Jon T. Brown

Gabapentin is not a GABAB receptor agonist.

Recent experiments have demonstrated that formation of functional type B gamma-aminobutyric acid (GABA(B)) receptors requires co-expression of two receptor subunits, GABA(B1) and GABA(B2). Despite the identification of these subunits and a number of associated splice variants, there has been little convincing evidence of pharmacological diversity between GABA(B) receptors comprising different subunit combinations. However, Ng et al. [Mol. Pharmacol., 59 (2000) 144] have recently suggested a novel and important pharmacological difference between GABA(B) receptor heterodimers expressing the GABA(B1a) and GABA(B1b) receptor subunits. This study suggested that the antiepileptic GABA analogue gabapentin (Neurontin) is an agonist at GABA(B) receptors expressing the GABA(B1a) but not the GABA(B1b) receptor subunit. The importance of this finding with respect to identifying novel GABA(B) receptor subunit specific agonists prompted us to repeat these experiments in our own [35S]-GTPgammaS binding and second messenger assay systems. Here we report that gabapentin was completely inactive at recombinant GABA(B) heterodimers expressing either GABA(B1a) or GABA(B1b) receptor subunits in combination with GABA(B2) receptor subunits. In addition, in both CA1 and CA3 pyramidal neurones from rodent hippocampal slices we were unable to demonstrate any agonist-like effects of gabapentin at either pre- or post-synaptic GABA(B) receptors. In contrast, gabapentin activated a GABA(A) receptor mediated chloride conductance. Our data suggest that gabapentin is not a GABA(B)-receptor agonist let alone a GABA(B) receptor subunit selective agonist.

Little or no ability of obestatin to interact with ghrelin or modify motility in the rat gastrointestinal tract

Obestatin, encoded by the ghrelin gene may inhibit gastrointestinal (GI) motility. This activity was re‐investigated.

LPA1 receptor-deficient mice have phenotypic changes observed in psychiatric disease

Several psychiatric diseases, including schizophrenia, are thought to have a developmental aetiology, but to date no clear link has been made between psychiatric disease and a specific developmental process. LPA(1) is a G(i)-coupled seven transmembrane receptor with high affinity for lysophosphatidic acid. Although LPA(1) is expressed in several peripheral tissues, in the nervous system it shows relatively restricted temporal expression to neuroepithelia during CNS development and to myelinating glia in the adult. We report the detailed neurological and behavioural analysis of mice homozygous for a targeted deletion at the lpa(1) locus. Our observations reveal a marked deficit in prepulse inhibition, widespread changes in the levels and turnover of the neurotransmitter 5-HT, a brain region-specific alteration in levels of amino acids, and a craniofacial dysmorphism in these mice. We suggest that the loss of LPA(1) receptor generates defects resembling those found in psychiatric disease.

Characterization of a CNS penetrant, selective M1 muscarinic receptor agonist, 77-LH-28-1

M1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimers disease and schizophrenia. However, the discovery of subtype‐selective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh‐binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC‐42 (4‐n‐butyl‐1‐[4‐(2‐methylphenyl)‐4‐oxo‐1‐butyl]‐piperidine), which bind to an allosteric site and selectively activate the M1 mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs.

Targeting Gut T Cell Ca2+ Release-Activated Ca2+ Channels Inhibits T Cell Cytokine Production and T-Box Transcription Factor T-Bet in Inflammatory Bowel Disease

Prolonged Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels is crucial in activating the Ca2+-sensitive transcription factor NFAT, which is responsible for directing T cell proliferation and cytokine gene expression. To establish whether targeting CRAC might counteract intestinal inflammation, we evaluated the in vitro effect of a selective CRAC inhibitor on T cell cytokine production and T-bet expression by lamina propria mononuclear cells (LPMC) and biopsy specimens from inflammatory bowel disease (IBD) patients. The inhibitory activity of the CRAC blocker was investigated through patch-clamp experiments on rat basophilic leukemia cells and fluorometric imaging plate reader intracellular Ca2+ assays using thapsigargin-stimulated Jurkat T cells and its detailed selectivity profile defined using a range of in vitro radioligand binding and functional assays. Anti-CD3/CD28-stimulated LPMC and biopsy specimens from 51 patients with IBD were cultured with a range of CRAC inhibitor concentrations (0.01–10 μM). IFN-γ, IL-2, IL-8, and IL-17 were analyzed by ELISA. T-bet was determined by immunoblotting. We found that the CRAC blocker concentration-dependently inhibited CRAC current in rat basophilic leukemia cells and thapsigargin-induced Ca2+ influx in Jurkat T cells. A concentration-dependent reduction in T-bet expression and production of IFN-γ, IL-2, IL-17, but not IL-8, was observed in IBD LPMC and biopsy specimens treated with the CRAC inhibitor. In conclusion, we provide evidence that the suppression of CRAC channel function may dampen the increased T cell response in the inflamed gut, thus suggesting a promising role for CRAC inhibitor drugs in the therapeutic management of patients with IBD.

Altered intrinsic neuronal excitability and reduced Na+ currents in a mouse model of Alzheimer's disease

Transgenic mice that overproduce beta-amyloid (Aβ) peptides can exhibit central nervous system network hyperactivity. Patch clamp measurements from CA1 pyramidal cells of PSAPP and wild type mice were employed to investigate if altered intrinsic excitability could contribute to such network hyperfunction. At approximately 10 months, when PSAPP mice have a substantial central nervous system Aβ load, resting potential and input resistance were genotype-independent. However, PSAPP mice exhibited a substantially more prominent action potential (AP) burst close to the onset of weak depolarizing current stimuli. The spike afterdepolarization (ADP) was also larger in PSAPP mice. The rate of rise, width and height of APs were reduced in PSAPP animals; AP threshold was unaltered. Voltage-clamp recordings from nucleated macropatches revealed that somatic Na(+) current density was depressed by approximately 50% in PSAPP mice. K(+) current density was unaltered. All genotype-related differences were absent in PSAPP mice aged 5-7 weeks which lack a substantial Aβ load. We conclude that intrinsic neuronal hyperexcitability and changes to AP waveforms may contribute to neurophysiological deficits that arise as a consequence of Aβ accumulation.

Old and New Pharmacology: Positive Allosteric Modulation of the α7 Nicotinic Acetylcholine Receptor by the 5-Hydroxytryptamine2B/C Receptor Antagonist SB-206553 (3,5-Dihydro-5-methyl-N-3-pyridinylbenzo[1,2-b:4,5-b′]di pyrrole-1(2H)-carboxamide)

The α7 nicotinic acetylcholine receptor (nAChR) has been implicated in Alzheimers disease and schizophrenia, leading to efforts targeted toward discovering agonists and positive allosteric modulators (PAMs) of this receptor. In a Ca2+ flux fluorometric imaging plate reader assay, SB-206553 (3,5-dihydro-5-methyl -N-3-pyridinylbenzo [1, 2-b:4,5 -b′]-di pyrrole-1(2H)-carboxamide), a compound known as a 5-hydroxytryptamine2B/2C receptor antagonist, produced an 8-fold potentiation of the evoked calcium signal in the presence of an EC20 concentration of nicotine and a corresponding EC50 of 1.5 μM for potentiation of EC20 nicotine responses in GH4C1 cells expressing the α7 receptor. SB-206553 was devoid of direct α7 receptor agonist activity and selective against other nicotinic receptors. Confirmation of the PAM activity of SB-206553 on the α7 nAChR was obtained in patch-clamp electrophysiological experiments in GH4C1 cells, where it failed to evoke any detectable currents when applied alone, yet dramatically potentiated the currents evoked by an EC20 (17 μM) and EC100 (124 μM) of acetylcholine (ACh). Native nicotinic receptors in CA1 stratum radiatum interneurons of rat hippocampal slices could also be activated by ACh (200 μM), an effect that was entirely blocked by the α7-selective antagonist methyllycaconitine (MLA). These ACh currents were potentiated by SB-206553, which increased the area of the current response significantly, resulting in a 40-fold enhancement at 100 μM. In behavioral experiments in rats, SB-206553 reversed an MK-801 (dizocilpine maleate)-induced deficit in the prepulse inhibition of acoustic startle response, an effect attenuated in the presence of MLA. This latter observation provides further evidence in support of the potential therapeutic utility of α7 nAChR PAMs in schizophrenia.

TRPM2 Is Elevated in the tMCAO Stroke Model, Transcriptionally Regulated, and Functionally Expressed in C13 Microglia

We report the detailed expression profile of TRPM2 mRNA within the human central nervous system (CNS) and demonstrate increased TRPM2 mRNA expression at 1 and 4 weeks following ischemic injury in the rat transient middle cerebral artery occlusion (tMCAO) stroke model. Microglial cells play a key role in pathology produced following ischemic injury in the CNS and possess TRPM2, which may contribute to stroke-related pathological responses. We show that TRPM2 mRNA is present in the human C13 microglial cell line and is reduced by antisense treatment. Activation of C13 cells by interleukin-1β leads to a fivefold increase of TRPM2 mRNA demonstrating transcriptional regulation. To confirm mRNA distribution correlated with functional expression, we combined electrophysiology, Ca2+ imaging, and antisense approaches. C13 microglia exhibited, when stimulated with hydrogen peroxide (H2O2), increased [Ca2+]i, which was reduced by antisense treatment. Moreover, patch-clamp recordings from C13 demonstrated that increased intracellular adenosine diphosphoribose (ADPR) or extracellular H2O2 induced an inward current, consistent with activation of TRPM2. In addition we confirm the functional expression of a TRPM2-like conductance in primary microglial cultures derived from rats. Activation of TRPM2 in microglia during ischemic brain injury may mediate key aspects of microglial pathophysiological responses.

Synaptic activation of GABAB receptors regulates neuronal network activity and entrainment

In the mammalian central nervous system, GABAB receptors mediate slow pre‐ and postsynaptic inhibition. Using rat hippocampal slices we investigated the role of synaptic GABAB receptors in regulating kainate‐induced subthreshold neuronal network oscillations in the gamma frequency range (25–80 Hz). The GABAB receptor agonist baclofen largely eliminated gamma oscillations. The GABAB receptor antagonist CGP55845 reversed this action of baclofen but alone did not alter the power or frequency of ongoing oscillations. To examine the role of synaptically released GABA on network activity, we electrically stimulated stratum radiatum of CA3 whilst recording gamma oscillations from stratum pyramidale. Single stimuli produced a pronounced transient (up to 1 s in duration) inhibition of gamma frequency oscillations. This stimulus‐induced shutdown of network activity was enhanced by the GABA uptake inhibitor tiagabine and largely inhibited by CGP55845. Multiple stimuli delivered at frequencies of 1–3 Hz resulted in an activity‐dependent fatigue of the inhibition of gamma activity, such that, after a number of stimuli, oscillations could be detected tens of milliseconds after the stimulus. Interestingly, this activity‐dependent fatigue of inhibition uncovered a stimulus‐dependent temporal entrainment of the gamma oscillations. Furthermore, the amount of repetitive synaptic input that was required to cause this entrainment was dramatically reduced by GABAB receptor antagonism such that it was evident within just a few stimuli. These data suggest that convergent afferent synaptic activity can alter the precise temporal arrangement of neuronal network activity. Furthermore, the flow of such information into a functioning neuronal network is highly regulated by GABAB receptor‐mediated synaptic inhibition.

The functional neurophysiology of the amyloid precursor protein (APP) processing pathway

Amyloid beta (Abeta) peptides derived from proteolytic cleavage of amyloid precursor protein (APP) are thought to be a pivotal toxic species in the pathogenesis of Alzheimers disease (AD). Furthermore, evidence has been accumulating that components of APP processing pathway are involved in non-pathological normal function of the CNS. In this review we aim to cover the extensive body of research aimed at understanding how components of this pathway contribute to neurophysiological function of the CNS in health and disease. We briefly outline changes to clinical neurophysiology seen in AD patients before discussing functional changes in mouse models of AD which range from changes to basal synaptic transmission and synaptic plasticity through to abnormal synchronous network activity. We then describe the various neurophysiological actions that are produced by application of exogenous Abeta in various forms, and finally discuss a number or other neurophysiological aspects of the APP pathway, including functional activities of components of secretase complexes other than Abeta production.