Memantine block depends on agonist presentation at the NMDA receptor in substantia nigra pars compacta dopamine neurones.

NMDA glutamate receptors (NMDARs) have critical functional roles in the nervous system but NMDAR over-activity can contribute to neuronal damage. The open channel NMDAR blocker, memantine is used to treat certain neurodegenerative diseases, including Parkinson's disease (PD) and is well tolerated clinically. We have investigated memantine block of NMDARs in substantia nigra pars compacta (SNc) dopamine neurones, which show severe pathology in PD. Memantine (10 µM) caused robust inhibition of whole-cell (synaptic and extrasynaptic) NMDARs activated by NMDA at a high concentration or a long duration, low concentration. Less memantine block of NMDAR-EPSCs was seen in response to low frequency synaptic stimulation, while responses to high frequency synaptic stimulation were robustly inhibited by memantine; thus memantine inhibition of NMDAR-EPSCs showed frequency-dependence. By contrast, MK-801 (10 µM) inhibition of NMDAR-EPSCs was not significantly different at low versus high frequencies of synaptic stimulation. Using immunohistochemistry, confocal imaging and stereological analysis, NMDA was found to reduce the density of cells expressing tyrosine hydroxylase, a marker of viable dopamine neurones; memantine prevented the NMDA-evoked decrease. In conclusion, memantine blocked NMDAR populations in different subcellular locations in SNc dopamine neurones but the degree of block depended on the intensity of agonist presentation at the NMDAR. This profile may contribute to the beneficial effects of memantine in PD, as glutamatergic activity is reported to increase, and memantine could preferentially reduce over-activity while leaving some physiological signalling intact.

P2Y1 receptor modulation of Ca2+-activated K+ currents in medium-sized neurons from neonatal rat striatal slices.

ATP signaling to neurons and glia in the nervous system occurs via activation of both P2Y and P2X receptors. Here, we investigated the effects of P2Y(1) receptor stimulation in developing striatal medium-sized neurons using patch-clamp recordings from acute brain slices of 7- and 28-day-old rats. Application of the selective P2Y(1) receptor agonist 2-(Methylthio) ADP trisodium salt (2-MeSADP; 250 nM) increased outward K(+) currents evoked by a ramp depolarization protocol in voltage-clamp recordings. This effect was observed in 59 out of 82 cells (72%) and was blocked completely by the P2Y(1) antagonist, 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate. The averaged 2-MeSADP-sensitive conductance was fitted by the sum of a linear conductance and a Boltzmann relation, giving one-half activation voltage of -14.2 mV and an equivalent charge of 2.91. The 2MeSADP-mediated effect was sensitive to submillimolar concentrations of tetraethylammonium (TEA; 200 µM), to 200 nM iberiotoxin and to 100 nM apamin, suggesting the involvement of both big and small potassium (BK and SK, respectively) calcium-activated channels. In current-clamp experiments, 2-MeSADP decreased depolarization-evoked action potential (AP) firing in all 26 cells investigated, and this effect was reversed by TEA and by apamin but not by iberiotoxin. We conclude that the stimulation of P2Y(1) receptors in developing striatal neurons leads to activation of calcium-activated potassium channels [I(K(Ca))] of both BK and SK subtypes, the latter responsible for decreasing the frequency of AP firing in response to current injection. Therefore, P2Y(1) signaling leading to activation of I(K(Ca)) may be important in regulating the activity of medium-sized neurons in the striatum.

Functional heterogeneity of NMDA receptors in rat substantia nigra pars compacta and reticulata neurones.

The nigra substantia nigra pars compacta (SNc) and substantia pars reticulata (SNr) form two major basal ganglia components with different functional roles. SNc dopaminergic (DA) neurones are vulnerable to cell death in Parkinson's disease, and NMDA receptor activation is a potential contributing mechanism. We have investigated the sensitivity of whole-cell and synaptic NMDA responses to intracellular ATP and GTP application in the SNc and SNr from rats on postnatal day (P) 7 and P28. Both NMDA current density (pA/pF) and desensitization to prolonged or repeated NMDA application were greater in the SNr than in the SNc. When ATP levels were not supplemented, responses to prolonged NMDA administration desensitized in P7 SNc DA neurones but not at P28. At P28, SNr neurones desensitized more than SNc neurones, with or without added ATP. Responses to brief NMDA applications and synaptic NMDA currents were not sensitive to inclusion of ATP in the pipette solution. To investigate these differences between the SNc and SNr, NR2 subunit-selective antagonists were tested. NMDA currents were inhibited by ifenprodil (10 microM) and UBP141 (4 microM), but not by Zn(2+) (100 nm), in both the SNr and SNc, suggesting that SNc and SNr neurones express similar receptor subunits; NR2B and NR2D, but not NR2A. The different NMDA response properties in the SNc and SNr may be caused by differences in receptor modulation and/or trafficking. The vulnerability of SNc DA neurones to cell death is not correlated with NMDA current density or receptor subtypes, but could in part be related to inadequate NMDA receptor desensitization.

Influence of the M3-M4 intracellular domain upon nicotinic acetylcholine receptor assembly, targeting and function.

BACKGROUND AND PURPOSE: The aim of this study was to investigate the influence of the intracellular domain of nicotinic acetylcholine receptor (nAChR) subunits upon receptor assembly, targeting and functional properties. EXPERIMENTAL APPROACH: Because most nAChR subunits form functional receptors only as heteromeric complexes, it can be difficult to examine the influence of individual subunits or subunit domains in isolation. A series of subunit chimaeras was constructed which contain the intracellular loop region (located between the M3 and M4 transmembrane domains) from nAChR subunits alpha1-alpha10 or beta1-beta4. All of these chimaeras contain common extracellular and transmembrane domains (from the nAChR alpha7 subunit and the 5-hydroxytryptamine receptor 5-HT(3A) subunit, respectively), thereby facilitating both homomeric receptor assembly and detection with radiolabelled or fluorescent alpha-bungarotoxin. KEY RESULTS: The nAChR M3-M4 intracellular loop domain had no significant effect upon levels of total subunit protein detected in transfected cells but had a significant influence upon levels of both cell surface and intracellular assembled receptors. Comparisons of functional properties revealed a significant influence of the intracellular loop domain upon both single-channel conductance and receptor desensitization. In addition, studies conducted in polarized epithelial cells demonstrate that the nAChR loop can influence receptor targeting, resulting in either polarized (apical) or non-polarized distribution. CONCLUSIONS AND IMPLICATIONS: Evidence has been obtained which demonstrates that the large intracellular loop domain of nAChR subunits can exert a profound influence upon receptor assembly, targeting and ion channel properties.

NR2B- and NR2D-containing synaptic NMDA receptors in developing rat substantia nigra pars compacta dopaminergic neurones.

NMDA receptors are present at glutamatergic synapses throughout the brain, and are important for the development and plasticity of neural circuits. Their subunit composition is developmentally regulated. We have investigated the developmental profile of functional synaptic NMDA receptor subunits in dopaminergic neurones of the substantia nigra pars compacta (SNc). In SNc dopaminergic neurones from rats aged postnatal day (P)7, ifenprodil inhibited NMDA-EPSCs with an estimated IC(50) of 0.36 microm and a maximum inhibition of 73.5 +/- 2.7% (10 microm), consistent with a substantial population of NR1/NR2B-containing diheteromeric receptors. UBP141, a novel NR2D-preferring antagonist, inhibited NMDA-EPSCs with an estimated IC(50) of 6.2 microm. During postnatal development, the maximum inhibitory effect of 10 microm ifenprodil significantly decreased. However, NMDA-EPSCs were not inhibited by Zn(2+) (200 nM) or potentiated by the Zn(2+) chelator TPEN (1 microm), and the effect of UBP141 did not increase during development, indicating that NR2B subunits are not replaced with diheteromeric NR2A or NR2D subunits. The time course of the decay of NMDA-EPSCs was not significantly changed in ifenprodil at any age tested. Together, these data suggest that diheteromeric NR1/NR2A or NR1/NR2D receptors do not account for the ifenprodil-resistant component of the NMDA-EPSC. We propose that NR1/NR2B/NR2D triheteromers form a significant fraction of synaptic NMDA receptors during postnatal development. This is the first report of data suggesting NR2D-containing triheteromeric NMDA receptors at a brain synapse.

Identification of domains influencing assembly and ion channel properties in alpha 7 nicotinic receptor and 5-HT3 receptor subunit chimaeras.

BACKGROUND AND PURPOSE: Nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT(3)Rs) are members of the superfamily of neurotransmitter-gated ion channels. Both contain five subunits which assemble to form either homomeric or heteromeric subunit complexes. With the aim of identifying the influence of subunit domains upon receptor assembly and function, a series of chimaeras have been constructed containing regions of the neuronal nAChR alpha 7 subunit and the 5-HT(3) receptor (3A) subunit. EXPERIMENTAL APPROACH: A series of subunit chimaeras containing alpha 7 and 5-HT(3A) subunit domains have been constructed and expressed in cultured mammalian cells. Properties of the expressed receptors have been examined by means of radioligand binding, agonist-induced changes in intracellular calcium and patch-clamp electrophysiology. KEY RESULTS: Subunit domains which influence properties such as rectification, desensitization and conductance have been identified. In addition, the influence of subunit domains upon subunit folding, receptor assembly and cell-surface expression has been identified. Co-expression studies with the nAChR-associated protein RIC-3 revealed that, in contrast to the potentiating effect of RIC-3 on alpha 7 nAChRs, RIC-3 caused reduced levels of cell-surface expression of some alpha 7/5-HT(3A) chimaeras. CONCLUSIONS AND IMPLICATIONS: Evidence has been obtained which demonstrates that subunit transmembrane domains are critical for efficient subunit folding and assembly. In addition, functional characterization of subunit chimaeras revealed that both extracellular and cytoplasmic domains exert a dramatic and significant influence upon single-channel conductance. These data support a role for regions other than hydrophobic transmembrane domains in determining ion channel properties.

The single-channel properties of purinergic P2X ATP receptors in outside-out patches from rat hypothalamic paraventricular parvocells.

The aim of this study was to characterize functionally the single-channel properties of rat hypothalamic paraventricular medial parvocell purinergic P2X receptors. Single-channel recordings were made from outside-out patches from coronal whole-brain slices from 4-day-old-rats. A heterogeneous population of functional P2X receptors were observed, possibly reflecting a mixture of homomeric P2X2 receptors and heteromers containing P2X2, P2X3, P2X4 or P2X6 subunits. "Flickery" P2X receptor channels with a high open probability (Po) and chord conductances of 14.4+/-1.75 and 29.6+/-0.5 pS at -80 mV were observed in nine patches. The mean open time was 2.51+/-1.30 ms. In the presence of 1 mM ATP, and excluding long periods of apparent desensitization, these channels had a mean Po of 0.58+/-0.29. "Non-flickery" P2X receptors with chord conductances of 16.9+/-3.48 and 32.4+/-3.75 pS, a mean open time of 0.63+/-0.15 ms and a mean Po of 0.01+/-0.004 at -80 mV were observed in the presence of 1 mM ATP in three patches. P2X receptors showed little apparent concentration dependence in their activity at ATP concentrations of 0.01-1 mM, probably reflecting considerable desensitization. The mean Po (for all data records) at 10 microM was 0.030+/-0.017 and 0.030+/-0.028 at 1 mM.

Characterization of the single-channel properties of NMDA receptors in laminae I and II of the dorsal horn of neonatal rat spinal cord.

The single-channel properties of native NMDA receptors in laminae I and II of the dorsal horn of the neonatal rat spinal cord were studied using outside-out patch-clamp techniques. These receptors were found to have several features that distinguish them from native NMDA receptors elsewhere in the CNS. Single-channel currents activated by NMDA (100 nm) and glycine (10 microm) exhibited five distinct amplitude components with slope-conductance values of 19.9 +/- 0.8, 32.9 +/- 0.6, 42.2 +/- 1.1, 53.0 +/- 1.0 and 68.7 +/- 1.5 pS. Direct transitions were observed between all conductance levels but transitions between 69-pS openings and 20-, 33- and 42-pS openings were rare. There was no significant difference in the frequency of direct transitions from 42- to 20-pS compared to 20- to 42-pS transitions. The Kb (0 mV) for Mg2+ was 89 microm. The Mg2+ unblocking rate constant was similar to other reported values. However, the Mg2+ blocking rate constant was larger than other reported values, suggesting an unusually high sensitivity to Mg2+. The NR2B subunit-selective antagonist, ifenprodil, had no significant effect on overall channel activity but significantly decreased the mean open time of 53-pS openings. These results suggest neonatal laminae I and II NMDA receptors are not simply composed of NR1 and NR2B subunits or NR1 and NR2D subunits. It is possible that these properties are due to an as yet uninvestigated combination of two NR2 subunits with the NR1 subunit or a combination of NR3A, NR2 and NR1 subunits.

Single channel properties of P2X ATP receptors in outside-out patches from rat hippocampal granule cells.

The single channel properties of P2X ATP receptors were investigated in outside-out patches from hippocampal granule cells in brain slices from 12-day-old rats. The results demonstrate that functional P2X ATP receptors are expressed in hippocampal granule cells and, combined with previously published information on the P2X subunits expressed in the hippocampus, suggest that the receptors may be heteromers of the P2X4 and P2X6 subunits or P2X1, P2X2, P2X4 and P2X6 subunits. Two distinct types of P2X channel openings were observed. A flickery P2X receptor channel was observed in three patches with a mean chord conductance of 32 +/- 6 pS, a mean open time of 1.0 +/- 0.3 ms and a mean burst length of 11 +/- 5 ms at a membrane potential of -60 mV. A large conductance P2X receptor was observed in 19 out of 98 patches with a mean conductance of 56 +/- 1. 8 pS, a linear current-voltage relationship between -80 and +60 mV with a reversal potential around 0 mV, a mean open time of 2.6 +/- 0. 2 ms and a mean burst length of 8.8 +/- 1.8 ms at -60 mV. At an ATP concentration of 1 mM, these channels exhibited a low steady-state open probability (Popen, 0.07 +/- 0.008; n = 15), little apparent desensitisation and were also activated by alpha,beta-methylene ATP (alpha,beta-meATP, 40 microM; Popen, 0.007 +/- 0.0002; conductance, 57 +/- 1.1 pS; n = 3). No decrease in the single channel conductance was observed on increasing the free extracellular calcium concentration from 0.3 to 0.85 mM. Channel closed time distributions were fitted with five exponential components with time constants (and relative areas) of 90 micros (20 %), 0.77 ms (32 %), 10 ms (15 %), 90 ms (18 %) and 403 ms (15 %) at 1 mM ATP. Of these, the first two components are suggested to represent gaps within single activations of the receptor based on the lack of agonist concentration dependence of these two shut time components between 1 microM and 1 mM ATP. Suramin (40 microM) significantly increased the single channel conductance (19 +/- 7 %; n = 5) and produced a small decrease in Popen (39 +/- 9 %; n = 5) by decreasing mean open time, burst length and total open time per burst. These actions of suramin are not consistent with simple competitive antagonism.

Properties of ATP receptor-mediated synaptic transmission in the rat medial habenula.

The properties of central ATP-mediated synaptic currents were studied using whole-cell patch-clamp recording in rat medial habenula slices. Release was shown to be calcium dependent with a Hill coefficient of approximately 2. The voltage dependence of synaptic current amplitudes was approximately linear. Some reduction of the synaptic current amplitudes was observed at 10 mM extracellular calcium, suggesting calcium block/permeability of the channels. This was confirmed by observation of current-voltage reversal potentials in different calcium concentrations. We estimate that the channels underlying half the synapses showed a negligible calcium permeability. In the other four out of eight synapses the results suggest a very high calcium permeability with an estimated PCa/PCs of > 10. Thus, at -70 mV, in 1 mM calcium, more than 15% of the ATP-mediated synaptic current is estimated to be carried by calcium, but only at synapses with calcium-permeable channels. Net current through these synaptic channels is also controlled by the voltage dependence of synaptic current decay time constants (increasing e-fold for 158 mV depolarization) and by a strong dependence of transmitter release on the frequency of stimulation of the presynaptic neurone, with failure rates increasing 3-fold as stimulation rates were increased from 1 to 10 Hz.

Neuropharmacology: a part for purines in pattern generation.

Purinergic transmission has been found to play a key role in the neural control of rhythmic swimming behaviour in Xenopus embryos: it may have similar importance in other vertebrate motor behaviours.

Persistence: a new statistic for characterizing ion-channel activity.

We introduce and illustrate by examples a new statistical technique, the persistence function, for characterizing ion-channel activity in a single-channel patch-clamp recording. Persistence is a function of both current and time. It is the probability that the current is at a given level (conditional on it having been at that level at an earlier time). Viewed as a function of current it exhibits the prominent conductance levels present in the recording, and viewed as a function of time for a conductance level it portrays the kinetics at that level.

Heterogeneity of neuronal nicotinic acetylcholine receptors in thin slices of rat medial habenula.

1. Neuronal nicotinic acetylcholine receptors in slices of rat medial habenula were studied using patch clamp recording techniques. 2. Whole cell current responses to cytisine could be blocked by hexamethonium, as expected for nicotinic receptors. The whole cell current-voltage relations were linear at negative membrane potentials, but showed strong inward rectification when chloride currents were minimized. 3. When 1 mM Ca2+ (0 mM Mg2+) was present in the external recording solution, the single channel conductances elicited by acetylcholine or nicotine in twenty patches were in the range 39-58 pS, with a mean of 47 pS. There appeared to be at least two groups of conductances. 4. In the open point amplitude distributions of three patches, the most common amplitude corresponded to 41 pS (81% of the area). In another four patches the most common amplitude corresponded to a mean conductance of 51 pS (83% of the area). Direct transitions between open levels were rare. 5. Channel closed times were not significantly different for the two conductance groups. However, for the four patches with predominantly 51 pS openings, the means of the distributions of open times longer than two filter rise times averaged 5.8 ms. Those patches with predominantly 41 pS openings averaged 14 ms. Also, for patches with predominantly 51 pS openings the overall mean burst length was 5.8 ms, whereas for patches with predominantly 41 pS openings it was 16.1 ms. 6. These observations suggest that 51 and 41 pS openings result from the activity of at least two, but possibly more, different receptor subtypes. We conclude that nicotinic receptors in the rat ventral medial habenula are heterogeneous.

Comparison of neuronal nicotinic receptors in rat sympathetic neurones with subunit pairs expressed in Xenopus oocytes.

1. The agonist sensitivity of nicotinic acetylcholine receptors in rat superior cervical ganglion (SCG) neurones was compared with that of cloned receptors expressed in Xenopus oocytes by pairwise injections of alpha 3-beta 2 or alpha 3-beta 4 neuronal nicotinic subunit combinations. 2. Agonist responses in rat SCG neurones indicated that cytisine was the most potent agonist and lobeline the least potent (rank order of potency: cytisine > dimethylphenylpiperazinium iodide (DMPP) > nicotine > ACh > carbachol > lobeline). 3. Receptors expressed in oocytes by injection of alpha 3 and beta 2 subunits had a relatively high sensitivity to DMPP and low sensitivity to cytisine (rank order of potency: DMPP > ACh > lobeline > carbachol > nicotine > cytisine), whereas receptors composed of alpha 3 and beta 4 subunits had a high sensitivity to cytisine and low sensitivity to DMPP (rank-order of potency: cytisine > nicotine approximately ACh > DMPP > carbachol > lobeline). 4. With the exception of responses to DMPP, agonist sensitivity measurements suggest that nicotinic receptors in the rat SCG are composed of alpha 3 and beta 4 subunits. The results are discussed in terms of the receptor subunit mRNAs known to be expressed in the rat SCG and previous evidence of functional heterogeneity of rat SCG nicotinic acetylcholine receptors.

ATP--a fast neurotransmitter.

ATP receptor-mediated responses in peripheral and central neurons have many characteristics which suggest that ATP may act as a fast neurotransmitter. While the receptors underlying these responses have properties which are similar to other ligand-gated ion channels which mediate fast neurotransmission, the nature of their calcium permeability and the rapid breakdown of ATP to adenosine may confer unique properties on ATP mediated synaptic transmission. The evidence that ATP acts as a fast neurotransmitter is reviewed and the properties of ATP and its receptor channels are discussed in terms of synaptic transmission.

Identification of M-channels in outside-out patches excised from sympathetic ganglion cells.

We have identified the species of K+ channel that underlies the neuronal M-current in rat sympathetic ganglion cells. The channels were kinetically and pharmacologically defined using outside-out and cell-attached patches. They exhibited multiple conductance levels, predominantly 3-9 pS. Their slow gating in response to voltage change in outside-out patches was exhibited only in the presence of AIF-4 or GTP gamma S on the inner membrane surface and when the lower conductance states were dominant. In the absence of AIF-4 or GTP gamma S, the channels exhibited rapid activation and deactivation. We conclude that M-channel gating may be controlled by an associated GTP-binding protein.

Activation of N-methyl-D-aspartate receptors by L-glutamate in cells dissociated from adult rat hippocampus.

1. Single channel recording techniques were used to study the ion channel openings resulting from activation of N-methyl-D-aspartate (NMDA) receptors by the agonist glutamate. Patches were from cells acutely dissociated from adult rat hippocampus (CA1). Channel activity was studied at low glutamate concentrations (20-100 nM) with 1 microM-glycine, in the absence of extracellular divalent cations. 2. Channel openings were to two main conductance levels corresponding to 50 pS and 40 pS openings in extracellular solution with 1 mM-Ca2+. Around 80% of openings were to the large conductance level. The single channel conductances increased as extracellular Ca2+ was reduced. 3. Distributions of channel open times were described by three exponential components of 87 microseconds, 0.91 ms and 4.72 ms (relative areas of 51, 31 and 18%). Most long openings were to the large conductance level. 4. The channel closed time distribution was complex, requiring five exponential components to describe it adequately. Of these five components, at least three, with time constants of 68 microseconds, 0.72 ms and 7.6 ms (relative areas of 38, 12 and 17%) represent gaps within single activations of the receptor. The presence of a component with a mean of 7.6 ms is notable because gaps of this length have not previously been identified as being within single NMDA receptor channel activations. 5. Channel activations were identified as including gaps underlying at least the first three closed time components. Activations consisted of clusters of channel openings. Distributions of the length of these clusters had mean time constants of 88 microseconds, 3.4 ms and 32 ms (relative areas of 45, 25 and 30%). Long clusters contained short, intermediate and long duration openings as well as subconductance openings. The open probability within clusters averaged 0.62. Three components were evident in distributions of the number of openings per cluster. These had mean values of 1.22, 3.2 and 11 openings per cluster. 6. An inverse correlation was evident between the length of adjacent open and closed times. When open intervals were separated into groups based on the length of adjacent gaps, the time constants of the exponential components in these conditional open time distributions were independent of the length of the adjacent gap. This supports the idea that the NMDA receptor channel gating has the properties of a discrete Markov process. 7. The long duration of NMDA receptor channel clusters suggests that they contribute to the slow time course of the NMDA receptor-mediated synaptic current.