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Departments of Neurology and Neurological Sciences, Stanford University Medical Center, Stanford, California
Submitted 30 June 2004; accepted in final form 30 August 2004
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Hernandez and Naritoku 1997; Salazar et al. 1985). A number of mechanisms may contribute to development of hyperexcitability or epileptiform activity after such injuries, including enhanced excitatory synaptic activities, loss of GABAergic inhibitory control of network activities, changes in membrane properties of individual neurons that increase their excitability, and other alterations in regulatory processes (Asprodini et al. 1992; Buhl et al. 1996; Esplin et al. 1994; Furtinger et al. 2001; McKinney et al. 1997; reviewed in Prince 1999). Partially isolated neocortex is a well-established chronic model of epileptogenesis following direct cortical trauma (Halpern 1972; Hoffman et al. 1994; Jacobs et al. 2000; Prince and Tseng 1993). Neocortical islands with intact pial circulation, prepared by placing undercutting lesions and severing connections to surrounding cortex, become hyperexcitable after a latent period and generate epileptiform activity both in vivo (Sharpless and Halpern 1962) and when studied using in vitro slice preparations (Hoffman et al. 1994; Prince and Tseng 1993; Salin et al. 1995). Interictal epileptiform discharges in these neocortical slices originate in layer V (Hoffman et al. 1994; Prince and Tseng 1993) where results from several experiments indicate that significant changes occur in excitatory synaptic processes affecting pyramidal neurons. Axonal sprouting in pyramidal cells of undercut cortex leads to development of extensive arbors, principally within layer V, which may be associated with new excitatory connectivity (Salin et al. 1995). An increase in the frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) and in the slope of the input-output relation for AMPA/kainate receptor-mediated excitatory currents evoked in layer V pyramidal neurons suggest an increase in the number of excitatory synapses and/or the probability of release at terminals (Li and Prince 2002). Layer V pyramidal cells within the partially isolated area have also undergone changes in their intrinsic electrophysiological properties that render them more excitable, including an increased input resistance and membrane time constant, a reduction in spike frequency adaptation, and a steeper slope of the relationship between spike frequency and applied depolarization (f/I slope) (Prince and Tseng 1993). The latter changes in action potential generation are similar to those seen in axotomized corticospinal neurons and likely due in part to reductions in Ca2+-activated K+ currents and slow spike afterhyperpolarizations (Tseng and Prince 1996).
These effects of injury on membrane properties have been detected with somatic recordings; it is not known whether they are also present in axons and their terminals where anatomical changes such as sprouting are known to take place and where alterations might result in significant changes in the efficacy of synaptic transmission (Ferhat et al. 2003; Nadler 2003; Salin et al. 1995; Scharfman et al. 2003; see Jacobs et al. 2000 for review). Axonal terminals in both acute (Gutnick and Prince 1972) and chronic epileptogenic cortical foci (Pinault and Pumain 1985) may themselves generate bursts of action potentials that propagate antidromically and presumably orthodromically to evoke transmitter release, further suggesting that presynaptic functional alterations are present (see Pinault 1995 for review).
Direct electrophysiological assessment of the properties of presynaptic terminals has only been possible at a few restricted sites within the CNS (Sakaba et al. 2002; Zhang and Jackson 1993) and is not feasible in cerebral cortex with currently available techniques. However, studies of short-term synaptic plasticity can yield information about transmitter release from axonal terminals (Dobrunz and Stevens 1997; Gil et al. 1999) that might be affected by chronic neuronal injury. Electrophysiological results from several experiments in models of epileptogenesis suggest that alterations in short-term plasticity of transmitter release from presynaptic terminals are present (see DISCUSSION and references therein). We therefore analyzed evoked EPSCs in layer V pyramidal neurons to determine whether responses to pairs and trains of stimuli differed in control versus partially isolated cortex.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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3 x 5-mm bone window centered on the coronal suture was removed, leaving the dura intact and exposing a portion of the frontoparietal cortex unilaterally. Transcortical cuts in sensorimotor cortex were made as previously described (Hoffman et al. 1994). A 30-gauge needle, bent at approximately a right angle 2.5–3 mm from the tip, was inserted parasagittally 1–2 mm from the interhemispheric sulcus, advanced under direct vision tangentially through the dura and just beneath the pial vessels, and lowered to a depth of 2 mm. The needle then was rotated through 120–135° to produce a contiguous white matter lesion, elevated to a position just under the pia to make a second transcortical cut, and removed. An additional transcortical lesion was placed 2 mm lateral and parallel to the initial parasagittal cut in a similar manner. The skull opening was then covered with sterile plastic wrap (Saran Wrap), and the skin was sutured. Lesioned animals recovered uneventfully from the surgery and were reanesthetized for slice experiments 2 wk later.
Techniques for preparing and maintaining brain slices in vitro were as previously described (Li and Prince 2002). Neocortical slices (300 µm) were transferred to a recording chamber where they were minimally submerged and maintained at 32 ± 1°C (mean ± SD). Slices were perfused at the rate of 1–2 ml/min with a solution [artificial cerebrospinal fluid (ACSF)] containing (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 2 MgSO4, 26 NaHCO3, and 10 glucose; pH 7.4 when saturated with 95% O2-5% CO2. Patch electrodes were pulled from borosilicate glass tubing (1.5 mm OD) and had an impedance of 2–3 M
when filled with intracellular solution containing (in mM): 120 Cs-gluconate, 5 CsCl, 5 ethylene glycolbis (
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.5 CaCl2, 2 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1 QX-314, 3 ATP-Na, and 0.2 GTP; pH 7.3 adjusted with 1M CsOH. The osmolarity of the pipette solution was adjusted to 275–285 mosM. Whole cell voltage-clamp recordings were made from visually identified layer V pyramidal cells using infrared video microscopy and differential interference contrast optics (Zeiss Axioskop) and an Axopatch 200 amplifier (Axon Instruments). An estimated liquid junction potential of 7 mV was subtracted from the recorded membrane potentials. The estimated ECl was –62 mV based on the Nernst equation, taking into account the permeability of gluconate through Cl– channels. Access resistance was measured in voltage-clamp mode from responses to 2 mV hyperpolarizing voltage pulses using software provided by Dr. J. Huguenard. Only data from neurons in which access resistance was <15 M and stable for the duration of recording were used for analysis.
Evoked cortico-cortical EPSCs were obtained with monopolar focal extracellular stimulation using a patch pipette filled with ACSF, located 80 ± 10 µm from the recorded soma in the same lamina. Layer V stimulation was used in all experiments because this produced reliable EPSCs in all cells, undercut and control, and likely activated inputs from sprouted axonal arbors of adjacent layer V neurons with altered intrinsic properties (Prince and Tseng 1993; Salin et al. 1995). Because of the nature of the lesion producing the partially isolated cortex, other presumably undamaged excitatory inputs to the layer V pyramidal cells, such as thalamocortical fibers or layer II/III pyramidal cell axons (Melchitzky et al. 2001) could not be tested.
Stimulus trains consisted of 10 stimuli of constant frequency with inter-stimulus intervals of 15–100 ms (66.7–10 Hz). In some experiments, a paired-pulse protocol was used. Stimulus intensity was adjusted to 1.3–1.5 times the threshold for evoking detectable EPSCs. Trains and paired pulses were delivered at a repetition rate of 0.07–0.1 Hz. To decrease variability, data from 9 to 15 trains in each condition were averaged, and mean values are presented. The amplitude of each EPSC evoked by the stimulus train was measured and normalized to that of the first EPSC. The amplitude decay for 
10 responses was fitted with a single exponential to obtain the value of the time constant of depression and assess the synaptic efficacy (Markram and Tsodyks 1996). The paired-pulse ratio (PPR) was calculated as the amplitude of the second EPSC/amplitude of first EPSC. Statistical significance was determined by a two-tailed Student's t-test (P < 0.05) unless otherwise indicated. Data are expressed as means ± SE.
The following pharmacological agents were used: from Sigma/RBI (St Louis, MO): 2-amino-5-phosphonovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 1,2,3,6,7,8-hexahydro-3-(hydroxyimino)-N,N,7-trimethyl-2-oxo-benzo[2,1-b:3,4-c']dipyrrole-5-sulfonamide hydrochloride (NS 257); from Tocris (Ellisville, MO): 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY341495), bicuculline (10 µM), (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845. These drugs were prepared as a concentrated stock solution in distilled water, in 0.1 M NaOH solution (for LY341495), or in a 50% water, 50% DMSO solution (for CNQX), and diluted to a final concentration in ACSF. Drugs were stored in aliquots, each of which was used only once to avoid loss of potency by the freeze-thaw process. Drugs were either added to the bathing medium (CNQX, APV, and bicuculline) or applied locally to the slice using a perfusion pipette placed 500 µm from the recording pipette. For local perfusion experiments, typically two barrels of the pipette were filled, one with ACSF and the other with the desired drug dissolved in ACSF. The area of the slice containing the recorded cell was perfused with a stream of ACSF from one barrel in the control and wash conditions, and with a stream of drug-containing ACSF from the second barrel for drug conditions. The time to "wash on" the drug by such local perfusion was typically 1–4 s as judged by alterations in EPSCs with some variation due to placement of the perfusion pipette.
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RESULTS |
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). To exclude a possible contribution of N-methyl-D-aspartate (NMDA) receptors to the responses, 50 µM APV was added to the bath solution in most of the experiments in the following text. Injury decreases the PPR
Repetitive stimulation can elicit both paired-pulse facilitation (PPF) and paired-pulse depression (PPD). These phenomena can be described by the PPR that is regarded as an index of presynaptic efficacy (Markram and Tsodyks 1996; Thomson et al. 1993a) (but see DISCUSSION). We examined the PPR at different inter-stimulus intervals in control and undercut cortex to test the hypothesis that the synaptic efficacy in undercut cortex may undergo an alteration after injury. As expected from previous reports in other cortical regions (Hempel et al. 2000), the PPR was closest to 1 at lower stimulus frequencies and smaller than 1 at higher stimulus frequencies, regardless of experimental condition (Table 1). However, there was a significant reduction in the PPR in undercut versus control cortex at 40, 50, and 66.7 Hz that was greater at higher frequencies (Figs. 1, B and C, and 2; Table 1). In control cortex, the PPR ranged from 1.01 at 10 Hz to 0.90 at 66.7 Hz, whereas in the undercut cortex, PPR was 0.92 at 10 Hz and 0.62 at 66.7 Hz.
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We had previously shown that the peak amplitude of the AMPA/kainate receptor-mediated component of EPSCs is significantly larger in undercut versus control cortex (Li and Prince 2002). To exclude the possibility that the observed decrease in PPR in the undercut was due to a larger initial EPSCs amplitude (e.g., Thomson et al. 1993a), we adjusted the stimulus intensity so that there was no significant difference in initial EPSC amplitude in the two groups of cells (100 pA). In addition, we plotted the amplitude of the initial EPSC against the PPR and found that there was no correlation between these parameters (Table 1). These data suggest that the difference in PPR between control and partially isolated cortex cannot be accounted for by differences in EPSC amplitude.
To further evaluate the decrease in PPR in neurons of partially isolated cortex, we tested individual cells in control and in undercut cortex at 10, 20, 40, and 66.7 Hz. Control cells showed PPR ranging between 1.03 (10 Hz) and 0.89 (66.7 Hz), whereas PPR values in undercut cells ranged between 0.92 (10 Hz) and 0.60 (66.7 Hz) (Table 2).
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In addition to the PPR, synaptic efficacy can be measured by the time constant of decay of amplitudes of successively evoked EPSCs, here termed the time constant of depression or 
, and by the steady state amplitude of EPSCs during high-frequency stimulation. Synaptic responses elicited by trains of 10 stimuli were measured in neurons from control and isolated cortex. At stimulus frequencies of 20, 40, 50 and 66.7 Hz, the time constant of depression in neurons of injured cortex was significantly shorter than in control cortex (Table 3; Fig. 2, A1, A2, B1 and B2) (see also Doherty and Dingledine 2001). A similar trend was also present with 10 Hz stimulus trains; however, it did not reach statistical significance (Table 3; Fig. 2B3). An analysis of the average steady-state amplitude of the last three EPSCs evoked by stimulus trains revealed that, at frequencies of 20, 40, and 50 Hz, neurons of injured cortex had a lower steady-state EPSC amplitude than those in control cortex (Fig. 2, A2 and B1 and B2; P < 0.01 for 20, 40, and 50 Hz), but there was no difference with stimuli of 10 and 66.7 Hz (Fig. 2, B3 and A1).
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A large number of pre- and postsynaptic mechanisms may underlie the alterations in PPR, steady-state amplitude, and time constant of depression found in injured cortex, including those affecting presynaptic terminals and transmitter release (reviewed in Zucker and Regehr 2002). We tested several candidate mechanisms that might influence these properties. The effects of [Ca2+]o on the PPR are well-established (Cummings et al. 1996; Zucker 1993). Increases in Ca2+ entry into terminals during depolarization leads to an increased probability of release at a synapse and depletion of the releasable pool of vesicles such that subsequent evoked synaptic currents/potentials are smaller. To determine the degree to which PPD in neurons of control and undercut slices in the present experiments was Ca2+ dependent, we recorded responses to pairs of stimuli in ACSF containing increased [Ca2+] or in Ca2+-free, Sr2+-containing solution (Fig. 3, A and B). Increasing [Ca2+]o from 2 to 4 mM significantly decreased the PPR at 10 Hz by 23 ± 4% in control (n = 7, P < 0.01) and by 12 ± 3% in cells of undercuts (n = 9, P < 0.01). This change in PPR in neurons of injured cortex was significantly less than that in control cells (Fig. 3 B, left, and C). The effect of high calcium on mean peak amplitude of the initial EPSC in the train was, however, not significantly different between control and undercut (1st response in control was 129 ± 19 pA in 2 mM Ca2+ and 135 ± 21 pA in 4 mM Ca2+, 5% increase; 1st response in undercut was 127 ± 18 pA in 2 mM Ca2+ and 136 ± 29 pA in 4 mM Ca2+, 7% increase). This result may have been due to a large variability in effects of elevated [Ca2+]o on peak EPSC amplitude from cell to cell; in both groups, some initial responses increased in high calcium while others were not affected. Thus changes in PPR appeared to be a more sensitive index of alterations in [Ca2+]o than response amplitude. When extracellular Ca2+ was replaced with equimolar Sr2+, the PPR was increased by an average of 35 ± 9% (n = 6, P < 0.01) in control and 26 ± 4% in undercut neurons (n = 4, P < 0.05, Fig. 3B). The shifts in PPR in control versus undercut cortex with 4 mM Sr2+ were not significantly different (Fig. 3B, right). In both control and undercut cortex, there was a strong correlation between the PPR in control solution and the change in PPR, defined as PPR in 2 mM [Ca2+]o minus PPR in 4 mM [Ca2+]o (Fig. 3D). Thus larger initial PPR values were associated with greater decreases in 4 mM [Ca2+]o.
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Another possibility for the decrease in PPR, steady-state EPSC amplitude and time constant of depression between undercut and control cells is a differential expression/activation of presynaptic receptors on excitatory terminals in the isolated cortex. For example, spillover of GABA onto presynaptic GABAB receptors on glutamatergic terminals might produce such effects, particularly if GABA reuptake was reduced in the injured tissue (Isaacson et al. 1993). To test this possibility, we studied the effects of a GABAB receptor antagonist on the amplitudes of EPSCs evoked by stimulus trains in neurons of control and undercut cortical slices (Fig. 4A). The PPR of control cells was 0.91 ± 0.10 and 0.92 ± 0.11 in standard ACSF and ACSF containing CGP55845(0.2–1 µM), respectively (n = 13, NS). In undercut cells, the PPR was 0.54 ± 0.07 predrug and 0.52 ± 0.05 in CGP55845(n = 14, NS). There was also no change in time constant of depression or steady-state EPSC amplitude after drug application in control or undercut cells (Fig. 4A). Thus in this population, activation of presynaptic GABAB receptors did not contribute to alterations in EPSC amplitude during trains of stimuli.
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; Stefani et al. 1996) and therefore might contribute to the PPD, the decrease in the steady-state of EPSCs, or the time constant of depression in the undercut cortex. Perfusion of ACSF containing the group II mGluR antagonist LY341495 (2 µM) did not significantly affect the PPR in control cells (0.95 ± 0.07 in normal ACSF and 1.00 ± 0.09 after LY341495 application, n = 11, P > 0.05; Fig. 4Bi) or in undercut cells (0.69 ± 0.6 before drug and 0.73 ± 0.7 in LY341495, n = 12, P > 0.05; Fig. 4Bii). Although LY341495 did not affect the time constant of depression in control neurons, a small but significant shift in the exponential decay of EPSC amplitudes was observed in undercut neurons (Fig. 4Bii). Activation of metabotropic glutamate autoreceptors thus makes a small contribution to the steady-state amplitude of EPSCs evoked by stimulus trains in neurons from undercut but not control adult cortex. A similar reduction in amplitude decay due to activation of metabotropic glutamate receptors has recently been found in immature neocortical slices (Bandrowski et al. 2002). Potential role of postsynaptic receptors
Injury-related alterations in postsynaptic glutamate receptors could play a role in the changes in short-term dynamics of evoked EPSC amplitude described in the preceding text. Anatomical data do suggest that there are chronic changes in AMPA receptor subunit composition in layer V pyramidal neurons of the partially isolated cortex, including a reduction in immunoreactivity for the GluR2 subunit (Kharazia and Prince 2001). A downregulation of GluR2 has also been reported in the amygdaloid kindling model of epilepsy (Prince et al. 1995), and mice lacking GluR2 show a greater amount of long-term potentiation (LTP) and increased mortality (Jia et al. 1996). Because the presence of GluR2 in the AMPA receptor complex is linked to reduced Ca2+ permeability (Hollmann et al. 1991), a decrease in GluR2 could result in increased Ca2+ flux into neurons during glutamate receptor activation that might, in turn, influence the short-term plasticity of evoked EPSCs. As one test of such potential postsynaptic receptor alterations, we studied the effects of spermine, a biogenic polyamine that reduces the amplitude of EPSCs by specifically binding to those AMPA receptors lacking GluR2 (Donevan and Rogawski 1995; Kumar et al. 2002; Washburn and Dingledine 1996; Washburn et al. 1997). No significant effect of spermine (5–10 µM) on EPSC amplitude was detected in seven control or seven undercut cells during 10 min of bath perfusion (Fig. 5), although in five additional undercut cells exposed to spermine over 12 min, there was a slight decrease in EPSC amplitude that did not reach statistical significance (not shown), suggesting that reduction in the expression of postsynaptic GluR2 subunits is not a major factor underlying the change in PPD in undercut cells.
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), suggesting that significant changes in rectification properties of postsynaptic AMPA receptors were not present (Kumar et al. 2002). The EPSC reversal potentials were 0.12 ± 2.8 mV (n = 9) and 2.97 ± 3.2 mV (n = 7) in the control and undercut groups, respectively (P = 0.52).
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). To determine whether concurrent activation of GABAA receptor-mediated postsynaptic inhibition during stimulus trains might be shunting EPSCs (Tseng and Haberly 1988; Ulrich 2003) and accounting in part for changes in PPD or the time constant of depression, we examined the effects of perfusing slices with ACSF containing bicuculline (10 µM). Near-threshold focal stimulation was used, and APV (50 µM) included in the perfusate to avoid evoking epileptiform activity in slices after blockade of GABAA currents with bicuculline (Fig. 7). Under these conditions, blockade of GABAA receptors had no significant effect on PPR, steady-state EPSC amplitude, or the time constant of depression at any frequency tested in control or undercut cortex (Fig. 7). As shown in the representative traces of Fig. 7, C and C1 and D and D1, bicuculline also had no significant effect on the amplitudes of EPSCs evoked by 20-Hz trains in either control (n = 4) or undercut cells (n = 5). Together with the lack of effects of GABAB receptor blockade (Fig. 4A), these data suggest that GABAergic mechanisms are not involved in the differences in short term plasticity between control and undercut cells.
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DISCUSSION |
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; Goussakov et al. 2000) and exposure to the convulsant drug, 4-aminopyridine in vivo and in vitro (Pena et al. 2002). Increased PPD of excitatory field potentials reported in epileptogenic human limbic structures (Wilson et al. 1998) might relate to changes in the functional properties of transmitter release, but this is difficult to evaluate due to potential concurrent activation of inhibitory circuits and other factors. Anatomical and biochemical alterations in axons and presynaptic terminals are a ubiquitous feature in models of both acute and chronic epileptogenesis (Arellano et al. 2004; Cavazos et al. 2003; Goussakov et al. 2000; Hinz et al. 2001; Hovorka et al. 1989; Marin-Padilla et al. 2003; Pierce and Milner 2001; Salin et al. 1995). In addition, alterations in excitability of axon terminals leading to spontaneous action potential generation and "backfiring" has been reported in both acute (Gutnick and Prince 1972; Noebels and Prince 1978; Rosen et al. 1973; Schwartzkroin et al. 1975a, b; Stasheff et al. 1993) and chronic models of epileptiform discharge (Pinault and Pumain 1985; see Pinault 1995 for review).
The present results show that, at stimulus frequencies >20 Hz, there is a significant decrease in the PPR (increase in PPD), as well as a more rapid time constant of decay of amplitudes of successively evoked EPSCs in presumably axotomized and otherwise damaged layer V pyramidal neurons of chronic partial cortical isolations (Fig. 2). The lesion producing the partial cortical isolation severs thalamocortical and other extrinsic inputs to cortex, and we assume that stimuli evoking EPSCs predominantly activate adjacent intracortical pyramidal cells and axons morphology and intrinsic properties of which have been altered by the chronic injury (Prince and Tseng 1993; Salin et al. 1995). Thalamocortical neurons and their axons projecting into the undercut cortex would be long gone 2–3 wk after the lesion (Barron et al. 1973). Assuming that layer V stimuli in controls activated both intracortical and thalamocortical fibers and that intracortical stimuli generate PPF while thalamocortical inputs predominantly evoke PPD (Gil et al. 1999), a loss of the latter component in the lesioned cortex would tend to reduce rather than enhance PPD responses. Therefore the enhanced PPD in the undercut cells in our experiments (Fig. 1) is unlikely due to removal of the thalamic inputs by the lesion.
The decreases in PPR might involve a number of both pre- and postsynaptic mechanisms (reviewed in Zucker and Regehr 2002). There was a small but significant prolongation of the time constant for decreases in amplitudes of successively evoked EPSCs, as well as for steady-state EPSC amplitudes, when presynaptic group II metabotropic glutamate receptors were blocked by LY341495 (Fig. 4B), suggesting that these autoreceptors were activated by glutamate released during the later parts of the stimulus train. Such delayed effects might be expected from autoreceptor activation (Zucker and Regehr 2002). We found no significant differences in the amplitude of the first or second EPSC in control versus LY341495-containing perfusates, indicating that the same presynaptic receptors were not activated by ambient agonist levels. The small LY341495 effect suggests that activation of metabotropic glutamate autoreceptors makes only a minor contribution to the final amplitude of EPSCs in neurons from the injured cortex and does not affect PPD. Preliminary experiments with group I and III mGluR antagonists did not reveal any effect on the time constant or final steady-state amplitude (A. E. Bandrowski and D. A. Prince, unpublished observations), and therefore effects of other selective agonists of mGluRs were not tested. Presynaptic GABAB receptors did not appear to be involved in the PPD (Fig. 4A); however possible contributions by other mediators of presynaptic inhibition, such as adenosine (Phillis et al. 1979; Prince and Stevens 1992) and neuropeptide Y (Bacci et al. 2002; Colmers et al. 1988), were not explored.
Increases in [Ca2+]o decreased the PPR (increased PPD) in both control and undercut neurons (Fig. 3, B and C), while substitution of Sr2+ for Ca2+ had the opposite effects (Fig. 3). These results support a presynaptic mechanism contributing to the PPD in both cell groups. PPD reflects a decrease in the available readily releasable pool of vesicles after the first stimulus, as a consequence of Ca2+ influx. Increases in the probability of release (P) at involved presynaptic terminals should be reflected as increases in PPD (Gil et al. 1999; Thomson et al. 1993a; Zucker and Regehr 2002). Thus increases in PPD seen in the neurons of partially isolated cortex suggest that P is increased in excitatory glutamatergic terminals. Such an increase in P could contribute, in part, to the increases in miniature EPSC frequency and larger peak amplitude of the AMPA/kainate receptor-mediated component of EPSCs as well as an increase in the slope of the input–output relation for evoked EPSCs in layer V pyramidal cells of partially isolated cortex found in previous experiments (Li and Prince 2002). However, other mechanisms, such as increased numbers of excitatory synapses (Salin et al. 1995), cannot be excluded.
Increases in [Ca2+]o caused a greater decrease of the PPR in control versus undercut neurons (Fig. 3B). One likely explanation for this finding would be a saturation or ceiling effect as a consequence of the presence of only a few (1–3) release sites for each synaptic terminal (Lisman and Harris 1993; Thomson 1998). For example, if cortical injury in some way increased resting [Ca2+]o (e.g., Raza et al. 2001) and enhanced influx of Ca2+ after the first evoked action potential, more of the releasable transmitter pool would be utilized and increases in [Ca2+]o would have less effect on the PPR. It is known that decreases in [Ca2+]o during activity are larger in epileptogenic brain (Heinemann et al. 1986) and that Ca2+ influx increases after injury (Barmashenko et al. 2003), presumably due to movement into terminals and soma-dendritic compartments. Such changes have been demonstrated directly in calcium imaging studies during epileptiform events (Badea et al. 2001). Changes in regulation of [Ca2+] in terminals might be a result of injury- or activity-related plastic alterations in a variety of presynaptic calcium channels, a change in calcium buffering (Yang et al. 1997; but see Lowenstein et al. 1994; Mattson et al. 1995), effects on presynaptic proteins that regulate transmitter release (Hinz et al. 2001), or indirect effects of injury-induced reductions in potassium currents. The latter possibility may be particularly relevant as CNS nerve terminals possess a variety of K channels (Wilke et al. 1998) and previous sharp electrode somatic recordings in axotomized corticospinal neurons (Tseng and Prince 1996), and hippocampal CA1 cells in the kainic acid model of chronic epileptogenesis (Franck and Schwartzkroin 1985) have shown a downregulation of slow spike afterpolarizations that are mediated by a Ca-activated K current (Hotson and Prince 1980). Injury that globally increased membrane resistivity and decreased K+ currents might enhance Ca2+ influx during action potential invasion of the axonal terminal region and alter short-term plasticity.
It has been generally accepted that presynaptic mechanisms such as depletion of the readily releasable pool of vesicles make important contributions to synaptic depression (Zucker and Regehr 2002). However, receptor desensitization can also play a role in determining the PPR because, in several cortical cell types, stimulus frequencies as low as 20 Hz can result in some desensitization of AMPA receptors (Rozov et al. 2001). At other CNS synapses in hippocampus (Hjelmstad et al. 1999) and cerebellum (Hashimoto and Kano 1998), desensitization does not contribute significantly to PPD. The hypothesis that acute injury results in enhanced AMPA receptor desensitization has been directly tested in cell cultures of neocortical neurons subjected to stretch injury (Goforth et al. 1999). In this model, the opposite effect was found, i.e., injured neurons showed decreased desensitization of AMPA currents. Further experiments are required to directly assess potential alterations in AMPA receptor desensitization in models of chronic cortical injury or induced epileptiform activity, where increases in short-term synaptic depression have been found.
The decrease GluR2 subunit immunoreactivity in pyramidal neurons of layer V in partially isolated cortex (Kharazia and Prince 2001) might have a significant effect on the functional properties of AMPA receptors (Washburn et al. 1997), including an increase in Ca2+ permeability and a resulting increase in PPD. However, the immunocytochemically demonstrated reduction in GluR2 subunit expression did not appear to influence our results as the I-V relations of evoked EPSCs in control and undercut were relatively linear (Fig. 6) and EPSCs were relatively insensitive to external polyamine (Fig. 5). This discrepancy may be related to the relative abundance of this subunit remaining in the layer V pyramidal neurons after injury (Washburn et al. 1997).
Changes in short-term plasticity have been observed during maturation of neocortical circuits. For example, PPD is a prominent feature of monosynaptic connections in immature cortex (Kriegstein et al. 1987; Ramoa and Sur 1996; Thomson and West 1993); however, in adults, both PPF and PPD are reported (Thomson et al. 1993a, b; for review see Reyes and Sakmann 1999; Thomson and Deuchars 1994). If, as has been suggested (Cramer and Chopp 2000; Wood et al. 1990), injury to mature CNS can recapitulate some developmental processes, changes in the PPD and the decay time constant of successive EPSC amplitudes might be expected in the injured cortex.
The functional consequences of enhanced PPD in chronically injured epileptogenic cortex are hard to predict. Because the emergence of sustained epileptiform (ictal) activity is closely associated with high-frequency neuronal discharges (Matsumoto and Ajmone-Marsan 1964), enhanced PPD and increased falloff in EPSC amplitude at high frequencies might be regarded as mechanisms that serve as brakes on generation of such activity. This effect would be counterbalanced by the increased P, implied from the increased PPD, and increased excitatory connectivity within the cortical network (Salin et al. 1995) that would enhance the frequency and amplitude of spontaneous excitatory events and facilitate emergence of epileptiform discharges. Like other forms of short-term plasticity, synaptic depression causes the response of a cortical neuron to depend on the previous history of afferent signals. As a result, neuronal responses reflect the current state of presynaptic activity within the context of previous activity. However, unlike the other mechanisms, such as postsynaptic somatic inhibition that can reduce responsiveness to all inputs, synaptic depression is input-specific, leading to a dramatic increase in the selectivity and sensitivity of a neuron to subtle changes in the firing patterns of its afferents. It has been recently reported that synaptic depression greatly increased the sensitivity of a model cell to sudden small changes in the frequency of presynaptic inputs (Abbott et al. 1997). In this connection, it is interesting that increased baseline synaptic activity frequently precedes generation of spontaneous epileptiform activity in models of partial epileptogenesis (e.g., Chamberlin et al. 1990).
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Address for reprint requests and other correspondence: D. A. Prince, Dept. of Neurology and Neurological Sciences, Room M016, 300 Pasteur Dr., Stanford, CA 94305-5122 (E-mail: daprince{at}Stanford.EDU)
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, control; 
, undercut.
) and after perfusion with ACSF containing drug (
) were normalized to the 1st response and subjected to repeated-measures ANOVA analysis. A, 1 and 2: application of (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845 0.2–1 µM) did not affect EPSC amplitudes in either control (A1, control, F = 0.664, P = 0.740) or undercut neurons (A2, undercut, F = 1.911, P = 0.056). B1: control: in control neurons there was no significant difference between normal ACSF and 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY341495, 2 µM) conditions (F = 1.347, P = 0.223). B2: undercut: there was a significant difference between normal ACSF and LY341495 conditions in undercut neurons (F = 2.368, P = 0.016). For both A and B, —, single-exponential fits. Error bars: SE. Numbers of neurons (n) shown at bottom of each graph. Note difference in slopes in A, 1 vs. 2, and B, 1 vs. 2, also seen in 
