A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

A list of Equipment used by this laboratory for recording electrophysiological signals can be found in Table 3. Following recovery from surgery, single-units are recorded by plugging a unity-gain headstage into the implanted connector. This headstage is connected via a cable to a commutator, which is capable of free rotation without breaks in the electrophysiological recording through the use of electrical slip rings. The commutator allows subjects to freely move while recording during behavior, which is one of the principle advantages of this preparation. Signals are then fed through a preamplifier (10x gain) that is used to differentially amplify the signal on each electrode against the ambient noise on an electrode selected because it does not exhibit a unit signal. Differential recordings are used to amplify the instantaneous difference between the two voltages. Because both wires record ambient noise approximately equally, their opposite polarities effectively enable subtraction of ambient noise from neural recordings (Figure 2). Finally, signals are band-pass filtered (450 Hz to 10 kHz; roll off -1.5 dB/octave at 1 kHz and -6 dB/octave at 11 kHz) and amplified 700x before being digitized using a computer with an A/D card (50 kHz sampling frequency/wire) and stored for offline analysis.

In software, waveforms are often sampled using a threshold detection method. Thus, signals which pass a given voltage threshold are digitized and stored. Still, there is an inherent rate of false-positives, in which electrical artifacts pass through the threshold, filtering and differential amplification, and are sampled. These non-neural waveforms are subtracted post-hoc using spike-sorting software which allows sampled discharges to be sorted based on their parameters (e.g. amplitude, peak and valley voltages, etc.) and subsequently allows for the elimination of electrical "noise" and the selection of the neuronal signal (Figure 3). Waveforms are included for analysis if 1) Waveforms present with canonical patterns of neural activity including a clearly defined action potential and after hyperpolarization (Figure 3; right panel); 2) the amplitude of a putative neural waveform exhibits at least a 2:1 signal:noise ratio (i.e. is clearly separable from the channel's noise band; amplitude of noise band = approx.. 50 µV); 3) Parameters remain stable throughout the entire session and 4) an interspike interval (ISI) histogram shows that no discharges occurred during the neuron's natural refractory period (i.e. ~2 msec; Figure 3 left panel).

Neural discharges can then be correlated with behavioral events (e.g. lever responding) that are recorded as timestamps via the use of a digital input-output (I/O) card. These time-stamps can be used to create peri-event time histograms (PETHs; Figure 4A), which are used to display neuronal discharges that occur in a specified time range around a particular behavioral event. Our laboratory has typically examined neural firing on the order of hours (Tonic¹⁰), minutes (Slow-Phasic¹¹), and seconds/milliseconds (Rapid-Phasic¹²). Tonic firing is typically used to examine the most global events in a session. For example, we often use this to compare firing rates of neurons when animals are self-administering drug, i.e. predrug versus on-drug firing rates. Slow phasic analyses are often used to examine firing during slow changing events like the pharmacological decay of a drug over minutes. Finally, rapid phasic analyses are used for more instantaneous events like an operant response or the onset of an experimental cue.

Figure 4 shows a histogram of tonic firing with a count of discharges in 30 second bins across an entire recording session during cocaine self-administration. The example cell shown is sensitive to changes in body levels of cocaine and becomes inhibited (Figure 4A) as the animal's body level of cocaine increases (Figure 4B), but returns to its original firing rate as the level of cocaine falls after the self-administration contingencies have ended. Depending on the target region, neurons may be sensitive to pharmacological effects, or any number of behavioral events including reward-associated cues⁶, sensorimotor input¹³, or motivated approach³.

Under the correct conditions, it is possible to record the same neuron for many sessions. In the striatum, neurons are homogeneously distributed (80% of wires yield only a single unit and cells are not layered or tightly grouped) and produce relatively small signals that quickly decay over distance. Under these conditions, our laboratory has been able to record the same neuron in the striatum for multiple weeks. Importantly, not all neurons can be verified across sessions and not all neurons are maintained over time. Determining one's ability to obtain these types of longitudinal recordings requires careful testing of the electrophysiological system. For example, it is possible to estimate the recording distance of microwires by using drivable electrodes. When considered alongside the physical properties of the wires, regional anatomy, and the waveform parameters of the neuron across sessions, one can determine with reasonable confidence that a waveform has been maintained across sessions^7,8,13.

Figure 1. Representative placements for the "skull window" (i.e. craniotomy), skull screws, and ground wire. A) A dorsal view of the skull showing the placement of a skull screw on each plate of the skull (red), a skull window (square), and a contralateral hole for the ground wire (green). When planning the skull window, the coordinates should be defined by determining the B) anterior and C) posterior medial and lateral coordinates for the target region (e.g. dorsolateral striatum). D) These coordinates can then be used to determine the placement of the window on the dorsal surface of the skull. Note that the skull window corresponds to the medial and lateral targets identified in B and C and also spans the specified range in the anteroposterior plane between B and C. These coordinates will be specific to each individual target region. Representative locations of the microwires in a 2 x 8 array are shown using small dots within the skull window shown in D. The final dorsoventral coordinate of the wires will depend on the depth to which they are lowered. Figures modified from Paxinos & Watson¹⁴. Click here to view larger image.

Figure 2. An illustration of the process of differential amplification. The topmost panel represents a neural signal prior to differential amplification (Signal + Noise). Following differential amplification, extraneous electrical signals that are found on all channels (Noise; middle panel) can be effectively subtracted from recording wires via the use of a specified differential electrode in order to improve isolation of the neural waveform (Signal; bottom panel).

Figure 3. An example neural waveform (right) recorded from one wire of a microwire array. The histogram (left) shows the number of action potentials in the milliseconds preceding each observed neural discharge in the session. Each bin represents a 1 millisecond period. The histogram demonstrates that no action potentials occurred in the neuron's natural refractory period (2 msec).

Figure 4. An example of a neuron that is tonically inhibited during cocaine self-administration. A) The firing rate (Spikes/30 sec) of a neuron across a long-access cocaine self-administration session in 30 second bins. B) The corresponding calculated drug level across the same session. In combination, increases in drug level are correlated with a depression in neural firing. Accordingly, when the session ends at ~425 min, neural firing recovers as body levels of cocaine fall.

Figure 5. Histological slices from two animals with microwire implants. A) A Nissl stained section using neutral red combined with a potassium ferrocyanide counter stain revealing lesioned microwire tips (blue). A microwire can be traced from the cortex down to the wire tips (blue) by following the wire tracks in the tissue. Tracks may be traced in a single slice or a series of adjacent slices. B) A section stained for Substance P, which reveals the ventral pallidum, coupled with a potassium ferrocyanide counter stain. Using immunohistological techniques, individual micro-wires can be localized within specific brain regions, or even within specific sub-regions of a particular nucleus. Figure shows a microwire tip (blue) localized within the ventral pallidum using the Substance P stain.