Long-term depression (LTD) is a persistent, use-dependent decrease of synaptic efficacy that is displayed in the hippocampus as well as elsewhere in the brain (Dudek & Bear, 1992; Mulkey & Malenka, 1992; Linden & Connor, 1995). In the hippocampus, LTD is divided into three categories: homosynaptic, heterosynaptic and associative LTD (Linden & Connor, 1995). Homosynaptic LTD is most readily induced in Schaffer collateral-pyramidal cell synapses of the area CA1 by prolonged (2-15 min), low-frequency (1-5 Hz) afferent stimulation, and is dependent on postsynaptic Ca2+ increases (Mulkey & Malenka, 1992; Bolshakov & Siegelbaum, 1994). It is generally acknowledged that this low-frequency induction protocol is most robust in young rats (< 35 days) (Mulkey & Malenka, 1992; Izumi & Zorumski, 1993; Dudek & Bear, 1993; Bolshakov & Siegelbaum, 1994; Mulkey et al. 1993, 1994; Errington et al. 1995). Similar protocols do not consistently induce LTD in older animals (> 35 days or ∼200 g) (Errington et al. 1995; Otani & Connor, 1995; Wagner & Alger, 1995; but see Dudek & Bear, 1992; Heynen et al. 1996).
We have recently reported that low-frequency stimulation applied asynchronously to two separate sets of synapses on the same CA1 pyramidal neuron induces LTD of these synapses in adult hippocampus (Otani & Connor, 1995). Induction of this LTD requires activation of both N-methyl-D-aspartate (NMDA) and metabotropic glutamate (mGlu) receptors, postsynaptic Ca2+ increases, and nitric oxide (NO) synthesis. Although more detailed mechanisms of this LTD have not been investigated, the asynchronous stimulation may generate a sufficient postsynaptic excitation without exceeding a LTD-inducible input frequency at one set of synapses (<10 Hz; Dudek & Bear, 1992). This notion predicts that in adult CA1, a pharmacological manipulation that enhances postsynaptic excitation lowers the threshold for LTD induction in single-pathway condition. In fact, 2 Hz single-pathway stimulation can induce LTD if it is combined with exposure to the mGluR agonist (1S, 3R/1S, 3S)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) (Otani & Connor, 1995). Furthermore, 1 or 2 Hz single-pathway stimulation induces LTD in the condition where γ-aminobutyric acid-A (GABAA)-mediated synaptic inhibition is blocked (Kerr & Abraham, 1995; Wagner & Alger, 1995; Otani & Connor, 1996a). The second protocol points to the significant role of the balance between synaptic excitation and inhibition in the generation of LTD, as has been long proposed for long-term potentiation (LTP) induction (Wigström & Gustafsson, 1983; Artola et al. 1990).
In the present study, we have measured Ca2+ changes by fura-2 ratio imaging simultaneously combined with intracellular electrical recording to investigate the mechanisms underlying LTD induction during block of GABAA synaptic inhibition in the adult hippocampus. Combining these techniques with pharmacological manipulations, we demonstrate a unique co-operativity between moderate dendritic Ca2+ entry and mGluR activation in the induction of LTD. Some of the results have appeared in the form of a short communication (Otani & Connor, 1996b).
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Male adult Sprague-Dawley rats (180-220 g, ∼50 days old) were anaesthetized by intramuscular injection of ketamine (40 mg kg−1) and xylazine (5 mg kg−1) and decapitated. The brain was rapidly removed from the skull, and the right hippocampus was dissected out in fully oxygenated (95 % O2-5 % CO2) artificial cerebrospinal fluid (ACSF) of the following composition (mM): 124 NaCl, 3 KCl, 26 NaHCO3, 2 CaCl2, 1 MgSO4, 10 D-glucose, and 1.25 NaH2PO4. Using a vibratome, transverse slices (400 μm) were prepared from the dorsal-mid portion of the hippocampus in chilled oxygenated ACSF. The slices were allowed to recover for at least 2 h at room temperature (∼20°C). A slice was then transferred to an interface recording chamber and continuously perfused with ACSF (1 ml min−1) at 32.5 ± 0.3°C. The CA1 region was separated from CA3 by a knife-cut to the Schaffer collaterals to reduce spontaneous presynaptic activity.
Stable intracellular recordings were made from over seventy CA1 pyramidal neurons using standard glass microelectrodes filled with 3 M potassium acetate (80-100 MΩ tip resistance). The mean resting membrane potential of the cells was -67 ± 0.8 mV, with an input resistance of 48 ± 1.0 MΩ. A spike amplitude of at least 85 mV was required for acceptable data. The membrane potential was held between -70 and -75 mV (-73 ± 0.5 mV on average) by negative current injection using a Neurodata amplifier (IR 283; New York) and monitored with a digital voltage meter (Precision 280). Monosynaptic excitatory postsynaptic potentials (EPSPs) of about 10 mV amplitude were evoked orthodromically by bipolar stimulating electrodes (25 μm twisted tungsten wires; monophasic square pulses of 150 μs duration) placed on the Schaffer collateral pathway. The responses were digitized at 4 kHz and stored on an IBM computer (model 5170; Armonk, NY, USA), using a TL-1 DMA interface (Axon Instruments) and the pCLAMP acquisition program (Axon Instruments).
During experiments, test EPSPs were evoked at 0.017 Hz. Standard LTD-inducing, low-frequency stimulation consisted of 900 pulses delivered at 2 Hz. Test stimulation was resumed 30 s after the end of 2 Hz stimulation. Only neurons in which spike height, input resistance and resting potential remained within 10 % of initial values were included in the analysis. Responses were averaged for each 5 min period, and changes were expressed as a percentage increase/decrease of the EPSP slope (mV per fixed time) from baseline level (a 5 min period just prior to conditioning). Student's two-tailed t test or analysis of variance (ANOVA) repeated measures was used for statistical analysis, with the level P < 0.05 considered as significant. All data are expressed as means ±s.e.m.
For the measurement of Ca2+ concentration ([Ca2+]), cells were impaled with microelectrodes initially containing 20 mM fura-2 (Molecular Probes) in the tip and 3 M potassium acetate in the barrel. After penetration, hyperpolarizing current (0.2-0.4 nA) was applied for 10–20 min to eject fura-2 which then diffused throughout the neuron. During this time, mixing of fura-2 and potassium acetate within the electrode also occurred, lowering the resistance from several hundred megaohms to ∼100 MΩ. Neurons near the top surface of the slice were examined for epifluorescence, using an upright microscope (Zeiss Axioskop, Thornburg, NY, USA) and a long-distance × 20 dry objective lens (Zeiss) in the interface configuration. A CCD camera system (Photometrics, Tucson, AZ, USA) was used in the frame transfer mode to acquire image pairs at 350 and 380 nm excitation wavelengths (100-150 ms exposure time). An image pair was recorded approximately every 60 s before and after 2 Hz conditioning stimuli and every 10–20 s during the 7.5 min conditioning protocol. Once the occurrence of consistent rises in [Ca2+] shortly after the onset of conditioning stimuli was established, the acquisition rate was accelerated up to 2–4 Hz during the first 20–30 s of conditioning. For imaging during 100 Hz stimuli, a logic signal from the camera controller served as a trigger to synchronize image acquisition and the delivery of the stimuli. [Ca2+] was determined from background corrected image pairs using the ratio method (Grynkiewicz et al. 1985).
All drugs were applied in the bathing medium, except BAPTA (Molecular Probes) and the protein kinase C inhibitor peptide PKC(19-36) (Gibco BRL), which were loaded into cells via the recording electrode (20 mM BAPTA and 250 μm PKC inhibitor in electrode). Other drugs used were dl-2-amino-5-phosphonovaleric acid (APV, Sigma), (+)-α-methyl-4-carboxyphenylglycine (MCPG, Tocris Cookson) and picrotoxin (Sigma).
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Both homosynaptic LTD and reversal of LTP (depotentiation) can be induced in the hippocampus by long (∼2-15 min), relatively low-frequncy (1-5 Hz) afferent stimulation. Our measurements showed that LTD-associated increases in postsynaptic [Ca2+] are transient. They occurred only during the first 20–30 s of 2 Hz conditioning, but conditioning periods this short were insufficient to induce LTD (Fig. 4A, inset). It has been shown that, for a significant depression, at least about 200 synaptic inputs have to be applied if the stimulus frequency is set at 2 Hz (1.7 min) (Fujii et al. 1991). On the other hand, we showed that exposure to MCPG during conditioning, which itself did not affect the postsynaptic [Ca2+] increases, blocked LTD. These results suggest the possibility that, for LTD induction, the rapid influx of Ca2+ is co-operative with a more prolonged episode of synaptic activation of mGluRs.
Postsynaptic hyperpolarization, but not the blockade of NMDA receptors, abolished the [Ca2+] rises and significantly reduced LTD, suggesting the involvement of voltage-sensitive Ca2+ channels in LTD induction. However, we did not attempt to block LTD by abolishing only the rapid [Ca2+] rises, i.e. the rises during the first 20–30 s of the conditioning. Therefore the possibility that undetectable [Ca2+] increases later in the conditioning period contribute to LTD induction cannot be ruled out. Also, we did not determine which types of Ca2+ channels are involved in LTD. In this respect, Oliet et al. (1997) recently showed involvement of T-type Ca2+ channels in mGluR-dependent induction of LTD in CA1 neurons prepared from young rats.
There are significant contrasts between the factors necessary for LTD induction shown in the present study and those found in other studies, as well as contrasts among these other studies. We suggest that the most important experimental variables contributing to these differences are conditioning stimulus frequency and amplitude, and age of the experimental animal. Using a 2 Hz protocol, we showed that a level of Ca2+ entry critical for LTD induction in the adult hippocampus can occur via the activation of voltage-gated dendritic channels rather than NMDA receptor-linked channels, so that LTD is still inducible in the absence of NMDA receptor activation. Bolshakov & Siegelbaum (1994), using neonatal rats and a 5 Hz protocol, also showed that LTD induction was not blocked by APV, but could be blocked by holding neurons at -70 mV during conditioning, and by bath-application of MCPG. Similarly, Oliet et al. (1997) showed, using neonatal and juvenile rats and the same 5 Hz stimuli, that MCPG-sensitive, APV-resistant LTD depends on Ca2+ influx via Ni2+-sensitive channels. In contrast, other studies that used 1 Hz protocols in either adult or immature hippocampus showed a requirement for NMDA receptors in LTD induction (Dudek & Bear, 1992; Mulkey & Malenka, 1992; Selig et al. 1995; Kerr & Abraham, 1995; Heynen et al. 1996; Staubli & Ji, 1996). We have indeed observed that, in the presence of APV, 1 Hz stimuli did not induce LTD while 2 or 5 Hz stimuli did, in the same slices prepared from immature hippocampus (S. Otani & J. A. Connor, unpublished observations). Thus, the higher frequency conditioning protocols appear to lower the dependency on NMDA receptor activation. These previous and present results suggest that methodological differences determine which induction factor (i.e. NMDA receptors or voltage-gated channels/mGluRs) is more dominantly involved in LTD. Alternatively, it may be that there are two separate mechanisms for LTD induction that coexist, one controlled by NMDA receptor activation and the other by mGluRs (Oliet et al. 1997). Variations in induction protocols may give one more importance than the other.
The stimulus strength used to induce LTD has also varied between different experiments. With the exception of our present study, which employed intracellular recording, all cases of synaptic LTD induction in adult preparations have been shown by measuring field responses. This complicates the comparison of results from different laboratories because of electrode placement and other factors. However, Kerr & Abraham (1995) have noted that LTD induction in adults fails to occur at a field EPSP amplitude of 0.5 mV, but is successfully induced at 1.0 mV (without GABAA block). Staubli & Ji (1996) showed that weak conditioning pulses, which produced no appreciable population spike, induced LTD in only about 20 % of preparations, while larger stimuli, which triggered population spikes, raised the success rate to 90–100 %. Stimulation in our experiments was subthreshold, or gave only sporadic firing, without GABAA block. Although their use of field recording makes it difficult to detect poorly synchronized firing, the stimulus employed by Wagner & Alger (1995) was probably also in the low range, and they too failed to observe LTD induction without GABAA block in slices from adults using a 1 Hz, 900 pulse protocol. Thus, we propose that the factors of primary importance in LTD induction in adult preparations are whether the cell fires in response to the conditioning stimulus and whether GABAA block increases the effectiveness of low amplitude stimuli to elicit firing.
In normal saline, CA1 neurons fire at most once in response to a moderate presynaptic stimulus. The conjunction of this depolarization with NMDA receptor activation should allow localized Ca2+ entry at the dendritic spines as well as less focal, voltage-gated entry. It has been directly shown in this laboratory that Ca2+ changes are most intense at the spines during tetanic stimulation and remain fairly localized there, even during tetani of several hundred milliseconds (Müller & Connor, 1991; Petrozzino et al. 1995). It is presumed, although not directly shown to our knowledge, that this pattern holds for single spikes. If a large part of the Ca2+ signalling in LTD derives from NMDA receptor influx, we would expect a homosynaptic induction as in the 1 Hz protocols, although, at high stimulus intensities, a wider region of Ca2+ spread, encompassing neighbouring synapses, might occur, supporting a heterosynaptic component (see Otani & Connor, 1996a; Staubli & Ji, 1996).
LTD induction in our study was dependent on postsynaptic PKC activation (see also Oliet et al. 1997), which has also been implicated in LTP induction (Lovinger et al. 1987; Malinow et al. 1989). We suggest that there may be a difference in the manner and location of PKC activation between two conditions. It is known that Ca2+-dependent activation of PKC subspecies expressed in CA1 pyramidal cell dendrites (α, β-II, γ) is facilitated by the presence of DAG (Nishizuka, 1992; Tanaka & Nishizuka, 1994): when arachidonic acid is available together with DAG, PKC may be activated even at basal [Ca2+] levels. Importantly, the action of these co-factors can sustain activation of PKC (Nishizuka, 1992). It is therefore possible that dendritic production of DAG and arachidonic acid (Dumuis et al. 1990) by repeated mGluR activation during prolonged low-frequncy orthodromic stimulation may help activate PKC persistently at low or basal [Ca2+] levels. In contrast, large [Ca2+] increases during LTP-inducing high-frequncy stimulation (see Fig. 6), especially the NMDA-dependent increases in dendritic spines (Petrozzino et al. 1995), may activate synaptic PKC with a limited access to DAG and arachidonic acid.
However, we should be still cautious in interpreting the effect of the mGluR antagonist MCPG on LTD induction. MCPG blocks both group I mGluRs (i.e. mGluR1 and 5), which are positively coupled to phospholipase C cascades, and group II mGluRs (i.e. mGluR2 and 3), which are known to be negatively coupled to cAMP pathways (Watkins & Collingridge, 1994; Nicoletti et al. 1996). Although CA1 pyramidal neurons express mGluR5 (Lujan et al. 1996), antagonism of MCPG to mGluR5 is somewhat controversial (Joly et al. 1995vs.Saugstad et al. 1995). It is thus possible that MCPG acted on group II mGluRs to block LTD induction in a manner unrelated to inhibition of the phospholipase C pathways. It has been shown in dentate gyrus that a group II mGluR antagonist blocks LTD induction by 1 Hz stimuli (Huang et al. 1997). In mossy fibre-pyramidal neuron synapses in area CA3, lack of functional mGluR2 resulted in the impairment of LTD with 1 Hz conditioning (Yokoi et al. 1996). It is interesting to note here that Petralia et al. (1996) recently showed the existence of group II mGluRs in CA1 postsynaptic neurons, as well as in presynaptic components.
The modulatory role of GABAA inhibition in LTD induction appears to be developmentally regulated; block of GABAA inhibition enables LTD induction in adult hippocampus (see also Wagner & Alger, 1995; Otani & Connor, 1996a), while none is necessary in younger animals (Mulkey & Malenka, 1992, Dudek & Bear, 1993; Izumi & Zorumski, 1993; Bolshakov & Siegelbaum, 1994; Wagner & Alger, 1995; Oliet et al. 1997). GABAergic inhibition develops slowly in the postnatal rat hippocampus (>12 days; Ben-Ari et al. 1989; Muller et al. 1989), lessening the need for (or effect of) exogenous blockers at early stages. Also, depotentiation in adult hippocampus requires no GABAA block (Barrionuevo et al. 1980; Staubli & Lynch, 1990; Fujii et al. 1991). As pointed out by Wagner & Alger (1995), in this case, the balance between synaptic excitation and inhibition is probably shifted towards a downregulation of synaptic GABAA inhibition by high-frequncy stimulation applied earlier to induce LTP (see also Abraham et al. 1987; Stelzer et al. 1994).