral (Ad) vectors are one of the most widely used tools for modelling gene therapy strategies. However, they have not been used in long‐term models of neurological disease, as the period of time for which they mediate strong transgene expression is limited and/or variable. In this study we investigated the longevity of transgene expression in the brain when the powerful neuron‐specific Ad‐synapsin (Sy)‐EGFP‐woodchuck hepatitis virus post‐transcriptional regulatory element (WPRE) vector cassette is used at titres that do not elicit an immune response.

Identification and characterization of a novel splice variant of the metabotropic glutamate receptor 5 gene in human hippocampus and cerebellum

The G-protein coupled metabotropic glutamate receptor mGlu5 plays a pivotal role as a modulator of synaptic plasticity, ion channel activity and excitotoxicity. Two splice variants, hmGlu5a and -5b have been reported previously. During screening of a human brain cDNA library for hmGlu5a, we identified a novel variant (hmGlu5d) generated by alternative splicing at the C-terminal domain. The predicted hmGlu5d protein has a C-terminal 267 amino acid shorter than that of hmGlu5a. The pattern of mRNA expression of mGluR5 variants in human brain were analyzed by RT-PCR and in situ hybridization histochemistry. RT-PCR analysis demonstrated the presence of the hmGlu5d transcript, although at low level, in human whole brain, cerebellum, cerebral cortex and hippocampus. [3H]Quisqualate displayed similar affinity at the hmGlu5 splice variants (K(D) values of 80+/-8 and 54+/-17 nM for hmGlu5a and -5d receptors, respectively). For the five mGlu agonists studied, a similar rank order of potency was observed on both hmGlu5a and -5d receptors: quisqualate>glutamate>DHPG>L-CCGI approximately ACPD. MPEP inhibited the glutamate (2 microM)-induced [Ca(2+)](i) response in hmGlu5a and -5d-HEK293 cells also with similar potency (IC(50) values 25+/-1.5 and 20+/-1.4 nM, respectively). Therefore, the large truncation of the C-terminal tail of mGlu5 does not have any apparent major effect on the potency and efficacy of agonists as measured by the [Ca(2+)](i) responses or by activation of recombinant G-protein coupled inwardly rectifying K(+) (GIRK) channel currents. The only major functional difference is the increased sensitivity of hmGlu5d to protein kinase C (PKC)-mediated desensitization, relative to hmGlu5a.