NMDA receptor-dependent long-term potentiation in mouse hippocampal interneurons shows a unique dependence on Ca 2+ /calmodulin-dependent kinases

Transcription

1 J Physiol (7) pp NMDA receptor-dependent long-term potentiation in mouse hippocampal interneurons shows a unique dependence on Ca + /calmodulin-dependent kinases Karri Lamsa, Elaine E. Irvine, K. Peter Giese and Dimitri M. Kullmann Institute of Neurology and Wolfson Institute for Biomedical Research, University College London Long-term potentiation (LTP) of excitatory synaptic transmission plays a major role in memory encoding in the cerebral cortex. It can be elicited at many synapses on principal cells, where it depends on Ca + influx through postsynaptic N-methyl-D-aspartic acid (NMDA) receptors. Ca + influx triggers phosphorylation of several kinases, in particular Ca + /calmodulindependent kinase type II (CaMKII). Auto-phosphorylation of CaMKII is a key step in the LTP induction cascade, as revealed by the absence of LTP in hippocampal pyramidal neurons of αcamkii T86A-mutant mice, where auto-phosphorylation of the α isoform at residue T86 is prevented. A subset of hippocampal interneurons mediating feed-forward inhibition also exhibit NMDA receptor-dependent LTP, which shows all the cardinal features of Hebbian LTP in pyramidal neurons. This is unexpected, because αcamkii has not been detected in interneurons. Here we show that pathway-specific NMDA receptor-dependent LTP is intact in hippocampal inhibitory interneurons of αcamkii T86A-mutant mice, although in pyramidal cells it is blocked. However, LTP in interneurons is blocked by broad-spectrum pharmacological inhibition of Ca + /calmodulin-dependent kinases. The results suggest that non-α Ca + /calmodulin-dependent kinases substitute for the α isoform in NMDA receptor-dependent LTP in interneurons. (Resubmitted 5 May 7; accepted after revision 3 September 7; first published online September 7) Corresponding author D. M. Kullmann: Institute of Neurology, University College London, Queen Square, London, WCN 3BG, UK. d.kullmann@ion.ucl.ac.uk Ca + /calmodulin-dependent kinase type II (CaMKII) is an abundant protein in the postsynaptic densities of excitatory synapses, and plays a central role in NMDA receptor-dependent long-term potentiation (LTP) (Malinow et al. 989; Lisman et al. ). It is thought to exist as a dodecameric holoenzyme consisting of α and β subunits in a variable ratio (Bennett et al. 983; Hudmon & Schulman, ). It is unusual in that its catalytic activityistriggeredinaca + -dependent manner, but following auto-phosphorylation (at T86 and T87 of the α and β isoforms, respectively) it becomes independent of Ca +. An obligatory role of auto-phosphorylation of the α subunit in NMDA receptor-dependent LTP is demonstrated by the finding that LTP in hippocampal pyramidal cells is abolished in α-isoform CaMKII T86A-mutant mice (Giese et al. 998; Cooke et al. 6). Because these mice also show major defects in tests of spatial learning (Giese et al. 998; Need & Giese, 3), This paper has online supplemental material. they have provided some of the most compelling evidence linking LTP to memory encoding. Although LTP has been extensively studied in synapses connecting principal (glutamatergic) cells in several areas of the cerebral cortex, evidence that it also occurs in glutamatergic synapses onto GABAergic inhibitory interneurons has been scanty (Maccaferri & McBain, 996; Mahanty & Sah, 998; Alle et al. ; Perez et al. ). We recently reported that NMDA receptor-dependent LTP could be readily elicited in approximately half of GABAergic interneurons whose somata locate in stratum radiatum of the area CA area in rat hippocampus (Lamsa et al. 5). Many interneurons in this area mediate feed-forward inhibition of CA pyramidal cells. Plasticity in glutamatergic synapses that drive these interneurons plays an important role in modulating disynaptic inhibition, and shaping the temporal window for input summation and spike generation in pyramidal neurons (Lamsa et al. 5). This form of synaptic plasticity is distinct from NMDA receptor-independent LTP, which can be elicited principally in interneurons in stratumoriens(perezet al. ; Kullmann & Lamsa, C 7 The Authors. Journal compilation C 7 The Physiological Society DOI:.3/jphysiol

2 886 K. Lamsa and others J Physiol ; Lamsa et al. 7), and which will not be considered further in this study. LTP at glutamatergic synapses on interneurons in stratum radiatum shows all the cardinal properties of LTP described in pyramidal cells: a requirement for postsynaptic depolarization to activate NMDA receptors for induction, pathway-specific expression, and no robust change in short-term synaptic facilitation (Lamsa et al. 5). However, LTP in interneurons shows a strikingly different sensitivity to disruption of cellular integrity than LTP in pyramidal cells: it can only be reliably elicited when recording with methods that avoid dialysis of intracellular content, such as the perforated patch technique. Conventional whole cell recording rapidly prevents LTP induction in these cells (Lamsa et al. 5). LTP in GABAergic interneurons potentially has major implications for our understanding of signal processing in the feed-forward circuitry during memory encoding, but it also challenges two widely held assumptions about the cellular substrate of NMDA receptor-dependent LTP. First, the finding that pathway specific LTP occurs in cells with few or no dendritic spines (Lamsa et al. 5) implies that compartmentalization of the postsynaptic induction cascade by these structures is not an absolute requirement for synaptic plasticity to be spatially restricted. Evidence that synaptically evoked Ca + microdomains can be elicited in smooth dendrites (Goldberg et al. 3; Goldberg & Yuste, 5) provides a possible resolution of this paradox. Second, as mentioned above, NMDA receptor-dependent LTP in CA principal cells of adult rats and mice requires auto-phosphorylation of αcamkii, which is not detected in hippocampal interneurons (Liu & Jones, 996; Sik et al. 998). However, the Ca + calmodulin signalling pathway has been shown to strongly up-regulate glutamatergic synaptic transmission in non-pyramidal neurons in the hippocampal CA subfield (Wang & Kelly, ), much as it does in pyramidal cells (Pettit et al. 994; Lledo et al. 995; Otmakhov et al. 997). The potentiation is blocked by calmodulin-binding peptides as well as by an autoinhibitory peptide of Ca + /calmodulin-dependent protein kinases (Wang & Kelly, ). We therefore examined the role of Ca + calmodulin-dependent signalling downstream of NMDA receptors in Hebbian LTP in hippocampal interneurons, through the use of αcamkii T86A-mutant mice and pharmacological manipulation of Ca + /calmodulin-dependent kinases. Methods Knock-in mice All animal procedures followed the Animals (Scientific Procedures) Act, 986. Heterozygous αcamkii T86Amutants backcrossed into a hybrid C57BL6 9/Sv genetic background were interbred to obtain homozygous and wild-type (WT) littermates. Genotyping was carried out by PCR analysis, as previously described (Giese et al. 998), with DNA obtained from tail biopsies on postnatal day, the day of weaning. Male mice homozygous for αcamkii T86A and WT littermates were used in all experiments. Hippocampal slices Three- to five-week-old homozygous αcamkii T86Amutant mice and WT littermates were killed by cervical dislocation and decapitated, and the brain was rapidly removed and placed in ice-cold ( to +4 C) sucrose cutting solution containing (mm): 75 sucrose, 87 NaCl,.5 KCl,.5 CaCl, 7 MgCl,.5 NaH PO 4, 5 NaHCO 3, 5 glucose, ph 7.4 and bubbled with 95% O 5% CO. Transverse hippocampal slices (5 μm thick) were cut using a vibrating microtome (Leica VT S, Leica Microsystems, Germany), and transferred to an interface chamber where they were maintained in artificial cerebrospinal fluid (ACSF) at room temperature ( 5 C) for at least 6 min before starting the experiments. The composition of the ACSF was (mm): 9 NaCl, 3.5 KCl,.5 CaCl,.3 MgSO 4,.5 NaH PO 4, 5 NaHCO 3, glucose, ph 7.4 (equilibrated with 95% O 5% CO ). Hippocampal slices were recorded in a chamber (Luigs & Neumann, Germany) mounted on the stage of an upright microscope (Olympus BX5WI, Japan), where they were held under a nylon mesh grid and superfused (at 3 ml min ) with ACSF at 3 C. Slices were visualized using a immersion objective with 4 zoom and infra-red differential interference contrast (DIC) optics or epifluorescence imaging (Till Photonics, Germany). Electrophysiology A cut was made in the slice between CA and CA3 to prevent propagation of recurrent excitation in Schaffer collaterals in response to stimulation. The GABA receptor blockers picrotoxin (PiTX, μm) and CGP543 (5 μm) were routinely added to the physiological solution during experiments. Cells were recorded with a Multiclamp 7 amplifier (Molecular Devices, CA, USA). Somatic perforated-patch recordings were obtained from CA pyramidal cells in stratum pyramidale and from non-pyramidal cells in mid or distal stratum radiatum using electrodes made from borosilicate glass capillaries (GC5F,.5 mm o.d., Harvard Apparatus, UK), pulled on a Sutter microelectrode puller (Novato, CA, USA). Pipette resistances were typically 8 M. Gramicidin (mixture of gramicidins A, B, C and D from Bacillus aneurinolyticus, Sigma) stock of C 7 The Authors. Journal compilation C 7 The Physiological Society

3 J Physiol NMDA receptor-dependent LTP in interneurons is independent of αcamkii 887 mg ml was prepared in dimethyl sulphoxide daily and kept at +4 C. The gramicidin-containing pipette filling solution was prepared by diluting the stock solution : in filtered (<. μm pore diameter) potassium gluconate pipette solution. The potassium gluconate pipette solution contained (mm): 45 potassium gluconate, 8 NaCl, 5 KOH-HEPES,. EGTA, and 5 QX-34 Br (ph 7., osmolarity 95 mosmol l ). The final gramicidin-containing solution was sonicated in an ultrasound bath several times for short (3 5 s) periods. This was repeated every min. New gramicidin-containing pipette solution was prepared every h. The electrode tip was filled with gramicidin-free filtered potassium gluconate solution. The series resistance was continuously monitored throughout the experiment, and recordings were started when it was < M and stable. Bridge balance was adjusted throughout the recordings. Depolarizing currents were intermittently injected to evoke action potentials to verify patch integrity. (Inadvertent patch rupture was detected by inability to evoke action potentials, because of diffusion of QX-34 into the cell. If this happened, the experiment was stopped.) In some experiments QX-34 was omitted and the fluorescent dye Alexa Fluor 488 ( μm) was included in the pipette, and the neuron was imaged with epifluorescence to monitor that the dye did not penetrate the patch and diffuse into the cell. If fluorescence was detected in the cell, the experiment was discontinued. Monosynaptic excitatory postsynaptic potentials (EPSPs) were evoked by alternately stimulating Schaffer collaterals via two bipolar electrodes positioned in stratum radiatum in area CA, connected to constant current isolated stimulators (Digitimer model DS3, stimulus pulse intensity μa, duration 5 μs). The stimulators were controlled by a custom data acquisition program (LabView, National Instruments). Evoked EPSPs were recorded from a resting membrane potential (liquid junction potential corrected), low-pass filtered (5 khz) and acquired at khz on a PC for off-line analysis. For LTP induction one of the pathways was stimulated at Hz for s (delivered twice with a s interval). Simultaneously, the postsynaptic cell was depolarized nominally to mv with a positive current step injected in current clamp mode. In some experiments EPSPs were recorded during a brief (5 ms) hyperpolarizing step (5 mv) to avoid action potential generation. The initial slope (3 5 ms from onset) of the EPSPs was analysed, and all EPSPs were visually verified for the analysis to restrict attention to monosynaptic excitatory inputs in LTP experiments. The electrophysiological properties of recorded interneurons were characterized at the end of the experiment. Cell input resistance was measured using a pa ( s) current pulse in bridge-balanced current clamp mode. Membrane time constant was determined from the negative slope of a mv step from resting membrane potential. Delay to spike was estimated from three or more responses as the average delay to the first spike generated by a depolarizing step slightly above the firing threshold. Maximal firing frequency was taken from the first ms during maximal action potential firing in response to a > 5 ms depolarizing step. Percentage adaptation was obtained from the reduction in the spiking frequency when a period 4 5 ms after the start of current injection was compared with the initial ms period during maximal firing frequency. Afterhyperpolarization (AHP) amplitude was measured from the first action potential generated by depolarization beyond firing frequency. Membrane potential sag (V-sag) was measured from a hyperpolarizing step to 9 mv. Rebound spikes indicate the average number of action potentials on release from hyperpolarization to 9 mv. Chemicals were purchased from Sigma. Receptor antagonists were purchased from Tocris Cookson. Statistical analysis Data are shown as means ± s.e.m., normalized by baseline values, and analysed with Student s paired or unpaired t test against a cut-off significance level (P) of.5. The experimenter was blind to the genotype in the experiments for Fig. A F. Results In order to shed light on the Hebbian LTP induction cascade in interneurons, we recorded from interneurons in stratum radiatum with the gramicidin perforated patch method, with GABA receptors blocked throughout. A limitation of the perforated patch method is that, because the gramicidin pores are only permeable to small monovalent cations, it does not allow introduction of chemical markers into the cell for post hoc anatomical identification. In order to discriminate interneurons from CA pyramidal cells we therefore systematically documented the passive and active electrophysiological properties of the recorded neurons at the end of experiments. This allowed interneurons in stratum radiatum to be clearly distinguished from displaced pyramidal cells (Gulyas et al. 998) and further allowed them to be classified into six categories following the guidelines of the Petilla convention (Yuste, 5). The six different electrophysiological non-pyramidal cell types were named after their action potential firing patterns: regular-spiking non-pyramidal cells (RSNP), further subdivided into rebounding (R-), non-rebounding (NR-), and rapidly adapting (RA-) RSNPs; burst-spiking non-pyramidal cells (BSNP); fast-spiking interneurons (FS); irregularly spiking interneurons (IS); and delayed C 7 The Authors. Journal compilation C 7 The Physiological Society

4 888 K. Lamsa and others J Physiol Table. Electrophysiological properties of non-pyramidal cell classes were significantly different from those of pyramidal cells PC NR-RSNP RA-RSNP R-RSNP FS DS IS (n = 6) (n = 36) (n = 6) (n = 6) (n = 5) (n = 3) (n = ) RMP (mv) 6.8 ± ± ± ± ± ± R in (MOhm) 7 ± 6 35 ± 3 33 ± 36 ± ± ± τ ( mv) (ms) 9.8 ± ± ± ± ± ± Delay to spike (ms) 48. ± ±.8 3. ± ± 6.7. ± ± Max firing freq. (Hz) 86 ± 9 4 ± 36 8 ± 5 8 ± 39 6 ± ± 5 7 % freq. adaptation 49 ± 5 4 ± 7 ± 3 4 ± 7 6 ± 34 ± 3 8 AHP amplitude (mv) 4. ±. 8.3 ±.8. ± ± 3.7. ± ± V-sag ( 9 mv) (mv).3 ±.7.3 ±..9 ± ± 4.8. ±.9. Rebound spikes. ±.4.6 ±.9.5 ±.6 The table shows a summary of the analysed electrophysiological properties of the CA pyramidal cells (in stratum pyramidale) and the six subtypes of non-pyramidal cells in s. radiatum (including all cells where electrophysiological characterization was successfully studied at the end of the experiment). Non-pyramidal cells are grouped according to their firing profiles. RMP, resting membrane potential; R in, input resistance; τ, membrane time constant; AHP, afterhyperpolarization. Each parameter of every interneuron group was compared to the corresponding value of the pyramidal cells. Asterisks indicate parameters that differed significantly (P <.5) from those of the pyramidal cells (unpaired t test). spiking interneurons (DS) (online supplemental material, Supplemental Fig. ). All interneuron classes in stratum radiatum differed significantly from CA pyramidal cells in their electrophysiological properties (Table ). Comparing the electrophysiological properties of the interneurons in the mutants and WT littermates revealed no difference in the occurrence of the interneuron types in between the genotypes (Fig. ). In both mutants and WT animals the predominant category was regular-spiking non-pyramidal cells, which accounted for approximately 8% of interneurons in stratum radiatum. We asked whether Hebbian long-term potentiation occurs in inhibitory interneurons in αcamkii T86A- mutant mice, in which autophosphorylation of the α subunit at T86 is prevented. In parallel we studied WT littermates, and the experimenter was blind to the genotype. After recording EPSPs in two Schaffer collateral pathways for a stable (> min) baseline, high-frequency stimulation was applied to one pathway ( Hz, s, delivered twice with a s interval), paired with postsynaptic depolarization (nominally to a somatic potential of mv). In order to take into account non-specific drift in recording conditions, EPSP initial slopes were not only normalized within each pathway by the baseline slope, but also compared between pathways: only if the paired pathway showed a > 5% increase in A B RSNP DS Normalized occurrence RSNP FS MUT n = 4 WT n = 6 IS DS Vm - 6 mv Ic Vm - 66 mv Ic IS Vm - 68 mv + pa + pa Ic + pa -5 pa - pa PC 5 mv Vm 5 ms +5 pa - 64 mv +3 pa -5 pa Ic -3 pa Figure. Occurrence of electrophysiologically defined interneuron types in stratum radiatum of CA in αcamkii T86A-mutant mice is similar to WT A, histogram showing the distribution of different interneuron spike firing categories in mutant and WT animals (RSNP, regular-spiking non-pyramidal cells; FS, fast spiking interneurons; IS, irregular-spiking interneurons DS, delayed-spiking interneurons;). B, sample membrane potential (V m ) traces with step pulses in current clamp (I c ) obtained from three interneurons representing the major spike pattern categories, and from a pyramidal cell (PC) for comparison. C 7 The Authors. Journal compilation C 7 The Physiological Society

5 J Physiol NMDA receptor-dependent LTP in interneurons is independent of αcamkii 889 slope relative to the control pathway, lasting 5 min, was LTP deemed to have been elicited. We have previously shown that NMDA receptor-dependent LTP can be elicited in approximately half of the interneurons in stratum radiatum in the rat (Lamsa et al. 5). We therefore subdivided cells according to whether Hebbian LTP induction was successful. Pairing high-frequency stimulus with depolarization led to persistent pathway-specific LTP in 3 out of interneurons recorded from homozygous mutant mice (average potentiation: 84 ± %). The time course of the potentiation is shown for these LTP-exhibiting interneurons in Fig. A and B. In seven other interneurons no change in synaptic strength occurred. The normalized A D αcamkii T86A-mutant interneurons 3 n = 3-3 WT interneurons P <.5 n= 5 - P <.5 B E Initial slope -5 min -5-- min mv 3.5 ms -5 min -5- min mv 4 ms 5 ms 5 ms C F 3 3 G 3 αcamkii T86A-mutantPCs n = 6-5 H -5 min -5- min mv 5 ms 5 ms I 3 3 Figure. αcamkii T86A-mutant mice show loss of Hebbian LTP in CA pyramidal cells but not in stratum radiatum interneurons A, EPSP initial slope (mean ± S.E.M., normalized by the baseline value) plotted for the paired and control pathways (filled and open symbols, respectively), in 3 non-pyramidal cells in stratum radiatum from αcamkii T86A-mutant mice. A high-frequency train (arrow) was delivered to one Schaffer collateral pathway (filled symbols) while the interneuron was depolarized to evoke a train of action potentials. B, traces (average of consecutive EPSPs) taken from a representative experiment before (blue) and after LTP (red). Seven other interneurons did not exhibit LTP. The initial slope of the EPSPs was measured between the vertical lines. C, average EPSP slopes in the two pathways, each normalized by the baseline value, plotted against one another for all cells tested. The points clustered on the line of identity (open triangles) showed no LTP. The grey squares represent 3 cells showing significant (P <.5) pathway-specific LTP and plotted in A. D F, data obtained from interneurons in WT mice, plotted in the same way, showing LTP in 5/9 cells. G I, data obtained from pyramidal neurons, showing post-tetanic potentiation only following pairing. For the non-pyramidal cell experiments, the experimenter was blind to the genotype. C 7 The Authors. Journal compilation C 7 The Physiological Society

6 89 K. Lamsa and others J Physiol EPSP initial slopes in the two pathways are plotted against one another for all cells in Fig. C. The points to the right of the line of identity (grey symbols) correspond to the 3 cells showing pathway-specific LTP. The fraction of cells showing LTP was not significantly different from that obtained in hippocampal slices taken from interleaved WT littermates (LTP observed in 5 out of 9 cells; Fig. D F). In contrast, with the same induction protocol and recording method, LTP could not be elicited in any of six electrophysiologically characterized pyramidal cells of αcamkii T86A-mutant mice (Fig. G I). In WT mice or rats, LTP was obtained in all pyramidal cells tested (data not shown). We thus conclude that, although prevention of α subunit auto-phosphorylation blocks LTP in pyramidal cells (Giese et al. 998), it has no effect on Hebbian LTP in interneurons in stratum radiatum. Because NMDA receptor-dependent LTP has previously only been studied in rats, we determined whether LTP in mouse interneurons shares similar induction and expression properties. These experiments were done in WT hippocampal slices. We designed an experiment to determine simultaneously whether LTP (i) is restricted to the conditioned afferent pathway, and (ii) remains confined to the interneuron where it is induced. We recorded EPSPs simultaneously from two interneuron in stratum radiatum, evoked by alternately stimulating via two bipolar stimulation electrodes also positioned in stratum radiatum. After collecting a stable baseline (> min), high-frequency stimulation of one of the two Schaffer collateral pathways ( Hz, s, delivered twice) was paired with postsynaptic depolarization of one of the two interneurons. The depolarized interneuron fired a train of action potentials during the tetanic stimulation. The other interneuron was hyperpolarized during the tetanic stimulation, in order to prevent firing (Fig. 3A). This protocol led to LTP in the tetanized pathway in the depolarized interneuron in four out of 7 slices, as witnessed by a persistent increase in the initial slope of the EPSP (6 ± %, P <.5) and lasting at least 3 min (Fig. 3B and C). The potentiation was always restricted to the tetanized pathway, and to the depolarized interneuron: the initial slope of the EPSP in the control pathway underwent no change ( 3 ± 9% of control). Moreover, neither pathway in the hyperpolarized interneuron underwent a long-term change in EPSP slope (tetanized: 9 ± %; control: 8 ± 9%). In three further slices, no potentiation was elicited in the tetanized pathway in either interneuron. Thus, we conclude that, in WT mice, pathway-specific LTP can be elicited by pairing presynaptic activity of Schaffer collaterals with postsynaptic depolarization, in about half of interneurons in stratum radiatum. LTP does not spread either Figure 3. Hebbian LTP at Schaffer collateral stratum radiatum interneuron synapses does not spread to other interneurons A, schematic showing experimental design. Two interneurons (IN and IN ) were recorded simultaneously with perforated patch pipettes. One cell (IN ) was hyperpolarized to 9 mv while the other cell (IN ) was made to fire a high-frequency train of action potentials at the same time as one pathway (filled symbol) was stimulated at Hz (the protocol was delivered twice, with a s interval). B, traces showing EPSP and initial slope in a depolarized cell and in a hyperpolarized cell 5 min before and 5 min after high-frequency afferent stimulation. The window for the initial slope (3 ms) is shown by dotted lines. C, EPSP initial slope (mean ± S.E.M., normalized by the baseline) plotted for depolarized (IN ) and hyperpolarized (IN ) cells in four similar experiments. The difference between the tetanized and control pathways (filled and open symbols, respectively) was tested by a paired t test at the time windows indicated. C 7 The Authors. Journal compilation C 7 The Physiological Society

7 J Physiol NMDA receptor-dependent LTP in interneurons is independent of αcamkii 89 to the un-tetanized pathway or to simultaneously recorded interneurons that were hyperpolarized. The proportion of interneurons where this form of LTP could be elicited was similar to the success rate in rat hippocampal slices (Lamsa et al. 5). The finding that Hebbian LTP is induced in interneurons of the αcamkii T86A-mutant mice indicates that α subunit auto-phosphorylation is not required for LTP in interneurons in stratum radiatum, in contrast to CA pyramidal cells. This implies that the molecular events triggered by pairing in interneurons are different from those in pyramidal cells. Hebbian LTP induction in pyramidal cells and in rat interneurons requires activation of NMDA receptors (Lamsa et al. 5, 7). However, a possible explanation for intact LTP in αcamkii-mutant mice is that it relies on a different induction mechanism that does not rely on NMDA receptors (Perez et al. ; Lamsa et al. 7). We therefore examined the role of NMDA receptors. EPSPs were recorded as above, but NMDA receptors were blocked with d,l--amino-5-phosphonopentanoic acid (dl-apv, μm). After a baseline (> min) one of the pathways was paired with postsynaptic depolarization as in Fig.. The LTP induction protocol was uniformly unsuccessful in nine interneurons tested in αcamkii T86A-mutant mice when NMDA receptors were blocked (Fig. 4A and B). LTP was similarly prevented by APV in interneurons of WT mice (with only one cell meeting the criterion of > 5% persistent increase in synaptic strength, Fig. 4C). Thus, NMDA receptor activation is necessary for Hebbian LTP induction in interneurons in CA stratum radiatum of the mouse hippocampus, as previously reported in rats (Christie et al. ; Lamsa et al. 5). Further, the similar results in αcamkii T86A-mutant and WT mice argue against a compensatory switch in mutant mice to an NMDAR-independent mechanism. Intact LTP in interneurons in αcamkii-mutant mice suggests that autophosphorylation at T86 is not necessary for LTP, in striking contrast to pyramidal cells. Three possible explanations for the results can be proposed: (i) the signalling cascade downstream of NMDA receptors in interneurons is not dependent on CAMKII activity at all; (ii) LTP requires CAMKII activity as in pyramidal cells, but depends on an isoform other than α; (iii) basal activity of αcamkii in the mutant is sufficient for LTP, which does not require autophosphorylation at T86. We tested the involvement of Ca + /calmodulin-dependent kinases in the LTP induction, by adding the broad spectrum blocker KN-6 ( μm) to the ACSF. Pre-incubation of the slice with KN-6 (> min) blocked induction of pathway-specific LTP in interneurons of both αcamkii T86A-mutant and WT mice (with only /9 cell meeting the criterion for LTP in each case, Fig. 4D F). LTP was similarly blocked by the alternate CaMKII inhibitor KN-93 μm, Fig. 4 G and H. When the inactive analogue KN-9 (5 μm) was applied for the same duration prior to pairing, LTP was elicited in 4/9 cells (Fig. 4I and J). Thus, blockade of LTP by KN-6 and KN-93 is unlikely to reflect non-specific actions of this group of compounds (Rezazadeh et al. 6). Although these results imply that LTP in interneurons in stratum radiatum depends on NMDA receptors and Ca + /calmodulin-dependent kinase activity, a possible (albeit unlikely) explanation is that, by chance, the interneuron samples were biased towards those that do not normally show LTP. We have previously shown that LTP-competent interneurons can be identified on-line by a sequential pairing protocol: LTP is reliably induced in two Schaffer collateral pathways targeting the cell when the pathways are paired consecutively (Lamsa et al. 5). We verified that this was also the case in αcamkii T86A-mutant mice: in 7 out of 7 interneurons where LTP was elicited in one pathway, subsequent pairing of the second pathway also resulted in LTP (Fig. 5A). This result argues strongly that failure to induce LTP in all interneurons reflects their heterogeneity. (Nevertheless, we have not been able to find a subset of interneurons, characterized electrophysiologically, that defines whether NMDAR-dependent LTP can be induced.) We then applied the sequential pairing experimental design to test the role of NMDA receptors and Ca + /calmodulin-dependent kinases. In 4 out of 4 cells from αcamkii T86A-mutant mice where pairing the first pathway elicited LTP, pairing the second pathway after blocking NMDA receptors was uniformly unsuccessful (Fig. 5B). This result confirms that NMDA receptors are required to induce LTP. Finally, in 4 out of 4 cells from αcamkii T86Amutant mice where pairing the first pathway elicited LTP, pairing the second pathway after blocking Ca + / calmodulin-dependent kinases was again uniformly unsuccessful (Fig. 5C). Thus, we conclude that LTP in WT mice does indeed depend strictly on Ca + /calmodulindependent kinases, although auto-phosphorylation of the α subunit is clearly not necessary. Discussion The striking result of the present study is that Hebbian LTP in glutamatergic synapses on inhibitory interneurons in stratum radiatum of CA is intact in αcamkii T86A-mutant mice, and yet depends on NMDA receptors and Ca + /calmodulin-dependent kinases. This is a unique pattern, which distinguishes NMDA receptor-mediated LTP in interneurons from that seen in adult pyramidal cells. It is also different from a form of LTP that occurs early in development in CA pyramidal cells (Yasuda et al. 3), and in dentate granule cells of αcamkii T86A-mutant mice (Cooke et al. 6), and which depends on cyclic AMP-dependent signalling. The present results on their own do not rule out the possibility that basal activity of the α isoform is C 7 The Authors. Journal compilation C 7 The Physiological Society

8 89 K. Lamsa and others J Physiol A MUT in APV B MUT in APV C WT in APV Hz, s - D E F MUT in KN-6 Hz, s - MUT in KN- 6 WT in KN- 6 G in KN- 93 Hz, s - H in KN- 93 I in KN- 9 J in KN- 9 Hz, s P <. - Figure 4. Pathway-specific LTP in non-pyramidal cells in stratum radiatum of αcamkii T86A-mutant mice requires NMDA receptors and Ca + /calmodulin-dependent kinase activity A, LTP could not be elicited in any of the αcamkii T86A-mutant (MUT) interneurons studied when NMDA receptors were blocked with DL-APV ( μm). B, baseline-normalized EPSP initial slopes of the cells in the paired and control pathways, plotted against one another (5 min following pairing). C, LTP was similarly blocked by APV in WT mice. Only out of 9 interneurons showed significant (P <.5) potentiation in the paired pathway (grey symbol). D and E, LTP in αcamkii T86A-mutant interneurons was blocked by the broad spectrum Ca + /calmodulin-dependent C 7 The Authors. Journal compilation C 7 The Physiological Society

9 J Physiol NMDA receptor-dependent LTP in interneurons is independent of αcamkii 893 A Hz, s Hz, s B Hz, s Hz, s C Hz, s Hz, s APV KN-6 n = 7 P <.5 P = n = 4 P <.5 P <.5 n = 4 P <.5 P < Figure 5. Sequential pairing of two pathways in LTP-competent interneurons shows that blockade of NMDA receptors or inhibition of Ca + /calmodulin-dependent kinase activity prevents LTP A, EPSP initial slope plotted in 7 interneurons in αcamkii T86A-mutant mice. LTP was first elicited in the pathway plotted with filled symbols. After > min, the second pathway (open symbols) was subsequently paired to induce LTP. P-values were obtained by comparing the two pathways. B, addition of APV after LTP was evoked in the first pathway prevented LTP induction in the second pathway. C, addition of KN-6 also prevented LTP in the second pathway. Neither manipulation reversed established LTP. neurons are not changed, makes the T86A-mutant mouse a potentially powerful tool to dissect the relative importance of use-dependent changes in synaptic strength at synapses on interneurons and pyramidal cells. Clearly, because the mutant mice exhibit major defects in tests of spatial learning (Giese et al. 998; Need & Giese, 3), the findings confirm that LTP at Schaffer collateral interneuron synapses cannot on its own sustain episodic memory encoding. Nevertheless, αcamkii T86Amutant mice exhibit some residual ability to learn tasks that are thought to depend on hippocampal and amygdalar circuits, albeit more slowly than WT animals (Irvine et al. 5). Plasticity in interneurons is a candidate mechanism that could contribute to the residual hippocampal memory acquisition, in addition to its role in preserving the ability of the feed-forward circuitry to mediate precise temporal pattern discrimination (Lamsa et al. 5). References AlleH,JonasP&GeigerJR(). PTP and LTP at a hippocampal mossy fiber-interneuron synapse. Proc Natl AcadSciUSA98, necessary for LTP induction. Nevertheless, we consider this highly unlikely because αcamkii has not been detected in interneurons (Liu & Jones, 996; Sik et al. 998). A more plausible explanation is that another isoform of CaMKII sustains the induction of LTP in these cells. α and β subunits show very high sequence homology. Indeed, the β subunit is an especially strong candidate since it has been reported to exhibit a higher sensitivity to Ca + /calmodulin, and to be phosphorylated by the α isoform (Brocke et al. 999). Some degree of interchange in the roles of the α and β isoforms is consistent with the report that intracellular application of activated αcamkii leads to potentiation of transmission in non-pyramidal hippocampal neurons (Wang & Kelly, ), much as it does in principal cells (Pettit et al. 994; Lledo et al. 995; Otmakhov et al. 997). We are, however, unable to test this hypothesis directly in the absence of β-isoform CaMKII-mutant mice or of selective pharmacological agents that discriminate between the isoforms. Expression of βcamkii in T86A-mutant mice is unchanged relative to WT mice (Giese et al. 998; Cooke et al. 6). This, together with the finding that the electrophysiological properties of the interkinase inhibitor KN-6 ( μm). EPSP initial slopes plotted as in (A and B). Only out of 9 tested interneurons showed potentiation in the presence of KN-6. F, pathway-specific potentiation was similarly blocked by the presence of KN-6 in WT littermates. A non-specific drift in EPSP slope occurred in some experiments, which affected both pathways equally. G and H, LTP was also blocked by the alternate CaMKII inhibitor KN-93. I, LTP was elicited normally in mutant mice in the presence of the analogue KN-9 (5 μm), which is inactive at CaMKII. Slices were exposed to KN-9 at least 3 min before testing LTP. All 9 cells studied are plotted in the figure. P-value obtained by comparison of the two pathways at the time point indicated shows a significant difference. J, baseline-normalized EPSP initial slopes of the 9 cells plotted as above. C 7 The Authors. Journal compilation C 7 The Physiological Society

10 894 K. Lamsa and others J Physiol Bennett MK, Erondu NE & Kennedy MB (983). Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain. J Biol Chem 58, Brocke L, Chiang LW, Wagner PD & Schulman H (999). Functional implications of the subunit composition of neuronal CaM kinase II. J Biol Chem 74, Christie BR, Frank KM, Seamans JK, Saga K & Sejnowski TJ (). Synaptic plasticity identified CA stratum radiatum interneurons and giant projection cells. Hippocampus, Cooke SF, Wu J, Plattner F, Errington M, Rowan M, Peters M, Hirano A, Bradshaw KD, Anwyl R, Bliss TV & Giese KP (6). Autophosphorylation of αcamkii is not a general requirement for NMDA receptor-dependent LTP in the adult mouse. J Physiol 574, Giese KP, Fedorov NB, Filipkowski RK & Silva AJ (998). Autophosphorylation at Thr86 of the α calcium-calmodulin kinase II in LTP and learning. Science 79, Goldberg JH & Yuste R (5). Space matters: local and global dendritic Ca + compartmentalization in cortical interneurons. Trends Neurosci 8, Goldberg JH, YusteR&Tamas G (3). Ca + imaging of mouse neocortical interneurone dendrites: contribution of Ca + -permeable AMPA and NMDA receptors to subthreshold Ca + dynamics. J Physiol 55, Gulyas AI, Toth K, McBain CJ & Freund TF (998). Stratum radiatum giant cells: a type of principal cell in the rat hippocampus. EurJNeurosci, Hudmon A & Schulman H (). Structure-function of the multifunctional Ca + /calmodulin-dependent protein kinase II. Biochem J 364, Irvine EE, Vernon J & Giese KP (5). αcamkii autophosphorylation contributes to rapid learning but is not necessary for memory. Nat Neurosci 8, 4 4. Kullmann DM & Lamsa KP (7). Long-term synaptic plasticity in hippocampal interneurons. Nat Rev Neurosci 8, Lamsa K, Heeroma JH & Kullmann DM (5). Hebbian LTP in feed-forward inhibitory interneurons and the temporal fidelity of input discrimination. Nat Neurosci 8, Lamsa KP, Heeroma JH, Somogyi P, Rusakov DA & Kullmann DM (7). Anti-Hebbian long-term potentiation in the hippocampal feedback inhibitory circuit. Science 35, Lisman J, Schulman H & Cline H (). The molecular basis of CaMKII function in synaptic and behavioural memory. Nat Rev Neurosci 3, Liu XB & Jones EG (996). Localization of α type II calcium calmodulin-dependent protein kinase at glutamatergic but not γ -aminobutyric acid (GABAergic) synapses in thalamus and cerebral cortex. ProcNatlAcadSciUSA93, Lledo PM, Hjelmstad GO, Mukherji S, Soderling TR, Malenka RC & Nicoll RA (995). Calcium/calmodulin-dependent kinase II and long-term potentiation enhance synaptic transmission by the same mechanism. Proc Natl Acad Sci USA9, MaccaferriG&McBainCJ(996). Long-term potentiation in distinct subtypes of hippocampal nonpyramidal neurons. JNeurosci6, Mahanty NK & Sah P (998). Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala. Nature 394, Malinow R, Schulman H & Tsien RW (989). Inhibition of postsynaptic PKC or CaMKII blocks induction but not expression of LTP. Science 45, Need AC & Giese KP (3). Handling and environmental enrichment do not rescue learning and memory impairments in αcamkii (T86A) mutant mice. Genes Brain Behav, Otmakhov N, Griffith LC & Lisman JE (997). Postsynaptic inhibitors of calcium/calmodulin-dependent protein kinase type II block induction but not maintenance of pairinginduced long-term potentiation. JNeurosci7, Perez Y, Morin F & Lacaille JC (). A hebbian form of long-term potentiation dependent on mglura in hippocampal inhibitory interneurons. Proc Natl Acad Sci USA98, PettitDL,PerlmanS&Malinow R (994). Potentiated transmission and prevention of further LTP by increased CaMKII activity in postsynaptic hippocampal slice neurons. Science 66, Rezazadeh S, Claydon TW & Fedida D (6). KN-93 (-[N-(- hydroxyethyl)]-n-(4-methoxybenzenesulfonyl)]amino-n- (4-chlorocinn amyl)-n-methylbenzylamine), a calcium/ calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels. J Pharmacol Exp Ther 37, Sik A, Hajos N, Gulacsi A, ModyI&FreundTF(998). The absence of a major Ca + signaling pathway in GABAergic neurons of the hippocampus. ProcNatlAcadSciUSA95, Wang JH & Kelly P (). Calcium-calmodulin signalling pathway up-regulates glutamatergic synaptic function in non-pyramidal, fast spiking rat hippocampal CA neurons. J Physiol 533, Yasuda H, Barth AL, Stellwagen D & Malenka RC (3). A developmental switch in the signaling cascades for LTP induction. Nat Neurosci 6, 5 6. Yuste R (5). Origin and classification of neocortical interneurons. Neuron 48, Acknowledgements This work was supported by the Wellcome Trust, the Academy of Finland (K.P.L.), the Medical Research Council (D.M.K.), and the BBSRC (IABB grant to K.P.G.). We are grateful to Jeff Vernon for help genotyping. Author s present address K. P. Giese: Institute of Psychiatry, King s College London. Supplemental material Online supplemental material for this paper can be accessed at: and C 7 The Authors. Journal compilation C 7 The Physiological Society