Cannabinoids Potentiate Emotional Learning Plasticity in Neurons of the Medial Prefrontal Cortex through Basolateral Amygdala Inputs

Abstract

Cannabinoids represent one of the most commonly used hallucinogenic drug classes. In addition, cannabis use is a primary risk factor for schizophrenia in susceptible individuals and can potently modulate the emotional salience of sensory stimuli. We report that systemic activation or blockade of cannabinoid CB1 receptors modulates emotional associative learning and memory formation in a subpopulation of neurons in the mammalian medial prefrontal cortex (mPFC) that receives functional input from the basolateral amygdala (BLA). Using in vivo single-unit recordings in rats, we found that a CB1 receptor agonist potentiated the response of medial prefrontal cortical neurons to olfactory cues paired previously with a footshock, whereas this associative responding was prevented by a CB1 receptor antagonist. In an olfactory fear-conditioning procedure, CB1 agonist microinfusions into the mPFC enabled behavioral responses to olfactory cues paired with normally subthreshold footshock, whereas the antagonist completely blocked emotional learning. These results are the first demonstration that cannabinoid signaling in the mPFC can modulate the magnitude of neuronal emotional learning plasticity and memory formation through functional inputs from the BLA.

Figure 1.

Olfactory associative learning in single mPFC neurons responding to BLA excitatory input. A, Left, Schematic presentation of neuronal recording sites in the mPFC. For clarity, only nonoverlapping sites are shown representing the anatomical distribution of recording in the mPFC. Black circles, 0.5 mg/kg WIN 55,212-2; open circles, 0.05 mg/kg WIN 55,212-2; open squares, 1.0 mg/kg AM-251; gray circles, 0.1 mg/kg AM-251; open polygons, saline controls. Right, Photomicrograph of a coronal section of the mPFC showing a representative recording site, located within the white circle. B, Left, Representative stimulation sites in the BLA corresponding to the mPFC recording sites shown in A. Symbols are the same as in A. Right, Photomicrograph of a coronal section of the BLA showing a representative stimulation site, indicated by black arrowheads. CeA, Central nucleus of amygdala; LaN, lateral nucleus of amygdala. C, Left, Orthodromic spike evoked by stimulation of the BLA. Right, The evoked response latency for the same neuron over 100 stimulations of the BLA at 600 μA.

Figure 2.

BLA-responsive mPFC neurons exhibited a stimulus-locked discharge in response to CS+ presentations. A, Rate histogram showing the firing frequency during baseline, CS+, and CS− olfactory stimulus presentations. Activity increases specifically in response to CS+ presentation and not during CS− presentation. B, The firing activity of the same neuron, immediately before CS+ presentation onset and during the course of the CS+ presentation, again illustrating CS+ stimulus-locked responding.

Figure 3.

Activation or blockade of CB1 receptors with WIN 55,212-2 (0.05–0.5 mg/kg, i.v.) or AM-251 (0.1–1.0 mg/kg, i.v.) modulates emotional learning in BLA-responsive neurons of the mPFC. A, After a subthreshold dose of WIN 55,212-2 (0.05 mg/kg), mPFC neurons display associative responding to the CS+ at a similar magnitude to saline controls. A higher dose (0.5 mg/kg) causes a potentiation in the neuronal associative response to the CS+ presentation. Pharmacological inactivation of the BLA with muscimol (500 ng) before olfactory conditioning but after WIN 55,212-2 (0.5 mg/kg) administration prevents neuronal associative responding at testing. However, if the BLA is inactivated after conditioning in animals given WIN 55,212-2 (0.5 mg/kg) before conditioning, BLA-responsive mPFC neurons still display robust potentiation of associative responding to the CS+ presentation relative to saline control animals. Pre-Cond, Preconditioning; Post-Cond, postconditioning. B, Comparing preconditioning and postconditioning 10 s odor presentations demonstrates that CB1 receptor activation with WIN 55,212-2 (0.5 mg/kg) potentiates associative neuronal responding specifically to CS+ presentations relative to saline control or animals pretreated with a lower dose of WIN 55,212-2 (0.05 mg/kg). C, Blockade of CB1 receptors with a competitive CB1 receptor antagonist, AM-251, blocked neuronal associative learning in BLA-responsive neurons of the mPFC at an intravenous dose of 1.0 mg/kg. A lower dose (0.1 mg/kg, i.v.) had no effect on associative neuronal responding relative to saline control animals. Pretreatment with AM-251 (1.0 mg/kg, i.v.) blocks the associative learning potentiation induced by WIN 55,212-2 (0.5 mg/kg, i.v.). D, Comparing preconditioning and postconditioning 10 s odor presentations after AM-251 pretreatment (0.1 or 1.0 mg/kg, i.v.) demonstrates that CB1 receptor blockade at the higher dose (1.0 mg/kg) prevents neuronal associative responding in response to CS+ presentations. E, Neither administration of the CB1 antagonist AM-251 (0.1 and 1.0 mg/kg, i.v.) nor the CB1 receptor agonist WIN 55,212-2 (0.05–0.5 mg/kg, i.v.) caused any significant alterations in spontaneous neuronal activity after injection. Pre, Preconditioning; Post, postconditioning. Error bars indicate mean ± SEM.

Figure 4.

Effects of WIN 55,212-2 (0.5 mg/kg, i.v.) on neuronal activity during conditioned cue presentations. A, Recording from an mPFC neuron in a control rat showing CS+-specific responding during presentation of the footshock-paired odor. B, Recording from the same neuron during the CS− odor presentation. C, Recording from an mPFC neuron in a WIN 55,212-2 (0.5 mg/kg) pretreated rat showing CS+-specific responding during presentation of the footshock-paired odor reveals robust potentiation of the conditioned neuronal response only in the presence of the CS+ olfactory stimulus. D, Recording from the same neuron during the CS− odor presentation.

Figure 5.

Cannabinoid CB1 receptors modulate associative neuronal bursting in BLA-responsive mPFC neurons. A, WIN 55,212-2 potentiates associative neuronal bursting in BLA-responsive mPFC neurons at the intravenous dose of 0.5 mg/kg compared with a lower dose of WIN 55,212-2 (0.05 mg/kg, i.v.) and saline controls. B, In contrast, AM-251 blocked neuronal associative bursting reflected in the percentage of spontaneous spikes occurring in burst events at a dose of 1.0 mg/kg but not a lower intravenous dose of 0.1 mg/kg. Pretreatment with the effective dose of AM-251 (1.0 mg/kg) competitively blocked the ability of WIN 55,212-2 (0.5 mg/kg) to potentiate neuronal associative bursting in BLA-responsive mPFC neurons. C, Activation of CB1 receptors potentiated the number of spikes per burst event specifically in response to CS+ presentations at a dose of 0.5 mg/kg but not at a lower dose of 0.05 mg/kg, both given intravenously. D, In contrast, AM-251 prevented this associative increase in the number of spikes per burst at the dose of 1.0 mg/kg but not at the lower dose of 0.1 mg/kg. Pretreatment with the effective dose of AM-251 (1.0 mg/kg) blocked the ability of WIN 55,212-2 (0.5 mg/kg) to potentiate the number of spikes per burst event in response to the CS+ presentation. E, Left, In a saline-pretreated control animal, the percentage of bursting during baseline spontaneous activity of mPFC neuronal responding compared with that during CS+ and CS− presentation is shown. Control neurons increase the percentage of bursting during baseline specifically in response to the CS+ presentation. Right, Activity trace for this neuron during the first second of CS+ odor presentation. In control neurons, bursts typically take place in doublets or triplets. F, Left, A BLA-responsive mPFC neuron from a WIN 55,212-2 (0.5 mg/kg, i.v.) pretreated animal showing the percentage of bursting activity over baseline, CS+, and CS− presentations. This neuron displays a potentiation in the percentage of bursting in response to the CS+ presentation. Right, Activity trace for this same neuron during the first second of CS+ presentation. G, Left, In a BLA-responsive mPFC neuron from an AM-251 (1.0 mg/kg) pretreated animal, there is a blockade of associative neuronal bursting in response to CS+ and CS− presentations. Right, The neuronal activity trace from this same neuron during the first second of CS+ presentation; no bursting activity is present. H, I, There is no significant correlation between firing frequency and percentage of spike events occurring in bursts. The difference between baseline firing frequency and CS+ firing frequency is plotted as a function of the difference between baseline percentage of bursting and CS+ percentage of bursting in the same neurons for either saline (H) or WIN 55,212-2 (I; 0.5 mg/kg) pretreated neurons. *p < 0.01. Error bars indicate mean ± SEM.

Figure 6.

CB1 receptor activation potentiates the frequency of burst events specifically in response to CS+ odor presentations. A, ISI histogram from a single BLA-responsive mPFC saline control rat showing the distribution of ISIs over the 2 min baseline, CS−, and CS+ recording epochs. A moderate shift in ISI frequency occurring below the 45 ms burst criterion (see Materials and Methods) takes place in response to the CS+ presentation relative to baseline. B, An ISI histogram from a single BLA-responsive mPFC neuron pretreated with the effective dose of WIN 55,212-2 (0.5 mg/kg) shows a strong shift toward ISI frequencies occurring below the 45 ms burst ISI criterion relative to baseline and CS− recording epochs, demonstrating that CB1 receptor activation can strongly potentiate associative bursting in response to emotionally salient conditioned stimuli. For all panels, the black arrows indicate the 45 ms burst ISI threshold cutoff point (see Materials and Methods).

Figure 7.

CB1 receptor activation or blockade in the mPFC modulates the behavioral expression of associative olfactory fear conditioning; the effects of intra-mPFC CB1 receptor modulation on neuronal and behavioral sensitivity to footshock presentation are shown. A, Rats show conditioned freezing behavior to an olfactory cue paired with a suprathreshold level of footshock (0.8 mA) 24 h after conditioning. Relative to saline controls, intra-mPFC microinfusions of AM-251 (50 ng/0.5 μl) before conditioning blocks olfactory fear conditioning relative to saline controls, whereas WIN 55,212-2 (50 ng/0.5 μl) has no effect on olfactory fear conditioning. Rats tested in the presence of intra-mPFC AM-251 (50 ng) still demonstrated a block in olfactory fear-conditioning expression, thus ruling out state-dependency effects. B, Similarly, AM-251 dose-dependently attenuates spontaneous exploratory behavior measures (see Materials and Methods) in response to CS+ odor presentations, whereas WIN 55,212-2 (50 ng) has no effect at this level of footshock (0.8 mA). C, A subthreshold level of footshock (0.4 mA) produces no fear conditioning in saline-pretreated control animals. However, intra-mPFC infusions of WIN 55,212-2 (25 or 50 ng/0.5 μl) potentiated the effects of this subthreshold level of footshock by enabling freezing to CS+ presentations relative to saline controls and a subthreshold dose of intra-mPFC WIN 55,212-2 (5 ng). This effect was blocked by simultaneous administration of the CB1 antagonist AM-251 (50 ng) with the highest effective dose of intra-mPFC WIN 55,212-2 (50 ng). Animals tested in the presence of intra-mPFC WIN 55,212-2 (50 ng) demonstrated strong olfactory fear-conditioning expression to subthreshold footshock, thus ruling out state-dependency effects. D, Subthreshold footshock failed to induce conditioned attenuation in exploratory behavior in response to postconditioning presentations of the CS+. However, in animals receiving intra-mPFC WIN 55,212-2 (25 or 50 ng), strong conditioned attenuation of exploratory behavior was observed during presentation of the CS+. This effect was blocked by coadministration of the CB1 antagonist AM-251 (50 ng) with the highest effective dose of intra-mPFC WIN 55,212-2 (50 ng). E, There were no significant differences in neuronal responsiveness in animals pretreated with effective systemic doses of either WIN 55,212-2 (0.5 mg/kg) or AM-251 (1.0 mg/kg). F, Bilateral intra-mPFC microinfusions of either WIN 55,212-2 (50 ng) or AM-251 (50 ng) produce no change in the percentage of freezing in response to suprathreshold footshock (0.8 mA) presentations during conditioning. G, H, Similarly, no differences were observed between intra-mPFC WIN 55,212-2 (50 ng) or AM-251 (50 ng) in terms of the number of jumps in response to footshock presentations (G) nor in the amount of defecation or in the percentage of animals displaying rearing behavior in response to footshock (H). Error bars indicate mean ± SEM.