Dissociated Role of Thalamic and Cortical Input to the Lateral Amygdala for Consolidation of Long-Term Fear Memory.

Post-encoding coordinated reactivation of memory traces distributed throughout interconnected brain regions is thought to be critical for consolidation of memories. However, little is known about the role of neural circuit pathways during post-learning periods for consolidation of memories. To investigate this question, we optogenetically silenced the inputs from both auditory cortex and thalamus in the lateral amygdala (LA) for 15 min immediately following auditory fear conditioning (FC) and examined its effect on fear memory formation in mice of both sexes. Optogenetic inhibition of both inputs disrupted long-term fear memory formation tested 24 h after FC. This effect was specific such that the same inhibition did not affect short-term memory and context-dependent memory. Moreover, long-term memory was intact if the inputs were inhibited at much later time points after FC (3 h or 1 d after FC), indicating that optical inhibition for 15 min itself does not produce any nonspecific deleterious effect on fear memory retrieval. Selective inhibition of thalamic input was sufficient to impair consolidation of auditory fear memory. In contrast, selective inhibition of cortical input disrupted remote fear memory without affecting recent memory. These results reveal a dissociated role of thalamic and cortical input to the LA during early post-learning periods for consolidation of long-term fear memory.SIGNIFICANCE STATEMENT Coordinated communications between brain regions are thought to be essential during post-learning periods for consolidation of memories. However, the role of specific neural circuit pathways in this process has been scarcely explored. Using a precise optogenetic inhibition of auditory input pathways, either thalamic or cortical or both, to the LA during post-training periods, we here show that thalamic input is required for consolidation of both recent and remote fear memory, whereas cortical input is crucial for consolidation of remote fear memory. These results reveal a dissociated role of auditory input pathways to the LA for consolidation of long-term fear memory.

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse.

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this purpose, an in vivo imaging technique that detects calcium transients in neurons using genetically encoded calcium indicators (GECIs), a miniaturized fluorescence microscope, and a gradient refractive index (GRIN) lens has been developed and successfully applied to many brain structures1 , 2 , 3 , 4 , 5 , 6. This imaging technique is particularly powerful because it enables chronic simultaneous imaging of genetically defined cell populations for a long-term period up to several weeks. Although useful, this imaging technique has not been easily applied to brain structures that locate deep within the brain such as amygdala, an essential brain structure for emotional processing and associative fear memory7. There are several factors that make it difficult to apply the imaging technique to the amygdala. For instance, motion artifacts usually occur more frequently during the imaging conducted in the deeper brain regions because a head-mount microscope implanted deep in the brain is relatively unstable. Another problem is that the lateral ventricle is positioned close to the implanted GRIN lens and its movement during respiration may cause highly irregular motion artifacts that cannot be easily corrected, which makes it difficult to form a stable imaging view. Furthermore, because cells in the amygdala are usually quiet at a resting or anesthetized state, it is hard to find and focus the target cells expressing GECI in the amygdala during baseplating procedure for later imaging. This protocol provides a helpful guideline for how to efficiently target cells expressing GECI in the amygdala with head-mount miniaturized microscope for successful in vivo calcium imaging in such a deeper brain region. It is noted that this protocol is based on a particular system (e.g., Inscopix) but not restricted to it.

Scolopendra subspinipes mutilans Extract Suppresses Inflammatory and Neuropathic Pain In Vitro and In Vivo.

BACKGROUND: Sciatic nerve injury develops from a variety of pathological causes, including traumatic injury and neuroinflammatory disorders, which are accompanied by pathological changes that have a critical impact on neuropathic pain and locomotor activity. Extracts of Scolopendra subspinipes mutilans (SSM) are used in traditional medicine for the treatment of a wide range of neuropathic diseases, including lower back pain, peripheral neuropathy, and sciatic nerve injury. Although SSM shows anti-inflammatory, antibacterial, and anticonvulsant activities, its diverse mechanisms of action remain unclear. Thus, the present study examined the effects of SSM in vitro and in vivo. METHODS: To estimate the anti-inflammatory effects of SSM, inflammatory conditions were induced using lipopolysaccharide (LPS) in RAW 264.7 cells, and inflammatory-related factors were evaluated by enzyme-linked immunosorbent assay (ELISA) and western blotting analyses. Sciatic nerve crush injury (SNCI) was induced in rats using a surgical clip instrument. The effects of SSM in the SNCI model were evaluated in behavioral tests by calculating the sciatic functional index (SFI) and measuring thermal hyperalgesia sensitivity and by monitoring inflammatory factors expression in western blotting analyses. RESULTS: We observed the anti-inflammatory effects of SSM treatment both in vitro and in vivo. The PGE2 and NO production were suppressed by SSM. Protein analyses indicated that expression of NF-κB and degradation of IκBα were suppressed by SSM treatment. In addition, the levels of iNOS, TNF-α, IL-6, and COX-2 expression were reduced by SSM treatment in RAW 264.7 cells and in the SNCI-induced animals. In behavioral studies, SSM treatment enhanced the SFI and improved the thermal sensitivity test results. CONCLUSIONS: Our results suggest that SSM suppresses the production of inflammatory factors via the NF-κB pathway and accelerates the morphological and functional recovery of the peripheral nervous system. Hence, SSM may be a useful therapeutic candidate for treatment of neuropathic pain diseases.

Memory recall and modifications by activating neurons with elevated CREB.

Memory is supported by a specific ensemble of neurons distributed in the brain that form a unique memory trace. We previously showed that neurons in the lateral amygdala expressing elevated levels of cAMP response-element binding protein are preferentially recruited into fear memory traces and are necessary for the expression of those memories. However, it is unknown whether artificially activating just these selected neurons in the absence of behavioral cues is sufficient to recall that fear memory. Using an ectopic rat vanilloid receptor TRPV1 and capsaicin system, we found that activating this specific ensemble of neurons was sufficient to recall established fear memory. Furthermore, this neuronal activation induced a reconsolidation-like reorganization process, or strengthening of the fear memory. Thus, our findings establish a direct link between the activation of specific ensemble of neurons in the lateral amygdala and the recall of fear memory and its subsequent modifications.

Brain region-specific activity patterns after recent or remote memory retrieval of auditory conditioned fear.

Memory is thought to be sparsely encoded throughout multiple brain regions forming unique memory trace. Although evidence has established that the amygdala is a key brain site for memory storage and retrieval of auditory conditioned fear memory, it remains elusive whether the auditory brain regions may be involved in fear memory storage or retrieval. To investigate this possibility, we systematically imaged the brain activity patterns in the lateral amygdala, MGm/PIN, and AuV/TeA using activity-dependent induction of immediate early gene zif268 after recent and remote memory retrieval of auditory conditioned fear. Consistent with the critical role of the amygdala in fear memory, the zif268 activity in the lateral amygdala was significantly increased after both recent and remote memory retrieval. Interesting, however, the density of zif268 (+) neurons in both MGm/PIN and AuV/TeA, particularly in layers IV and VI, was increased only after remote but not recent fear memory retrieval compared to control groups. Further analysis of zif268 signals in AuV/TeA revealed that conditioned tone induced stronger zif268 induction compared to familiar tone in each individual zif268 (+) neuron after recent memory retrieval. Taken together, our results support that the lateral amygdala is a key brain site for permanent fear memory storage and suggest that MGm/PIN and AuV/TeA might play a role for remote memory storage or retrieval of auditory conditioned fear, or, alternatively, that these auditory brain regions might have a different way of processing for familiar or conditioned tone information at recent and remote time phases.

Increasing CREB in the auditory thalamus enhances memory and generalization of auditory conditioned fear.

Although the lateral nucleus of the amygdala (LA) is essential for conditioned auditory fear memory, an emerging theme is that plasticity in multiple brain regions contributes to fear memory formation. The LA receives direct projections from the auditory thalamus, specifically the medial division of the medial geniculate nucleus (MGm) and adjacent posterior intralaminar nucleus (PIN). While traditionally viewed as a simple relay structure, mounting evidence implicates the thalamus in diverse cognitive processes. We investigated the role of plasticity in the MGm/PIN in auditory fear memory. First we found that auditory fear conditioning (but not control manipulations) increased the levels of activated CREB in both the MGm and PIN. Next, using viral vectors, we showed that exogenously increasing CREB in this region specifically enhanced formation of an auditory conditioned fear memory without affecting expression of an auditory fear memory, formation of a contextual fear memory, or basic auditory processing. Interestingly, mice with increased CREB levels in the MGm/PIN also showed broad auditory fear generalization (in contrast to control mice, they exhibited fear responses to tones of other frequencies). Together, these results implicate CREB-mediated plasticity in the MGm/PIN in both the formation and generalization of conditioned auditory fear memory. Not only do these findings refine our knowledge of the circuitry underlying fear memory but they also provide novel insights into the neural substrates that govern the degree to which acquired fear of a tone generalizes to other tones.