Inositol 1,3,4,5-tetrakisphosphate enhances long-term potentiation by regulating Ca²⁺ entry in rat hippocampus

Abstract

1. The effect of inositol 1,3,4,5-tetrakisphosphate (InsP₄) on long-term potentiation (LTP) was investigated in the CA1 region of rat hippocampal slices. Intracellular application of InsP₄ and EPSP recordings were carried out using the whole-cell configuration.

2. Induction of LTP in the presence of InsP₄ (100 μM) resulted in a substantial enhancement of the LTP magnitude compared with control potentiation. Using an intrapipette perfusion system, it was established that application of InsP₄ was required during induction of potentiation for this enhancement to occur. An enhancement of LTP was not observed if a non-metabolizable inositol 1,4,5-trisphosphate (InsP₃) analogue (2,3-dideoxy-1,4,5-trisphosphate, 100 μM) was applied intracellularly.

3. Current-voltage relations of NMDA receptor-mediated EPSCs were not altered by InsP₄ application. The presence of InsP₄ was slightly effective in relieving a D-(—)-2-amino-5-phosphonopentanoic acid (D-APV)-induced block of LTP.

4. The peak current amplitude of voltage-gated calcium channels (VGCCs) was increased by InsP₄. ω-Conotoxin GVIA inhibited the InsP₄-induced LTP facilitation.

5. These data indicate that InsP₄ can modify the extracellular Ca²⁺ entry through upregulation of VGCCs, which may in turn contribute to the observed enhancement of LTP induced by InsP₄.

6. To investigate the possible involvement of intracellular Ca²⁺ release in the facilitatory effect of InsP₄ on LTP, different inhibitors of the endoplasmic reticulum-dependent Ca²⁺ release were applied (heparin, ryanodine, cyclopiazonic acid). The results suggest that InsP₄ activates postsynaptic InsP₃-dependent Ca²⁺ release which normally does not contribute to the calcium-induced calcium release-dependent LTP.

Several different postsynaptic mechanisms contribute to the necessary Ca²⁺ entry during induction of long-term potentiation (LTP) in hippocampal CA1 pyramidal cells (Malenka et al. 1992). The role of N-methyl-D-aspartate (NMDA) receptors (Bliss & Collingridge, 1993), of voltage-gated calcium channels (VGCCs) (Cavus & Teyler, 1996; Wilsch et al. 1998) and of calcium-induced calcium release (CICR) (Obenaus et al. 1989; Wang & Kelly, 1997) in LTP is well established. However, the evidence indicating a role for the inositol polyphosphates especially for inositol 1,4,5-trisphosphate (InsP₃) and inositol 1,3,4,5-tetrakisphosphate (InsP₄) has not yet been delivered, although InsP₃ is known to initiate Ca²⁺ entry from the endoplasmic reticulum (ER) and InsP₄ is also thought to act as a second messenger modulating Ca²⁺ signals (Berridge & Irvine, 1989; Irvine, 1991; Berridge, 1993).

We and others have established that group I metabotropic glutamate receptors (mGluRs) are involved in LTP (Bashir et al. 1993; Wilsch et al. 1998). In this process phospholipase C is activated through G proteins to hydrolyse the lipid precursor phosphatidylinositol 4,5-bisphosphate to give diacylglycerol and InsP₃ (Pin & Duvoisin, 1995). Further metabolism of InsP₃ leads to the formation of other inositol polyphosphates (Berridge & Irvine, 1989). Activation of an InsP₃ 3-kinase, the messenger RNA of which is enriched in the CA1 region (Mailleux et al. 1991), causes production of InsP₄ (Berridge & Irvine, 1989). Accordingly, an increased InsP₄ production was observed after agonist stimulation of the group I mGluRs (Baird et al. 1991).

Many effects of InsP₄ were characterized in several cell preparations derived, mainly, from cells other than from the central nervous system of mammals (Irvine, 1991). It seems that InsP₄ acts either on different cell membrane conductances (Morris et al. 1987; Sawada et al. 1990; Wu et al. 1991; Fadool & Ache, 1994) or activates Ca²⁺ release primarily from InsP₃-dependent internal stores alone, or facilitates responses to InsP₃ (Gawler et al. 1990; Parker & Ivorra, 1991; Wilcox et al. 1993; Mills et al. 1997). In the central nervous system, InsP₄ was shown to modulate VGCC conductances (De Waard et al. 1992; Tsubokawa et al. 1996). In contrast, it is not yet established whether InsP₄ binding proteins have a distinct cellular function (Theibert et al. 1991; Cullen et al. 1995; Kreutz et al. 1997; Aggensteiner et al. 1998).

An earlier study in our laboratory indicated that using 2,3-diphosphoglyceric acid, an inhibitor of the InsP₃ 5-phosphatase and of InsP₃ 3-kinase, inhibited LTP (Behnisch & Reymann, 1994). Blocking InsP₃ metabolism leads to the termination of InsP₄ and of inositol bisphosphate production. This result, and the unclear role of InsP₄ in cellular function with respect to LTP, has led us to investigate whether an increased concentration of InsP₄ would be able to modulate the EPSP potentiation and if so, what are the possible underlying cellular mechanisms.

METHODS

Experiments were performed on hippocampal slices from 4- to 5-week-old male rats of the Wistar outbred strain MOL: WIST (SHOE) and carried out according to German animal welfare guidelines. After decapitation the brain was quickly removed and placed in a medium kept at 0°C containing the following concentrations of chemicals (mM): NaCl, 124; KCl, 0.75; CaCl₂, 0.5; Mg₂SO₄, 10; KH₂PO₄, 1.25; glucose, 20; Hepes, 10; and pH was adjusted to 7.25 with 1 M NaOH. Using a CAMPDEN vibroslicer (Loughborough, UK), 300 μm thick transverse slices were cut and transferred to a pre-chamber containing artificial cerebrospinal fluid (ACSF) at 30°C with the following composition (mM): NaCl, 124; KCl, 0.75; CaCl₂, 2.5; Mg₂SO₄, 1.3; KH₂PO₄, 1.25; glucose, 10; NaHCO₃, 24. Acidity was adjusted with a gas mixture of 95 % O₂-5 % CO₂. The CA3 region was always removed to avoid epileptiform activity since bicuculline (10 μM) was routinely added to the solution to suppress GABA_A receptor-mediated inhibition. One hour later, the slices were transferred to the recording chamber which was integrated into an upright Zeiss microscope (Axioskop FS). Perfusion rate of the ACSF was 2 ml min⁻¹. A × 10 objective was used to help the adjustment of a monopolar platinum stimulation electrode in the stratum radiatum. Thin walled, low resistance (4 MΩ) borosilicate glass patch pipettes were pulled with a DMZ Universal Puller, (Zatz-Instrumente, Augsberg, Germany). For an accurate positioning of the pipette on CA1 cells, a × 40 water immersion objective was used together with a WPI micromanipulator. The cell was obtained using the ‘blind’ approach of the whole-cell configuration.

In experiments of current-clamp recording of EPSPs, the internal solution contained (mM): potassium gluconate, 135; KCl, 5; MgCl₂, 2; Hepes, 10; glucose, 20; and pH was adjusted to 7.2 using 1 M KOH. An Axoclamp 2A amplifier in bridge mode was used for recording EPSPs. The signals were filtered with a low-pass WPI filter at 2 kHz and digitized at 10 kHz with a CED 1401 AD/DA board. For sampling and stimulation the program Intracell for Windows (1.0) was used. Data were stored on a 386 PC. After rupturing the patch membrane, the stimulation strength was adjusted with bipolar 0.2 ms stimulation pulses to obtain, on average, EPSPs of 3-5 mV in amplitude. Responses were recorded at 0.033 Hz and averaged every minute. Values of EPSP amplitudes were normalized to the mean of the baseline values recorded in the first 15 min and are presented as percentage of control. A uniform adjustment of the stimulation electrode meant a need for a similar stimulation strength to evoke similar potentials (3-5 mV) in the experiment. To induce EPSP potentiation after 15 min of baseline recording, two tetani each 400 ms long at 100 Hz were delivered 20 s apart using 0.4 ms bipolar pulses and paired with a +0.5 nA somatic current injection.

If the cell had an unusually increased input resistance or fast, non-accommodating firing pattern with evoked polysynaptic fast EPSPs then on suspicion of being an interneurone, the recording was terminated. The membrane potential of the control cells measured -68 ± 3 mV (n = 19) and the input resistance was 68 ± 2 MΩ (n = 19, recorded with a -0.1 nA, 400 ms current pulse; experiments represented in Fig. 1A and B). In cases where either the membrane potential or the input resistance changed more than 20 % during the experiment, the cell was not taken into account during statistical analysis. However, since a drug effect could not be excluded, it was always checked to see if a change in these basic parameters was consistent with a drug application. During our different drug applications, however, no change was observed with respect to the membrane potential or the input resistance (not shown).

Figure 1. Recording of baseline synaptic transmission and LTP during intracellular application of InsP₄.

A, the amplitude of monosynaptic EPSPs of CA1 pyramidal cells was recorded for 75 min in control media using the whole-cell configuration (○, n = 7). In parallel the effect of intracellularly applied InsP₄ (100 μM) on baseline synaptic transmission is presented (•, n = 5). Aa-d represent analog unaveraged traces of EPSPs. Responses are represented at 10 and 45 min, respectively, of control recording (Aa and b) and recording under InsP₄ treatment (Ac and d). B describes the time course of control LTP (○, n = 12) and of the LTP recorded with InsP₄ application (•, n = 13). Ba-d describe analog traces as A. There is no difference in access resistance (C and D). Potentiation was induced after 15 min of recording as indicated by the arrow. Scale bar applies to both A and B.

In some series of experiments InsP₄ was applied intracellularly at a desired time point. For this purpose, a special intrapipette perfusion system was set up. A Hamilton syringe was connected to a thin flame-pulled quartz capillary through a plastic tube which contained the required internal solution (Alford et al. 1993). The tip of the capillary was advanced close to the tip of the patch pipette (about 150 μm). To check the dynamics of the diffusion perfusion Lucifer Yellow was injected. Five to ten minutes after application of the dye, fluorescence signals of even dendritic origin could be observed.

During the voltage-clamp recording of Ca²⁺ currents and of EPSCs, the internal pipette solution had the following composition (mM): CH₃O₃SCs, 140; MgCl₂, 2; Hepes, 10; glucose, 20; EGTA, 0.2; and pH was adjusted to 7.2 with 1 M CsOH. Where Ca²⁺ currents were measured, 1 μM tetrodotoxin was added to the ACSF to block the voltage-gated Na⁺ channels. For stimulation and sampling CED Patch and Voltage Clamp software was used. Special care was always taken to keep the series resistance (R_s) low during experiments (e.g. Fig. 6B, control: n = 11, R_s= 10 ± 0.6 MΩ). Difference in R_s between groups was not observed. Signals were amplified with an Axopatch-1D amplifier. Capacitative currents but not series resistance were compensated. (In one series of experiments (Fig. 6A) the signals were out of the acquisition range of the AD/DA board and therefore a × 0.5 gain had to be used for reduction of the currents. The measured values were later doubled.) Leakage subtraction was automatically performed. In the case of experiments where synaptically evoked NMDA receptor-mediated currents were measured, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 μM) was added to the ACSF to block α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation. For stimulation and sampling of currents, the same electrode and software were used as in the current-clamp experiments except for the amplifier.

Figure 6. The influence of InsP₄ on VGCCs.

A shows the means of peak amplitudes of Ca²⁺ currents evoked with depolarizing voltage steps from a holding potential of -80 to +10 mV either under control conditions (○, n = 6) or under InsP₄ treatment (•, n = 6, 100 μM). Aa and b show analog current traces 6 min after rupturing the patch membrane in control and under InsP₄ treatment, respectively. B shows means from experiments where the Ca²⁺ currents were evoked with depolarizing voltage steps from a holding potential of -40 to +10 mV either under control conditions (○, n = 11) or under InsP₄ treatment (•, n = 11). Ba and b represent the analog current traces as in A, but under these recording conditions.

For statistical analysis two-tailed Mann-Whitney U-test was applied. Populations were regarded as significantly different if P was less than 0.05. Data are expressed as means ± standard error of mean (s.e.m.). Values corresponding to a particular set of experiments are presented in some cases on more than one graph and repeated in the text for clarity.

The following drugs were applied during the experiments. Extracellular applications: (-)-bicuculline methobromide (stock: 1 mM in distilled water (dw), Tocris); CNQX disodium salt (stock: 500 μM in dw, Tocris); D(-)-2-amino-5-phosphonopentanoic acid (d-APV, stock: 5 mM in 1 equiv of NaOH, Tocris); tetrodotoxin citrate (stock: 0.5 mM in dw, Tocris); ω-conotoxin GVIA (dissolved directly in the ACSF, Alamone Labs); cyclopiazonic acid (CPA; stock: 100 mM in DMSO, final concentration of DMSO 0.02 %, v/w, ICN).

Intracellular applications: D-myo-inositol 1,3,4,5-tetrakisphosphate, octapotassium salt (Alexis); D-myo-inositol 1,4,5-trisphosphate, 2,3-dideoxy-hexasodium salt (Calbiochem); ryanodine (Tocris); heparin, low molecular weight, sodium salt, molecular mass ∼3 kD (Sigma). All four drugs were dissolved directly in the internal solution.

RESULTS

Application of InsP₄ enhances LTP without affecting baseline EPSPs

When recording baseline synaptic transmission and comparing values from experiments where InsP₄ was applied intracellularly (n = 5, 100 μM) with control recordings (n = 7), no difference was found (Fig. 1A). At 45 min in control experiments mean normalized EPSPs were 97 ± 18 % and InsP₄ treatment had no effect with normalized EPSPs measuring 105 ± 20 %. Tetanic stimulation of the Schaffer collaterals 15 min after baseline recording resulted in a 260 ± 19 % potentiation of EPSPs which declined to 130 ± 16 % by the end of the experiment, at 60 min post-tetanization (n = 12, Fig. 1B). Intracellular application of InsP₄ (100 μM) resulted in an increase of the LTP magnitude. Values that belong to the InsP₄ treatment (n = 13) were different from the initial value after induction and throughout the recording remained significantly different from control potentiation. After induction in control experiments the initial mean was 260 ± 19 %, whereas under InsP₄ treatment the potentiation was increased to 343 ± 19 %. At 30 min post-tetanization the mean normalized EPSP control measurement was 146 ± 11 %, compared with InsP₄ treatment of 231 ± 15 %. At 60 min post-tetanization the values were 130 ± 16 and 199 ± 12 %, respectively.

An increased InsP₄ concentration during induction is required for the enhancement of LTP

An intrapipette perfusion system made it possible to apply InsP₄ (100 μM) at any desired time point (Fig. 2). If InsP₄ was perfused into the pyramidal cell 2 min after the baseline recording started (n = 5), a significant enhancement of the potentiation could be observed immediately after induction compared with control experiments (n = 12). The first mean after induction reached 260 ± 19 % in control experiments and 371 ± 31 % under InsP₄ treatment. In another set of experiments, the perfusion started 10-12 min after induction of LTP (n = 5). Recordings of EPSP showed no difference compared with control LTP experiments (n = 12). At 30 min post-tetanization the mean of normalized EPSPs for control was 146 ± 11 %, whereas that for the late InsP₄ perfusion was 147 ± 15 %.

Figure 2. InsP₄ is perfused intracellularly before and after tetanization.

The graph shows LTP experiments performed using an intrapipette perfusion system. ○, values of control LTP (n = 12). Perfusing InsP₄ (100 μM) into the patch pipette 2 min after rupturing the patch membrane, allowed an increased InsP₄ concentration in the cell during tetanization (□, n = 5). The effect of an InsP₄ perfusion, which was initiated 10-12 min after tetanization, on LTP is presented (•, n = 5). Potentiation was induced after 15 min of recording indicated by the arrow.

A non-metabolizable InsP₃ derivative has no effect on LTP

An InsP₃ derivative, 2,3-dideoxy-inositol 1,4,5-trisphosphate (2,3-dideoxy-InsP₃), that is non-metabolizable to InsP₄ and shows biological activity similar to InsP₃ (Kozikowski et al. 1993) was applied intracellularly (100 μM). Under 2,3-dideoxy-InsP₃ treatment, neither values of baseline recording (Fig. 3A, n = 7) nor values in LTP experiments (Fig. 3B, n = 8) showed a difference compared with values of control baseline (Fig. 3A, n = 7) and control LTP (Fig. 3B, n = 12), respectively. At 45 min, the mean normalized baseline EPSP was 97 ± 18 % in control experiments and 99 ± 10 % in experiments under 2,3-dideoxy-InsP₃ treatment. In LTP experiments 30 min post-tetanization control potentiation reached 146 ± 11 % compared with 144 ± 24 % in LTP experiments with application of 2,3-dideoxy-InsP₃.

Figure 3. Recording of baseline synaptic transmission and LTP during intracellular application of 2,3-dideoxy-InsP₃.

In A, baseline synaptic transmission in control experiments (○, n = 7) and in experiments where 2,3-dideoxy-InsP₃ was applied intracellularly (•, n = 7, 100 μM) is represented. In B the effect of this InsP₃ analogue on LTP is represented. ○, represent control LTP measurements (n = 12) whereas • describe LTP experiments under 2,3-dideoxy-InsP₃ treatment (n = 8). Potentiation was induced after 15 min of recording as indicated by the arrow.

Current-voltage relations of synaptically evoked NMDA receptor-mediated EPSCs are not altered if InsP₄ is applied

Current-voltage (I-V) relations of NMDA receptor-mediated EPSCs were investigated under control conditions and in experiments where InsP₄ (100 μM) was applied intracellularly. CNQX (5 μM) was applied extracellularly and in some experiments D-APV (50 μM) was used to make sure that the currents under investigation were NMDA receptor-mediated ones without contamination with an AMPA receptor-mediated component (Fig. 4e). EPSCs were recorded at seven different membrane potentials every 4 min for 22 min (Fig. 4). A run-down of the NMDA receptor-mediated synaptic current could be observed within 22 min. At -15 mV holding potential and 2 min after the recording started, control responses were measuring -96 ± 6 pA (not shown) and at 14 min -67 ± 6 pA (Fig. 4). There was no difference between the populations at any of the recorded time points (control, n = 10; InsP₄, n = 8). For example at -15 mV holding potential and 14 min after the recording began control responses measured -67 ± 6 pA and the mean was -72 ± 12 pA in InsP₄ treated cells (Fig. 4).

Figure 4. Plot of current-voltage relation of synaptically-evoked NMDA receptor-mediated EPSCs during intracellular application of InsP₄.

NMDA receptor-mediated synaptic currents were evoked at different holding potentials in control experiments (○, n = 10) and under InsP₄ treatment (•, n = 8, 100 μM). AMPA receptors were blocked with CNQX (5 μM). The panel shows the I-V relation after 14 min of rupturing the patch membrane. Subpanels a-d represent unaveraged analog EPSCs. Both traces a (○) and b (•) belong to measurements at +40 mV whereas c (○) and d (•) represent current traces at -15 mV. Trace e shows the response after wash-in of D-APV (50 μM).

Application of D-APV blocks LTP but a post-tetanic potentiation (PTP) remains; InsP₄ application strongly facilitates this PTP and the block of LTP is slightly relieved

When applying the NMDA receptor antagonist D-APV (50 μM) extracellularly, LTP was inhibited to 108 ± 4 % at 30 min post-tetanization (n = 8). However, an initial increase in the value (to 160 ± 10 %) immediately after tetanization remained (Fig. 5). Intracellular application of InsP₄ (n = 8, 100 μM) proved to be slightly effective in relieving the D-APV-induced block. At 30 min post-tetanization the mean normalized EPSP was 108 ± 4 % for the D-APV-treated LTP. With application of InsP₄ the corresponding value was 122 ± 7 %. However, this difference was not significant. The first mean after tetanization of normalized EPSPs measured from InsP₄-treated cells (230 ± 16 %) was significantly different from the control one.

Figure 5. The effect of the intracellularly applied InsP₄ on the D-APV-inhibited LTP.

D-APV (50 μM) was applied extracellularly to inhibit NMDA receptors and LTP was recorded under this condition (○, n = 8). •, values corresponding to experiments where InsP₄ was applied intracellularly (n = 8, 100 μM). Potentiation was induced after 15 min of recording as indicated by the arrow.

Peak amplitude of VGCCs evoked from a holding potential of -40 mV is increased by InsP₄

We have tested the effect of intracellularly applied InsP₄ (100 μM) on Ca²⁺ currents evoked by depolarizing voltage steps. Ca²⁺ currents were evoked either from a holding potential of -80 mV or from -40 mV using a depolarizing step to +10 mV for 150 ms. The peak as well as late amplitude (at 100 ms) were measured every 2 min for 24 min. A run-down of the Ca²⁺ current was observed. At -80 mV holding potential and 2 min after the recording started, currents from control experiments measured -3.59 ± 0.12 nA (Fig. 6A, n = 6) and at 22 min the value was -1.84 ± 0.39 nA. Applying InsP₄ intracellularly and measuring Ca²⁺ currents evoked from a holding potential of -80 mV (n = 6) resulted in no difference throughout the experiment, compared with control recordings, either in peak amplitude (Fig. 6A at 6 min, control -3.41 ± 0.15 nA, InsP₄ -3.56 ± 0.24 nA) or in late amplitude (not shown).

However, using a depolarizing step from -40 mV to +10 mV resulted in an increased amplitude of peak Ca²⁺ currents under InsP₄ treatment (Fig. 6B, n = 11). Compared with control measurements, there was a significant increase from the 6th to the 16th minute (n = 11). Six minutes after recording began, the control measures yielded -1.78 ± 0.1 nA whereas InsP₄ treatment increased the currents to -2.14 ± 0.1 nA. At 16 min the mean control was -0.81 ± 0.1 nA; InsP₄ treatment similarly led to an increased current amplitude of -1.16 ± 0.14 nA. The mean amplitudes of late components were again found not to be different (not shown).

InsP₄ cannot enhance LTP if ω-conotoxin GVIA is applied

We have investigated whether the facilitatory effect of InsP₄ could be inhibited by extracellularly applied ω-conotoxin GVIA (1 μM), a toxin known to inhibit the N-type Ca²⁺ channels. The results in Fig. 7 show that intracellular application of InsP₄ (100 μM) does not potentiate LTP if the channel inhibitor is present (n = 7) compared with experiments carried out under ω-conotoxin GVIA treatment (n = 8). At 30 min post-tetanization normalized EPSPs of ω-conotoxin GVIA-treated cells averaged 127 ± 13 % whereas InsP₄ treatment led to a potentiation of 158 ± 16 %.

Figure 7. Effect of intracellular InsP₄ on LTP in the presence of ω-conotoxin GVIA.

ω-Conotoxin GVIA (1 μM) was applied extracellularly and LTP was measured (○, n = 8). The effect of the intracellular application of InsP₄ on this LTP is represented by • (n = 7, 100 μM). Potentiation was induced after 15 min of recording as indicated by the arrow.

ω-Conotoxin GVIA did not significantly inhibit control LTP (not shown). Potentiation for control LTP 30 min post-tetanization was 146 ± 11 % whereas for ω-conotoxin GVIA treatment it was 127 ± 13 %. It should be noted that application of ω-conotoxin GVIA required a significantly increased stimulation strength to achieve the same range of EPSP amplitude (n = 15) compared with control experiments (n = 19). The mean stimulation strength for control experiments was 1.8 ± 0.1 V and in experiments under ω-conotoxin GVIA treatment the strength had to be increased to 3.6 ± 0.3 V.

Role of InsP₃-dependent Ca²⁺ release in the CICR-dependent LTP

We investigated whether the inhibition of Ca²⁺ release from the endoplasmic reticulum (ER) could modify the facilitatory effect of InsP₄ on LTP. For this purpose ryanodine (200 μM) and low-molecular weight heparin (10 mg ml⁻¹, molecular mass ∼3 kD) were applied as the most common inhibitors of ryanodine- and InsP₃ receptor-mediated Ca²⁺ release, respectively. Intracellular application of ryanodine significantly inhibited LTP from the 15th minute after induction (n = 8) compared with control LTP experiments (Fig. 8A, n = 12). At 30 min post-tetanization the mean normalized EPSP in control experiments was 146 ± 11 % but ryanodine inhibited the potentiation to 94 ± 11 %. Heparin applied intracellularly (n = 8), however, had no effect on the control potentiation of EPSPs (Fig. 8B, n = 12). At 30 min after induction the mean value from control experiments was 146 ± 11 % compared with a potentiation of 160 ± 14 % under heparin treatment.

Figure 8. The effects of different ER Ca²⁺ release inhibitors on control potentiation and on LTP recorded with intracellular application of InsP₄.

In A, the control LTP (○, n = 12) can be compared with the effect of intracellularly applied ryanodine on LTP (•, n = 8, 200 μM). In B the effect of heparin on LTP (▪, n = 8, 10 mg ml⁻¹) is presented together with values of control LTP (○, n = 12). C shows the results of experiments where ryanodine was co-applied with InsP₄ in LTP experiments (▵, n = 9). In this graph the InsP₄ (□, n = 13) and the ryanodine effect (•, n = 8) on LTP are presented as well. In D, the effect of co-application of heparin with InsP₄ on LTP can be seen (⋄, n = 7). Values for LTP experiments in the presence of InsP₄ (□, n = 13) and of heparin (▪, n = 8) are also presented. E describes the effect of the combined application of ryanodine plus heparin plus InsP₄ (▴, n = 7). For comparison, the values from the experiments for InsP₄ plus ryanodine co-application are plotted (▵, n = 9). F, experiments on LTP are presented where either CPA was applied extracellularly alone (▿, n = 6, 20 μM) or together with InsP₄ (▾, n = 7). Potentiation was induced after 15 min of recording as indicated by the arrow in each panel.

The next set of experiments (Fig. 8C and D) was carried out to investigate the possible effect of InsP₄ on the actions of the antagonists described above. Figure 8C shows that co-application of ryanodine and InsP₄ (n = 9) relieves the ryanodine-induced inhibition of LTP (n = 8). The populations were significantly different, e.g. at 30 min post-tetanization co-application of ryanodine and InsP₄ increased the potentiation to 175 ± 11 % compared with the inhibition of ryanodine on control LTP (94 ± 11 %). However, the reversal was only partial since a maximal enhancement could not be observed (Fig. 8C, n = 13, 231 ± 15 %). The substantial InsP₄ effect on the magnitude of the control LTP was not reached except for the first 8 min after tetanization. The first mean after tetanization with InsP₄ treatment was 343 ± 19 % and a similar potentiation of 354 ± 33 % was achieved when InsP₄ and ryanodine are co-applied. Conversely, co-applying InsP₄ and heparin (Fig. 8D, n = 7) resulted in no difference compared with the potentiation under heparin treatment alone (n = 8). At 30 min post-tetanization heparin treatment gave a potentiation of 160 ± 14 %; whereas heparin inhibited the enhancement by InsP₄ of LTP with the potentiation reaching a similar 173 ± 29 % for the co-application of heparin and InsP₄. Enhancement of control LTP by InsP₄ application at the same time point gave a mean of 231 ± 15 % (Fig. 8D, n = 13).

Combined intracellular application of ryanodine, heparin and InsP₄ (Fig. 8E, n = 7) resulted in an inhibition compared with results from experiments where ryanodine and InsP₄ were applied together (n = 9). At 30 min after induction, the EPSP was potentiated to 175 ± 11 % under co-treatment with InsP₄ and ryanodine whereas the combined application of InsP₄ plus ryanodine plus heparin inhibited this effect to 94 ± 15 %. To achieve similar conditions where both intracellular stores were simultaneously but not independently inhibited, CPA, an ER Ca²⁺ uptake inhibitor (Seidler et al. 1989) was applied extracellularly (Fig. 8F, n = 6, 20 μM). CPA prevented InsP₄ from enhancing the EPSP potentiation (n = 7) except for the first mean after tetanization which was 298 ± 26 % compared with a potentiation of 183 ± 20 % in the CPA experiment. At 30 min post-tetanization, however, the mean normalized EPSP was 103 ± 10 % in the CPA experiment, whereas the potentiation in experiments where CPA and InsP₄ were co-applied was 126 ± 17 %. A slight but not statistically significant increase appeared in the experiments if InsP₄ was applied in addition to the CPA treatment. Furthermore, CPA inhibited LTP from the 6th to the 34th minute after induction of LTP (not shown). At 30 min post-tetanization the normalized mean EPSP was 146 ± 11 % in control experiments, whereas CPA inhibited the potentiation to 103 ± 10 %.

DISCUSSION

In our study, we have shown that postsynaptic intracellular application of InsP₄ enhances LTP in CA1 pyramidal cells. We propose that the effect of this non-membrane permeable inositol polyphosphate analogue is postsynaptic. To our understanding, it is not conceivable that a signalling pathway modulated by InsP₄ exists that could upregulate presynaptic transmitter release through diffusible messengers.

Possible involvement of mGluRs

A G protein-coupled receptor activation is mostly involved in the production of inositol polyphosphates (Berridge & Irvine, 1989). On application of quisqualate, a mGluR group I agonist, an increased InsP₄ production was readily observed (Baird et al. 1991). Furthermore, mGluR group I activation with the agonist 3,5-dihydroxyphenylglycine (DHPG) 30 min before tetanization has been shown to facilitate LTP in the CA1 region of hippocampus (Cohen et al. 1998). Since the metabolism of InsP₄ has been found to be slow, compared with that of InsP₃ (Sims & Albritton, 1998), a long-lasting elevation of InsP₄ level in the cell can sensitize molecular pathways, which contribute to the LTP induction with an increased efficacy during tetanization. Therefore a physiological mechanism of mGluR group I action (or of other G protein-coupled receptors such as muscarinic ones) is possibly partially mediated through an increase of intracellular InsP₄ concentration. Carrying out experiments using the group I mGluR agonist and InsP₄ could help to answer the question whether the facilitatory effect of group I mGluR activation and of InsP₄ application on LTP shares a common mechanism.

The importance of InsP₄ in the Ca²⁺ homeostasis

InsP₄ has been frequently implicated in cellular Ca²⁺ regulation (Irvine, 1991). In a previous study an influence of InsP₄ on Ca²⁺ entry has already been suggested for CA1 neurons in gerbil hippocampal slices. Contrary to what we have shown, a different effect of InsP₄ was observed since an initial potentiation was converted gradually into a depression of EPSCs and the cells irreversibly depolarized (Tsubokawa et al. 1994). The authors suggest deterioration in Ca²⁺ accumulation is responsible for the observed effect. This could be due to a technical difference compared with our results, since in that study the recording took place at 35°C. It has been shown that a reduction in temperature (Δt of 5°C) is protective against ischaemic insults (Miyazawa et al. 1993) and a prevention of cell death may occur. Our results, as discussed later, indicate that InsP₄ can modify the overall Ca²⁺ entry during induction of LTP. Since the involvement of Ca²⁺ in LTP is not questioned (Malenka et al. 1992), this can lead to either an enhanced potentiation as we have observed, or under different environmental conditions to an altered Ca²⁺ homeostasis and subsequent cell death.

Basic properties of the InsP₄-induced enhancement of LTP

InsP₄ had no effect on long-term baseline synaptic transmission indicating that InsP₄ itself cannot evoke LTP-like mechanisms. It was important to rule this out since activation of mGluR group I and II using 1-amino-cyclopentane-1,3-dicarboxylate (ACPD) leads to a slow onset potentiation (Bashir et al. 1993). InsP₄ could have initiated such a potentiation since it can be produced through mGluR group I activation (Baird et al. 1991).

To investigate if the potentiating InsP₄ effect was involved in the induction mechanisms of LTP or in the maintenance processes, InsP₄ was applied at various time points. Applying InsP₄ before tetanization led to an enhancement of potentiation, but a later application (after induction) showed no effect. This result indicates that the functional effect of InsP₄ is linked to the induction processes. Experiments investigating the effect of group I mGluRs on LTP revealed a similar time course. DHPG application 30 min before tetanization led to an enhancement of potentiation of field EPSPs and similar to our observation, application of this drug 10 min after induction of potentiation did not influence the maintenance processes (Cohen et al. 1998). This does not yet allow us to draw the conclusion that the two mechanism should be exclusively identical.

We tested the possibility that an increased InsP₃ concentration could mimick the InsP₄ effect, although an InsP₄ 3-phosphatase activity in brain has not yet been reported (Berridge & Irvine, 1989). Hypothetically, an increased InsP₄ concentration by favouring the reverse reaction of the InsP₃ 3-kinase, should lead to an increased InsP₃ concentration, that may make this suspected metabolite responsible for the observed InsP₄ effect. This possibility is excluded by our finding that application of a non-metabolizable InsP₃ analogue (2,3-dideoxy-InsP₃) which shows biological activity similar to InsP₃ did not affect either baseline synaptic transmission or LTP. Since a non-metabolizable InsP₄ analogue is not yet available, the further metabolism of InsP₄ in the inositol polyphosphate pathway and the possible effects of other metabolic products cannot be excluded completely.

Since we assume that the potentiating effect of InsP₄ on LTP could involve a group I mGluR-mediated pathway, it is important to note that the downstream effect of the mGluR agonist ACPD was reported to be a direct regulation of the NMDA receptors (O'Connor et al. 1994). This effect could contribute to the enhancement of LTP. In our further experiments, the results show that the intracellular application of InsP₄ does not change the I-V relations of synaptically evoked NMDA receptor-mediated currents (Fig. 6). Thus, NMDA receptors are not involved directly in the InsP₄ effect.

Regarding mechanisms leading to LTP, it is well documented that synaptic potentiation induced by tetanization in the frequency range of 100 Hz depends on the Ca²⁺ entry mediated by NMDA receptors (Bliss & Collingridge, 1993). In our experiments, InsP₄ was only able to relieve the D-APV-induced block of LTP slightly. This indicates a dependence of the InsP₄ regulatory mechanism on the NMDA receptor-dependent Ca²⁺ entry that occurs during tetanization.

VGCCs and their interaction with InsP₄

To investigate the mechanism of the InsP₄ effect further, we have carried out experiments investigating the role of VGCCs in the facilitatory InsP₄ effect on LTP. A study describing the enhancement of single Ca²⁺ channel activity after InsP₄ application in the CA1 neurones of the gerbil (Tsubokawa et al. 1996) has led us to carry out more experiments in this direction. Furthermore, in cerebellar granule neurones from the rat, InsP₄ could elicit increased extracellular Ca²⁺ entry from a holding potential of -40 mV, but not from -110 mV, probably by shifting the steady-state inactivation curve of the Ca²⁺ current towards more positive values (De Waard et al. 1992). Our result (Fig. 6) validates these findings, that VGCCs can be upregulated at a depolarized membrane potential (at -40 mV) by intracellular application of InsP₄.

An increased Ca²⁺ influx during tetanization generated by InsP₄ could in fact explain the enhancement of InsP₄ on the PTP in LTP experiments under D-APV-induced NMDA receptor block; the proposed origin of PTP is postulated to be presynaptic (Malenka et al. 1992). The slight increase in potentiation in these experiments (Fig. 5) is not significantly different from the D-APV-treated LTP experiments, and could well support the assumption that InsP₄ facilitates a pathway not dependent on NMDA receptor activation. This pathway includes possibly VGCCs in the expression of LTP, since it has already been described that certain forms of LTP, e.g. those using an increased tetanization frequency of 200 Hz, are sensitive to the inhibition of VGCCs (Cavus & Teyler, 1996, and references cited within). The contribution of InsP₄-generated enhancement of VGCCs to the LTP expression could be more clearly defined if other induction protocols of LTP are applied which favour the Ca²⁺ entry through VGCCs during induction.

Previous studies have established that the currents that are sensitive to InsP₄ are either exclusively ω-conotoxin GVIA sensitive in the CA1 neurones (Tsubokawa et al. 1996) or partially sensitive to the N-type Ca²⁺ channel blocker in cerebellar granule cells (De Waard et al. 1992). If the primary action of InsP₄ was to upregulate Ca²⁺ channels that are sensitive to this channel blocker, then it is proposed that an inhibition of these channels would result in an inhibition of the InsP₄-generated enhancement of LTP. Our experiments demonstrate that applying ω-conotoxin GVIA prevented InsP₄ from exerting its facilitatory effect. Furthermore, LTP was not inhibited by ω-conotoxin GVIA. In this context we have to consider two other findings. Firstly, LTP was inhibited in another study in the CA1 region using this N-type Ca²⁺ channel blocker if a concentration was used that did not have an effect on population spike amplitude (Frank et al. 1991). Secondly, the fact that in our experiments we needed higher stimulation strengths to evoke a similar amplitude of EPSPs, could mean that other populations of fibres and synapses, with possibly different properties, were involved during induction (Reid et al. 1997) which contributed to an LTP that might have properties different from our control potentiation. The alternative suggestion to reduce the ω-conotoxin GVIA concentration so that it had no inhibition on baseline EPSPs could have meant that the postsynaptic Ca²⁺ channels would not have been fully antagonized during induction of LTP.

Role of ER Ca²⁺ stores in LTP and their modulation through InsP₄

Many studies indicate that InsP₄ activates Ca²⁺ release primarily from InsP₃-dependent internal stores alone, or facilitates responses via InsP₃ (Gawler et al. 1990; Parker & Ivorra, 1991; Wilcox et al. 1993; Mills et al. 1997). Therefore, we investigated how the inhibition of the ER-dependent Ca²⁺ release might modify the facilitatory InsP₄ effect on LTP.

Other laboratories have studied the effects of the inhibition of ryanodine-sensitive CICR on LTP expression in the CA1 area of the hippocampus. Using the ryanodine receptor antagonist dantrolene, an inhibition of LTP was reported (Obenaus et al. 1989). In a recent study LTP was inhibited in the CA1 region with postsynaptic co-application of dantrolene and heparin (Wang & Kelly, 1997). Regarding the role of the other ER Ca²⁺ release mechanism in LTP, this is the only evidence that the InsP₃-sensitive stores might participate in LTP induction. However, heparin, an InsP₃ receptor antagonist, was not applied alone. Indirect evidence shows that during inhibition of endoplasmic Ca²⁺ release, by blocking the Ca²⁺ uptake pump with thapsigargin, LTP is inhibited (Harvey & Collingridge, 1992; Behnisch & Reymann, 1995). This approach does not differentiate between InsP₃- and ryanodine-sensitive stores.

Based on our results, we conclude: an inhibition of CICR leads to a reduction of the control potentiation. This effect and earlier indications (Wang & Kelly, 1997) might suggest a possible postsynaptic regulation of LTP through CICR. Intracellular application of heparin elicited no influence on LTP, but inhibited the potentiating InsP₄ effect on LTP, probably by blocking InsP₃-dependent Ca²⁺ release. The specificity of heparin, however, remains unclear especially concerning the possible target of InsP₄ as well. When the InsP₃-sensitive pathway is intact, InsP₄ relieves the inhibition of LTP induced by postsynaptic application of the CICR antagonist ryanodine but only up to control level, i.e. not as substantially as in experiments where InsP₄ was applied under control conditions.

In experiments where CICR and InsP₃-dependent Ca²⁺ release antagonists were co-applied with InsP₄, an inhibition of LTP occurred. These results suggest that as soon as the InsP₃-dependent release is blocked, InsP₄ loses its LTP potentiating ability and the remaining potentiation is inhibited. When these two internal Ca²⁺ release pathways were simultaneously, but not independently blocked we observed similar effects. CPA, a drug known to empty internal Ca²⁺ stores by blocking Ca²⁺ uptake (Seidler et al. 1989) inhibited LTP (Fig. 8). InsP₄ was similarly ineffective in relieving the inhibition, except for a post-tetanic part. A slight, but statistically not significant facilitatory effect of InsP₄ is suggested by the data in Fig. 8F. This result indicates that an upregulated Ca²⁺ entry through VGCCs, enhanced by InsP₄, during induction of LTP could take place leading to the induction of an NMDA receptor-independent form of potentiation.

Thus, our results suggest that InsP₄ is ineffective in producing a significant facilitation when InsP₃-dependent Ca²⁺ release is blocked. For the substantial enhancement of potentiation in the presence of InsP₄, CICR and InsP₃-dependent release seems to be required. Furthermore, under control conditions CICR primarily determines the amount of resulting potentiation after tetanization; and InsP₃-dependent Ca²⁺ release does not contribute to the potentiation of EPSPs.

In summary, we conclude that InsP₄ can mediate Ca²⁺ entry through VGCCs which might have a more pronounced effect if the potentiation is strongly dependent on VGCCs. Furthermore, our results emphasize the importance of postsynaptic ER Ca²⁺ stores in LTP and a possible physiological function of InsP₄ could be to activate InsP₃-dependent Ca²⁺ release, in such a way that it can participate in the induction of the LTP which is normally dependent on the CICR (Fig. 9).

Figure 9. Scheme illustrating the possible mechanisms of injected InsP₄ on LTP facilitation in hippocampal CA1 pyramidal cells.

A, under control LTP conditions NMDA and ryanodine receptors contribute to the potentiation. In contrast, InsP₃-dependent Ca²⁺ release probably does not. B, after the whole-cell application of InsP₄, InsP₃-dependent Ca²⁺ release is activated as well as Ca²⁺ influx through VGCCs. This is enhanced and leads to a facilitated potentiation. Under physiological conditions, InsP₄ is produced after activation of G protein-coupled receptors, such as mGluRs or muscarinic receptors. According to previous studies, InsP₄ can interact with N-type VGCCs. The action of InsP₄ on proteins in the plasma membrane or in the ER is possibly mediated by InsP₄ binding proteins (InsP₄BPs), such as p42^IP4 (Aggensteiner et al. 1998). See text for further details. Text used in this scheme: AMPAR, AMPA receptor; NMDAR, NMDA receptor; InsP₃R, InsP₃ receptor; InsP₄R, InsP₄ receptor; RyanodineR, ryanodine receptor.