An Empathic Pain-Regulated Neural Circuit

Empathy is a general term in psychology that refers to the correct understanding of the feelings and emotions of others, so as to achieve mutual understanding, care, and harmony. Empathic behavior is common in rodents [1], and various emotions such as pain, happiness and fear can be transmitted in the form of empathy. Empathy is a step-by-step process. Initially, it may only manifest as motor imitation, then develop into emotional contagion influenced by others' experience, and the highest level of empathy is altruistic behavior [2]. Moreover, this behavior differs due to the degree of emotional identity between individuals, although some disputes exist in the literature. Empathic pain is defined as empathy with pain observed in another individual and producing effects comparable to the self-pain that is experienced in the observer’s body. By comparing behavioral indicators, researchers have demonstrated that rats that have had similar pain experiences, can induce empathic pain in both familiar and stranger rat observers [3]. Social contacts with a familiar conspecific in pain result in empathic pain which is driven by activation of the medial prefrontal cortex–locus coeruleus/norepinephrine–sympathoadrenomedullary axis (SAM), whereas social contacts with unfamiliar conspecifics in pain result in stress-associated analgesia which is co-driven by hypothalamo–pituitary–adrenocortical and SAM hyperactivity [4]. A better understanding of the neural circuits mediating empathic pain will greatly facilitate the development of therapies that target pathological forms of empathy, or its absence, in a variety of neuropsychiatric disorders.

Studies have confirmed the existence of empathic pain induced in animal models by formalin, complete Freund's adjuvant, and spared nerve injury and found that multiple brain regions are involved in empathic pain behaviors [5]. With advances in experimental techniques, neuroimaging, and interdisciplinary research, numerous studies have focused on exploring the neural mechanisms of empathic pain. By using techniques such as functional magnetic resonance imaging, several brain regions like the anterior insula and anterior medial cingulate cortex/dorsal anterior cingulate cortex (ACC) have been shown to be involved in subserving empathy for pain [6]. Moreover, Geng et al. have demonstrated that the medial prefrontal cortex plays an important role in mediating the empathic pain of spinal nociception [7]. Smith et al. revealed, using optogenetic techniques, that the ACC and its projections to the nucleus accumbens (NAc) are selectively involved in the social transmission of pain perception [8], providing a structural basis for uncovering the neural circuits of pain empathy. However, the factors that influence pain empathy have not been fully elucidated, and the exact neural circuits of the transmission of pain empathy and its molecular regulatory mechanism remain unclear.

Recently, Professor Tao Chen’s team reported that glutamate synaptic transmission projecting from the insular cortex (IC) to the basolateral amygdala (BLA) is involved in regulating pain empathy, and they further revealed the synaptic and molecular mechanisms of pain empathy [9]. Mechanistically, they found that the dynamic expression of presynaptic synaptophysin-2 (Syt2) and postsynaptic Rab3-interacting molecule-3 (RIM3) play important roles in regulating synaptic strength in the IC-BLA circuits (Fig. 1).

Fig. 1.

Increasing release of glutamate in IC–BLA excitatory projections is closely related to the formation and regulation of pain empathy in sibling mice. And presynaptic Syt2 and postsynaptic RIM3 are important signaling molecules regulating enhanced synaptic transmission in the IC–BLA pathway. The expression levels of Syt2 and RIM3 are positively correlated with the development of empathic pain in sibling mice, and inhibition of these synaptic proteins reduces glutamate release and rescues empathic pain.

First of all, sibling mice and stranger mice were allowed to observe mice with left common peroneal nerve ligation for a period of time. By testing the hind paw withdrawal mechanical threshold (PWMT), they found that not only the demonstration mice but also the sibling observer mice showed significant pain sensitivity. This phenomenon was not found in the stranger observer mice, suggesting the specific presence of empathic pain in sibling mice. Fos-CreER^T2/Ai9 mice were further used to identify the brain regions activated by empathic pain in the sibling observer mice, and the results showed that the expression of FOS protein was increased in some areas, such as the ACC and NAc, with the greatest ratio of FOS⁺ neurons in the right IC (IC^R) and right BLA (BLA^R) compared with the stranger group. The authors next confirmed the presence of the IC-BLA projection using tracing approaches. The results suggested that BLA-projecting pyramidal cells in the IC might be involved in the regulation of empathic pain. Moreover, the electrophysiological properties of neurons projecting from the IC to the BLA in siblings showed that both the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were increased while the paired pulse ratio (PPR) was decreased, all of which indicated enhanced activity of IC-BLA-projecting neurons in observed pain.

To further determine whether glutamatergic projections from the IC to the BLA in sibling mice were increased, adeno-associated virus AAV2/9-CaMKIIα- ChR2-mCherry was injected into layer V of the IC^R. They found that the EPSCs and inhibitory postsynaptic currents of neurons in the BLA were induced by optostimulation, and each was blocked by the corresponding receptor antagonist. Moreover, decreased PPR of both pyramidal cells and interneurons in the BLA of siblings but not strangers was recorded when modulating optostimulation.

To further investigate how the IC–BLA pathway works in empathic pain, the authors first injected an anterograde virus, AAV2/1-hSyn-Cre-EGFP that is transmitted across synapses, into the IC^R, then the Cre-dependent AAV2/9 virus CaMKIIa-DIO-taCaspase3-mCherry was injected into the BLA^R; this strategy induced the apoptosis of pyramidal cells in the BLA. They found that the PWMT of sibling mice was significantly increased after pyramidal neuron ablation. Furthermore, the authors established a Cre-FLEX system by injecting AAV2/1-hSyn-FLEX-Flpo into the IC^R of GAD2-Cre mice, then the Flp-dependent virus AAV2/9-Ef1α-fDIO-taCasp3-TEVp-WPRE was injected into the BLA^R. In contrast to the phenomenon in pyramidal neurons, the ablation of postsynaptic interneurons aggravated the mechanical pain response of sibling mice. The authors further ablated postsynaptic pyramidal cells of the left IC (IC^L)-to-BLA^R pathway and found that there were no significant changes in the mechanical threshold of siblings, suggesting that empathic pain is characterized by lateral preferential modulation. In conclusion, the results demonstrated that pyramidal neurons play an important role in regulating empathic pain, while GABAergic interneurons indirectly regulate observational pain behaviors by inhibiting the activity of pyramidal cells in the BLA.

Next, the authors used designer receptors exclusively activated by designer drugs technology to specifically inhibit or activate neurons projecting from the IC^R to the BLA^R. Activation of the IC^R-BLA^R projection neurons induced a marked reduction in the mechanical threshold of stranger observers, whereas inhibition of this pathway reduced the ability of the sibling observers to express empathic pain. However, activation or inhibition of the IC^L-BLA^L pathway had little effects on the regulation of empathic pain in stranger or sibling mice. These results implied that IC^R neurons projecting to the BLA^R are important for mice to generate empathic pain.

However, the neural mechanisms and molecular basis of empathic pain are still unclear. To solve this problem, the authors first obtained homogenates of IC^R and BLA^R from sibling and stranger mice, and then acquired ribosome-bound mRNAs by phosphorylated translating ribosome affinity purification. The RNA-sequencing results revealed that synaptic transmission-related genes including Snap25, RIM1, Syt2, and RIM3 were significantly enriched in the sibling group. Moreover, western blot results confirmed the upregulated expression of Syt2 and RIM3 in the IC^R and BLA^R of sibling mice, suggesting that these two proteins in the IC and BLA participate in the regulation of empathic pain in mice.

To test the above hypothesis, the authors used immunoelectron-microscopy to detect the location of Syt2 and RIM3 at the ultrastructural level. As expected, Syt2 was presynaptic and RIM3 was postsynaptic. Furthermore, using the RNAscope approach, the authors confirmed that the expression of Syt2 mRNA in IC^R pyramidal cells was significantly increased in sibling mice. Next, they applied a virus-mediated gene knockdown strategy to downregulate the presynaptic Syt2 and postsynaptic RIM3, and found that their down-regulation significantly reduced empathic pain in sibling mice. Then AAV2/9-CaMKIIα-ChR2-mcherry was injected into the IC^R of sibling mice with down-regulated presynaptic Syt2 (shSyt2) or postsynaptic RIM3 (shRIM3), so that fibers projecting from IC^R to BLA^R were modulated by optical stimulation. Whole-cell patch recordings showed that the PPR of both pyramidal cells and interneurons in shSyt2-injected mice was larger than that in controls, but the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/N-methyl-D-aspartate (AMPA/NMDA) receptor ratio did not change. On the contrary, the PPR in shRIM3-injected mice remained stable, while the AMPA/NMDA ratio was significantly increased.

Previous research has demonstrated that mutual regulation of the ACC–NAc pathway can affect the generation of empathic pain [10]. Therefore, the authors further explored the difference between the generation of empathic pain mediated by IC nerve cells and that mediated by ACC. By labeling IC–BLA and ACC–NAc pathway neurons with retrobeads, the authors found that, compared with that in IC^R-BLA^R projecting neurons, the synaptic transmission in the ACC^R-NAc^R pathway was significantly enhanced in sibling mice with one day of observational pain training. However, after seven days of training, the electrical signals of neurons in the IC^R–BLA^R pathway were significantly increased but not those in the ACC^R–NAc^R pathway. These results suggest that the ACC^R–NAc^R pathway might be more likely to participate in the generation of early empathic pain, while the IC^R–BLA^R pathway is involved in its maintenance.

In summary, in this study, they identified a neural circuit and synaptic modulation mechanisms of empathic pain: enhanced glutamatergic synaptic transmission from the IC to the BLA pathway dependent on synaptic proteins Syt2 and RIM3. Previous evidence has shown that the rodent brain functions in an asymmetric way, such as the ACC in empathy [10]. The authors also confirmed this phenomenon that empathic pain is hemispherically lateralized. Besides, in vivo recording of neuronal firing indicated that the ACC and IC play different roles in the induction and maintenance of empathic pain.

However, there are still some issues that need to be addressed. For example, as the familiarity of the sibling observation mice with the demonstration mice increased, the empathic pain generated by the IC–BLA pathway also increases accordingly. What is the possible mechanism for this change? In addition, the results showed that many other brain regions were activated in the observer mice; whether these regions also play roles in empathic pain regulation needs further exploration. The results showed that BLA^R also received projections from IC^L, but ablation of the IC^L–BLA^R postsynaptic pyramidal cells did not affect empathic pain in sibling mice. What mediates the lateral preferential regulation in empathic pain? Previous researchers have shown that an image-forming visual function pathway is directly involved in the mediation of empathic pain [7]. The role of the visual pathway in empathic pain and its connections to pain-related neural circuits remain to be further elucidated. Vision is not the only sensory source that mediates pain empathy; researchers have found that olfactory cues released by mice experiencing hyperalgesia rapidly provoke hypersensitivity in co-housed mice [11], implicating an important role of olfaction in the development of empathy for pain. Future efforts are needed to identify the underlying structural and functional interconnectivity between olfactory input and other higher brain regions for the development of empathic pain at the circuit level. Whether other behaviors such as fear and happiness can be transmitted to siblings through the same neural circuits is worth studying. Researches have shown that chronic pain can put extra stress on the body, and it is also associated with heightened symptoms of depression and anxiety [12]. Can depression, anxiety, and other adverse emotions be induced in siblings accompanied by empathic pain? Is its mediated neural circuit consistent with that of pain? Although the authors have established the role of excitatory and inhibitory neurons in the IC and BLA during the regulation of empathic pain, is empathic pain mediated by a specific neuronal subtype? Previous studies on empathic pain mainly focused on the ACC, however, what is the difference between empathic pain and other empathic behavior? The relationship between the ACC–NAc and IC–BLA pathways is worthy of study in the regulation of empathic pain. In conclusion, this paper clarifies the special loop mechanisms that regulate empathy in animal models, and a more refined understanding of the brain mechanisms of empathy will also accelerate the development of new treatments for empathy deficits associated with neuropsychiatric disorders.