Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

The overall goal of the following experiment is to assess and to manipulate neural mass action and neurodynamics during learning. This is achieved by first implanting multi electrode arrays that allow site-specific simultaneous stimulation and recording of neuronal activity in the auditory cortex. As a second step implanted, animals are trained in a two compartment shuttle box in a go no-go paradigm while recording the ongoing neuronal activity in the auditory cortex and using intracortical micro stimulation for conditioning.

Next, analysis of the learning behavior and learning related spatiotemporal activity patterns are performed on a trial by trial basis offline. The results show how site-specific cortical micro stimulation can be used to evoke spatiotemporal activity patterns that allow characterizing internal subjective cognitive states. Thereby this method establishes links between cortical circuit activity patterns and observed behavioral states of learning and perception.

The here described techniques demonstrate that it is not only possible to relate individual cortical activity patterns to defined behavioral and cognitive states, but that it is also possible to evoke by electrical simulation defined perceptions that can be associated with different behaviors. The extension of this findings is towards the development of neuroprosthetic substitution for lost sensory function. This method can also be applied to other rodent species such as rats and mice.

Generally, Individuals new to this method will struggle because experience and well-developed standards in handling and training of animals are required to obtain good results from these learning experiments. First stimulatory multi-channel electrode arrays are prepared and then surgically implanted into the auditory cortex. According to the instructions in the written portion of the protocol, the rest of the procedure commences only after the animals have fully recovered from surgery.

Epidural surface arrays are made from stainless steel wire arranged in a three by six matrix with an inter electrode distance of around 600 microns. Plug the head stage amplifier into the recording cable. Use a harness of thin flexible cables wrapped by a metal mesh to protect the connection from damage by biting animals.

Use a pre amplifier in the shielded box to increase signal to noise ratio and band pass. Filter the signal in the desired frequency range for local field potential recordings. Sample data at more than one kilohertz sampling frequency with a two to 300 hertz filter For action potential recordings, use a sampling frequency of at least 40 kilohertz and a 300 to 4, 000 hertz filter.

To begin the behavioral test, use a shuttle box in an acoustically and electric shielded chamber. The box contains two compartments separated by a hurdle. Use hurdle heights appropriate for specific species as the height of the hurdle influences the behavioral bias of the response.

After connecting the animal to be tested to the recording and stimulation cable, gently place the animal in the training chamber and allow it to habituate for three minutes before the start of a session, record all compartment changes in the habituation phase. Carefully check the quality of the recording before the start of training. Apply an online fast Fourier transform filter to the signal to determine the amplitude of 50 hertz noise.

Finally, double check all connections between the head connector, the adapters, the head stages, the cable harness, and the amplifiers. Use a multi-channel stimulator to deliver intracortical micro stimulation to the stimulation electrodes for conditioned stimulation. Deliver the conditioned stimulus and then deliver the foot shock uncondition stimulus through the grid floor, ensure that the strength of the shock delivered is aversive, but not painful.

As seen here. Present blank trials without unconditional stimulus and condition stimulus interspersed between the trials in order to correct via subtle behavior. Classify a compartment change after the conditioned stimuli onset within a critical time window of four seconds as a hit response.

If a conditioned reaction does not occur in response to the conditioned stimulus within the critical time window, immediately deliver a mild foot shock for six to 10 seconds. As uncondition stimulus classify this as a miss for conditioned stimulus negative trials, a compartment change within the critical time window is classified as a false alarm response. Apply the unconditioned stimulus for up to 10 seconds immediately after this inappropriate conditioned response.

Do not apply the foot shock unconditioned stimulus after conditioned stimulus negative trials when the animal stays in the compartment during the critical time window. Classify this as correct rejection after the whole experiment is complete control for the stable position of the stimulation electrode array, providing surgical anesthesia and then applying monopolar cathartic current delivered through all stimulation channels to obtain iron deposits in the tissue at the position of implantation. Carefully determine the escape latencies online increased foot shock strength step if escape latencies are longer than two seconds.

After the first 20 trials to analyze the training data, calculate conditioned reaction rates for conditioned stimulus positive and conditioned stimulus negative trials as shown on screen plot conditioned reaction rates as a function of session or trial block for evaluating the training progress and learning dynamics. Finally perform deep prime analysis according to the instructions in the written portion of the protocol. A sensitivity index with the deep prime of more than one can be used as the threshold criterion for successful discrimination response latencies during condition stimuli positive trials are plotted here for individual trials Over all training sessions, all responses with latencies below seconds correspond to successful hit responses.

Histograms of response latencies are bimodal corresponding to HIIT responses at less than six seconds and escape responses at six to eight seconds. This is a typical example of an electrically evoked potential or EEP from a single animal averaged across condition stimulus positive trials in a single session of training, the EEP is shown before the removal of single pulse stimulus artifacts in black, and after the removal of artifacts in red. The early prominent negative peak known as N two zero can be seen at a latency of 20 milliseconds.

This figure shows further analysis of the spatial distributions of the N two zero amplitude in response to a positive condition stimulus at the rostral stimulation electrode in the top panel, and to a negative condition stimulus at the coddle stimulation electrode. In the bottom panel, this reveals the spatial resolution of evoked states throughout the auditory cortex. While attempting this procedure, it is important to remember that the grounding of the grid floor is critical to obtain high quality electrophysiological recording, as well as a proper delivery of electrical stimulation Following this procedure.

Other methods like optogenetic and pharmacological manipulations can be included in the protocol in order to investigate specific cell types and transmitter systems in the cortical circuitry during perception and learning. This technique paves the way for researchers in the field from a mere correlative to more causal description of the role of neuronal dynamics for learning and memory functions.