Transient Synaptic Potentiation in the Visual Cortex. I. Cellular Mechanisms

Krisztina Harsanyi and Michael J. Friedlander

Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Harsanyi, Krisztina and Michael J. Friedlander. Transient synaptic potentiation in the visual cortex. I. Cellular mechanisms. J. Neurophysiol. 77: 1269-1283, 1997. The cellular mechanisms that underlie transient synaptic potentiation were studied in visual cortical slices of adult guinea pigs (>= age 5 wk postnatal). Postsynaptic potentials (PSPs) elicited by stimulation of the white matter/layer VI border were recorded with conventional intracellular techniques from layer II/III neurons. Transient potentiation (average duration 23 ± 3 min, mean ± SE) was evoked by 60 low-frequency (0.1 Hz) pairings of weak afferent stimulation with coincident intracellular depolarizing pulses (80 ms) of the postsynaptic cell. Fifty-one percent (47 of 92) of the pairing protocols led to significant enhancement (+26 ± 3%) of the PSP peak amplitude. Blockade of action potential output from the recorded neuron during pairing with Lidocaine, N-ethyl bromide quaternary salt in the recording micropipette did not reduce the likelihood of potentiation (7 of 14 protocols = 50%). Thus transient synaptic potentiation does not require action potential output from the paired cell or recurrent synaptic activation in the local cortical circuit. Rather, the modification occurs at synaptic sites that directly impinge onto the activated neuron. Intracellular postsynaptic blockade of inhibitory PSPs only onto the paired cell with the chloride channel blocker 4,4'-dinitro-stilbene-2,2'-disulfonic acid and the potassium channel blocker cesium in the micropipette also did not reduce the likelihood of induction of potentiation (6 of 9 protocols = 67%). These results suggest that the potentiation is due to a true up-regulation of excitatory synaptic transmission and that it does not require a reduction of inhibitory components of the compoundPSP for induction. Chelation of postsynaptic intracellular calciumwith 1,2-bis-2-aminophenoxy ethane-N,N,N',N'-tetraacetic acid (BAPTA) in all cases effectively blocked the induction of potentiation (no change in the PSP, 9 of 13 protocols; induction of synaptic depression, 4 of 13 protocols), suggesting that a rise in the intracellular postsynaptic calcium level is critical for the pairing-induced synaptic potentiation to occur. Bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (APV) reversibly blocked potentiation of the PSP peak amplitude in most cells (14 of 16) that were capable of significant potentiation in control solution. Blockade of nitric oxide production with bath application of the competitive inhibitor of nitric oxide synthase, L-nitro-arginine (LNA), did not significantly affect the likelihood of synaptic potentiation (11 of 20 cells). It did, however, block subsequent enhancement for several cells (2 of 4) that had previously had their inputs potentiated. Moreover, LNA increased the overall average magnitude of synaptic potentiation (with an additional +28%) when induction was successful. These results suggest that endogenous cortical nitric oxide production can both positively and negatively modulate this NMDA receptor-mediated type of synaptic plasticity.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The mammalian visual cortex is capable of profound structural and functional reorganization in response to altered patterns of sensory input during a critical period of postnatal development (Hubel and Wiesel 1970; Wiesel 1982). Manipulations such as imbalanced binocular vision in neonates cause dramatic structural modification in synapses (Friedlander et al. 1991; LeVay et al. 1978) that may contribute to the functional shift in ocular dominance of cortical neurons. In recent years, the capacity for functional reorganization of the visual cortex (Chino et al. 1992; Gilbert and Wiesel 1992; Kaas et al. 1990) and the cortical representation of other sensory modalities (somatosensory cortex: Allard et al. 1991; Merzenich et al. 1988; Recanzone et al. 1990; Wall et al. 1992; auditory cortex: Ahissar et al. 1992; Edeline et al. 1993; Weinberger et al. 1993) in older animals has also been well documented.

The capacity to rapidly and dynamically alter synaptic throughput in the cerebral cortex (Brasil-Neto et al. 1992; Edeline et al. 1993; Kapadia et al. 1994; Pettet and Gilbert 1992) has been suggested to contribute to early stages of sensory memory formation (Ishai and Sagi 1995) as well as functional reorganization in response to injury (Darian-Smith and Gilbert 1994; Garraghty and Kaas 1991). Although the long-lasting changes that underlie functional reorganization, such as ocular dominance column shifts after early postnatal monocular visual deprivation, are largely attributed to anatomic changes, more rapid and dynamic alterations in signaling efficiency in cortical networks are thought to result from modification of the strength of existing synaptic connections (Ahissar et al. 1992; Recanzone et al. 1990). Thus it is of interest to understand the algorithms and cellular mechanisms whereby imposed patterns of activity can rapidly and transiently modify the efficiency of pathways within the sensory cerebral cortex.

Fregnac et al. (1988, 1992) and Shulz and Fregnac (1992) showed that brief periods of imposed low-frequency covariance of pre- and postsynaptic activity in vivo can alter the effective properties of visual cortical neurons in both kittens and adult cats. With the use of an in vitro cortical slice preparation, we demonstrated (Fregnac et al. 1994) that 1) this type of transient synaptic potentiation can occur in visual cortex of mature guinea pigs; 2) it is spatially specific, modifying only the particular synaptic pathway on the cell that participated in the pre/postsynaptic pairing; 3) it is reversible and it can repeatedly be induced in the same cell; and 4) it only occurs if synaptic activation and postsynaptic depolarization temporally overlap, consistent with the Bienenstock, Cooper and Munro model of synaptic plasticity (Bienenstock et al. 1982). The cellular mechanisms that underlie this form of synaptic plasticity in the mature visual cortex, however, have not been explored.

In addition to serving as a potential substrate for rapid and dynamic modulation of the efficiency of cortical circuits, the protocol leading to transient synaptic potentiation has several distinct experimental advantages. Because it requires simultaneous pre- and postsynaptic activity and only a weak low-frequency synaptic input along with postsynaptic depolarization for its induction, potentiation can be induced in a single neuron in a cortical slice and does not require high-frequency stimulation of afferents or pharmacological intervention such as bath application of blockers of synaptic inhibition. Moreover, because the potentiation is reliably induced repeatedly in the same synaptic pathway in a neuron and is transient in nature (Fregnac et al. 1994; Friedlander et al. 1993), a number of experimental protocols can be reversibly induced in the same cell to test the underlying molecular mechanisms.

In the present study we evaluated the cellular mechanisms of this type of transient synaptic plasticity in mature animals. The visual cortex of the relatively precocious species, the guinea pig, was used from animals >5 wk of age. Tricolored guinea pigs were used because these animals do not have the anomalous visual anatomic projection of albinos (Cucchiaro and Guillery 1984), they have a topographically organized visual cortex (Spatz et al. 1991) and laminar distribution of cell types and projection (see Fig. 1) similar to those of other mammalian species, and the capacity of their synapses to undergo long-term (Abraham et al. 1987; Zalutsky and Nicoll 1990) and short-term plasticity (Fregnac et al. 1994; Friedlander et al. 1993) is well established. Properties evaluated include determination of where in the cortical network the synaptic modification occurs, determination of the specific synapse types that are modified by selective intracellular blockade of inhibitory synaptic inputs, and the role of N-methyl-D-aspartate (NMDA) receptor activation, postsynaptic intracellular calcium, and nitric oxide (NO) production in the induction process. Preliminary accounts of these results have been presented (Harsanyi and Friedlander 1993-1995).


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FIG. 1. Cortical location of neurons included in the study. Top: guinea pigs (n = 3) were injected intraocularly with wheat germ agglutinin-horseradish peroxidase (5 µl, 5%, see METHODS). Animals were killed 7 days after the injection. The marker was transported transneuronally to the primary visual cortex: note the horseradish peroxidase reaction product in layer 4 (see numbered layers). Scale bar: 500 µm. Bottom left: morphology of a typical pyramidal neuron in layer II/III. At the end of recordings obtained with micropipettes containing biocytin (2%), further diffusion of biocytin into the neurons was facilitated by the use of negative intracellular step current pulses superimposed on continuous negative current (see METHODS). Scale bar: 200 µm. Bottom right: same cell as in bottom left, viewed at higher magnification. Note the spiny dendrites, the projection axon (filled arrow), and the recurrent axon (open arrow). Scale bar: 20 µm. Background stain in all panels: cresyl violet (Nissl).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Preparation of slices for electrophysiology

Experiments were performed on visual cortical slices of pigmented guinea pigs aged 34-180 days postnatal (weight 300-900 g). Dissection procedures to prepare brain slices have been described in detail previously (Fregnac et al. 1994). Briefly, animals were deeply anesthetized with ether. The occipital pole of one hemisphere was rapidly removed and was maintained and sliced in artificial cerebrospinal fluid (ACSF) at 4°C. Coronal slices (400 µm thick) containing area 17 were cut with a vibroslicer (Campden Instruments, London, UK), and transferred to an interface-type recording chamber (Medical Systems, Greenvale, NY). Slices were maintained in the humidified recording chamber undisturbed for >1.5 h before the start of experiments. During this period the temperature of the recording chamber was slowly warmed up to, and then maintained at, 35°C. Additional slices were kept in an oxygenated holding chamber (Medical Systems, Greenvale, NY) at room temperature in ACSF for <5 h.

Solutions

The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (DL-APV; Sigma, St. Louis, MO), and the NO synthase (NOS) inhibitor N-nitro-L-Arginine (Sigma) were dissolved in ACSF and bath applied. In some experiments only the D isomer of APV was used, and this gave results similar to those obtained with the use of the DL form. Composition of ACSF was as follows (in mM): 124 NaCl, 2 KCl, 2 MgSO4, 2 CaCl2, 1.25 KH2PO4, 26 NaHCO3, and 11 glucose. Superfusates were saturated with 95% O2-5% CO2 to maintain pH at 7.4. Flow rate of the solution was 1 ml/min.

Recording techniques and stimulation

Recording micropipettes were pulled from glass capillary filaments (1.5 mm OD, 0.86 mm ID; A-M Systems) with a horizontal puller (Sutter Instruments, San Rafael, CA). Pipettes filled with 2.0 M potassium acetate (pH adjusted to 7.1 with 1.0 M acetic acid) had resistances between 90 and 140 MOmega . In some experiments, 100 mM Lidocaine, N-ethyl bromide quaternary salt (QX-314; Research Biochemicals, Natick, MA), 200 mM 1,2-bis-2-aminophenoxy ethane-N,N,N',N'-tetraacetic acid (BAPTA; Sigma, St. Louis, MO), or 1.5% biocytin (Sigma) was included in the pipette filling solution. In some experiments inhibitory postsynaptic potentials (IPSPs) were intracellularly blocked by filling the recording micropipettes with the chloride channel blocker 4,4'-dinitro-stilbene-2,2'-disulfonic acid (DNDS) (500 µM) dissolved in 1.0 M cesium acetate. Bipolar stimulating electrodes (FHC, Brunswick, ME) were positioned at the white matter/layer VI border. Recording micropipettes (resistance 90-150 MOmega ) were placed in the supragranular cortical layers on beam with the stimulating electrode. Input resistance (Rin) of the cells was determined with hyperpolarizing step current injections (from 0.02 to 0.1 nA; duration 80 ms) through the recording micropipette. Conventional intracellular recordings were obtained with an Axoclamp 2 A amplifier (Axon Instruments, Foster City, CA) in bridge mode. Data were digitized at 4 kHz, collected, and subsequently analyzed on a Macintosh II computer with the use of customized software. First, the intensity of stimulation (driven by a pulse generator: Master 8; AMPI, Jerusalem, Israel) through the bipolar electrode was adjusted to evoke postsynaptic potentials (PSPs) of 30-35% of the value required to obtain an action potential. Frequency of the afferent stimulation was 0.1 Hz throughout all experiments. In a 10- to 20-min baseline period, these low-frequency stimulation epochs (50 µs, 30-100 µA) were delivered to the layer VI/white matter border and PSPs were recorded from a layer II/III neuron. The pairing protocol (60 pairings over 10 min at 0.1 Hz) consisted of intracellular postsynaptic depolarizing current pulses (duration 80 ms; intensity +0.5 to +3.5 nA) delivered to the neuron, with coincident stimulation of the afferents (following the onset of the depolarizing current pulse with a 25-ms delay; Fig. 2A). Thus pre- and postsynaptic activation always overlapped during the pairing protocol. With the exception of experiments in which QX-314 was used in the recording micropipette, neurons generated action potentials during the pairing depolarizations (Fig. 2A). During the postpairing period, control stimulation and recording of PSPs were resumed.


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FIG. 2. Example of pairing protocol and data analysis. A: example of 3 consecutive conjunctions of pre- and postsynaptic activity during pairing protocols. The impaled neuron is depolarized by a current pulse (80 ms, +2.0 nA) injected through the recording micropipette. A burst of action potentials is evoked by the depolarizing pulse overlapping with the delivery of the afferent stimulation (arrows). The white matter stimulation follows the onset of the intracellular depolarizing pulse by 25 ms. Sixty pairing trials are delivered at 0.1 Hz for 10 min. This frequency of white matter stimulation, and the strength of the afferent stimulation, are identical to those used during control stimulation in the pre- and postpairing periods. B: analysis of the postsynaptic potentials (PSPs). Down arrow: time of white matter afferent stimulation. Double-headed vertical arrow: PSP peak amplitude. The initial slope (IS; thick line) is determined by analyzing the change in voltage during the 1st ms of the PSP. "Slope to peak" (S) is calculated between 10 and 90% of the maximum PSP amplitude. Dashed line: half-width of the PSP at half-height of the peak amplitude (HWHH). C: analysis of the significance of the effects of pairing. The pairing protocol takes places in the time period (10 min) between the 2 arrows. Averages obtained from trials in the last 5 min of the baseline period are considered as the control group. An unpaired t-test statistics is utilized to compare this control group with the average PSP peak amplitude in the 1st 2 min of the postpairing group (1); then the analysis window moves forward in 1-min increments (2, 3, 4, 5. . .). The time point at which the t-test provides a P value of 0.05 marks the end of significant effect on the PSP. Similar statistical analysis was carried out on the IS, S, and HWHH of the PSP.

Data analysis

Only neurons with stable resting membrane potential (Er), Rin, and baseline period were included in the analysis. Several aspects of the PSPs were analyzed. The start of the PSP was defined as the point in which the recorded voltage was >2 SD from the baseline. The peak amplitude was determined at the highest point of the PSP. With the use of customized software, PSP peak amplitude, initial slope (slope in the 1st ms of the PSP), slope to peak (slope of the PSP between 10 and 90% of the peak amplitude), and HWHH (half-width measured at half-height of the PSP) were measured off-line (Fig. 2B). To analyze statistically significant effects of pairings on peak amplitude, initial slope, "slope to peak" and HWHH, PSPs in the pre- and postpairing periods of each experiment were evaluated with the use of unpaired t-tests. The significance level was set at 0.05 (2-tailed test). Series of t-tests were carried out on a given experiment. Choosing the last 30 PSPs (last 5 min of the baseline) immediately before the pairings as a control group, the t-tests were conducted on the first 12 postpairing trials (subgroup 1); then the analysis moved forward in 1-min increments (subgroups 2, 3, 4, 5. . . ; Fig. 2C). With this "sliding window" method, we evaluated the existence and duration of significant potentiation quantitatively. Direct comparisons of pre- and postpairing epochs were made by superimposing the averages of 30 PSPs recorded immediately preceding versus immediately following the pairing trials (Fig. 3). Quantitative analysis of the magnitude of synaptic potentiation was performed on these epochs. Uncorrected chi 2 test was used to analyze possible effects on the likelihood of potentiation.


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FIG. 3. Phenomenon of transient synaptic potentiation. These records were obtained from a neuron of a 40-day-old animal [resting membrane potential (Er): -89 mV; input resistance (Rin): 36 MOmega ]. A: effects of a pairing protocol (carried out in the time period showing no recorded PSPs) on the peak amplitude of the PSP. Intensity of the postsynaptic depolarizing pulse during pairing was 2.3 nA. Duration of the depolarizing current pulses in all experiments was 80 ms. Note the transient increase (+36%) in the PSP peak amplitude occurring after the pairing procedure. A significant enhancement of the PSP peak amplitude persisted for 25 min after the pairing ended. Averages of 30 PSPs immediately preceding and following the pairing trials are shown superimposed at right. B: PSP peak amplitude values normalized to the baseline average. C: effects of pairing on the initial slope (+33% enhancement). The initial rising phases of the pre- and postpairing PSPs are illustrated at right. D: effects of pairing on the slope to peak (+64%) in the same experiment. Note the effect of the pairing protocol on the waveform of the averaged PSP (right). In this experiment no change of HWHH was detected. E: likelihood (black columns) and magnitude (white columns) of pairing-induced transient potentiation of PSP peak amplitude, initial slope, and slope to peak. In the majority of cases, potentiation of the initial slope (n = 19 of 92) or the slope to peak (n = 39 of 92) occurred in conjunction with PSP peak amplitude potentiation. Changes of initial slope and slope to peak not accompanied by a change in peak amplitude were also detected (n = 3 of 92 and n = 1 of 92, respectively). Error bars in this and all subsequent figures: means ± SE.

Histology

The localization of primary visual cortex in the guinea pig brain was confirmed before the study with the method of intraocular injection (5.0 µl) and further transneuronal transport of wheat germ agglutinin-horseradish peroxidase complex (5%) to the visual cortex (Fig. 1, top). Animals (n = 3) were anesthetized during the injection procedure with 2.0% halothane in 70% N2O-30% O2. Seven days after the injections, animals were killed and perfused with saline followed by 2% glutaraldehyde. The whole brains were blocked out and sectioned at 100 µm. Sections were then serially processed for the peroxidase reaction, with hydrogen peroxide in the presence of diaminobenzidine or tetramethylbenzidine. Sections were also processed with the traditional cresyl violet (Nissl) method.

To verify laminar localization, several cells were filled with biocytin. Although most of the electrophysiological recordings were made with standard 2.0 M potassium acetate-filled micropipettes, in these experiments microelectrodes filled with 2% biocytin (in 2.0 M potassium acetate) were used (Fig. 1, bottom). Intracellular injections of biocytin were made after recording, with the use of negative current pulses superimposed on continuous negative current (-0.5 to -1.0 nA DC with 200-ms pulses of -0.5 to -1.0 nA at 3.3 Hz for 10-20 min). After overnight immersion in 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer, the tissue was reimbedded in albumin/gelatin and sectioned at 100 µm on a vibratome. Sections were incubated in an avidin-biotin-horseradish peroxidase complex (ABC, Vector), then reacted with 0.3% hydrogen peroxide and diaminobenzidine [50 mg in 100 ml of 0.05 M tris(hydroxymethyl)aminomethane buffer]. The tissue was counterstained with cresyl violet.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Database: overview

The analysis of the mechanisms of transient synaptic potentiation was carried out for 74 supragranular neurons (92 pairing tests; Table 1) in guinea pig primary visual cortex. Data were obtained from cortical slices of animals aged >= 34 days postnatal.

 
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TABLE 1. Properties of neurons included in the analysis of the likelihood and magnitude of transient synaptic potentiation

The basic membrane properties of the tested neurons were relatively homogenous. The overall mean value of the Er of all cells studied was -83 ± 1 (SE) mV (see Table 1). The Rin of the cells was also similar (45 ± 3 MOmega , mean ± SE). The average amplitude of the intracellular depolarizing current pulses applied during pairing protocols was +2.3 ± 0.1 nA. Figure 1 illustrates the cortical location of the guinea pig primary visual cortex and one of the layer II/III pyramidal neurons that was filled with intracellularly injected biocytin after completion of the experimental protocol. Note that there is a rich thalamocortical projection to layer IV and that the supragranular spiny pyramidal neurons have projection axons (filled arrow) that issue recurrent collaterals (open arrow).

Phenomenon of transient synaptic potentiation

The basic procedure for inducing and evaluating the potentiation is illustrated in Fig. 2. The responses of a neuron to three cycles (of a total of 60 pairings applied at 0.1 Hz) of paired pre- and postsynaptic activation are illustrated in Fig. 2A. Note that in these protocols the cell was "allowed" to generate a train of spikes during the pairing. An example of a synaptic response elicited before the pairing protocols is shown in Fig. 2B. Various components of the PSP, including peak amplitude, initial slope, slope to peak, and HWHH, were analyzed before and after application of the pairing protocol as indicated (see METHODS and Fig. 2 legend). The type of analysis used for one of the measured parameters (PSP peak amplitude) over an entire epoch is illustrated in Fig. 2C. An example of a typical result is demonstrated in Fig. 3. Note that the pairing resulted in an increase in the average PSP peak amplitude from 5.9 to 8.0 mV (Fig. 3A) or a +36% relative increase (Fig. 3B) immediately after the pairing and that the PSP remained significantly enhanced above baseline 25 min after completion of the pairing protocol. However, unlike conventional long-term potentiation (LTP) in the hippocampus (Bliss and Lomo 1973; Lynch et al. 1983; Malenka et al. 1988), particularly when induced with more robust protocols, this potentiation is transient; it generally decays back to baseline over a 15- to 45-min period (25 min in the experiment shown in Fig. 3). In the example illustrated in Fig. 3, both the initial slope of the PSP (Fig. 3C), indicative of an early monosynaptic component of the compound synaptic response, and the slope to peak (Fig. 3D), which also likely includes other components of the compound synaptic response, increased, although by differing relative amounts (+33 and +64%, respectively). The HWHH of the PSP remained unchanged (not illustrated).

Transient synaptic potentiation of the PSP peak amplitude occurred in 51% of the cases (n = 47 of 92 of the PSPs tested). The mean magnitude of PSP peak amplitude potentiation was an increase of +26 ± 3% (Fig. 3E). The actual pre- and postpairing PSP peak amplitude values are demonstrated in Fig. 4A. A cumulative histogram (Fig. 4B) illustrates the normalized effect of the PSP peak amplitude. The overall mean duration on effect (as evaluated by the change in the peak amplitude of the PSP) is 23 ± 3 min. As indicated in Figs. 2 and 3, a number of components of the evoked compound PSP can change because of the pairing protocol. Potentiation of HWHH occurred in only a few instances (n = 3 of 92; not illustrated). Potentiation of the initial slope, that is indicative of effects on the monosynaptic component of the compound PSP, occurred in 24% (n = 22 of 92) of the PSPs tested; 19 of the 22 PSPs also demonstrated amplitude potentiation. Enhancement of the slope to peak component of the PSP occurred more frequently (43%, n = 40 of 92 of the PSPs tested; 39 of these 92 also showed amplitude potentiation; see Fig. 3E). Thus, of the 51% (47 of 92) of the PSPs sampled that showed amplitude potentiation, 40% (n = 19 of 47) also had their initial slope potentiated. These results suggest that both early monosynaptic and later polysynaptic components of the PSPs are susceptible to this form of synaptic plasticity.


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FIG. 4. Analysis of the magnitude of transient synaptic potentiation. A: scatter plot illustrating the actual pre- and postpairing PSP peak amplitude values. In each case, the average magnitude of 30 trials recorded immediately preceding and immediately following the pairing protocol is plotted. The 47 cases in which significant potentiation of the PSP peak amplitude occurred are shown. Diagonal line (slope = 1.0): case in which pre- and postpairing PSP amplitudes are equivalent. B: cumulative histogram of the magnitude of the PSP peak amplitude potentiation (n = 47).

Network versus synaptic contributions

In our previous study of visual cortical neurons of adult animals (Fregnac et al. 1994), the maintenance phase of transient potentiation was found to be due to a modification in synaptic efficiency (vs. changes in the driving force on the synaptic currents due to an alteration in Er or to changes in Rin) and to be spatially restricted to only the synaptic pathway that was coactive with the postsynaptic depolarization. However, the exact site of potentiation induction within the activated cortical network has not been established unequivocally. During the pairing protocols, the intracellularly applied depolarizing pulses usually cause the postsynaptic neuron to spike (Fig. 2). Thus there is uncertainty as to the site of the synaptic potentiation. The generation of bursts of action potentials by the coactivated neuron is also likely to depolarize other neighboring cortical neurons that share common convergent synaptic elements with the activated cell, and thus the pairing procedure could potentially alter the efficacy of other synapses in a polysynaptic circuit. To discriminate modifications imposed at synaptic sites directly on the recorded neuron from those that occur elsewhere in the cortical network, the lidocaine derivative QX-314 was applied intracellularly in a series of control experiments to block spike output (Fig. 5, inset at top) from the activated neuron (Connors and Prince 1982) during the pairing protocol. An example of one such experiment is illustrated in Fig. 5B. Figure 5A illustrates 2 of the 60 pairings. Note that, unlike our standard protocol, in which the neuron generates a train of action potentials during the pairing (Fig. 2), this cell shows only graded depolarizations (Fig. 5A). However, the synaptic potentiation still is reliably induced (Fig. 5B) and was so in 7 of 14 cases tested (50%) with intracellular QX-314 application that blocked spike production. Thus the likelihood of potentiation (51%) was not significantly affected (chi 2 = 0.006, P = 0.939) by the absence of action potentials in the paired neuron. Moreover, intracellular QX-314 did not have any effect on the magnitude of potentiation (+30 ± 6% vs. +26 ± 3%, P = 0.595). These results indicate that transient synaptic potentiation does not require spike output from the neuron participating in the pairing, and that the modification of synaptic efficiency occurs at synaptic contacts made by the stimulated afferents directly impinging onto that cell.


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FIG. 5. Transient synaptic potentiation in the absence of sodium action potentials in the postsynaptic cell. The filling solution in the recording micropipette contained 100 mM Lidocaine, N-ethyl bromide quaternary salt (QX-314) in 2.0 M potassium acetate. Recording started 20 min after impalement of the cell. A: note that the intracellular current pulses (+1.7 nA) used in the pairing protocol did not lead to action potentials in the cell (Er: -84 mV; Rin: 55 MOmega ); instead, only graded depolarizations occurred. Arrows: time of afferent stimulation. B: effects of pairing in the absence of sodium spikes (records are from the same cell whose pairing is illustrated in A). Following the control baseline and completion of the pairing protocol, potentiation (+40%) of the PSP peak amplitude was observed, similar to that observed for cells that were allowed to spike during pairing. C: likelihood (black columns; 50%) and magnitude (white columns; increase of +30 ± 6%, mean ± SE) of potentiation did not differ significantly from control (51% likelihood and +26 ± 3% increase) during intracellular blockade of action potentials in the neuron.

Site of plasticity

The compound PSPs evoked by stimulation at the white matter/layer VI border can consist of several excitatory and inhibitory components that overlap temporally. Consequently, the pairing-induced enhancement of the PSP peak amplitude could reflect a potentiation of excitatory synaptic transmission and/or a reduced strength of synaptic inhibition. To discriminate between these alternatives in the mature cortex, we blocked IPSPs only in the recorded neuron by applying intracellular cesium to block potassium currents, and a compound that blocks gamma -aminobutyric acid-A(GABAA)-mediated chloride currents by intracellular application, the disulfonic stilbene DNDS (Bridges et al. 1989; Dudek and Friedlander 1996a,b; Matsuoka et al. 1990; Singh et al. 1991). This approach is preferable to the bath application of GABAA receptor antagonists such as bicuculline, which would affect IPSPs in all cells and could lead to hyperexcitability in the entire cortical slice. Examples of these experiments are illustrated in Fig. 6. Blockade of the inhibitory component of the PSP required a time period of ~15 min. This intracellular blockade of the Cl- conductance eliminated the early IPSP, and resulted in a PSP with a single monotonic decay (Fig. 6A). In 6 of 9 experiments (67%) with cesium and DNDS in the microelectrode, synaptic potentiation was successfully induced in the neurons. The likelihood of potentiation during intracellular DNDS/cesium application was not significantly greater than that observed in control (51%) cases (chi 2 = 0.798, P = 0.372). Similarly, the magnitude of potentiation was not significantly affected by intracellular blockade of IPSPs (+32 ± 6% vs. +26 ± 3%, P = 0.419). Thus our pairing protocol induces an enhancement of compound PSPs by potentiating excitatory synaptic transmission.


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FIG. 6. Transient synaptic potentiation occurs during intracellular blockade of postsynaptic inhibition in cortical neurons. Recordings were obtained with micropipettes filled with 4,4'-dinitro-stilbene-2,2'-disulfonic acid (DNDS) (500 µM) in cesium acetate (1.0 M). A: at the start, the membrane potential (Em) of the neuron was depolarized by current injection during recording of the synaptic responses to visualize the early inhibitory PSP (IPSP). Bottom arrow: average of 4 PSPs with an apparent early IPSP at 5 min after the impalement of the cell (same as in B). The IPSP is blocked in 15 min (top arrow). B: pairing protocol on the cell shown in A (Em: -80 mV; Rin: 50 MOmega ), with the use of intracellular current pulses of +2.2 nA and then +2.3 nA, reliably induced epochs of potentiation. C: likelihood (black columns) of potentiation was not significantly different (67 vs. 51%; P = 0.372) when IPSPs were blocked by intracellular DNDS/cesium. The magnitude (white columns) of potentiation during intracellular blockade of IPSPs did not differ significantly from control (+32 ± 6% vs. +26 ± 3% increase, P = 0.419).

Involvement of NMDA receptors

The NMDA class of glutamate receptors have been shown to play a key role in a variety of forms of synaptic plasticity throughout the CNS (Artola and Singer 1987; Cline and Constantine-Paton 1990; Collingridge et al. 1983; Dudek and Bear 1992; Hahm et al. 1991; Morris et al. 1986). Moreover, the activation of these receptors can contribute to the generation of excitatory PSPs (EPSPs) during nonpotentiated synaptic transmission (Sutor and Hablitz 1989a-c). To investigate the role of NMDA receptors in the induction of visual cortical transient synaptic potentiation, we evaluated the ability of the competitive blocker of NMDA receptors, APV (50 µM), to block the effects of the pairing protocol.

Two types of experiments in which APV was used in the superfusate were conducted. Typical results are illustrated in Fig. 7. One protocol utilized initial bath application of APV, with attempted potentiation induction followed by APV washout and return to control ACSF. A subsequent second attempt at potentiation induction was then made (Fig. 7A). The other protocol utilized initial pairing protocol in normal ACSF followed by superfusion with APV and a second attempt at potentiation induction (Fig. 7B). With both protocols, NMDA receptor blockade consistently and reversibly blocked the induction of transient potentiation. For 12 of 14 cells tested that were capable of generating transient potentiation in normal ACSF (Fig. 7D), APV blocked the induction. Regardless of the order of experimental manipulations, that is, ACSF superfusion first followed by APV application, or vice versa (Fig. 7D), APV very efficiently blocked the induction of potentiation. Note that the presence of APV reduced the amplitude of the baseline PSP whether it had already been potentiated (Fig. 7B) or not (Fig. 7A). This is indicative of an NMDA receptor-mediated component to the EPSP in the nonpotentiated and potentiated states, which was verified by evaluating the voltage dependence of the PSP and the susceptibility of the late, depolarization-dependent phase of the EPSP to APV blockade (Fig. 7C).


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FIG. 7. Effects of bath applied 2-amino-5-phosphonovaleric acid (APV) on transient synaptic potentiation. Two examples (A and B) demonstrate the absence of potentiation during N-methyl-D-aspartate (NMDA) receptor blockade. Horizontal bars: application of APV (50 µM). Experiments were carried out by either initial APV application with attempted potentiation induction (A) followed by normal artificial cerebrospinal fluid (ACSF) replacement, or by initial attempted potentiation induction in normal ACSF (B) followed by APV. A: recording of PSPs started in the 60th min of APV bath application. Pairing protocol (+2.0-nA depolarizing pulses) was conducted on the cell (Er: -90 mV; Rin: 60 MOmega ) 1st during superfusion of APV, and did not lead to synaptic potentiation. Note that washout of APV resulted in a slight increase in the PSP size. The 2nd pairing protocol, in the presence of control ACSF, in which intracellular depolarizing pulses were used at the same intensity as before (+2.0 nA), evoked potentiation (+33% increase) of the PSP peak amplitude. B: pairing paradigm with +2.3 nA intracellular depolarizing pulses was carried out on the neuron (Er: -87 mV; Rin: 36 MOmega ) 1st in control ACSF, where a potentiation of the PSP peak amplitude occurred (+33%). Superfusion of 50 µM APV followed. The subsequent pairing protocol conducted in the presence of APV did not result in potentiation. Arrows: short breaks in the recordings when the voltage dependence of the PSPs was tested (shown in C). C: effect of APV on the late component of the PSP. The voltage dependence of the PSP (same cell as in B) was evaluated 1st in control ACSF, then during continuous superfusion of APV (50 µM). The cell was subjected to depolarization (Em: -70 mV; top) and hyperpolarization (Em: -98 mV; bottom) by injection of current with appropriate polarity through the recording micropipette. PSPs were also collected at Er = -87 mV (middle). Each trace is an average of 5 PSPs. The effect of APV application on the late component of the PSP, most visible at depolarized Ems (top), is demonstrated by the overlaid traces (inset at bottom; Em: -70 mV). D: effect of APV on the likelihood of potentiation as a function of the order of ACSF and APV superfusion. Blockade of induction of synaptic potentiation by APV did not depend on the order of ACSF and APV superfusion.

Role of calcium

The influx of calcium ions through NMDA channels and the subsequent control of intracellular calcium levels are crucial for the induction of various forms of synaptic plasticity including induction of hippocampal (Lynch et al. 1983; Malenka et al. 1988) and cortical (Baranyi and Szente 1987; Kimura et al. 1990) LTP and long-term depression. Thus the role of calcium in triggering this more dynamic and reversible form of cortical plasticity was evaluated. In this series of experiments, intracellular recording micropipettes containing the calcium chelator BAPTA were used. On impalement of a neuron with a BAPTA-containing micropipette, stimulus-evoked PSPs initially were not elicited for >= 20-30 min. During this time period, the slow afterhyperpolarization which is mediated by calcium-induced K+ currents (Madison and Nicoll 1984; Schwartzkroin and Stafstrom 1980), was monitored by evoking a train of action potentials with long (250 ms) depolarizing current pulses (Fig. 8A). Elimination of the Ca2+-activated K+ current-dependent slow afterhyperpolarization was taken as an indication of successful diffusion of BAPTA into the cell (Lancaster and Nicoll 1987). This type of chelation of calcium ions in the postsynaptic neuron reliably prevented the occurrence of potentiation (Fig. 8B) in all tested neurons (n = 13 of 13; Fig. 8D). Interestingly, in several experiments(n = 4 of 13) with intracellular BAPTA application, the pairing protocol not only blocked the induction of transient potentiation, but it resulted in a significant synaptic depression (Fig. 8C; the average reduction in the PSP size for these four cells was -44 ± 3%), which was never observed after pairing protocols in which depolarizing pulses were used (Fregnac et al. 1994). The average change in the PSP peak amplitude following pairing protocols in the presence of intracellular BAPTA, including cases with no potentiation or synaptic depression, was a decrease of -11 ± 5% (Fig. 8D).


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FIG. 8. Role of calcium. Recordings with micropipettes containing 200 mM 1,2-bis-2-aminophenoxy ethane-N,N,N',N'-tetraacetic acid (BAPTA) (see drawing in A). A: to monitor the diffusion of BAPTA into the cell, intracellular depolarizing test pulses (+0.7 nA; 250 ms) were applied 1st at 5 min after the impalement of the neuron, while the Em was held at -71 mV. Fifteen minutes later (20 min after impalement of the cell), applying the same test pulses, there was an apparent reduction of the slow afterhyperpolarization that followed the train of action potentials, signifying successful diffusion of the calcium chelator into the cell. B: pairing protocol, in which +1.8-nA depolarizing current was used, conducted on the same cell (Er: -85 mV; Rin: 15 MOmega ) as in A. No change in the PSP peak amplitude was observed. C: example of depression of the PSP peak amplitude (-35%) in a neuron (Er: -86 mV; Rin: 30 MOmega ) evoked by the same pairing protocol (+2.0-nA pulses) in the presence of intracellular BAPTA. D: in no cases (0 of 13) was potentiation of the PSPs observed when pairing protocols were conducted in the presence of intracellular BAPTA. The average change in PSP peak amplitude following the pairing protocol during intracellular chelation of calcium was a decrease by -11 ± 5% (white columns). This is due to the 4 of 13 cases in which significant synaptic depression was detected after the pairing paradigm.

Role of NO

The induction of transient potentiation in the mature cortex, like other models of synaptic plasticity, requires postsynaptic NMDA receptor activation and a rise in intracellular calcium. Previous studies have demonstrated that this process can lead to activation of neuronal NOS (Garthwaite et al. 1988) and the production of NO, which in turn facilitates presynaptic release of glutamate (Montague et al. 1994) and the induction of at least one form of synaptic potentiation (hippocampal CA1/LTP) (Schuman and Madison 1991, 1994; but see Williams et al. 1993). Thus we evaluated the role of NO production in induction of transient potentiation. The competitive NOS inhibitor L-nitro-arginine (LNA) (100 µM) was bath applied at the outset of the recording. To be certain that the LNA was effectively distributed in the cortical slice, several (2-4) hours of preincubation were allowed before testing for potentiation induction. Despite the presence of LNA in the bath, synaptic potentiation was inducible in 11 of 20 (55%) of cells tested (Fig. 9) in mature animals. The average duration of potentiation during bath application of LNA was not significantly different from the duration of effect in control ACSF (17 ± 3 min vs. 23 ± 3 min; P = 0.2171). Thus LNA application did not have a significant effect on the likelihood (control: 47 of 92, 51%; chi 2 = 0.101, P = 0.751; Fig. 9C) or duration of potentiation of naive slices. Interestingly, the average magnitude of effect in the presence of LNA was significantly greater than in control ACSF (+44 ± 7% vs. +26 ± 3%, P < 0.01, Fig. 9C). However, in another series of experiments in which reliable transient synaptic potentiation was first induced in the cell to serve as an internal control, in half (n = 2 of 4) of those cases tested (Fig. 9B), subsequent application of LNA to the bath prevented a second epoch of potentiation in slices that had already been potentiated and recovered.


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FIG. 9. Nitric oxide (NO) and transient synaptic potentiation. The NO synthase (NOS) inhibitor L-nitro-arginine (LNA) (100 µM) was present in all experiments (A-C) in the bath. A: potentiation of the PSP peak amplitude following a pairing protocol applied during continuous bath application (>3 h) of LNA. This case is a representative of experiments in which, compared with control, significantly greater magnitude of potentiation (+67%) was obtained during blockade of NO production. B: 1st pairing protocol (not shown) on this cell was conducted in ACSF and evoked a potentiation of the PSP peak amplitude (+27% increase). After recovery, superfusion of LNA was initiated. After 45 min of application of the NOS inhibitor a pairing protocol identical to that used in control ACSF (+1.5-nA pulses) was applied, and did not evoke potentiation of the PSPs. C: summary plot of the likelihood (black columns) and the magnitude (white columns) of transient potentiation during superfusion of ACSF or LNA. Note that the overall proportion of cells that were able to be potentiated (11 of 20 = 55 vs. 51%) was not different, although the average magnitude of the effect is significantly greater (P < 0.01) in the presence of LNA (+44 ± 7% increase in PSP peak amplitude).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Summary of findings

The major new findings of this report are that transient synaptic potentiation in the visual cortex of mature animals 1) does not require sodium action potential output from the postsynaptic neuron for its induction, implicating the synapses that directly impinge on the tested neuron as the site of plasticity, 2) requires NMDA receptor activation for its induction, 3) is maintained by a true up-regulation of excitatory synaptic transmission (vs. a down-regulation of inhibitory synaptic transmission) onto the activated neuron, 4) requires a rise in postsynaptic intracellular calcium for its induction, and 5) can be modulated by endogenous NO production. These results are summarized schematically in Fig. 10. The shaded area indicates that afferent excitatory synapses on the recorded neuron are sufficient targets of the potentiation mechanism(s) versus feedback pathways, or inhibitory inputs onto the same neuron.


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FIG. 10. Site of transient synaptic potentiation in the local cortical circuit. Because intracellular blockade of IPSPs does not prevent the occurrence of synaptic potentiation (see Fig. 6), we conclude that it takes place at excitatory (+) synaptic sites (shaded areas), and that modulation at inhibitory synapses (-) during the pairing protocol is not necessary for this plasticity. Also, excitation through positive feedback loops (bottom) is not a necessary step for the induction of potentiation, because it can be evoked in the absence of action potentials in the postsynaptic neuron (intracellular QX-314 experiments; see Fig. 5).

Sustained versus transient potentiation

The requirements for induction of transient synaptic potentiation in the mature visual cortex share some similarities with conventional LTP in the hippocampal CA1 region and in the neocortex, where more robust experimental stimulation protocols are generally applied (Andersen et al. 1980; Bear et al. 1992; Dunwiddie and Lynch 1978; Kimura et al. 1989; Malenka et al. 1989). These similar requirements include NMDA receptor activation (Fig. 7) (Artola and Singer 1987; Harris et al. 1984; Larson and Lynch 1988; Sutor and Hablitz 1989c), an increase in postsynaptic intracellular calcium (Fig. 8) (Lynch et al. 1983; Malenka et al. 1988), and the sufficiency of subthreshold postsynaptic depolarization with synaptic activation for induction (Baranyi et al. 1991; Gustafsson et al. 1987). In our paradigm, a period of temporal covariance of pre- and postsynaptic activity is essential for the transient synaptic potentiation (Fregnac et al. 1994; Friedlander et al. 1993; our control data), in concordance with the Bienenstock, Cooper and Munro model (Bear et al. 1987; Bienenstock et al. 1982; Clothiaux et al. 1991). This implies the necessity for a coincidence detector, for which NMDA receptors are likely candidates (Collingridge et al. 1988; Herron et al. 1986). The requirement for NMDA receptor activation for induction of transient potentiation (Fig. 7) in layer II/III neurons of mature visual cortex is also consistent with the known laminar localization of NMDA receptors (Currie et al. 1994; Fox et al. 1989). It is interesting to note that a small but significant NMDA receptor-mediated component of the evoked synaptic response is evident in this visual cortex supragranular pyramidal neuron (Fig. 7, A-C) during control periods, even at holding potentials of -60 to -70 mV. Other reports (Artola and Singer 1990; Burgard and Hablitz 1995) have indicated similar contributions of NMDA receptors to the PSP at membrane potentials close to rest. This suggests that any degree of NMDA receptor activation is not sufficient to induce the plasticity but that it has a threshold for the induction of transient synaptic potentiation.

A notable difference between transient synaptic potentiation and more conventionally described LTP that often is induced by tetanization protocols (Andersen et al. 1980; Berry et al. 1989; Malenka et al. 1989; Perkins and Teyler 1988) and with bath application of pharmacological blockers of synaptic inhibition (Clark and Collingridge 1995; Nowicky and Bindman 1993; Yamamoto et al. 1980) in both cortex and hippocampus is the kinetic profile of the potentiation (mean duration of a decaying transient potentiation = 23 min, range 8-110 min, vs. conventional LTP, which may remain in a steady state for many hours). Thus, although the transient potentiation may share some common mechanisms for the induction and the early stages of expression with conventional LTP, there must be differences in the maintenance phase. These differences may be quantitative, because conventional LTP protocols activate long-lasting biochemical cascades more strongly (Dash et al. 1991; Frey et al. 1993; Huang et al. 1994), or qualitative, because the low-frequency pairing protocol accesses only more transient acting local modifiers of synaptic efficiency.

A phenomenon with analogous kinetics has been observed in the hippocampus, termed short-term potentiation (Clark and Collingridge 1995; Colino et al. 1992; Malenka et al. 1992). This is in contrast to the well-described "true LTP" in hippocampus (Bliss and Lomo 1973; Malenka 1991). It is of interest to know whether there is something fundamentally different about the visual cortex and the hippocampus with respect to their ability to maintain a steady-state enhancement of excitatory synaptic transmission or whether the differences between hippocampal short-term potentiation and LTP are similar to differences between synaptic potentiation evoked by low-frequency pairing (the transient synaptic potentiation described in our study) versus tetanus-induced LTP in the visual cortex (Artola and Singer 1987; Kimura et al. 1989; Perkins and Teyler 1988). It is worth noting that pairing protocols in the hippocampus (Gustafsson et al. 1987; Malenka et al. 1988) are more likely to result in LTP, although in many of these protocols the induction parameters are stronger than those used in our experiments. Thus the local intracortical circuitry may more effectively dampen the capacity for the induction of LTP. Indeed, in another series of experiments (Dudek and Friedlander 1996a,b) we found that synaptic inhibition passing through layer IV of guinea pig visual cortex is pronounced and correlates with the likelihood of inducing another type of synaptic plasticity, long-term depression. The pairing protocol applied at 0.1-0.3 Hz (this study; Fregnac et al. 1994) does not lead to a steady-state LTP in visual cortex supragranular neurons, even when the number of pairings is extended to over 100 and when the depolarization of the postsynaptic cell is suprathreshold (as in most of our experiments when QX-314 was not included in the recording micropipette). Kirkwood and Bear (1994) referred to the strong inhibitory circuit in layer IV as the "plasticity gate." Thus it appears that in this respect there is a difference between hippocampal CA1 and cortical layer II/III plasticity.

It is important to note, however, that LTP induction in mature animals generally requires conditions such as pharmacological blockade of intracortical synaptic inhibition in the entire cortical slice by bath application of bicuculline (Artola and Singer 1987, 1990; Bear et al. 1992) or direct stimulation in layer IV to bypass the local inhibitory circuitry (Kirkwood and Bear 1994). The transient synaptic potentiation, described here, can be successfully obtained without these requirements.

Role of NO

Biochemical data from our laboratory (Montague et al. 1994) and others (Pittaluga and Raiteri 1994) suggest that NMDA receptor activation in cortical synapses leads to a transient enhancement of glutamate release consistent with a retrograde signaling mechanism. Moreover, this effect is dependent on NO production (Hanbauer et al. 1992; Meffert et al. 1994; Montague et al. 1994; Rowley et al. 1993). Indeed, our electrophysiology results demonstrate that inhibition of NOS in mature animals blocks transient potentiation for some supragranular cortical neurons (Fig. 9B). However, NOS blockade consistently enhances the magnitude (nearly a doubling) of potentiation when it is induced (Fig. 9C). One possible interpretation is that there are subpopulations of supragranular cortical neurons/synapses: those that require NO for induction of synaptic potentiation and those that do not require NO. The other possibility is that the capacity for NO production and its ability to modify synapses is ubiquitous in supragranular cortex, and a combination of actions of NO is at work at any given domain in the cortical circuit. In favor of the first interpretation is the observation that even though the cortical neuropil is richly invested with NOS-positive profiles (Aoki et al. 1993; Picanco-Diniz et al. 1993), only a subpopulation of cortical neuronal somata is positive for NADPH diaphorase (Vincent and Kimura 1992), reflecting NOS activity (Bredt et al. 1991; Dawson et al. 1991; but see Wendland et al. 1994). In favor of the second interpretation are the diverse physiological actions of NO, including inhibition of calcium fluxes through NMDA receptors (Hoyt et al. 1992; Lei et al. 1992; Lipton et al. 1993; Manzoni and Bockaert 1993; Manzoni et al. 1992) and NMDA receptor NO-mediated enhancement of release of glutamate (Montague et al. 1994). Thus endogenous NO production is capable of both upward and downward modulation of cortical synaptic plasticity and these competing processes are probably in delicate balance.

Sites and functional implications of cortical synaptic plasticity

Experiments that utilize pairing protocols, including our own and others (Baranyi et al. 1991; Fregnac et al. 1994; Gustafsson et al. 1987), demonstrate the necessity for temporal coincidence of postsynaptic depolarization and synaptic input to the same cell. These studies, however, do not directly address the sufficiency of these processes for triggering the potentiation. This is a limitation of any stimulation protocol in a conventional brain slice preparation, where activation of synapses occurs not only on the target cell but also on neighboring cells. Moreover, paradigms that employ "minimal stimulation" (Foster and McNaughton 1991; Liao et al. 1995; Schuman and Madison 1994) or even dual intracellular recording (Friedlander et al. 1990; Mason et al. 1991; Sayer et al. 1990) are subject to this type of diffuse activation (albeit on a smaller scale) because single afferents diverge widely and can influence many postsynaptic neurons (Buhl et al. 1994; Freund et al. 1985a,b; Friedlander and Martin 1989; Friedlander et al. 1991; Li et al. 1994; Martin and Whitteridge 1984; Sorra and Harris 1993). Thus, in addition to the contribution of conventional synaptic activation, diffusible signals such as NO, carbon monoxide, brain-derived neurotrophic factor, platelet activating factor, or arachidonate (Korte et al. 1995; Williams and Bliss 1989; Zhuo et al. 1993) from neighboring cells may contribute to the potentiation in the milieu of a slice or the intact brain. Such messengers could act in an anterograde or lateral as well as a retrograde direction (Aoki et al. 1997; Schuman and Madison 1994).

The lack of necessity for sodium spikes for potentiation induction demonstrates that graded depolarization of the postsynaptic neuron, in conjunction with synaptic input, is sufficient for the induction of transient potentiation (Fig. 5). Recent reports (Jaffe et al. 1992; Spruston et al. 1995; Stuart and Sakmann 1994) have shown that action potentials can back-propagate from the soma, evoking dendritic calcium transients that may play a role in pairing-induced synaptic potentiation. Whether dendritic spikes occur in our experiments with QX-314 is uncertain. However, if they do, such spikes would likely be seen as electrotonically degraded transients at the somatic microelectrode. Alternatively, calcium-mediated spikes due to locally activated voltage-gated calcium channels could still occur in the dendrites (Denk et al. 1995).

The experiments with intracellular application of QX-314 or DNDS (Figs. 5 and 10) indicate that our pairing protocol induces an enhancement of compound PSPs by potentiating excitatory synaptic transmission. Because 0.1-Hz stimulation of the afferent pathway alone, without coincident direct depolarization of the postsynaptic cell, does not lead to potentiation (Fregnac et al. 1994; our control data), a temporally contiguous signal from the depolarized cell is necessary to contribute to the induction process. Thus it is unlikely that synaptic contacts distal to the final input neuron in a polysynaptic pathway would be affected by the pairing paradigm.

In summary, our data demonstrate that coincident pre- and postsynaptic activation in layer II/III of the visual cortex can induce transient and dynamic modification of synaptic efficiency due to a true up-regulation of synaptic efficiency at excitatory synapses on the postsynaptic cell that participates in the pairing protocol. Transient synaptic potentiation mediated by NMDA receptor activation and a postsynaptic intracellular calcium signal may provide a mechanism to quickly alter the functional pattern of connectivity in a domain of primary sensory cortex subserving processes such as selective attention, figure-from-ground separation, or even brief iconic visual memory (Gegenfurtner and Sperling 1993), in mature animals, in which a number of studies have demonstrated a marked capacity for plasticity (Fregnac et al. 1994; Gilbert and Wiesel 1992; Ishai and Sagi 1995; Kaas et al. 1990).

    ACKNOWLEDGEMENTS

  We thank Drs. John Hablitz, Robin Lester, and Serena Dudek for reading drafts of the manuscript and providing insightful suggestions, and J. Neville and D. Burton for secretarial assistance. F. Hester provided excellent technical assistance with histological preparations. K. Ramer wrote the customized software used for data acquisition.

  This work was supported by National Eye Institute Grant EY-05116, HFSP Grant RG 69193, and National Institutes of Health Training Grant T32 EY-07033-18.

    FOOTNOTES

  Address for reprint requests: M. J. Friedlander, Department of Neurobiology, 516 Civitan International Research Bldg., University of Alabama at Birmingham, Birmingham, AL 35294.

  Received 16 July 1996; accepted in final form 26 November 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society