Electrophysiological recordings were conducted for the tissue/macro-electrode electrical model and NA functionality experiments on eight adult male Sprague-Dawley rats weighing 460 ± 151 g. Surgical and experimental procedures were performed in accordance with the European Community’s Council Directive (EU Directive 2010/63/EU) and the National Institute of Health Guide for Care and Use of Laboratory Animals. A concentric bipolar stimulation electrode (NEX100, Phymep, France) was implanted in the right STN of the brain using stereotaxic procedures according to the atlas of Paxinos and Watson (1986) [28].

Electrophysiological recordings of neuronal signals were done under two conditions: animal under anaesthesia (terminal acute study) or awake and freely moving (acute study). The tissue/macro-electrode electrical model characterization used three anesthetized animals. The NA functionality was first assessed with an anesthetized animal but the in vivo recording setup required improvements to avoid punctate excessive 50 Hz power line interference (Section 4.4). After improving the in vivo recording setup, NA functionality was assessed in two anesthetized animals and three animals under awake conditions (one animal was used for recording under both conditions).

Spontaneous recordings under the first condition were made under anaesthesia induced using urethane (10 mL/kg). In order to record LFP signals in freely moving conditions, animals were implanted with a chronic stimulating electrode in the right STN under anaesthesia with a 1.9 mg.kg⁻¹ solution of 52% ketamine (Virbac, France), 22% xylazine (Rompun, Bayer Vital, Germany) and 26% saline solution (B. Braun, France). Body temperature was maintained at 37 °C ± 0.5 S.D. with the use of a homeothermic heating blanket. Cortanmycétine cream (Intervet, France) was applied to the eyes to prevent corneal dehydration and infection. Rats were placed in a stereotaxic frame and a craniotomy was performed over the right STN (AP: −3.8, ML: 2.5) according to the rat brain atlas [28]. Six screws were placed around the craniotomy (A2 DIN 84, Fixnvis, France) and covered with dental composite resin (SuperBond C&B, Sun Medical, Japan). A bipolar electrode (MS306/SP, PlasticsOne, USA) was then lowered into the perforation to a depth of 8 mm below the cortical surface. The electrode base and screws were finally covered with dental cement (Dentalon Plus, HeraeusKulzer, USA). After suturing, wounds were infiltrated with analgesic (Ketofen, Centravet, France), Fucidin cream (Fucidine 2%, Laboratoires Leo, France) and iodine solution (BetadineDermique 10%, MedaPharma, France). All rats followed a post-operative antibiotic treatment (Duphamox, Fort Dodge, France). Behavioral experiments were initiated 21 days post-surgery to allow recovery of the animals from surgery.

In order to characterize the tissue/macro-electrode interface in the LFP band and develop the model, data were obtained from a bench top in vitro and in vivo setup. The setup and measurement details are described in detail elsewhere [29]. Briefly, Figure 4a presents the setup based on a voltage divider formed by a reference resistor and the electrode. In order to mimic biological conditions, the generated stimulus amplitude was in the range of LFPs. Preliminary experiments with a commercial recording setup led to identification of the maximum LFP amplitude. The commercial recording setup included a Neurolog (Digitimer, UK) acquisition system and a Powerlab (AD Instruments, USA) interface. The NEX100 and MS306/SP macro-electrodes were used to record the neuronal signals with COS low noise amplifiers (INA114AP, TI) in both in vitro solution (Ovalbumin, Ovatransferrin and Ovomucoid simulating the brain medium) and terminal rodent studies.

Figure 4b presents the setups for bench testing and in vivo experiments. For electrical characterization, the integrated NA was followed by an adaptation board (IIA), and by commercial instruments (e.g., oscilloscope). Because the signal range is too small to be measured with our instruments (at least 2 μV for the spectrum analyzer, 2 mV for the oscilloscope), a second low noise 40 dB amplification stage was required, and was designed with COS components on the IIA board. Passive component sizing relied on a trade-off between DC rejection and noise attenuation. The polarization capacitor C_I2 on the IIA board was required for DC match with the following display and analysis instrumentation. The paths a0 and a1 were used respectively for frequency response and power spectral density (PSD) characterizations. Eight NA samples on eight chips were characterized. The overall measurement bandwidth was divided into two bands to improve the measurement resolution: 0.5–500 Hz and 0.1–10 kHz. The bandwidth resolution was defined as the tenth of the lowest frequency in those bands. The low frequency characterization down to DC (in the order of milli-hertz) was performed with an HP89410A spectrum analyzer. In order to measure the expected low cut-off frequency (1 Hz) for the frequency response characterization, a high value of C_I2 (6.8 µF) was used.

Figure 4c describes the noise measurement setup and its linear noise model. The IIA board was needed because the spectrum analyzer noise was higher than the NA output noise. First, we measured the overall amplification system noise PSD (with NA and IIA board connected, and with NA inputs grounded), then we measured the IIA board noise PSD (without NA, and with IIA inputs grounded). Assuming that each contribution to noise (boards and instruments) is uncorrelated, the NA noise PSD is defined as in (4). Integration of discrete measured data lead to estimates of the equivalent input-referred noise over the measurement bandwidth as defined in Equation (5).

The figure of merit noise efficiency factor (NEF) introduced in [30] has been widely used in the neuronal amplifier designer community since Harrison used it to present his topology [20], and is defined in (6).

(6) with BW the NA band (Hz); the input referred noise (V_RMS) usually integrated on the BW band; I_tot the total supply current (A); U_t the thermal voltage; k the Boltzmann constant, and T the temperature (300.15 K).

To illustrate the ability of the integrated NA to record spike signals, we used a test-bench setup with synthesized waveforms as NA input signals. Synthesized waveforms generated by a waveform generator consisted of the superposition of a 500Hz-sinusoid wave (SW) on peak-to-peak amplitude-controlled noise. Four data sets were generated, one with no noise and three with different noise levels. The signal-to-noise ratio (SNR) is defined as the peak amplitude ratio of the SW signal and the noise.

For this first test-bench validation, the waveform data were loaded into a waveform generator (Agilent 33250A, Agilent Producer, Santa Clara, CA, USA), and the generator output was attenuated using two 30dB attenuators (Agilent 8493B, Agilent Producer, Santa Clara, CA, USA) resulting in a SW signal with a 100 µVpp amplitude for input to the NA. This input signal was amplified and filtered with the integrated NA, a second amplification stage and an additional filter (dashed line, Figure 4b). An operational amplifier comparator (TL082CP) was used as a simple threshold comparator to demonstrate that the recording system SNR did not corrupt the low amplitude sine waves.

The three reference SNR ratios were measured at the waveform generator (i.e., before attenuation), at 3.0 V/V, 2.2 V/V and 1.9 V/V. For each of the four data sets, the SNR results were averaged over 70 measurements at the waveform generator and additional filter outputs.

We used the integrated NA in vivo neural recording experiments on awake rodents to verify operation of the circuit when connected in situ. Figure 4d presents the in vivo setup used in this study. This was a modified version of that used in a preliminary study with an analog programmable circuit (instead of this integrated circuit) [31]. The NA records the raw signal between one of the two contacts of the NEX100 macro-electrode and the ground connected under the skull of the animal. The path a1 referred to in Figure 4b was used for in vivo low amplitude signal measurement and power spectral density analysis. The same power spectral density measurement method described for the noise characterization was used for in vivo experiments.

First, we conducted preliminary experiments with a commercial recording setup. We measured LFP signals of an awake rat with a maximum amplitude of 700 μV using a concentric bipolar NEX100 macro-electrode. The amplitude of the neuronal background activity was determined, on a deeply anesthetized rat, to be 10 μV. These measures confirmed the data provided in the literature and gave us a basis to conduct the in vivo tissue/electrode interface characterization.

Figure 5 shows in vivo measures of the magnitude of the NEX100 electrode impedance versus different levels of voltage stimulus over the LFP band. Randle’s model was used in which the electrode is approximated as a resistance R_S followed by a parallel capacitor C_e with resistor R_P. Below ~20 Hz, the electrical behavior of this macro-electrode is capacitive. Above ~20Hz, it is equivalent to a resistor.

Table 2 compares the measured values for Randle’s model of NEX100 and MS360 macro-electrodes with two other electrodes: the Medtronic 3387 DBS electrode and a multi-electrode array (using micro-electrodes) [10,32]. For an acutely implanted electrode, the electrode impedance in the LFP band was approximated by a 34nF-C_e capacitance.

In this section, we present the measurement results for the frequency, noise and power neuronal amplifier performances that meet the required NA specification for future use in a closed-loop implanted neuroprosthesis prototype.

Figure 6 presents the neuronal amplifier frequency response. Figure 6a presents the low cut-off frequency f_low measure, which is lower than expected (41.2 mHz vs.1 Hz). Figure 6b presents the overall bandwidth of the neuronal amplifier for three different current bias levels. As expected, the high cut-off frequency f_high is tuned by the bias current I_bias, as the f_high definition in Equation (2) is linear with the transconductance G_m, and itself, linearly dependent on I_bias since the input differential transistors are in weak inversion.

Table 3 reports the mean and standard deviations of the NA gain and high cut-off frequency averages for Monte Carlo simulation (300 iterations, process and mismatch variations) and measurements (8 chips from the same fabrication run). The measured standard deviations are smaller than the simulated ones.

The noise characterization and in vitro and in vivo measurements performed on an awake animal are illustrated in Figure 7. Figure 7a (bottom curve) plots the NA input-referred noise density vs. frequency. To be easily compared with in vivo LFP measurements, the input-referred noise is plotted on the 1–500 Hz band only. From this noise figure, we extract the corner frequency f′_corner of 39 Hz for the NA and IIA board (path a0, Figure 4b). Because of the IIA board polarization (i.e. an equivalent low cut-off frequency of 1 Hz, cf. methods) and according to Equation (3), we conclude that this value gives a maximum limit for the neuronal amplifier’s intrinsic f_corner. As a consequence, the resultant NA input-referred noise is also a maximum value, and was found to be 3.99 μV_RMS over 1–10 kHz. Figure 7b illustrates the amplified input-referred in vivo signal.

Table 4 summarizes measured performances of neuronal amplifiers from the literature based on [20]. We selected NA measured performances that present a noise characterization computed at low frequencies with a maximal low frequency of 10 Hz [17,18,19,33,34,35,36,37]. This is not the case in most neuronal amplifiers presented in the literature due to the necessary measurement time and effort. The first part of Table 4 presents fabricated circuits meeting the following criteria: they use a 0.35 μm CMOS process; the low cut-off frequency f_low is inferior to 10 Hz and the NA has a bandwidth that covers LFP and spike bands. The second part of Table 4 presents some of the latest neuronal amplifiers (period 2009–2013) that are built using other technologies.

Figure 8 illustrates the ability of the integrated NA to record spike signals generated using a waveform generator. Table 5 presents SNR measurements at the waveform generator output (input to the NA) and at the recording system (NA) output. When no noise was added on the loaded SW signal, a 2.9 V/V maximum SNR was measured from the recording system. When noise was added on the loaded SW signal, the recording system SNR was close to the input SNR, with constant standard deviation for different noise levels. Figure 8a illustrates a spike output waveform from the generator with added noise while Figure 8b shows the ability to record this activity and identify spikes using a comparator. The overall gain for the recording system was set at 76 dB (Figure 4b: integrated low noise 20 dB amplifier, INA board second 40dB amplifier, and a 16 dB filter).

After the NA characterization, we conducted several series of in vivo measurements to validate the device in situ. Our long term goal is to demonstrate that our system can be used for in vivo experiments of LFP recording with awake and moving rats.

The in vivo power spectral density was measured and was referred to the NA input (Figure 7a, top curve). It is one example among the four measurements made on different days. Animal condition (deeply anesthetized or awake) LFP recording were similar (not shown). In vivo input-referred measured PSD was 18.34 μV_RMS, integrated on 1–10 kHz.

The NEX100 is a macro-electrode (100 kΩ at 1 kHz); therefore mostly LFPs could be recorded and one oscilloscope hardcopy (Figure 7b) is shown. If the neuronal amplifier output is filtered by a 00 Hz low-pass filter, it provides the same shape as the output of the NA (not shown). The in vivo input-referred signal amplitude was 100 µVpp. These results were confirmed in experiments performed on four different animals and over an average of four consecutive hours.

We designed a setup to characterize the tissue/macro-electrode interface using an electrical model by appropriate stimulation of the deep brain subthalamic nucleus region with a small macro-electrode. The stimulation amplitude was chosen to reflect those observed under recording conditions. Indeed, high stimulation amplitude could lead to non-linear effects, which would not reflect the in vivo recording conditions. Dispersion of the different measures versus the stimulation voltage level (Figure 5) is not significant (less than 2%): these measures suggest a linear model. This confirms the relevance of Randle’s linear model for the tissue/macro-electrode model in the LFP band. The impedance characteristics for NEX100 and MS306/SP (Table 1) are similar, which is not surprising given their similar geometrical characteristics. This comparison proves the robustness of the characterization setup. The extracted values from in vitro measurements are under-estimated compared to those from in vivo studies, suggesting the need for an in situ characterization. The characterization of the tissue/macro-electrode interface provides guidelines for the impedance matching between the tissue/macro-electrode interface and the NA, which was addressed by appropriate component sizing in our NA design.

The NA topology addresses the required specifications and meets changes in the biological system input dynamics. Because we are also interested in determining if analog linear processing of the recorded signal could generate a stimulation signal, the heterodyning method is not used.

The front-end design should then consider the electrode DC level and its drift, which are classical NA issues that can result in output saturation. Various techniques have been described [7,38] to eliminate this DC component: high-pass filtering (blocking or subtracting low frequencies), intrinsic low gain at low frequency, stabilization, chopper modulation or offset cancelling. The NA topology presented by Harrison in [20] is one of the most popular for various recording applications (electroencephalogram, electrocardiogram, deep brain recordings with in vivo multielectrode array, e.g., [22,39]) because of its low noise and potential low power/area consumptions. The DC level is handled with high-pass filtering with this topology. We chose to use this NA topology and adapt the sizing of the components to meet our challenging specifications regarding the noise, gain and power/area consumptions. We used transistors in moderate and strong inversion to meet noise and power specifications.

Frequency and noise responses are some of the critical specifications for the NA. As shown in Figure 6b, the first frequency response parameter of interest is the low cut-off frequency value. The measured frequency is lower than the simulated value. This can be explained by discrepancies in the model of the lateral PNP in the 0.35 μm CMOS process design kit which overestimates the cut-off frequencies. This was also observed in the original design [20] with the AMI 1.35μm CMOS process. Moreover, we demonstrated in an awake rat experiment (see following sub-section) that the f_low limited change has no impact on the proper functioning of our design.

Mismatch in the circuit fabrication process and parasitic capacitors may be responsible for the difference between simulated and measured performances of the gain and the high cut-off frequency f_high. Variation in f_high will have little impact on spike recording because this frequency can be chosen in the 5–10 kHz band [14,15] and can still be tuned together with the NA transconductance G_m (I_bias) with Equation (2).

Even with a low amplification gain, the NA noise and power/area performances are reasonable compared to that of other high gain amplifiers with the same topology [17,18,19,33,34,35,36,37]. Moreover, we achieve the best NEF among all technology process designs. Note that a recent paper [18] has presented the same recording circuit strategy in an integrated circuit but no bandwidth characterization over the continuous LFP and spike band and with no in vivo LFP recordings presented. Moreover, their NA gain was set at a value (50V/V) that may be too high to be used in a closed-loop system such as that presented in the Kent and Grill study [8].

Additional test-bench experiments illustrate the ability of the NA to record spikes with several SNRs. The true SNR at the NA input cannot be determined because the NA input can also record uncontrolled external thermal noise. However, this demonstration shows that the output SNR decreased in proportion to the input SNR, and with a low standard variation (Table 5). The detection rate was acceptable because we used a simple threshold method. This extension of the NA bandwidth over the spike band provides the opportunity for a large variety of closed-loop paradigm validations.

Because the LFP signal shares many features with the noise signal, the following measurement shows that the in vivo recorded signal is indeed an in vivo spontaneous neural recording, and not NA intrinsic noise. In Figure 7a, the in vivo PSD referred to the NA input (18.34 μV_RMS) is compared with the NA input-referred noise (3.99 μV_RMS): this demonstrates that our in vivo recordings are indeed in vivo extracellular brain measurements.

As an example, Figure 7b presents the NA recording output signal amplitude and shape. The NA recordings are equivalent to the LFPs recorded in the same condition using a commercial recording setup. Our NA not only provides similar characteristics but our integrated circuit is also portable because of its small size and operability using a battery source. These successful in vivo validations with awake and freely moving rats lead the way for extended investigations of LFP control within a closed-loop system for various applications.

This recording circuit strategy requires careful noise consideration for the cascade of low gain second-order filter amplifier stages in the neuronal amplifier in order to maximize the SNR as defined in Friis formula [40]. A recent manuscript [18] also assesses that such a design is achievable. The recording circuit architecture with the artifact reduction strategy we used has proven successful for in vivo recording and stimulation of neuronal activity by [6] in the subthalamic nucleus (STN) of two idiopathic parkinsonian patients, and by [8], in the ventral lateral thalamus of an adult cat, both using COS components [6,8]. Thus, we believe that this NA is suitable for recording LFPs and spikes for a future implantable closed-loop system using a single electrode.