QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online August 6, 2004
Journal of Experimental Biology 207, 3201-3212 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01106

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JEB Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ballanyi, K. Search for Related Content PubMed PubMed Citation Articles by Ballanyi, K.

Protective role of neuronal K_ATP channels in brain hypoxia

Klaus Ballanyi

Department of Physiology & Pediatrics, Perinatal Research Centre, University of Alberta, 232 HMRC, Edmonton, Alberta, T6G 2S2, Canada

View larger version (45K): [in a new window]   Fig. 1. Model systems for analysis of the involvement of neuronal ATP-sensitive K+ (KATP) channels in brain hypoxia–anoxia. Coronal brain slices from rodents are used to study the electrophysiological response to oxygen depletion in three types of central neurons. Anoxia-vulnerable cerebellar Purkinje neurons from 16–20-day-old mice with a characteristic flat dendritic tree show pronounced rises of intracellular Ca2+ (Cai) during rhythmic or tonic activity of membrane potential (Vm). Cai is monitored in superficial cells filled via the recording patch-electrode with 50–200 µmol l–1 of the Ca2+-sensitive dye fura-2. The same techniques are applied to tonically active dorsal vagal neurons in medullary slices from juvenile rats or mice. These neurons innervate organs of the gastrointestinal tract, such as pancreatic ß-cells, in which KATP channel properties and functions are being thoroughly explored. Whole-cell patch-clamp recording is done in neurons of the ventral respiratory group (VRG) including the rhythmogenic pre-Bötzinger complex (PBC) or inspiratory active hypoglossal motoneurons (XII-MN) in coronal medullary slices or brainstem–spinal cord preparations from neonatal rodents. The cells can be labelled with dyes such as lucifer-yellow or biocytin for subsequent (immuno)histochemical analysis of their structure and neurotransmitter receptors. Rhythmic inspiratory activity is recorded with glass suction electrodes from hypoglossal (XII) nerve rootlets in the slices or from cervical nerve rootlets in an en bloc preparation. Reconstructed respiratory neuron data from K. Ballanyi and S. Schwarzacher. Brain section taken from Paxinos (1982).

View larger version (36K): [in a new window]   Fig. 2. KATP channels in dorsal vagal neurons of juvenile rodents. (A) Superfusion of nitrogen-gassed hypoxic saline causes tissue anoxia in the dorsal vagal nucleus of medullary slices kept at 30°C. In dorsal vagal neurons of rats, such anoxia results in a sustained hyperpolarisation and concomitant suppression of tonic action potential discharge that are reversed by the sulfonylurea KATP channel blocker tolbutamide (200 µmol l–1). Whole-cell recordings were done using patch-electrodes containing (in mmol l–1) 140 K-gluconate, 1 MgCl2, 0.5 CaCl2, 1 NaCl, 10 Hepes, pH 7.4. The electrodes also contained Na2ATP at different concentrations, in most cases 1 mmol l–1. However, varying the ATP concentration between 0 and 20 mmol l–1 did not affect the membrane response to anoxia (Müller et al., 2002). (B) The anoxic hyperpolarisation is due to opening of single KATP channels, as revealed in this example for chemical anoxia due to bath application of 1 mmol l–1 cyanide (CN–). The sharp deflections on the cell-attached current (Im) trace during control, CN– plus tolbutamide (200 µmol l–1) and wash are caused by tonic spiking. Holding potential: 0 mV. (C) Current traces of the recording in B at higher time resolution. (D) In an inside-out patch from a mouse dorsal vagal neuron, KATP channel activity is abolished by addition of 20 µmol l–1 ATP to the superfusate mimicking the intracellular solution. Holding potential: –50 mV. (E) Antisense RNA-polymerase chain reaction (aRNA-PCR) analysis of cytoplasm obtained during whole-cell recording reveals that three dorsal vagal neurons (DVN1–3) of rats coexpress mRNA for the inward-rectifying K+ (Kir) channel isoform, Kir6.2, and the sulfonylurea receptor (SUR) isoform, SUR1. Obviously, dorsal vagal neurons express the same type of KATP channels as pancreatic ß-cells innervated by a subpopulation of these neurons. A, reproduced from Ballanyi and Kulik (1998); B, C and E, reproduced from Karschin et al. (1998); D, data from K. Ballanyi and J. Brockhaus.

View larger version (19K): [in a new window]   Fig. 3. Relationship between intracellular Ca2+ concentration (Cai) and anoxic KATP channel activation in dorsal vagal neurons from juvenile rats. (A) Chemical anoxia due to 1 mmol l–1 CN– produces a persistent hyperpolarisation and block of tonic spiking while Cai increases by <50 nmol l–1. Tolbutamide (200 µmol l–1) reverses the anoxic hyperpolarisation. The concomitant reappearance of spiking induces a further stable increase of Cai that is, nevertheless, not much larger than Cai levels during physiological activity of these cells. (B) The persistence of the anoxic Cai rise in Ca2+-free superfusate suggests that this Ca2+ signal is due to release from intracellular stores. (C) The Cai rise associated with chemical anoxia is not substantially attenuated following depletion of endoplasmic reticulum Ca2+ stores with the Ca2+ pump blocker cyclopiazonic acid (CPA; 30 µmol l–1), while the anoxia response is mimicked and occluded by the mitochondrial blocker FCCP (1 µmol l–1). (D) Simultaneous recording of Cai, mitochondrial potential () and membrane current (Im) in a voltage-clamped dorsal vagal neuron filled via the patch-electrode with both 100 µmol l–1 fura-2 and 5 mg ml–1 rhodamine-123. A rapid increase of Cai during an outward current due to depolarisation from –50 to 0 mV (20 s) is followed by a modest increase in rhodamine-123 fluorescence, indicating a depolarisation of . In response to CN– (1 mmol l–1), a considerably larger mitochondrial depolarisation whose onset kinetics correlate with that of the KATP outward current is observed, while the rise of Cai is notably slower. A, reproduced from Ballanyi and Kulik (1998); B–D, data from K. Ballanyi and A. Kulik.

View larger version (26K): [in a new window]   Fig. 4. KATP channels delay a progressive anoxic Cai rise due to Ca2+ influx in whole-cell-recorded Purkinje neurons of cerebellar slices from juvenile mice. (A) CN– (1 mmol l–1) blocks spontaneous spiking due to a hyperpolarisation and concomitant increase in membrane conductance as measured in response to regular injection of hyperpolarising dc current pulses (see insets). During this phase of chemical anoxia, Cai shows a stable increase by <50 nmol l–1. Several minutes after the onset of a spontaneous repolarisation of membrane potential in the presence of CN–, a secondary progressive rise of Cai starts to develop. (B) The anoxic hyperpolarisation is caused by a prominent tolbutamide-sensitive outward current. In the presence of tolbutamide, the onset of the secondary progressive phase of the anoxic Cai rise develops almost immediately at the beginning of a CN–-induced inward current that is usually masked by the KATP outward current. Holding potential: –60 mV. (C) In a Purkinje neuron, filled via the patch-electrode with 100 mmol l–1 Cs+ and 30 mmol l–1 TEA+ to block K+ currents, CN– evokes an inward current accompanied by a major rise of Cai. These responses are not notably affected by bath application of 20 µmol l–1 CNQX and 100 µmol l–1 APV to block ionotropic glutamate receptors. (D) In a Cs+/TEA+ filled cell, the CN–-induced Cai rise is abolished by Ca2+-free superfusate that also contains 5 mmol l–1 Mg2+ to block Ca2+ channels and 1 mmol l–1 EGTA to buffer extracellular Ca2+. All recordings from K. Ballanyi, M. Lückermann and D. W. Richter.

View larger version (63K): [in a new window]   Fig. 5. Spatiotemporal relation of anoxic Cai rises in Purkinje neurons. Digital imaging was used to measure Cai in two regions of interest (pink, dendritic tree; red, soma). The continuous Cai traces illustrate that administration of CN– (1 mmol l–1) in the presence of tolbutamide (200 µmol l–1) induces instantly a similar moderate Cai rise in both the soma and dendrites. During the following 60 s, a secondary progressive Cai increase occurs first in the dendritic tree. Subsequently, a Ca2+ wave progresses from the dendrites to the soma as shown by the sequence of individual images whose numbers correspond to those on the continous Cai trace. Data and images from K. Ballanyi et al.

View larger version (23K): [in a new window]   Fig. 6. Effects of anoxia on rhythmic bursting of respiratory neurons in isolated medulla preparations from neonatal rats. (A) Bursting of rhythmogenic pre-Bötzinger complex (PBC) neurons is possibly caused by the cooperative interaction between regenerative intrinsic ion conductances such as persistent Na+ channels (NaP) or intermediate-voltage-activated (P/Q-type) Ca2+ channels (CaP) with Kir channels (including KATP channels) or leak (e.g. TASK-1 channels) that contribute to resting membrane potential (Vm) and are affected by various neuromodulators, CO2/H+ and/or O2. These neuromodulators may also indirectly affect PBC neurons via an action on Kir (or TASK-1) channels of pacemaker cells within the reticular formation proposed to provide excitatory drive to rhythmogenic PBC cells. Spike firing during individual bursts is mediated by Hodgkin-Huxley-type Na+ channels (NaHH) plus L- and N-type Ca2+ channels (CaL,N). (B) In a minor subpopulation of inspiratory (PBC) neurons in a brainstem–spinal cord preparation, a hyperpolarisation induced by anoxia does not block the rhythmic drive potential, as also evident from persistence of inspiratory-related cervical (C4) nerve rootlet activity. (C) In other respiratory neurons, such as this inspiratory cell in a brainstem–spinal cord preparation, anoxia depresses the drive potential and abolishes spiking. This effect is antagonised by the Kir and KATP channel antagonist Ba2+. The downward deflections on the membrane potential traces in B and C are responses to injection of dc current for measurement of membrane conductance. Data from K. Ballanyi.

View larger version (31K): [in a new window]   Fig. 7. Involvement of KATP channels, but not adenosine receptors, in anoxic hyperpolarisation of neonatal respiratory neurons and frequency depression of respiratory rhythm. (A) Chemical anoxia due to 1 mmol l–1 CN– notably depresses respiratory frequency in the brainstem–spinal cord preparation from newborn rats. In the presence of CN–, this effect is reversed by the KATP channel blocker gliclazide (200 µmol l–1). (B) In a neuron in the region of the pre-Bötzinger complex (PBC) of a non-rhythmic medullary slice, tolbutamide blocks the outward current and conductance increase underlying the anoxic hyperpolarisation. Membrane conductance is measured by injection of hyperpolarising dc current pulses. (C) In a pre-inspiratory neuron (also classified as `biphasic-expiratory'; Ballanyi et al., 1999), the anoxia-induced hyperpolarisation and conductance increase abolishes rhythmic fluctuations of membrane potential. After recovery from anoxia (wash), administration of adenosine (500 µmol l–1) fails to mimic the anoxic hyperpolarisation, conductance increase or block of respiratory-related membrane potential fluctuations. A, data from L. Secchia and K. Ballanyi; B,C, data from K. Ballanyi.