Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.
While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons' action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.
Learning new stimulus-response-outcome associations engages a wide range of neural plasticity processes. These processes are ultimately reflected in the changing spike output of individual neurons. Arguably, one of the most frequently employed learning paradigms is Pavlovian fear conditioning conducted with rodents. In this setting, the acquisition and extinction of a conditioned response take place within a few dozen trials2. The rapid development of conditioned fear can be advantageous because it allows running a large number of animals within a short time. Also, acquisition and extinction can be observed within a few tens of trials on a single day in naive animals3,4 or spread across 2 to 3 days2,5-8. However, the insights gained about the changes of neural activity during learning in these experiments do not necessarily apply outside the domain of fear conditioning. For example, goal-directed behavior driven by positive reinforcement is more adequately modeled by operant rather than Pavlovian conditioning procedures, and may in part depend on different neural substrates9,10. Also, fear conditioning develops so rapidly that neural responses to the CS can only be observed for a few dozen trials, placing severe limits on the analysis of changes of neural activity during learning.
Unfortunately, the acquisition and extinction of operant responding usually takes many days. This is detrimental for neurophysiological investigations, because it is notoriously difficult to record the activity of single cells over more than a few hours. Due to the high similarity of the waveforms of extracellularly recorded action potentials, it is problematic to claim that spikes recorded on one day are generated from the same cell as spikes with similar waveforms recorded on the next11,12, especially in areas with a high cell density such as the hippocampus.
To address these issues, we developed a novel behavioral paradigm utilizing 3 learning conditions within one experimental session on a single day. This requires that the experimental animal is willing to perform hundreds of trials under varying conditions on a thin schedule of reinforcement. Homing pigeons (Columbia livia forma domestica) are classic model organisms in experimental psychology13-17. These birds are able to perform complex visual discriminations18, can flexibly adapt behavior to changing reinforcement contingencies19,20, and are uniquely avid workers, performing 1,000 trials with minimal amount of reinforcement. These characteristics make them especially suitable for the experiments described below.
This protocol describes a complex behavioral task suitable for concurrent single-unit recordings. We have described the SIFC task for pigeons, but it can be easily adapted to rodents by requiring nose pokes or lever pressing rather than key pecks, and substituting visual by olfactory, auditory, or tactile stimuli.
Perhaps the most critical steps during the training procedure are 1) gradual reduction of reward probability and 2) increase in trial number. Regarding intermittent reinforcemen…
The authors have nothing to disclose.
This research was supported by grants from the German Research Foundation (DFG) to MCS (FOR 1581, STU 544/1-1) and OG (FOR 1581, SFB 874). The website of the DFG is http://www.dfg.de/en/index.jsp. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors thank Thomas Seidenbecher for providing us with the gold-plating protocol as well as Tobias Otto for help with setting up the electrophysiological recording equipment.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Resistance wire (for use as electrodes) | California Fine Wire, Grover Beach (CA), USA | Stablohm 675; formvar-coated nichrome wires (outer diameter 25 µm) | |
Microconnectors | Ginder Scientific, Nepean, Ontario, Canada | GS18PLG-220 (plug) & GS18SKT-220 (socket to build headstage) | |
Cannulae | Henke Sass Wolf, Tuttlingen, Germany | 0.4x20mm/ 27Gx3/4" | |
Gold solution for plating | Neuralynx, Bozeman (MT), USA | SIFCO Process Gold Non-Cyanide, Code 5355 | |
Solution for ultrasonic bath | Alconox, Inc., New York, USA | 1304 | Tergazyme |
Conductive glue | Henkel Loctite | LOCTITE 3888 Silver filled, conductive, adhesive | |
Stainless steel screws | J.I. Morris, Southbridge (MA), USA | F0CE125 self-tapping miniature screws, body length 1/8 inches | |
Light-curing dental cement | van der Ven Dental, Duisburg, Germany | Omniceram Evo Flow A2 | |
Light-curing unit | van der Ven Dental, Duisburg, Germany | Jovident Excelled 215 Curing Light (wireless LED light curing unit) | |
Filter amplifiers | npi electronic GmbH, Germany | DPA-2FS | |
A/D converter | Cambridge Electronic Design, Cambridge, UK | power 1401 | |
Spike2 software | Cambridge Electronic Design, Cambridge, UK | Version 7.06a | |
Matlab | The Mathworks, Natick (MA), USA | R2012a |