Hyperpolarization-activated currents in presynaptic terminals of mouse cerebellar basket cells.

Using patch-clamp techniques, a hyperpolarization-activated current (I(h)) was recorded from synaptic terminals of mouse cerebellar basket cells. Ih was blocked quickly and reversibly by 2 mM Cs(+), and subtraction revealed a rapidly activating and deactivating I(h) current. Similar gating and block of presynaptic I(h) were also seen with the more selective inhibitor ZD 7288 (10 microM). The time constant of activation (tau (a))of presynaptic I(h) current became faster with membrane hyperpolarization, being approximately 74 ms at -130 mV, changing e-fold for a 33 mV change in membrane potential. Whole-cell recordings from basket cell somata also revealed an I(h) current, which was similarly sensitive to block by ZD 7288. Inhibition of I(h) by 10 microM ZD 7288 reduced the frequency ( approximately 34 %) and amplitude ( approximately 26 %) of spontaneous IPSCs (sIPSCs) recorded in Purkinje cells, one of the principal synaptic targets of basket neurones. This is the first report of an I(h) current in mammalian inhibitory presynaptic terminals, which may be an important target for neuromodulation in the cerebellum. Comparing the biophysical properties and distribution of cloned hyperpolarization-activated cation channels, we also suggest a molecular candidate underlying I(h) at these synapses.

A functional role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons.

Cerebellar granule neurons (CGNs) are one of the most populous cells in the mammalian brain. They express an outwardly rectifying potassium current, termed a "standing-outward" K(+) current, or IK(SO), which does not inactivate. It is active at the resting potential of CGNs, and blocking IK(SO) leads to cell depolarization. IK(SO) is blocked by Ba(2+) ions and is regulated by activation of muscarinic M(3) receptors, but it is insensitive to the classical broad-spectrum potassium channel blocking drugs 4-aminopyridine and tetraethylammonium ions. The molecular nature of this important current has yet to be established, but in this study, we provide strong evidence to suggest that IK(SO) is the functional correlate of the recently identified two-pore domain potassium channel TASK-1. We show that IK(SO) has no threshold for activation by voltage and that it is blocked by small extracellular acidifications. Both of these are properties that are diagnostic of TASK-1 channels. In addition, we show that TASK-1 currents expressed in Xenopus oocytes are inhibited after activation of endogenous M(3) muscarinic receptors. Finally, we demonstrate that mRNA for TASK-1 is found in CGNs and that TASK-1 protein is expressed in CGN membranes. This description of a functional two-pore domain potassium channel in the mammalian central nervous system indicates its physiological importance in controlling cell excitability and how agents that modify its activity, such as agonists at G protein-coupled receptors and hydrogen ions, can profoundly alter both the neuron's resting potential and its excitability.

Electrophysiological characterization of voltage-gated K(+) currents in cerebellar basket and purkinje cells: Kv1 and Kv3 channel subfamilies are present in basket cell nerve terminals.

To understand the processes underlying fast synaptic transmission in the mammalian CNS, we must have detailed knowledge of the identity, location, and physiology of the ion channels in the neuronal membrane. From labeling studies we can get clues regarding the distribution of ion channels, but electrophysiological methods are required to determine the importance of each ion channel in CNS transmission. Dendrotoxin-sensitive potassium channel subunits are highly concentrated in cerebellar basket cell nerve terminals, and we have previously shown that they are responsible for a significant fraction of the voltage-gated potassium current in this region. Here, we further investigate the characteristics and pharmacology of the voltage-dependent potassium currents in these inhibitory nerve terminals and compare these observations with those obtained from somatic recordings in basket and Purkinje cell soma regions. We find that alpha-DTX blocks basket cell nerve terminal currents and not somatic currents, and the IC(50) for alpha-DTX in basket cell terminals is 3.2 nM. There are at least two distinct types of potassium currents in the nerve terminal, a DTX-sensitive low-threshold component, and a second component that activates at much more positive voltages. Pharmacological experiments also reveal that nerve terminal potassium currents are also markedly reduced by 4-AP and TEA, with both high-sensitivity (micromolar) and low-sensitivity (millimolar) components present. We suggest that basket cell nerve terminals have potassium channels from both the Kv1 and Kv3 subfamilies, whereas somatic currents in basket cell and Purkinje cell bodies are more homogeneous.

Modulation of inhibitory post-synaptic currents (IPSCs) in mouse cerebellar Purkinje and basket cells by snake and scorpion toxin K+ channel blockers.

Using an in vitro mouse cerebellar slice preparation and whole-cell electrophysiological recording techniques we have characterized Purkinje and basket cell inhibitory post-synaptic currents (IPSCs), and examined the effects of a number of selective peptidergic K+ channel blockers. Spontaneous IPSC amplitude ranged from approximately 10 pA up to approximately 3 nA for both cell types [mean values: Purkinje cells -122.8+/-20.0 pA (n = 24 cells); basket cells -154.8+/-15.9 pA (n = 26 cells)]. Frequency varied from approximately 3 up to approximately 40 Hz, [mean values: basket cells 14.9+/-1.7 Hz (n=26 cells); Purkinje cells 17.9+/-2.2 Hz (n=24 cells)]. 5 microM bicuculline eliminated virtually all spontaneous currents. IPSC rise times were fast (approximately 0.6 ms) and the decay phase was best fit with the sum of two exponential functions (tau1 and tau2: approximately 4 ms and approximately 20 ms, n=40; for both cell types). The snake toxins alpha-dendrotoxin (alpha-DTX) and toxin K greatly enhanced IPSC frequency and amplitude in both cell types; the closely related homologues toxin I and gamma-dendrotoxin (gamma-DTX) produced only marginal enhancements (all at 200 nM). Two scorpion toxins, margatoxin (MgTX) and agitoxin-2 (AgTX-2) had only minor effects on IPSC frequency or amplitude (both at 10 nM). Low concentrations of tetraethylammonium (TEA; 200 microM) had no overall effect on cerebellar IPSCs, whilst higher concentrations (10 mM) increased both the frequency and amplitude. The results suggest that native K+ channels, containing Kv1.1 and Kv1.2 channel subunits, play an influential role in controlling GABAergic inhibitory transmission from cerebellar basket cells.

Patch-clamp recordings from cerebellar basket cell bodies and their presynaptic terminals reveal an asymmetric distribution of voltage-gated potassium channels.

Cerebellar basket cells form highly specialized inhibitory synaptic contacts with Purkinje cells, namely the pericellular basket and pinceau nerve terminal structures, wrapping around the Purkinje cell somatic and axon hillock regions. These inhibitory synaptic contacts are ideally located to control the ultimate output of the cerebellar cortex. Previous immunohistochemical studies have shown that these synaptic structures possess a very high density of the dendrotoxin (DTX)-sensitive potassium channel subunit, Kv1.2. We have taken advantage of this unique anatomical arrangement offering a high concentration of identified Kv channel subunits by combining whole-cell patch-clamp recording and fluorescence microscopy to establish a novel preparation and perform the first recordings from unambiguously identified mammalian CNS inhibitory presynaptic terminals. We report that DTX-sensitive potassium channels are present in basket cell terminals but not in the basket cell soma. This selective cellular distribution suggests that these channels play an important role in modulating cerebellar inhibitory synaptic transmission.

The contrasting effects of dendrotoxins and other potassium channel blockers in the CA1 and dentate gyrus regions of rat hippocampal slices.

1. The effects of potassium channel blocking compounds on synaptic transmission in the CA1 and dentate gyrus regions of the rat hippocampus were examined by means of simultaneous field potential recording techniques in brain slices. 2. 4-Aminopyridine (4-AP) enhanced the excitatory postsynaptic potential (e.p.s.p.) and induced multiple population spike responses in both regions. EC50 values were 6.7 microM in the CAI (n = 5) and 161.7 microM (n = 5) in the dentate gyrus. 3. Tetraethylammonium (TEA) increased the amplitude and induced broadening of the population spike in both regions. In the dentate gyrus (n = 5) a single slow spike response was introduced (EC50 12.8 mM) and in the CA1 region (n = 5) the response was transformed into two wide spikes (EC50 2.6 mM). 4. In the CA1 region all of the dendrotoxins (toxin I, toxin K, alpha-Dtx and delta-Dtx) induced multiple population spikes and enlarged e.p.s.p. responses. Potentials recorded simultaneously in the dentate gyrus exhibited comparatively minor enhancements. The EC50 value for toxin 1 in the CA1 was calculated to be 237 nM (n = 4). Estimated EC50 values were obtained for alpha-Dtx (1.1 microM, n = 3), toxin K (411 nM, n = 4) and delta-Dtx (176 nM, n = 3). 5. In the presence of toxin 1, DL-2-amino-5-phosphonovaleric acid (APV) induced slight reduction of the late e.p.s.p. phase (n = 3). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) abolished all population spikes leaving a late slow positive waveform (n = 3). Co-application of APV and CNQX abolished all postsynaptic responses. 6. Charybdotoxin (CbTx) was significantly less potent than the dendrotoxins and had mixed actions in the CA1 region (n = 3). Again the dentate gyrus exhibited reduced sensitivity (n = 3). 7. In the presence of mast cell degranulating peptide (MCDP), enhancement of the CA1 field potential response (n = 5) was greater than that observed in the dentate gyrus (n = 5). 8. The results show that some potassium channel modulators can profoundly enhance CA1 region synaptic responses in the absence of notable changes in dentate gyrus excitability. Selective enhancement of defined synaptic pathways by potassium channel modulators may prove to have considerable therapeutic potential.

Modification of rat brain Kv1.4 channel gating by association with accessory Kvbeta1.1 and beta2.1 subunits.

We have examined the effects of co-expression of Kvbeta1.1 and Kvbeta2.1 subunits on the gating of rat brain Kv1.4 channels, expressed in Xenopus oocytes. Expression of Kv1.4 subunits alone produced a rapidly inactivating "A" type current, which activated at potentials beyond -60 mV in a solution containing high levels of rubidium. Current activation curves obtained from tail current measurements were fitted with a Boltzmann function, with V1/2 = -47 mV and k = 10 mV. Neither the Kvbeta1.1 nor Kvbeta2.1 subunits altered the voltage dependence of activation. Both subunits accelerated the activation time constant of Kv1.4, without affecting its voltage dependence. Surprisingly, the Kvbeta2.1 subunit, which lacks an N-terminal inactivation domain, was almost as effective as the Kvbeta1.1 subunit in speeding up Kv1.4. Steady-state inactivation of Kv1.4 was unchanged upon co-expression with either Kvbeta1.1 or Kvbeta2.1 subunits. Kv1.4 recovered from inactivation with two time constants; apart from an approximately 50% lengthening of the slow time constant with a high Kvbeta2.1 injection ratio, neither time constant was altered by either the Kvbeta1.1 or Kvbeta2.1 subunits, suggesting little interaction with recovery from C-type inactivation. Clearly, beta subunits have the potential to modify the gating of Kv1.4 channels in the brain more subtly than has been suggested previously.

Effects of high helium pressure on intracellular and field potential responses in the CA1 region of the in vitro rat hippocampus.

We have investigated the effects of high helium pressure (up to 13.3 MPa) on the electrophysiological responses of CA1 neurons in rat hippocampal slices using a purpose built pressure chamber, designed to facilitate field potential and intracellular recording. In field potential experiments, near threshold orthodromic responses were depressed by modest pressure (0.4 MPa). At higher stimulus intensities, orthodromic and antidromic population spike amplitudes were not increased at 5 MPa but were significantly enhanced at 10 MPa, and multiple population spikes were observed in some experiments. Orthodromic paired pulse potentiation was not affected by pressure up to 10 MPa. In intracellular experiments there were no significant differences between mean values obtained for resting membrane potential and input resistance at atmospheric pressure and pressures of up to 10 MPa. However, spontaneous depolarizing membrane potential excursions, decreased slow after-hyperpolarization responses and reduced accommodation properties were observed at pressure (5-10 MPa). One possibility is that the SK and M potassium channel systems may be more sensitive to high pressure than other membrane ion channels. These effects may contribute towards the high pressure neurological syndrome observed in vivo.

The action of anaesthetics and high pressure on neuronal discharge patterns.

1. The membrane actions of both anaesthetics and high pressure have been studied in rat hippocampal slices in experiments where either field potential (orthodromic or antidromic) responses from the CA1 region are recorded or intracellular measurements in CA1 neurones are made. 2. It is clear that anaesthetics have multiple post-synaptic actions in CA1 pyramidal neurones. For example, the amplitude of antidromic field potential responses are depressed (e.g. 2.5% enflurane) or increased (20 mM ketamine) or recruitment of a second population spike occurs (2.5% halothane). 3. In separate intracellular experiments anaesthetics have been shown to hyperpolarize [e.g. inhalations (2.5%), or methohexitone, 50-100 microM), or depolarize (ketamine, > or = 20 microM), CA1 pyramidal neurones decreasing or increasing respectively the responses to weak depolarizing synaptic input, or direct stimulation. 4. In the case of some anaesthetics (inhalation agents or ketamine), the accommodation of action potential discharge is also decreased, this being accompanied by a reduction in the amplitude of the associated slow after hyperpolarization (AHP). Consequently many neurones fire repetitively in response to long lasting, strong depolarizing inputs. Sub-classes of K+ channel such as the M (channel closed by muscarinic agonists) and the SK (Ca(2+)-dependent K+ channel of small conductance) type are currently believed to control the accommodation of spike discharge and the AHP. Consistent with this idea is the finding that in voltage-clamp experiments enflurane decreased reversibly the M current. 5. Methohexitone (50 microM) does not block the accommodation of action potential discharge but interestingly induces a long lasting depolarization and action potential bursting. 6. In comparison, high pressure (51-101 ATA) can induce a second population spike in antidromic field potential recordings, indicative also of a post-synaptic action. 7. There is no apparent change in the resting membrane potential or input resistance of CA1 neurones at high pressure, however 51 or 101 ATA produced a reduction in the accommodation of action potential discharge and the associated AHP leading to repetitive discharge in response to strong (0.7 nA) depolarizing currents. 8. In a few neurones spontaneous depolarizations and action potential discharge also occurred during compression between 51-101 ATA. 9. Our working hypothesis is that certain subclasses of neuronal K+ channel represent interesting targets for both anaesthetic and high pressure action.

Methods for intracellular recording from hippocampal brain slices under high helium pressure.

A method for intracellular recording from rat hippocampal brain slices under helium pressure is described. The preparation is mounted on a horizontal mobile platform that is rolled into the pressure chamber and can be viewed at pressure. Remote manipulation of the glass microelectrodes is achieved by a high-resolution electrically driven commercially available system. The slice is superfused continuously from a closed system within the chamber. Temperature is maintained at 37 degrees C and PO2 at 0.5 atm within the pressure chamber. A pressure of 200 ATA can be obtained, although thus far recordings have been made up to only 130 ATA. The experiments demand that a number of sample recordings be made from the same slice at both ambient and high pressure, and tests have proved that, although difficult, this can be achieved. The resting membrane potential, the current-voltage relationship, and the action potential responses to short (8 ms), medium (80 ms), and long (800 ms) depolarizing current pulses have all been measured in CA1 pyramidal neurons.

Inhalation anaesthetics block accommodation of pyramidal cell discharge in the rat hippocampus.

The effects of the three inhalation anaesthetics enflurane, isoflurane and halothane were tested in vitro on accommodation of rat CA1 neurones. At near clinical concentrations (approximately 2.5%) the anaesthetics slightly depressed antidromic field potential responses. At the same concentrations the anaesthetics also blocked accommodation reversibly and reduced the after hyperpolarization of CA1 neurones. No significant changes in the threshold potential were observed, although the resting membrane potential was often increased in the presence of the anaesthetics. The action of enflurane was not blocked by propranolol 20 mumol litre-1 and enflurane had no obvious effect on the duration of Ca2+ spikes of CA1 neurones. It is concluded that the anaesthetics may have a direct effect on membrane K+ channels such as the Ca2+-activated K+ conductance and that the block of accommodation is unlikely to account for the proconvulsant action of enflurane.

In vitro actions of ketamine and methohexitone in the rat hippocampus.

The effects of ketamine and methohexitone have been tested in vitro on rat CA1 pyramidal neurones using conventional extracellular and intracellular recording techniques. Ketamine 20-200 mumol litre-1 predominantly increased excitability by a postsynaptic action: it enhanced the amplitude of the antidromic (field) potential response in extracellular recordings; in intracellular studies depolarized or did not change the resting membrane potential; increased intrinsic excitability (assessed by direct stimulation); and reduced accommodation properties of CA1 neurones. Methohexitone 10-100 mumol litre-1 did not affect the amplitude of the antidromic field potential responses, tended to hyperpolarize and reduce the intrinsic excitability, but did not alter accommodation properties. At these concentrations these agents either did not affect or, in the case of ketamine, enhanced excitatory synaptic transmission on to the CA1 pyramidal neurones. Methohexitone 50 and 100 mumol litre-1 also induced a large, slow (several seconds) after depolarization which followed the conventional orthodromic response and may lead to action potential discharge. It is clear that these agents have multiple actions on CA1 pyramidal neurones in vitro and that ketamine and methohexitone in vitro influence excitability by different mechanisms.