Differential Maintenance and Frequency-Dependent Tuning of LTP at Hippocampal Synapses of Specific Strains of Inbred Mice

Peter V. Nguyen,1,2 Steven N. Duffy,1,2 and Jennie Z. Young2

 1Department of Physiology and  2Division of Neuroscience, University of Alberta School of Medicine, Edmonton, Alberta T6G 2H7, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nguyen, Peter V., Steven N. Duffy, and Jennie Z. Young. Differential Maintenance and Frequency-Dependent Tuning of LTP at Hippocampal Synapses of Specific Strains of Inbred Mice. J. Neurophysiol. 84: 2484-2493, 2000. Transgenic and knockout mice are used extensively to elucidate the molecular mechanisms of hippocampal synaptic plasticity. However, genetic and phenotypic variations between inbred mouse strains that are used to construct genetic models may confound the interpretation of cellular neurophysiological data derived from these models. Using in vitro slice stimulation and recording methods, we compared the membrane biophysical, cellular electrophysiological, and synaptoplastic properties of hippocampal CA1 neurons in four specific strains of inbred mice: C57BL/6J, CBA/J, DBA/2J, and 129/SvEms/J. Hippocampal long-term potentiation (LTP) induced by theta-pattern stimulation, and by repeated multi-burst 100-Hz stimulation at various interburst intervals, was better maintained in area CA1 of slices from BL/6J mice than in slices from CBA and DBA mice. At an interburst interval of 20 s, maintenance of LTP was impaired in CBA and DBA slices, as compared with BL/6J slices. When the interburst interval was reduced to 3 s, induction of LTP was significantly enhanced in129/SvEms slices, but not in DBA and CBA slices. Long-term depression (LTD) was not significantly different between slices from these four strains. For the four strains examined, CA1 pyramidal neurons showed no significant differences in spike-frequency accommodation, membrane input resistance, and number of spikes elicited by current injection. Synaptically-evoked glutamatergic postsynaptic currents did not significantly differ among CA1 pyramidal neurons in these four strains. Since the observed LTP deficits resembled those previously seen in transgenic mice with reduced hippocampal cAMP-dependent protein kinase (PKA) activity, we searched for possible strain-dependent differences in cAMP-dependent synaptic facilitation induced by forskolin (an activator of adenylate cyclase) and IBMX (a phosphodiesterase inhibitor). We found that forskolin/IBMX-induced synaptic facilitation was deficient in area CA1 of DBA/2J and CBA/J slices, but not in BL/6J and 129/SvEms/J slices. These defects in cAMP-induced synaptic facilitation may underlie the deficits in memory, observed in CBA/J and DBA/2J mice, that have been previously reported. We conclude that hippocampal LTP is influenced by genetic background and by the temporal characteristics of the stimulation protocol. The plasticity of hippocampal synapses in some inbred mouse strains may be "tuned" to particular temporal patterns of synaptic activity. From a broader perspective, our data support the notion that strain-dependent variation in genetic background is an important factor that can influence the synaptoplastic phenotypes observed in studies that use genetically modified mice to explore the molecular bases of synaptic plasticity.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Understanding the cellular and molecular mechanisms of activity-dependent synaptic plasticity is an important goal of neuroscience. Transgenic and gene-targeting techniques are potent tools for defining the functions of individual genes in certain forms of synaptic plasticity in the mammalian brain (Hogan et al. 1994). Within particular brain regions, genes may be expressed in elevated amounts (Mayford et al. 1996), or the expression of specific genes may be eliminated by targeted deletion (Capecchi 1989; Soriano 1995). These molecular strategies have been effectively applied to generate genetically modified mice, and these animals have been used extensively to explore the molecular mechanisms of synaptic plasticity (Chen and Tonegawa 1997; Mayford et al. 1995a; Picciotto and Wickman 1998).

A prolific number of studies have tried to elucidate the functions of specific genes and signaling molecules in particular forms of activity-dependent synaptic plasticity, learning, and memory (Micheau and Riedel 1999; Picciotto and Wickman 1998). In these studies, a common strategy has been to use two inbred strains of mice to generate genetically modified mice. An inbred strain is one in which matings between siblings have been performed for at least 20 generations, resulting in a population of genetically homogeneous animals (Lyon and Searle 1989). One inbred strain supplies a viable genetic background for breeding and survival, while a second inbred strain provides stem cells for genetic manipulation (Hogan et al. 1994). An important consideration inherent in all of these studies is that disruption or overexpression of a single gene can lead to compensatory changes in the expression of other genes, the presence or absence of which can vary according to the genetic backgrounds of the mouse strains used to generate a genetically modified line of mice (Crawley et al. 1997; Gerlai 1996). Valid interpretation of the neurophysiological phenotypes that emerge from genetically modified mice therefore requires a knowledge of the synaptoplastic and electrophysiological properties of relevant neurons in the parent strains used to produce genetically modified lines of mice. Hence, the characterization of cellular electrophysiological and synaptoplastic properties of neurons and synapses in relevant brain structures of inbred strains of mice is an important step toward defining the genetic and molecular bases of synaptic plasticity.

Much is known about the behavioral characteristics and cognitive performance capabilities of some strains of inbred mice, particularly with reference to learning and memory (Crawley et al. 1997; Owen et al. 1997). For example, DBA/2 and C57BL/6 mice show markedly different performance capabilities in tests of spatial learning and memory. DBA/2 mice are impaired in spatial learning (Paylor et al. 1993), and they show deficient hippocampal synaptic plasticity that may compromise their performance on tasks requiring integration of spatial and contextual information (Bampton et al. 1999; Matsuyama et al. 1997; Nguyen et al. 2000). In contrast, C57BL/6 mice show robust hippocampus-based learning and memory that are correlated with intact synaptic physiology in area CA1 (Nguyen et al. 2000). Some of the learning deficits in DBA/2 mice may be linked to reduced levels of hippocampal protein kinase C (PKC) activity (Matsuyama et al. 1997; Wehner et al. 1990). Also, in another inbred strain, CBA/J, profound deficits in spatial and nonspatial learning and memory are correlated with deficiencies in the expression of some forms of hippocampal synaptic plasticity in area CA1 (Nguyen et al. 2000). However, a conjoint characterization of cellular biophysical properties, activity-dependent plasticity, and the expression of signal transduction-mediated synaptic plasticity has not been performed on hippocampal CA1 neurons in these strains. It is important to examine and compare these functional properties of hippocampal neurons in these mouse strains, because the deficits in learning and memory in DBA/2 and CBA/J mice (Nguyen et al. 2000; Paylor et al. 1993) may be linked to alterations in some or all of these cellular attributes in these strains.

Hippocampal long-term potentiation (LTP) and long-term depression (LTD) constitute activity-dependent enhancement and reduction, respectively, of excitatory synaptic strength (Bear and Abraham 1996; Madison et al. 1991). These two types of synaptic plasticity are believed to play important roles in some forms of learning and memory in the mammalian brain (Martin et al. 2000). In humans and mice, area CA1 of the hippocampus is vital for the formation of long-term memory (Tsien et al. 1996; Zola-Morgan et al. 1986). Genetic modifications of key signaling molecules within area CA1 of the mouse hippocampus can impair long-term memory and LTP (reviewed by Micheau and Riedel 1999). Some data on mouse strain-specific impairments of memory and of hippocampal LTP in area CA1 have been reported (Nguyen et al. 2000; see also Bampton et al. 1999, for dentate gyrus data). However, the cellular and molecular mechanisms underlying these strain-dependent defects in LTP are undefined. For example, enhanced glutamatergic postsynaptic currents have been implicated in the induction of hippocampal LTP in area CA1 (reviewed by Kullmann and Siegelbaum 1995 and Malenka and Nicoll 1993), and differences in the magnitudes of these currents may exist among particular inbred mouse strains. Also, modified membrane excitability or altered spike frequency accommodation in hippocampal neurons may influence the sensitivity of these neurons to excitatory synaptic input and might thereby regulate the thresholds for induction of LTP and LTD in these neurons. Strain-specific variations in these cellular electrophysiological properties may impart differential expression of cellular excitability and spike firing efficacy in hippocampal neurons, and these electrophysiological alterations, in turn, might modify the signal processing capabilities of hippocampal circuits.

Signal transduction pathways are also important for memory formation and for modulating hippocampal synaptic strength (reviewed by Elgersma and Silva 1999; Martin et al. 2000; Micheau and Riedel 1999; Soderling and Derkach 2000). Strain-dependent variations in hippocampal protein kinase expression may influence LTP and learning and memory (see Wehner et al. 1990 for PKC data). cAMP-dependent protein kinase (PKA) is vital for hippocampus-dependent long-term memory and for maintenance of LTP in area CA1 of C57BL/6 mice (Abel et al. 1997; Nguyen and Kandel 1997), but cAMP-induced forms of synaptic facilitation have not been examined in inbred strains that display deficits in hippocampus-dependent long-term memory (e.g., CBA/J, DBA/2J strains, Paylor et al. 1993; Nguyen et al. 2000).

The primary aim of the present study was to compare the membrane biophysical, cellular electrophysiological, and synaptoplastic properties of hippocampal neurons in particular inbred strains of mice. A secondary objective of our study was to compare the expression of cAMP-induced synaptic facilitation in these same strains, since PKA and cAMP are known to play important roles in the expression of hippocampus-based long-term memory and hippocampal LTP (Abel et al. 1997; Huang and Kandel 1994; Slack and Pockett 1991; Weisskopf et al. 1994; Wong et al. 1999). The strains examined here were selected because their performance capabilities on hippocampus-dependent learning and memory tasks have been previously documented (Crawley et al. 1997; Nguyen et al. 2000; Owen et al. 1997; Paylor et al. 1993), and also because these strains have been used extensively to generate genetically modified mice for studies of synaptic plasticity and cognitive functions, including learning and memory (Piciotto and Wickman 1998). We addressed the following fundamental questions: Is hippocampal LTP in some inbred strains optimally "tuned" to, or induced by, specific temporal patterns of imposed synaptic activity? Are there strain-dependent differences in spike firing efficacy, postsynaptic glutamatergic currents, and membrane biophysical properties in hippocampal neurons? Do hippocampal neurons show significant strain-dependent variations in cAMP-induced synaptic facilitation? Some of these data have been published in abstract form (Duffy et al. 2000).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

All mice were male, aged 4-8 wk (Jackson Labs, Maine). We examined four inbred strains: C57BL/6J, CBA/J, DBA/2J, and 129/SvEms-+Ter?/J. For LTD experiments, we used hippocampal slices from mice aged 4-5 wk, while all other experiments were performed on slices cut from mice aged 7-8 wk. Animals were housed on a 12-h light/dark cycle at the University of Alberta Health Sciences Animal Care Facility, using guidelines approved by CCAC (Canadian Council on Animal Care).

Electrophysiology: extracellular field recordings

Transverse hippocampal slices (400-µm thickness) were cut and maintained in an interface chamber at 28°C (for more details, see Nguyen and Kandel 1997). The artificial cerebrospinal fluid (ACSF) used for dissection and superfusion contained (in mM) 125 NaCl, 4.4 KCl, 1.5 MgSO4, 1 NaH2PO4, 26 NaHCO3, 10 glucose, 2.5 CaCl2. Extracellular excitatory postsynaptic field potentials (fEPSP) were recorded in stratum radiatum of area CA1 with glass microelectrodes (2-4 MOmega resistances, filled with ACSF). The Schaffer collateral pathway was stimulated with bipolar nickel chromium electrodes positioned in stratum radiatum, and evoked fEPSPs were recorded, digitized, and analyzed using pClamp 7 software (Axon Instruments). Baseline fEPSPs were elicited once per minute by applying a stimulus intensity (0.08-ms pulse width) sufficient to evoke fEPSP amplitudes that were 40% of maximal sizes. For some experiments, fEPSPs were measured during stimulation (once per minute at test intensity) of a second, independent pathway in stratum radiatum (Nguyen et al. 1994). This control pathway did not receive high-frequency stimulation and it was monitored before and after induction of LTP in a neighboring pathway in stratum radiatum of the same slice. Various stimulation protocols were used to induce LTP and LTD; these are described at appropriate parts of the RESULTS section. Rp-cAMPS, an inhibitor of PKA, was dissolved in ACSF. Forskolin and IBMX (from RBI) were prepared as 50 mM stock solutions in DMSO and were diluted in ACSF to obtain a final DMSO concentration of 0.1%. This DMSO concentration did not affect synaptic responses under our experimental conditions.

Whole-cell patch-clamp recordings

Hippocampal CA1 neurons were patch-clamped in the "blind" whole-cell mode (Blanton et al. 1989) using an Axopatch-1D amplifier and pClamp-7 acquisition software (Axon Instruments). Whole-cell patch-clamp electrodes were produced on a multistage puller (Sutter Instruments) to yield resistances of 6-9 MOmega . All cells were in the CA1 pyramidal cell layer, as judged by visual placement of the patch-clamp electrode. Spike-frequency accommodation was used as the physiological criterion for identifying these neurons as pyramidal cells (Kandel and Spencer 1961; Schwartzkroin 1977; Schwartzkroin and Mathers 1978). Cells with resting potentials more depolarized than -60 mV, or spike amplitudes <80-90 mV, were rejected. gamma -aminobutyric acid-A (GABAA) currents were blocked with bath-applied 10 µM picrotoxin (RBI). Cellular resting potential (Em), input resistance (Rin), spike-frequency accommodation (SFA), and membrane depolarization (following 100-Hz, 1-s stimulation) were measured in current-clamp mode with an electrode solution containing (in mM) 130-140 potassium gluconate, 10 NaCl, 5 MgCl2, 10 HEPES, 2 NaATP, 0.3 NaGTP, 0.5 EGTA, 0.05 CaCl2, pH 7.3. Synaptically evoked glutamatergic currents were measured in voltage-clamp mode using an electrode solution containing (in mM) 130-140 KF, 10 HEPES, 10 NaCl, 10 EGTA, pH 7.3.

Data analysis

We assessed induction and maintenance of LTP or LTD in slices from the four strains. "Induction" was assessed as the fEPSP slope measured 5 min after the end of high-frequency stimulation (for LTP experiments) or immediately after 1-Hz stimulation (for LTD experiments). Interstrain comparisons of induction efficacy were done by pooling and averaging the fEPSP slopes measured at these time points within each strain and by comparing the resulting mean slopes across strains. "Maintenance" was assessed by calculating the ratio of the fEPSP slope measured 50 min (for theta-burst LTP experiments), 100 min (for all other LTP protocols), or 45 min (for LTD experiments) after high- or low-frequency stimulation, to the fEPSP slope measured 5 min after initiation of potentiation or immediately after initiation of depression. The 50- and 100-min time points were chosen because the inhibitory effects of PKA antagonists, which block maintenance (but not induction) of LTP, are clearly evident at these time points (Frey et al. 1993; Huang and Kandel 1994; Nguyen and Kandel 1997). The ratio ("maintenance index") of the fEPSP slopes measured at these early and late time points was calculated for each slice and expressed as a percentage, and the mean ratio was obtained for each strain by pooling and averaging the ratios produced by a given stimulation protocol. Hence, a maintenance index of zero would signify complete decay of fEPSP slopes to control baseline values, and an index of 100 would signify no decay from initial fEPSP slope values measured soon after high- or low-frequency stimulation. Averaged data for induction and maintenance of LTP and LTD are presented in Tables 1 and 2. Biophysical data are summarized in Table 3.


                              
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Table 1. Induction of LTP and LTD in inbred strains


                              
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Table 2. Maintenance of LTP and LTD in inbred strains


                              
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Table 3. Summary of biophysical measurements from CA1 pyramidal neurons

A single-factor analysis of variance (ANOVA) was used to assess whether at least one group (strain) was significantly different from the others, within a given stimulation regimen. To identify which particular pairs of strains showed significant differences in our measured electrophysiological parameters, we applied Student's t-test to those cases where the ANOVA revealed a significant difference between groups.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CBA, DBA, and 129/SvEms mice are deficient in theta-burst LTP

Theta patterns of electrical activity in the hippocampus can occur during environmental exploration and behavioral conditioning (Grastyan et al. 1959; Green et al. 1960; Vanderwolf 1969), and LTP induced by theta-burst patterns of stimulation (TBS) may be linked to the exploratory behavior of rodents in spatially novel environments (Bland 1986; Larson et al. 1986; Otto et al. 1991; Staubli and Lynch 1987).

In CBA, DBA, and 129/SvEms slices, TBS (15 bursts of four pulses at 100-Hz, delivered at an interburst interval of 200 ms) elicited initial increases in fEPSP slopes (measured 5 min after theta-burst stimulation) that were significantly less than those measured in BL6 slices (Fig. 1, Table 1). In slices from CBA and DBA mice, maintenance of theta-burst LTP was also significantly less robust than in BL/6J slices (Fig. 1, Table 2). These results indicate that genetic background may significantly influence the induction and persistence of theta-burst LTP in area CA1.



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Fig. 1. Long-term potentiation (LTP) evoked by theta-burst stimulation is reduced in 129/SvEms, DBA, and CBA slices. A: plot of extracellular excitatory postsynaptic field potential (fEPSP) slope versus time, comparing averaged responses from BL/6J (n = 14 mice, 14 slices) and 129/SvEms (n = 6 mice, 7 slices). B and C: the BL/6J data in A are shown in B and C for comparison with average responses from DBA (n = 8 mice, 8 slices) and CBA (n = 9 mice, 10 slices) strains. Sample fEPSP traces from individual experiments at t = 65 min (45 min post-induction) are shown on the right. TBS = theta-burst stimulation.

Strain-specific rescue of LTP by a temporally compressed pattern of stimulation

Synaptoplastic processes in the hippocampus are believed to be critically modulated by the temporal pattern of synaptic stimulation (Bear and Abraham 1996; Bito et al. 1996; Hawkins et al. 1993; Madison et al. 1991). Multiple bursts of high-frequency stimulation can elicit long-lasting potentiation in the rodent hippocampus (see review by Huang et al. 1996). Previous studies have shown strain-dependent deficits in the expression of LTP following temporally spaced patterns of stimulation (e.g., "tetra-burst" stimulation: four 1-s bursts of 100-Hz, with bursts spaced 5 min apart; Nguyen et al. 2000). However, temporally compressed patterns of stimulation can also induce robust and long-lasting LTP in mouse hippocampal slices (Woo et al. 2000), and these compressed patterns more closely resemble the firing patterns of hippocampal neurons in vivo (Kandel and Spencer 1961).

We tested the hypothesis that there may be strain-dependent differences in the responsiveness of hippocampal synapses to distinct temporal patterns of synaptic activity. Slices from the four strains were stimulated with four 100-Hz bursts (1-s burst duration) at 20-s interburst intervals, and fEPSPs were tracked for 100 min after high-frequency stimulation. As shown in Fig. 2 and Table 1, induction of LTP was significantly less robust in slices from 129/SvEms mice than in BL6 slices. In CBA and DBA slices, the level of potentiation seen 5 min following tetra-burst stimulation with a 20-s interburst interval was not significantly different from the levels observed in BL6 slices (Table 1). In contrast, CBA and DBA slices showed significantly less robust maintenance of LTP than BL6 slices following tetra-burst stimulation with a 20-s interburst interval (Table 2). Furthermore, CBA slices showed significantly less robust maintenance of LTP than slices from 129/SvEms mice (Table 2).



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Fig. 2. LTP evoked by multiple bursts of stimulation is deficient in some strains. LTP elicited by four, 1-s bursts of 100-Hz, interburst interval of 20 s ("4 × 100 Hz @ 20 s") is reduced in 129/SvEms (A, n = 7 mice, 7 slices), DBA (B, n = 7 mice, 7 slices), and CBA (C, n = 10 mice, 12 slices) slices as compared with BL/6J (n = 5 mice, 5 slices). Baseline fEPSPs measured during stimulation of a neighboring pathway that did not experience high-frequency stimulation ("no HFS") were unaffected in each strain tested. The BL/6J curve in A is repeated in B and C for comparison with other strains. Sample sweeps to the right are synaptic responses measured at t = 110 min (90 min post-induction).

Extremely compressed tetra-burst stimulation (with just a 3-s interburst interval) can induce long-lasting and robust LTP in hippocampal slices prepared from PKA mutant mice (Woo et al. 2000). These mice show deficient LTP following temporally spaced patterns of stimulation (Abel et al. 1997). This suggests that this compressed pattern of stimulation may be optimal for recruiting subcellular processes that are important for LTP expression at CA1 synapses. To test this hypothesis on these four inbred strains, we applied the same tetra-burst protocol as before, but we further reduced the interburst interval to just 3 s. With this protocol, we observed significantly greater induction and maintenance of LTP in 129/SvEms slices than with the 20-s interburst interval in this same strain (Tables 1 and 2, see also Fig. 3A; compare with Fig. 2A). Furthermore, the initial level of potentiation seen in 129/SvEms slices with the 3-s protocol was no longer significantly different from that seen in BL6 slices (Table 1). Thus, the defect in LTP induction seen in 129/SvEms slices with an interburst interval of 20 s can be rescued by reducing the interburst interval to just 3 s, without changing the total amount of imposed activity. In contrast, this 3-s protocol decreased (though not significantly) the average level of initial potentiation in CBA and DBA slices, as compared with values measured following the 20-s protocol (Table 1). Also, like the 20-s protocol, the 3-s regimen elicited significantly less robust maintenance of LTP in CBA and DBA slices as compared with BL6 and 129/SvEms slices (Table 2). These results show that the plasticity of hippocampal synapses in some inbred mouse strains is sensitive to the temporal pattern of imposed activity, and they support the notion that some temporally compressed patterns of stimulation may be optimal for recruiting subcellular processes important for the induction of robust and persistent LTP.



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Fig. 3. Strain-selective enhancement of LTP by temporally compressed stimulation. A: there was no significant difference in LTP between BL/6J (n = 7 mice, 7 slices) and 129/SvEms slices (n = 5 mice, 5 slices) when the interburst interval was reduced to 3 s. B and C: LTP in DBA (n = 6 mice, 6 slices) and CBA (n = 7 mice, 7 slices) strains remained deficient relative to BL/6J slices. The BL/6J curve in A is repeated in B and C for comparison with other strains. Sample sweeps were recorded at t = 130 min (110 min post-induction).

Strain-independent induction of LTD

In contrast to LTP, induction and maintenance of LTD, elicited by 1-Hz stimulation for 15 min, were not significantly different between slices from BL/6J and each of the other strains (Fig. 4, Tables 1 and 2). Thus, LTP induction and maintenance were impaired in some of the strains examined here, while LTD was not significantly different between these same strains.



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Fig. 4. Expression of LTD is not strain-dependent. A-C: prolonged low-frequency stimulation results in small reductions in average fEPSP slopes that are not significantly different between all strains tested (BL/6J, n = 6 mice, 6 slices; 129/SvEms, n = 8 mice, 8 slices; DBA, n = 6 mice, 6 slices; CBA, n = 5 mice, 5 slices).

Biophysical properties of hippocampal CA1 pyramidal cells are similar between strains

The observed differences in LTP expression among these four mouse strains may result from strain-dependent variations in the biophysical and electrophysiological properties of hippocampal neurons. Strain-selective alterations in cellular biophysical and electrophysiological properties could modify LTP expression by altering Ca2+ influx through postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptor channels. For example, lower membrane input resistances would produce smaller postsynaptic membrane depolarizations for a given synaptic input, leading to weaker LTP induction and attenuated LTP expression. Similarly, a smaller contribution of NMDA currents to the total postsynaptic glutamatergic current, or a fewer number of action potentials evoked during sustained postsynaptic membrane depolarization (i.e., greater spike-frequency accommodation), may reduce postsynaptic Ca2+ influx and attenuate LTP expression.

We measured membrane resting potential (Em), membrane input resistance (Rin), spike-frequency accommodation, membrane depolarization in response to 100-Hz stimulation, and synaptically evoked glutamatergic currents in CA1 pyramidal neurons (in slices) using blind whole-cell patch-clamp techniques. Measurements of these biophysical properties are illustrated in Fig. 5, and the data are summarized in Fig. 6 and Table 3.



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Fig. 5. Biophysical measurements recorded from hippocampal CA1 pyramidal cells. Sample records from BL/6J and 129/SvEms strains are shown. A: current-clamp recordings during injection of current through the patch electrode, showing spike frequency accommodation in BL/6J (left) and 129/SvEms neurons (right). Top traces: response to 300 pA current injection; middle: 200 pA; bottom: 100 pA. B, left: recordings of evoked excitatory postsynaptic currents at holding potentials of -100 mV and +40 mV. Symbols demarcate time points of current measurements shown in the right panel of B. Peak current is an estimate of the non-NMDA current component. Current at 35-ms post-peak is an estimate of the NMDA component. B, right: I-V plots for the two cells recorded in the left panel. C: current-clamp recordings showing voltage responses in BL/6J and 129/SvEms neurons during 100-Hz stimulation of presynaptic Schaffer collateral fibers.



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Fig. 6. Biophysical properties of CA1 pyramidal neurons are not strain-dependent. A: membrane excitability: plot of number of action potentials produced during a 1-s current pulse versus current amplitude. B, left: spike frequency accommodation: plot of spike number versus delay from onset of current injection. These are averages of fifth-order polynomial curve fits of data obtained from BL/6J (n = 13 cells), 129/SvEms (n = 28), DBA (n = 14), and CBA (n = 21) neurons. B, right: plot of the spike frequency over the final 400 ms of the current pulse relative to the initial spike frequency over the first 50 ms of the pulse. There were no significant differences between strains. C: ratios of NMDA to non-NMDA current were not significantly different between strains (BL6, n = 14 cells; 129/SvEms, n = 12; DBA, n = 5; CBA, n = 5). D: plot of the time integral of membrane depolarization during a single 1-s burst of 100-Hz presynaptic stimulation, expressed relative to BL/6J responses (BL/6J, n = 8 cells; 129/SvEms, n = 9; DBA, n = 7; CBA, n = 7). No significant differences were evident between the strains.

Membrane excitability can be assessed by determining the spike firing efficacy of a cell over a range of injected current amplitudes. To measure membrane excitability in CA1 pyramidal neurons, we injected current pulses (50-300 pA) (Fig. 5A) and counted the number of spikes evoked at each current magnitude. Spike frequency accommodation is a reduction in spike firing rate during a sustained stimulus (Schwartzkroin 1977) and accommodation was assessed by determining the delay of each spike in a spike train evoked by 300-pA current injection.

Non-NMDA and NMDA receptor-mediated glutamatergic currents were measured by stimulating the presynaptic Schaffer collateral pathway (with an extracellular electrode) while varying the holding membrane potential between -100 and +40 mV (Fig. 5B, left). The non-NMDA receptor-mediated current was measured as the early peak current, while the NMDA receptor-mediated current was identified by measuring the evoked current magnitude 35 ms after the peak. The I-V graph (Fig. 5B, right) displays the rectifying current (NMDA component) measured 35 ms after the peak, and the early linear current (non-NMDA component).

Finally, we measured the membrane depolarizations of CA1 pyramidal neurons in response to a single 1-s burst of 100-Hz stimulation of Schaffer collateral fibers (Fig. 5C). This measurement assesses the degree of membrane depolarization experienced by the soma following activation of dendritic excitatory synapses. The amount of somal depolarization seen after a single brief burst of stimulation is dependent on the amount of cable filtering of EPSPs imposed by the dendritic conductance load of a cell. The degree of dendritic filtering of EPSPs, and thereby the net depolarization experienced by the soma following brief stimulation, may vary according to genetic background. Repeated bursts of 100-Hz stimulation were not used because such repeated patterns of synaptic stimulation can lead to rapid postsynaptic receptor modifications (e.g., AMPA receptor mobilization/phosphorylation, Banke et al. 2000; Shi et al. 1999), which may alter the degree of somal depolarization in a manner that may be independent of genetic background.

The number of action potentials counted at any given magnitude of injected current was not statistically different between strains (Fig. 6A, P > 0.2, single-factor ANOVA). To determine the average spike frequency accommodation, we fitted individual spike delay plots (Fig. 6B, left) with a fifth-order polynomial (R2 was always >0.98 after using a fifth-order polynomial) and averaged the plots for each strain (Fig. 6B, left). The ratio of initial slope (t = 0-50 ms) to late slope (t = 600-1000 ms) of these plots is a measure of the mean reduction in average spike frequency (Fig. 6B, right) during current injection, and the ratios obtained for these strains were not statistically different from each other (P > 0.1, single-factor ANOVA).

The ratio of the peak size of the synaptically evoked glutamatergic current (measured at Em = -100 mV) to the current amplitude 35 ms after this peak was used as an estimate of the relative contribution of NMDA current to the total evoked glutamatergic current (Fig. 6C, Table 3). Again, no strain-dependent differences were observed (P > 0.2, single-factor ANOVA), suggesting that altered non-NMDA/NMDA receptor currents do not account for the LTP deficiencies seen in some of the tested strains.

Membrane depolarizations during a single 1-s burst of 100-Hz stimulation (Figs. 5C and 6D) were variable, but they were not significantly different among the tested strains (P > 0.2, single-factor ANOVA; Table 3). This result suggests that strain-specific reduction of postsynaptic depolarization during high-frequency presynaptic stimulation cannot explain the observed strain-dependent differences in LTP induction.

cAMP-induced synaptic facilitation is deficient in selected strains

Transgenic mice with reduced hippocampal PKA activity have defective maintenance of LTP (Abel et al. 1997). Are there strain-specific differences in cAMP-dependent synaptic facilitation? We explored this question by measuring the magnitudes of synaptic facilitation induced by chemical activation of the cAMP-PKA pathway (Chavez-Noriega and Stevens 1992). Transient co-application of the adenylate cyclase activator, forskolin, and the phosphodiesterase inhibitor, IBMX (50 µM each), elicited variable increases in fEPSP slope in some strains (Fig. 7). While 129/SvEms slices showed an increase in fEPSP slope that was not significantly different from BL/6J slices (BL/6J, 264 ± 37%; 129/SvEms, 212 ± 34%; P > 0.1), DBA and CBA slices showed significantly smaller synaptic facilitation than BL/6J slices (DBA: 123 ± 15%, P < 0.01; CBA: 152 ± 30%, P < 0.05). The forskolin/IBMX-induced facilitation in BL/6J slices was blocked by Rp-cAMPS (100 µM, an inhibitor of PKA) (Fig. 7A), thereby confirming that this form of synaptic facilitation is mediated by activation of PKA. Hence, these data point to a strain-selective deficit in PKA-dependent synaptic facilitation.



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Fig. 7. Forskolin/IBMX-induced synaptic facilitation is impaired in some strains. A: forskolin (FSK) and IBMX (50 µM each) elicited similar amounts of synaptic facilitation in BL/6J (n = 5 mice, 5 slices) and 129/SvEms slices (n = 6 mice, 6 slices). This facilitation was blocked in BL/6J slices (n = 5 mice, 5 slices) by pretreatment with RpcAMPS (100 µM), an inhibitor of PKA. B and C: forskolin/IBMX-induced facilitation was diminished in DBA (n = 6 mice, 6 slices) and in CBA (n = 6 mice, 6 slices) strains. Sample sweeps were recorded at t = 70 min.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Our study has revealed significant variations in the induction and maintenance of LTP in area CA1 of four selected strains of inbred mice. Hippocampal slices from CBA/J and DBA/2J mice showed less robust induction and maintenance of LTP than BL/6J slices following either tetra-burst high-frequency stimulation or theta-burst stimulation. Hence, defective induction and maintenance of LTP observed in mutant mice generated from CBA/J or DBA/2J strains may result from the genetic background of the parent strain rather than from the genetic manipulation per se.

The deficient induction of theta-burst LTP seen in DBA/2J, CBA/J, and 129/SvEms/J slices correlates with these strains' poor performance on the Barnes circular platform maze, a test of spatial learning and memory (Nguyen et al. 2000). LTP induced by theta-burst stimulation may be linked to the exploratory behavior of rodents in spatially novel environments, and theta patterns of electrical activity in the hippocampus can occur during such exploratory behavior (Bland 1986; Larson et al. 1986; Otto et al. 1991). Although we have demonstrated that maintenance of theta-burst LTP is strain-dependent, the exact mechanisms responsible for these strain-specific deficits are undefined. One possible cellular basis for the strain-specific variation in theta-burst LTP seen here is that strain- and activity-dependent differences in the strength or duration of feed-forward synaptic inhibition (Alger and Nicoll 1982; McCarren and Alger 1985) within the in vitro slice preparation may affect theta-burst LTP induction. Further research is needed to explore the role of synaptic inhibition in regulating LTP in these inbred strains.

Previous studies have shown differential expression of LTP in the dentate gyrus of specific inbred strains of anesthetized mice (129 Ola, C3H, C57 albino, DBA/2, and FVB/N strains: see Bampton et al. 1999). These studies used temporally compressed theta-pattern stimulation for longer periods of time than the regimen used in our present study. The study by Bampton et al. (1999), and our present findings, suggest that a strain-specific deficit in theta-burst LTP induction may exist in multiple distinct regions of the hippocampus both in in vitro and in vivo preparations. The collective results suggest that the learning and memory deficits reported for DBA/2 mice may be linked to defective theta-burst LTP in more than one sub-region of the hippocampus.

Although we observed no significant differences in LTD among slices of these four strains, it should be noted that we have only used one stimulation protocol to induce LTD (1 Hz, 15 min). This protocol was examined because of its reliability in producing measurable LTD at rodent hippocampal synapses (Dudek and Bear 1992). We did not examine the effects of different temporal patterns of low-frequency stimulation on LTD in these strains, since delivering the same number of stimulus pulses at higher frequencies may engage processes that elicit LTP (Dudek and Bear 1992) in a manner that may be dependent on other factors, such as kinase activation dynamics (see Mayford et al. 1995b).

Hippocampal synapses may be "tuned" to particular temporal patterns of electrical activity, and the subcellular processes responsible for activity-dependent plasticity at these synapses may be optimally engaged by select patterns of electrical activity. An important finding presented here is the observation that the temporal pattern of synaptic stimulation can critically modulate the induction and maintenance of hippocampal LTP in a strain-specific manner. Induction and maintenance of LTP were enhanced in 129/SvEms/J slices following repeated stimulation using 3-s interburst intervals, while the same total amount of imposed activity produced less robust induction and maintenance of LTP when a 20-s interburst interval was applied. In the other three strains tested, changing the interburst interval from 20- to 3-s, while keeping the total imposed activity constant, did not significantly affect the induction or maintenance of LTP. Extremely compressed stimulation patterns, such as the 3-s interval protocol used here, might be more effective (than spaced stimulation) at recruiting subcellular processes important for LTP induction. Our results indicate that such activity-sensitive processes may be expressed in a manner that is dependent on genetic background. In other words, the hippocampal neurons of some mouse strains may be more optimally tuned to particular temporal patterns of synaptic activity because of strain-related variations in genetic background. Our data also underscore the importance of using various temporal patterns of synaptic stimulation when searching for altered synaptic plasticity in genetically modified mice.

The biophysical membrane properties and cellular electrophysiological attributes of hippocampal CA1 pyramidal neurons did not significantly vary between the four strains examined here. Our data complement and further extend previous research that showed no marked strain-dependent variations in synaptic input-output coupling in area CA1 (Nguyen et al. 2000; see also Bampton et al. 1999 for dentate gyrus data). We observed no significant strain-specific differences in membrane input resistance, spike frequency accommodation, and membrane depolarization (during 100-Hz stimulation) in CA1 pyramidal cells. Thus, variation in genetic background among these four inbred strains does not appear to cause significant variations in membrane biophysical properties or spike firing efficacy of CA1 neurons in these strains.

Glutamatergic receptors, such as NMDA and non-NMDA receptors, are important for the induction of hippocampal LTP (Malenka and Nicoll 1993), but no significant strain-dependent variation in the sizes of synaptically evoked NMDA and non-NMDA currents was observed in the present study. These data are consistent with those reported by Jia et al. (1998), which showed that, for some inbred and hybrid strains (129 Sv, 129 Sv × C57BL/6, and 129 Sv × CD1), LTP of AMPA- and NMDA-type currents was independent of genetic background. We conclude that the genetic backgrounds of the strains examined here do not significantly alter the amplitudes of synaptically evoked glutamatergic currents as measured under the experimental conditions used here. However, there may be more subtle strain-dependent differences in the expression or modulation of these receptors which we have not examined in the present study.

The most intriguing finding of the present study is the lack of robust cAMP/PKA-dependent synaptic facilitation in CBA/J and DBA/2J slices. Forskolin enhances synaptic transmission in hippocampal area CA1 in a manner that requires adenylate cyclase activity (Chavez-Noriega and Stevens 1992). Co-application of forskolin with IBMX was used in the present study because such treatment increases the reliability of induction of robust synaptic facilitation in area CA1 (Chavez-Noriega and Stevens 1992). IBMX blocks adenosine receptors and inhibits phosphodiesterases in the hippocampus (Smellie et al. 1979). Both actions can increase cAMP levels, since tonic activation of adenosine receptors in area CA1 (Dunwiddie and Hoffer 1980) has been correlated with inhibition of adenylate cyclase (Chavez-Noriega and Stevens 1992; Dunwiddie and Fredholm 1989; Fredholm et al. 1983). Our block of forskolin/IBMX-induced synaptic facilitation by a specific inhibitor of PKA (Rp-cAMPS) confirms that this enhancement of synaptic transmission was mediated through PKA.

It is noteworthy that CBA/J and DBA/2J mice are defective in some forms of hippocampus-dependent spatial and nonspatial memory (Nguyen et al. 2000). PKA is critical for the maintenance of hippocampal LTP in area CA1 and for hippocampus-based memory consolidation (Abel et al. 1997). Our data suggest that the deficits in spatial and nonspatial memory seen in CBA/J and DBA/2J mice (Nguyen et al. 2000) may be correlated with deficient cAMP-induced (and PKA-dependent) synaptic facilitation in hippocampal area CA1. Ours is the first study to demonstrate natural, genotype-related variations in PKA-dependent forms of synaptic facilitation in the hippocampus. Further research is needed to determine whether this strain-specific deficit in PKA-dependent synaptic facilitation is correlated with altered hippocampal expression of particular PKA subunits (for preliminary data, see Duffy et al. 2000) or modified levels of hippocampal PKA activity.

In summary, our findings underscore the importance of careful selection of strains for use in generating genetically modified mice. These inbred strains should be screened for physiological phenotypes that may confound the interpretation of results derived from the electrophysiological analysis of genetically modified mice produced through the cross-breeding of these strains. Our study has shown that activity-dependent synaptic plasticity is significantly influenced by genetic background and by the particular temporal pattern of stimulation used for examining synaptoplastic mechanisms in hippocampal slices. Hippocampal synapses in particular strains, such as 129/SvEms/J, appear to be tuned to specific temporal patterns of synaptic activity. In contrast, hippocampal synapses in other strains, such as C57BL/6J, CBA/J, and DBA/2J, appear to be less susceptible to subtle changes in the temporal pattern of imposed synaptic activity. Pyramidal neurons in area CA1 of the four strains tested here showed no significant differences in membrane biophysical properties, spike firing efficacy, or glutamatergic synaptic transmission (as measured with whole-cell patch-clamp techniques). However, in some strains, cAMP-dependent synaptic facilitation was attenuated, suggesting that some of the observed strain-specific deficits in LTP may (directly or indirectly) result from altered signaling through the cAMP-PKA pathway. We suggest that genetic background can critically influence the efficacy or expression of intracellular synaptoplastic mechanisms involving signal transduction processes such as phosphorylation and dephosphorylation. These mechanisms may include components of specific kinase/phosphatase cascades, or substrate proteins regulated by, but located downstream from, these modulatory cascades. In contrast, the electrophysiological responses of synaptic glutamatergic receptors and membrane excitability appear to be less susceptible to significant alterations caused by variations in genetic background. Hence, strain-dependent variations in genetic background may selectively affect intracellular processes in hippocampal neurons, and alterations of some of these processes (e.g., cAMP-mediated signal transduction) may underlie some of the documented strain-dependent deficits in hippocampus-based learning and memory (Crawley et al. 1997; Nguyen et al. 2000; Paylor et al. 1993; Wehner et al. 1990).


    ACKNOWLEDGMENTS

P. V. Nguyen held Scholarship awards from the Canadian Institutes of Health Research (CIHR) and the Alberta Heritage Foundation for Medical Research (AHFMR). S. N. Duffy is a Postdoctoral Fellow of the AHFMR.

This work was supported by grants and funds (to P. V. Nguyen) from the AHFMR, the CIHR, and the University of Alberta Faculty of Medicine.


    FOOTNOTES

Address for reprint requests: P. V. Nguyen, Dept. of Physiology, University of Alberta School of Medicine, Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada.

Received 26 April 2000; accepted in final form 4 August 2000.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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