Depolarisation and suppression of burst firing activity in the mouse subthalamic nucleus by dopamine D1/D5 receptor activation of a cyclic-nucleotide gated non-specific cation conductance

Neuronal burst firing in the subthalamic nucleus (STN) is one of the hallmarks of dopamine depletion in Parkinson's disease. Here, we have determined the postsynaptic effects of dopamine in the STN and the functional consequences of dopamine receptor modulation on burst firing in vitro. STN cells displayed regular spiking activity at a rate of 7.9 +/- 0.5 Hz. Application of dopamine (30 mu M) induced membrane depolarisations accompanied by an increase in firing rate of mean 12.0 +/- 0.6 Hz in all 69 cells. The dopamine effect was mimicked by the dopamine D1/D5 receptor agonist SKF38393 (10 mu M, 17 cells) and the dopamine D2-like receptor agonist quinpirole (10 mu M, 35 cells), partly reduced by D1/D5 antagonist SCH23390 (2 mu M, seven cells), but unaffected by the D2 antagonists sulpiride (10 mu M, seven cells) or eticlopride (10 mu M, six cells). Using voltage ramps, dopamine induced an inward current of 69 +/- 9.4 pA at a holding potential of -60 mV (n = 17). This current was accompanied by an increase in input conductance of 1.55 +/- 0.35 nS which reversed at -30.6 +/- 2.3 mV, an effect mimicked by SKF38393 (10 AM, nine cells). Similar responses were observed when measuring instantaneous current evoked by voltage steps and in the presence of the I-h blocker, ZD7288, indicating effects independent of I-h. The increase in conductance was blocked by SCH23390 (2 mu M, n = 4), mimicked by the activator of adenylyl cyclase forskolin (10 mu M, n = 7) and blocked by H-89, an inhibitor of cyclic AMP dependent protein kinase A (10 PM, n = 6). These results indicate that the dopamine depolarisation is in part mediated by D1/D5 receptor mediated activation of a cyclic-nucleotide gated (CNG) non-specific cation conductance. This conductance contributes to the membrane depolarisation that changes STN neuronal bursting to more regular activity by significantly increasing burst duration and number of spikes per burst.

Functional presynaptic HCN channels in the rat globus pallidus

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are expressed postsynaptically in the rodent globus pallidus (GP), where they play several important roles in controlling GP neuronal activity. To further elucidate the role of HCN channels in the GP, immunocytochemical and electrophysiological approaches were used to test the hypothesis that HCN channels are also expressed presynaptically on the local axon collaterals of GP neurons. At the electron microscopic level, immunoperoxidase labelling for HCN1 and HCN2 was localized in GP somata and dendritic processes, myelinated and unmyelinated axons, and axon terminals. One population of labelled terminals formed symmetric synapses with somata and proximal dendrites and were immunoreactive for parvalbumin, consistent with the axon collaterals of GABAergic GP projection neurons. In addition, labelling for HCN2 and, to a lesser degree, HCN1 was observed in axon terminals that formed asymmetric synapses and were immunoreactive for the vesicular glutamate transporter 2. Immunogold labelling demonstrated that HCN1 and HCN2 were located predominantly at extrasynaptic sites along the plasma membrane of both types of terminal. To determine the function of presynaptic HCN channels in the GP, we performed whole-cell recordings from GP neurons in vitro. Bath application of the HCN channel blocker ZD7288 resulted in an increase in the frequency of mIPSCs but had no effect on their amplitude, implying that HCN channels tonically regulate the release of GABA. Their presence, and predicted role in modulating transmitter release, represents a hitherto unidentified mechanism whereby HCN channels influence the activity of GP neurons. © The Authors (2007).

Simultaneous estimation of global background synaptic inhibition and excitation from membrane potential fluctuations in layer III neurons of the rat entorhinal cortex in vitro

It is becoming clear that the detection and integration of synaptic input and its conversion into an output signal in cortical neurons are strongly influenced by background synaptic activity or "noise." The majority of this noise results from the spontaneous release of synaptic transmitters, interacting with ligand-gated ion channels in the postsynaptic neuron [Berretta N, Jones RSG (1996); A comparison of spontaneous synaptic EPSCs in layer V and layer II neurones in the rat entorhinal cortex in vitro. J Neurophysiol 76:1089-1110; Jones RSG, Woodhall GL (2005) Background synaptic activity in rat entorhinal cortical neurons: differential control of transmitter release by presynaptic receptors. J Physiol 562:107-120; LoTurco JJ, Mody I, Kriegstein AR (1990) Differential activation of glutamate receptors by spontaneously released transmitter in slices of neocortex. Neurosci Lett 114:265-271; Otis TS, Staley KJ, Mody I (1991) Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release. Brain Res 545:142-150; Ropert N, Miles R, Korn H (1990) Characteristics of miniature inhibitory postsynaptic currents in CA1 pyramidal neurones of rat hippocampus. J Physiol 428:707-722; Salin PA, Prince DA (1996) Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex. J Neurophysiol 75:1573-1588; Staley KJ (1999) Quantal GABA release: noise or not? Nat Neurosci 2:494-495; Woodhall GL, Bailey SJ, Thompson SE, Evans DIP, Stacey AE, Jones RSG (2005) Fundamental differences in spontaneous synaptic inhibition between deep and superficial layers of the rat entorhinal cortex. Hippocampus 15:232-245]. The function of synaptic noise has been the subject of debate for some years, but there is increasing evidence that it modifies or controls neuronal excitability and, thus, the integrative properties of cortical neurons. In the present study we have investigated a novel approach [Rudolph M, Piwkowska Z, Badoual M, Bal T, Destexhe A (2004) A method to estimate synaptic conductances from membrane potential fluctuations. J Neurophysiol 91:2884-2896] to simultaneously quantify synaptic inhibitory and excitatory synaptic noise, together with postsynaptic excitability, in rat entorhinal cortical neurons in vitro. The results suggest that this is a viable and useful approach to the study of the function of synaptic noise in cortical networks. © 2007 IBRO.

An investigation into the synaptic conditions and celluar mechanisms underlying cerebellar synaptic plasticity

The direction of synaptic plasticity at the connection between parallel fibres (PFs) and Purkinje cells can be modified by PF stimulation alone. Strong activation (Hartell, 1996) or high frequency stimulation (Schreurs and Alkon, 1993) of PFs induced a long-term depression (LTD) of PF-mediated excitatory postsynaptic currents. Brief raised frequency molecular layer stimulation produced a cAMP-dependent long-temi potentiation (LTP) of field potential (FP) responses (Salin et al., 1998). Thin slices of cerebellar vermis were prepared from 14-21 day old male Wistar rats decapitated under Halothane anaesthesia. FP's were recorded from the Purkinje cell layer in response to alternate 0.2Hz activation of stimulating electrodes placed in the molecular layer. In the presence of picrotoxin, FPs displayed two tetrodotoxin-sensitive, negative-going components termed N1 and N2. EPs were graded responses with paired pulse facilitation and were selectively blocked by 101AM 6-cyano-7-nitroquinoxaline-2,3-dicne (CNQX) an antagonist at iy,-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type ionotropic glutamate receptors (AMPAR) suggesting that they were primarily PE-mediated. The effects of raised stimulus intensity (RS) and/or increased frequency (IF) activation of the molecular layer on FP responses were examined. In sagittai and transverse slices combined RS and IF molecular layer activation induced a LTD of the N2 component of FP responses. RSIF stimulation produced fewer incidences of LTD in sagittal slices when an inhibitor of nitric oxide synthase (NOS), guanylate cyclase (GC), protein kinase G (PKG) or the GABAB receptor antagonist CGP62349 was included into the perfusion medium. Application of a nitric oxide (NO) donor, a cyclic guanosine monophosphate (cGMP) analogue or a phosphodiesterase (PDE) type V inhibitor to prevent cGMP breakdown paired with IF stimulation produced an acute depression, Raised frequency (RF) molecular layer stimulation produced a slowly emerging LTD of N2 in sagittal slices that was largely blocked in the presence of NOS, cGMP or PKG inhibitors. In transverse slices RE stimulation produced a LTP of the N2 component that was prevented by an inhibitor of protein kinase A or NOS. Inhibition of cGMP-signalling frequently revealed an underlying potentiation suggesting that cGMP activity might mask the effects of cAMP. In sagittal slices RE stimulation resulted in a potentiation of FPs when the cAMP-specific PDE type IV inhibitor rolipram was incorporated into the perfusion medium. In summary, raised levels of PE stimulation can alter the synaptic efficacy at PF-Purkinje cell synapses. The results provide support for a role of NO/cGMP/PKG signalling in the induction of LTD in the cerebellar cortex and suggest that activation of GABAa receptors might also be important. The level of cyclic nucleotide-specific PDE activities may be crucial in determining the level of cGMP and CAMP activity and hence the direction of synaptic plasticity.

The heteromeric 5-HT3A/B receptor:the effect of 5-HT3B subunits on receptor structure and ligand recognition

The 5-HT3 receptors are members of the cys-loop family of ligand-gated ion channels. Two functional subtypes are known, the homomeric 5HT3A and the heteromeric 5HT3A/B receptors, which exhibit distinct biophysical characteristics but are difficult to differentiate pharmacologically. Atomic force microscopy has been used to determine the stoichiometry and architecture of the heteromeric 5HT3A/B receptor. Each subunit was engineered to express a unique C-terminal epitope tag, together with six sequential histidine residues to facilitate nickel affinity purification. The 5-HT3 receptors, ectopically expressed in HEK293 cells, were solubilised, purified and decorated with antibodies to the subunit specific epitope tags. Imaging of individual receptors by atomic force microscopy revealed a pentameric arrangement of subunits in the order BBABA, reading anti-clockwise when viewed from the extracellular face. Homology models for the heteromeric receptor were then constructed using both the electron microscopic structure of the nicotinic acetylcholine receptor, from Torpedo marmorata, and the X-ray crystallographic structure of the soluble acetylcholine binding protein, from Lymnaea stagnalis, as templates. These homology models were used, together with equivalent models constructed for the homomeric receptor, to interpret mutagenesis experiments designed to explore the minimal recognition differences of both the natural agonist, 5-HT, and the competitive antagonist, granisetron, for the two human receptor subtypes. The results of this work revealed that the 5-HT3B subunit residues within the ligand binding site, for both the agonist and antagonist, are accommodating to conservative mutations. They are consistent with the view that the 5-HT3A subunit provides the principal and the 5-HT38 subunit the complementary recognition interactions at the binding interface.

The control of mitosis in mammalian tissues

The question of which factors are central in determining whether a cell will undertake a new round of mitosis or will decycle has been examined in the isolated thymic lymphocyte model. Such cell populations possess both in vivo and in vitro a subpopulation of quiescent lymphoblasts which may be induced to reinitiate their mitotic programme. In the intact animal the major determinant of proliferative activity is the plasma ionised calcium concentration. However it has been established in culture that a variety of hormones, ions, cyclic nucleotides, plant lectins and ionophores may like calcium elicit a mitogenic response. These agents do not appear however to initiate DNA synthesis in an identical fashion. Rather there are two distinct intracellular mitogenic axes. The first axis includes a number of adenylate cyclase stimulants, cyclic AMP, phosphodiesterase inhibitors and magnesium ions. It was found that all these mitogens required extracellular magnesium ions to exhibit their stimulatory capacity. This dichotomy in mitogenic activity was further emphasised by the observation that these mitogens are all inhibited by testosterone, whilst the magnesium-independent mitogens were insensitive to this androgen. Indeed this second group of stimulatory factors required the presence of calcium ions in the extracellular milieu for activity, and were, in contrast to the magnesium-dependent mitogens inhibited by the presence of oestradiol in the culture. By examining the interrelationships between these various mitogens and inhibitors it has been possible to propose a mechanism to describe the activation process in the thymocyte. Studies of the metabolism of cyclic nucleotides, membrane potential and transmembrane ion fluxes indicate that there may be a complex relationship between membrane fluidity, ion balance and cyclic nucleotide levels which may individually or in concert promote the initiation of DNA synthesis. A number of possible mechanisms are discussed to account for these observations.

Diverse antiepileptic drugs increase the ratio of background synaptic inhibition to excitation and decrease neuronal excitability in neurones of the rat entorhinal cortex in vitro

Although most anti-epileptic drugs are considered to have a primary molecular target, it is clear that their actions are unlikely to be limited to effects on a single aspect of inhibitory synaptic transmission, excitatory transmission or voltage-gated ion channels. Systemically administered drugs can obviously simultaneously access all possible targets, so we have attempted to determine the overall effect of diverse agents on the balance between GABAergic inhibition, glutamatergic excitation and cellular excitability in neurones of the rat entorhinal cortex in vitro. We used an approach developed for estimating global background synaptic excitation and inhibition from fluctuations in membrane potential obtained by intracellular recordings. We have previously validated this approach in entorhinal cortical neurones [. Greenhill and Jones (2007a) Neuroscience 147:884-892]. Using this approach, we found that, despite their differing pharmacology, the drugs tested (phenytoin, lamotrigine, valproate, gabapentin, felbamate, tiagabine) were unified in their ability to increase the ratio of background GABAergic inhibition to glutamatergic excitation. This could occur as a result of decreased excitation concurrent with increased inhibition (phenytoin, lamotrigine, valproate), a decrease in excitation alone (gabapentin, felbamate), or even with a differential increase in both (tiagabine). Additionally, we found that the effects on global synaptic conductances agreed well with whole cell patch recordings of spontaneous glutamate and GABA release (our previous studies and further data presented here). The consistency with which the synaptic inhibition:excitation ratio was increased by the antiepileptic drugs tested was matched by an ability of all drugs to concurrently reduce intrinsic neuronal excitability. Thus, it seems possible that specific molecular targets among antiepileptic drugs are less important than the ability to increase the inhibition:excitation ratio and reduce overall neuronal and network excitability. © 2010 IBRO.

IUPHAR-DB:the IUPHAR database of G protein-coupled receptors and ion channels

The IUPHAR database (IUPHAR-DB) integrates peer-reviewed pharmacological, chemical, genetic, functional and anatomical information on the 354 nonsensory G protein-coupled receptors (GPCRs), 71 ligand-gated ion channel subunits and 141 voltage-gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes represent the targets of approximately one-third of currently approved drugs and are a major focus of drug discovery and development programs in the pharmaceutical industry. IUPHAR-DB provides a comprehensive description of the genes and their functions, with information on protein structure and interactions, ligands, expression patterns, signaling mechanisms, functional assays and biologically important receptor variants (e.g. single nucleotide polymorphisms and splice variants). In addition, the phenotypes resulting from altered gene expression (e.g. in genetically altered animals or in human genetic disorders) are described. The content of the database is peer reviewed by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR); the data are provided through manual curation of the primary literature by a network of over 60 subcommittees of NC-IUPHAR. Links to other bioinformatics resources, such as NCBI, Uniprot, HGNC and the rat and mouse genome databases are provided. IUPHAR-DB is freely available at http://www.iuphar-db.org. © 2008 The Author(s).

The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

The impact of the CACNA1C gene polymorphism on frontolimbic function in bipolar disorder

Genome-wide association studies in bipolar disorder (BD)1 have implicated a single-nucleotide polymorphism (rs1006737, G right arrow A) in the CACNA1C gene, which encodes for the alpha 1c (CAV1.2) subunit of the voltage-gated, L-type calcium channel. Neuroimaging studies of healthy individuals report that this risk allele modulates brain function within limbic (amygdala, anterior cingulate gyrus) and hippocampal regions during tasks of reward processing2, 3 and episodic memory. Moreover, animal studies suggest that the CaV1.2 L-type calcium channels influence emotional behaviour through enhanced neurotransmission via the lateral amygdala pathway. On the basis of this evidence, we tested the hypotheses that the CACNA1C rs1006737 risk allele will modulate neural responses within predefined prefrontal and subcortical regions of interest during emotional face processing and that this effect would be amplified in BD patients.