In vitro activities of novel oxapenems, alone and in combination with ceftazidime, against gram-positive and gram-negative organisms

Four novel oxapenem compounds (i.e., AM-112, AM-113, AM-114, and AM-115) were investigated for their β-lactamase inhibitory activity against a panel of isolated class A, C, and D enzymes, which included expanded-spectrum β-lactamase enzymes (ESBLs). The oxapenems were potent β-lactamase inhibitors. Activity varied within the group, with AM-113 and AM-114 proving to be the most active compounds. The 50% inhibitory concentrations for these agents were up to 100,000-fold lower than that of clavulanic acid against class C and D enzymes. As a group, the oxapenems were more potent than clavulanic acid against enzymes from all classes. The ability of these compounds to protect ceftazidime from hydrolysis by β-lactamase-producing strains was evaluated by MIC tests that combined ceftazidime and each oxapenem in a 1:1 or 2:1 ratio. The oxapenems markedly reduced the MICs for ceftazidime against class C hyperproducing strains and strains producing TEM- and SHV-derived ESBLs. There was little difference between the activity of 1:1 and 2:1 combinations of ceftazidime and oxapenem. The oxapenems failed to enhance the activity of ceftazidime against derepressed AmpC-producing Pseudomonas aeruginosa strains.

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.

Novel biological roles for pyrimidines

The development of classical and lipophilic inhibitors of dihydrofolate reductase (DHFR) as antitumour agents is reviewed and the advantages and problems associated with each class are discussed. The antitumour activity, pharmacokinetics and metabolism of m-azido-pyrimethamine (MZP), a novel lipophilic inhibitor, are considered and compared with metoprine, the prototype lipophilic antifolate. Evidence for a folate-independent target for lipophilic DHFR inhibitors is presented. Synthetic studies centred on three principal objectives. Firstly a series of structural analogues of MZP were prepared encompassing alkoxy, chloro and alkylamino substituents and evaluated, as the ethanesulphonate salts, for activity against mammalian DHFR. Inhibitory constant (KI) determinations were conducted by a Zone B analysis, the corresponding 4'-azido isomer of MZP proving more potent than the parent compound. Secondly, to facilitate metabolism and stability studies on MZP, a range of possible reference compounds were synthesised and characterised. Finally, a series of diaminopyrimidine derivatives were synthesised embracing structural features incompatible with DHFR inhibitory activity, in order that such compounds may serve as biochemical probes for the unidentified folate-independent target for lipophilic diaminopyrimidines discussed previously. Inactivity against DHFR was achieved via introduction of an ionic or basic group into a normally hydrophobic region of the molecule and compounds were screened against a mammalian DHFR and thymidylate synthase to confirm the abolition of activity. Several derivatives surprisingly proved potent inhibitors of DHFR exhibiting KI values comparable to that of methotrexate. Analogues were screened for antitumour activity in vitro and in vivo against murine leukaemia cell lines in order to identify potential lead compounds. Several derivatives virtually inactive against DHFR exhibited a disparate cytotoxicity and further biochemical studies are warranted. The nobreak hitherto unreported debenzylation of 2,4-diamino-5-(N-alkyl-benzylaminophenyl) pyrimidines was discovered during the course of the synthetic studies, treatment of these compounds with nitrous acid affording the corresponding benzotriazoles.

Synthesis and biological properties of 2-phenylbenzothiazole derivatives

2-Phenylbenzothiazoles have structural similarities to the antioestrogenic 2-phenylindole, zindoxifene and to the oestrogenic isoflavone, genistein which also inhibits tyrosine kinases. Hydroxylated 2-phenylbenzothiazole derivatives were therefore produced and tested for oestrogenic and tyrosine kinase inhibitory activity. Synthesis of methoxy substituted 2-phenylbenzothiazoles was via the Jacobson method, demethylation being effected by boron tribromide at -70oC. Three amino substituted 2-phenylbenzothiazoles were also synthesised and tested for activity. Data is presented for oestrogen receptor binding activity, aromatase inhibitory activity, epidermal growth factor receptor tyrosine kinase (EGFRTK) inhibitory activity and cytotoxicity to ANN-1, 3T3, MCF-7 and WIDR cells. Oestrogen receptor binding affinity (RBA) was shown by five of the nine compounds tested. 2-(4-hydroxy)-6-hydroxybenzo-thiazole was the most active of the benzothiazoles tested (RBA 0.7). This is low but comparable to that of genistein. EGFRTK inhibitory activity was shown by four of the six benzothiazole derivatives tested; activity was comparable to that of genistein. Cytotoxicity assays have shown no selective toxicity of 2-phenylbenzothiazoles to any of the cell lines tested. Toxicity to MCF-7 cells was similar to that for other cell lines despite some compounds showing oestrogen receptor binding capacity. Amino-substituted 2-phenylbenzothiazoles showed selective toxicity towards transformed ANN-1 cells compared to normal 3T3 cells but the mechanism of this selectivity has not been established. Molecular modelling techniques, including CHEM-X, QUANTA and MOPAC were used to compare known ATP-competitive tyrosine kinase inhibitors with a model of ATP built from the crystal structure of the ATP-phosphoglycerate kinase complex. Structural features thought to be important to kinase inhibition were found and used to suggest further 2-phenylbenzothiazole analogues which may have improved activity.

The design of novel antifolates against pneumocystis carinii

The pneumonia caused by Pneumocystis carinii is ultimately responsible for the death of many acquired immunodeficiency syndrome (AIDS) patients. Large doses of trimethoprim and pyrimethamine in combination with a sulphonamide and/or pentamidine suppress the infection but produce serious side-effects and seldom prevent recurrence after treatment withdrawal. However, the partial success of the aforementioned antifolates, and also trimetrexate used alone, does suggest dihydrofolate reductase (DHFR) as a target for the development of antipneumocystis agents. From the DHFR inhibitory activities of 3'-substituted pyrimethamine analogues it was suggested that the 3'-(3'',3''-dimethyltriazen-1''-yl) substituent may be responsible for the greater activity for the P.carinii over the mammalian enzyme. Crystallographic and molecular modeling studies revealed considerable geometrical and electronic differences between the triazene and the chemically related formamidine functions that may account for the differences in DHFR inhibitory profiles. Structural and electronic parameters calculated for a series of 3'-(3'',3''-disubstitutedtriazen-1''-yl) pyrimethamine analogues did not correlate with the DHFR inhibitory activities. However, the in vitro screening against P.carinii DHFR revealed that the 3''-hydroxyethyl-3''-benzyl analogue was the most active and selective. Models of the active sites of human and P.carinii DHFRs were constructed using DHFR sequence and structural homology data which had identified key residues involved in substrate and cofactor binding. Low energy conformations of the 3'',3''-dimethyl and 3''-hydroxyethyl-3''-benzyle analogues, determined from nuclear magnetic resonance studies and theoretical calculations, were docked by superimposing the diaminopyrimidine fragment onto a previously docked pyrimethamine analogue. Enzyme kinetic data supported the 3''-hydroxyethyl-3''-benzyl moiety being located in the NADPH binding groove. The 3''-benzyl substituent was able to locate to within 3 AA of a valine residue in the active site of P.carinii DHFR thereby producing a hydrophobic contact. The equivalent residue in human DHFR is threonine, more hydrophilic and less likely to be involved in such a contact. This difference may account for the greater inhibitory activity this analogue has for P.carinii DHFR and provide a basis for future drug design. From an in vivo model of PCP in immunosuppressed rats it was established that the 3"-hydroxyethyl-3"-benzyl analogue was able to reduce the.P.carinii burden more effectively with increasing doses, without causmg any visible signs of toxicity. However, equivalent doses were not as effective as pentamidine, a current treatment of choice for Pneumocystis carinii pneumonia.

Non-classical inhibitors of dihydrofolate reductase

This thesis comprises two main objectives. The first objective involved the stereochemical studies of chiral 4,6-diamino-1-aryl-1,2-dihydro-s-triazines and an investigation on how the different conformations of these stereoisomers may affect their binding affinity to the enzyme dihydrofolate reductase (DHFR). The ortho-substituted 1-aryl-1,2-dihydro-s-triazines were synthesised by the three component method. An ortho-substitution at the C6' position was observed when meta-azidocycloguanil was decomposed in acid. The ortho-substituent restricts free rotation and this gives rise to atropisomerism. Ortho-substituted 4,6-diamino-1-aryl-2-ethyl-1,2-dihydro-2-methyl-s-triazine contains two elements of chirality and therefore exists as four stereoisomers: (S,aR), (R,aS), (R,aR) and (S,aS). The energy barriers to rotation of these compounds were calculated by a semi-empirical molecular orbital program called MOPAC and they were found to be in excess of 23 kcal/mol. The diastereoisomers were resolved and enriched by C18 reversed phase h.p.l.c. Nuclear overhauser effect experiments revealed that (S,aR) and (R,aS) were the more stable pair of stereoisomers and therefore existed as the major component. The minor diastereoisomers showed greater binding affinity for the rat liver DHFR in in vitro assay. The second objective entailed the investigation into the possibility of retaining DHFR inhibitory activity by replacing the classical diamino heterocyclic moiety with an amidinyl group. 4-Benzylamino-3-nitro-N,N-dimethyl-phenylamidine was synthesised in two steps. One of the two phenylamidines indicated weak inhibition against the rat liver DHFR. This weak activity may be due to the failure of the inhibitor molecule to form strong hydrogen bonds with residue Glu-30 at the active site of the enzyme.

Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens

Plant oxylipins are a large family of metabolites derived from polyunsaturated fatty acids. The characterization of mutants or transgenic plants affected in the biosynthesis or perception of oxylipins has recently emphasized the role of the so-called oxylipin pathway in plant defense against pests and pathogens. In this context, presumed functions of oxylipins include direct antimicrobial effect, stimulation of plant defense gene expression, and regulation of plant cell death. However, the precise contribution of individual oxylipins to plant defense remains essentially unknown. To get a better insight into the biological activities of oxylipins, in vitro growth inhibition assays were used to investigate the direct antimicrobial activities of 43 natural oxylipins against a set of 13 plant pathogenic microorganisms including bacteria, oomycetes, and fungi. This study showed unequivocally that most oxylipins are able to impair growth of some plant microbial pathogens, with only two out of 43 oxylipins being completely inactive against all the tested organisms, and 26 oxylipins showing inhibitory activity toward at least three different microbes. Six oxylipins strongly inhibited mycelial growth and spore germination of eukaryotic microbes, including compounds that had not previously been ascribed an antimicrobial activity such as 13-keto-9(Z),11(Z),15(Z)- octadecatrienoic acid and 12-oxo-10,15(Z)-phytodienoic acid. Interestingly this first large-scale comparative assessment of the antimicrobial effects of oxylipins reveals that regulators of plant defense responses are also the most active oxylipins against eukaryotic microorganisms, suggesting that such oxylipins might contribute to plant defense through their effects both on the plant and on pathogens, possibly through related mechanisms. © 2005 American Society of Plant Biologists.

Human intervertebral disc aggrecan inhibits nerve growth in vitro

OBJECTIVE: To assess the effects of human intervertebral disc aggrecan on nerve growth and guidance, using in vitro techniques. METHODS: Aggrecan extracted from human lumbar intervertebral discs was incorporated into tissue culture substrata for the culture of the human neuronal cell line, SH-SY5Y, or explants of chick dorsal root ganglia. The effects on nerve growth of different concentrations of aggrecan extracted from the anulus fibrosus and nucleus pulposus, and of these aggrecan preparations following enzymic deglycosylation, were compared. RESULTS: Disc aggrecan inhibited the growth of neurites from SH-SY5Y cells and induced growth cone turning of chick sensory neurites in a concentration-dependent manner. Aggrecan isolated from the anulus fibrosus was more inhibitory than that isolated from the nucleus pulposus, but enzymic pretreatments to reduce the glycosylation of both types of disc aggrecan partially abrogated their inhibitory effects. CONCLUSION: Nerve growth into degenerate intervertebral discs has been linked with the development of low back pain, but little is known about factors affecting disc innervation. The finding that disc aggrecan inhibits nerve growth in vitro, and that this inhibitory activity depends on aggrecan glycosylation, has important implications for our understanding of mechanisms that may regulate disc innervation in health and disease.

Modulation of neuronal network activity in the primary motor cortex

In the present study I investigated the mechanisms of modulation of neuronal network activity in rat primary motor cortex using pharmacological manipulations employing the in vitro brain slice technique. Preparation of the brain slice in sucrose-based aCSF produced slices with low viability. Introducing the neuroprotectants N-acetyl-cysteine, taurine and aminoguanidine to the preparatory method saw viability of slices increase significantly. Co-application of low dose kainic acid and carbachol consistently generated beta oscillatory activity in M1. Analyses indicated that network activity in M1 relied on the involvement of GABAA receptors. Dose-response experiments performed in M1 showed that beta activity can be modulated by benzodiazepine site ligands. Low doses of positive allosteric modulators consistently desynchronised beta oscillatory activity, a mechanism that may be driven by a1-subunit containing GABAA receptors. Higher doses increased the power of beta oscillatory activity. Whole-cell recordings in M1 uncovered three interneuronal subtypes regularly encountered in M1; Fast-spiking, regular-spiking non-Pyramidal and low threshold spiking. With the paradoxical effects of positive allosteric modulators in mind, subsequent voltage-clamp recordings in FS cells revealed a constitutively active tonic inhibitory current that could be modulated by zolpidem in two different ways. Low dose zolpidem increased the tonic inhibitory current in FS cells, consistent with the desynchronisation of network oscillatory activity seen at this concentration. High dose zolpidem decreased the inhibitory tonic current seen in FS cells, coinciding with an increase in oscillatory power. These studies indicate a fundamental role for a tonic inhibitory current in the modulation of network activity. Furthermore, desynchronisation of beta activity in M1 decreased as viability of the in vitro brain slice increased, suggesting that the extent of desynchronisation is dependent upon the pathophysiological state of the network. This indicates that low dose zolpidem could be used as a therapeutic agent specifically for the desynchronisation of pathological oscillations in oscillopathies such as Parkinson’s disease.

Studies on the mode of action and beta-lactamase inhibitory properties of novel oxapenem compounds

Four novel oxapenem compounds were evaluated for their ß-lactamase inhibitory and antibacterial properties. Two (AM-112 and AM-113) displayed intrinsic antibacterial activity with MICs of between 2 to 16µg/ml and 0.5-2µg/ml against Escherichia coli and methicillin-sensitive and -resistant Staphylococcus aureus, respectively. The isomers of these compounds, AM-115 and AM-114 did not display significant antibacterial activity. Combination of the oxapenems with ceftazidime afforded protection against ß-lactamase-producing strains, including hyperproducers of class C enzymes and extended-spectrum ß-lactamase enzymes. A fixed 4µg/ml concentration of AM-112 protected a panel of eight cephalosporins against hydrolysis by class A and class C ß-lactamase producers. In vivo studies confirmed the protective effect of AM-112 for ceftazidime against ß-lactamase producing S. aureus, Enterobacter cloacae and E. coli strains in a murine intraperitoneal infection model. Each of the oxapenems inhibited class A, class C and class D ß-lactamases isolated from whole cells and purified by isoelectric focusing. AM-114 and AM-115 were as effective as clavulanic acid against class A enzymes. AM-112 and AM-113 were less potent against these enzymes. Class C and class D enzymes proved very susceptible to inhibition by the oxapenems. Molecular modelling of the oxapenems in the active site of the class A. TEM-1 and class C P99 enzymes identified a number of potential sites of interaction. The modelling suggested that Ser-130 in TEM-1 and Tyr-150 in P99 were likely candidates for cross-linking of the inhibitor, leading to inhibition of the enzyme. Morphology studies indicated that sub-inhibitory concentrations of the oxapenems caused the formation of round-shaped cells in E. coli DC0, indicating inhibition of penicillin-binding protein 2 (PBP2). The PBP affinity profile of AM-112 was examined in isolated cell membranes of E. coli DC0, S. aureus NCTC 6571, Enterococcus faecalis SFZ and E. faecalis ATCC 29213, in competition with a radiolabelled penicillin. PBP2 was identified as the primary target for AM-112 in E. coli DC0. Studies on S. aureus NCTC 6571 failed to identify a binding target. AM-112 bound to all the PBPs of both E. faecalis strains, and a concentration of 10µg/ml inhibited all the PBPs except PBP3.

Cellular uptake and biological activity of synthetic hammerhead ribozymes

Glioblastoma Multiforme (GBM) is a highly malignant form of brain cancer for which there is no effective cure. The over-expression of a number of genes, including the epidermal growth factor receptor (EGFr), has been implicated as a causative factor of tumourigenesis. Ribozymes are a class of ribonucleic acid that possess enzymatic properties. They can inhibit gene-expression in a highly sequence specific manner by catalysing the trans-cleavage of target RNA. The potential use of synthetic hammerhead ribozymes as novel anti-brain tumour agents was investigated in this study. The successful use of synthetic, exogenously administered ribozymes for such applications will require chemical modifications that improve biological stability and a fundamental understanding of cellular uptake mechanisms. Chimeric 2'-O-methylated hammerhead ribozymes proved to be significantly more stable (>4000-fold) in serum than unmodified RNA ribozymes and exhibited high in vitro catalytic activity. The cellular association of an internally [32P]-labelled 2'-O-methylated chimeric ribozyme in U87-MG human glioma cells was temperature-, energy- and pH-dependent and involved an active process that could be competed with a variety of polyanions. Indications are that the predominant mechanism of uptake is by adsorptive and / or receptor mediated endocytosis. Twenty 2'-O-methylated chimeric ribozymes were designed to cleave various sites along the EGFr mRNA. In vitro, 18 ribozymes exhibited high activity in cleaving a complementary short substrate. Using LipofectAMINETM as a delivery agent, the efficacy of these ribozymes was evaluated in the A431 cell line, which expresses amplified levels of EGFr. Studies revealed that although the ribozymes were taken up by the cells and remained stable over a period of 4 days, no significant reduction in either EGFr expression or cell proliferation was evident. The presence of telomerase, a ribonucleoprotein responsible for telomere elongation, has been strongly associated with tumour progression. The biological activity of a 2'-O-methylated ribozyme targeted against the RNA component of telomerase was determined. The ribozyme exhibited specific dose-dependent inhibition of telomerase activity in U87-MG cell lysates with an IC50 of –4μM. When 4μM ribozyme was delivered to intact U87-MG cells, complexed to LipofectAMINETM, telomerase activity was significantly reduced to 74.5±4.17% of the untreated control. Free ribozyme showed no significant inhibitory effect demonstrating the importance of an appropriate delivery system for optimum delivery of exogenously administered ribozymes.

Polarised secretion of leukaemia inhibitory factor

Leukaemia inhibitory factor (LIF) is a cytokine that is active on a wide variety of cells. Multiple LIF transcripts have been described. The transcripts LIF-D and LIF-M encode different signal peptides, which in mouse have been associated with differential localisation of the mature protein. LIF-D is associated with a freely diffusible protein, whereas the LIF-M is associated with the extracellular matrix. The polarity of LIF secretion has yet to be described and could illuminate the mechanisms of LIF localisation. Here the polarised endogenous secretion of human LIF and IL-6 in Caco-2 cells was characterised under normal culture conditions and following induction with IL-1b. Whether the apical or basolateral membrane was stimulated influenced the pattern of secretion (LIF: Unstimulated, 59% basolateral. Dual stimulation, 68% basolateral. Basolateral stimulation, 79% basolateral. Apical stimulation, 53% basolateral). IL-6 displayed a similar dependence on the site of stimulation but was predominantly secreted at the membrane that was stimulated. To determine the effect of the alternate signal peptides on the polarity of LIF secretion, LIF was epitope tagged with FLAG. Epitope-tagging with FLAG was used to separate endogenous from exogenous protein expression. However, despite the normal biological activity of LIF-FLAG and detection of the FLAG in a western blot, detection of the LIF-FLAG under non-reducing conditions was not observed, and therefore it was unsuitable for secretion studies. Untagged LIF was expressed exogenously in Madin-Darby canine kidney (MDCK) cells under the control of a tetracycline response promoter that allowed a variety of LIF expression levels to be tested. Exogenous murine LIF was secreted predominantly from the apical (60%) membrane of MDCK cells irrespective of the signal peptide expressed.

Inhibition of secretary activity in cells isolated from the rat stomach

The overall aim of this study was to further understanding of themechanisms by which inhibitors of secretory activity mediate their action inisolated stomach cells. One objective was to determine whether a G-proteinsensitive to inactivation by pertussis toxin was involved in the action of thefollowing inhibitors of histamine-stimulated acid secretion: prostaglandin E2(PGE2), somatostatin, epidermal growth factor (EGF) and 12-0-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C.The site and mechanism by which EGF inhibited acid secretion and itseffects on pepsinogen secretion were also of interest. Further objectiveswere to determine whether TPA could induce down-regulation of proteinkinase C in parietal cells and to examine the inhibitory action of cyclic GMPon acid secretion. Acid secretion was estimated by the accumulation of theweak base aminopyrine in parietal cells. Experiments in which cells were preincubated with pertussis toxinindicated that PGE2, somatostatin and EGF mediated their inhibitory actionagainst histamine-stimulation via an inhibitory G-protein of the "Gi·like"family. Stimulation of PGE2 production by EGF also involved a pertussistoxin-sensitive G-protein. EGF inhibited acid secretion stimulated byforskolin, but only in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This action of EGF was sensitive toinactivation by pertussis toxin. It is suggested that the effect of EGF was dueto an increase in low Km cyclic AMP phosphodiesterase activity, rather thanan effect on the histamine (H2) receptor. EGF did not inhibit pepsinogensecretion. TPA exerted only a small part of its inhibitory action by a mechanismsensitive to pertussis toxin. TPA was unable to induce detectable down-regulationof protein kinase C. Acid secretion stimulated by near-maximallyeffective concentrations of h1stamme plus IBMX, dibutyryl cyclic AMP(dbcAMP) and K+ was inhibited by dibutyryl cyclic GMP (dbcGMP).

The role of GABAergic modulation in motor function related neuronal network activity

At rest, the primary motor cortex (M1) exhibits spontaneous neuronal network oscillations in the beta (15–30 Hz) frequency range, mediated by inhibitory interneuron drive via GABA-A receptors. However, questions remain regarding the neuropharmacological basis of movement related oscillatory phenomena, such as movement related beta desynchronisation (MRBD), post-movement beta rebound (PMBR) and movement related gamma synchronisation (MRGS). To address this, we used magnetoencephalography (MEG) to study the movement related oscillatory changes in M1 cortex of eight healthy participants, following administration of the GABA-A modulator diazepam. Results demonstrate that, contrary to initial hypotheses, neither MRGS nor PMBR appear to be GABA-A dependent, whilst the MRBD is facilitated by increased GABAergic drive. These data demonstrate that while movement-related beta changes appear to be dependent upon spontaneous beta oscillations, they occur independently of one other. Crucially, MRBD is a GABA-A mediated process, offering a possible mechanism by which motor function may be modulated. However, in contrast, the transient increase in synchronous power observed in PMBR and MRGS appears to be generated by a non-GABA-A receptor mediated process; the elucidation of which may offer important insights into motor processes.

Beta frequency neuronal network activity in the primary motor cortex

In this study I investigated the mechanisms of neuronal network oscillatory activity in rat M1 using pharmacological manipulations and electrical stimulation protocols, employing the in vitro brain slice technique in rat and magnetoencephalography (MEG) in man. Co-application of kainic acid and carbachol generated in vitro beta oscillatory activity in all layers in M1. Analyses indicated that oscillations originated from deep layers and indicated significant involvement of GABAA receptors and gap junctions. A modulatory role of GABAB, NMDA, and dopamine receptors was also evident. Intracellular recordings from fast-spiking (FS) GABAergic inhibitory cells revealed phase-locked action potentials (APs) on every beta cycle. Glutamatergic excitatory regular-spiking (RS) and intrinsically-bursting (IB) cells both received phase locked inhibitory postsynaptic potentials, but did not fire APs on every cycle, suggesting the dynamic involvement of different pools of neurones in the overall population oscillations. Stimulation evoked activity at high frequency (HFS; 125Hz) evoked gamma oscillations and reduced ongoing beta activity. 20Hz stimulation promoted theta or gamma oscillations whilst 4Hz stimulation enhanced beta power at theta frequency. I also investigated the modulation of pathological slow wave (theta and beta) oscillatory activity using magnetoencephalography. Abnormal activity was suppressed by sub-sedative doses of GABAA receptor modulator zolpidem and the observed desynchronising effect correlated well with improved sensorimotor function. These studies indicate a fundamental role for inhibitory neuronal networks in the patterning beta activity and suggest that cortical HFS in PD re-patterns abnormally enhanced M1 network activity by modulating the activity of FS cells. Furthermore, pathological oscillation may be common to many neuropathologies and may be an important future therapeutic target.