In a group of 8 animals, we used laminar electrophysiology in the center of the forepaw somatosensory representation during contralateral forepaw electrical stimulation to measure local field potential (LFP), multi unit activity (MUA) in different cortical layers before and after topical application of 0.1 mM concentration of GABA in artificial cerebrospinal fluid (aCSF). The stimulation consisted of 14 four seconds trains of twelve 200-μs and <2 mA pulses delivered with hypodermic needles inserted into the forepaw. From the depth-resolved LFP, current source density (CSD) profiles were calculated using the software developed by A. Dale et al. (Pettersen et al., 2006). shows the CSD profile in a representative animal. Blue and red curves represent data obtained before and 16–24 minutes after GABA application, respectively. The initial sink (negative deflection) in layer IV and corresponding source (positive deflection) in the surface is not affected by GABA application, while the sink in the supragranular layers and the corresponding source in the infragranular layers are reduced, similar to the findings reported by Staba (Staba et al., 2004
). reports the LFP, MUA and CSD profiles in specific cortical layers obtained by averaging over eight animals. In each animal, we normalized the signals with respect to the initial peak in layer IV in the cycles before GABA application. Error bars shown at every 10 ms represent standard errors. While we did not observe significant changes in MUA, possibly due to the small concentration of GABA used here and the relatively short time we waited after GABA application, the LFP activity significantly changed in superficial, supragranular and infragranular layers. The small change in amplitude observed in the LFP in the granular layer does not reach statistical significance (p-value>0.05).
Fig. 1 Current source density profiles in a representative animal. The traces shown are from before (left panel) and after (right panel) GABA application. The two vertical lines in the left panel show the times of initial sink at ~900 um and the secondary sink (more ...)
Fig. 2 Average responses across the cortical column measured with a laminar electrode. Local field potential (left), multi-unit activity (middle) and current source density profiles (right) are obtained averaging across rats the electrodes on the surface (0–100 (more ...)
Laser Doppler during the laminar electrophysiology experiments and diffuse correlation spectroscopy (DCS) during DOI/EEG experiments were used to monitor whether the infusion of GABA increased the baseline blood flow. In both LD and DCS measurements, the changes in blood flow between pre-infusion and GABA cycles were not statistically significant (p-value > 0.1) and not correlated with the hemoglobin concentration changes during these conditions.
In 8 animals we did simultaneous EEG/DOI measurements. In each animal we performed 11 measurement cycles: 2 before, 2 during, and 2 after infusion of aCSF and after a 20 minute period, 1 before, 2 during, and 2 after infusion of 0.1 mM GABA dissolved in aCSF (, in the Methods Section). For each cycle, there were 2 minutes of DCS acquisition during rest, DOI and EEG acquisition of 14 electrical stimulation trains of the right forepaw (paw contralateral to infusion side, left SI), 2 min of DOI and EEG acquisition during 7 stimulation trains of the left forepaw (paw ipsilateral to the infusion side, right SI - control side). The data presented here are responses to contralateral stimulation only. Data represented as ‘infusion side’ refers to response from the left SI to right forepaw stimulation and data represented as ‘control side’ refers to response from the right SI to left forepaw stimulation.
Fig. 7 Schematic drawing of the optical probe and EEG electrode as positioned on the animal head (top) and schematic diagram of the experimental protocol (bottom). In all animals the infusion side was in the left hemisphere. The right and left forepaw were stimulated (more ...)
In all our experiments we verified that the animal systemic blood pressure and heart rate did not change significantly when infusing the cortex with 0.1 mM of GABA. For the DOI/EEG group, systemic blood pressure values were 118±11 mmHg, 121±17 mmHg, and 119±5 mmHg during pre-infusion, aCSF and GABA infusions, respectively. The respiration rate and body temperature were maintained at 44±1 bpm and 37 °C, respectively. The average values of the blood-gas analysis across all experiments were: pH = 7.36±0.01, pCO2 = 36.2±5.7 mmHg, and pO2 = 192.9±12 mmHg.
From the EEG data, for each cycle, we calculated the average SEP responses averaging both across trains and across stimuli. shows the grand averaged EEG responses across rats for the infusion side (panels a and b) and the control side (panels c and d) during contralateral forepaw stimulations normalized with respect to the maximum amplitude of P1. Panels a and c report mean responses during pre-infusion, aCSF and GABA infusion cycles (averages of the last infusion cycle and two post-infusion cycles) with error bars (standard errors) shown every 5 ms. Panels b and d report the responses for each cycle with error bars shown every 10 ms. As stated earlier, the EEG responses were broken down into individual SEP components – P1, N1, and P2. In the infusion side, the N1 and P2 SEP components (both peak amplitude and area under the curve) are significantly reduced with GABA with respect to the cycles before GABA infusion. P1 peak amplitude is also reduced by GABA, but to a lower extent than N1 and P2.
Fig. 3 Grand average of contralateral SEP responses on the infusion (panels a and b) and control sides (panels c and d). Panels a and c: averages during pre-infusion (green), aCSF (blue) and GABA (red) cycles. Stimulation occurred at t=0 ms. Panels b and d: (more ...)
shows the hemodynamic changes measured in the somatosensory cortex in response to contralateral forepaw stimulation averaged across rats for the infusion (panels a and b) and the control (panels c and d) sides. Oxy-hemoglobin concentration (HbO) changes are positive, and deoxy-hemoglobin (HbR) changes are negative. Similar to , in , panels a and c report the mean responses for pre-infusion, aCSF and GABA cycles (averages of the last infusion cycle and two post-infusion cycles) and panels b and d report the responses for each cycle. Error bars representing standard errors are shown every 1 second. Both HbO and HbR are significantly reduced with GABA with respect to the cycles before GABA infusion.
Fig. 4 Grand average of contralateral hemoglobin responses on the infusion (panels a and b) and control sides (panels c and d). Panels a and c: averages during pre-infusion (green), aCSF (blue) and GABA (red) cycles. Stimulation occurred from t=0 to t=4 s. Panels (more ...)
report the average percent changes of the area and the peak amplitudes of SEP and hemoglobin responses In the infusion side, while all quantities decreased in amplitude with GABA, P1 component become broader with GABA, so that the area under the curve of P1 did not change sufficiently with respect to the pre-infusion runs. As a result, reductions of ΣP1 were not statistically significant, while reductions of ΣN1, ΣP2, ΣHbO and ΣHbR with GABA were large and highly significant (p-values ≤0.002). Similarly, peak amplitudes of responses were also compared. Reductions in N1 and P2 were very close to reductions in HbO and HbR. Changes in P1 peak amplitude were less significant and smaller than changes in N1 and P2. In the control side, we did not observe any statistically significant reduction on the area or peak amplitude of N1, P2, HbO and HbR during GABA and aCSF cycles, but we observed a statistically significant reduction of P1 peak amplitude and area during the GABA cycles. Finally we compared the SEP and hemodynamic responses in the two hemispheres before infusion (pre-infusion cycles) and did not observe any statistically significant difference in the responses due to the surgery in the infusion side.
Table 1 Average percent changes and standard errors of the SEP and hemoglobin concentration areas (top) and peak amplitudes (bottom) with respect to pre-infusion cycles. Positive changes indicate increase, negative decrease of the responses with respect to the (more ...)
The scatter plots in show the correlation between the areas under the SEP components and the oxy-hemoglobin response curves for individual rats for the 11 cycles in the infusion side. A highly statistically significant correlation is achieved between either ΣN1 or ΣP2 and ΣHbO (see p-values in the figure) or ΣHbR (not shown for brevity). When comparing peak amplitudes, there is an improved correlation between P1 peak and hemodynamic response, but it is still statistically lower than correlations using N1 and P2 peak amplitudes.
Fig. 5 Scatter plots oxy-hemoglobin concentration and SEP components (P1, N1 and P2) for each cycle and each rat (shown with different colors) in the infusion side using area under the response. For each animal, the SEP and hemoglobin responses are normalized (more ...)
shows the hemoglobin responses measured and predicted by a linear convolution model (Franceschini et al., 2008
) using the three SEP components, the total SEP signal T, and the input stimuli (S) for the grand average over all animals in the infusion side during pre-infusion, aCSF and GABA stages. To compare results, we calculated the coefficients of determination (R2
) between the measured and predicted hemoglobin concentrations. Using area under the SEP components as inputs to the model, over the 11 cycles, the R2
values between the measured and predicted hemoglobin responses are 0.51–0.53 for P1 and input stimuli S, 0.65–0.67 for N1 and P2 for either HbO or HbR. The R2
values of N1, P2 and T are statistically significantly larger than the R2
obtained with P1 or S (p-value<0.02). Similarly, using the peak responses as inputs to the model (figure not shown), P1 has a statistically significant lower R2
than N1 (p-values < 0.02). The P2 SEP component is very broad and does not have a well-defined peak, and even if it predicts hemodynamic responses better than P1, it does not reach statistical significance (p-value = 0.09). For the control side, we found predictions were similar for the different SEP components without statistically significant differences. The coefficients of determinations ranged between 0.54 to 0.61 for HbO and 0.59–0.64 for HbR. This underlies the fact there is no variance in the control side during the experiment because of the single stimulation condition used and lack of effect of GABA on the non-infused side. Finally, while the predictions for deoxy-hemoglobin were higher than for oxy-hemoglobin, they did not reach statistical significance.
Fig. 6 Grand average of the measured oxy-hemoglobin (red) and deoxy-hemoglobin (blue) responses to right forepaw stimulation in the infusion side during pre-infusion, aCSF and GABA conditions. Corresponding predictions of Hbo and HbR using the input stimuli (more ...)