Impact of Persistent Cortical Activity (Up States) on Intracortical and Thalamocortical Synaptic Inputs

Abstract

The neocortex generates short epochs of persistent activity called up states, which are associated with changes in cellular and network excitability. Using somatosensory thalamocortical slices, we studied the impact of persistent cortical activity during spontaneous up states on intrinsic cellular excitability (input resistance) and on excitatory synaptic inputs of cortical cells. At the intrinsic excitability level, we found that the expected decrease in input resistance (high conductance) resulting from synaptic barrages during up states is counteracted by an increase in input resistance due to depolarization per se. The result is a variable but on average relatively small reduction in input resistance during up states. At the synaptic level, up states enhanced a late synaptic component of short-latency thalamocortical field potential responses but suppressed intracortical field potential responses. The thalamocortical enhancement did not reflect an increase in synaptic strength, as determined by measuring the evoked postsynaptic current, but instead an increase in evoked action potential (spike) probability due to depolarization during up states. In contrast, the intracortical suppression was associated with a reduction in synaptic strength, apparently driven by increased presynaptic intracortical activity during up states. In addition, intracortical suppression also reflected a reduction in evoked spike latency caused by depolarization and the abolishment of longer-latency spikes caused by stronger inhibitory drive during up states. In conclusion, depolarization during up states increases the success of excitatory synaptic inputs to reach firing. However, activity-dependent synaptic depression caused by increased presynaptic firing during up states and the enhancement of evoked inhibitory drive caused by depolarization suppress excitatory intracortical synaptic inputs.

INTRODUCTION

During quiescent states, such as natural sleep and certain forms of anesthesia, cortical networks are not silent but engage in spontaneous synchronized activity called “slow oscillations” (Metherate et al. 1992; Steriade et al. 1993b). Slow oscillations are characterized by rhythmic cycles of synaptically mediated depolarization and increased firing (up states), followed by decrease of synaptic inputs leading to membrane hyperpolarization and cessation of firing (down states). The short epochs of persistent activity during cortical up states resemble the activated state of cortex during arousal and cognition (Castro-Alamancos 2004b; Moruzzi and Magoun 1949; Steriade et al. 2001). Up states correspond to recurrent synaptic network activity generated within neocortex and maintained by balanced excitatory and inhibitory conductances (McCormick et al. 2003; Shu et al. 2003b; Steriade et al. 1993a). Activity in two distinct synaptic pathways of neocortex differentially affect up states. Thalamocortical inputs generally facilitate up states while intracortical inputs inhibit them (Rigas and Castro-Alamancos 2007). Conversely, it is unknown how up states affect thalamo- and intracortical synaptic inputs.

This issue has received some attention in vivo, but the findings are contentious. In anesthetized rats, up states suppress barrel cortex responses to brief whisker deflections (Petersen et al. 2003; Sachdev et al. 2004), and this resembles the sensory suppression of whisker responses observed during natural or artificially induced activated (arousal) states (Castro-Alamancos 2004a; Castro-Alamancos and Oldford 2002). During up states, the whisker sensory stimulus recruits less excitatory conductance and proportionally more inhibitory conductance in cortical cells. However, up states increase, rather than decrease, the responsiveness of barrel cortex cells to artificial excitatory postsynaptic potentials (EPSPs) in vivo (Hasenstaub et al. 2007), which indicates that barrel cortex cells are intrinsically more excitable during up states. In contrast, up states in anesthetized cat visual cortex are associated with stronger responses to visual stimuli under a variety of stimulus characteristics (Arieli et al. 1996; Azouz and Gray 1999; Haider et al. 2007).

To reach the cortex, most sensory inputs travel through thalamocortical synapses followed by intracortical synapses (Castro-Alamancos and Connors 1997b). Using thalamocortical slices of rodent somatosensory cortex, we tested the effects of spontaneous up states on thalamo- and intracortical synaptic inputs by measuring field potentials (FPs), PSPs, and postsynaptic currents (PSCs) evoked in cortical cells.

METHODS

Slices were prepared as previously described (Rigas and Castro-Alamancos 2007) from adult (>7 wk) CD-1 mice. Mice were deeply anesthetized with an overdose of ketamine hydrochloride (>100 mg/kg). On losing all responsiveness to a strong tail pinch, the animal was decapitated, and the brain was rapidly extracted. Slices (400 μm thick) were cut in the thalamocortical plane (Agmon and Connors 1991) using a vibratome. Slices were transferred to an interface chamber where they were bathed constantly (1–1.5 ml/min) with artificial cerebrospinal fluid (ACSF) at 32.5°C. The ACSF contained (in mM) 126 NaCl, 3.5 KCl, 1.25 NaH₂Po₄, 26 NaHCO₃, 1 MgSO₄ 7H₂O, 10 dextrose, and 1 CaCl₂ 2H₂O. FP recordings were made using low-impedance (∼0.5 MΩ) glass pipettes filled with ACSF. Blind whole cell recordings were obtained from layer IV–III cells of somatosensory cortex (SI) using patch electrodes of 4–12 MΩ impedance. For current-clamp recordings, the electrodes were filled with internal solution containing (in mM) 135 K-gluconate, 4 KCl, 2 NaCl, 0.2 EGTA, 10 Tris-phosphocreatine, 0.3 trisGTP, 10 HEPES, and 4 MgATP (290 mosM). For voltage-clamp recordings, the electrodes were filled with internal solution containing (in mM) 135 Cs-gluconate, 4 CsCl, 2 NaCl, 0.2 EGTA, 10 Tris-phosphocreatine, 0.3 trisGTP, 10 HEPES, 4 MgATP, and 5 QX-314 (290 mosM). Under our conditions during current-clamp recordings, the Nernst equilibrium potential for Cl⁻ is −81 mV and for K⁺ is −96.7 mV.

In some experiments, we used iontophoresis to inject neurobiotin in the cortex to reveal retrogradely labeled thalamic cells. Iontophoresis was performed as previously described (Rigas and Castro-Alamancos 2007) with a low-impedance glass pipette filled with neurobiotin (2% in 0.5 M K-acetate) and consisted of current pulses (1.5 μA) of 5-s duration delivered at 0.1 Hz during 15 min. To allow transportation of the neurobiotin after injection, the slices were incubated in the recording chamber for 2–3 h after the iontophoresis before placing them in fixative. The slices were fixated in 4% paraformaldehye with 1% glutaraldehyde, later cryoprotected with sucrose (30%) and re-sectioned on a cryostat (80 μm). Sections incubate in 3% hydrogen peroxide, followed by 0.2% Triton X-100 and by incubation in 2% goat serum. Incubation with ABC reagent (Vectorlabs) occurs overnight. The following day, diaminobenzidine is applied to the sections. After color development, sections are mounted and cleared in xylene. All procedures were reviewed and approved by the Animal Care Committee of Drexel University.

Concentric bipolar stimulating electrodes were used to electrically stimulate the thalamus (thalamocortical) and cortex (intracortical). The FP electrode was first used to identify the cortical region with the strongest and shortest latency response evoked by thalamic stimulation. Intracellular recordings were obtained from layers IV–III adjacent to the FP electrode. Thalamocortical EPSPs met two criteria; they depressed at 20 Hz and had short latencies (<2.5 ms). To identify spontaneous up states on-line and increase the chance of applying stimuli during the up state, an analog threshold detector was used to detect up states from the synchronous negative deflections of the FP recording. Also, once an up state was detected, a lockout period assured that nothing was detected for 5 s. Thus thalamo- and intracortical stimuli were tested on the up state at a rate <0.2 Hz. In addition, up and down states were further classified off-line.

Up/down state classification

All up and down states were classified off-line. We considered both the FP (network) activity and the single-cell membrane potential to classify up states. It is important to note that simultaneous intracellular and FP recordings revealed that FP recordings faithfully reflected up states (see Fig. 1 A). Thus up states consisted of long-lasting (>50 ms) negative and positive FP deflections (termed, for simplicity, network up state) that were always accompanied by barrages of synaptic potentials and some degree of depolarization in simultaneously recorded cells that led to occasional action potential firing. Within a network up state, individual cells were overall more depolarized than during down states but exhibited considerable variability in membrane potential, as previously reported (i.e., large V_m variance during up states) (McCormick et al. 2003). Thus as described in the following text, we considered both the membrane potential of the recorded cell and the state of the network defined with FP recordings. In fact, using only the membrane potential of a single cell can lead to erroneous classifications because on occasion cells may depolarize briefly due to incoming synaptic activity from one or a few inputs, but this is not at all reflected in the network activity and should not be considered an up state. By definition, up states are network events. In addition, the membrane potential of the intracellularly recorded cell must also be considered because of the large variability it can undergo during network up states. With this background in mind, we used the following methods to classify synaptic responses and current pulses:

FIG. 1.

Effect of up states on input resistance. A: typical intracellular and field potential (FP) recording and classification of spontaneous activity as up and down states based solely on FP network activity. Note, however, that to classify an event as up or down, both the V_m of the cell and the FP are considered (see methods for details). B: examples of 50-ms current pulses (−0.2 nA) delivered every 300 ms to the cell to measure input resistance during spontaneous activity. C: averaged traces from the cell in B corresponding to pulses delivered during the down or up state classified based on FP alone. D: input resistance values from 1 cell obtained during minutes of recordings plotted against the membrane potential of the cell before each current pulse. Each value is color coded according to the state of the network as up (red) or down (black) defined based on FP recordings. Moreover, these measurements were made when the cell was held at rest (no current injected; middle) and when the cell was either depolarized (right) or hyperpolarized (left) with current injection. E: same data as in D separated according to state. Left: plot of the down state values shown in the 3 panels in D (black filled circles) and color coded according to whether the cell was at rest (gray), depolarized (green), or hyperpolarized (blue) with current injection. Right: plot of Up state values shown in the 3 panels in D (red filled circles).

First, in some experiments where we tested the impact of up states on evoked FP responses (Figs. 3 and 9) we defined up states based on FP recordings alone. Specifically in these cases, a stimulus was considered to have occurred during an up state if the FP revealed long-lasting negative and positive deflections, typical of an up state (see Fig. 1A), within a time window of 500 ms before the stimulus, and the event had not yet recovered to baseline during the stimulus. Conversely, a stimulus was considered to have occurred during a down state if there was no evidence of a negative deflection in the FP within a time window of 500 ms before the stimulus. Thus as shown in Fig. 1, A and B, we marked the onset and offset of each network up state based on visual inspection, so that stimuli or pulses occurring within a network up state could be determined. Based on >100 simultaneous intracellular and FP recordings during up states, we are convinced that this is an accurate method of determining network up states.

Second, for experiments that also included intracellular recordings (Figs. 4–7), we used the FP criteria just described plus the membrane potential of the cell immediately before the stimulus. Thus a stimulus was considered to have occurred during an up state if the FP criteria was met (network up state), and the membrane potential of the cell was more depolarized than the midpoint potential between the steady hyperpolarized and depolarized state for that cell. This threshold was determined from plots of time spent at each membrane potential during minutes of spontaneous activity. In every experiment, the accuracy of the classification as down and up was carefully confirmed by visually inspecting each individual stimulus trial off-line. Average traces were calculated by averaging 30 stimulus trials.

Finally, for experiments that measured the effect of up states on input resistance (Figs. 1 and 2), we used the previous methods and took additional precautions when classifying current pulses as delivered during up and down states. To assure that the current pulses occurred during a steady up or down period, we measured the membrane potential (mean of 3 ms) immediately before and 45 ms after each current pulse and discarded all pulses in which there was more than a 3-mV change. This was found to be important because in those cases in which the change was >3 mV, there was clear evidence of a transition during the pulse from down to up or vice versa. To avoid the confound of action potentials (spikes) on input resistance measurements, those trials during which a spontaneous spike occurred between 50 ms before and after a current pulse were discarded.

FIG. 2.

Population data showing the effect of up states on input resistance. A: up states significantly suppress the mean but increase the coefficient of variation of input resistance (R_in) per cell. Top: data for each cell; bottom: averages (means ± SE; * P < 0.01). B: effect of depolarization and hyperpolarization during the down and up states on input resistance. Depolarization increases input resistance during the down and up states. (means ± SE; * P < 0.05 vs. down state at rest).

Data analyses were performed using Originlab and Sciworks software. For statistical analyses, data were first tested for normality using the Shapiro–Wilk test. If the data were considered normally distributed, parametric statistics were applied (ANOVA repeated measures or t-test paired). Otherwise, we applied nonparametric statistics (Wilcoxon signed ranks for paired comparisons, Mann-Whitney for nonpaired comparisons, Kruskal-Wallis for multiple groups).

RESULTS

Effect of up states on input resistance

Using thalamocortical slices from adult mice, we conducted simultaneous intracellular (whole cell) and extracellular (FP and multiunit) recordings from microelectrodes placed in layers IV–III of the somatosensory (SI) cortex. All the cells reported in this study had overshooting spikes and a resting membrane potential more negative than −60 mV. The cells described in this section, about the effect of up states on input resistance, were all regular spiking (RS) cells from layers IV and III. In these slices, spontaneous up and down states recur at ∼0.1 Hz. The up state is readily detected in the FP recording as a synchronous population (network) event that in the intracellular recording is characterized by barrages of postsynaptic potentials that depolarize the recorded neuron by 5–15 mV. During the down state, cortical cells are relatively hyperpolarized and there is little synaptic or network activity (Fig. 1A).

To examine the impact of up states on the excitability of cortical cells, we measured input resistance using a brief hyperpolarizing (0.1–0.2 nA, 50 ms) current pulse delivered at regular intervals (100–350 ms; Fig. 1B). Input resistance was calculated using Ohm's law, by dividing the steady-state voltage deflection at the end of the pulse by the current injected (Fig. 1C). In each cell, several hundred current pulses were delivered. Figure 1, D and E, plots the input resistance values measured from each of these pulses for one cell versus the membrane potential of the cell immediately before each pulse, when the cell was at resting membrane potential (no current injection) and when the cell was depolarized or hyperpolarized with positive or negative current injection, respectively. In this case, the FP was used to classify each recording period as down or up state, and pulses were classified accordingly depending on the period they occurred (Fig. 1; see methods for details). During network down states, each current pulse produced a similar voltage deflection. However, during network up states, the voltage deflections were very variable; the voltage deflection could be similar, smaller or larger compared with the down state. We conducted several analyses on this data.

First, we analyzed each cell (n = 13) individually by comparing the input resistance during both states defined using FPs. For each cell, statistical comparisons were computed between the hundreds of pulses delivered during the down and up states (Fig. 1D). In all but one cell, the input resistance measured during up states was significantly lower than during down states (P < 0.01; 12 of 13 cells). In the remaining cell, there was no significant difference between the two states (P > 0.2; 1 of 13 cells). Second, we performed a population analysis by using the mean input resistance values during up and down states from each cell. The mean input resistance was 10 ± 2% lower during up states than during down states (P < 0.01, n = 13; Fig. 2A). While the mean change in input resistance was relatively small between up and down states, we noticed that the variability of individual measurements was much larger during up states compared with down states (Fig. 1D). Indeed the coefficient of variation of input resistance values were on average three times larger during up states compared with down states (P < 0.01; n = 13; Fig. 2A). Thus up states produce a several-fold increase in input resistance variability which leads on average to a slightly reduced input resistance. Finally, we used an alternative method to measure input resistance by calculating the time constant (tau) of the decay of the voltage deflection after the end of the current pulse. This was accomplished by fitting a single-exponential curve to the decay. In agreement with the measures obtained using Ohm's law, the time constant (tau) was slower during down states than during up states (10 ± 0.6 vs. 7.9 ± 0.7 ms; P < 0.01), indicating that input resistance was reduced during up states.

The previous results indicate that on average input resistance is reduced during up states classified based on the network activity (FP). Therefore we tested if the same results would hold when up states are classified in addition considering the membrane potential of the recorded cells (see methods). Up and down states were first classified using the FP as in the preceding text. In addition, up states required cells to be more depolarized than a certain threshold, and down states required cells to be more hyperpolarized than this threshold. The threshold was selected as the midpoint between the up and the down states from plots of time spent at different membrane potentials. This approach limits the impact that the membrane potential variance within an up state has on the measurement and thus better reflects the impact of depolarization during network activity. Using this scheme, we then measured the effect of up states on input resistance and found that the mean input resistance was 3.4 ± 1.9% lower during up states than during down states (P < 0.05, n = 13). The fact that input resistance was less decreased when up states were classified considering also the membrane potential of the cell suggests that depolarization per se may affect input resistance.

There are two major components of up states, membrane potential fluctuations reflecting enhanced synaptic input and membrane depolarization. We tested the impact of membrane depolarization on input resistance using constant current injection to depolarize or hyperpolarize the cells by the same amount that they are depolarized during spontaneous up states. In all cells tested (n = 8), the input resistance was significantly increased by membrane depolarization (P < 0.01; Fig. 2B), while hyperpolarizing the cell by the same amount, had no significant effect. As reported in the preceding text, up states decreased input resistance compared with down states, and we found that depolarization significantly increased input resistance both during down and up states. Moreover, the increase in input resistance caused by depolarization during up states was sufficient to counteract the reduction caused by the up states (Fig. 2B). As later emphasized in discussion, the increase in input resistance caused by depolarization is likely due to activation of voltage-dependent currents. Thus this change may not be considered a true increase in input resistance per se, but it is a true increase in the effective input resistance since it can affect synaptic inputs. Taken together, these results indicate that network synaptic activity during up states decreases input resistance but this is counteracted by membrane depolarization resulting in a more variable, but on average slightly decreased, input resistance during up states.

Effect of up states on cortical FP (population) responses

The previous results indicate that input resistance is moderately decreased during Up states. This raises the question about how afferent synaptic inputs onto cortical cells may be affected by up states. To study this question, electrical stimulation was delivered via two different stimulating electrodes in the VB thalamus or in the cortex during down states and during the occurrence of spontaneous up states. To avoid stimulating thalamocortical fibers within the neocortex, the cortical stimulating electrode was placed in the upper layers, between layers II and III, and 0.5–1 mm away from the FP and whole cell recording electrodes. To deliver the stimuli during the up states, an analog threshold detector was used to detect the up states on-line from the FP recording. As described in methods, all stimuli were classified off-line to assure that they indeed occurred during up or down states. In these experiments, the stimulus intensity was adjusted so that measurable EPSP and FP responses were evoked from each of the electrodes. This meant that moderate intensity (20–60 μA) cortex and thalamus stimulation were used. As previously described (Rigas and Castro-Alamancos 2007), moderate intensity stimulation of the thalamus evokes a short-latency excitatory response that peaks between 3 and 6 ms. This short-latency thalamocortical response is followed by a long-latency (>10 ms) thalamocortical response that corresponds to an evoked up state. Note that measurement of the short-latency EPSP is not affected by the longer latency evoked up state, which occurs later than the peak of the EPSP. As discussed later, measurement of longer-latency thalamocortical inhibitory PSPs (IPSPs) evoked during the down state are obviously affected by the occurrence of long-latency evoked up states. Moderate intensity stimulation of the cortex also evokes a short-latency intracortical EPSP response that peaks between 3 and 8 ms, but as previously described, this response is not followed by up states but instead by a long-lasting IPSP (Rigas and Castro-Alamancos 2007). For simplicity, population analyses of extracellular FP and intracellular PSP responses were conducted separately.

First, we determined the effect of up states on FP thalamocortical responses. During down states, the short-latency thalamocortical FP response consists of a fiber volley component that peaks before 2 ms and is followed by a negative synaptic component that peaks after 2.5 ms (Fig. 3A ). The fiber volley is insensitive to stimulation frequency and glutamate receptor antagonists but is abolished by TTX (not shown). The synaptic component is strongly suppressed by high-frequency stimulation, is abolished by glutamate receptor antagonists (not shown), and corresponds to EPSPs and EPSCs recorded from nearby cells (see following text). We found that during up states, the peak amplitude of the thalamocortical response fiber volley (<2 ms) was not significantly affected compared with the down state (P = 0.8; n = 39). Moreover, the rising slope of the earliest thalamocortical synaptic response component that corresponds to the negativity between 2 and 3 ms poststimulus was also not significantly affected by up states (P = 0.4; n = 39). However, the peak amplitude of the thalamocortical synaptic response component measured between 2.5 and 10 ms was significantly enhanced during up states compared with down states (P < 0.01; n = 39; Fig. 3, A and B). The short-latency thalamocortical response peak enhancement caused by up states consisted in the development of a peak negativity at around 5 ms poststimulus (Fig. 3A). Hence the time to peak of the thalamocortical response shifted by ∼1 ms, from 4.3 ± 0.1 ms during down states to 5.3 ± 0.1 during up states (P < 0.01, n = 39). These results indicate that the early rising slope of the synaptic thalamocortical FP response (2–3 ms) is unaffected by up states, but the later peak amplitude of this response (∼5 ms) is enhanced by up states.

FIG. 3.

Effect of up states on FP responses. A: typical FP traces corresponding to average thalamocortical (TC) and intracortical (IC) responses evoked during down and up states. The baselines of the traces during both states are superimposed for comparison. B: population data showing the effect of up states on the peak amplitude of short-latency responses measured between 2 and 10 ms (TC) and 3 and 10 (IC) ms poststimulus. (means ± SE; *P < 0.01).

Second, we determined the effect of up states on intracortical FP responses. During down states, the short-latency intracortical FP response consists of an initial negative peak at ∼3 ms that reflects a nonsynaptic component (i.e., mix of fiber volley and antidromic spiking) that sums with a rising postsynaptic component (Fig. 3A). After 3 ms, the intracortical FP response reflects a purely postsynaptic component because it is strongly suppressed by high-frequency stimulation, is abolished by glutamate receptor antagonists (not shown), and corresponds to EPSPs and excitatory postsynaptic currents (EPSCs) recorded from nearby cells (see following text). We did not measure the slope of the early synaptic component because, unlike for thalamocortical responses, for intracortical responses, it is in part mixed with the nonsynaptic component, making slope measurements difficult. However, we found that the peak amplitude of the short-latency intracortical FP response measured between 3 and 10 ms was significantly smaller during up states compared with down states (P < 0.01; n = 24; Fig. 3C). The short-latency intracortical FP response suppression caused by up states was accompanied by a significant reduction in the time to peak from 6.8 ± 0.2 ms to 6.1 ± 0.2 (P < 0.01; n = 24). These results indicate that the short-latency intracortical FP response is suppressed by up states.

Effect of up states on evoked spikes

The previous results indicate that up states enhance the later component of short-latency thalamocortical responses (∼5 ms) and suppress intracortical responses. To determine the intracellular correlates of these effects, we performed whole cell recordings from cells located adjacent to the FP recording electrode in layers IV and lower III and measured the impact of up states on evoked spikes and on subthreshold PSPs. The cells described in this section, about the effects of up states on PSPs, were all regular spiking (RS) cells (n = 14). An additional small group of cells (n = 3), that had the defining features of fast spiking (FS) cells (Gibson et al. 1999), are briefly described separately. All the cells studied responded with a fast onset thalamocortical EPSP of <2.5 ms (seemingly monosynaptic). During down states, the membrane potential of the recorded cells was −71.1 ± 1 mV, while during up states, it was −61.1 ± 1 (P < 0.01; n = 17 cells). Spontaneous spike firing measured during a 1-s time window before each stimulus was <0.04 Hz (n = 17) during down states. Up states significantly increased spontaneous firing to 0.97 ± 0.3 Hz in RS cells (P < 0.01; n = 14) and to 7.4 ± 2 Hz in FS cells (n = 3). In this section, we consider the effects of up states on evoked spikes measured with intracellular recordings.

Figure 4 shows a typical example of the effects of up states on short-latency (2–10 ms) thalamo- and intracortical responses in an RS cell. During the down state, the thalamocortical EPSP never evoked a spike (Fig. 4A), even if the stimulation intensity was increased; consistent with previous studies (Cruikshank et al. 2007; Porter et al. 2001). During up states, the thalamocortical response reliably evoked spikes (Fig. 4B), and this was correlated with an enhanced FP response (Fig. 4C). During the down state, the intracortical EPSP can reliably evoke spikes (Fig. 4A), depending on the stimulus intensity. During up states, the latency of the spikes becomes shorter (Fig. 4, A and B) so that longer-latency spikes are absent. The abolishment of longer latency spikes was correlated with a suppressed FP response (Fig. 4C).

FIG. 4.

Effect of up states on PSPs. A: overlaid individual traces of short-latency thalamo- and intracortical evoked responses during down and up states. Note that up states enhanced firing probability of thalamocortical responses and shifted the spike onset of intracortical responses. B: peristimulus time histograms (PSTH) of spikes evoked by thalamo- and intracortical responses during down and up states. C: average PSPs obtained by averaging traces after spikes were eliminated using a median filter. Also shown are the simultaneously recorded FP responses. Average traces and PSTHs were computed from 30 stimulus trials per state.

Figure 5 shows population data on the effect of up states on thalamo- and intracortical evoked spikes. Regarding thalamocortical responses, during down states, RS cells (n = 8) only very rarely (<1%) produced a spike in response to thalamic stimulation within a short-latency time window poststimulus (2–10 ms), while FS cells (n = 3) consistently evoked a spike in response to almost every stimulus (>80%). Up states significantly increased the spike probability of thalamocortical responses during the short-latency time window in both RS cells (P < 0.01; n = 8; Fig. 5B) and FS cells (n = 3). In RS cells, this increase was still significant when the spontaneous firing was subtracted from the evoked response (evoked-spontaneous; P < 0.01; n = 8, Fig. 5B), but this was not the case for FS cells (not shown). Thus RS cells barely trigger any short-latency (<10 ms) spikes during down states but evoked firing increases significantly during up states. FS cells already respond robustly during down states and are less significantly enhanced by up states.

FIG. 5.

Population data showing the effect of up states on evoked spikes. A: PSTHs showing the effect of up states on thalamocortical (n = 8) and intracortical (n = 18) short-latency evoked spikes. B: effect of up states on spike probability measured during a short-latency response time window poststimulus (2–10 ms). Shown is the effect of up states on spontaneous firing (Spont), on evoked responses (Evoked) and on evoked responses in which the spontaneous firing has been subtracted (Evoked-Spont). (means ± SE; *P < 0.01).

The previous results show that up states enhance the thalamocortical response spike probability during a short-latency time window (2–10 ms). Next we considered how using a longer-latency time window (10–50 ms) to measure spike probability may impact the results. We measured thalamocortical evoked spike probability between 10 and 50 ms poststimulus and found no significant difference between down and up states (0.47 ± 0.1 vs. 0.48 ± 0.1 for down vs. up spikes per stimulus; P = 0.8, n = 8). However, when considering these results, it is important to remember that thalamic stimulation delivered during down states triggers up states that fall during the long-latency time window (Rigas and Castro-Alamancos 2007). This explains why the number of evoked long-latency spikes is not different during down and up states; in both states, an up state is present. Based on these results, one may erroneously conclude that up states do not affect thalamocortical responses, but this would be inaccurate since what is being measured is an evoked up state and not the short-latency thalamocortical response. In addition, one may attempt to correct the evoked responses by subtracting the contribution of up state spikes. This could be done by subtracting the number of spikes that occur during a spontaneous up state per se (within a 40-ms window) from the number of spikes that occur 10–50 ms after thalamic stimulation delivered during the up state. If this is done, up states appear to depress long-latency thalamocortical responses (0.47 ± 0.1 vs. 0.0006 ± 0.05 for down vs. up corrected spikes per stimulus; P < 0.01, n = 8), but this is because thalamic stimulation delivered during the down state triggers an up state that falls during the long-latency time window. We believe that such analyses using too wide a time window after the stimulus (with or without spike subtraction) are flawed because they are not measuring the true (i.e., short-latency) thalamocortical response. These considerations may well explain why some have argued that up states produce larger thalamocortical responses during down states and that up states disconnect the cortex from the thalamus (Watson et al. 2008).

Regarding intracortical responses, during down states, intracortical stimulation readily triggers short-latency spikes (2–10 ms). Up states had no significant effect on short-latency firing probability of intracortical responses (P = 0.4; n = 18; Fig. 5B). However, it is worth noting that when the spontaneous firing was subtracted from the evoked response, intracortical responses were close to significantly suppressed (P = 0.05; n = 18; Fig. 5B). Up states consistently reduced the latency of spikes evoked within the short-latency time window (P < 0.01; n = 18; Fig. 5A). Thus spikes evoked by intracortical stimulation shifted to shorter latencies during up states while longer-latency spikes, typical of down states, were abolished.

The previous results show that up states enhance spike firing probability for thalamocortical responses and reduce the spike latency for intracortical responses. We next tested if depolarization alone, equivalent to that produced by up states, was capable of reproducing the effects of up states on spike firing. Figure 6 shows thalamocortical (A) and intracortical (B) responses evoked during down states and during depolarization produced by current injection that mimicked spontaneous up state in those cells. For thalamocortical responses, depolarization was able to reproduce the effect of up states on evoked spikes. Thus thalamocortical responses triggered no spikes during down states but depolarization, equivalent to an up state, increased thalamocortical response spike probability (Fig. 6A). Moreover, the effect of depolarization was specific to the recorded (depolarized) cell because the population FP response was not changed during depolarization trials (Fig. 6, bottom). For intracortical responses, depolarization reduced the onset latency of evoked spikes (Fig. 6B) just like up states do. Similar effects were obtained in several cells (n = 5). These results indicate that depolarization accounts for the major effects of up states on evoked spikes.

FIG. 6.

Effect of depolarization during down states on evoked spikes. Overlaid individual traces of short-latency thalamocortical (A) and intracortical (B) evoked responses during down states and during depolarization produced by current injection equivalent to a spontaneous up state in the recorded cells. Bottom: a PSTH of evoked spikes and FP responses.

Effect of up states on subthreshold PSPs

We next considered the effects of up states on subthreshold EPSPs. In this case, we measured only responses that evoked no spikes during either down or up states within a poststimulus time window of 2–10 ms. Thalamocortical EPSPs had a short-latency onset of 2.15 ± 0.08 ms (n = 14), while intracortical EPSPs had a slower onset of 3.08 ± 0.1 ms (n = 17). During up states, thalamo- and intracortical EPSPs were suppressed (Fig. 7A; P < 0.01). This is expected due to a reduction of EPSP driving force caused by depolarization and also due to a shunt caused by a reduction in input resistance as described above. However, while intracortical EPSPs were reduced by 92 ± 7%, thalamocortical EPSPs were reduced by only 45 ± 8% (Fig. 7B). Moreover, depolarization alone suppressed both thalamo- and intracortical EPSPs similarly, and this effect is similar to the percent suppression observed on thalamocortical EPSPs (Fig, 7, A and B). To assure that the differential effects of up states on thalamo- and intracortical subthreshold EPSPs was not due to a difference in the amplitude of evoked EPSPs, we compared well matched EPSPs (∼3-mV amplitude during the down state) for both pathways (n = 6 cells) and still found that intracortical EPSPs were significantly more suppressed by up states than thalamocortical EPSPs (94 ± 8 vs. 40 ± 9%, respectively). Thus a reduction in driving force and input resistance may explain part of the suppression of thalamo- and intracortical EPSPs during up states. However, if we assume that thalamo- and intracortical synapses are spatially located in dendritic regions that are similarly affected by synapses active during up states, then driving force and input resistance changes caused by up states cannot explain the stronger suppression of intracortical EPSPs compared with thalamocortical EPSPs.

FIG. 7.

Effect of up states and depolarization on subthreshold excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs). A: average intracellular and FP traces evoked during down states (black), up states (red), and depolarization (green) for thalamo- and intracortical responses. The baselines are overlaid for comparison. B: effect of up states and depolarization on the peak amplitude of subthreshold EPSPs measured between 2 and 10 ms poststimulus. The EPSP amplitude is plotted as a percentage of the down state response (means ± SE; *P < 0.01). C: effect of up states and depolarization on the peak amplitude of IPSPs measured between the peak of the EPSPs and 50 ms poststimulus (means ± SE; *P < 0.01).

Finally, we determined the effects of up states on the long-latency membrane potential that follows the short-latency excitatory response. For simplicity, we term this measure IPSP, but we note that the long-latency thalamocortical responses measured during down states are contaminated by evoked up states. IPSPs were measured as the peak hyperpolarization in a time window between the peak of the short-latency EPSP and ≤50 ms poststimulus. During down states, the short-latency thalamocortical EPSP was followed by up states at longer latencies (>10 ms), as previously described (Rigas and Castro-Alamancos 2007), and there was little evidence of an evoked hyperpolarization, such as an IPSP (Fig. 7A). In contrast, the intracortical EPSP evoked during down states was not followed by an up state (Rigas and Castro-Alamancos 2007) but was instead followed by a hyperpolarizing potential, akin to an IPSP (Fig. 7A). During up states, IPSP driving force increases due to depolarization; this is clear by comparing down (black) and Depol (green) intracortical responses in Fig. 7C (IPSP amplitude increases with depolarization alone). Thus a larger amplitude IPSP was observed to follow both thalamo- and intracortical EPSPs during up states (Fig. 7A). However, the intracortical IPSP was still larger in amplitude than the thalamocortical IPSP (Fig. 7, A and C). Depolarization was able to closely reproduce the effects of up states on intracortical IPSPs, but only partially on thalamocortical IPSPs (Fig. 7, A and C). This is because up states are still being triggered in the network by thalamocortical stimulation, so that the recorded (depolarized) cell is still producing an up state on top of the larger IPSP driving force caused by depolarization.

Effect of up states on synaptic currents

Our measurements of thalamo- and intracortical EPSPs indicate that they are differentially affected by up states. Intracortical EPSPs are more suppressed than thalamocortical EPSPs. However, up states confound the measurement of synaptic strength because the membrane potential, and thus driving force, changes during current clamp. To determine if up states affect synaptic strength, we measured thalamo- and intracortical EPSCs with whole cell voltage-clamp recordings, which keep the voltage stable during up states. Simultaneous FP recordings were also performed. To assure that voltage clamp was maximally effective, we filled the cells with a Cs⁺ and QX-314-based solution, which makes the cells much more compact and somewhat more amenable to voltage clamp (Williams and Mitchell 2008). In addition, to assure that both thalamo- and intracortical EPSCs were under voltage-clamp control, we confirmed that voltage steps >0 mV were effective at reversing evoked EPSCs. As described in the following text, an additional concern with the following results arises from the possibility that thalamo- and intracortical synapses are localized at significantly different distances from the electrode. More distally located synapses will be less clamped and more affected by driving force changes caused by depolarization. Thus during all these experiments we conducted simultaneous FP recordings so that we could compare the population extracellular current reflected in the FP recordings with the intracellular current.

Figure 8 A shows a typical recording from one cell that was held at −75 mV; its resting membrane potential recorded in current clamp immediately before switching to voltage clamp. EPSCs were measured as the peak inward current during a 2- to 10-ms time window poststimulus. We found that thalamocortical EPSCs were not significantly suppressed by up states (P = 0.3; n = 12; Fig. 8B), whereas intracortical EPSCs evoked in those same cells were significantly suppressed by up states (P < 0.01; n = 12; Fig. 8B). These results indicate that thalamocortical synaptic strength is not affected by up states, but intracortical synaptic strength is suppressed by up states. Another interpretation of these results is that intracortical synapses are more distal and less clamped than thalamocortical synapses. While this is possible, it is worth noting that the EPSCs behaved as predicted from the FP responses. Regarding thalamocortical responses, the early rising component of the FP, which is unaffected by up states (Fig. 3), was correlated with the rising EPSC, which was also unaffected by up states. Also, as expected, the late component of the FP (∼5 ms), which is enhanced by up states and corresponds to increased spike probability (Figs. 4 and 5), was not corresponded with an enhanced late PSC component because sodium currents are blocked (QX-314), and they would be clamped anyway. Thus the late (∼5 ms) thalamocortical FP response component that is enhanced by up states reflects a population spike (i.e., summed action potentials of thalamocortical recipient cells) and also, perhaps, the local postsynaptic response of intracortical synapses driven by the increased firing of thalamocortical recipient cells (i.e., polysynaptic response) during up states. Regarding intracortical responses, the suppression of the peak amplitude of the FP response (>3 ms) was correlated with the suppression of the EPSC. In conclusion, assuming that intracortical synapses are equivalently clamped as thalamocortical synapses, the effect of up states on intracortical responses is independent of changes in driving force caused by depolarization.

FIG. 8.

Effect of up states on synaptic currents. A: typical example of the effect of up states on evoked synaptic currents recorded in voltage clamp. Average postsynaptic current (PSC) and FP traces evoked during the down and up states are overlaid for comparison. B: population data showing the peak amplitude of the EPSC measured 2–10 ms poststimulus during down and up states (means ± SE; *P < 0.01).

What suppresses intracortical synaptic inputs?

Thalamo- and intracortical excitatory synapses reaching layer 4 of somatosensory cortex are known to depress with increased activity both in slices and in vivo (Castro-Alamancos 1997; Castro-Alamancos and Connors 1996; Gil et al. 1997). We hypothesize that enhanced presynaptic intracortical activity during up states depresses intracortical synaptic strength, and this does not occur for thalamocortical responses because of the lack of major reciprocal connections between cortex and thalamus in the slice. For cortical up states to enhance presynaptic thalamocortical activity, the slice must have intact corticothalamic fibers that drive thalamocortical cells that have intact thalamocortical fibers reaching the cortex. To test these hypotheses, we conducted additional experiments.

First, we measured the effect of up states on paired-pulse responses. If presynaptic intracortical activity during up states depresses intracortical synaptic transmission, we would expect an increase in the paired-pulse ratio during up states (Castro-Alamancos and Connors 1997a). Figure 9A shows paired-pulse ratios during down and up states for intracortical FP responses measured as the peak amplitude between 3 and 10 ms poststimulus. Paired-pulse intracortical responses produced with a 100-ms interstimulus interval showed significantly less depression (or more facilitation) during up states than during down states (P < 0.01; n = 5). The effect of up states on paired-pulse ratio was similar for the second pulse and the fourth pulse in a 10-Hz train (Fig. 9A). This is consistent with a decrease in neurotransmitter release for intracortical responses during up states. However, we caution that paired-pulse data in intact networks (with inhibition intact) can be difficult to interpret; for example, due to the interplay between excitation and inhibition (Castro-Alamancos 1997). We did not test paired-pulses on thalamocortical responses because the first stimulus always evokes an up state, which affects the second stimulus, and impedes paired-pulse measurements during the down state.

FIG. 9.

Up states increase paired-pulse ratios of intracortical responses. A, top trace: overlaid FP responses evoked by intracortical stimulation consisting of a pair of pulses with a 100-ms interstimulus interval during down and up states. Bottom: population data of paired-pulse ratios calculated from the 2nd or the 4th stimulus in a 10-Hz train (means ± SE; *P < 0.01). B: example of retrogradely labeled thalamic cells after iontophoretic application of neurobiotin in the cortex of a thalamocortical slice. Four closely spaced injections were done in layer 4 around the area that produced the largest FP responses evoked by electrical stimulation of the thalamus. In all cases, cortical neurobiotin either labeled no cells or only a small cluster of cells in ventrobasal thalamus (see arrow). This example was the largest thalamic labeling observed.

Second, we addressed the integrity of thalamocortical connections between thalamus and cortex in the slice. If these connections are mostly severed, it would favor the argument that presynaptic thalamocortical activity is mostly mute in the slice, and this protects thalamocortical inputs from activity-dependent depression. Previous work in thalamocortical slices has shown that some thalamic cells in the slice receive intact inputs from corticothalamic cells and are driven by cortical up states, while other thalamic cells have intact thalamocortical axons that can trigger cortical up states when depolarized with glutamate puffs in the thalamus (Rigas and Castro-Alamancos 2007). However, the number of thalamic cells that receive inputs from corticothalamic cells and also send thalamocortical fibers that contact cortical cells appears to be quite small or inexistent in most thalamocortical slices based on two observations.

The first observation is that glutamate puffs applied in the ventrobasal thalamus only evoke cortical responses in fewer than half (42%; 20 of 47 slices) of thalamocortical slices that evoke cortical responses to electrical stimulation of the thalamus. Moreover, glutamate puffs are only effective at triggering cortical responses within a very small (limited) region of the ventrobasal thalamus in each slice; i.e., small displacements of the glutamate pipette within the ventrobasal thalamus eliminate the effect of glutamate on cortical responses (Rigas and Castro-Alamancos 2007). This indicates that in a thalamocortical slice few thalamic cells have intact thalamocortical axons reaching the cortex. Thus electrical stimulation mostly activates severed axons that contact cortical cells.

A second observation supported this conclusion as well. In several thalamocortical slices (n = 14) that produced effective cortical responses to electrical stimulation of the ventrobasal thalamus, we applied neurobiotin using iontophoresis in the middle layers of somatosensory cortex to determine if cells bodies would be retrogradely labeled in the ventrobasal thalamus. Multiple closely spaced injections (3–4) were made per slice to label a significant portion of the somatosensory cortex, and these were centered on the cortical areas that produced the strongest thalamocortical FP responses in those slices. Consistent with the glutamate puff results, we found that only 36% (5 of 14) of thalamocortical slices (responsive to electrical stimuli) had retrogradely labeled thalamocortical cells. Moreover, in all the cases there was only a very small cluster of cells that were labeled; a typical case is presented in Fig. 9B. Based on these observations we conclude that reciprocal connections between thalamus and cortex are limited in the thalamocortical slice. Therefore presynaptic thalamocortical activity driven by up states is mostly mute in the slice, particularly compared with the situation in vivo. In contrast, presynaptic intracortical activity during up states is obviously enhanced in the slice, as this is what allows up states to ensue.

DISCUSSION

We studied the impact of spontaneous up states on input resistance and on synaptic inputs in cortical cells. Up states produced a relatively small reduction in input resistance because the high conductance resulting from synaptic bombardment is counteracted by an increase in input resistance due to depolarization. In addition, up states affected differently thalamo- and intracortical synaptic inputs. Up states enhanced the late component of short-latency thalamocortical responses (∼5 ms) but suppressed short-latency intracortical responses. The thalamocortical enhancement was due to an increase in spike probability caused by depolarization and not by an increase in synaptic strength. The intracortical suppression reflected a shift to shorter-latency spikes caused by depolarization, a concomitant abolishment of longer-latency spikes attributable to an enhanced evoked IPSP driving force also caused by depolarization, and an input-specific reduction in synaptic strength caused by activity.

Effect of up states on input resistance

Due to their nature, up states are associated with cortical cell firing, which implies that synaptic activity is pervasive during up states. Although, the absolute estimates of conductance changes can vary considerably, there is good agreement that up states are associated with an enhancement of synaptic conductance (Destexhe et al. 2003; Haider et al. 2006; McCormick et al. 2003; Shu et al. 2003b; Waters and Helmchen 2006). However, there is less agreement about the nature of the synaptic conductance change. One group reports that up states consist of balanced inhibition and excitation (McCormick et al. 2003; Shu et al. 2003b). Others argue that the ratio favors excitation, and that inhibition is sparse (Waters and Helmchen 2006). Furthermore, others argue that the ratio favors synaptic inhibition during up states, so that up states can be defined as high-conductance states dominated by inhibition (Rudolph et al. 2005).

Apart from the enhanced synaptic conductance, up states are associated with several additional changes in cortical cells. Most notably, depolarization by 5–15 mV and an increase in membrane potential variance or noise. Depolarization per se increases the excitability of cortical cells making cells more responsive to current pulses. Moreover, injection of a small balanced excitatory and inhibitory synaptic conductance that increase membrane potential variance has been shown to produce a sharp increase in cell excitability that mimics changes observed during up states (Chance et al. 2002; McCormick et al. 2003), but these effects can depend on the duration and amplitude of the test stimulus (Shu et al. 2003a).

The mechanism underlying the effect of synaptic noise and depolarization on cell excitability is unclear. A recent study (Waters and Helmchen 2006), has proposed that the reason why changes in synaptic conductance and depolarization lead to enhanced excitability is because these changes are opposed by inward rectification (Connors et al. 1982; Stafstrom et al. 1982), resulting in a net increase in input resistance during up states. Basically, in many cortical cells, slope conductance is not linear but negative, and this can be explained because of activation of voltage-dependent noninactivating inward currents during subthreshold depolarization which sum with outward currents. The increased input resistance triggered by depolarization during up states makes these cells more compact and counteracts synaptically induced conductance increases.

Our results show that up states produce on average only a relatively small reduction in input resistance because depolarization alone leads to increases in input resistance. Thus the synaptic conductance increases during up states are counteracted by the input resistance increase due to depolarization. The net effect is on average only a relatively small change in input resistance during up states. However, we also note that the coefficient of variation of input resistance measurements was very large during up states, which implies that there may be different excitability states during up states ranging from high to low conductance depending on a variety of factors. In essence, our results indicate that excitability during up states is fairly dynamic because intrinsic cellular mechanisms counteract the shunt produced by enhanced synaptic conductance during up states.

What suppresses intracortical but not thalamocortical responses during up states?

The relatively small conductance increase during up states presumably shunts equally thalamo- and intracortical inputs. Consequently, what explains the differential effects of up states on these responses? There are three additional effects of up states on evoked synaptic responses. First, depolarization during up states makes EPSPs reach threshold faster and more successfully, which leads to faster onset spikes for intracortical responses and increased spike probability for thalamocortical responses.

Second, evoked IPSPs have larger amplitudes during up states because depolarization moves the membrane away from the reversal potential for IPSPs increasing their driving force. The IPSPs can inhibit excitatory responses that occur within the IPSP time window, such as long-latency intracortical spikes and intracortical EPSPs that have longer onset latencies. Indeed we found that longer-latency intracortical spikes were abolished during up states. Thalamocortical EPSPs are less vulnerable to this inhibitory effect because of their faster onsets (Gabernet et al. 2005). Also, intracortical IPSPs have larger amplitudes than thalamocortical IPSPs, which may lead to stronger suppression of intracortical EPSPs. The cause of the larger amplitude of intracortical IPSPs is currently unclear. It may be due to the fact that the intracortical electrical stimulus directly discharges inhibitory interneurons close to the stimulating electrode.

Finally, it is well accepted that many intracortical excitatory synapses show short-term activity-dependent depression (Castro-Alamancos 1997; Castro-Alamancos and Connors 1997a; Markram et al. 1998; Thomson and Deuchars 1997). We reason that enhanced presynaptic intracortical activity during up states depresses intracortical synaptic strength, and this does not occur for thalamocortical responses because of the lack of essential connections between cortex and thalamus in the slice, based on the effects of glutamate puffs and retrograde labeling. Thus presynaptic thalamocortical activity driven by up states is mostly mute in the slice, particularly compared with the situation in vivo. Presynaptic intracortical activity during up states is obviously enhanced in the slice, as this is what allows up states to ensue.

We conclude that activity-dependent synaptic depression of intracortical connections and enhanced evoked inhibition lead to intracortical suppression during up states. However, synaptic depression and enhanced inhibition are counteracted by depolarization during up states, resulting on balance in faster intracortical evoked spikes with similar probability compared with the Down state. In addition to the previously mentioned actions of up states, it is also possible that up states affect differentially thalamo- and intracortical responses because of the different dendritic location of these synapses on cortical cells. Future morpho- and electrophysiological work will be needed to address this possibility. It is also worth emphasizing that our results do not imply that thalamo- and intracortical synapses are intrinsically differently sensitive to up states. Instead, our results indicate that the responses they trigger in cortex are affected differently by up states because of other factors, such as the different presynaptic activity that they experience during up states (i.e., in the slice presynaptic thalamocortical activity is mute) and because of differences in the responses they recruit during down states (e.g., in the slice short-latency spikes are not evoked by thalamocortical stimulation in RS cells during down states but are readily evoked by intracortical stimulation).

Functional consequences of thalamocortical enhancement

In vivo, up states (Petersen et al. 2003; Sachdev et al. 2004) and activated states (Castro-Alamancos and Oldford 2002) lead to the suppression of sensory responses in rodent barrel cortex. In slices, we found that thalamocortical responses are instead enhanced by up states because depolarization brings ineffective thalamocortical EPSPs closer to firing threshold. It is important to note that thalamocortical responses recorded in slices differ considerably from sensory responses recorded in the barrel cortex in vivo. Based on our experience with both methods, there are two major differences. 1) In slices, thalamocortical responses rarely reach firing threshold during down states. However, in vivo, either thalamic electrical stimulation or sensory stimulation drive robust thalamocortical responses that effectively reach firing threshold even in urethane-anesthetized or sleeping (quiescent) rats (Castro-Alamancos and Oldford 2002; Hirata and Castro-Alamancos 2006, 2008). Thalamocortical EPSPs may be unable to reach firing threshold during down states in the slice because many of the thalamocortical synapses innervating a particular cortical cell are severed and not activated by the thalamic electrical stimulus. This argument makes sense in light of the need for thalamocortical convergence to trigger cortical spikes in vivo (Bruno and Sakmann 2006; Hirata and Castro-Alamancos 2008). 2) In slices, down states consist of long hyperpolarizations that are not typically observed in vivo during normal conditions. For example, during normal urethane anesthesia levels, which we call quiescent states, cortical cells do not undergo long hyperpolarizations, such as the steady down state periods observed in the slice (A. Hirata and M. A. Castro-Alamancos, unpublished observations); typically ketamine is used on top of urethane to attain longer down states in vivo (e.g., Hasenstaub et al. 2007). It is possible that the steady down states found in slices are more akin to a very deep state of anesthesia in vivo during which all activity is suppressed (e.g., burst suppression).

Our results indicate that up states have a facilitating effect on thalamocortical responses. This conclusion further raises the question about why sensory responses are suppressed by up states and activated states in vivo. The present slice study cannot directly answer that question, but we have proposed that sensory responses are suppressed because of increased thalamocortical firing that depresses thalamocortical connections (Castro-Alamancos and Oldford 2002). The fact that up states per se are facilitating supports this hypothesis. Limited reciprocal connections between thalamus and cortex in the slice assure that thalamocortical responses are not suppressed because there is little presynaptic thalamocortical activity during up states. Moreover, sensory responses in vivo effectively drive intracortical responses of thalamocortical-recipient cells (e.g., between layers 4 and 2/3), which are vulnerable to the intracortical suppression we found in the slice during up states. Thus intracortical suppression may also contribute to the suppression of long-latency sensory cortical responses during up and activated states in vivo.

GRANTS

This work was supported by the National Institutes of Health.