Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

Summary

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.

Abstract

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.

Introduction

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 animals^3,4 or spread across 2 to 3 days^2,5-8. 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 substrates^9,10. 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 next^11,12, 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 psychology^13-17. These birds are able to perform complex visual discriminations¹⁸, can flexibly adapt behavior to changing reinforcement contingencies^19,20, 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.

Protocol

Ethics Statement All experiments were conducted in accordance with the German guidelines for the care and use of animals in science. Procedures were approved by a national ethics committee of the state of North Rhine-Westphalia, Germany. System overview Operant Testing Chamber The operant chamber (Figure 1) measures 34 cm x 34 cm x 50 cm. Three translucent response keys (4 cm x&#…

Representative Results

Behavior Figure 4A shows the behavioral performance of an animal in one example session. The performance level of the animal reaches criterion for NS 2 within 180 trials (45 stimulus presentations) and is close to 100% for the NS 1 from the beginning. This strategy – first responding to the same key for both new stimuli, and then adjusting responses for one of the stimuli – is about as often observed as initial random responding to both NS. In this session, the NS 2 was randomly …

Discussion

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…

Materials

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