Neural signatures of opioid-induced risk-taking behavior in the prelimbic prefrontal cortex.

Opioid use disorder occurs alongside impaired risk-related decision-making, but the underlying neural correlates are unclear. We developed a novel approach-avoidance conflict model using a modified conditioned place preference paradigm to study neural signals of risky opioid seeking in the prefrontal cortex, a region implicated in executive decision making. Upon establishment of morphine conditioned place preference, rats underwent a subsequent conflict test in which fear-inducing cat odor was introduced in the previously drug-paired side of the apparatus. While the saline control group avoided the cat odor side, the morphine group maintained preference for the paired side despite the presence of cat odor. K-means clustering identified two subsets of morphine-treated rats that exhibited either persistent drug seeking (Risk-Takers) or increased avoidance (Risk-Avoiders) during conflict. Single-unit recordings from the prelimbic cortex (PL) revealed decreased neuronal firing rates upon acute morphine exposure in both Risk-Takers and Risk-Avoiders, but this firing rate suppression was absent after repeated administration. Risk-Avoiders also displayed distinct post-morphine excitation in PL which persisted across conditioning. During the preference test, subpopulations of PL neurons in all groups were either excited or inhibited when rats entered the paired side. Interestingly, while this inhibitory signal was lost during the subsequent conflict test in both saline and Risk-Avoider groups, these inhibitory responses persisted in Risk-Takers. Our results suggest that loss of PL inhibition after opioid conditioning is associated with the formation of contextual reward memory. Furthermore, persistent PL inhibitory signaling in the drug-associated context during conflict may underlie increased risk taking following opioid exposure.

Enhancing Associative Learning in Rats With a Computationally Designed Training Protocol.

Background: Learning requires the activation of protein kinases with distinct temporal dynamics. In Aplysia, nonassociative learning can be enhanced by a computationally designed learning protocol with intertrial intervals (ITIs) that maximize the interaction between fast-activated PKA (protein kinase A) and slow-activated ERK (extracellular signal-regulated kinase). Whether a similar strategy can enhance associative learning in mammals is unknown. Methods: We simulated 1000 training protocols with varying ITIs to predict an optimal protocol based on empirical data for PKA and ERK dynamics in rat hippocampus. Adult male rats received the optimal protocol or control protocols in auditory fear conditioning and fear extinction experiments. Immunohistochemistry was performed to evaluate pCREB (phosphorylated cAMP response element binding)\protein levels in brain regions that have been implicated in fear acquisition. Results: Rats exposed to the optimal conditioning protocol with irregular ITIs exhibited impaired extinction memory acquisition within the session using a standard footshock intensity, and stronger fear memory retrieval and spontaneous recovery with a weaker footshock intensity, compared with rats that received massed or spaced conditioning protocols with fixed ITIs. Rats exposed to the optimal extinction protocol displayed improved extinction of contextual fear memory and reduced spontaneous recovery compared with rats that received standard extinction protocols. Moreover, the optimal conditioning protocol increased pCREB levels in the dentate gyrus of the dorsal hippocampus, suggesting enhanced induction of long-term potentiation. Conclusions: These findings demonstrate that a computational model-driven behavioral intervention can enhance associative learning in mammals and may provide insight into strategies to improve cognition in humans.

Positioning the brainstem within the neural network of threat prediction.

In a recent study, Strickland and McDannald dissected the role of brainstem networks in threat prediction. Using probabilistic threat discrimination in rats, the authors demonstrated that brainstem neurons estimate threat probability and generate positive aversive prediction errors after unexpected outcomes. Their findings suggest that, beyond organizing defensive behaviors, brainstem neurons are involved in threat prediction computations.

Neural correlates and determinants of approach-avoidance conflict in the prelimbic prefrontal cortex.

The recollection of environmental cues associated with threat or reward allows animals to select the most appropriate behavioral responses. Neurons in the prelimbic (PL) cortex respond to both threat- and reward-associated cues. However, it remains unknown whether PL regulates threat-avoidance vs. reward-approaching responses when an animals' decision depends on previously associated memories. Using a conflict model in which male Long-Evans rats retrieve memories of shock- and food-paired cues, we observed two distinct phenotypes during conflict: (1) rats that continued to press a lever for food (Pressers) and (2) rats that exhibited a complete suppression in food seeking (Non-pressers). Single-unit recordings revealed that increased risk-taking behavior in Pressers is associated with persistent food-cue responses in PL, and reduced spontaneous activity in PL glutamatergic (PLGLUT) neurons during conflict. Activating PLGLUT neurons in Pressers attenuated food-seeking responses in a neutral context, whereas inhibiting PLGLUT neurons in Non-pressers reduced defensive responses and increased food approaching during conflict. Our results establish a causal role for PLGLUT neurons in mediating individual variability in memory-based risky decision-making by regulating threat-avoidance vs. reward-approach behaviors.

Understanding the dynamic and destiny of memories.

Memory formation enables the retention of life experiences overtime. Based on previously acquired information, organisms can anticipate future events and adjust their behaviors to maximize survival. However, in an ever-changing environment, a memory needs to be malleable to maintain its relevance. In fact, substantial evidence suggests that a consolidated memory can become labile and susceptible to modifications after being reactivated, a process termed reconsolidation. When an extinction process takes place, a memory can also be temporarily inhibited by a second memory that carries information with opposite meaning. In addition, a memory can fade and lose its significance in a process known as forgetting. Thus, following retrieval, new life experiences can be integrated with the original memory trace to maintain its predictive value. In this review, we explore the determining factors that regulate the fate of a memory after its reactivation. We focus on three post-retrieval memory destinies (reconsolidation, extinction, and forgetting) and discuss recent rodent studies investigating the biological functions and neural mechanisms underlying each of these processes.

A NeuroD1 AAV-Based Gene Therapy for Functional Brain Repair after Ischemic Injury through In Vivo Astrocyte-to-Neuron Conversion.

Adult mammalian brains have largely lost neuroregeneration capability except for a few niches. Previous studies have converted glial cells into neurons, but the total number of neurons generated is limited and the therapeutic potential is unclear. Here, we demonstrate that NeuroD1-mediated in situ astrocyte-to-neuron conversion can regenerate a large number of functional new neurons after ischemic injury. Specifically, using NeuroD1 adeno-associated virus (AAV)-based gene therapy, we were able to regenerate one third of the total lost neurons caused by ischemic injury and simultaneously protect another one third of injured neurons, leading to a significant neuronal recovery. RNA sequencing and immunostaining confirmed neuronal recovery after cell conversion at both the mRNA level and protein level. Brain slice recordings found that the astrocyte-converted neurons showed robust action potentials and synaptic responses at 2 months after NeuroD1 expression. Anterograde and retrograde tracing revealed long-range axonal projections from astrocyte-converted neurons to their target regions in a time-dependent manner. Behavioral analyses showed a significant improvement of both motor and cognitive functions after cell conversion. Together, these results demonstrate that in vivo cell conversion technology through NeuroD1-based gene therapy can regenerate a large number of functional new neurons to restore lost neuronal functions after injury.

Boosting of Thalamic D2 Dopaminergic Transmission: A Potential Strategy for Drug-Seeking Attenuation.

This commentary focuses on novel findings by Clark et al. (2017) published in eNeuro, which show that dopamine D2 receptors (D2Rs) in the paraventricular nucleus of the thalamus (PVT) are involved in cocaine sensitization. We extend the discussion on how their findings contribute to our understanding of the role of the PVT in drug seeking by providing new insight on the role of the PVT in the regulation of food-seeking and fear responses. We also consider the significance of the neuroanatomical findings reported by Clark et al., that the PVT is reciprocally connected with areas of the brain involved in addiction and discuss the implications associated with the source and type of dopaminergic fibers innervating this area of the thalamus.

Thalamic Regulation of Sucrose Seeking during Unexpected Reward Omission.

The paraventricular nucleus of the thalamus (PVT) is thought to regulate behavioral responses under emotionally arousing conditions. Reward-associated cues activate PVT neurons; however, the specific PVT efferents regulating reward seeking remain elusive. Using a cued sucrose-seeking task, we manipulated PVT activity under two emotionally distinct conditions: (1) when reward was available during the cue as expected or (2) when reward was unexpectedly omitted during the cue. Pharmacological inactivation of the anterior PVT (aPVT), but not the posterior PVT, increased sucrose seeking only when reward was omitted. Consistent with this, photoactivation of aPVT neurons abolished sucrose seeking, and the firing of aPVT neurons differentiated reward availability. Photoinhibition of aPVT projections to the nucleus accumbens or to the amygdala increased or decreased, respectively, sucrose seeking only when reward was omitted. Our findings suggest that PVT bidirectionally modulates sucrose seeking under the negative (frustrative) conditions of reward omission.

Bidirectional Modulation of Extinction of Drug Seeking by Deep Brain Stimulation of the Ventral Striatum.

BACKGROUND: Recent research in humans and rodents has explored the use of deep brain stimulation (DBS) of the ventral capsule/ventral striatum (VS) as a possible treatment for drug addiction. However, the optimum electrode placement and optimum DBS parameters have not been thoroughly studied. Here we varied stimulation sites and frequencies to determine whether DBS of the VS could facilitate the extinction of morphine-induced conditioned place preference in rats. METHODS: Rats were implanted with DBS electrodes in the dorsal or ventral subregions of the VS and trained to the morphine conditioned place preference. Subsequently, rats received extinction sessions over 9 days, combined with 60 min of either high- (130 Hz) or low- (20 Hz) frequency DBS. To study circuit-wide activations after DBS of the VS, c-fos immunohistochemistry was performed in regions involved in the extinction of drug-seeking behaviors. RESULTS: High-frequency DBS of the dorsal-VS impaired both extinction training and extinction memory, whereas high-frequency DBS of the ventral-VS had no effect. In contrast, low-frequency DBS of the dorsal-VS strengthened extinction memory when tested 2 or 9 days after the cessation of stimulation. Both DBS frequencies increased c-fos expression in the infralimbic prefrontal cortex, but only low-frequency DBS increased c-fos expression in the basal amygdala and the medial portion of the central amygdala. CONCLUSIONS: Our results suggest that low-frequency (rather than high-frequency) DBS of the dorsal-VS strengthens extinction memory and may be a potential adjunct for extinction-based therapies for treatment-refractory opioid addiction.

Revisiting the role of infralimbic cortex in fear extinction with optogenetics.

Previous rodent studies have implicated the infralimbic (IL) subregion of the medial prefrontal cortex in extinction of auditory fear conditioning. However, these studies used pharmacological inactivation or electrical stimulation techniques, which lack temporal precision and neuronal specificity. Here, we used an optogenetic approach to either activate (with channelrhodopsin) or silence (with halorhodopsin) glutamatergic IL neurons during conditioned tones delivered in one of two phases: extinction training or extinction retrieval. Activating IL neurons during extinction training reduced fear expression and strengthened extinction memory the following day. Silencing IL neurons during extinction training had no effect on within-session extinction, but impaired the retrieval of extinction the following day, indicating that IL activity during extinction tones is necessary for the formation of extinction memory. Surprisingly, however, silencing IL neurons optogenetically or pharmacologically during the retrieval of extinction 1 day or 1 week following extinction training had no effect. Our findings suggest that IL activity during extinction training likely facilitates storage of extinction in target structures, but contrary to current models, IL activity does not appear to be necessary for retrieval of extinction memory.

Enhancement of fear extinction with deep brain stimulation: evidence for medial orbitofrontal involvement.

Deep brain stimulation (DBS) of the ventral capsule/ventral striatum (VC/VS) reduces anxiety, fear, and compulsive symptoms in patients suffering from refractory obsessive-compulsive disorder. In a rodent model, DBS-like high-frequency stimulation of VS can either enhance or impair extinction of conditioned fear, depending on the location of electrodes within VS (dorsal vs ventral). As striatal DBS activates fibers descending from the cortex, we reasoned that the differing effects on extinction may reflect differences in cortical sources of fibers passing through dorsal-VS and ventral-VS. In agreement with prior anatomical studies, we found that infralimbic (IL) and anterior insular (AI) cortices project densely through ventral-VS, the site where DBS impaired extinction. Contrary to IL and AI, we found that medial orbitofrontal cortex (mOFC) projects densely through dorsal-VS, the site where DBS enhanced extinction. Furthermore, pharmacological inactivation of mOFC reduced conditioned fear and DBS of dorsal-VS-induced plasticity (pERK) in mOFC neurons. Our results support the idea that VS DBS modulates fear extinction by stimulating specific fibers descending from mOFC and prefrontal cortices.

A temporal shift in the circuits mediating retrieval of fear memory.

Fear memories allow animals to avoid danger, thereby increasing their chances of survival. Fear memories can be retrieved long after learning, but little is known about how retrieval circuits change with time. Here we show that the dorsal midline thalamus of rats is required for the retrieval of auditory conditioned fear at late (24 hours, 7 days, 28 days), but not early (0.5 hours, 6 hours) time points after learning. Consistent with this, the paraventricular nucleus of the thalamus (PVT), a subregion of the dorsal midline thalamus, showed increased c-Fos expression only at late time points, indicating that the PVT is gradually recruited for fear retrieval. Accordingly, the conditioned tone responses of PVT neurons increased with time after training. The prelimbic (PL) prefrontal cortex, which is necessary for fear retrieval, sends dense projections to the PVT. Retrieval at late time points activated PL neurons projecting to the PVT, and optogenetic silencing of these projections impaired retrieval at late, but not early, time points. In contrast, silencing of PL inputs to the basolateral amygdala impaired retrieval at early, but not late, time points, indicating a time-dependent shift in retrieval circuits. Retrieval at late time points also activated PVT neurons projecting to the central nucleus of the amygdala, and silencing these projections at late, but not early, time points induced a persistent attenuation of fear. Thus, the PVT may act as a crucial thalamic node recruited into cortico-amygdalar networks for retrieval and maintenance of long-term fear memories.

Long-range GABAergic neurons in the prefrontal cortex modulate behavior.

Cortical glutamatergic projections are extensively studied in behavioral neuroscience, whereas cortical GABAergic projections to downstream structures have been overlooked. A recent study by Lee and colleagues (Lee AT, Vogt D, Rubenstein JL, Sohal VS. J Neurosci 34: 11519-11525, 2014) used optogenetic and electrophysiological techniques to characterize a behavioral role for long-projecting GABAergic neurons in the medial prefrontal cortex. In this Neuro Forum, we discuss the potential implications of this study in several learning and memory models.

Hippocampal--prefrontal BDNF and memory for fear extinction.

Infusing brain-derived neurotrophic factor (BDNF) into the infralimbic (IL) prefrontal cortex is capable of inducing extinction. Little is known, however, about the circuits mediating BDNF effects on extinction or the extent to which extinction requires BDNF in IL. Using local pharmacological infusion of BDNF protein, or an antibody against BDNF, we found that BDNF in the IL, but not prelimbic (PL) prefrontal cortex, is both necessary and sufficient for fear extinction. Furthermore, we report that BDNF in IL can induce extinction of older fear memories (14 days) as well as recent fear memories (1 day). Using immunocytochemistry, we show that BDNF is increased in the ventral hippocampus (vHPC), but not IL or PL, following extinction training. Finally, we observed that infusing BDNF into the vHPC increased the firing rate of IL, but not PL neurons in fear conditioned rats. These findings indicate that an extinction-induced increase in BDNF within the vHPC enhances excitability in IL targets, thereby supporting extinction memories.

Deep brain stimulation of the ventral striatum increases BDNF in the fear extinction circuit.

Deep brain stimulation (DBS) of the ventral capsule/ventral striatum (VC/VS) reduces the symptoms of treatment-resistant obsessive compulsive disorder (OCD), and improves response to extinction-based therapies. We recently reported that DBS-like stimulation of a rat homologue of VC/VS, the dorsal-VS, reduced conditioned fear and enhanced extinction memory (Rodriguez-Romaguera et al., 2012). In contrast, DBS of the ventral-VS had the opposite effects. To examine possible mechanisms of these effects, we assessed the effects of VS DBS on the expression of the neural activity marker Fos and brain-derived neurotrophic factor (BDNF), a key mediator of extinction plasticity in prefrontal-amygdala circuits. Consistent with decreased fear expression, DBS of dorsal-VS increased Fos expression in prelimbic and infralimbic prefrontal cortices and in the lateral division of the central nucleus of amygdala, an area that inhibits amygdala output. Consistent with improved extinction memory, we found that DBS of dorsal-VS, but not ventral-VS, increased neuronal BDNF expression in prelimbic and infralimbic prefrontal cortices. These rodent findings are consistent with the idea that clinical DBS of VC/VS may augment fear extinction through an increase in BDNF expression.

Infusion of cannabidiol into infralimbic cortex facilitates fear extinction via CB1 receptors.

Previous studies have implicated cannabinoids in extinction of conditioned fear. We have recently showed that intraventricular infusion of the phytocannabinoid cannabidiol (CBD) facilitates fear extinction, but the brain regions underlying this effect remained unknown. Here we demonstrate that repeated microinjections of CBD into the infralimbic cortex (IL) facilitated fear extinction, as indicated by reduced levels of freezing during extinction test. Systemic administration of the CB1 receptor antagonist rimonabant blocked the effects of intra-IL CBD, suggesting that CBD acts through CB1 receptors to facilitate fear extinction. Our findings suggest a potential therapeutic use of CBD for extinction-based therapies of aversive memories in humans.

Systemic or intra-prelimbic cortex infusion of prazosin impairs fear memory reconsolidation.

The alpha-1 adrenergic antagonist prazosin has been used to alleviate the symptoms of PTSD, but the mechanism remains unclear. One possibility is that prazosin may disrupt fear memory reconsolidation, leading to attenuation of fear responses. To test this hypothesis, we administered a single systemic injection of prazosin during the reconsolidation of olfactory fear conditioning in rats. We found that a post-retrieval injection of prazosin disrupted subsequent retrieval of fear. Similarly, intra-prelimbic cortex infusion of prazosin during the reconsolidation period also disrupted subsequent retrieval of fear. These findings suggest that fear memory undergoes reconsolidation through activation of alpha-1 adrenergic receptors in the prelimbic cortex.

Deep brain stimulation of the ventral striatum enhances extinction of conditioned fear.

Deep brain stimulation (DBS) of the ventral capsule/ventral striatum (VC/VS) reduces symptoms of intractable obsessive-compulsive disorder (OCD), but the mechanism of action is unknown. OCD is characterized by avoidance behaviors that fail to extinguish, and DBS could act, in part, by facilitating extinction of fear. We investigated this possibility by using auditory fear conditioning in rats, for which the circuits of fear extinction are well characterized. We found that DBS of the VS (the VC/VS homolog in rats) during extinction training reduced fear expression and strengthened extinction memory. Facilitation of extinction was observed for a specific zone of dorsomedial VS, just above the anterior commissure; stimulation of more ventrolateral sites in VS impaired extinction. DBS effects could not be obtained with pharmacological inactivation of either dorsomedial VS or ventrolateral VS, suggesting an extrastriatal mechanism. Accordingly, DBS of dorsomedial VS (but not ventrolateral VS) increased expression of a plasticity marker in the prelimbic and infralimbic prefrontal cortices, the orbitofrontal cortex, the amygdala central nucleus (lateral division), and intercalated cells, areas known to learn and express extinction. Facilitation of fear extinction suggests that, in accord with clinical observations, DBS could augment the effectiveness of cognitive behavioral therapies for OCD.

A time-dependent role of midline thalamic nuclei in the retrieval of fear memory.

Increasing evidence indicates that the medial prefrontal cortex (mPFC) and the amygdala mediate expression and extinction of conditioned fear, but few studies have examined the inputs to these structures. The dorsal part of the midline thalamus (dMT) contains structures such as the mediodorsal nucleus, paraventricular nucleus, and paratenial nucleus that project prominently to mPFC, as well as to basal (BA) and central (Ce) nuclei of the amygdala. Using temporary inactivation with GABA agonist muscimol, we found that dMT was necessary for retrieving auditory fear memory that was 24 h old, but not 2-8 h old. However, pre-training infusions did not impair fear acquisition or extinction. To determine the possible targets of dMT that might modulate fear retrieval, we combined dMT inactivation with Fos immunohistochemistry. Rats with inactivation-induced impairment in fear retrieval showed increased Fos in the lateral division of Ce (CeL), and decreased Fos in the medial division of Ce. No differences in Fos expression were observed in the mPFC or BA. We suggest that the projections from the paraventricular nucleus to CeL are involved in retrieval of well consolidated fear memories. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Role of beta-adrenergic receptors in the ventromedial prefrontal cortex during contextual fear extinction in rats.

It has been reported that stress-related activation of the noradrenergic system strengthens the formation of aversive memories and that beta-adrenergic receptors seem to be involved in this emotional memory processing. In this study, the effects of beta-adrenergic compounds on the extinction of contextual conditioned fear responses were evaluated. Rats were trained with footshock in a conditioning box. In the 3 days following the training, the animals were re-exposed to the apparatus and received either a single or repeated intraperitoneal injections of the beta-adrenergic antagonist propranolol, the beta-adrenergic agonist isoproterenol, or saline 30 min before (acquisition of extinction) or immediately after (consolidation of extinction) the extinction sessions. A drug-free session was performed on the last day. While repeated isoproterenol treatment facilitated the consolidation of contextual fear extinction, repeated propranolol administration impaired the acquisition and the consolidation of this process. Further, the role of ventromedial prefrontal cortex (vmPFC) in the extinction of contextual conditioned fear was tested with an immunohistochemistry assay. Our results show a reduction in Fos-protein expression between the first and the last extinction session. In a follow-up experiment, intra-vmPFC microinjection of isoproterenol before the first extinction session facilitated the extinction of contextual fear. This facilitation was antagonized by pre-treatment with atenolol, suggesting that this change is mediated by beta-1-adrenergic activity. Our results reinforce the role of the vmPFC in fear extinction mechanisms, suggesting that vmPFC-beta-1-adrenergic receptor activation underlies part of the facilitation of the fear extinction processes.