Experiments were approved by the Institutional Animal Care and Use Committee, Zhejiang University. Seven adult male Sprague-Dawley rats (260-340 g) were each anesthetized with urethane (1.25 g/kg, i.p.) and placed in a stereotaxic apparatus (Stoelting Co., USA). The anesthetic state was monitored by pinching rat feet and observing rat responses. Body temperature was maintained at $\sim$ 37$^{\circ}$C. The nasal skin was cut and two stainless-steel screws were fixed in the nasal bone to serve as the reference and ground electrodes.

The scalp was opened and partial skull was removed to allow the placement of a recording electrode (RE) and a stimulating electrode (SE). The RE was a 16-channel (2*8) electrode array (Model Poly2, Neuro-Nexus Technologies, USA) positioned in hippocampus Area CA1 (AP: 3.5 mm, ML: 2.7 mm, DV: $\sim$ 2.5 mm). The SE was a concentric bipolar stainless-steel electrode (Model CBCSG75, FHC, USA) positioned in the Schaffer collaterals of the hippocampus Area CA1 (AP: 2.2 mm, ML: 2.0 mm, DV: $\sim$ 2.8 mm). According to the clear lamellar organizations of neuronal structures in hippocampus, the final positions of electrodes were determined based on the waveforms of orthodromically-evoked potentials as well as unit spike signals that appeared serially in the 16 channel recording array (Kloosterman:2001, Feng:2013). Specifically, a single stimulation pulse on the Schaffer collaterals could induce a population spike (PS) and a field excitatory postsynaptic potential (fEPSP) in the downstream pyramidal cell layer (Pyr. layer) and stratum radiatum (S. rad.), respectively. The distance between the two layers was $\sim$ 0.4 mm (Fig. 1A).

Figure 1.

Spontaneous theta rhythms of neuronal firing in hippocampus Area CA1 of anaesthetized rats. (A) Left: Schematic diagram of the rat hippocampus and electrode location. Recording electrode (RE) and stimulating electrode (SE) were respectively located in hippocampus Area CA1 and in the upstream Schaffer collaterals. Arrow indicates direction of stimulus-induced excitation. Electrical signals in the pyramidal layer (Pyr. layer) and in the stratum radiatum (S. rad.) were collected by two recording sites in the RE array (separated by 0.4 mm). Right: Typical waveforms of the population spike (PS) and field excitatory postsynaptic potential (fEPSP) evoked by a stimulus and recorded in Pyr. and S. rad. layers, respectively. Inverted triangles: Truncated stimulus artifacts. (B) Left: Segment of raw recording in the Pyr. layer together with associated local field potential (LFP) in theta range and multiple unit activity (MUA). Middle: Autocorrelation profile of the sequence of unit spikes extracted from the MUA. Right: Corresponding LFP-power spectrum density (top: LFP-PSD) and unit spike-power spectrum density (bottom: unit-PSD). (C) Left: Recording from S. rad. corresponding to the segment in (B) with its LFP in theta range. Right: Corresponding LFP-power spectrum density. Shaded area: Theta range (2-5 Hz).

At the end of experiments, the rats were euthanised by intraperitoneal over-dose injection of urethane followed by injection of a saturated KCl solution directly into the heart.

Raw signals from the recording electrode with a frequency range of 0.3-5000 Hz were amplified $\times$ 100 with a 16-channel extracellular amplifier (Model 3600, A-M System Inc., USA). The amplified signals were then sampled by a ML880 PowerLab data acquisition system (ADInstruments Inc., Australia) with a sampling rate of 20 kHz and an A/D conversion resolution of 16 bits.

Stimulations were 100 Hz biphasic current pulses with a pulse width of 0.1 ms and duration of 1 min, generated by an isolated pulse stimulator (Model 3800, A-M System Inc., USA). Current intensity was set at 0.3-0.5 mA to evoke an approximately 75% maximal amplitude PS according to the input-output curve of single pulse stimulation.

Four neighboring channels of the recording array located in the pyramidal layer were used to extract the unit spikes of neuronal firing as described previously (Feng:2017). Briefly, HFS stimulation artifacts were removed by a linear interpolation algorithm (Yu:2016): a signal segment of 1.5 ms around each artifact of a stimulation pulse was replaced by a short linear interpolation that connected the two endpoints of the artifact segment. Next, the artifact-free signals were filtered (> 500 Hz) by a high-pass digital filter (LabChart v7.0 software, ADInstruments Inc., Australia) to remove the local field potential (LFP) in the low-frequency band and to obtain multiple unit activity (MUA). Unit spikes in the MUA signals were then detected by an amplitude threshold of about $\times$ 3-5 the signal standard deviation (Vargas-Irwin:2007). Finally, feature vectors of the spike waveforms (first principal components and amplitudes) were used to sort the spikes of multiple neurons with open-source clustering software (SpikeSort 3D, Neuralynx Inc, www.Neuralynx.com). Histograms of inter-spike intervals of the sorted unit spikes were used to verify the clustering results of single unit spikes (Bartho:2004).

According to the waveform features of spikes of interneurons and pyramidal neurons, single unit spikes of different neurons were distinguished (Feng:2017). Briefly, half widths (measured from the negative peak to the post-positive peak) of the mean spike waveforms of different neurons were evaluated. Neurons with spike half widths of < 0.4 ms and > 0.7 ms were defined as putative interneurons and pyramidal neurons, respectively (Bartho:2004).

To investigate the effect of axonal HFS on the theta rhythms of neuronal firing, autocorrelation histograms of the binary sequences of unit spikes were generated to calculate power spectrum density (PSD) in the periods before, during, and after HFS (Kitchigina:2003, Kitchigina:2013). The length of autocorrelograms was three seconds with a temporal resolution of two milliseconds. The mean component in autocorrelograms was removed and the values were then normalized to eliminate the influence of spike rates. To analyze rhythms in the sequences of unit spikes by autocorrelogram, the PSD of autocorrelograms (unit-PSDs) was calculated by Welch's modified periodogram method with a Hanning window, an overlap of 50%, and a frequency resolution of 0.49 Hz.

Additionally, PSD curves of LFP in both the pyramidal layer and the stratum radiatum were calculated in the frequency range of 0-10 Hz to evaluate the theta rhythms in the LFP. The frequency resolution of LFP-PSD was 0.15 Hz.

Cumulative PSD power in the frequency range of theta rhythm (2-5 Hz) was calculated for both unit-PSD and LFP-PSD to evaluate the intensity of theta rhythm.

All statistical data were represented as mean $\pm$ standard deviation. Statistical significance of the difference between HFS data and the corresponding baseline data were tested by paired $t$-test. Pearson's correlation analysis was used to test the linear correlation between the theta rhythms of unit-PSD and the theta rhythms of LFP-PSD. The significance level was set at $p$ < 0.05.

Previous studies have shown that theta rhythms of the LFP commonly appeared in the hippocampus of urethane anesthetized rats with frequencies of 2-5 Hz (Buzsaki:2002). In the present study, the baseline recording in the pyramidal layer of Area CA1 clearly showed a rhythm with a peak of 2.3 Hz in the LFP-PSD (Fig. 1B). Meanwhile, the unit spikes of MUA fired in clusters and were obviously rhythmic in the autocorrelogram of the spike sequence. A peak at 2.7 Hz appeared in the unit-PSD of the autocorrelogram, indicating a theta rhythm in the spontaneous firing of neuronal action potentials (Fig. 1B). Additionally, the baseline recording in the stratum radiatum showed a clear rhythm with a peak of 2.75 Hz in LFP-PSD (Fig. 1C). The peak power density value 0.63 mV$^{2}$/Hz in the LFP-PSD of stratum radiatum was greater than the peak value 0.29 mV$^{2}$/Hz in the LFP-PSD of pyramidal layer, indicating a stronger theta rhythm in the LFP of stratum radiatum (Fig. 1B and Fig. 1C).

To investigate the effects of HFS on the rhythms of neuronal firing, one minute of 100 Hz HFS trains were applied to the Schaffer collaterals of the hippocampus Area CA1 (Fig. 2). During HFS, in the pyramidal layer, the MUA increased and the theta rhythms of LFP decreased (Fig. 2A). The theta rhythms of LFP in the stratum radiatum also decreased significantly during HFS (Fig. 2B).

Figure 2.

Changes in the multiple unit activity and local field potential evoked by stimulation (one minute, 100 Hz). (A) Example recording from Pyr. layer and associated multiple unit activity and local field potential in theta range. Typical spike waveforms of interneurons and pyramidal neurons are given in the two expanded insets. (B) Corresponding recording from S. rad. layer. Shaded area: High-frequency stimulation period, stimulus artifacts removed.

The late 45 s period of MUA signals during one minute HFS was used to analyze unit spikes to avoid interference from the large PS events at the onset of HFS (Feng:2013). Accordingly, the 45 s period of baseline signal immediately before HFS was used as a baseline control. A 45 s period of MUA signals starting from two minutes after the termination of HFS was selected to analyze firing recovery (Fig. 2A). The two minute period immediately following HFS was skipped because the firing of unit spikes would stop for tens of seconds and then gradually recove during this period (Feng:2017).

Unit spikes of interneurons and pyramidal neurons in seven rat experiments were analyzed. An example of the raster plots of unit spikes (Fig. 3A) shows that before HFS, the firing of an interneuron was clustered synchronously with the theta rhythm of LFP in the pyramidal layer and stratum radiatum, indicating a rhythmic firing. However, during HFS, this firing rhythm disappeared despite an increase in the firing rate of the interneuron. After HFS, the rhythm recovered. The suppression of rhythmic firing by HFS was more obvious in the autocorrelograms and in the unit-PSD plots of the interneuron (Fig. 3B). A peak appeared at 3.4 Hz in the unit-PSD before HFS disappeared during HFS and reappeared at 3.9 Hz after HFS. The mean power of theta rhythms in unit-PSD of interneurons was significantly decreased from 1.62 $\pm$ 0.79 before HFS to 0.12 $\pm$ 0.06 during HFS ($p$ < 0.01, paired $t$-test, $n$ = 7) and recovered to 1.39 $\pm$ 1.04 after HFS (Fig. 3C).

Figure 3.

Axonal stimulation suppressed theta rhythms in neuronal firing. (A) Top to bottom: Examples of theta signal recorded from Pyr. layer, raster plots of unit spikes of an interneuron and a pyramidal neuron, and theta signal from S. rad. during the three periods of: before, during, and after high-frequency stimulation, respectively. (B) Example autocorrelation profiles and their unit-power spectrum densities (unit-PSD) for interneuron spikes during the three different periods. (C) Statistical power of theta rhythms in unit-PSD of interneurons during the three different periods. Paired $t$-test was used to compare mean power during stimulation and baseline mean power prior to stimulation (** p < 0.01, $n$ = 7). (D) and (E) Corresponding data for pyramidal neurons.

Rhythmic firing of pyramidal neurons was not obvious in raster plots because of their low spontaneous firing rates (Fig. 3A). However, the theta rhythms in their firing before and after HFS were obvious in the corresponding autocorrelograms and unit-PSD plots (Fig. 3D). The suppression of rhythmic firing during HFS was statistically significant as the mean power of theta rhythms in the unit-PSD of pyramidal neurons decreased from 1.29 $\pm$ 0.65 before HFS to 0.11 $\pm$ 0.07 during HFS ($p$ < 0.01, paired $t$-test, $n$ = 7) and recovered to 0.99 $\pm$ 0.76 after HFS (Fig. 3E).

HFS also suppressed theta rhythms in LFP signals (Fig. 4). For LFP of baseline recordings before HFS, peaks of theta rhythms were obvious in LFP-PSD of both the pyramidal layer and the stratum radiatum. The peaks decreased during HFS and recovered after HFS (Fig. 4A and 4B). The mean power of theta rhythms in LFP-PSD of the pyramidal layer significantly decreased from 0.067 $\pm$ 0.019 mV$^{2}$ before HFS to 0.052 $\pm$ 0.016 mV$^{2}$ during HFS ($p$ < 0.05, paired $t$-test, $n$ = 7; Fig. 4C). Similarly, the mean power of theta rhythms in LFP-PSD of the stratum radiatum significantly decreased from 0.185 $\pm$ 0.058 mV$^{2}$ before HFS to 0.063 $\pm$ 0.043 mV$^{2}$ during HFS ($p$ < 0.01, paired $t$-test, $n$ = 7; Fig. 4D). Additionally, in baseline recordings, the mean power of theta rhythms in LFP-PSD of the stratum radiatum (0.185 $\pm$ 0.058 mV$^{2}$) was significantly larger than that of the pyramidal layer (0.067 $\pm$ 0.019 mV$^{2}$; $p$ < 0.01, paired $t$-test, $n$ = 7). However, there was no significant difference in the LFP theta rhythms of the two layers during HFS.

Figure 4.

Axonal stimulation suppressed theta rhythms in the local field potential. (A) and (B) Examples of local field potential-power spectrum densities in Pyr. layer (A) and S. rad. (B) for the three different periods: before, during, and after stimulation. (C) and (D) Statistical comparison of the power of theta rhythms (2-5 Hz) for the three different periods. $^{*}$p < 0.05, $^{**}$p < 0.01, paired $t$-test, $n$ = 7.

These results indicate that axonal HFS could suppress the theta rhythm activity of both downstream interneurons and pyramidal neurons. Simultaneously, HFS decreased theta rhythms in the LFP of different layers of the hippocampus Area CA1. This suggests that changes in the theta rhythms of unit spikes might be related to changes of LFP rhythms. Therefore, the relationship between the power of theta rhythms of neuronal firing and the power of theta rhythms of LFP was next evaluated.

To investigate the relationship between the theta rhythms of neuronal firing and those of LFP, the data of theta power in the three periods (before, during, and after HFS) were pooled together and Pearson's correlation coefficient was calculated (Fig. 5). For the LFP in the pyramidal layer, there were no significant correlations between the power of theta rhythms of neuronal firing (neither the interneurons nor the pyramidal neurons) and the power of LFP theta rhythms (Fig. 5A and 5B). However, for LFP in stratum radiatum, significant correlations were observed between the power of theta rhythms of both types of neuron and the power of LFP theta rhythms (Fig. 5C and 5D).

Figure 5.

Correlation between theta rhythm of neuronal firing and theta rhythm of local field potential. Scatter diagrams of theta power of unit spikes (A and C for interneurons, B and D for pyramidal neurons) against the corresponding theta power of local field potential (A and B for Pyr. Layer, C and D for S. rad.). Pearson's correlation coefficient (R) is shown above each plot together with the p-value. The data number ($N$ = 3 $\times$ 7 = 21) is the number of pooled data from the three different periods of before, during, and after stimulation within the seven experiments.

These results indicate that changes of neuronal firing rhythms were related to the LFP of the stratum radiatum (located hundreds of microns away from the neuronal somata) rather than the LFP of the pyramidal layer that immediately surrounded the neuronal somata. This was attributed to the fact that stratum radiatum contains excitatory synaptic terminals of afferent axons.