Abstract
Women are disproportionately affected by chronic pain compared with men. While societal and environmental factors contribute to this disparity, sex-based biological differences in the processing of pain are also believed to play significant roles. The central lateral nucleus of the amygdala (CeLC) is a key region for the emotional-affective dimension of pain, and a prime target for exploring sex differences in pain processing since a recent study demonstrated sex differences in CGRP actions in this region. Inputs to CeLC from the parabrachial nucleus (PB) play a causal role in aversive processing and release both glutamate and calcitonin gene-related peptide (CGRP). CGRP is thought to play a crucial role in chronic pain by potentiating glutamatergic signaling in CeLC. However, it is not known if this CGRP-mediated synaptic plasticity occurs similarly in males and females. Here, we tested the hypothesis that female CeLC neurons experience greater potentiation of glutamatergic signaling than males following endogenous CGRP exposure. Using trains of optical stimuli to evoke transient CGRP release from PB terminals in CeLC, we find that subsequent glutamatergic responses are preferentially potentiated in CeLC neurons from female mice. This potentiation was CGRP dependent and involved a postsynaptic mechanism. This sex difference in CGRP sensitivity may explain sex differences in affective pain processing.
Significance Statement
The central lateral nucleus of the amygdala (CeLC) receives a dense projection from parabrachial nucleus (PB) neurons that corelease calcitonin gene-related peptide (CGRP) and glutamate following aversive stimuli. This PBCGRP→CeLC projection plays a causal role in chronic pain. We show that endogenous CGRP release potentiates glutamate signaling in female, but not male, CeLC neurons. In the context of previous work in male CeLC, this suggests that females are more sensitive to even transient CGRP release events. Understanding how this sex difference in CGRP sensitivity arises could enhance strategies for treating chronic pain in both women and men.
Introduction
Women are disproportionately affected by pain, experiencing greater severity, duration, and incidence of chronic pain across many conditions (Mogil, 2009, 2012, 2021; Osborne and Davis, 2022). While societal and environmental factors can influence this sex bias (Fillingim, 2000; Bartley and Fillingim, 2013), genetic, neuroimmune, and neurobiological components are thought to be involved in these sex differences (Stratton et al., 2024). Identifying mechanisms that drive sex differences in chronic pain may aid in the development of novel diagnostics and therapies to better treat women and men with these conditions.
The central lateral nucleus of the amygdala (CeLC; the “nociceptive amygdala”) is a critical center for the emotional-affective dimension of pain (Neugebauer et al., 2020). Nociceptive inputs to CeLC originate primarily from parabrachial nucleus, whose afferents form large—presumably highly efficacious—perisomatic synapses in CeLC (Delaney et al., 2007; Chou et al., 2022). CeLC integrates nociceptive and aversive inputs (Neugebauer et al., 2003; Neugebauer, 2015) and interacts with other key nodes in the pain system (Janak and Tye, 2015; Neugebauer et al., 2020). That parabrachial nucleus inputs to CeLC are causally related to persistent pain is supported by studies showing that pain-like behaviors can be suppressed by manipulating this pathway (Neugebauer, 2015; Wilson et al., 2019; Chiang et al., 2020; Raver et al., 2020; Mazzitelli et al., 2021).
Parabrachial nucleus (PB) neurons that project to CeLC express both glutamate and calcitonin gene-related peptide (CGRP; Shimada et al., 1985; Schwaber et al., 1988; Neugebauer et al., 2020). While low-frequency firing predominantly facilitates glutamate signaling, high-frequency firing of PB CGRP neurons induces the fusion of large dense core vesicles (LDCVs; Tallent, 2008; Schöne et al., 2014; Qiu et al., 2016), releasing packaged neuropeptides including CGRP. PB neurons which express CGRP fire at these high frequencies in response to aversive input, especially in chronic pain conditions (Uddin et al., 2018; Raver et al., 2020; Smith et al., 2023). The subsequent release of CGRP upon CeLC neurons is causally related to chronic pain conditions (J. S. Han et al., 2005, 2010; Okutsu et al., 2017; Shinohara et al., 2017; Chou et al., 2022; Kang et al., 2022; Presto and Neugebauer, 2022; Allen et al., 2023; Kim et al., 2024b).
Despite known sex differences in pain conditions and in pain mechanisms, including sex differences in CGRP-related pain mechanisms in humans (Labastida-Ramírez et al., 2019; de Vries Lentsch et al., 2021), essentially all data on PB and its effects on CeLC are from studies of male animals. An important exception is a demonstration that CGRP RNA levels in the CeLC are upregulated at different stages of neuropathic pain in male and female rats and that CGRP receptor antagonist has sex-specific effects on pain behaviors (Presto and Neugebauer, 2022). Also relevant is a finding that the effects of CGRP on GABA transmission in the spinal cord, and on pain behaviors, is sex specific (Paige et al., 2022).
Here, we test the hypothesis that CGRP exerts a sex-dependent effect on glutamate signaling in the CeLC. By using a model system which combines optogenetics with patch electrophysiology, we evoke endogenous CGRP release from PBCGRP terminals in the CeLC in vitro. By relying on endogenous release of CGRP, rather than exogenous application of CGRP, we minimize the risk of off target effects by more closely mimicking physiologic release of neuropeptides. We first validate that single optic stimulation of channel rhodopsin (ChR2) expressing PBCGRP terminals induces glutamate release in the CeLC, while high-frequency stimulation is required to induce neuropeptide release in vitro. We then test the effect of endogenously released CGRP on glutamate signaling. We predicted that CeLC glutamate signaling is potentiated by CGRP signaling, in line with previous studies in male rodents (J. S. Han et al., 2010; Okutsu et al., 2017), in both sexes, but with a greater magnitude of potentiation in female neurons.
Materials and Methods
Animals
All procedures adhered to the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the University of Maryland School of Medicine. We used 25 CGRP-CRE heterozygous mice (13 female, 12 male), ages 3–12 months, that were bred in house from male B6.Cg-Calcatm1.1(cre/EGFP)Rpa/J (strain #033168) × female C57BL/6J mice (strain #000664). Breeding pairs were obtained from The Jackson Laboratory. Offspring were weaned at postnatal day (P)21 and housed two to five per cage in single-sex groups. Food and water were available ad libitum, and lights were maintained on a 12 h light/dark cycle. Two males (M1–2) and two females (F1–2) were used for fiber photometry experiments. The remaining mice were used for in vitro electrophysiology, where 1–3 neurons were recorded in each mouse from 1 to 3 CeLC slices.
Virus injection
We anesthetized the animals with isoflurane and placed them in a stereotaxic frame. Either left or right PB (−5.2 mm AP, ±1.5 mm ML, −2.9 mm DV) was targeted via a small craniotomy (∼1–2 mm). Only the right PB was targeted in LDCV photometry recordings. We injected 0.5 μl of adeno-associated virus generated by the University of Maryland School of Medicine's Viral Vector Core, AAV5-DIO-ChR2-eYFP or 0.25 μl AAVDJ-DIO-CYbSEP2 coinjected with AAV5-DIO-ChR2-eYFP. CybSEP2 is a presynaptic pH-sensitive sensor which is trafficked by LDCVs and which undergoes a shift in fluorescence upon LDCV fusion and neuropeptide release (Kim et al., 2024b). Viruses were injected using a MICRO2T SMARTouch controller and Nanoliter202 injector head (World Precision Instruments) at a flow rate of 100 nl/min. The pipette was left in place for 10 min before being slowly retracted over 5–10 min. Mice were given Rimadyl for postoperative analgesia. Injection sites were verified by visually confirming robust eYFP fluorescence in the external PB.
In vitro slice electrophysiology
We anesthetized adult mice (2–12 months old) and generated 300-µm-thick live coronal brain sections through the central nucleus of the amygdala using a modified slice collection method as described in Ting et al. (2014) and our prior studies. For recordings, we placed slices in a submersion chamber continuously perfused (2 ml/min) with artificial cerebrospinal fluid (ACSF; in mM): 119 NaCl, 2.5 KCl, 1.2 NaH2PO4, 2.4 NaHCO3, 12.5 glucose, 2 MgSO4·7H2O, and 2 CaCl2·2H2O. ACSF was adjusted to a pH of 7.4, mOsm of 305, and bubbled with carbogen (95% O2 and 5% CO2) throughout use.
We obtained whole-cell voltage-clamp recordings (−70 mV) from the capsular region of the CeLC using borosilicate pipettes with an impedance of 4–6 MΩ and containing the following (in mM): 130 cesium methanesulfonate, 10 HEPES, 1 magnesium chloride, 2.5 ATP-Mg, 0.5 EGTA, 0.2 GTP-Tris, 5 QX-314, and 2% biocytin, pH 7.3 (285 mOsm). Excitatory postsynaptic currents (EPSCs) were optically evoked by whole field illumination at 470 nm (Lambda LS light source, Sutter Instrument) and maximum power of 1.4 mW. Optical stimulation parameters, both high-frequency stimulation (10 or 20 Hz, 3 ms pulse duration) and single/paired exposures (3 ms pulse duration, 100 ms interval), were controlled by a SmartShutter system (Sutter Instrument). We monitored series resistance by measuring the current evoked by a −5 mV square pulse at ∼20 s intervals. Evoked oEPSC amplitudes were quantified using Clampfit 11.2 (Molecular Devices).
In vitro LDCV quantification
Acute brain sections containing the CeLC were collected as above (see above, In vitro slice electrophysiology) from adult mice previously injected with AAVDJ-DIO-CYbSEP2 (Kim et al., 2024b) and AAV5-DIO-ChR2-eYFP in the ipsilateral parabrachial nucleus. A fiber-optic probe (400 μM diameter, 0.39 NA; RWD Life Science) was placed over the visually identified fluorescent afferents from PB within CeLC to record LDCV sensor transients evoked by high-frequency optical stimulation of PBCGRP fibers in the CeLC at 470 nm (CoolLED). Sensor transients were recorded through the fiber-optic probe connected to a RZX10 LUX fiber photometry processor running Synapse software (Tucker-Davis Technologies) through a Doric mini cube (Doric Lenses). Fiber photometry LED power was calibrated to 15 µW using a digital optical power meter (Thorlabs).
We analyzed the data using customized Python scripts adapted from Tucker-Davis Technologies templates which calculated relative changes in fluorescence. Changes in sensor fluorescence were calculated by subtracting the scaled isosbestic signal (405 nm) from the sensor fluorescence (465 nm). Event-related changes in sensor fluorescence were converted to ΔF/F using the 5 s window prior to each stimulation as baseline. The area under the curve (AUC) for the average response was calculated for each mouse using the AUC analysis function in GraphPad Prism.
Experimental design and statistical analysis
Statistical tests were conducted using Prism 10 (GraphPad), and sample size was determined using G*Power software suite (Heinrich-Heine, Universität Düsseldorf). Parametric tests were used when appropriate assumptions were met; otherwise, we used nonparametric tests. Specific statistical tests are detailed in Table 1. Unless describing a time course, baseline versus post optic comparisons are shown as the average in the 90 s before and 90 s after optic tetanus delivery. Averages described in the text are formatted as mean ± SD unless otherwise stated. All figures were designed using a combination of Prism 10 (GraphPad) and Inkscape 1.3.2. Atlas images for Figure 5A were adapted from Franklin and Paxinos (2008) and accessed via a web-based tool (https://labs.gaidi.ca/mouse-brain-atlas/).
Statistics for showing the corresponding figure number, animals, metric, comparisons being made, test statistic, medians or means, sample size, and p values
Results
High-frequency optical stimulation of PBCGRP terminals induces LDCV release in the CeLC
Parabrachial nucleus (PB) neurons that express CGRP (PBCGRP) corelease glutamate and CGRP. To evoke glutamate and CGRP release from these terminals in CeLC, we injected a Cre-dependent channel rhodopsin (ChR2) virus into the PB of CGRP-Cre mice (Fig. 1A). The CeLC receives dense input from PB CGRP neurons (Fig. 1B), whose cell bodies densely localize in the lateral parabrachial nucleus (LPB, Fig. 1C). We first recorded optically induced excitatory postsynaptic currents (oEPSCs) from ipsilateral CeLC neurons evoked by a single pulse of 470 nm light (3 ms duration, 0.05 Hz; Fig. 1). Application of AMPA receptor antagonist (CNQX, 20 μM) suppressed oEPSCs, indicating these oEPSCs are dependent on glutamate signaling. Similar suppression was observed in four of four neurons. While these single optic stimuli reliably induced oEPSCs in CeLC neurons, high-frequency stimulus trains (10 Hz, 5 s), which mimic firing patterns of PB neurons during noxious stimulation in normal and chronic pain states (Uddin et al., 2018; Raver et al., 2020; Smith et al., 2023), resulted in rapid suppression in oEPSC amplitudes recorded in CeLC neurons from both male and female mice (Fig. 1E, 11 female neurons; 8 male neurons). This suggests that the strength of glutamatergic excitation of CeLC neurons postsynaptic to PB afferents rapidly diminishes during sustained activity.
Low-frequency optic stimulation of PBCGRP terminals in CeLC induces glutamate release, while high-frequency optic stimulation induces neuropeptide release. A, AAV5-DIO-ChR2-eYFP OR AAV5-DIO-ChR2-eYFP AND AAVDJ-DIO-CYbSEP2 virus was injected into the left or right lateral parabrachial of CGRP-Cre mice. CeLC slices were collected for either fiber photometry or voltage-clamp patch electrophysiology. B, Photomicrographs showing PBCGRP EYFP + terminals in ipsilateral central amygdala (CeLC). Scale bar = 200 μm. A biocytin filled CeLC neuron shown in inset. Scale bar, 20 μm. C, An injection site, with dense eYFP + PBCGRP neuronal cell bodies located in lateral parabrachial (LPB). Scale bar, 200 μm. SCP, superior cerebellar peduncle. D, A single optic stimulus induces an optically evoked EPSC (oEPSC) at baseline (black trace) which is suppressed by 20 μM CNQX (red). Blue arrow indicates delivery of single optic stimulus (3 ms duration). E, Rapid reduction in oEPSC amplitudes during delivery of a high-frequency optic tetanus. Example stimulus train and response in CeLC neuron shown in inset (cyan). F, Heat map depicting the change in CybSEP2 fluorescence (ΔF/F) in response to different durations of 20 Hz stimulus train of ChR2-expressing PBCGRP inputs in a male CeLC slice. Three trials averaged per row. G, Quantification of average fluorescence in 5 s pre- and poststimulus for trials shown in F.
While high-frequency activity causes a drop in glutamatergic drive, we reasoned that these same stimulus trains would be sufficient to drive release of CGRP from large dense core vesicles (LDCVs; Tallent, 2008; Schöne et al., 2014; Qiu et al., 2016). To test this, we directly measured neuropeptide release from PBCGRP afferents which co-express channelrhodopsin2 (ChR2) and the LDCV sensor CybSEP2. CybSEP2 is a pH-sensitive sensor transported by LDCVs, which undergoes a shift in fluorescence upon LDCV fusion and neuropeptide release (Kim et al., 2024b). We coinjected Cre-dependent CybSEP2 and Cre-dependent ChR2 into the PB of CGRP-cre mice (Fig. 1A) and performed fiber photometry in slices from ipsilateral CeLC to monitor LDCV release. Figure 1F depicts a heat map obtained from recordings where the duration of the stimulus train increased from 250 ms (5 pulses at 20 Hz) to 10 s (200 pulses at 20 Hz). As evidenced by the corresponding ΔF/F (Fig. 1F) and quantified as AUC (Fig. 1G), short duration stimulus trains failed to produce detectable changes in sensor fluorescence. However, increasing the stimulus duration progressively increased recorded fluorescent intensity (Fig. 1G). Similar responses were seen in slices from male and female mice (n = 2). In contrast, mice injected with a single virus, either CybSEP2 (n = 1) or ChR2 (n = 1), demonstrated no such increase. These data indicate that high-frequency optic stimulation is sufficient to induce PBCGRP LDCV fusion, and subsequent neuropeptide release, in vitro in the CeLC. This duality in PBCGRP neurotransmitter release, where glutamate signaling dominates at low frequencies while neuropeptides signaling is predominate at high frequencies, was recently independently confirmed in Kim et al. (2024a).
PB glutamatergic signal is potentiated in females following PB endogenous CGRP release
We took advantage of this ability to evoke PB neuropeptide release to determine if endogenous CGRP release potentiates PBCGRP→CeLC glutamatergic synapses. To measure the amplitude of this glutamatergic signaling, we recorded responses to single pulses of blue light (3 ms duration, 0.05 Hz) to induce ChR2-mediated glutamatergic oEPSCs in postsynaptic CeLC neurons (Fig. 2). After establishing the baseline oEPSC amplitude, we used high-frequency optical stimulation (10 Hz, 5 s) to evoke neuropeptide release in the CeLC and then remeasured the glutamatergic response to single-pulse stimulation. Figure 2A depicts oEPSC recorded from a CeLC neuron from a female mouse, before and after CGRP release, demonstrating a 44% increase in the amplitude of the glutamatergic response after the tetanus stimulation. In contrast, Figure 2B shows that the same procedure failed to potentiate oEPSCs in a CeLC neuron from a male. Group data for female and male mice are quantified in Figure 2C, where CeLC neurons from female mice displayed an average potentiation of 136% ± 29% (p = 2 × 10−3, N = 16 cells from 11 female mice) following high-frequency tetanus, while male CeLC neurons showed no net potentiation, with an average postoptic tetanus magnitude of 106% ± 29% (p = 0.59, N = 8 cells from 5 male mice). The time course of this response is shown in Figure 2D where oEPSCs were significantly potentiated only in female mice (RM two-way mixed-effects sex F(1,314) = 9.794, p < 0.01; time F(6,229) = 2.629, p = 0.02; sex × time F(14,314) = 2.080, p = 0.01) and up to 60 s after the tetanus (Dunnet's multiple-comparisons test, p < 0.05). Additionally, in female CeLC neurons, there was a small reduction of 1.5 ± 2.6 pA in holding current (p = 0.04; 15 neurons, 10 mice) in the 5 s following high-frequency optic tetanus (Fig. 2E). This suggests there was a temporary change in membrane ion conductance induced by the high-frequency optic tetanus. In males there was no net change in holding current (p = 0.16; 15 neurons, 9 mice; Fig. 2E).
Optic stimulation of PB CGRP induces a transient, CGRP-dependent potentiation of glutamate sensitivity, preferentially in females. PBCGRP neurotransmitter release was induced via single (glutamate) or high-frequency (CGRP) optic tetanus. oEPSCs evoked by single-pulse optic stimulation from CeLC neurons from a female (A) and a male (B) neuron recorded at baseline (black traces) and post PB CGRP release (red traces). Note the potentiation in the female, but not the male neuron. Blue arrows indicate delivery of single optic stimulus (3 ms duration). C, Amplitudes of optically evoked PBCGRP glutamate responses (oEPSC) in the 80 s period following high-frequency optic in female and male CeLC neurons. Dashed lines depict averages. D, Amplitude of oEPSC, normalized to baseline, transiently potentiated for ∼60 s in female CeLC neurons (pink). E, A shift in baseline holding current specifically in females accompanied this high-frequency optic stimulus. Using a higher-frequency optical train to enhance PB CGRP release (20 Hz, 10 s) and recording in 0 mg solution to enhance NMDA currents failed to evoke oEPSC potentiation (F, G). H, Significant difference in the effects of endogenous CGRP release on female and male neurons. Increases and decreases were defined as >20% change in oEPSC amplitudes.
To test whether the observed sex differences in potentiation arise from a higher threshold for potentiation in CeLC neurons from males versus females, we use a higher frequency, longer optical tetanus to encourage greater endogenous CGRP release (20 Hz, 10 s) while recording from a population of CeLC neurons patched in separate male mice (N = 7 neurons, 5 mice). In addition, as previous studies demonstrated that CGRP acts through a NMDA-dependent mechanism in the CeLC (J. S. Han et al., 2010; Okutsu et al., 2017), we amplified the signal from recruited NMDA channels by excluding Mg2+ in the ACSF. Even under these conditions there was no net potentiation in male CeLC neurons, as postoptic tetanus oEPSC amplitude was 89% ± 46% of baseline (p = 0.57; Fig. 2F). Figure 2G depicts the time course of this response (RM one-way mixed-effect of time F(2.203,12.75) = 0.6122, p = 0.57).
Across both experiments, only 13% of recorded male CeLC neurons (2/15 neurons, 10 mice) were potentiated (>20% elevation of oEPSC amplitude) following high-frequency stimulation. In contrast, potentiation occurred in 62% of female CeLC neurons (10/16 neurons, 11 mice). Figure 2H depicts the responses across all recorded neurons, where male and female CeLC neurons respond differently to high-frequency optic stimulation (p = 0.003).
Potentiation in females was reproducible within a neuron; once response amplitudes returned to baseline, a similar level of potentiation (p = 0.61 compared with the previous potentiation) was achieved by a second delivery of high-frequency optic stimulation (N = 8 neurons, 5 mice; Fig. 3A). Repotentiation in female CeLC neurons was suppressed by preapplication of 1 μM of the CGRP receptor antagonist (CGRP8–37; p = 0.02), suggesting that CGRP signaling contributes to this increase in PBCGRP→CeLC glutamate signaling (N = 8 neurons, 6 mice; Fig. 3B).
Potentiation is CGRP dependent and influences presynaptic release probability in females. A, Potentiation of PBCGRP glutamate signaling in female neurons is reproducible, as a second PB CGRP release event (CGRP2) induces a similar level of potentiation as an initial PB CGRP release event (CGRP1). B, This potentiation is CGRP dependent as evidenced by its suppression by 1 μM CGRP8–37, CGRP receptor antagonist. C, The potentiation is not associated in with a change in paired pulse ratios, indicating that it does not involve presynaptic mechanisms. Black dashed lines indicate group averages. Inset shows traces of paired pulses (100 ms interstimulus interval) at baseline (black) and after high-frequency optic tetanus (red).
Effect of high-frequency optic tetanus on presynaptic release probability and passive membrane properties
We tested whether CGRP-dependent potentiation in female neurons is driven by a presynaptic mechanism by comparing oEPSC responses to paired pulse stimulation and calculated a paired pulse ratio (PPR), the ratio of the amplitude of the second pulse divided by the amplitude of the first pulse; changes in paired pulse ratio implicate the involvement of a presynaptic mechanism (Manabe et al., 1993; Debanne et al., 1996; Dobrunz and Stevens, 1997; J. Kim and Alger, 2001). A PPR above one is associated with a low probability of vesicle release (i.e., “weaker” synapses), whereas a PPR below one is associated with a high probability of release. Example paired pulse oEPSCs are shown in Figure 3C (inset). We compared PPR before and after optic tetanus delivery. At baseline, the oEPSC PPR was <1 in all female CeLC neurons, suggesting these are high release probability synapses. Female CeLC neurons (10 neurons, 7 mice) did not exhibit a change in PPR following CGRP-mediated potentiation (p = 0.15; Fig. 3C). This suggests that CGRP acts postsynaptically.
To test if CGRP-mediated potentiation reflects postsynaptic mechanisms, we compared holding current before and after potentiation. While there was a small reduction in holding current in female CeLC neurons immediately following the high-frequency optic tetanus (Fig. 2E), there was no change in holding current PB CGRP release compared with baseline in either male (p = 0.16; 14 neurons, 9 mice) or female (p = 0.21; 16 neurons, 11 mice) CeLC neurons. This suggests that while female CeLC neurons are transiently depolarized in response to high-frequency optic tetanus, the potentiated glutamatergic response outlasts the change in driving potential evoked by the tetanus itself. There was no change in series resistance following PB CGRP release in either male (p = 0.6; 14 neurons, 9 mice) or female (p = 0.62; 16 neurons, 11 mice) CeLC neurons. Therefore, the effects of endogenous CGRP release upon CeLC neurons likely reflect a localized change to the post synapse and not a more far-reaching alteration to the overall intrinsic membrane properties of the CeLC neurons.
CGRP-dependent potentiation and input strength are consistent between hemispheres and across the A-P axis of the CeLC
Lateralization in the function of CeLC has been reported in several preclinical models of pain (Carrasquillo and Gereau, 2008; Ji and Neugebauer, 2009; Allen et al., 2023). Therefore, we examined whether lateralization of PBCGRP optic tetanus-evoked potentiation occurs in female neurons. There was no difference (p = 0.45; 15 neurons, 10 mice) in potentiation magnitude between left and right CeLC (Fig. 4A). Additionally, there was no difference in baseline oEPSC amplitude (p = 0.99; 18 neurons, 12 mice) or in baseline paired pulse ratio (p = 0.76; 18 neurons, 12 mice) between recordings from the left and right CeLC (Fig. 4B,C). These findings indicate that there is no lateralization in PBCGRP→CeLC glutamatergic input strength or synaptic release probability in females.
CGRP-dependent potentiation and input strength is not lateralized, and response to CGRP is not due to experimental variables. All data from female CeLC neurons. Potentiation is indistinguishable in neurons in the left or right CeLC (A). Two measures of PBCGRP glutamatergic input strength, oEPSC baseline amplitudes (B) and paired pulse ratios at baseline (C) also show no lateralization. Potential experimental sources of heterogeneity are not correlated with potentiation—animal age (D); virus incubation time (E) do not differ between responder neurons (>20% potentiation) and nonresponder neurons. Similarly, access resistance (F), baseline current (G), membrane resistance (H), or cell capacitance (I) shows no difference between responders and nonresponders.
CeLC may differ functionally not only between hemispheres but also across the anterior-posterior axis. For example, optogenetic stimulation of PBCGRP inputs to the anterior and posterior CeLC induces different behaviors; stimulating PBCGRP in posterior CeLC inputs induces freezing, while stimulating PBCGRP in anterior CeLC predominantly induces changes in respiration and vasoconstriction (Bowen et al., 2020). Therefore, we compared the sensitivity of the female CeLC across the A-P axis to glutamatergic potentiation following PB CGRP release. In female CeLC neurons where oEPSCs were detected in response to optic stimulation of PBCGRP terminals (direct PBCGRP glutamatergic input), wide-field images of the CeLC were acquired following patching. The neuronal anterior-posterior position relative to the bregma was then determined using a stereotaxic atlas (Franklin and Paxinos, 2008). The location of recorded neurons was not related to PB CGRP-driven glutamate potentiation (r = 0.26, p = 0.36; 14 neurons, 8 mice), baseline PB glutamate amplitude (r = 0.16, p = 0.53; 17 neurons, 10 mice), or baseline PB glutamate paired pulse ratio (r = 0.10, p = 0.74; 13 neurons, 8 mice) in females. This suggests both PB CGRP and PB glutamate exert comparable effects on glutamatergic input from PB across anterior and posterior CeLC in females.
To determine if failure of neurons to potentiate results from experimental variables, we compared several experimental properties between female CeLC neurons that exhibited CGRP-dependent potentiation (>20%) and those that did not. We compared animal age (p = 0.79; 16 neurons, 11 mice; Fig. 4D), viral incubation times (p = 0.17; 16 neurons, 11 mice; Fig. 4E), and access resistance (p = 0.44; 16 neurons, 11 mice; Fig. 4F). Experimental parameters did not differ between potentiated and nonpotentiated neurons. We also compared passive membrane properties: baseline holding current (p = 0.96; 16 neurons, 11 mice; Fig. 4G), membrane resistance (p = 0.40; 15 neurons, 10 mice; Fig. 4H), and cell capacitance (p = 0.98; 15 neurons, 10 mice; Fig. 4I); variations in cell capacitance and resistance in particular can reflect differences in the electrical “control” of a patch and resulting resolution. Passive membrane properties did not differ between potentiated and nonpotentiated neurons.
Parabrachial glutamate inputs to central amygdala show no sex differences
We considered the possibility that the sex differences in synaptic potentiation are related to sex differences in the underlying glutamatergic component of the PBCGRP→CeLC signaling pathway. Figure 5A displays the location of CeLC neurons receiving glutamatergic PBCGRP input. There were no sex differences in the proportion of CeLC neurons receiving PBCGRP input (p = 0.44; 23 female neurons, 11 mice; 21 male neurons, 10 mice; Fig. 5B); over 75% of both male and female CeLC neurons exhibited evoked oEPSCs following optic stimulation of PBCGRP glutamate release. There was also no sex difference in baseline oEPSC amplitudes (p = 0.95, 15 male neurons, 10 mice; 19 female neurons, 11 mice; Fig. 5C). This suggests that the strength of PBCGRP→CeLC synapses is similar in males and females.
Male and female CeLC neurons recording locations and membrane properties. A, Location of male (blue) and female (pink) CeLC neurons that received direct PBCGRP glutamatergic input recorded in the lateral capsule of the CeLC across a number of coronal slices. Positions indicated relative to bregma. B, There was no sex difference in the proportion of recorded CeLC neurons that received direct PBCGRP glutamatergic input. C, There was no sex difference in oEPSC amplitudes at baseline, a metric of input strength. Male and female CeLC neurons also had similar baseline currents (D), access resistance (E), membrane resistance (F), or membrane capacitance (G).
The CeLC contains a variety of GABAergic neuronal classes (Schiess et al., 1999; Xu et al., 2003; Lopez De Armentia and Sah, 2004; Chieng et al., 2006; Amano et al., 2012; Lu et al., 2015; Wilson et al., 2019). The sex differences in response to endogenous CGRP release may relate to sex difference in the subpopulations of CeLC neurons receiving direct PBCGRP input. While functionally distinct central amygdala subclasses are primarily defined by their firing pattern (Schiess et al., 1999; Lopez De Armentia and Sah, 2004; Chieng et al., 2006; Amano et al., 2012; Wilson et al., 2019), subtle differences in intrinsic neuronal properties between these classes have also been observed, particularly resting membrane potential (Schiess et al., 1999; Chieng et al., 2006). We observed no sex differences in intrinsic neuron properties including holding current (p = 0.27; 14 male neurons, 10 mice; 18 female neurons, 11 mice, Fig. 5D), which is the current required to maintain a constant membrane potential. Similarly there was no sex difference in access resistance (p = 0.76, 13 male neurons, 9 mice; 17 female neurons, 12 mice; Fig. 5E) membrane resistance (p = 0.69; 15 male neurons, 10 mice; 17 female neurons, 12 mice; Fig. 5F), nor cell capacitance (p = 0.65; 15 male neurons, 10 mice; 17 female neurons, 12 mice; Fig. 5G).
Discussion
Endogenous CGRP release
The central amygdala (CeLC) expresses many neuropeptides (Neugebauer et al., 2020), including CGRP. CGRP modulates CeLC neuronal activity, especially in chronic pain conditions (J. S. Han et al., 2005, 2010; Shinohara et al., 2017; Neugebauer et al., 2020). Previous studies used exogenous CGRP to investigate its effects on CeLC neuronal functions. However, these results are confounded by the fact that the physiological concentration and clearance rate of CGRP in CeLC is unknown, making it unclear if exogenously applied CGRP accurately mimics physiological conditions. Here, we circumvented these limitations by endogenously inducing neuropeptide release using optogenetics to drive parabrachial CGRP-expressing (PBCGRP) activity.
High-frequency firing is necessary for neuropeptide release (Schöne et al., 2014; Qiu et al., 2016). We reasoned high-frequency firing of PBCGRP neurons, which occurs following aversive stimuli (Uddin et al., 2018; Smith et al., 2023), would induce CGRP release. Indeed, driving PBCGRP activity optogenetically resulted in large dense core vesicle (LDCV) release from PBCGRP terminals in the CeLC, evidenced by LDCV sensor signal (Fig. 1F). Further, these stimuli trains resulted in potentiation of CeLC PBCGRP glutamate response (Fig. 2C). That potentiation was suppressed by a CGRP antagonist confirms involvement of CGRP receptors (Fig. 3B). These findings indicate that endogenous release of CGRP can potentiate the activity of CeLC neurons. Whether other neuropeptides which are coexpressed in PBCGRP neurons (Pauli et al., 2022) also contribute to this phenomenon remains to be determined.
Sex differences
There was a sex difference in CeLC neuron response to endogenous CGRP release. In females, most (62%) of CeLC neurons exhibit transient, CGRP-dependent potentiation to PBCGRP glutamatergic signaling after high-frequency stimulation of PBCGRP inputs, compared with 13% of male neurons. In contrast, previous studies (J. S. Han et al., 2010; Okutsu et al., 2017) describe a similar level of glutamatergic potentiation (∼140%) in male CeLC neurons with exogenous CGRP. This discrepancy may reflect lower CGRP sensitivity in males or insufficient CGRP release to activate receptors. This hypothesis is supported by the kinetics of CGRP-driven potentiation in male CeLC, which required >7 min of continuous CGRP exposure (J. S. Han et al., 2010; Okutsu et al., 2017), compared with female CeLC, where 5 s of CGRP release were sufficient to induce glutamate potentiation. No potentiation was observed in males even with increased PBCGRP stimulation. Behavioral studies also support enhanced CGRP signaling in females in pain and anxiety (Avona et al., 2019; Paige et al., 2022; Presto and Neugebauer, 2022), while clinical data demonstrates greater efficacy of CGRP-targeting therapies in women (Porreca et al., 2024).
Several mechanisms may underlie this sex difference. CGRP clearance may be more active in males. CGRP degradation remain a field of active study (Russo and Hay, 2023), with several enzymes implicated in CGRP clearance (Katayama et al., 1991; Davies et al., 1992; McDowell et al., 1997; Fernandez-Patron et al., 2000). Sex differences exist in some enzymes (Zhao et al., 2011; Howe et al., 2019; Omori et al., 2020), but not all (Reuveni et al., 2017; Bronisz et al., 2023).
Males may release less CGRP than females in CeLC. Both sexes show similar PB neuron activation to noxious stimuli (Smith et al., 2023), and no sex difference in CeLC LDCV release was observed in vitro (observation from Kim et al., 2024b). However, male PB neurons may release fewer LDCVs in response to equivalent activation, or CGRP may be less densely packaged among the many neuropeptides contained within LDCVs. This remains untested, although we did not observe potentiation in males upon increasing the “dose” of CeLC CGRP by escalating PBCGRP stimulation.
Finally, considering the longer CGRP potentiation timescale in males (J. S. Han et al., 2010; Okutsu et al., 2017), bath application of CGRP may exert potentiating effects via signaling from an intermediate cell. CGRP receptors are robustly expressed various glial cells, including endothelial cells (Crossman et al., 1990), where they promote vasodilation (Brain et al., 1985). CGRP's effects on other glial types remain to be determined. Whatever the cause, future research should explore whether these sex differences are linked to genetic sex differences (i.e., sex hormone exposure during development) or environmental conditions. This is challenging to address due to the influence of sex on cage environment, including social play (Cox and Rissman, 2011) and fighting (Meakin et al., 2013) in group housing.
There were no sex differences in PBCGRP glutamatergic input. The majority of CeLC neurons in both sexes received PBCGRP glutamatergic input, in line with previous literature describing robust PB input to the “nociceptive amygdala” (J. S. Han et al., 2010; Okutsu et al., 2017). Input strength was also similar between sexes, measured by optically evoked PBCGRP glutamate current amplitude. This suggests low-frequency PB activity, dominated by glutamate signaling, has similar effects on CeLC neurons of both sexes. However, high-frequency PB firing during chronic pain (Helassa et al., 2018; Uddin et al., 2018; Raver et al., 2020) shifts signaling to neuropeptide predominance, a finding independently supported by recent work from Kim et al. (2024a). Thus, functional consequences of a sex difference in CGRP's effect in the CeLC may be restricted to aversive conditions.
Synaptic mechanisms
Short-term potentiation describes transient (seconds to minutes) potentiation of glutamatergic synapses (Fioravante and Regehr, 2011), as described here in female CeLC neurons following endogenous CGRP release. A postsynaptic mechanism has been implicated in male CeLC, where CGRP engages PKA-dependent NMDA receptor recruitment to the PBCGRP synapse (J. S. Han et al., 2010; Okutsu et al., 2017). Our findings are consistent with these data, as we observed no change in paired pulse ratio in female CeLC neurons following CGRP release (Fig. 3C).
Potentiation kinetics
Endogenous CGRP release transiently potentiates glutamatergic input for (∼60 s following a 5 s stimulus train), whereas exogenous CGRP maintains potentiation throughout CGRP application (J. S. Han et al., 2010; Okutsu et al., 2017) and up to 30 min afterward (Okutsu et al., 2017). This difference in potentiation kinetics may be due to clearance dynamics. Endogenous CGRP release results in physiological concentrations, subject to clearance and diffusion away from CGRP receptors. Exogenous application of CGRP may overwhelm endogenous clearance mechanisms, allowing for continuous activation of CGRP receptors and maintained potentiation of glutamate signaling.
Response heterogeneity
In females, 62% of CeLC neurons showed potentiation after CGRP release. Response heterogeneity was not explained by experimental factors such as animal age, viral incubation time, passive membrane properties, or patch access. While neither estrous cycle stage or sex hormone levels were measured in this study, we predict these are not sufficient to explain the observed response heterogeneity. In naturally cycling females, estrous cycle does not affect fear generalization (Keiser et al., 2017) or pain responses (Mogil and Chanda, 2005). Activation of anterior/posterior CeLC induces divergent behavioral/physiological responses (Bowen et al., 2020), while pain lateralizing to the right CeLC (Neugebauer and Li, 2003; J. S. Han and Neugebauer, 2004; Carrasquillo and Gereau, 2008; Ji and Neugebauer, 2009; Sadler et al., 2017; Allen et al., 2023). However, CeLC neuron location also did not explain this observed response heterogeneity; we found no difference in the magnitude of glutamate potentiation, nor PBCGRP glutamatergic input strength, between hemispheres or across the anterior-posterior axis.
Heterogeneity in female CeLC responses may reflect molecular diversity in CeLC GABAergic neurons, such as PKCδ+ and somatostatin (SST+) GABAergic neurons. While both subpopulations receive PB glutamatergic input (Wilson et al., 2019) and express CGRP receptors (S. Han et al., 2015; Chou et al., 2022), they play opposing roles in nociception (Wilson et al., 2019). It is possible that these populations have different sensitivities to endogenous CGRP signaling. This could arise from differences in the subcellular locations of CGRP receptors relative to the site of CGRP release. Differences in PBCGRP terminal contacts onto PKCδ+ and SST+ neurons support this possibility (Shimada et al., 1989; Ye and Veinante, 2019); perisomatic CGRP+ terminals closely surround PKCδ+ CeLC neurons, but rarely SST+ neurons. However, these studies did not specifically investigate the location of CGRP receptors, nor underlying sex differences in receptor localization, which may contribute to the observed sex difference in CeLC CGRP sensitivity. CGRP-induced changes in proximal synapses, such as those observed on PKCδ+ neurons, would be more easily resolved due to superior voltage-clamp control (Spruston et al., 1993). Alternatively, there could be differences in signaling downstream from the CGRP receptor, while PKA-dependent (Gαs) signaling is confirmed in male neurons (J. S. Han et al., 2010; Okutsu et al., 2017), both Gαi and Gαq signaling have been implicated in other cell types (Walker et al., 2010).
Even transient aversive PB hyperactivity induces PB neuropeptide release in the CeLC (Kim et al., 2024b). Our findings suggest that the female nociceptive amygdala is particularly vulnerable to excitation during these CGRP release events, whereas males require sustained elevated CGRP to induce similar potentiation (J. S. Han et al., 2010; Okutsu et al., 2017). This sex difference in CGRP sensitivity may underlie sex differences in affective pain processing, with females showing heightened vulnerability to pain and its related affective sequalae (Mogil, 2009, 2012, 2021; Osborne and Davis, 2022).
Footnotes
This work was supported by National Institutes of Health–National Institute of Neurological Disorders and Stroke Grants R01NS099245, R01NS127827, and Fellowship F31NS134126 (to R.L.).J.B.A. performed work on this project while in the Department of Neurobiology. He is currently affiliated with the Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts 02118.
The authors declare no competing financial interests.
- Correspondence should be addressed to Rebecca Lorsung at rebecca.lorsung{at}som.umaryland.edu.
