In vivo evidence for functional NMDA receptor blockade by memantine in rat hippocampal neurons.

Antagonising the NMDA (N-methyl-D: -aspartate) receptor complex is a widely hypothesised therapeutic approach in several neurodegenerative conditions, such as Alzheimer's disease. Memantine, a moderate affinity uncompetitive NMDA receptor antagonist, has been in clinical use for several years and numerous experimental data support its NMDA receptor blocking effects. It has recently been reported in transfected HEK293T cells that physiological concentrations of Mg(2+) may impart partial NMDA receptor subtype selectivity and weaken the overall inhibitory actions of memantine in NMDA receptor-mediated cellular events. In the present study, we set out to investigate the effect of intravenously applied memantine on iontophoresed NMDA-evoked firing of hippocampal CA1 neurons using in vivo conditions. Cumulative doses of memantine in the rat (4, 8 and 16 mg/kg i.v.) caused the firing rate to decrease in a dose-dependent manner to 77 ± 7, 58 ± 8 and 34 ± 12% of control, respectively, while saline application had no significant effect. We show that therapeutic doses of memantine are able to antagonize NMDA receptor-mediated activity in the principal cells of the hippocampus in vivo, i.e. in the presence of physiological concentrations of Mg(2+).

An intraperitoneally administered pentapeptide protects against Abeta (1-42) induced neuronal excitation in vivo.

The underlying cause of Alzheimer's disease (AD) is thought to be the accumulation and aggregation of a misfolded protein, amyloid-beta (Abeta). A promising strategy against AD is the application of protective, peptide-based neuroprotective agents that selectively bind to Abeta. We recently described a pentapeptide, LPYFDa, which recognizes Abeta (1-42) and protects neurons against the toxic effects of aggregated Abeta (1-42) both in vitro and in vivo. Our previous work indicated that the in vivo ejection of fibrillar Abeta (1-42) into the hippocampal CA1 region resulted in a massive increase in the NMDA-evoked neuronal firing rate. Our current aim was to study whether intraperitoneally administered LPYFDa is capable of protecting against the synaptotoxic action of fibrillar Abeta (1-42) administered by iontophoresis. Our investigations of the in vivo biodistribution of tritium-labelled LPYFDa and single-unit electrophysiology revealed that LPYFDa readily crosses the blood-brain barrier, and protects the synapses against the excitatory action of fibrillar Abeta (1-42) in a relatively wide temporal window in rat. This pentapeptide may serve as a lead compound for the design of novel drug candidates for the prevention of AD.

Facilitation and inhibition of firing activity and N-methyl-D-aspartate-evoked responses of CA1 hippocampal pyramidal cells by alpha7 nicotinic acetylcholine receptor selective compounds in vivo.

Alpha7 nicotinic acetylcholine receptors (nAChRs) are promising novel targets for the treatment of neurocognitive disorders. Although the cognitive enhancer potential of alpha7 nAChR agonists and positive allosteric modulators (PAMs) has been confirmed in several preclinical animal models, there are only sparse in vivo electrophysiological data on their effects on the firing activity and excitability of neurons. The present study investigated and compared local effects of alpha7 nAChR agonist PHA-543613 and PAMs PNU-120596 and NS-1738 on the spontaneous and N-methyl-D-aspartate-evoked (NMDA-evoked) firing rate of rat CA1 hippocampal pyramidal cells, in vivo. Furthermore, effects of alpha7 nAChR antagonist methyllycaconitine (MLA) and GABA were also tested. Results showed substantially different effects of the alpha7 nAChR agonist and PAMs. While PNU-120596 and NS-1738 predominantly and significantly increased both spontaneous and NMDA-evoked firing rate of the neurons, application of PHA-543613 resulted in almost equal distribution of facilitatory and inhibitory effects. The increase of the NMDA-evoked firing rate exerted by NS-1738 was superadditive over the sum of the single effects of NMDA and NS-1738. The simultaneous application of alpha7 nAChR agonist PHA-543613 and PAM NS-1738 resulted in additive increase of both spontaneous and NMDA-evoked firing rate. However, NS-1738 counteracted inhibitory effects of PHA-543613 in 5 out of 6 neurons, resulting in a synergistic potentiation of their firing responses to NMDA. Our results suggest that alpha7 nAChR PAMs increase neuronal excitability more potently than agonists, while the remarkable occurrence of inhibitory effects of PHA-543613 (possibly originating from receptor desensitization) implies that agonists may exert neuroprotective effects.

Integrin activation modulates NMDA and AMPA receptor function of CA1 cells in a dose-related fashion in vivo.

The large family of heterodimeric, transmembrane cell adhesion receptors, integrins mediate numerous functions in the immature and adult CNS. Integrins are described to modulate basic synaptic function and plasticity, and to modulate the activity of the two major excitatory ionotrophic receptor subclass, NMDA and AMPA receptors. We further addressed the role of integrin activation in the normal excitatory synaptic function by utilizing in vivo single-unit recordings combined with microiontophoretic drug application in the CA1 region of the rat. Cells were excited by alternating NMDA and AMPA ejection, while integrin activation was achieved by the ejection of an RGD sequence containing pentapeptide in low and high concentration. Low integrin activation resulted in increased NMDA and decreased AMPA induced firing rate, while high RGD concentration enhanced both types of elicited responses. The control pentapeptide, pentaglycine had no effect on NMDA or AMPA evoked firing rate in either low or high concentration. These results suggest a bidirectional, dose dependent action of integrin activation on basic synaptic transmission, which may underlie the long term synaptic plasticity changes modulated by integrins.

A novel carbon tipped single micro-optrode for combined optogenetics and electrophysiology.

Optical microelectrodes (optrodes) are used in neuroscience to transmit light into the brain of a genetically modified animal to evoke and record electrical activity from light-sensitive neurons. Our novel micro-optrode solution integrates a light-transmitting 125 micrometer optical fiber and a 9 micrometer carbon monofilament to form an electrical lead element, which is contained in a borosilicate glass sheathing coaxial arrangement ending with a micrometer-sized carbon tip. This novel unit design is stiff and slender enough to be used for targeting deep brain areas, and may cause less tissue damage compared with previous models. The center-positioned carbon fiber is less prone to light-induced artifacts than side-lit metal microelectrodes previously presented. The carbon tip is capable of not only recording electrical signals of neuronal origin but can also provide valuable surface area for electron transfer, which is essential in electrochemical (voltammetry, amperometry) or microbiosensor applications. We present details of design and manufacture as well as operational examples of the newly developed single micro-optrode, which includes assessments of 1) carbon tip length-impedance relationship, 2) light transmission capabilities, 3) photoelectric artifacts in carbon fibers, 4) responses to dopamine using fast-scan cyclic voltammetry in vivo, and 5) optogenetic stimulation and spike or local field potential recording from the rat brain transfected with channelrhodopsin-2. With this work, we demonstrate that our novel carbon tipped single micro-optrode may open up new avenues for use in optogenetic stimulation when needing to be combined with extracellular recording, electrochemical, or microbiosensor measurements performed on a millisecond basis.

Endomorphin-2, an endogenous tetrapeptide, protects against Abeta1-42 in vitro and in vivo.

The underlying cause of Alzheimer's disease (AD) is thought to be the beta-amyloid aggregates formed mainly by Abeta1-42 peptide. Protective pentapeptides [e.g., Leu-Pro-Phe-Phe-Asp (LPFFD)] have been shown to prevent neuronal toxicity of Abeta1-42 by arresting and reversing fibril formation. Here we report that an endogenous tetrapeptide, endomorphin-2 (End-2, amino acid sequence: YPFF), defends against Abeta1-42 induced neuromodulatory effects at the cellular level. Although End-2 does not interfere with the kinetics of Abeta fibrillogenesis according to transmission electron microscopic studies and quasielastic light scattering measurements, it binds to Abeta1-42 during aggregation, as revealed by tritium-labeled End-2 binding assay and circular dichroism measurements. The tetrapeptide attenuates the inhibitory effect on cellular redox activity of Abeta1-42 in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazolyl-2)-2,-5-diphenyltetrazolium bromide (MTT) assay. In vitro and in vivo electrophysiological experiments show that End-2 also protects against the field excitatory postsynaptic potential attenuating and the NMDA-evoked response-enhancing effect of Abeta1-42. Studies using [D-Ala (2), N-Me-Phe (4), Gly (5)-ol]-enkephalin (DAMGO), a mu-opioid receptor agonist, show that the protective effects of the tetrapeptide are not mu-receptor modulated. The endogenous tetrapeptide End-2 may serve as a lead compound for the drug development in the treatment of AD.

Divergent effects of Abeta1-42 on ionotropic glutamate receptor-mediated responses in CA1 neurons in vivo.

Aggregated Abeta1-42 is hypothesized to be the central cause of Alzheimer's disease. However, early changes in synaptic activity may be detected in the disease long before a significant cell loss is manifested. Despite the fact that Abeta1-42 interference with long-term potentiation (LTP) and the field excitatory postsynaptic potential (fEPSP) is well documented, the exact mechanism of these events remains to be clarified. Here we studied the effects of iontophoretically applied Abeta1-42 on the neuronal firing evoked in vivo on the CA1 hippocampal neurons of Wistar rats by different agonists of the ionotropic glutamate receptors: N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainic acid (KA). NMDA elicited firing enhanced in all of the measured cells; in contrast, the AMPA-mediated responses decreased significantly after Abeta1-42 ejection. The changes in KA-evoked responses to Abeta1-42 revealed two types of cells. In the first type, the KA-mediated firing remained at the control level, while in the second type, Abeta1-42 attenuated the KA-evoked responses. A protective pentapeptide, Leu-Pro-Tyr-Phe-Asp-amide, was used to verify the specificity of these beta-amyloid-elicited effects. The pentapeptide protected against the modulatory effects of Abeta1-42 on the NMDA and AMPA responses. In conclusion, we have shown that Abeta1-42 exerts divergent effects on the activity of the ionotropic glutamate receptors in vivo. These results suggest that the LTP disruption and fEPSP attenuation seen after Abeta1-42 application are in part due to the altered function of these receptors.

Enhancement of NMDA responses by beta-amyloid peptides in the hippocampus in vivo.

The effects of Alzheimer's disease-related beta-amyloid (Abeta) peptides on the N-methyl-D-aspartate (NMDA)-evoked cell firing rate were studied in hippocampal CA1 neurons of the rat. Extracellular single-unit recordings were combined with iontophoretic applications that allowed quantitative analyses of the interactions between Abeta peptides and NMDA receptor-mediated events in vivo. The NMDA responses were significantly increased both by the full length Abeta1-42 and by its model fragment Abeta25-35. Enhancements of the NMDA responses by the Abeta peptides lasted about 15 min and were irreversible. The effects of Abeta25-35 were prevented by the pentapeptide Lys-Leu-Val-Gly-Phe-amide (KLVGF) and were not evoked when its reversed sequence (Abeta35-25) was applied.

Divalent heavy metal cations block the TRPV1 Ca(2+) channel.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel involved in pain sensation and in a wide range of non-pain-related physiological and pathological conditions. The aim of the present study was to explore the effects of selected heavy metal cations on the function of TRPV1. The cations ranked in the following sequence of pore-blocking activity: Co(2+) [half-maximal inhibitory concentration (IC(50)) = 13 µM] > Cd(2+) (I (50) = 38 µM) > Ni(2+) (IC(50) = 62 µM) > Cu(2+) (IC(50) = 200 µM). Zn(2+) proved to be a weak (IC(50) = 27 µM) and only partial inhibitor of the channel function, whereas Mg(2+), Mn(2+) and La(3+) did not exhibit any substantial effect. Co(2+), the most potent channel blocker, was able not only to compete with Ca(2+) but also to pass with it through the open channel of TRPV1. In response to heat activation or vanilloid treatment, Co(2+) accumulation was verified in TRPV1-transfected cell lines and in the TRPV1+ dorsal root ganglion neurons. The inhibitory effect was also demonstrated in vivo. Co(2+) applied together with vanilloid agonists attenuated the nocifensive eye wipe response in mice. Different rat TRPV1 pore point mutants (Y627W, N628W, D646N and E651W) were created that can validate the binding site of previously used channel blockers in agonist-evoked (45)Ca(2+) influx assays in cells expressing TRPV1. The IC(50) of Co(2+) on these point mutants were determined to be reasonably comparable to those on the wild type, which suggests that divalent cations passing through the TRPV1 channel use the same negatively charged amino acids as Ca(2+).

Fibrillar Abeta (1-42) enhances NMDA receptor sensitivity via the integrin signaling pathway.

The aggregated form of amyloid-beta (Abeta) (1-42) has been shown to increase N-methyl-D-aspartic acid (NMDA) evoked neuronal activity in vivo. Here we further characterized this phenomenon by investigating the role of integrin activation and downstream Src kinase activity using in vivo electrophysiology and in vitro intracellular Ca (2+) measurements. Pretreatment of differentiated SH-SY5Y cells with fibrillar Abeta (1-42) markedly enhanced the intracellular calcium increases caused by NMDA receptor (NMDA-R) stimulation. Function blocking antibody against beta1 integrin depressed the facilitatory effects of Abeta (1-42). Similarly, Abeta (1-42) facilitated NMDA-R driven firing of hippocampal neurons in vivo, and this effect was reduced by neutralizing antibody against beta1 integrins. The positive action of Abeta (1-42) on NMDA-R dependent responses was also depressed by an inhibitor known to block Src kinase. These results support the hypothesis that aggregated Abeta (1-42) is recognized by the beta1 subunit containing integrins and may induce a Src kinase dependent NMDA receptor phosphorylation.

NK-1 receptors modulate the excitability of ON cells in the rostral ventromedial medulla.

The role of neurokinin-1 (NK-1) receptors in the rostral ventromedial medulla (RVM) was studied using extracellular single-unit recording combined with microiontophoresis. In rats, on- and off-type neurons were identified using noxious heat or mechanical stimuli applied to the tail. Responses evoked by iontophoretic application of N-methyl-d-aspartate (NMDA) were determined before and after intraplantar injection of capsaicin or iontophoretic application of substance P. In off cells, capsaicin produced an extended pause in ongoing activity but did not alter the subsequent spontaneous discharge rate or NMDA-evoked responses. In contrast, spontaneous discharge rates of on cells increased after capsaicin, and their responses to NMDA increased >100% above control values. The increased responses to NMDA after capsaicin were attenuated by iontophoretic application of the selective NK-1 receptor antagonist L-733,060. Similarly to capsaicin, iontophoretic application of the selective NK-1 receptor agonist, [Sar(9),Met(O(2))(11)]-substance P (SM-SP), increased the spontaneous discharge rate and NMDA-evoked responses of on cells by >100% of control values. These effects were antagonized by L-733,060. Immunohistochemical studies showed that a subset of neurons in the RVM labeled NK-1 receptors and that nearly all of these neurons were immunoreactive for the NMDAR1 subunit of the NMDA receptor. These results demonstrate that activation of NK-1 receptors in the RVM enhances responses of on cells evoked by NMDA. It is suggested that activation of NK-1 receptors in the RVM and the ensuing sensitization of on cells may contribute to the development of central sensitization and hyperalgesia after tissue injury and inflammation.

Anti-calmodulins and tricyclic adjuvants in pain therapy block the TRPV1 channel.

Ca(2+)-loaded calmodulin normally inhibits multiple Ca(2+)-channels upon dangerous elevation of intracellular Ca(2+) and protects cells from Ca(2+)-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca(2+). Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca(2+)-uptake via the vanilloid inducible Ca(2+)-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca(2+) entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced (45)Ca(2+)-uptake at microM concentrations: calmidazolium (broad range) > or = trifluoperazine (narrow range) chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca(2+) or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca(2+)-uptake in intact TRPV1(+) cells, and suggests an extracellular site of inhibition. TRPV1(+), inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca(2+)-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca(2+)-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca(2+)-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca(2+)-channels but not affecting motoneurons.

CD59 blocks not only the insertion of C9 into MAC but inhibits ion channel formation by homologous C5b-8 as well as C5b-9.

Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b-9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b-9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59(-) human B-cells, which formed non-selective ion channels with a total conductance of 360 +/- 24 pS as measured at the beginning of the steady-state phase of the inward currents. C5b-8 and small-size MAC (MAC containing only a single C9) can also form ion channels. Nevertheless, in CD59(+) human B-cells in spite of small-size MAC formation, an ion current could not be detected. In addition, restoring CD59 to the membrane of the CD59(-) cells inhibited the serum-evoked inward current. The ion channels formed by the small-size MAC were therefore sealed, indicating that CD59 directly interfered with the pore formation of C5b-8 as well as that of small-size C5b-9. These results offer an explanation as to why CD59-expressing cells are not leaky in spite of a buildup of homologous C5b-8 and small-size MAC. Our experiments also confirmed that ion channel inhibition by CD59 is subject to homologous restriction and that CD59 cannot block the conductivity of MAC when generated by xenogenic (rabbit) serum.