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NEUROSCIENCE |
1 Department of Physiology and Biophysics Program, University of Wisconsin - Madison, 1300 University Ave, Madison WI 53706, USA
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Abstract |
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(Received 23 April 2006;
accepted after revision 24 July 2006;
first published online 27 July 2006)
Corresponding author M. B. Jackson: Department of Physiology, University of Wisconsin - Madison, 1300 University Ave, Madison, WI 53706, USA. Email: mjackson{at}physiology.wisc.edu
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Introduction |
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Different synaptic pathways generally have different forms of plasticity with fundamentally different properties. For a given pathway within a particular region, one might expect synapses to exhibit uniform behaviour. This hypothesis has yet to be thoroughly tested, but recordings from different locations indicated that LTP induced by high-frequency stimulation in the stratum radiatum (SR) tended to be much weaker near the stimulation site (Kauer, 1999). This could reflect the local action of inhibitory interneurons, which are activated together with the long-range excitatory Schaffer collaterals and commissural fibres. Voltage imaging can reveal spatial heterogeneity over a wide area, and thus provide insight into how hippocampal circuitry regulates the expression of activity-induced changes in function. The present study used voltage imaging to investigate the spatial distribution of LTP in the CA1 region of the hippocampal slice, and to relate this distribution to the spread of spikes and EPSPs evoked by electrical stimulation. We found that LTP expression varied within a slice. This spatial heterogeneity bears little resemblance to the distribution of responses to single shocks, but depends on which pathway was activated and on whether the induction stimulus elicited postsynaptic spikes.
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Methods |
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Male Sprague-Dawley rats (3–5 weeks old) were rendered unconscious by elevating levels of CO2, and decapitated, in accordance with the guidelines of the National Institutes of Health, as approved by the Animal Care and Use Committee of the University of Wisconsin - Madison. The brains were then removed and chilled in ice-cold cutting solution consisting of (mM): 124 NaCl, 3.2 KCl, 26 NaHCO3, 1.25 NaH2PO4, 1 CaCl2, 6 MgSO4, and 10 glucose, bubbled with 95% O2–5% CO2. Horizontal hippocampal slices 350–400 µm thick were cut with a tissue slicer (HR2, Sigmann Elektronik, Germany), selecting sections midway along the dorsal–ventral axis. A cut at the CA2 region was made to prevent epileptiform activity. Slices were stored in a vial of bubbled artificial cerebrospinal fluid (aCSF) at 29°C for 1 h. aCSF was identical to the cutting solution but contained 2.5 mM CaCl2 and 1.3 mM MgSO4. Slices were stained with bubbled aCSF containing 0.02 mg ml–1 of the voltage-sensitive absorbance dye RH482 (NK3630, Hayashibara Biochemical Laboratories, Okayama, Japan) (Momose-Sato et al. 1999; Chang & Jackson, 2003) for 1 h at 29°C, and returned to aCSF for later use. During recording, hippocampal slices were continuously perfused with bubbled aCSF in a submerged recording chamber and maintained at 29–31°C. (±)-2-amino-5-phosphonovaleric acid (APV) was applied by bath application in some experiments to test the role of N-methyl-D-aspartic acid (NMDA) receptors. All chemicals (other than RH482) were purchased from Sigma.
Electrophysiology
Stimulating and field potential (FP) recording electrodes were made from borosilicate glass capillaries (1.15 mm ID, 1.50 mm OD) filled with aCSF. Stimulating electrodes had 10–30 µm tips; recording electrodes had resistances of 2–5 M
. Slices were stimulated with 200 µs current pulses delivered by a monopolar constant-current stimulus isolator (Model A365, World Precision Instruments, Sarasota, FL, USA) to the SR every 30 s. The recording electrode was placed in the middle of the SR, 250–400 µm away from the stimulating electrode. A current pulse was applied 50 ms after the beginning of data acquisition. The theta burst stimulation (TBS) protocol used to induce LTP included three theta bursts (TB) separated by 10 s. Each TB consisted of 10 bursts at 5 Hz and each burst consisted of six pulses at 100 Hz. This induction protocol approximates that used to induce LTP 2, which has been shown to depend on IP3 receptor-sensitive Ca2+ stores (Raymond & Redman, 2006). Field potentials were recorded with an Axopatch 200B amplifier (Axon Instruments).
Voltage imaging and data analysis
The instrumentation follows Wu & Cohen (1993) and is very similar to the commercial photodiode array system Neuroplex-II from RedShirtImaging LLC (Fairfield, CT, USA) (Jackson & Scharfman, 1996; Demir et al. 1998). A set of 464 optical fibres was bundled into a hexagonal array, and each fibre was coupled to a Hamamatsu photodiode. Signals were amplified to 5 V nA–1 of photocurrent, low-pass filtered with a four-pole Bessel filter at 500 Hz, multiplexed, and digitized at a frame rate of 1.63 kHz with eight expansion boards (MSXB 002-02, Microstar Laboratories, Bellevue, WA, USA) and a DAP3200e/214 data acquisition board. Data were acquired in 500 ms segments, and traces were displayed as either single trials or averages of up to four trials.
An upright Reichert Jung Diastar microscope (Leica, Deerfield, IL, USA) was illuminated from below with a 100 W tungsten–halogen bulb driven by a Kepco ATE 36-30 DM power supply (Flushing, NY, USA). Illuminating light passed through a 702 ± 18 nm bandpass filter (Omega, Brattleboro, VT, USA), and transmitted light was collected with a 10 x Olympus UPlanApo objective (NA = 0.40). The centre-to-centre distance between neighbouring photodiode fields was 67 µm. An electronically controlled shutter was opened 200 ms before the start of data acquisition. Optical signals 
I/I (where I is recorded light intensity) were presented as the change in transmitted light divided by the resting light intensity I/I measured before LTP induction. A movable mirror directed images to a CCD camera; images were captured with a frame grabber (Data Translation, Marlboro, MA, USA).
Data acquisition, signal processing and analysis were performed with a Pentium 4 computer running a program written in C++. Additional analysis and plotting were performed with the computer program Origin (Microcal, Northampton, MA, USA). We used a 3-point binomial temporal filter, in which signal amplitude S(t) was replaced by S(t – 1)/4 + S(t)/2 + S(t +1)/4, to reduce noise in the traces. The baseline drift of each signal was corrected by fitting the baseline to a third-order polynomial and subtracting this function; the baseline was taken as the time before the stimulus and 150 ms after the stimulus. Stimulus artifacts were removed from displayed FP recordings. Peak signals were used to create colour-encoded maps of responses and of LTP. The colours in pixels between centres of photodiodes were linearly interpolated. A Gaussian spatial filter with a space constant of half the interphotodiode distance reduced noise in these maps. LTP was defined as the increase in peak responses of optical signals. LTP data were only used if the pre-induction baseline remained stable for 20 min or more.
Analysis of optical EPSPs
The optical signals reported here generally consisted of EPSPs and spikes. In order to assess the magnitudes of these two contributions, we fitted the EPSP component of an optical signal (oEPSP) to a computer-simulated function. The optical signal, I/I, is proportional to the membrane potential change within the physiological range of voltages (Wu & Cohen, 1993). Therefore:
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Vm is the deviation of the membrane potential from rest, averaged over all the cell membrane at a particular location, and ß is a constant. The membrane potential, Vm(t), is then determined by numerical integration of the differential equation:
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-function.
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| (3) |
are free parameters. The simulated EPSP depends on , the product of ß and Gsyn, and the ratios of Cm to gleak and of Gsyn to gleak. Varying Gsyn/gleak in the range of 0.01–20.0 has little impact on the time course of Vm(t), so we fixed it at a constant value of 1.0. The best fit was determined by varying , ßxGsyn, and Cm/gleak to minimize the root-mean-square error (see Fig. 1E). A narrow window was set around the spike to exclude this part of the record from the fit of the oEPSP. The spike amplitude was taken as the peak amplitude minus the amplitude of the oEPSP at that time point.
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Results |
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Stimulation electrodes were positioned in the SR as indicated in Fig. 1A. A single pulse evokes optical signals over a broad area within the CA1 region of a slice (Fig. 1B), with the largest responses in the SR and stratum oriens (SO) (Fig. 1B and Ci). Although the voltage-sensitive dye stains the membranes of pyramidal cells, interneurons and glia cells, we attribute the optical signals primarily to voltage changes in pyramidal cells for the following reasons. In every instance optical signals were consistent with FP recordings from the same site. Figure 1E shows an example (compare FP with site 1), and this general parallel held in a large number of experiments. FP recordings in different regions of hippocampal slices are widely interpreted in terms of voltage changes in pyramidal cells (Andersen et al. 1980a). Interneurons are not expected to contribute significantly because of their lower density; pyramidal cells outnumber interneurons by an estimated ratio of 10 : 1 (Traub et al. 1999). Glia cells are numerous, but the dye we used, RH482, preferentially stains neurons over glia cells (Konnerth et al. 1987; Kojima et al. 1999). Furthermore, evoked responses in hippocampal glia cells have much smaller amplitudes and slower time courses as demonstrated by patch clamp recording (Bergles & Jahr, 1997), field potential recording (Diamond et al. 1998), and voltage imaging (Kojima et al. 1999).
Stimulation of the SR with theta bursts induced enduring increases in the magnitudes of both optical signals and FPs. Figure 1D shows plots of the time course of the slope of the field EPSP (fEPSP) and of the optical signals at four different locations (as pictured in Fig. 1A). After TBS, the amplitudes of the optical signals at all four locations increased, reaching a plateau within 10 min, and then remaining above baseline for the duration of the experiment (50 min), although the optical signal at the stimulation electrode (SE – location 3) declined slightly before stabilizing. The fEPSP slope and the optical signal at the same site both showed potentiation (location 1). Thus, the voltage-sensitive dye provides a signal that allows us to evaluate LTP throughout a slice.
Figure 1Ci shows a colour-encoded intensity map of the initial peak response to a single pulse, and Fig. 1Cii shows the map of peak responses to the same stimulus current 50 min after TBS. The post-TBS intensity map clearly shows larger responses extending over a wider area. Subtracting the peak intensity before TBS from that after TBS renders a map of LTP (Fig. 1Ciii). This map shows a spatially complex distribution, with seven distinct foci (white arrows). Figure 1Ci–Ciii illustrate a major finding of this study, that LTP shows a complex non-uniform spatial distribution, which bears little resemblance to the distribution of single-pulse responses.
The foci of strong LTP in the spatial map of Fig. 1Ciii can be highlighted by plotting the peak response amplitude along contours through a slice. A plot of response amplitude along the pyramidal cell somato-dendritic axis before TBS showed that responses were strongest in the SR (Fig. 2A, top, open squares). Responses in the SO were weaker, and the stratum lacunosum-moleculare (SLM) had the weakest responses. The weak responses in the SP cannot be compared with those in neighbouring SO and SR because the cell body layer has a lower density of plasma membrane, and the density of dye is therefore also lower. With a smaller fraction of light absorbed by dye the optical signals emanating from the SP will be reduced (Chang & Jackson, 2003), but the extent of this reduction is difficult to quantify. After TBS the responses grew over the entire contour (Fig. 2A, top, filled circles). Taking the difference between the pre- and post-TBS contours (Fig. 2A, below, filled triangles) showed that LTP was greatest in the SO, smaller but pronounced in the SR, and barely visible in the SLM.
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LTP of optical signals depends on NMDA receptors
LTP detected optically depended on the activation of NMDA receptors. In the presence of the NMDA receptor antagonist APV (50 µM), electrical stimulation evoked large responses that spread over considerable distances (Fig. 3Aii), but TBS produced no LTP (Fig. 3Aiii). APV was then removed and the same TBS protocol induced LTP (Fig. 3Aiv). Figure 3B illustrates these results by plotting the time course of responses at two locations. Similar results were obtained in seven slices, and these experiments demonstrate that the LTP observed with voltage imaging shows the expected dependence on NMDA receptors.
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Different stimulus currents produced qualitatively different spatial patterns of LTP expression. With 20 µA (n = 6), only the area around the SE expressed LTP (Fig. 4A, right), and this simple spatial pattern was seen in all six slices tested with this weak stimulus. The larger potentiation in the SO appeared primarily in the spike component (data presented below), indicating that neurons close to the SE became suprathreshold for action potentials after TBS. Similar results were seen with 40 µA (n = 7). LTP expression remained confined to the region around the SE, but the area was wider (Fig. 4B, right).
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With 80 µA (n
= 9), TBS produced a heterogeneous spatial pattern of LTP expression (Fig. 4D, right). Regions showing more LTP generally were found within a few hundred microns of the SE or near the CA2 region, which was usually 
1 mm from the SE. Thus, increasing the stimulus intensity did not simply enlarge the area of LTP expression, but increased the complexity of the LTP map and changed the qualitative appearance of the potentiated regions. For all stimulation intensities tested, areas with strong responses (Fig. 4, middle panels) and strong LTP expression (Fig. 4, right panels) were usually confined to the SR and the SO. The SLM showed weak responses and essentially no LTP.
Counting the number of peaks in the maps of LTP and plotting this number versus stimulus current shows the increase in the spatial heterogeneity of LTP (Fig. 4E). Peaks in LTP maps were generally taken as regions of at least three photodiodes where the peak signal was at least 6% higher than that of surrounding photodiodes. The total number of peaks increased with stimulus strength and reached a plateau at about five. (The dependence on stimulus was significant by one-way ANOVA; P < 0.005.) The increase in number of peaks was evident in both the SO and SR. Peaks in the SO and SR were generally in register (i.e. at mirror locations on either side of the SP), and this parallel has a simple explanation that neurons expressing strong LTP in the SR usually generated action potentials that invaded the basal dendrites in the SO.
Contribution of spikes to LTP
Spikes and EPSPs both contribute to optical signals, and the appearance of a rapid spike on top of the broader oEPSP corresponds closely with the appearance of the population spike inflection in a FP recording from the same site (Fig. 1E, compare FP top traces with optical traces from location 1). Spikes in the dendritic regions are generally somewhat broader than in the cell body layer, and the durations of dendritic spikes in our recordings of 3–4 ms are very similar to those recorded by patch clamping dendrites (Magee & Johnston, 1995; Spruston et al. 1995; Magee, 1999). By decomposing optical signals into these two basic components (see Methods) we can evaluate their roles in the LTP observed at different locations. (It should be noted that the spikes are postsynaptic as they always appear on top of the oEPSP. In the SR we can usually see a presynaptic axonal volley just before the onset of the oEPSP; data not shown.) To examine the contributions of spikes and oEPSPs we selected representative sites for detailed analysis from five experiments in which LTP was induced with 60 µA, a stimulus intensity strong enough to induce a spatially varied pattern of LTP (Fig. 4). In the SR, we chose one location at the SE, a second location 270 µm away (4 x the photodiode spacing of our detector), and a third location where LTP expression was maximal. These three locations were complemented by three locations in the SO directly across the SP from the locations selected in the SR.
For each location in the SR (Fig. 5A) and the SO (Fig. 5B), the overall signal, the spike component, and oEPSP component all increased significantly, except for the EPSP at the stimulation site in the SR. The changes in each parameter associated with TBS-induced LTP are shown in Fig. 5C and D. In general, spikes increased more than oEPSPs at locations expressing the greatest increase in overall signal. These results indicate that either the small changes in oEPSP amplitude bring a large number of cells above the threshold for action potentials, or that a change in dendritic excitably also contributes to the increase in spike amplitude (Daoudal et al. 2002; Wang et al. 2003; Frick et al. 2004; Fan et al. 2005). Conversely, at locations where LTP was weaker, the differences between the amplitude increases of spikes and oEPSPs were smaller and not significant. These results reflect a general trend that at sites showing the greatest LTP, spikes made the largest contribution.
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One form of spatial variation consistently seen with strong stimulation intensities was suppression of LTP near the stimulating electrode. To determine if this was due to saturation of responses, we varied the stimulation intensity and examined the responses near the SE and 270 µm away, in the SR and SO. Near the SE in the SR, the total response was close to saturation with stimulation intensities above 50 µA, while responses at the other three locations continued to increase with stronger stimulation (Fig. 6A). Decomposing responses into spikes and oEPSPs showed that both components increased with stimulus current, and reached a plateau as the responses saturated. The response curves of spikes were sigmoidal (Fig. 6B), reflecting the cooperative activation of action potentials by synaptic inputs. By contrast, the stimulus–response curves of oEPSPs were hyperbolic, with no sigmoidal character (Fig. 6C). Both oEPSPs and spikes near the SE saturated at lower stimulus currents than at more distant locations. Thus, near the site of stimulation, strong stimulus currents produce saturating responses that appear to occlude LTP, but 270 µm away the less than saturating responses could still be potentiated. This may be relevant to aspect of the spatial heterogeneity in the LTP maps such as Figs 1Ciii, and 4C and D, but leaves unexplained the peaks and valleys seen at greater distances from the site of stimulation.
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The spatial heterogeneity of LTP could arise from the specific circuitry within a hippocampal slice or from a subtle spatial variation in the condition of the tissue. The uniformity in condition is a concern for work in a brain slice, where tissue recovers from the trauma of cutting. To address this possibility, we performed experiments with two stimulating electrodes. In these experiments, SE1 (red arrow in Fig. 7) was in the inner third of the SR (one third the distance between the SP and border with SLM), and near the subiculum. We placed SE2 (yellow arrow in Fig. 7) 600 µm away in the SR, from half-way to two-thirds of the distance to the SLM, and close to the CA2 region (Fig. 7A). In view of the parallel trajectories of SR axons (Andersen et al. 1980a), this positioning insured the stimulation of different pathways. Stimuli were applied alternately to SE1 and SE2 every 30 s (50 µA) and Fig. 7Bi and Ci display the spatial distributions of these two responses. TBS applied first to SE1 potentiated responses evoked by SE1 but not responses evoked by SE2 (Fig. 7Bii and Cii). One hour after the TBS at SE1, TBS applied to SE2 potentiated responses at sites where potentiation by SE1 failed. Figure 7Ciii displays the map of LTP induced by SE2, and Fig. 7Biii shows that responses evoked by SE1 were unchanged. The time course of responses illustrates these results by showing a location where the response to SE1 was potentiated by the first TBS at SE1 but not by the second TBS at SE2 (Fig. 7D, location 1). Likewise, responses at a second location show no change after the first TBS but were potentiated by the second (Fig. 7D, location 2). Thus, LTP expression was pathway-specific, with no detectable increases in responses evoked by stimulation at a site different from the site to which TBS was applied.
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Spikes during TBS predict the spatial extent of LTP
Postsynaptic depolarization serves as a critical signal in the induction of LTP and postsynaptic action potentials play an essential role in providing this depolarization (Magee & Johnston, 1995; Spruston et al. 1995; Bi & Poo, 1998; Linden, 1999; Bi & Poo, 2001). We therefore examined the relation between the spatial distribution of LTP and the spatial distribution of spikes elicited by the TBS during induction. Figure 8 shows the results for two locations that failed to express LTP in response to TBS (location 1 in the SR and 2 in the SO) and two locations that showed potentiation (location 3 in the SR and 4 in the SO). Superimposed signals recorded 2 min before and 1 h after induction show how TBS affects the responses from each of these locations (Fig. 8B). The FP and the optical signal recorded at the same location did not change after TBS (Fig. 8B and D, FP and location 1). Signals at the adjacent location in the SO also did not change (Fig. 8B and D, location 2). The first pulse train of each TB failed to evoke spikes in the optical signals at locations 1 or 2, or in the FP recordings from location 1 (Fig. 8C). During the second and the third TB both the FP and the optical signals increased, but only transiently, and returned to baseline within 2 min. For the two locations expressing LTP in the SR and the SO (Fig. 8B, locations 3 and 4, respectively), spikes could be clearly identified during the first pulse trains of the second and the third TB (Fig. 8C). Analysis of optical signals at these two locations revealed that spikes contributed substantially to the amplitude increases (Fig. 8D, locations 3 and 4). We examined signals from several photodiodes in 76 different slices from 26 animals, and found that whenever an area showed LTP above a threshold of 6% of the maximum for that slice, spikes were always evident during TBS at those locations. Regions where spikes could not be seen during TBS induction generally failed to express LTP above our detection threshold.
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Discussion |
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Spread of oEPSPs and spikes
Optical signals observed by voltage imaging reflect a sum of voltage changes contributed by spikes and EPSPs (Figs 1E and 8B) (Grinvald et al. 1982; Kasuga et al. 2003). FP recordings from the same site show the same components, and intracellular voltage recordings in this preparation are consistent with these two forms of population activity (Golding & Spruston, 1998; Gasparini et al. 2004). With weak stimulation in the middle of the SR, EPSPs dominated the optical signals. Locally evoked EPSPs in the SR were largely restricted to this layer, with little spread to dendrites in the adjacent SLM and SO. Thus, stimulation of Schaffer collateral/commissural fibres predominantly activated the proximal apical dendrites of pyramidal cells within this stratum (Andersen et al. 1980a). Stronger stimulation evoked larger EPSPs and spikes in the SR and the SO, but the responses in the SLM remained weak. Action potentials do not propagate effectively to the remote parts of apical dendrites in CA1 pyramidal cells (Spruston et al. 1995; Magee & Johnston, 1995). The distal apical dendrites have higher densities of various potassium channels (Magee, 2000; Reyes, 2001), and these could limit the spread of voltage changes into the SLM. Since dendrites in the SLM receive inputs from the entorhinal cortex through the temporoammonic pathway and dendrites in the SR receive inputs from Schaffer collaterals and commissural fibres, the electrical segregation of the distal and proximal apical dendrites suggests that these two compartments can process their respective inputs with some degree of independence (Magee, 1998; Remondes & Schuman, 2002; Bernard & Johnston, 2003; Nolan et al. 2004; Ang et al. 2005).
At locations expressing strong LTP, the spike component showed an increase that was significantly greater than the increase evident in the oEPSP. The increase in the spike component may indicate that a greater fraction of the postsynaptic cells at a location are driven past their firing threshold, or, alternatively, that the firing of cells at that location becomes more synchronous. With a dye that senses membrane potential, crossing the action potential threshold and generating a spike gives rise to a larger change in the optical signal than does a graded increase in an EPSP. However, in some experiments, increases in spike amplitude in the SR were not accompanied by detectable increases in the amplitude of oEPSPs, suggesting that in these cases the potentiation arose primarily from the modulation of voltage-gated channels (Daoudal et al. 2002; Wang et al. 2003; Frick et al. 2004; Fan et al. 2005). Regardless of the origin of the increase in spike signals, it is significant that TBS-induced LTP allows the same stimulus current to produce a larger population spike, indicating either increased spike synchrony or firing of a greater number of postsynaptic cells (Andersen et al. 1980b; Taube & Schwartzkroin, 1988; Chavez-Noriega et al. 1990).
Role of spikes in the induction of LTP
LTP depended critically on the generation of postsynaptic spikes during TBS. Areas without detectable spikes during TBS failed to express stable LTP. These observations support the hypothesis that LTP depends on postsynaptic spikes during induction. Regions near the stimulus electrode generated spikes during TBS without subsequent LTP, but this probably reflected the occlusion of LTP, either through saturation or some other mechanism. Elsewhere, the appearance of postsynaptic spikes during induction faithfully predicts the expression of LTP. Postsynaptic spikes provide postsynaptic depolarization, which enables Ca2+ to enter a cell and activate signalling cascades that modify synaptic strength and excitability (Grover & Teyler, 1990; Bliss & Collingridge, 1993; Malenka & Bear, 2004). Back-propagating action potentials play critical roles in providing this postsynaptic depolarization (Magee & Johnston, 1995; Spruston et al. 1995), but spikes originating in dendrites can also perform this function (Golding et al. 2002; Lisman & Spruston, 2005). Although in the present data we cannot distinguish dendritic spikes from back-propagating action potentials unequivocally, our results strengthen the case for an essential role of postsynaptic spikes in LTP (Magee & Johnston, 1997; Markram et al. 1997; Pike et al. 1999; Linden, 1999; Bi & Poo, 2001; Golding et al. 2002). The correlation between postsynaptic spikes and LTP thus suggests that the spatial complexity is a manifestation of a requirement for concurrent pre- and post-synaptic firing, as postulated by Hebb.
Spatial heterogeneity of LTP
The distribution of LTP in the CA1 region revealed several forms of spatial heterogeneity. Comparing different layers, we detected strong LTP in the SR and SO but not in the SLM. The absence of LTP in the SLM clearly results from the failure of evoked responses to spread to this layer, and indicates that even after the potentiation of synapses in the SR, the SLM remains functionally isolated (as discussed above). While responses in the SO are stronger, the large signal increases there cannot be attributed to the strengthening of synapses on basal dendrites. Since the activated Schaffer collateral/commissural fibres predominantly innervate dendrites in the SR (Andersen et al. 1980a), the increased signals in the SO probably resulted from more effective transmission of EPSPs and spikes from the apical dendrites into and through the cell bodies in the SP. The increase in the spike component in the SO could reflect both more pyramidal cells generating action potentials and increases in the amplitudes of dendritic action potentials. These changes would originate in the SR dendrites in the vicinity of the activated synapses, triggered by localized Ca2+ changes in dendritic shafts (Raymond & Redman, 2006). It will be interesting to test induction protocols that produce more extensive Ca2+ rises to see if this can potentiate synapses and dendrites over greater distances.
Increasing the stimulation intensity altered the spatial pattern of LTP (Fig. 4). Variations in stimulus frequency and in the statistical properties of stochastic stimuli have also been shown to influence the spatial pattern of both LTP and LTD in the CA1 region (Aihara et al. 2005). We found that increasing the stimulus current enlarged the size of the potentiated region and converted the map from a focus in the SR around the SE and an adjacent focus in the SO to a complex distribution with several local maxima. Strong stimulation saturated responses near the SE and occluded LTP. Although the release of a local extracellular signal that blocks LTP expression remains a possibility (Kauer, 1999; Aihara et al. 2005), saturation of both spikes and EPSPs is clearly an important factor in the suppression of LTP near the stimulating electrode. Thus, the evaluation of a contribution from local circuits will depend on controlling for saturation circuits and inhibitory interneurons are more likely to play a role in the other spatial features in the complex LTP maps. Although we attempted to address the role of inhibition by blocking GABAA receptors with picrotoxin or bicuculline, the resulting multi-spike activity made analysis and interpretation of the optical signals more difficult. Nevertheless, this remains an important possibility that we hope to address with pharmacological tools. It would be especially interesting to use pharmacological or genetic methods to target a specific group of interneurons, such as calbindin-containing bistratified interneurons, which have dendritic and axonal arbors in the SR (Dingledine et al. 1987; Freund & Buzsaki, 1996).
Since the LTP map so closely resembles the spike map during TBS, elucidating the mechanism underlying the spatial complexity of LTP may reduce the problem of determining how postsynaptic spikes are regulated and what forms of short-term facilitation allow synaptic inputs to bring cells above threshold. It is important to bear in mind that the spatial heterogeneity of LTP was not matched by the spatial heterogeneity in the response to a single shock applied prior to TBS. Thus, the distant local maxima in the distribution of LTP reflect the distribution of some other signal. The lack of overlap of LTP distributions for different stimulus sites in the same slice (Fig. 7) indicates that this other signal depends on TBS, and that it is not an ambient property of the slice. One possibility is that the distribution of LTP reflects the activity of another circuit parallel to the Schaffer collateral/commissural fibre pathway with a sparse and non-uniform projection field. This circuit would then facilitate the primary excitatory pathway during TBS to enable postsynaptic cells to spike and thus initiate potentiation.
The heterogeneous spatial distribution of LTP in the CA1 region of rat hippocampal slices implies an organization of hippocampal circuits specifically associated with synaptic plasticity. This organization could be related to the columns of neurons in the primary somatosensory cortex (Woolsey & Van der Loos, 1970; Petersen et al. 2003; Sun et al. 2006) and the primary visual cortex (Hubel & Wiesel, 1962; Grinvald et al. 1984; Bonhoeffer & Grinvald, 1991; Ohki et al. 2005). It is also possible that different ensembles of neurons, such as the ensembles responsible for theta and the gamma oscillations (Chrobak et al. 2000; Buzsaki, 2002; Remondes & Schuman, 2002), are activated and potentiated during TBS. The architecture revealed by the spatial heterogeneity in TBS-induced LTP may be an essential element in the selective modification of specific subsets of neural circuits during the storage of information by the nervous system.
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) and after (•) TBS. Bottom, profile of amplitude increases (LTP). B, profiles of peak amplitudes and amplitude increases along a contour through the SR. Top, profiles of responses before and after TBS. The black arrow indicates the stimulation site. Bottom, profile of amplitude increases, with three local maxima indicated by white arrows (as in 
