The role of K2p channels in anaesthesia and sleep.

Tandem two-pore potassium channels (K2Ps) have widespread expression in the central nervous system and periphery where they contribute to background membrane conductance. Some general anaesthetics promote the opening of some of these channels, enhancing potassium currents and thus producing a reduction in neuronal excitability that contributes to the transition to unconsciousness. Similarly, these channels may be recruited during the normal sleep-wake cycle as downstream effectors of wake-promoting neurotransmitters such as noradrenaline, histamine and acetylcholine. These transmitters promote K2P channel closure and thus an increase in neuronal excitability. Our understanding of the roles of these channels in sleep and anaesthesia has been largely informed by the study of mouse K2P knockout lines and what is currently predicted by in vitro electrophysiology and channel structure and gating.

Cerebellar granule cell Cre recombinase expression.

The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells.

CNQX increases GABA-mediated synaptic transmission in the cerebellum by an AMPA/kainate receptor-independent mechanism.

GABA(A) receptor-mediated inhibitory synaptic transmission within the CNS is often studied in the presence of glutamate receptor antagonists. However, for nearly a decade it has been known that, in the hippocampus, one of the most commonly used alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), can increase the frequency of spontaneous GABA(A) receptor-mediated postsynaptic currents (sIPSCs). In the present study we examined the effect of CNQX and related compounds on GABA-mediated synaptic transmission in the cerebellum. At various stages of development, low concentrations of CNQX increased the frequency of sIPSCs recorded from granule cells. This effect was independent of the blocking action of CNQX on ionotropic glutamate receptors, as it was not observed with the broad-spectrum glutamate receptor antagonist kynurenate. No increase in sIPSC frequency was observed with the NMDA receptor antagonists D-AP5 or 7-ClK, the selective AMPA receptor antagonists GYKI 52466 or GYKI 53655, or the kainate receptor antagonist NS-102. In contrast, two other quinoxaline derivatives, NBQX and DNQX, were capable of increasing sIPSC frequency. These results demonstrate that the novel excitatory action of CNQX, unrelated to blockade of ionotropic glutamate receptors, is not restricted to the hippocampus and can be observed with structurally related compounds.

NMDA receptor subunits: diversity, development and disease.

N-methyl-D-aspartate receptors (NMDARs) are present at many excitatory glutamate synapses in the central nervous system and display unique properties that depend on their subunit composition. Biophysical, pharmacological and molecular methods have been used to determine the key features conferred by the various NMDAR subunits, and have helped to establish which NMDAR subtypes are present at particular synapses. Recent studies are beginning to address the functional significance of NMDAR diversity under normal and pathological conditions.

Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance.

Many neurons receive a continuous, or 'tonic', synaptic input, which increases their membrane conductance, and so modifies the spatial and temporal integration of excitatory signals. In cerebellar granule cells, although the frequency of inhibitory synaptic currents is relatively low, the spillover of synaptically released GABA (gamma-aminobutyric acid) gives rise to a persistent conductance mediated by the GABA A receptor that also modifies the excitability of granule cells. Here we show that this tonic conductance is absent in granule cells that lack the alpha6 and delta-subunits of the GABAA receptor. The response of these granule cells to excitatory synaptic input remains unaltered, owing to an increase in a 'leak' conductance, which is present at rest, with properties characteristic of the two-pore-domain K+ channel TASK-1 (refs 9,10,11,12). Our results highlight the importance of tonic inhibition mediated by GABAA receptors, loss of which triggers a form of homeostatic plasticity leading to a change in the magnitude of a voltage-independent K + conductance that maintains normal neuronal behaviour.

Slow deactivation kinetics of NMDA receptors containing NR1 and NR2D subunits in rat cerebellar Purkinje cells.

We have examined the deactivation kinetics of native N-methyl-D-aspartate receptors (NMDARs) containing NR1 and NR2D subunits by patch-clamp recording from Purkinje cells in cerebellar slices from young rats. NMDAR-mediated whole-cell currents were elicited in response to bath application of 20 microM NMDA and 50 microM glycine. The NMDAR-mediated currents were small, with an average whole-cell conductance of approximately 750 pS. Following the rapid application of brief pulses (1-10 ms) of 1 mM glutamate to outside-out membrane patches, we observed a low-conductance type of single-channel activity which lasted up to 30 s after the removal of agonist. Analysis of individual channel openings revealed asymmetry of transitions between the main- and subconductance states - a characteristic of NR1/NR2D-containing NMDARs. The averaged macroscopic current exhibited a decay time course which was well described by a single exponential function with a time constant of approximately 3 s. We conclude that native NR1/NR2D-containing NMDARs, like their recombinant counterparts, display very slow deactivation kinetics. This feature should provide a means for identification of these receptors at synapses, and indicates that they do not contribute to the synaptic NMDAR currents so far described.

Identification of subunits contributing to synaptic and extrasynaptic NMDA receptors in Golgi cells of the rat cerebellum.

1. To investigate the properties of N-methyl-D-aspartate receptors (NMDARs) in cerebellar Golgi cells, patch-clamp recordings were made in cerebellar slices from postnatal day 14 (P14) rats. To verify cell identity, cells were filled with Neurobiotin and examined using confocal microscopy. 2. The NR2B subunit-selective NMDAR antagonist ifenprodil (10 microM) reduced whole-cell NMDA-evoked currents by approximately 80 %. The NMDA-evoked currents were unaffected by the Zn2+ chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN; 1 microM) suggesting the absence of NMDARs containing NR2A subunits. 3. Outside-out patches from Golgi cells exhibited a population of 'high-conductance' 50 pS NMDAR openings. These were inhibited by ifenprodil, with an IC50 of 19 nM. 4. Patches from these cells also contained 'low-conductance' NMDAR channels, with features characteristic of NR2D subunit-containing receptors. These exhibited a main conductance of 39 pS, with a sub-conductance level of 19 pS, with clear asymmetry of transitions between the two levels. As expected of NR2D-containing receptors, these events were not affected by ifenprodil. 5. The NMDAR-mediated component of EPSCs, evoked by parallel fibre stimulation or occurring spontaneously, was not affected by 1 microM TPEN. However, it was reduced (by approximately 60 %) in the presence of 10 microM ifenprodil, to leave a residual NMDAR-mediated current that exhibited fast decay kinetics. This is, therefore, unlikely to have arisen from receptors composed of NR1/NR2D subunits. 6. We conclude that in cerebellar Golgi cells, the high- and low-conductance NMDAR channels arise from NR2B- and NR2D-containing receptors, respectively. We found no evidence for NR2A-containing receptors in these cells. While NR2B-containing receptors are present in both the synaptic and extrasynaptic membrane, our results indicate that NR1/NR2D receptors do not contribute to the EPSC and appear to be restricted to the extrasynaptic membrane.

Single-channel properties of synaptic and extrasynaptic GABAA receptors suggest differential targeting of receptor subtypes.

Many neurons express a multiplicity of GABAA receptor subunit isoforms. Despite having only a single source of inhibitory input, the cerebellar granule cell displays, at various stages of development, more than 10 different GABAA subunit types. This subunit diversity would be expected to result in significant receptor heterogeneity, yet the functional consequences of such heterogeneity remain poorly understood. Here we have used single-channel properties to characterize GABAA receptor types in the synaptic and extrasynaptic membrane of granule cells. In the presence of high concentrations of GABA, which induced receptor desensitization, extrasynaptic receptors in outside-out patches from the soma entered long-lived closed states interrupted by infrequent clusters of openings. Each cluster of openings, which is assumed to result from the repeated activation of a single channel, was to one of three main conductance states (28, 17, or 12 pS), the relative frequency of which differed between patches. Such behavior indicates the presence of at least three different receptor types. This heterogeneity was not replicated by individual recombinant receptors (alpha1beta2gamma2S or alpha1beta3gamma2S), which gave rise to clusters of a single type only. By contrast, the conductance of synaptic receptors, determined by fluctuation analysis of the synaptic current or direct resolution of channel events, was remarkably uniform and similar to the highest conductance value seen in extrasynaptic patches. These results suggest that granule cells express multiple GABAA receptor types, but only those with a high conductance, most likely containing a gamma subunit, are activated at the synapse.

NMDA receptor diversity in the cerebellum: identification of subunits contributing to functional receptors.

Recent studies of N-methyl-D-aspartate (NMDA) receptors have led to the suggestion that there are two distinct classes of native NMDA receptors, identifiable from their single-channel conductance properties. 'High-conductance' openings arise from NR2A- or NR2B-containing receptors, and 'low-conductance' openings arise from NR2C- or NR2D-containing receptors. In addition, the low-conductance channels show reduced sensitivity to block by Mg2+. The readily identified cell types and simple architecture of the cerebellum make it an ideal model system in which to determine the contribution of specific subunits to functional NMDA receptors. Furthermore, mRNA for all of these four NR2 subunits are represented in this brain region. We have examined NMDA channels in Purkinje cells, deep cerebellar nuclei (DCN) neurons and Golgi cells. First we find that NR2D-containing NMDA receptors give rise to low-conductance openings in cell-attached recordings from Purkinje cells. The characteristic conductance of these events cannot, therefore, be ascribed to patch excision. Second, patches from some DCN neurons exhibit mixed populations of high- and low-conductance openings. Third, Golgi cells also exhibit a mixed population of high- and low-conductance NMDA receptor openings. The features of these low-conductance openings are consistent with the presence of NR2D-containing NMDA receptors, as suggested by in situ hybridization data. On the other hand the existence of high-conductance channels, with properties typical of NR2B-containing receptors, was not expected. Our results provide new evidence about the subunit composition of NMDA receptors in identified cerebellar cells, and suggest that examination of single-channel properties is a potentially powerful approach for determining the possible subunit composition of native NMDA receptors.

Synchronizing retinal activity in both eyes disrupts binocular map development in the optic tectum.

Spatiotemporal correlations in the pattern of spontaneous and evoked retinal ganglion cell (RGC) activity are believed to influence the topographic organization of connections throughout the developing visual system. We have tested this hypothesis by examining the effects of interfering with these potential activity cues during development on the functional organization of binocular maps in the Xenopus frog optic tectum. Paired recordings combined with cross-correlation analyses demonstrated that exposing normal frogs to a continuous 1 Hz of stroboscopic illumination synchronized the firing of all three classes of RGC projecting to the tectum and induced similar patterns of temporally correlated activity across both lobes of the nucleus. Embryonic and eye-rotated larval animals were reared until early adulthood under equivalent stroboscopic conditions. The maps formed by each RGC class in the contralateral tectum showed normal topography and stratification after strobe rearing, but with consistently enlarged multiunit receptive fields. Maps of the ipsilateral eye, formed by crossed isthmotectal axons, showed significant disorder and misalignment with direct visual input from the retina, and in the eye-rotated animals complete compensatory reorientation of these maps usually induced by this procedure failed to occur. These findings suggest that refinement of retinal arbors in the tectum and the ability of crossed isthmotectal arbors to establish binocular convergence with these retinal afferents are disrupted when they all fire together. Our data thus provide direct experimental evidence that spatiotemporal activity patterns within and between the two eyes regulate the precision of their developing connections.

Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors.

1. To investigate the origin and functional significance of a recently described tonic GABAA receptor-mediated conductance in cerebellar granule cells we have made recordings from cells in cerebellar slices from rats of different ages (postnatal days P4 to P28). 2. During development there was a dramatic change in the properties of GABA-mediated synaptic transmission. The contribution to GABAA receptor-mediated charge transfer from the tonic conductance (GGABA), relative to that resulting from discrete spontaneous postsynaptic currents (sPSCs), was increased from 5% at P7 to 99% at P21. GGABA was reduced by bicuculline, tetrodotoxin and by lowering extracellular Ca2+, and was initially present only in those cells which exhibited sPSCs. 3. At P7 sPSCs were depolarizing, occasionally triggering a single action potential. By P18 the GABA reversal potential was shifted close to the resting potential and GGABA produced a shunting inhibition. Removal of GGABA by bicuculline increased granule cell excitability in response to current injection. 4. This novel tonic inhibition is present despite the low number of Golgi cell synapses on individual granule cells and appears to result from 'overspill' of synaptically released GABA leading to activation of synaptic and extrasynaptic GABAA receptors.

Experience-dependent mechanism of binocular map plasticity in Xenopus: incongruent connections are masked by retinal input.

When one eye in larval Xenopus is rotated to change the orientation of its visual map on the contralateral tectum, the map from the other eye to the same tectum can subsequently alter its orientation to match the rotated input. This plasticity re-establishes binocular congruence between the two maps and involves an experience-dependent reorganization of functional connections in the crossed isthmotectal projection, which delivers the ipsilateral eye's map to the tectum. Here, recordings were made at the same tectal sites in 8 eye-rotated frogs immediately before and after removing the rotated retina. All had congruent binocular maps prior to enucleation, with matching receptive fields in the two eyes at each recording site. In 5 frogs, eye removal unmasked additional, incongruent receptive fields in the ipsilateral eye, recorded from previously undetectable crossed isthmotectal connections occupying their original positions in the tectum. This result suggests that the plasticity also involves functional suppression of binocularly-incongruent inputs from the ipsilateral eye by retinotectal afferents.

The Maturation of the Superior Collicular Map of Auditory Space in the Guinea Pig is Disrupted by Developmental Auditory Deprivation.

Guinea pigs, reared from birth in an environment of omnidirectional white noise, fail to develop a map of auditory space in the deeper layers of the superior colliculus. Collicular responses from such noise-reared animals reveal large auditory spatial receptive fields. The representation of auditory space in the colliculus shows no topographic order. Exposing developing animals to the noise environment only for restricted time periods showed that animals reared normally up to 26 days after birth (DAB) and then placed in the noise chamber could not construct spatial maps, whereas animals reared normally to 30 DAB and then placed in the noise chamber until the terminal mapping experiment could construct topographically organized spatial maps with local receptive fields. Limiting the noise exposure to the period between 26 and 30 DAB was sufficient to prevent spatial map formation. The failure to form a map of auditory space did not reflect environmental damage to the cochlea or the functional organization of the primary auditory pathway. The response thresholds of cochlear microphonics and of auditory responses in both the inferior and superior colliculus were normal in noise-reared animals. Similarly normal were the tonotopic organization and frequency tuning characteristics of inferior collicular neurons. The rearing environment thus appears to exert a selective effect upon the maturation of the superior collicular map of auditory space. We attribute this effect to the masking, by the omnidirectional broad-band noise, of discrete localized auditory stimuli. Cues deriving from these latter stimuli would appear to be necessary for the elaboration of the map of auditory space. This auditory experience operates during a 4 day crucial developmental period from 26 to 30 DAB. This is the same developmental time window as that during which visual experience is required for the construction of the map.

Endomyocardial biopsy after heart transplantation in children.

Clinical evaluation, physical examination, and noninvasive testing have been suggested as adjuncts to endomyocardial biopsy for diagnosing acute graft rejection in children after heart transplantation. Because the consequences of delayed diagnosis or unnecessary treatment of rejection may be serious in pediatric transplant recipients, we reviewed our experience with repeated endomyocardial biopsy in seven children (aged 6 months to 19 years) and assessed the sensitivity and specificity of clinical and noninvasive data for diagnosing acute rejection. There were no serious complications in 71 biopsy procedures. In no patient did the presence of abnormal clinical findings or noninvasive testing coincide with treatable rejection that was proved on biopsy. However, there were nine episodes of treatable rejection in the absence of abnormal physical findings or noninvasive studies. At this time we consider repeated endomyocardial biopsy to be a feasible and safe procedure in infants and children and do not consider clinical findings and noninvasive testing sufficient to make therapeutic judgments in regard to acute graft rejection.

Successful percutaneous balloon valvuloplasty of the aortic valve in an infant.

Percutaneous balloon aortic valvuloplasty was attempted in an eight-month-old infant with severe aortic valve stenosis. The procedure resulted in a fall in the resting transvalvular systolic pressure gradient from 106 mmHg to 40 mmHg and no aortic regurgitation. Clinical and Doppler echocardiography findings suggest persistent improved status 3 1/2 months after the procedure.