Differential Effects of Lateral and Medial Entorhinal Cortex Lesions on Trace, Delay and Contextual Fear Memories

The entorhinal cortex (EC), with connections to the hippocampus, amygdala, and neocortex, is a critical, yet still underexplored, contributor to fear memory. Previous research suggests possible heterogeneity of function among its lateral (LEC) and medial (MEC) subregions. However, it is not well established what unique roles these subregions serve as the literature has shown mixed results depending on target of manipulation and type of conditioning used. Few studies have manipulated both the LEC and MEC within the same experiment. The present experiment systematically manipulated LEC and MEC function to examine their potential roles in fear memory expression. Long-Evans rats were trained using either trace or delay fear conditioning. The following day, rats received an N-methyl-D-aspartate (NMDA)-induced lesion to the LEC or MEC or received a sham surgery. Following recovery, rats were given an 8-min context test in the original context. The next day, rats were tested for tone freezing in a novel context with three discrete tone presentations. Further, rats were tested for hyperactivity in an open field under both dark and bright light gradient conditions. Results: Following either LEC or MEC lesion, freezing to context was significantly reduced in both trace and delay conditioned rats. LEC-lesioned rats consistently showed significantly less freezing following tone-offset (trace interval, or equivalent, and intertrial interval) in both trace and delay fear conditioned rats. Conclusions: These data suggest that the LEC may play a role in the expression of a conjunctive representation between the tone and context that mediates the maintenance of post-tone freezing.

Muscimol-induced inactivation of the anterior cingulate cortex does not impair trace fear conditioning in the rat

Previous studies suggest that trace conditioning depends on the anterior cingulate cortex (ACC). To examine the role of ACC in trace fear conditioning further, 48 rats were surgically prepared for infusion with saline or 62.5 or 125 µg/side muscimol to inactivate ACC reversibly prior to conditioning. A noise stimulus was followed by a 1 mA footshock, with or without a 10-second trace interval between these events in a conditioned suppression procedure. The trace-conditioned groups (10 seconds) showed less test suppression than the control-conditioned groups (0 seconds). Counter to prediction, there was no effect of muscimol infusion on suppression to the noise stimulus in the 10-second trace groups.

How the Cerebellum and Prefrontal Cortex Cooperate During Trace Eyeblinking Conditioning

Several data have demonstrated that during the widely used experimental paradigm for studying associative learning, trace eye blinking conditioning (TEBC), there is a strong interaction between cerebellum and medial prefrontal cortex (mPFC). Despite this evidence, the neural mechanisms underlying this interaction are still not clear. Here, we propose a neurophysiologically plausible computational model to address this issue. The model is constrained on the basis of two critical anatomo-physiological features: (i) the cerebello-cortical organization through two circuits, respectively, targeting M1 and mPFC; (ii) the different timing in the plasticity mechanisms of these parallel circuits produced by the granule cells time sensitivity according to which different subpopulations are active at different moments during conditioned stimuli. The computer simulations run with the model suggest that these features are critical to understand how the cooperation between cerebellum and mPFC supports motor areas during TEBC. In particular, a greater trace interval produces greater plasticity changes at the slow path synapses involving mPFC with respect to plasticity changes at the fast path involving M1. As a consequence, the greater is the trace interval, the stronger is the mPFC involvement. The model has been validated by reproducing data collected through recent real mice experiments.

The effects of stimulus distribution form during trace conditioning

Three experiments examined the effect of distribution form of the trace interval on trace conditioning. In Experiments 1 and 2, two groups of rats were conditioned to a fixed-duration conditioned stimulus (CS) in a trace interval procedure; rats in Group Fix received a fixed-duration trace interval, whereas for rats in Group Var the trace interval was of variable duration. Responding during the CS was higher in Group Var than in Group Fix, whereas during the trace interval this difference in responding reversed—Group Fix showed higher response rates than Group Var. Experiment 3 examined whether the greater response rate observed during the CS in Group Var was due to a performance effect or the acquisition of greater associative strength by the CS. Following trace conditioning, the rats from Experiment 1 underwent a second phase of delay conditioning with the same CS; a 5-s auditory stimulus was presented in compound with the last 5 s of the 15-s CS, and the unconditioned stimulus (US) was delivered at the offset of the CSs. On test with the auditory stimulus alone, subjects in Group Var showed lower response rates during the auditory stimulus than subjects in Group Fix. We interpreted these findings as evidence that the superior responding in Group Var during the CS was a result of it acquiring greater associative strength than in Group Fix.

Persistent activity in a cortical-to-subcortical circuit: bridging the temporal gap in trace eyelid conditioning

We have addressed the source and nature of the persistent neural activity that bridges the stimulus-free gap between the conditioned stimulus (CS) and unconditioned stimulus (US) during trace eyelid conditioning. Previous work has demonstrated that this persistent activity is necessary for trace eyelid conditioning: CS-elicited activity in mossy fiber inputs to the cerebellum does not extend into the stimulus-free trace interval, which precludes the cerebellar learning that mediates conditioned response expression. In behaving rabbits we used in vivo recordings from a region of medial prefrontal cortex (mPFC) that is necessary for trace eyelid conditioning to test the hypothesis that neurons there generate activity that persists beyond CS offset. These recordings revealed two patterns of activity during the trace interval that would enable cerebellar learning. Activity in some cells began during the tone CS and persisted to overlap with the US, whereas in other cells, activity began during the stimulus-free trace interval. Injection of anterograde tracers into this same region of mPFC revealed dense labeling in the pontine nuclei, where recordings also revealed tone-evoked persistent activity during trace conditioning. These data suggest a corticopontine pathway that provides an input to the cerebellum during trace conditioning trials that bridges the temporal gap between the CS and US to engage cerebellar learning. As such, trace eyelid conditioning represents a well-characterized and experimentally tractable system that can facilitate mechanistic analyses of cortical persistent activity and how it is used by downstream brain structures to influence behavior.

Eyeblink conditioning contingent on hippocampal theta enhances hippocampal and medial prefrontal responses

Trace eyeblink classical conditioning (tEBCC) can be accelerated by making training trials contingent on the naturally generated hippocampal 3- to 7-Hz theta rhythm. However, it is not well-understood how the presence (or absence) of theta affects stimulus-driven changes within the hippocampus and how it correlates with patterns of neural activity in other essential trace conditioning structures, such as the medial prefrontal cortex (mPFC). In the present study, a brain-computer interface delivered paired or unpaired conditioning trials to rabbits during the explicit presence (T+) or absence (T−) of theta, yielding significantly faster behavioral learning in the T+-paired group. The stimulus-elicited hippocampal unit responses were larger and more rhythmic in the T+-paired group. This facilitation of unit responses was complemented by differences in the hippocampal local field potentials (LFP), with the T+-paired group demonstrating more coherent stimulus-evoked theta than T−-paired animals and both unpaired groups. mPFC unit responses in the rapid learning T+-paired group displayed a clear inhibitory/excitatory sequential pattern of response to the tone that was not seen in any other group. Furthermore, sustained mPFC unit excitation continued through the trace interval in T+animals but not in T−animals. Thus theta-contingent training is accompanied by 1) acceleration in behavioral learning, 2) enhancement of the hippocampal unit and LFP responses, and 3) enhancement of mPFC unit responses. Together, these data provide evidence that pretrial hippocampal state is related to enhanced neural activity in critical structures of the distributed network supporting the acquisition of tEBCC.

Temporal Patterns of Inputs to Cerebellum Necessary and Sufficient for Trace Eyelid Conditioning

Trace eyelid conditioning is a form of associative learning that requires several forebrain structures and cerebellum. Previous work suggests that at least two conditioned stimulus (CS)-driven signals are available to the cerebellum via mossy fiber inputs during trace conditioning: one driven by and terminating with the tone and a second driven by medial prefrontal cortex (mPFC) that persists through the stimulus-free trace interval to overlap in time with the unconditioned stimulus (US). We used electric stimulation of mossy fibers to determine whether this pattern of dual inputs is necessary and sufficient for cerebellar learning to express normal trace eyelid responses. We find that presenting the cerebellum with one input that mimics persistent activity observed in mPFC and the lateral pontine nuclei during trace eyelid conditioning and another that mimics tone-elicited mossy fiber activity is sufficient to produce responses whose properties quantitatively match trace eyelid responses using a tone. Probe trials with each input delivered separately provide evidence that the cerebellum learns to respond to the mPFC-like input (that overlaps with the US) and learns to suppress responding to the tone-like input (that does not). This contributes to precisely timed responses and the well-documented influence of tone offset on the timing of trace responses. Computer simulations suggest that the underlying cerebellar mechanisms involve activation of different subsets of granule cells during the tone and during the stimulus-free trace interval. These results indicate that tone-driven and mPFC-like inputs are necessary and sufficient for the cerebellum to learn well-timed trace conditioned responses.