| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Related Rapid Report |
1 Laboratory of Cellular and Synaptic Neurophysiology, NICHD, NIH, Bethesda, MD 20892, USA
2
Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, CA 92037, USA
3
Nobel Institute for Neurophysiology, Department of Neuroscience, Retzius väg 8, A3:5, Karolinska Institute, SE-17177 Stockholm, Sweden
Abstract
Kainate receptors (KARs) play an important role in synaptic physiology, plasticity and pathological phenomena such as epilepsy. However, the physiological implications for single cells and neuronal networks of the distinct expression patterns of KAR subunits are unknown. One intriguing effect of KAR activation is a long-term change to intrinsic neuronal excitability and neuronal firing patterns, such as single-spike and spike-burst firing. In this study, we describe the role of kainate receptor subunits in the metabotropic regulation of the slow and medium afterhyperpolarization (AHP) currents (IsAHP, ImAHP). Using whole-cell patch-clamp recordings from CA3 pyramidal cells of wild-type (WT) and KAR knockout mice, we show that the kainate-induced decrease of IsAHP and ImAHP amplitude is protein-kinase-C-dependent and absent in GluR6–/– but not GluR5–/– pyramidal neurones. Our findings suggest that activation of GluR6-containing KARs modulates AHP amplitude, and influences the firing frequency of pyramidal neurones.
(Received 14 October 2004;
accepted after revision 5 November 2004;
first published online 11 November 2004)
Corresponding author A. Fisahn: Nobel Institute for Neurophysiology, Department of Neuroscience, Retzius väg 8, A3:5, Karolinska Institute, SE-17177 Stockholm, Sweden. Email: andre.fisahn{at}neuro.ki.se
Kainate is a well-known excitotoxic compound that increases excitability, generates seizure-like activity (Westbrook & Lothman, 1983; Fisher & Alger, 1984; Ben-Ari & Cossart, 2000) as well as gamma oscillations in the hippocampus (Hormuzdi et al. 2001). Changes in excitability have consequences for action potential firing characteristics of neurones, which in turn are important for the overall behaviour and output of neuronal networks such as synchronized oscillations or seizure-like activity. Prolonged depolarization of neurones above their firing threshold leads to initially sustained generation of action potentials. In most principal neurone types in the hippocampus, however, the generation of action potentials slows down and stops before the depolarizing stimulus has abated (Hotson & Prince, 1980). This spike frequency accommodation is brought about by a number of conductances, including a Ca2+-activated K+ current with slow decay time, which hyperpolarize the cell membrane (IsAHP; Lancaster & Adams, 1986; Faber & Sah, 2002; Sah & Faber, 2002; Stocker, 2004). The slow AHP current therefore governs the length and frequency of bursts of action potentials (Madison & Nicoll, 1984; Traub et al. 1993). Besides the slow AHP phase (5–10 s), a medium (100–300 ms) and a fast phase (5–10 ms) have been characterized. The fast AHP is generated by I(C), a voltage-and Ca2+-dependent current mediated by BK channels (Brown & Griffith, 1983; Storm, 1987). The medium AHP results from the activation of several conductances, one of which is the calcium-activated hyperpolarizing K+ current ImAHP (Stocker et al. 1999). These currents with fast and medium decay times modulate the length and frequency of single action potentials. A recent study showed that IsAHP is decreased by direct activation of KARs on CA1 pyramidal neurones (Melyan et al. 2002). Moreover, this action of kainate was shown to be metabotropic, activating a protein kinase C (PKC)-based signalling pathway (Melyan et al. 2002, 2004).
Kainate receptors are widely expressed in the hippocampal formation, with the various subunits (GluR5-7, KA1-2) showing distinct expression patterns (Wisden & Seeburg, 1993). A recent study showed distinct roles for the GluR5 and GluR6 subunits in kainate-induced gamma oscillations and the underlying synaptic physiology in hippocampal area CA3 (Fisahn et al. 2004). Changes in pyramidal neurone firing patterns in area CA3, i.e. from burst firing to single action potential firing, are considered essential components for the induction of gamma oscillations. In this study, we investigated the putative roles of native kainate receptors containing GluR5 or GluR6 in the modulation of the slow and medium AHP, and hence the action potential firing patterns of pyramidal neurones.
Methods
Mice (n = 76) were anaesthetized by isoflurane inhalation and decapitated according to NIH guidelines. Horizontal hippocampal slices of 250 µm thickness were prepared from 13- to 16-day-old animals of either sex, as previously described (Fisahn et al. 2002). For patch-clamp recordings, slices were superfused with artificial cerebrospinal fluid (ACSF) containing (mM): 130 NaCl, 3.5 KCl, 24 NaHCO3, 1.25 NaH2PO4, 1.5 CaCl2, 1.5 MgCl2 and 10 glucose.
Kainate receptor knockout mice originated from a mixed 129/Sv and C57/Bl6 background that were backcrossed for at least 10 generations to 129Sv/Ev mice to provide an isogenic 129Sv/Ev strain. After six generations of mating to 129Sv/Ev, more than 99% of the genetic background is 129Sv/Ev. To minimize the numbers of animals used, knockout mice from homozygote crossings were compared to control recordings taken from 129Sv/Ev wild-type (WT) crossings.
Patch-clamp recordings were made from somata of pyramidal cells of the hippocampal area CA3, as previously described (Fisahn et al. 2002). Briefly, glass microelectrodes (resistance 3–4 M
) were made from thin-walled borosilicate glass TW150F (WPI). The recording solution contained (mM): 140 KOH, 140 methanesulphonic acid, 10 Hepes, 0.6 EGTA, 2 sodium ATP, 0.3 sodium GTP, 2 MgCl2. Osmolarity was adjusted to 270–280 mosmol l–1 using KOH, and pH was adjusted to 7.3. Chemicals were purchased from Tocris Cookson or Sigma. Patch-clamp recordings (20 KHz sampling; 1–5 kHz filtering) were made using an Axopatch-1D amplifier with pClamp acquisition software (Axon Instruments).
Commercially available and in-house algorithms using Axograph (Axon Instruments) and KaleidaGraph software (Synergy Software) were used for analyses. Slow and medium AHP currents were recorded as tail currents activated following a 200 ms step from –50 mV to 0 mV and back to –50 mV (Melyan et al. 2002). Slow and medium AHP currents usually occurred together in a recording and were clearly discernable in the tail current trace as two separate peaks, with the ImAHP time course over before the onset of IsAHP, ensuring adequate separation of each current component. The slow AHP was evoked by depolarizing current pulses (1 s, 100 pA; Melyan et al. 2002). Displayed recordings are averages of 10 traces (pulses given every 20 s). Different time frames were used during analysis to isolate the currents (1–20 s for IsAHP, 0–500 ms for ImAHP). Charge transfer was computed by integrating the area under a current trace between two time points. Data are represented as means ± S.E.M. Student's t test was used for statistical analyses.
Results
Kainate reduces IsAHP in a PKC-dependent manner
In a first set of experiments, we evoked IsAHP as a tail current following a depolarizing voltage step in CA3 pyramidal neurones of WT mice under voltage-clamp conditions (holding potential (Vh) = –50 mV; 200-ms-long depolarizing pulse to 0 mV). The IsAHP charge transfer in control conditions (102.5 ± 10.5 pC; 100 µM picrotoxin, 50 µM D-aminophosphonovalerate(D-APV), 1 µM TTX present) was decreased by 39.9 ± 5.2% after superfusion with 200 nM kainate (57.8 ± 4.7 pC; n = 11; Fig. 1A). Kainate increased the holding current by 67.1 ± 11.9 pA (n = 11; data not shown). When repeating this experiment with hippocampal slices previously incubated for 3 h with the PKC antagonist calphostin C (2 µM), kainate had a significantly reduced effect on IsAHP charge transfer (9.6 ± 4.4%; control, 245.7 ± 68.9 pC; kainate, 224.9 ± 61.4 pC; n = 5; Fig. 1A). This shows that the kainate-induced IsAHP decrease involves a second messenger pathway depending on PKC.
|
Next we evoked IsAHP in pyramidal neurones of GluR5–/– and GluR6–/– mice. Similar to WT, kainate (200 nM) decreased IsAHP charge transfer in GluR5–/– by 43.6 ± 5.9% (control, 83.2 ± 12.7 pC; kainate, 47.5 ± 10.3 pC; n = 9; Fig. 1A) and increased the holding current by 66.5 ± 17.2 pA (n = 9; data not shown). Excluding the GluR5 subunit by pharmacological means in WT slices (500 nM LY 293558) led to nearly identical results: charge transfer decreased by 41.76 ± 4.88% (control, 99.3 ± 13.9 pC; kainate, 56.1 ± 6.7 pC; n = 6; data not shown). The kainate-induced decrease of IsAHP charge transfer in GluR5–/– was significantly reduced by preincubation of slices with the PKC antagonist calphostin C (2 µM; 1.1 ± 4.4%; control, 71.8 ± 11.3 pC; kainate, 71.5 ± 14.3 pC; n = 3; Fig. 1A). This shows that the GluR5 KAR subunit is not likely to participate in the modulation of IsAHP.
Unlike in WT and GluR5–/– neurones, kainate had a significantly smaller effect in GluR6–/– neurones. Kainate (200 nM) decreased IsAHP charge transfer by only 10.2 ± 4.5% (control, 78.4 ± 15.2 pC; kainate, 72.1 ± 14.5 pC; n = 8; Fig. 1A), and increased the holding current by only 2.1 ± 3.5 pA (n = 8; data not shown). Subsequent to kainate application, we applied noradrenaline (10 µM), which is known to suppress IsAHP via a PKA-dependent pathway (Madison & Nicoll, 1986; Pedarzani & Storm, 1993). This resulted in a marked decrease of IsAHP charge transfer by 54.8 ± 15.2% (control, 80.9 ± 16.8 pC; noradrenaline, 36.6 ± 8.4 pC; n = 4; Fig. 1A). Our data suggest that the GluR6 KAR subunit is involved in modulating IsAHP via a second messenger pathway involving PKC.
ImAHP reduction is absent in GluR6–/– but not GluR5–/–
Analysing evoked IsAHP traces with expanded time scale revealed a second AHP current with faster decay kinetics – reminiscent of the ImAHP (Stocker et al. 1999) – immediately following the depolarizing pulse. In WT and GuR5–/– pyramidal neurones, kainate (200 nM) decreased ImAHP charge transfer by 58.5 ± 4.4% in WT (control, 2.1 ± 0.3 pC; kainate, 0.9 ± 0.2 pC; n = 14; Fig. 1B), and by 58.4 ± 4.4% in GluR5–/– (control, 1.9 ± 0.5 pC; kainate, 0.9 ± 0.3 pC; n = 7; Fig. 1B). In contrast, the kainate-induced decrease of ImAHP charge transfer was significantly reduced in GluR6–/– neurones (27.7 ± 9.5%; control, 1.9 ± 0.4 pC; kainate, 1.5 ± 0.4 pC; n = 8; Fig. 1B). As was the case with IsAHP, the kainate-induced modulation of ImAHP was PKC-dependent, as shown by a significant reduction in kainate-induced charge transfer decrease in WT neurones after preincubation with the PKC antagonist calphostin C (2 µM; 18.5 ± 4.6%; control, 4.0 ± 1.2 pC; kainate, 3.3 ± 1.1 pC; n = 5; Fig. 1B).
Firing frequency increase induced by AHP reduction is absent in GluR6–/–
In a further set of experiments, WT and GluR6–/– pyramidal neurones were recorded under current-clamp conditions (held at resting membrane potential; Vm = –58.7 ± 3.5 mV), and slow AHPs were evoked by injecting 1-s-long pulses of 100 pA. Kainate (200 nM) reduced the AHP (integrated) in WT by 44.4 ± 5.6% but not GluR6–/– neurones (2.9 ± 4.6%; Fig. 2A; n = 6 each; 10 sweep averages shown). Finally, we recorded from WT and GluR6–/– pyramidal cells held at their firing threshold so as to generate action potentials continuously. In control conditions, WT neurones fired at 0.8 ± 0.3 Hz, and GluR6–/– neurones at 0.2 ± 0.2 Hz. Kainate (200 nM) increased firing frequency in WT by 460% to 4.4 ± 1.9 Hz, but failed to do so in GluR6–/– (–11%; 0.2 ± 0.1 Hz; n = 3 each; data not shown). Averages of the action potentials triggered at the rising face of the spike showed a kainate-induced reduction of the AHP, and smaller interspike interval in WT but not GluR6–/– neurones (Fig. 2B; n = 3 each).
|
Our data demonstrate that the metabotropic action of kainate on the Ca2+-activated K+ currents that underlie the AHP occurs via GluR6-containing KARs. It effects the reduction of both IsAHP and ImAHP via a PKC-dependent second messenger pathway, and causes pyramidal neurones to fire single action potentials at increased frequency. The PKA pathway, in turn, is unlikely to be involved in the metabotropic action of kainate. It is interesting to note that the kainate-induced decrease of ImAHP in CA3 is not found in CA1 pyramidal neurones (Melyan et al. 2002). We would argue that the discrepancies between ImAHP modulation in CA1 and CA3 pyramidal neurones reflect the differences of action potential firing patterns in these neurones.
Long-term changes of neuronal excitability as well as firing patterns resulting from modulation of intrinsic conductances have been studied extensively (Madison & Nicoll, 1984; Traub et al. 1993; Giese et al. 1998; Saar et al. 2001). In the context of rhythmic network activity like gamma oscillations, a switch from burst- to single-spike mode in pyramidal neurones is a crucial ingredient, besides an increased tonic excitation, to generate such activity (Fisahn, 1999). Our data demonstrate a GluR6-dependent mechanism that accomplishes changes in pyramidal neurone firing pattern. Our results showing a lack of AHP modulation in GluR6–/– neurones complement findings that kainate-induced depolarization is absent in GluR6–/– pyramidal neurones as well as interneurones, and that GluR6–/– hippocampal slices are unable to generate gamma oscillation when superfused with kainate (Fisahn et al. 2004). Furthermore, our data help to explain why GluR6–/– mice are more resistant to kainate-induced seizures than their WT counterparts (Mulle et al. 1998). The GluR6-containing KARs modulating the AHP are likely to be the same receptors involved in synaptic transmission. For future studies it will be of interest to determine whether synaptic activation of GluR6-containing receptors can modulate action potential AHP.
References
Ben-Ari Y & Cossart R (2000). Kainate, a double agent that generates seizures: two decades of progress. Trends Neurosci 23, 580–587.[CrossRef][Medline]
Brown DA & Griffith WH (1983). Calcium-activated outward current in voltage-clamped hippocampal neurones of the guinea-pig. J Physiol 337, 287–301.
Faber ES & Sah P (2002). Physiological role of calcium-activated potassium currents in the rat lateral amygdala. J Neurosci 22, 1618–1628.
Fisahn A (1999). An investigation into cortical gamma frequency oscillations in vitro. PhD Thesis, Oxford University.
Fisahn A, Contractor A, Traub RD, Buhl EH, Heinemann S & McBain CJ (2004). Distinct roles for the kainate receptor subunits GluR5 and GluR6 in kainate-induced hippocampal gamma oscillations. J Neurosci 24, 9658–9668.
Fisahn A, Yamada M, Duttaroy A, Gan JW, Deng CX, McBain CJ & Wess J (2002). Muscarinic induction of hippocampal gamma oscillations requires coupling of the M1 receptor to two mixed cation currents. Neuron 33, 615–624.[CrossRef][Medline]
Fisher RS & Alger BE (1984). Electrophysiological mechanisms of kainic acid-induced epileptiform activity in the rat hippocampal slice. J Neurosci 4, 1312–1323.[Abstract]
Giese KP, Storm JF, Reuter D, Fedorov NB, Shao LR, Leicher T, Pongs O & Silva AJ (1998). Reduced K+ channel inactivation, spike broadening, and after-hyperpolarization in Kvbeta1.1-deficient mice with impaired learning. Learn Mem 5, 257–273.
Hormuzdi S, Pais I, LeBeau FEN, Towers SK, Rozov A, Buhl EH, Whittington M & Monyer H (2001). Impaired electrical signaling disrupts gamma frequency oscillations in connexin 36-deficient mice. Neuron 31, 487–495.[CrossRef][Medline]
Hotson JR & Prince DA (1980). A calcium-activated hyperpolarization follows repetitive firing in hippocampal neurons. J Neurophysiol 43, 409–419.
Lancaster B & Adams PR (1986). Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons. J Neurophysiol 55, 1268–1282.
Madison DV & Nicoll RA (1984). Control of the repetitive discharge of rat CA1 pyramidal neurones in vitro. J Physiol 354, 319–331.
Madison DV & Nicoll RA (1986). Actions of noradrenaline recorded intracellularly in rat hippocampal CA1 pyramidal neurones, in vitro. J Physiol 372, 221–244.
Melyan Z, Wheal HV & Lancaster B (2002). Metabotropic-mediated kainate receptor regulation of IsAHP and excitability in pyramidal cells. Neuron 34, 107–114.[CrossRef][Medline]
Melyan Z, Wheal HV & Lancaster B (2004). Metabotropic regulation of intrinsic excitability by synaptic activation of kainate receptors. J Neurosci 24, 4530–4534.
Mulle C, Sailor A, Perez-Otano I, Dickinson-Anson H, Castillo PE, Bureau I et al. (1998). Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice. Nature 392, 601–605.[CrossRef][Medline]
Pedarzani P & Storm JF (1993). PKA mediates the effects of monoamine transmitters on the K+ current underlying the slow spike frequency adaptation in hippocampal neurons. Neuron 11, 1023–1035.[CrossRef][Medline]
Saar D, Grossman Y & Barkai E (2001). Long-lasting cholinergic modulation underlies rule learning in rats. J Neurosci 21, 1385–1392.
Sah P & Faber ES (2002). Channels underlying neuronal calcium-activated potassium currents. Prog Neurobiol 66, 345–353.[CrossRef][Medline]
Stocker M (2004). Ca2+-activated K+ channels: molecular determinants and function of the SK family. Nat Rev Neurosci 5, 758–770.[CrossRef][Medline]
Stocker M, Krause M & Pedarzani P (1999). An apamin-sensitive Ca2+-activated K+ current in hippocampal pyramidal neurons. Proc Natl Acad Sci U S A 96, 4662–4667.
Storm JF (1987). Action potential repolarization and a fast after-hyperpolarization in rat hippocampal pyramidal cells. J Physiol 385, 733–759.
Traub RD, Miles R & Jefferys JGR (1993). Synaptic and intrinsic conductances shape picrotoxin-induced synchronized after-discharges in the guinea-pig hippocampal slice. J Physiol 461, 525–547.
Westbrook GL & Lothman EW (1983). Cellular and synaptic basis of kainic acid-induced hippocampal epileptiform activity. Brain Res 273, 97–109.[CrossRef][Medline]
Wisden W & Seeburg PH (1993). A complex mosaic of high-affinity kainate receptors in rat brain. J Neurosci 13, 3582–3598.[Abstract]
Acknowledgements
This research was supported by a HFSP Long-Term Fellowship (A.F.), NIH grant NS28709, the McKnight Foundation, the John Adler Foundation (S.F.H.) and an NICHD intramural award (C.J.M.).
This article has been cited by other articles:
|
|
 |
|
  G. Grabauskas, B. Lancaster, V. O'Connor, and H. V. Wheal Protein kinase signalling requirements for metabotropic action of kainate receptors in rat CA1 pyramidal neurones J. Physiol., March 1, 2007; 579(2): 363 - 373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. D. Salinas, L. A. C. Blair, L. A. Needleman, J. D. Gonzales, Y. Chen, M. Li, J. D. Singer, and J. Marshall Actinfilin Is a Cul3 Substrate Adaptor, Linking GluR6 Kainate Receptor Subunits to the Ubiquitin-Proteasome Pathway J. Biol. Chem., December 29, 2006; 281(52): 40164 - 40173. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. F. de Sevilla, J. Garduno, E. Galvan, and W. Buno Calcium-Activated Afterhyperpolarizations Regulate Synchronization and Timing of Epileptiform Bursts in Hippocampal CA3 Pyramidal Neurons J Neurophysiol, December 1, 2006; 96(6): 3028 - 3041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. V. Negrete-Diaz, T. S. Sihra, J. M. Delgado-Garcia, and A. Rodriguez-Moreno Kainate Receptor-Mediated Inhibition of Glutamate Release Involves Protein Kinase A in the Mouse Hippocampus J Neurophysiol, October 1, 2006; 96(4): 1829 - 1837. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
