Frequency (khz) Intensity (db SPL) Frequency (khz)

Transcription

1 a Before After No. of spikes b No. of spikes Time (ms) 5 5 Time (ms) Intensity (db SPL) Intensity (db SPL) Frequency (khz) Frequency (khz) Spike no. Spike no. 8 c Percent change BW6 * No. of Spikes Supplementary Figure. Effects of SCH59 on cortical responses. Tone-evoked spiking activity and spike TRF recorded from the same cortical site before (a) and after (b) cortical injection of SCH59 alone. Left, Peri-stimulus spike-timing histogram (PSTH). Right, spike TRF presented as a gray-scale map. (c) Percentage change in the bandwidth of spike TRF (measured at 6dB) and in the number of tone-evoked spikes (at 6 db) after SCH59 application. N 5 sites. Bar SD. Paired t-test, * P<..

2 Intensity (db SPL) Spike No. # 4 Frequency (khz) Supplementary Figure. Intensity threshold of spike TRFs. Multi unit spike TRF before cortical silencing recorded from the same site as the example cell in Figure. Arrow indicates the level of intensity threshold for subthreshold responses of the example cell, which is db SPL below the spike TRF.

3 Frequency range (octave) Before After Intensity (db SPL) Supplementary Figure 3. Responding frequency ranges after cocktail application. Frequency range for excitatory synaptic responses before (blue) and after (red) the cocktail application. Data are grouped according to the tone intensity. N 5 cells. Bar SD.

4 Cortex Intracortical Sum Thalamocortical Thalamus Supplementary Figure 4. A proposed model of cortical circuitry underlying excitatory synaptic TRFs in layer IV. The recorded cortical neuron receives broadly tuned thalamocortical input and narrowly-tuned intracortical input from other cortical neurons with a similar response property. The summed tuning curve is sharper than that of thalamocortical input.

5 a b V act (mv) g m e-5 S/cm g m e-4 S/cm I syn / I syn µm µm Synapse location (µm) g max (µs) Supplementary Figure 5. The modeling of membrane leakage effect on recorded synaptic currents and holding potentials. (a) The actual holding potential along the dendrite V act is not significantly changed by the increase of leakage conductance and the reasonable holding condition is maintained for voltage clamping recording. (b) The attenuation (I syn /I syn ) caused by the increase of leakage conductance changes only slightly with the change of synaptic current amplitude. g max is the maximum synaptic conductance for alpha synapse. Synaptic sites at µm and µm from the soma were calculated.

6 a mv -3 mv -7 mv - mv 65 pa 5 ms b r Synaptic current (pa) Membrane potential (mv) Supplementary Figure 6. The I V curve after cocktail application. (a) Synaptic currents (average of four repeats) evoked by a tone of.9 khz at 7 db, recorded at different holding potentials: mv (cyan), -3 mv (red), -7 mv (green) and - mv (blue). (b) I V curves (after correction) for synaptic currents averaged within a.5 ms window after the stimulation onset. Bar SD.

7 Before correction After correction V h (mv) V act (mv) Cell I' syn (na) I syn (na) V h (mv) V act (mv) Cell I' syn (na) I syn (na) V h (mv) V act (mv) Cell I' syn (na) I syn (na) Supplementary Figure 7. The correction of I V curves of three recordings. Left column are the I V curves before correction, which do not pass the origin. Right column are the I V curves after correction, whose x intercepts are very close to the origin, which indicate that the cells were reasonably clamped under our experimental condition. Each row represents one cell. Bar SD.

8 Supplementary Table. Derived whole-cell parameters of three recordings. Cell (77) Cell (56) Cell 3 (66) g r (before) 6.96 ns 8.9 ns 9.4 ns g r (after) 9.44 ns 9.9 ns 7. ns g r.48 ns ns 7.7 ns R in (before) 44 MΩ MΩ 6 MΩ R in (after) 6 MΩ MΩ 58 MΩ R in 38 MΩ MΩ 48 MΩ Rs(before) MΩ 7 MΩ 8 MΩ Rs(after) 3 MΩ 4 MΩ 3 MΩ R s 9 MΩ 3 MΩ 5 MΩ

9 Defining Cortical Frequency Tuning with Recurrent Excitatory Circuitry Baohua B. Liu *,, Guangying K. Wu *,3, Robert Arbuckle #, Huizhong W. Tao,4 and Li I. Zhang, Note I. Derive the application concentration for mixed SCH 59 and muscimol. Equations for the competitive binding with GABA receptors A Gb A Gb [ A Gb] [ A] [ Gb] [ ] () B Gb B Gb A Ga A Ga B Ga B Ga [ Gb] [ Gb] [ ] B () [ Ga] [ A] [ Ga] [ ] A (3) [ Ga] [ Ga] [ ] B (4) A: SCH95; B: muscimol; Gb: GABA B receptor; Ga: GABA A receptor. [A], [B], [Ga] and [Gb] represent the concentration of A, B, Ga and Gb, respectively. Here, and refer to functional binding constants. EC 5 or IC 5 values were recovered from the literatures, and used to calculate K values, as we considered functional effects of bindings: opening or blocking channels: µ M (Ref and ) 5µ M (Ref 3) (Ref 4).7µ M was estimated according to the instruction that SCH59 has no binding affinity to GABA A receptors at concentration up to µm (Tocris). To estimate the value of, we assumed that less than % of GABA A receptors are bound by SCH59 at µm. According to equation (3), and setting [Ga total ] [Ga] [A Ga], [ A Ga] [ Ga ] total [ A] [ A] 9µ M.

10 . Estimate the activation effect of muscimol application alone Derived from the two reactions ( and 4): [ B Gb] [ B Ga] [ Gb] [ Ga] (5) (6) At the effective silencing concentration of muscimol ( 5 µm, Ref. 5), [ B Gb] [ Gb] [ B Ga] [ Ga] 5µ M 5µ M.7µ M 5µ M 5% 94% Thus, considerable portion of GABA B receptors will be activated at the effective concentration of muscimol for activating GABA A receptors..3 To derive the ratio of SCH59 and muscimol for effectively activating GABA A receptors, while blocking GABA B receptors Derived from equations () (4), we can obtain: [ A Gb] [ B Gb] [ A Ga] [ B Ga] [ A] [ Gb ] total [ A] [ Gb ] total [ A] [ A] [ Ga ] total [ A] [ Ga ] total [ A] (7) ( [ ] [ Gb] [ A Gb] [ B Gb] (8) Gb total ) (9) ( [ ] [ Ga] [ A Ga] [ B Ga] () Ga total )

11 (i) To achieve that less than 5% of GABA B receptors are activated by muscimol. From eq (8), we can derive [ B Gb] [ Gb ] If [ B Gb ] [ ] Gb total [ A] 5 [ A] total 5.5, then 5 [ ] 5 A [ A ] As [A]>>µM in its application, , or.95 [ B ].5 ( [ A] ) (ii) To achieve that the majority GABA A receptor will not be blocked by SCH59. In other words, () less than 5% of GABA A receptors are bound with SCH59. From eq (9), we can derive [ A Ga] [ Ga ].95 total [ A] [ A ] 45 6 [ A] 9 [ A] 9 [ A].7.5 As effective [B] (µm) is around 5, 6 [ B ] >> 45, then.95 [ A ] 6 [ A] () Combining eq () and eq () together, we get [ ] [ ] A B (3).4 Activation of GABA A receptors under our experimental condition At the ratio used in the current project ([ A ] 3/ ), and considering muscimol concentration at 5 µm, the effects on GABA A receptors would be [ B Ga] [ Ga ] [ A].7µ M.7µ M.5 total 9µ M 93% i.e. more than 93% of GABA A receptors will be activated.

12 Note II. Space clamp and effects of cortical cocktail application on synaptic responses Here, we want to provide a quantitative estimation of nonspecific effects of drug application on voltage clamp and synaptic currents.. Correcting amplitude of synaptic responses according to changes in presumptive pure thalamic inputs In this study, we estimated the level of nonspecific reduction in the recorded synaptic responses after drug application according to changes in the presumptive pure thalamocortical inputs at the subthreshold intensity threshold. The assumption is supported by two lines of evidence: first, the multiunit TRF recorded at the same site before silencing has higher intensity threshold (Supplementary Fig. ), suggesting that the synaptic inputs at the subthreshold intensity threshold are unlikely contributed by local intracortical connections; second, the kinetics of response currents (Fig. d and Fig. 3a, c, e, g) also supports this assumption. We analyzed the kinetics of the rising phase of response currents before and after cocktail microinjection. The kinetics of an input consisting of both thalamocortical and intracortical components is likely characterized by two or multiple phases due to differences in the onset timing: an early fast rising phase representing the monosynaptic thalamic inputs, and later phases for integrated local and thalamic inputs. On the other hand, there will be one phase for pure thalamic inputs. For all the five cells obtained, we averaged inputs evoked by best frequencies at 6 db SPL, as well as at the intensity threshold. As shown in the figures, the inputs at 6 db are clearly bi phased before cortical silencing and become mono phased after drug application, suggesting that the later phase can be attributed to intracortical inputs. On the other hand, those at the intensity threshold remain mono phased before and after silencing, consistent with the assumption.. Can isopotential of the cell for the recorded synaptic inputs be achieved in our voltage clamp recordings? In this study, we have assumed linear, isopotential neurons for the recorded excitatory synaptic

13 inputs, as described in previous studies 6-9. Potential deviations due to space clamp error and cable attenuation for synaptic inputs at the distal dendrites were discussed in several recent studies 7-9. Here, we provide further estimation of our space clamp before and after drug application: First, cortical cells appear to be reasonably clamped under our experimental condition. This is supported by the linearity of I V curves (Fig. b) and the fact that when cells were clamped at mv, no significant excitatory currents were observed (Fig. b), except the outward Cl - mediated currents. In addition, the derived reversal potential for the early component of tone evoked currents ( ms window after response onset, mainly excitatory) was ± 5mV (Fig. b), close to the known reversal potential for glutamatergic currents. These data suggest that under our voltage clamp recording conditions, the tone evoked synaptic currents can be detected with a reasonable clamping accuracy, with a deviation within 5 mv. This may be attributed to the use of intracellular cesium, TEA, QX 34, and ketamine anesthesia, which together block most voltage dependent currents (though K and Na channels, and NMDA receptors). Furthermore, the overlap of the frequency range for excitatory response with that for tone evoked membrane potential depolarization (Fig. 4b, right) suggests that excitatory responses recorded at -7mV reflect the effective inputs made onto the recorded cell, consistent with our previous observation 9. Second, the reasonable voltage clamp can be achieved in the dendrites close to soma (<µm), as simulated with a Neuron model (ref, downloaded from neuron.duke.edu). Model description: Equivalent cylinder neuron model We used simplified equivalent cylinder model, which consists of a cylinder of micron length and micron diameter as soma and a cable of micron length and.8 micron diameter as dendrite. Other physical parameters used in the simulation were the specific intracellular resistivity R a

14 Ω cm, the specific membrane capacitance C m µf/cm, the specific leakage conductance at normal condition g m e-5 S/cm, resting potential E r -6mV. The soma was clamped at -7mV, which is the holding potential used in the experiment. We used alpha synapse, whose conductance ( t / τ ) has the form of g g ( t / τ e, to simulate the synaptic input. τ was set as 4ms, max ) g max as.5 µs, and the excitatory reversal potential (E e ) as mv. In this model, we estimated the effects of increasing leakage conductance by muscimol, by assuming g m e-4 S/cm after drug application. As shown in Supplementary Fig. 5a, the synapses located micron away from the soma can be clamped well, which is not significantly affected by the increase of membrane leakage. This model study suggests that the tone evoked synaptic inputs we examined may be from dendritic regions close to soma, which had been reasonably clamped. Third, I V curve remains linear and crosses the origin ( ). An example I V curve obtained from a neuron after cortical silencing is shown (Supplementary Fig. 6). In this case, both clamping voltages, synaptic currents, and a junction potential of mv were corrected according to the discussion below..3 The potential effects of cocktail drug application on voltage clamp and synaptic currents Two effects were observed for cocktail drug application: ) the drug microinjection caused gentle mechanical effects, resulting in an increase of series resistance (R s ) after application (see the table below), which then remained stable (with change <%); ) Muscimol in the cocktail increases the leakage of the cell by constitutively activating GABAa receptors, as reflected by an increase in leakage current (g leak ). Both effects will affect I V curve. However, they can be corrected given that isopotential condition for voltage-clamping is maintained, which is supported by linear I V curve after drug application and the simulation with increased membrane permeability. Here, we first derived the effects of drug application on clamping voltage (V h ) and synaptic currents (I syn ):

15 i. Derivation of equations Simplified isopotential neuron model Based on the isopotential neuron model, the actual voltage applied on the soma V act can be expressed by equation (). V h is the holding potential provided by the amplifier; I e is the recorded current through the electrode, which is the sum of real synaptic currents I syn and leakage currents I r ; R s is the series resistance. The amplitudes of I syn and I leak (inward current) are determined by the equation (3) and (4) respectively. g syn is the synaptic conductance and g r is the leakage conductance; E syn is the synaptic reversal potential (here mv for excitatory synapse) and E r is the resting potential; R in is the input resistance. V act ( t) V I ( t) R h e s () I ( t) I ( t) I ( t) () I I e syn syn syn leak ( V ( t E ) ( t) g ( t) ) (3) act ( V ( t E ) leak ( t) g r * act ) r ; g r / Rin syn (4) Thus, the V act in the presence of synaptic inputs will be significantly different from that in the absence of synaptic inputs, and both the clamping voltage and the measured synaptic currents need to be corrected according to the change of V act. Without correction, the measured synaptic current is simply derived by: I syn (t) I e (t) I leak (t), which does not consider that I leak changes with input current. Using the equations () (4), we can derive the relationship between I syn and I syn as following.

16 I syn Rin Rs ( t) I' syn ( t) (5) R And the relationship between V h and I syn, V I t in R R R R R in s in s h ' syn ( ) * s syn R in R ( ) in g Rs t R in As E syn mv, comparing equation (3) and (6), the curve (V act I syn ) will cross the origin ( ), while the curve (V h I syn ) will not. E r R in R R in s E syn (6) ii. Calculation of input resistance (R in ) from our experiments Based on the value of I leak, we can estimate leakage conductance and input resistance according to Equation (4). By holding at two different voltages V act and V act, and measuring the corresponding leakage currents I leak and I leak, then we can calculate R in. g r ( I I ) ( V V ) (7) leak leak act act.4 Correction of synaptic currents according to changes in R in and R s (Supplementary Table ) For three of the five cells in this study, we applied two clamping voltages after cocktail application. I syn can then be corrected by the factor (R in R s )/R in. For these three cells, the correction factor based on presumptive pure thalamic inputs (which is what we used) is.66 ±.3, while it is.36 ±.7 if based on the changes in R in and R s, which contributes to 73% of the correction we used (see.6 below for further discussion on other potential non specific effects)..5 Correction of I V curves after drug application According to equations () and (5), we calculated V act and I syn, and replotted the curves of V act vs I syn (Supplementary Fig. 7, right column), and compared them with the V h I syn curves (Supplementary Fig. 7, left column). After corrections, the I V curves almost pass the origin, which also confirms that ) reasonable clamping is conserved; and ) isopotential model is valid..6 Other potential nonspecific effects of drug application i. Change in cable effects due to increase in membrane permeability

17 The attenuation of the recorded synaptic currents caused by the increase of membrane permeability is a function of synaptic position, and increases as the synapse becomes farther away from the soma. When the synapse is at micron distance from the soma, this gives about % decrease in measured peak current. Moreover this attenuation is only slightly affected by the increase of amplitude of synaptic currents (Supplementary Fig. 5b). ii. The pharmacological methods are highly dependent on the specificity of the pharmacological agents used. However, our current understanding of these agents may still be limited as they may not have been tested in a broad spectrum for potential side effects. For example, the effects of muscimol on presynaptic GABAb receptors are only described recently 3. There may be other non specific effects of the cocktail drugs, which can contribute to the correction factors. In addition, the application of the cocktail at the ratio in our experiments, still results in the activation of GABAb receptors (~ 5%). Its potential effects on presynaptic transmission can not be excluded. Taken together, we think that the changes in R s and R in caused the major non specific effect of the cocktail application, since they largely explain the observed reduction of excitatory synaptic responses. It should also be noted that this study was carried out in anesthetized animals. The anesthesia will reduce the general activity level since ketamine blocks NMDA receptors, and can result in a scaling down of the tuning curves. This type of effects may not qualitatively affect our conclusion. Although ketamine anesthesia will also effect the temporal pattern of activity, e.g. long-latency (>ms) and oscillatory responses, these effects normally will not affect the short-latency responses examined in this study, as NMDA receptors are mostly blocked by Mg before spikes are generated 3-4. References. Ong, J., Marino, V., Parker, D.A., Kerr, D.I. & Blythin, D.J. The morpholino-acetic acid analogue Sch 59 is a selective GABA b receptor antagonist in rat neocortical slices. Eur J Pharmacol, 36, 35-4, (998).

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