Skip to main content

Peripheral nerve injury reduces the excitation-inhibition balance of basolateral amygdala inputs to prelimbic pyramidal neurons projecting to the periaqueductal gray


Cellular and synaptic mechanisms underlying how chronic pain induces maladaptive alterations to local circuits in the medial prefrontal cortex (mPFC), while emerging, remain unresolved. Consistent evidence shows that chronic pain attenuates activity in the prelimbic (PL) cortex, a mPFC subregion. This reduced activity is thought to be driven by increased inhibitory tone within PL circuits. Enhanced input from the basolateral amygdala (BLA) to inhibitory neurons in PL cortex is one well-received mechanism for this circuit change. In mice, we used retrograde labeling, brain slice recordings, and optogenetics to selectively stimulate and record ascending BLA inputs onto PL neurons that send projections to the periaqueductal gray (PAG), which is a midbrain structure that plays a significant role in endogenous analgesia. Activating BLA projections evoked both excitatory and inhibitory currents in cortico-PAG (CP) neurons, as we have shown previously. We measured changes to the ratio of BLA-evoked excitatory to inhibitory currents in the spared nerve injury (SNI) model of neuropathic pain. Our analysis reveals a reduced excitation-inhibition (E/I) ratio of BLA inputs to PL-CP neurons 7 days after SNI. The E/I ratio of BLA inputs to CP neurons in neighboring infralimbic (IL) cortex was unchanged in SNI animals. Collectively, this study reveals that the overall E/I balance of BLA inputs to PL neurons projecting to the PAG is reduced in a robust neuropathic pain model. Overall, our findings provide new mechanistic insight into how nerve injury produces dysfunction in PL circuits connected to structures involved in pain modulation.

Circuit and cellular dysfunction in the medial prefrontal cortex (mPFC) has been well-documented in rodent models of chronic pain [1,2,3,4,5,6,7,8,9]. Extensive evidence indicates that alterations to long-term plasticity of synaptic inputs within the mPFC contributes to chronic pain states [10]. Both arthritic and neuropathic pain models augment basolateral amygdala (BLA) inputs that drive feed-forward inhibition of layer 5 (L5) pyramidal neurons in mPFC [3, 5]. A major target of L5 neurons in mPFC is the periaqueductal gray (PAG), which is a key midbrain structure involved in descending inhibition of ascending nociceptive inputs [11, 12]. Additional work reports that nerve injury enhances inhibition of mPFC output to the PAG [4]. Here we aimed to determine whether the spared-nerve injury (SNI) model of neuropathic pain attenuates the ratio of excitatory to inhibitory (E/I) currents in cortico-PAG (CP) neurons in mPFC following optogenetic excitation of BLA axons in acute brain slice.

As we’ve done previously in our lab [13], we injected Alexa Flour-647 conjugated Cholera toxin B (Thermo-Fisher; ~ 100 nL) into the PAG and AAV1.CAG.ChR2-Venus.WPRE.SV40 (Addgene 20,071, ~ 100–200 nL) into the BLA of male or female C57BL6/J mice (stock# 00664; Jackson Labs; Fig. 1a). Mice were allowed to recover for at least 7 days before introducing the spared nerve injury (SNI) that involved removing ~ 2 mm sections of the peroneal and tibial nerve while leaving the sural nerve intact (Fig. 1b). Seven to 8 days following surgery, animals in the SNI group displayed robust mechanical allodynia while sham surgery animals showed no change in pain behavior (Fig. 1c). Following behavioral analysis, mice were briefly anesthetized (15–20 s) with isoflurane, decapitated and brains were rapidly removed. Coronal sections (300 μm) of the mPFC were prepared for optogenetic and electrophysiological recordings and clearly showed overlap of BLA axons and retrogradely labeled CP somata (Fig. 1d). Fluorescent retrogradely labeled CP neurons were selectively visualized for this study using a far red filter (660 nm emission) in line with a coolLED system (Scientifica, UK). Broad field excitation of ChR2+ BLA axons was performed using a 470 nm LED stimulus through the same coolLED system at ~ 40 mW output to the slice for three milliseconds. Series resistance was required to be < 35 MΩ and to have ≤15% variation between the initial and final reading to be included in the analysis. Recordings were filtered at 2 kHz and digitized at 10 kHz. Excitatory (EPSC) and inhibitory (IPSC) responses to photoactivation of ChR2+ BLA projections were recorded at command voltages of − 70 and + 10 mV, respectively in the presence of NMDA receptor antagonist CPP (5 μM, Tocris, UK). Data analysis was performed offline using Matlab routines (Mathworks, Inc.).

Fig. 1
figure 1

Nerve injury alters excitation-inhibition balance of inputs to cortic-PAG neurons in mPFC

Whole-cell slice recordings from layer 5 (L5) CP neurons in PL cortex contralateral to sham/SNI revealed that the ratio of peak excitatory input to peak inhibitory input (E/I ratio) evoked by optogenetic stimulation of ChR2+ BLA axons was significantly lower (t (14) = 2.3, p = 0.04, Student’s unpaired t-test) in SNI animals (Fig. 1e-h). However, this decrease in E/I ratio of BLA inputs was not detected (t (11) = 0.96, p = 0.36, Student’s unpaired t-test) in recordings from L5 CP neurons in IL cortex, which is ventrally adjacent to PL cortex (Fig. 1i-l). As in our previous work [11], we distinguished IL from PL by differences in cytoarchitecture including a distinguishable narrowing of layer 3 and an infiltration of layer 2 cells into layer 1 of IL. Peak amplitudes for EPSCs and IPSCs did not differ in PL-CP and IL-CP neurons recorded from sham and SNI mice (Fig. 1m, n). There was no statistical difference (t (10) = 1.14, p = 0.28, Student’s unpaired t-test) in E/I ratios between PL-CP and IL-CP neurons recorded from sham mice (Fig. 1o). However, in SNI mice, the E/I ratio recorded from PL-CP neurons was significantly reduced compared to IL-CP neurons (t (15) = 2.53, p = 0.02, Student’s unpaired t-test; Fig. 1p). While we did not observe sex dependent effects, further experiments are needed to substantiate this observation.

We have shown that BLA disynaptically drives inhibitory inputs to CP neurons via activation of local GABAergic neurons in both PL and IL cortex [13]. Previous work has shown that the BLA preferentially targets parvalbumin-expressing inhibitory neurons (PVINs) in mPFC over pyramidal neurons and other inhibitory neuron subtypes [14]. Following SNI, BLA inputs onto PVINs in PL cortex are augmented due to reduced presynaptic endocannabinoid signaling [4]. These findings align with our current data showing that SNI reduces the E/I ratio of BLA-driven inputs on CP neurons in PL cortex. This reduction in E/I ratio is likely driven by an enhancement of local inhibitory inputs from PVINs onto PL-CP neurons thereby reducing output to the PAG, which has been shown to contribute to neuropathic pain [4]. Activating the PL-PAG pathway may be a novel therapeutic strategy for treating pain as optogenetic stimulation of PL axons in the PAG has been shown to alleviate pain behavior in SNI mice [4]. Indeed this may be feasible as low-frequency electrical stimulation of the PL cortex has been shown to reduce aversive responses in a persistent inflammatory model [15], which likely involves increasing the activity of the PL-PAG pathway.

We have previously shown that SNI reduces excitability of CP neurons in PL, but not IL, cortex [1], which is consistent with our data showing no change to the E/I ratio of BLA-driven inputs onto IL-CP neurons. This dichotomy could be because BLA neurons targeting the PL and IL are distinct populations [16]. This raises the possibility that BLA neurons targeting IL do not express presynaptic endocannabinoid receptors, but this still needs to be elucidated. Nonetheless, SNI drives a separation in E/I balance between CP neurons in PL and IL.

Overall, our data build upon an emerging mechanism for how nerve injury drives dysfunction in cortical circuits upstream of structures essential for endogenous analgesia. While these circuits are likely critical for immediate top-down control of pain, it is becoming clearer that maladaptive changes to these same circuits may be a significant contributing factor to neuropathic pain following nerve injury.

A) Schematic and bright-field images of retrograde tracer injection into periaqueductal gray (PAG) and AAV-ChR2-venus virus injection into basolateral amygdala (BLA). B) Diagrammatic representation of spared nerve injury (SNI) model of neuropathic pain in the mouse. C) Mechanical allodynia measured using von Frey test in sham (F(1,20) = 1.24; p = 0.28) and SNI (F (1, 13) = 28.2; p < 0.0001; Two-way ANOVA) animals at baseline and post-operative day (POD) 7 or 8. D) Representative image of ChR2+ BLA axons (green) retrogradely-labeled cortico-PAG (CP) neurons (red) in coronal section of the prelimbic (PL) and infralimbic (IL) regions of medial prefrontal cortex (mPFC). E) Representative (4x) image showing recording from a CP neuron in PL cortex. F) Schematic showing recording configuration for measuring synaptic currents in PL-CP neurons following stimulation (470 nm, 3 milliseconds) of ChR2+ BLA axons. G) Overlay of total excitatory and inhibitory post-synaptic currents from PL-CP neurons from sham (grey) and SNI (red) mice. Thick line trace is the average of individual traces. H) Ratio of excitatory and inhibitory (E/I) inputs to PL-CP neurons from sham and SNI mice (*p < 0.05; Student’s unpaired t-test). I) Representative (4x) image showing recording from a CP neuron in IL cortex. J) Schematic showing recording configuration for measuring BLA input to IL-CP neurons. K) Overlay of total excitatory and inhibitory post-synaptic currents from IL-CP neurons. Thick line trace is the average of individual traces. L) Ratio of excitatory and inhibitory inputs to IL-CP neurons. M) Peak EPSC values (solid circle: mean ± SEM) for PL-CP (sham: 428 ± 162.5; SNI: 271.3 ± 56; t (14) = 1.01, p = 0.3305) and IL-CP (sham: 441 ± 188.2; SNI: 665 ± 180; t (11) = 0.82, p = 0.43, Student’s unpaired t-test) neurons. N) Peak IPSC values for PL-CP (sham: 1562.4 ± 685.5; SNI: 1284.2 ± 214; t (14) = 0.43, p = 0.67) and IL-CP (sham = 1531 ± 465.3; SNI: 1462.6 ± 293; t (11) = 0.13, p = 0.9, Student’s unpaired t-test) neurons. O, P) E/I ratio comparisons of PL and IL cortex from (O) sham and (P) SNI mice (*p < 0.05; Student’s unpaired t-test).

Availability of data and materials

All data generated or analyzed during this study are included in this published article. Data files used for this manuscript are available via a direct and reasonable request to the corresponding author and approval from Indiana University.


  1. Cheriyan J, Sheets PL. Altered excitability and local connectivity of mPFC-PAG neurons in a mouse model of neuropathic pain. J Neurosci. 2018;38(20):4829–39.

    Article  CAS  Google Scholar 

  2. Metz AE, Yau HJ, Centeno MV, Apkarian AV, Martina M. Morphological and functional reorganization of rat medial prefrontal cortex in neuropathic pain. Proc Natl Acad Sci U S A. 2009;106(7):2423–8.

    Article  CAS  Google Scholar 

  3. Ji G, Sun H, Fu Y, Li Z, Pais-Vieira M, Galhardo V, et al. Cognitive impairment in pain through amygdala-driven prefrontal cortical deactivation. J Neurosci. 2010;30(15):5451–64.

    Article  CAS  Google Scholar 

  4. Huang J, Gadotti VM, Chen L, Souza IA, Huang S, Wang D, et al. A neuronal circuit for activating descending modulation of neuropathic pain. Nat Neurosci. 2019;22(10):1659–68.

    Article  CAS  Google Scholar 

  5. Zhang Z, Gadotti VM, Chen L, Souza IA, Stemkowski PL, Zamponi GW. Role of Prelimbic GABAergic circuits in sensory and emotional aspects of neuropathic pain. Cell Rep. 2015;12(5):752–9.

    Article  CAS  Google Scholar 

  6. Mitric M, Seewald A, Moschetti G, Sacerdote P, Ferraguti F, Kummer KK, et al. Layer- and subregion-specific electrophysiological and morphological changes of the medial prefrontal cortex in a mouse model of neuropathic pain. Sci Rep. 2019;9(1):9479.

    Article  Google Scholar 

  7. Kelly CJ, Huang M, Meltzer H, Martina M. Reduced Glutamatergic currents and dendritic branching of layer 5 pyramidal cells contribute to medial prefrontal cortex deactivation in a rat model of neuropathic pain. Front Cell Neurosci. 2016;10:133.

    PubMed  PubMed Central  Google Scholar 

  8. Lee M, Manders TR, Eberle SE, Su C, D'Amour J, Yang R, et al. Activation of corticostriatal circuitry relieves chronic neuropathic pain. J Neurosci. 2015;35(13):5247–59.

    Article  CAS  Google Scholar 

  9. Cordeiro Matos S, Zhang Z, Seguela P. Peripheral neuropathy induces HCN Channel dysfunction in pyramidal neurons of the medial prefrontal cortex. J Neurosci. 2015;35(38):13244–56.

    Article  Google Scholar 

  10. Bliss TV, Collingridge GL, Kaang BK, Zhuo M. Synaptic plasticity in the anterior cingulate cortex in acute and chronic pain. Nat Rev Neurosci. 2016;17(8):485–96.

    Article  CAS  Google Scholar 

  11. Ferreira AN, Yousuf H, Dalton S, Sheets PL. Highly differentiated cellular and circuit properties of infralimbic pyramidal neurons projecting to the periaqueductal gray and amygdala. Front Cell Neurosci. 2015;9:161.

    PubMed  PubMed Central  Google Scholar 

  12. Basbaum AI, Fields HL. Endogenous pain control mechanisms: review and hypothesis. Ann Neurol. 1978;4(5):451–62.

    Article  CAS  Google Scholar 

  13. Cheriyan J, Kaushik MK, Ferreira AN, Sheets PL. Specific Targeting of the Basolateral Amygdala to Projectionally Defined Pyramidal Neurons in Prelimbic and Infralimbic Cortex. eNeuro. 2016;3(2):1-13.

  14. McGarry LM, Carter AG. Inhibitory gating of Basolateral amygdala inputs to the prefrontal cortex. J Neurosci. 2016;36(36):9391–406.

    Article  CAS  Google Scholar 

  15. Zhou H, Zhang Q, Martinez E, Dale J, Robinson E, Huang D, et al. A novel neuromodulation strategy to enhance the prefrontal control to treat pain. Mol Pain. 2019;15:1744806919845739.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Senn V, Wolff SB, Herry C, Grenier F, Ehrlich I, Grundemann J, et al. Long-range connectivity defines behavioral specificity of amygdala neurons. Neuron. 2014;81(2):428–37.

    Article  CAS  Google Scholar 

Download references


The authors thank Charlie T. Anderson (West Virginia University) for constructive comments on the manuscript.


The work was supported by National Institutes of Health Grant R01-NS094389 (P.L.S.).

Author information

Authors and Affiliations



P.L.S. designed research with input from J.C..; J.C. performed all experiments; P.L.S. and J.C. analyzed data and wrote the paper. The author(s) read and approved the final manuscript.

Corresponding author

Correspondence to Patrick L. Sheets.

Ethics declarations

Ethics approval and consent to participate

All experiments were approved by the Indiana University School of Medicine Animal Care and Use Committee and adhered to the animal welfare guidelines of the National Institutes of Health.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cheriyan, J., Sheets, P.L. Peripheral nerve injury reduces the excitation-inhibition balance of basolateral amygdala inputs to prelimbic pyramidal neurons projecting to the periaqueductal gray. Mol Brain 13, 100 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: