Research report
Behavioral and paired-pulse facilitation analyses of long-lasting depression at excitatory synapses in the medial prefrontal cortex in mice

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Abstract

Accumulative evidence indicates that acute (before extinction) and long-lasting (during extinction) depression can occur at excitatory synapses in mouse medial prefrontal cortex (mPFC) during re-exposure to a tone (conditioned stimulus: CS), previously paired with footshock (unconditioned stimulus: US). As recently shown, the long-term depression (LTD)-like plasticity in the mPFC does not interfere with extinction of CS-evoked freezing but predicts spontaneous recovery of this fear response. Here, the objectives were to investigate: (i) whether a resistance to extinction without any prefrontal acute synaptic plasticity could produce LTD-like changes, and (ii) by the use of paired-pulse facilitation (PPF) analyses, whether pre- or post-synaptic mechanisms were involved in this LTD phenomenon. Preliminary analyses indicated that levels of acute depression did not correlate with the degree of fear acquisition (effects of number of CS–US pairings). As a consequence, mice conditioned with 2CS+ or 2CS+/2CS− (partial reinforcement of the CS known to induce resistance to extinction) exhibited CS-associated freezing without any acute synaptic depression in the mPFC. However, during further CS-alone presentations, the 2CS+/2CS− group developed LTD-like changes that accompanied their resistance to extinguish freezing to the CS. In contrast, the 2CS+ group normally extinguished their conditioned freezing with synaptic transmission remaining at baseline levels. PPF analyses revealed that facilitation was unchanged following prefrontal LTD. These data, combined with our previous findings, (i) support a critical involvement of prefrontal LTD-like changes in spontaneous recovery of fear responses, and (ii) suggest a post-synaptic site for these changes.

Introduction

Following auditory fear conditioning (AFC; e.g. tone–footshock pairing), the presentation of the conditioned stimulus (CS; tone) without the unconditioned stimulus (US; footshock) evokes fear responses such as freezing behavior and increased heart rate that decrease in magnitude if the CS continues to be presented alone. This inhibitory process, defined as extinction [18], can be accompanied in mice medial prefrontal cortex (mPFC) by a conversion from an acute depression to long-term enhancement of synaptic responses [10], which, because of its resemblance to the artificially induced long-term potentiation (LTP), is termed LTP-like changes. Based on the involvement of the mPFC in cognitive processes [6], such changes in synaptic responses may, in part, encode some of the cognitive attributes of the CS. In particular, since the certainty of the impending US is high at the beginning of extinction, the acute decrease in prefrontal synaptic transmission may represent such processing at each CS presentation [7]. However, because the certainty of danger weakens with repeated presentations of the CS without the US, the subsequent conversion of acute depression of synaptic efficacy to LTP-like changes in the mPFC may be related to predicting the lack of US [10]. In striking contrast, our more recent findings [11] indicate that during repeated CS-alone presentations the prefrontal acute depression can also be transformed into long-term depression (LTD)-like changes (resembling to the artificially induced LTD). Remarkably, this conversion did not interfere with extinction of freezing behavior to the CS, but was predictive of spontaneous recovery of fear responses. This effect was also confirmed in mice that received thalamic low-frequency stimulation (LFS) during extinction, as this treatment, which elicited LTD in the mPFC, facilitated recovery of fear responses [11]. Taken together, these data suggest the involvement of long-lasting depression at prefrontal excitatory synapses in the reinforcement of processes underlying the spontaneous recovery of fear responses. To further test this hypothesis, we determined whether development of LTD-like changes depends on the initial acute depression (in our previous experiments, both LTD and LTD-like changes developed from acute depression during extinction [10], [11]).

As thresholds for fear conditioning are lower emotionally than cognitively [16], [17], and knowing that fear is mostly mediated by the amygdala, which also modulates prefrontal cognitive activity relative to fear [7], we hypothesized that there is a threshold at which the mPFC glutamatergic synapses become sensitive to fear-related amygdala activity. The first experiment, therefore, determined conditioning parameters that could result in CS-evoked freezing (amygdala-dependent activity) without any associated acute synaptic modification in the mPFC. The ‘sub-cognitive’ parameters were then used in the following experiment with a partial reinforcement protocol known to produce resistance to extinction [4], [20]. In this condition, we hypothesized that the progressive development of uncertainty about the predictive value of the CS regarding US occurrence would be associated with LTD-like changes in the mPFC. We also examined with a paired-pulse facilitation (PPF) procedure whether pre- or post-synaptic mechanisms were implicated in prefrontal LTD.

Section snippets

Subjects and surgery

Subjects were 4-month-old male C57BL/6 mice (IFFA Credo, Lyon, France). They were individually housed in Plexiglas cages and were maintained on a free-feeding regimen with a 12:12 h light/dark schedule. All studies took place during the light portion of the cycle. The experiments were performed in accordance with the European Communities Council Directive (86/609/EEC). The animals were anesthetized with avertin (made up as 1.25 ml avertin concentrate, i.e. 100 g tribromoethanol dissolved in 62 ml

Experiment I

In this experiment we tested whether low level of conditioning could induce freezing to CS without any acute synaptic modification in the mPFC.

Experiment II

Since conditioning with 2 CS–US parings was adequate in our experimental conditions to produce CS-associated freezing without any prefrontal synaptic response (N1–P2) changes, we tested whether a partial reinforcement performed with 2 CS–US pairings mixed with 2 CS-alone presentations during the conditioning phase could induce resistance to extinction-associated prefrontal LTD-like changes.

Experiment III

We have established that, independently of the mode of induction of long-lasting depression of synaptic efficacy in the mPFC (behavioral induction or following thalamic LFS: [11]), this form of synaptic plasticity is strongly linked to spontaneous recovery of fear responses in mice. However, the mechanisms (pre- or post-synaptic) by which this form of synaptic plasticity is expressed remain unknown. The third experiment addressed this issue by using a PPF procedure; PPF is a transient form of

General discussion

In contrast to a previous study showing a strong correlation between acute depression of prefrontal excitability and conditioned fear responses [7], the present study analyzing the effects of the number of CS–US pairings on subsequent CS-associated depression of prefrontal synaptic efficacy did not indicate such a relationship. This discrepancy may be related to the different behavioral protocols used and their cognitive consequences. In the first experiment [7], mice had to learn that a

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    Present address: Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058, Basel, Switzerland.

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