Customer Article｜Restoring the Function of Thalamocortical Circuit Through Correcting Thalamic Kv3.2 Channelopathy Normalizes Fear Extinction Impairments in a PTSD Mouse Model
Release time：2024-10-15 17:20:07
Once bitten by a snake, one is afraid of a well rope for ten years" is a typical example of impaired fear memory extinction. Many war veterans, survivors of traffic accidents, and individuals with social and public speaking phobias are prone to develop disorders related to impaired fear memory extinction. Impaired fear memory extinction is one of the most common symptoms of post-traumatic stress disorder (PTSD), and due to limited understanding of its underlying neural mechanisms, effective therapeutic strategies are also limited. In this study, functional screening revealed hyperactivity in the mediodorsal thalamic nucleus (MD) during fear extinction. Furthermore, various machine learning algorithms were used to analyze the encoding patterns of MD during persistent fear responses. The anterior cingulate cortex (ACC) was identified as a functional downstream region of MD that mediates the extinction of fear memory. This study uncovered the role of the thalamocortical circuit in PTSD-related impaired fear memory extinction and proposed potential therapeutic strategies targeting ion channels.
1. Hyperactivated Neuronal Activity of the MD in a PTSD Mouse Model.
Researchers established a PTSD mouse model using electric shocks, which has been well-characterized and effectively replicates the clinical symptoms of PTSD patients. By training the PTSD mice to associate a conditioned stimulus (CS, represented by a tone) with an unconditioned stimulus (US, represented by an electric shock), they assessed the ability of fear memory extinction. Through the expression of immediate early genes and whole-cell patch-clamp recordings, the researchers observed activation in higher-order thalamic regions, including the MD, paraventricular thalamus, and posterior thalamus. They found that the MD neurons in PTSD mice were hyperactive.
Figure 1. MD neurons were hyperactivated in a mouse model of PTSD.
2. Hyperactivated MD Encoded Impaired Fear Extinction
The researchers employed various machine learning algorithms, such as random forest models, polynomial regression models, support vector machine models, and principal component analysis, to decode the relationship between MD activity and fear behaviors, finding that MD calcium signals could effectively decode the fear behaviors of mice on a frame-by-frame basis. They analyzed the peak amplitudes of the calcium signals following the tone and discovered that the peak amplitude in PTSD mice was significantly higher than that in control mice. Additionally, they used a support vector machine model to decode the mice's states based on their MD neuronal calcium signals and corresponding behaviors. To better understand the differences between PTSD mice and control mice, they utilized principal component analysis to simplify and visualize the multifaceted datasets, including MD neuronal activity, mouse fear behavior patterns, and general mouse conditions. By calculating the pairwise distances between the data points from the two groups, they found that the distance increased as the number of extinction trials grew, particularly in the last 10 trials. This difference was also evident when plotting the MD calcium responses for each trial. These results indicate that the abnormal activity of the MD in PTSD mice encodes persistent fear behaviors during the fear extinction test.
Figure 2. Neuronal activity in the MD correlated with the freezing levels in mice.
3. Optogenetic inhibition of the MD promotes fear extinction
Researchers delivered the light-activated chloride-pumping halorhodopsin eNpHR 3.0 to MD neurons using a wireless optogenetic device, finding that optogenetic inhibition of MD neurons significantly reduced freezing time in PTSD mice during fear extinction and decreased fearful responses in the retrieval test. This result suggests that inhibiting MD neuronal activity may facilitate fear extinction in PTSD mice.
Figure 3. Wireless optogenetic inhibition of MD neurons induced decreased freezing levels during the extinction test in mice with PTSD.
4. Hyperactivated MD Functionally and Structurally Reshaped the Local Microcircuitry in the ACC
The researchers first verified the anatomical connections between the MD and ACC, then examined the effects of hyperactivated MD on synaptic strength in the thalamocortical circuit. Using optogenetic stimulation, they recorded the input strength from the MD in pyramidal neurons, PV+ interneurons, and somatostatin-expressing (SST+) interneurons (Figure 4G–L). They also tested whether enhanced MD synaptic inputs reshaped local synaptic connectivity in the ACC using mammalian GFP reconstitution across synaptic partners (mGRASP). In PTSD mice, an increase in thalamocortical synapses was observed in PV+ interneurons. Next, they investigated whether the enhanced connectivity between the MD and ACC affected local microcircuits in the ACC, finding that the paired-pulse ratio (PPR) of PV+ cells was lower than that of pyramidal neurons in both control and PTSD mice. The cell pairs from PTSD mice showed a lower PPR than those from control mice, indicating relatively increased thalamic input to PV+ interneurons in the ACC of PTSD mice. These results suggest that hyperactivated MD neurons enhance local inhibition in the ACC by preferentially increasing synaptic inputs to PV+ interneurons, and that the increased thalamocortical synaptic inputs reshape local circuitry in the ACC, enhancing local inhibition in PTSD mice. Finally, they confirmed through optogenetics and chemogenetics that PV+ interneurons are essential for fear extinction regulation in the MD-ACC circuit in PTSD mice.
Figure 4. Excessive excitation of PV+ neurons in the ACC projecting from the MD was sufficient and necessary for the higher freezing levels of micewith PTSD.
5. Decreased Phosphorylation of Kv3.2 in the MD
Since hyperactivated MD is the main contributor to circuit alterations in PTSD, they examined the substrates of neuronal hyperactivity and found that the most evident change in MD neurons during activity was a reduced half-width, which has been reported to be mediated by voltage-gated K+ and Kv3 channels. Phosphorylation of Kv3 channels may also affect channel currents. Changes in Kv3 channels were detected by recording Kv3 currents in MD neurons from PTSD mice and wild-type (WT) mice using whole-cell patch-clamp techniques. They further analyzed the phosphorylation levels of Kv3.1 and Kv3.2 and found that the phosphorylation level of the Kv3.2 channel in MD neurons of PTSD mice was significantly lower than that in WT mice, while the phosphorylation level of Kv3.1 remained unchanged. They conducted immunoprecipitation-mass spectrometry analysis to identify potential molecules regulating Kv3.2 phosphorylation. Two related upregulated proteins, casein kinase II beta subunit (CSNK2B) and PPP6C, were identified, and their levels were significantly increased in PTSD mice. Protein kinases and phosphatases primarily regulate phosphorylation, suggesting that the upregulation of PPP6C may lead to decreased phosphorylation of Kv3.2.
Figure 5. Increased PPP6C expression led to enhanced Kv3.2 channel function.
6. Conditional Knockdown PPP6C in the MD Effectively Facilitated Fear Extinction
Next, the researchers attempted to knock down PPP6C expression in the MD by packaging the siRNA into a non-viral lipid nanoparticle (LNP) system and bilaterally infusing it into the MD using a cannula. They validated the protein levels of PPP6C and the phosphorylation levels of Kv3.2 and Kv3 after 48 hours of local infusion, finding that the Kv3.2 current intensity in the LNP-siRNA group was significantly lower than that in the LNP-empty group. The siRNA also suppressed neuronal firing by reducing the half-width of action potentials. Additionally, they recorded PV+ neurons using PV-tdTomato mice and tested their connectivity with optogenetic stimulation. In the PTSD LNP-siRNA group, they detected an increased paired-pulse ratio (PPR), indicating that LNP-siRNA reduced the thalamic inputs to ACC PV+ neurons. In behavioral experiments, they observed that LNP-siRNA administration significantly promoted fear extinction in the PTSD mouse model compared to the empty-LNP group. Thus, their results demonstrate that LNP-siRNA can inhibit PPP6C in MD neurons and rescue the impairment of fear extinction in a PTSD mouse model.
Figure 6. LNP-siRNA treatment reversed high freezing levels in a mouse model of PTSD
Original article link: https://onlinelibrary.wiley.com/doi/10.1002/advs.202305939
The RV virus combination used to verify the anatomical structure of the MD-ACC and the AAV viral vectors used for optogenetics, chemogenetics, and the mGRASP system were all provided by Brain Case Biotech. If you are interested in these products or encounter any issues during your experiments, please contact BD@ebraincase.com
