Activation of Ih is necessary for patterning of mGluR and mAChR induced network activity in the hippocampal CA3 region.

Neuronal networks of the hippocampal CA3 region generate stereotyped patterns of electrical activity in response to activation of metabotropic glutamate receptors (mGluRs) or muscarinic acetylcholine receptors (mAChRs) that consist of intermittent episodes of prolonged oscillatory activity. In light of the slow kinetics of such network responses, we investigated the possible contribution of the hyperpolarisation-activated inward current (I(h)) in the generation and maintenance of hippocampal oscillatory states. Hippocampal 'mini-slice' experiments in which the main subfields of the hippocampus were isolated by transection of the connecting afferents revealed that the CA3 region was the primary generator of both mGluR and mAChR-mediated network responses. Subsequent patch-clamp experiments confirmed the presence of a prominent hyperpolarisation-activated inward current in the principal cells of the CA3 region that was sensitive to caesium chloride and the selective I(h) blocker ZD-7288.Furthermore, in the presence of mAChR or mGluR agonists these cells exhibited a slow membrane potential oscillation that was independent of AMPA receptor-mediated synaptic transmission. Blockade of I(h) suppressed this oscillation as well as mGluR and mAChR-induced theta based intermittent network oscillatory behaviour. These data support the idea that the I(h) pacemaker current is important in the generation of patterned neuronal activities in the hippocampus.

Modulation of the hyperpolarisation-activated current, Ih, in rat facial motoneurones in vitro by ZD-7288.

ZD-7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride] and Cs(+) have been used to distinguish the currents contributing to inward rectification in neonatal rat facial motoneurones (FMs). ZD-7288 (0.1-10 microM) inhibited a current that reversed at -43.7+/-3.7 mV in artificial cerebrospinal fluid (ACSF) containing 3 mM K(+) (n=9), and displayed the time and voltage dependence of the hyperpolarisation-activated current, I(h). Depolarisation-activated transient (I(K(A))) and sustained outward currents were unaffected by ZD-7288. The IC(50) for block of I(h) by ZD-7288 was around 0.2 microM. Onset of inhibition was slow and no recovery was seen after washing in ZD-7288-free ACSF for up to 4 h. In the presence of ZD-7288, Ba(2+) and Rb(+) blocked an inwardly rectifying potassium (K(+)) current, confirming both the presence of I(K(IR)) and its insensitivity to ZD-7288. Cs(+) rapidly and reversibly blocked both I(h) and I(K(IR)). Inhibition of I(h) by ZD-7288 showed no use dependence, internally applied ZD-7288 also blocked I(h), and tail current analysis indicated inhibition to be voltage-independent. In the presence of internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and after previous exposure to ZD-7288, 5-hydroxytryptamine (5-HT), but not noradrenaline, promoted a recovery of I(h) that was not observed if ZD-7288 was present throughout the recording period. This interaction between ZD-7288 and irreversible 5-HT-receptor activation may be related to the mechanism underlying ZD-7288-mediated block of these channels.

Characterization of 5-HT-sensitive potassium conductances in neonatal rat facial motoneurones in vitro.

1. The properties of the 5-HT-sensitive K+ conductance of neonatal rat facial motoneurones were examined in brainstem slices using whole-cell patch-clamp techniques. 2. In a small proportion of motoneurones, 5-hydroxytryptamine (5-HT) evoked an inward current mediated solely by a decrease in K+ conductance. The reversal potential (V5-HT) was dependent on the external K+ concentration and the 5-HT-evoked current (I5-HT) displayed a linear current-voltage (I-V) relationship. 3. In the remaining motoneurones, the 5-HT-evoked decrease in K+ conductance could only be observed in isolation once a concomitant 5-HT-mediated enhancement of the hyperpolarization-activated current, Ih, had been abolished with the Ih blocker, ZD-7288. 4. External Cs+ also abolished the Ih-mediated component of I5-HT but, in addition, blocked part of the 5-HT-sensitive K+ current. At potentials hyperpolarized to V5-HT, Cs+ voltage dependently blocked I5-HT while at potentials depolarized to V5-HT, I5-HT was largely unaffected. Ba2+ and Rb+ had identical actions to Cs+ on the 5-HT-sensitive K+ current. 5. The Ba2+-, Rb+- and Cs+-sensitive component of the 5-HT-sensitive K+ current inwardly rectified with a reversal potential that was dependent on the K+ equilibrium potential (EK). 6. Replacing external Na+ with N-methyl-D-glucamine, blocking Ca2+ entry, or preventing an increase in intracellular [Ca2+] with BAPTA, all failed to alter I5-HT at potentials depolarized to EK. 7. I5-HT at depolarized potentials was reversibly blocked by 4-aminopyridine (4 mM) but not tetraethylammonium chloride (30 mM) and did not show inactivation during depolarizing voltage pulses (1.5 s duration). 8. The results suggest that, in addition to enhancing Ih, 5-HT modulates two distinct K+ conductances in neonatal rat facial motoneurones. The actions of Cs+, Ba2+ and Rb+ support the involvement of a member of the inwardly rectifying family of K+ channels while the other K+ channel may belong to the voltage-gated family.

Modulation of IH by 5-HT in neonatal rat motoneurones in vitro: mediation through a phosphorylation independent action of cAMP.

The depolarization of adult and neonatal rat facial and spinal motoneurones by 5-hydroxytryptamine (5-HT) in part involves an enhancement of the hyperpolarization-activated, inward-rectifier, IH. Under experimental conditions which promote this action, 5-HT evokes an inward current which can be mimicked by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP) and potentiated by the cAMP-specific phosphodiesterase inhibitor Ro 20-1724. In this study, we show that this action of 5-HT can be blocked by the adenylyl cyclase inhibitors 2'3'-dideoxyadenosine (2',3'-DDA). 5'-adenylimidodiphosphate (AMP-PNP) and SQ-22536 (9-(tetrahydro-2-furyl)adenine), but not by external or internal application of the protein kinase inhibitors H-7, staurosporine and chelerythrine. The most recently cloned 5-HT receptor subtypes, 5-HT4, 5-HT6 and 5-HT7, can all stimulate adenylyl cyclase when activated. In the presence of internal GTP-gamma-S, 5-HT irreversibly enhanced IH. The 5-HT-induced inward current could be reversibly blocked by methysergide, but not by the 5-HT4 receptor antagonist GR-113808A, the 5-HT6 and 5-HT7 antagonist clozapine and the 5-HT1A antagonist WAY-100365. 5-Methoxytryptamine (5-MeOT) and 5-carboxamidotryptamine (5-CT) mimicked the action of 5-HT with a rank order of potency of 5-HT = 5MeOT > 5-CT. Surprisingly, 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH DPAT), a 5-HT1A and 5-HT7 agonist was inactive on facial motoneurones unlike its reported agonist action on spinal motoneurones. It is proposed that cAMP produced by 5-HT-mediated stimulation of adenylyl cyclase acts in a phosphorylation-independent manner, possibly directly, on the IH channel. The 5-HT receptor subtype mediating this response cannot be correlated with any of the classified 5-HT receptor subtypes that stimulate adenylyl cyclase.

Adenosine 3':5'-cyclic monophosphate mediates a 5-hydroxytryptamine-induced response in neonatal rat motoneurones.

5-Hydroxytryptamine (5-HT) is present in nerve fibres descending from the brainstem Raphe nuclei to motoneurones and its release is thought to exert excitatory actions. 5-HT, applied from the outside, directly depolarizes spinal and cranial motoneurones in slices. This action of 5-HT is mediated, in part, by an inwardly rectifying cationic current, Ih. In cardiac cells, an equivalent current, if, has been shown to be directly activated by adenosine 3':5'-cyclic monophosphate (cAMP) applied to the inside of the patch membrane. By applying the whole-cell method to thin slices of brainstem and spinal cord, we have shown that intracellularly applied camp and extracellularly applied dibutyryl camp or forskolin mimics the inward current induced by 5-ht. The selective cAMP phosphodiesterase inhibitor, Ro 20-1724, clearly prolonged the 5-HT-induced current. Maximal doses of dibutyryl cAMP or forskolin occluded the 5-HT-induced current. The broad spectrum protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methlypiperazine (H-7) and N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8) had no effect on the currents induced by 5-HT and forskolin. From these results, we suggest that activation of 5-HT receptors on the motoneuronal membrane stimulates formation of intracellular cAMP, thereby inducing the inward current, possibly by a direct action on Ih.

Preparative methods for brain slices: a discussion.

Criteria for slice health and factors that affect slice health were discussed by many of the participants in the conference. In addition to the standard parameters of slice health (energy metabolism, morphology, electrophysiological responsiveness) more subtle but possibly equally important manifestations of slice health were discussed. These included protein synthesis, and more subtle changes, of which we are becoming increasingly aware. The latter include synthesis of stress-related proteins, altered levels of phosphorylation, altered levels of proteolysis. These last were only touched on, but it is becoming apparent they do in fact constitute important manifestations of differences between the slice preparation and the in vivo tissue. They may well lead to quite different responses in slices from those that occur in vivo. While many ways of optimizing slice wellness were discussed, there was a fair consensus that certain adjustments will optimize the most widely measured aspects of cell function. These include the following, wherever possible. Use of young animals, use of the interface chamber, preparing slices with the vibratome, pre-treating animals with ice-cold cardiac perfusion before sacrificing, using pre-incubation media which reduce NMDA receptor activation, free radical formation and cell swelling. When possible these treatments should perhaps be continued into the normal incubation. This being said, many viewpoints were actually expressed in the discussion, and it should be read to get a feel for the usefulness of the different approaches.

The use of brain slices and dissociated neurones to explore the multiplicity of actions of 5-HT in the central nervous system.

The cellular actions of 5-hydroxytryptamine (5-HT) on adult and neonatal rat central neurones have been investigated in detail using a combination of in vitro slice and dissociated neurone preparations. Patch-clamp recordings from acutely dissociated adult rat dorsal raphe neurones confirm data obtained using conventional slice preparations that 5-HT activates an inwardly rectifying potassium channel through a 5-HT1A receptor leading to hyperpolarization of the cell. Single-channel recordings indicate that this pathway requires only the involvement of a pertussis toxin-sensitive G-protein. Adult rat facial motoneurones in conventional slices are depolarized by 5-HT through a combination of mechanisms, closure of potassium channels and enhancement of the hyperpolarization-activated, cationic current, IH. Distinct receptors appear to mediate these two actions. Both mechanisms are present in visually indentified neonatal rat facial motoneurones in thin brain slices. Whole-cell patch-clamp recordings show the action of 5-HT on IH to mediate a caesium-sensitive inward current which can be mimicked by the adenylate cyclase activator, forskolin. The experimental data illustrate how a range of complimentary in vitro electrophysiological techniques can be employed to unravel neurotransmitter mechanisms and pharmacology.

Ionic mechanisms mediating 5-hydroxytryptamine- and noradrenaline-evoked depolarization of adult rat facial motoneurones.

1. The actions of 5-hydroxytryptamine (5-HT) and noradrenaline (NA) on the membrane properties of facial motoneurones in slices from the adult rat brainstem in vitro were examined using intracellular recording techniques. 2. In voltage clamp recording, hyperpolarizing voltage steps (> 20 mV), from holding potentials at or close to the resting potential, induced a slowly activating, voltage-dependent inward current possessing properties similar to the hyperpolarization-activated current (Ih) seen in other cell types. From tail current analysis two groups of facial motoneurones can be distinguished in terms of the activation range for Ih, one with a half-maximal activation at -81 mV and the other at -94 mV but with similar shapes. 3. 5-HT (120/126) and NA (21/21) depolarized facial motoneurones. The reversal potentials (Em) obtained from peak voltage amplitude I-V plots in varying extracellular potassium concentrations suggested mechanisms involving a decrease in K+ conductance. 4. Under voltage clamp, close to the resting potential, both 5-HT (39/41) and NA (13/13) evoked inward currents. 5. I-V plots and plots of 5-HT-sensitive current at different membrane potentials, obtained from currents evoked by voltage steps and measured before the development of Ih (instantaneous current), indicated that the 5-HT-evoked inward current was predominately associated with a decrease in conductance but with a range of reversal potentials for 5-HT (E5-HT) from close to, to much more negative than the reversal potential for a potassium conductance (EK). In some cases no change or increases in instantaneous conductance were observed. 6. Steady-state I-V relationships and plots of 5-HT-sensitive current, measured after development of Ih, indicated a 5-HT-associated conductance increase with a time and voltage dependence close to that of Ih, which could be abolished by extracellular caesium (2-5 mM). 7. The NA-evoked inward current was always associated with a decrease in conductance. Instantaneous and steady-state I-V relationships as well as plots of NA-sensitive current indicated a reversal potential at EK. 8. The activation curve for Ih was shifted to more positive potentials in the presence of 5-HT. The time constant for activation of Ih showed a similar shift. 9. 5-Carboxamidotryptamine (5-CT), a 5-HT receptor agonist, was selective for the enhancement of Ih and only evoked an inward current when the holding potential was within the activation range of Ih.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiology of adult rat facial motoneurones: the effects of serotonin (5-HT) in a novel in vitro brainstem slice.

Studies of adult rat motoneurones using in vitro slice preparations are rare. We here describe a novel brainstem slice of the adult rat containing the facial motor nucleus (FMN). Data obtained for facial motoneurones (FM) by intracellular recording indicate that they display several passive and active properties seen in other rat cranial and spinal motoneurones. Bath application of serotonin (5-HT) evokes a reversible depolarization of FMs which is associated with an increase in input resistance due to a reduction in potassium permeability. This effect is unaffected by tetrodotoxin indicating a postsynaptic site of action.