Adolescent alcohol exposure promotes mechanical allodynia and alters synaptic function at inputs from the basolateral amygdala to the prelimbic cortex

Alcohol is sometimes used as a temporary form of pain relief. However, heavy and regular consumption can have serious side effects, including altering how the brain processes pain. Over time, this may lead to more frequent and intense episodes of pain, creating a vicious cycle in which individuals drink more to counteract their heightened discomfort.

Recent studies in rodents have shown that binge drinking during adolescence also increases sensitivity to pain, with this change often persisting into adulthood. Yet, how alcohol use in teenagers impacts the parts of the brain that process pain remains poorly understood.

To investigate this question, Obray et al. compared the brains of adolescent rats that had either been exposed or not exposed to alcohol. The rats were subjected to two types of pain stimuli: mechanical pressure using the end of a metal wire and thermal pain via a heated surface. The team found that less pressure was needed for the alcohol-exposed rats to pull their paws away, suggesting they were more sensitive to pain. However, both groups of rats exhibited similar responses to the heat-related stimulus.

Next, Obray et al. explored the connections between the parts of the brain that process pain. A region of the brain known as the prefrontal cortex integrates the sensory and emotional aspects of pain by sending information to and from the amygdala and periaqueductal gray areas. Obray et al. found that inhibitory interneurons in the prefrontal cortex, which may reduce the transmission of pain, were not as well connected to the amygdala in the alcohol-exposed rats. In addition, neurons linking the prefrontal cortex to the periaqueductal gray areas were more excitable in these animals compared to the non-exposed rats.

These findings suggest that alcohol use during adolescence may make the brain more reactive to pain while impairing its ability to modulate pain signals. Understanding how early alcohol exposure alters pain sensitivity could help scientists develop strategies that disrupt the harmful cycle between alcohol use and pain. However, further studies are needed to determine whether the effects observed in this study also occur in humans.

Alcohol misuse and pain exhibit a bidirectional relationship. Due to its analgesic properties, alcohol is often used to self-medicate for pain relief (Alford et al., 2016; Riley and King, 2009). However, the dose required for this effect results in blood alcohol levels akin to binge drinking (Neddenriep et al., 2019; Thompson et al., 2017), heightening the risk of alcohol-related harm. While acute alcohol consumption can temporarily alleviate pain, chronic misuse promotes hyperalgesia (Dina et al., 2000; Dudek et al., 2020; Edwards et al., 2012; Jochum et al., 2010; Julian et al., 2019; You et al., 2020). Notably, frequent acute pain and pain interference are associated with an elevated risk of being diagnosed with an alcohol use disorder (AUD) (Barry et al., 2013; Edlund et al., 2013; McDermott et al., 2018). This dynamic extends to adolescents where untreated pain correlates with earlier initiation of alcohol use (Chau and Chau, 2023), and alcohol consumption is associated with heightened ongoing and future pain (Hestbaek et al., 2006; Horn-Hofmann et al., 2018; Pascale et al., 2022). Recent preclinical studies in rodents further indicate that adolescent intermittent ethanol (AIE) exposure induces persistent hyperalgesia spanning into adulthood (Bertagna et al., 2024; Kelley et al., 2024; Khan et al., 2023; Secci et al., 2024). Given the association between pain and AUD, understanding the impact of adolescent alcohol use on nociceptive circuits is crucial for improving AUD treatments.

Nociception involves multimodal processing across a distributed brain network. Within this network, the medial prefrontal cortex (mPFC) is a key node for evaluating and responding to pain (Bastuji et al., 2016; Garcia-Larrea and Peyron, 2013; Ong et al., 2019). The prelimbic (PrL) subregion of the mPFC receives inputs from the basolateral amygdala (BLA) (Cunningham et al., 2002; Gabbott et al., 2006; Krettek and Price, 1977) and sends projections to the ventrolateral periaqueductal gray (vlPAG) (An et al., 1998; Floyd et al., 2000), both regions involved in nociception. Within this circuit, glutamatergic inputs from the BLA drive parvalbumin interneuron (PVIN)-mediated feedforward inhibition of pyramidal neurons projecting from the PrL to the vlPAG (PrL^PAG neurons) to promote nociception (Cheriyan et al., 2016; Dilgen et al., 2013; Gadotti et al., 2019; Huang et al., 2019; McGarry and Carter, 2016). Of note, activation of PrL PVINs has been shown to be pronociceptive (Yin et al., 2020; Zhang et al., 2015), while activation of PrL^PAG neurons generally exhibits antinociceptive effects (Drake et al., 2021; Gao et al., 2023; Huang et al., 2019; Yin et al., 2020), although this finding has not been universally observed (Fan et al., 2018). These findings highlight the involvement of a BLA–PrL–vlPAG circuit in modulating nociception.

Adolescence marks a critical period of continuing development of the mPFC. This period is characterized by heightened plasticity rendering this region particularly vulnerable to environmental insults, including repeated binge alcohol consumption (Crews et al., 2019; Spear, 2000; Spear, 2018). Notably, this developmental phase is accompanied by significant alterations in excitatory and inhibitory neurotransmission within the mPFC. Excitatory changes include pruning of both synapses and dendritic spines (Drzewiecki et al., 2016; Koss et al., 2014; Mallya et al., 2019; Petanjek et al., 2011; Rakic et al., 1994), maturation of AMPA and NMDA receptor trafficking (Flores-Barrera et al., 2014; Miller et al., 1990; Murphy et al., 2012), and increased innervation from the BLA (Cunningham et al., 2002; Cunningham et al., 2008). Concurrently, there is a substantial increase in excitatory input onto PVINs (Caballero et al., 2014; Cunningham et al., 2008), leading to a shift toward greater inhibition in the excitatory/inhibitory (E/I) balance at pyramidal neurons (Caballero et al., 2021; Cass et al., 2014; Klune et al., 2021). Preclinical rodent models have demonstrated that AIE exposure disrupts the normative developmental trajectory of the mPFC, inducing persistent alterations in intrinsic excitability and synaptic function. Specifically, adolescent alcohol exposure leads to decreased intrinsic excitability of PVINs (Trantham-Davidson et al., 2017), reduced excitatory input onto PVINs (Trantham-Davidson et al., 2017), diminished inhibition at pyramidal neurons (Centanni et al., 2017), and augmented intrinsic excitability of pyramidal neurons in the mPFC (Galaj et al., 2020; Salling et al., 2018). These findings underscore the sensitivity of mPFC circuitry to long-lasting AIE-induced changes. Building upon this understanding, the present study investigated how AIE exposure, in conjunction with a carrageen-induced inflammatory paw pain challenge, alters synaptic function at BLA inputs onto PVINs and PrL^PAG neurons.

The procedure for adolescent alcohol exposure used in this study is a well-characterized model designed to simulate the effects of repeated episodes of binge-like alcohol exposure. Rats were subjected to eight intermittent cycles of ethanol vapor from PD 28 to PD 54. Behavioral intoxication and blood ethanol concentrations (BECs) were assessed at the end of each cycle. The average behavioral intoxication score using the 5-point rating scale was 2.2 ± 0.1 for male rats in the AIE-saline treatment condition, 2.2 ± 0.1 for male rats in the AIE-carrageenan treatment condition, 2.2 ± 0.1 for female rats in the AIE-saline treatment condition, and 2.1 ± 0.1 for female rats in the AIE-carrageenan treatment condition, which represents a moderate level of intoxication. The corresponding BEC values were 196.4 ± 34.8 for male rats in the AIE-saline treatment condition, 240.0 ± 33.8 for male rats in the AIE-carrageenan treatment condition, 184.8 ± 38.8 for female rats in the AIE-saline treatment condition, and 177.7 ± 31.2 for female rats in the AIE-carrageenan treatment condition. Male rats had significantly higher intoxication scores than female rats (Wilcoxon rank-sum test: z = 1.967, p = 0.0492). There was no difference in average intoxication scores between rats assigned to the saline and carrageenan pain conditions (Wilcoxon rank-sum test: z = 0.450, p = 0.6529). There was no difference in BEC level between male and female rats (Wilcoxon rank-sum test: z = 1.365, p = 0.1724) or for rats assigned to the saline and carrageenan pain conditions (Wilcoxon rank-sum test: z = –0.955, p = 0.3395). Average intoxication scores and BEC levels were positively correlated (r_s = 0.499, p = 0.0001).

AIE exposure augmented mechanical sensitivity

The first set of studies evaluated the impact of AIE on mechanical and thermal sensitivity from adolescence to early adulthood. Weekly assessments were conducted using electronic Von Frey and Hargreaves apparatuses. The initial evaluation was performed at PD 24 prior to the first cycle of ethanol vapor exposure, and the final assessment at PD 80, which was approximately 4 weeks after the last cycle of exposure.

For mechanical sensitivity, analysis of data from the electronic Von Frey test revealed that AIE significantly reduced paw withdrawal threshold, indicating increased sensitivity to mechanical touch (main effect of AIE: F_(1,115) = 12.81, p = 0.0005, partial η² = 0.1002 [0.0203, 0.2105]; Figure 1A, B). Additionally, female rats exhibited greater sensitivity to mechanical touch compared to male rats (main effect of sex: F_(1,115) = 5.98, p = 0.0160, partial η² = 0.0494 [0.0014, 0.1434]). There was no significant interaction between AIE and sex (AIE × sex interaction: F_(1,115) = 1.02, p = 0.3147). Mechanical touch sensitivity decreased with age (main effect of age: F_(8,920) = 75.08, p = 0.0000, partial η² = 0.3950 [0.3444, 0.4328]), with no significant interactions between age and AIE or sex (AIE × age interaction: F_(8,920) = 0.70, p = 0.6327; sex × age interaction: F_(8,920) = 1.50, p = 0.1829; AIE × sex × age interaction: F_(8,920) = 1.10, p = 0.3602). The data were also averaged for each rat starting after the first cycle of AIE (PD 31–PD 80). Consistent with the full dataset analysis, the averaged data showed that AIE increased sensitivity to mechanical touch (main effect of AIE: F_(1,115) = 13.33, p = 0.0004, partial η² = 0.1039 [0.0221, 0.2149]; Figure 1C) and female rats were more sensitive to mechanical touch than male rats (main effect of sex: F_(1,115) = 5.63, p = 0.0193, partial η² = 0.0467 [0.0008, 0.1393]). There was no significant interaction between AIE and sex (AIE × sex interaction: F_(1,115) = 1.28, p = 0.2608).

Figure 1

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Numerical data for mechanical and thermal sensitivity of rats during adolescence.

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For thermal sensitivity, analysis of data from the Hargreaves test indicated no significant effect of AIE on paw withdrawal latency (main effect of AIE: F_(1,115) = 1.21, p = 0.2738; Figure 1D, E). Additionally, there were no significant sex differences or interactions with AIE (main effect of sex: F_(1,115) = 0.59, p = 0.4446; AIE × sex interaction: F_(1,115) = 1.85, p = 0.1767). Thermal sensitivity was found to decrease with age (main effect of age: F_(8,920) = 11.45, p = 0.0000, partial η² = 0.0906 [0.0520, 0.1197]). Furthermore, no significant interactions between age and AIE or sex were observed (AIE × age interaction: F_(8,920) = 1.11, p = 0.3567; sex × age interaction: F_(8,920) = 2.02, p = 0.0546; AIE × sex × age interaction: F_(8,920) = 0.70, p = 0.6626). The data were also analyzed as the average paw withdrawal latency for each rat beginning after the first cycle of AIE (PD 31–PD 80). Consistent with the results from the full dataset, analysis of the averaged data showed no significant effect of AIE, sex, or interaction between the two on thermal sensitivity (main effect of AIE: F_(1,115) = 1.09, p = 0.2977; main effect of sex: F_(1,115) = 1.57, p = 0.2128; AIE × sex interaction: F_(1,115) = 2.20, p = 0.1408; Figure 1F).

Carrageenan-induced inflammatory paw pain was unaltered by AIE exposure

As AIE was found to increase baseline mechanical sensitivity, the subsequent studies assessed its impact on carrageenan-induced hyperalgesia in adult rats. Carrageenan is a well-known proinflammatory agent that induces edema and transient hyperalgesia in the carrageenan-induced inflammatory paw pain model (Benitz and Hall, 1959; Neves et al., 2020; Vazquez et al., 2015; Winter et al., 1962; Yang and Tsaur, 2023).

Prior to administering carrageenan or saline into the hindpaw, baseline mechanical and thermal sensitivity was assessed using the electronic Von Frey and Hargreaves tests, respectively. Analysis revealed that AIE-driven reductions in paw withdrawal threshold persisted for more than 8 weeks after discontinuing ethanol vapor exposure (main effect of AIE: F_(1,109) = 5.50, p = 0.0209, partial η² = 0.0480 [0.0006, 0.1441]; Figure 2A). In addition, female rats continued to display greater mechanical sensitivity than males, with no significant interactions between AIE and sex (main effect of sex: F_(1,109) = 10.66, p = 0.0015, partial η² = 0.0891 [0.0139, 0.1998]; AIE × sex interaction: F_(1,109) = 0.93, p = 0.3367). Baseline analysis of the thermal sensitivity data showed no significant effects of AIE, sex, or an interaction between AIE and sex on paw withdrawal latency (main effect of AIE: F_(1,109) = 1.69, p = 0.1968; main effect of sex: F_(1,109) = 1.27, p = 0.2616; AIE × sex interaction: F_(1,109) = 0.38, p = 0.5384; Figure 2B).

Figure 2

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Numerical data for mechanical and thermal sensitivity of rats in response to a carrageenan paw pain challenge.

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Following carrageenan or saline administration, mechanical and thermal sensitivity were evaluated, and data were averaged across assessments occurring at three timepoints – 2-, 6-, and 24-hr post-injection. The averaged data were expressed as a percentage of each rat’s pre-injection baseline score. Examination of paw withdrawal threshold revealed a carrageenan-induced increase in mechanical sensitivity (main effect of carrageenan: F_(1,105) = 116.41, p = 0.0000, partial η² = 0.5258 [0.3934, 0.6201]; Figure 2C–F). Analysis further indicated that female rats displayed a greater increase in sensitivity than males across both the saline and carrageenan treatments, with no significant effects of AIE, or interactions between AIE, sex, and carrageenan (main effect of sex: F_(1,105) = 6.22, p = 0.0142, partial η² = 0.0559 [0.0020, 0.1578]; main effect of AIE: F_(1,105) = 0.93, p = 0.3366; AIE × sex interaction: F_(1,105) = 0.03, p = 0.8706; AIE × carrageenan interaction: F_(1,105) = 0.59, p = 0.4441; sex × carrageenan interaction: F_(1,105) = 1.72, p = 0.1925; AIE × sex × carrageenan interaction: F_(1,105) = 1.18, p = 0.2790). Similarly, examination of paw withdrawal latency revealed a carrageenan-induced increase in thermal sensitivity (main effect of carrageenan: F_(1,105) = 132.70, p = 0.0000, partial η² = 0.5583 [0.4308, 0.6470]; Figure 2G–J). No significant effects of AIE, sex, or interactions between AIE, sex, and carrageenan were observed (main effect of AIE: F_(1,105) = 0.00, p = 0.9625; main effect of sex: F_(1,105) = 0.00, p = 0.9510; AIE × sex interaction: F_(1,105) = 0.23, p = 0.6307; AIE × carrageenan interaction: F_(1,105) = 0.01, p = 0.9352; sex × carrageenan interaction: F_(1,105) = 0.14, p = 0.7120; AIE × sex × carrageenan interaction: F_(1,105) = 3.85, p = 0.0524).

AIE exposure and carrageenan enhanced the intrinsic excitability of PrL^PAG neurons

Subsequently, the impact of AIE exposure- and carrageenan-induced hyperalgesia on PrL^PAG neuron intrinsic excitability was examined through current-clamp recordings of current evoked firing obtained from green retrobead labeled cells in the PrL cortex. Labeling with green retrobeads indicated that the neuron projected ipsilateral from the left hemisphere of the PrL cortex to the vlPAG (Figure 3). For each electrophysiological experiment, values are reported per animal and reflect the average value of 1–5 neurons recorded from each rat. Table 1 contains a summary of the biophysical properties of the recorded PrL^PAG neurons. No significant differences between treatment conditions or sex were observed for these properties.

Figure 3

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Table 1

Condition	Sex	Vrest (mV)	Rinput (MΩ)
AIR: SAL	Male	–66.6 ± 0.9	75.3 ± 3.5
	Female	–65.0 ± 1.7	76.3 ± 2.4
AIR: CAR	Male	–66.2 ± 1.4	83.2 ± 5.4
	Female	–65.2 ± 1.5	83.9 ± 4.1
AIE: SAL	Male	–65.0 ± 1.4	86.9 ± 7.8
	Female	–65.6 ± 1.5	76.6 ± 2.9
AIE: CAR	Male	–65.6 ± 1.0	88.6 ± 4.3
	Female	–66.5 ± 1.2	83.4 ± 6.0

Analysis of the firing data indicated that both AIE and carrageenan enhanced intrinsic excitability (main effect of AIE: F_(1,72) = 7.24, p = 0.0089, partial η²=0.0914 [0.0059, 0.2292]; main effect of carrageenan: F_(1,72) = 4.07, p = 0.0474, partial η²=0.0535 [0.0000, 0.1776]; Figure 4A–G). Moreover, the effect of AIE on intrinsic excitability became more pronounced with increasing current step size (AIE × current step interaction: F_(20,1440) = 3.82, p = 0.0117, partial η² = 0.0503 [0.0194, 0.0604]). Although the number of action potentials (APs) fired increased alongside the amount of injected current (main effect of current step: F_(20,1440) = 230.22, p = 0.0000, partial η² = 0.7618 [0.7404, 0.7747]), no other significant effects of sex or interactions between AIE, carrageenan, sex, or current step were observed (main effect of sex: F_(1,72) = 1.78, p = 0.1861; AIE × carrageenan interaction: F_(1,72) = 0.00, p = 0.9760; AIE × sex interaction: F_(1,72) = 0.17, p = 0.6823; sex × carrageenan interaction: F_(1,72) = 0.00, p = 0.9686; AIE × sex × carrageenan interaction: F_(1,72) = 0.46, p = 0.4985; sex × current step interaction: F_(20,1440) = 1.27, p = 0.2851; carrageenan × current step interaction: F_(20,1440) = 1.13, p = 0.3377; AIE × sex × current step interaction: F_(20,1440) = 0.66, p = 0.5694; AIE × carrageenan × current step interaction: F_(20,1440) = 2.46, p = 0.0656; sex × carrageenan × current step interaction: F_(20,1440) = 0.20, p = 0.8924; AIE × sex × carrageenan × current step interaction: F_(20,1440) = 1.13, p = 0.3383).

Figure 4

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Numerical data for the intrinsic excitability and selected biophysical properties of PrL^PAG neurons.

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To better understand the observed changes in intrinsic excitability, additional analyses were performed to assess the effects of AIE, carrageenan, and sex on AP threshold, rheobase, voltage sag, and afterhyperpolarization in PrL^PAG neurons. This analysis revealed that carrageenan reduced the AP threshold of PrL^PAG neurons, with the largest reduction observed in AIE exposed rats (main effect of carrageenan: F_(1,72) = 7.01, p = 0.0100, partial η² = 0.0887 [0.0051, 0.2258]; AIE × carrageenan interaction: F_(1,72) = 5.52, p = 0.0215, partial η² = 0.0713 [0.0007, 0.2029]; Figure 4H, I). No additional effects of treatment condition or sex on the AP threshold were found (main effect of sex: F_(1,72) = 3.23, p = 0.0764; main effect of AIE: F_(1,72) = 3.48, p = 0.0662; AIE × sex interaction: F_(1,72) = 0.17, p = 0.6794; sex × carrageenan interaction: F_(1,72) = 0.15, p = 0.7004; AIE × sex × carrageenan interaction: F_(1,72) = 1.31, p = 0.2561). Despite the observed change in AP threshold, there were no significant effects of treatment condition or sex on rheobase in these neurons (main effect of AIE: F_(1,72) = 0.71, p = 0.4014; main effect of carrageenan: F_(1,72) = 0.71, p = 0.4014; main effect of sex: F_(1,72) = 0.13, p = 0.7186; AIE × carrageenan interaction: F_(1,72) = 1.76, p = 0.1889; AIE × sex interaction: F_(1,72) = 0.01, p = 0.9044; sex × carrageenan interaction: F_(1,72) = 0.71, p = 0.4014; AIE × sex × carrageenan interaction: F_(1,72) = 1.18, p = 0.2814; Figure 4J, K). Hyperpolarization-activated cation current (I_h) linked voltage sag was reduced by carrageenan (main effect of carrageenan: F_(1,72) = 5.03, p = 0.0281, partial η² = 0.0652 [0.0000, 0.1946]; Figure 4L, M), with the largest reduction occurring in male rats (sex × carrageenan interaction: F_(1,72) = 5.20, p = 0.0256, partial η² = 0.0673 [0.0000, 0.1975]). As a result, male carrageenan treated rats had smaller I_h-induced hyperpolarization sag than female rats (main effect of sex: F_(1,72) = 20.92, p < 0.0001, partial η² = 0.2252 [0.0758, 0.3746]). Afterhyperpolarization, in contrast, was reduced by AIE exposure (main effect of AIE: F_(1,72) = 53.33, p < 0.0001, partial η² = 0.4255 [0.2518, 0.5535]; Figure 4N, O) with the largest reduction occurring in females (AIE × sex interaction: F_(1,72) = 21.94, p < 0.0001, partial η² = 0.2335 [0.0817, 0.3827]). Notably, PrL^PAG neurons from female rats displayed greater afterhyperpolarization than those from male rats in all treatment conditions except for AIE exposure paired with carrageenan (main effect of sex: F_(1,72) = 12.92, p = 0.0006, partial η² = 0.1522 [0.0310, 0.2998]; AIE × sex × carrageenan interaction: F_(1,72) = 6.81, p = 0.0110, partial η² = 0.0864 [0.0045, 0.2228]). The analysis revealed no further effects of treatment or sex on either hyperpolarization sag (main effect of AIE: F_(1,72) = 0.03, p = 0.8528; AIE × carrageenan interaction: F_(1,72) = 2.18, p = 0.1446; AIE × sex interaction: F_(1,72) = 0.04, p = 0.8500; AIE × sex × carrageenan interaction: F_(1,72) = 0.50, p = 0.4797) or afterhyperpolarization (main effect of carrageenan: F_(1,72) = 0.04, p = 0.8333; AIE × carrageenan interaction: F_(1,72) = 0.25, p = 0.6203; sex × carrageenan interaction: F_(1,72) = 2.53, p = 0.1160).

AIE exposure enhanced the E/I balance at inputs from the BLA onto PrL^PAG neurons

The next set of studies assessed the impact of AIE and carrageenan on synaptic function at BLA inputs to PrL^PAG neurons. This involved recording from green retrobead labeled pyramidal neurons in the PrL cortex while optically stimulating terminals from the BLA (Figure 5A).

Figure 5

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Numerical data characterizing optically evoked postsynaptic excitatory and inhibitory currents onto PrL^PAG neurons.

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To evaluate the E/I balance at BLA inputs to PrL^PAG neurons, voltage-clamp recordings of optically evoked excitatory postsynaptic currents (oEPSCs) and optically evoked inhibitory postsynaptic currents (oIPSCs) were obtained from green retrobead labeled cells in the PrL. Analysis of oEPSC peak amplitudes indicated no significant effects of AIE, carrageenan, or sex (main effect of AIE: F_(1,69) = 2.36, p = 0.1294; main effect of carrageenan: F_(1,69) = 0.35, p = 0.5572; main effect of sex: F_(1,69) = 3.64, p = 0.0604; AIE × carrageenan interaction: F_(1,69) = 0.00, p = 0.9757; AIE × sex interaction: F_(1,69) = 1.45, p = 0.2334; sex × carrageenan interaction: F_(1,69) = 0.00, p = 0.9617; AIE × sex × carrageenan interaction: F_(1,69) = 0.76, p = 0.3851; Figure 5B, C).

Conversely, oIPSC amplitude was significantly reduced in AIE exposed rats (main effect of AIE: F_(1,69) = 37.66, p = 0.0000, partial η² = 0.3531 [0.1771, 0.4942]; Figure 5D, E). Additionally, carrageenan was found to enhance oIPSC amplitude (main effect of carrageenan: F_(1,69) = 4.17, p = 0.0449, partial η² = 0.0570 [0.0000, 0.1858]); however, this increase was attenuated in AIE exposed rats (AIE × carrageenan interaction: F_(1,69) = 5.16, p = 0.0262, partial η² = 0.0696 [0.0000, 0.2036]). No significant effects of sex, or interactions between sex and AIE or carrageenan on oIPSC amplitude were found (main effect of sex: F_(1,69) = 0.83, p = 0.3644; AIE × sex interaction: F_(1,69) = 2.90, p = 0.0929; sex × carrageenan interaction: F_(1,69) = 0.97, p = 0.3287; AIE × sex × carrageenan interaction: F_(1,69) = 1.84, p = 0.1788).

To quantify the resulting E/I balance at PrL^PAG neurons, the ratios of oEPSCs to oIPSCs were compared. This revealed that AIE enhanced the E/I balance (main effect of AIE: F_(1,69) = 52.48, p = 0.0000, partial η² = 0.4320 [0.2545, 0.5611]; Figure 5F, G) and female rats exhibited larger E/I ratios than male rats (main effect of sex: F_(1,69) = 5.21, p = 0.0255, partial η² = 0.0703 [0.0000, 0.2045]). No significant effects of carrageenan or interactions between AIE, sex, and carrageenan were found (main effect of carrageenan: F_(1,69) = 0.96, p = 0.3307; AIE × carrageenan interaction: F_(1,69) = 0.01, p = 0.9158; AIE × sex interaction: F_(1,69) = 0.05, p = 0.8190; sex × carrageenan interaction: F_(1,69) = 1.72, p = 0.1935; AIE × sex × carrageenan interaction: F_(1,69) = 0.08, p = 0.7741).

The AMPA/NMDA ratio at direct inputs from the BLA onto PrL^PAG neurons was unaltered by AIE exposure or carrageenan

To assess the AMPA/NMDA ratio at monosynaptic inputs from the BLA to PrL^PAG neurons, voltage-clamp recordings of oAMPA and oNMDA currents were obtained from green retrobead labeled cells in the PrL during application of tetrodotoxin (TTX) and 4-aminopyridine (4-AP) (Figure 6A). Analysis of oAMPA current amplitude revealed no significant effects of AIE, carrageenan, or sex (main effect of AIE: F_(1,72) = 0.50, p = 0.4817; main effect of carrageenan: F_(1,72) = 0.31, p = 0.5806; main effect of sex: F_(1,72) = 3.19, p = 0.0784; AIE × carrageenan interaction: F_(1,72) = 0.03, p = 0.8652; AIE × sex interaction: F_(1,72) = 0.20, p = 0.6576; sex × carrageenan interaction: F_(1,72) = 0.29, p = 0.5889; AIE × sex × carrageenan interaction: F_(1,72) = 0.26, p = 0.6122; Figure 6B, C). Likewise, evaluation of the amplitude of oNMDA currents revealed no effects of treatment condition or sex (main effect of AIE: F_(1,72) = 1.25, p = 0.2680; main effect of carrageenan: F_(1,72) = 0.29, p = 0.5932; main effect of sex: F_(1,72) = 2.86, p = 0.0952; AIE × carrageenan interaction: F_(1,72) = 0.34, p = 0.5635; AIE × sex interaction: F_(1,72) = 0.42, p = 0.5204; sex × carrageenan interaction: F_(1,72) = 1.55, p = 0.2176; AIE × sex × carrageenan interaction: F_(1,72) = 2.40, p = 0.1257; Figure 6D, E). The resultant ratios of oAMPA to oNMDA currents were compared and also were found to be unaltered by AIE, carrageenan, or sex (main effect of AIE: F_(1,72) = 1.34, p = 0.2513; main effect of carrageenan: F_(1,72) = 1.00, p = 0.3199; main effect of sex: F_(1,72) = 2.81, p = 0.0982; AIE × carrageenan interaction: F_(1,72) = 0.25, p = 0.6173; AIE × sex interaction: F_(1,72) = 0.75, p = 0.3907; sex × carrageenan interaction: F_(1,72) = 0.01, p = 0.9328; AIE × sex × carrageenan interaction: F_(1,72) = 1.30, p = 0.2582; Figure 6F, G).

Figure 6

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Numerical data characterizing optically evoked AMPA and NMDA currents at monosynaptic inputs from the basolateral amygdala (BLA) onto PrL^PAG neurons.

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Asynchronous excitatory postsynaptic currents at direct inputs from the BLA onto PrL^PAG neurons were unaffected by AIE exposure or carrageenan

To examine pre- and postsynaptic alterations in glutamatergic neurotransmission at BLA inputs onto PrL^PAG neurons, voltage-clamp recordings of optically evoked asynchronous excitatory postsynaptic currents (aEPSCs) were obtained from green retrobead labeled cells while bath applying TTX and 4-AP (Figure 7A). Analysis showed that female rats exhibited larger aEPSCs than male rats (main effect of sex: F_(1,70) = 11.75, p = 0.0010, partial η² = 0.1437 [0.0256, 0.2925]). No other significant effects of AIE, carrageenan, or any interactions on aEPSC amplitude were observed (main effect of AIE: F_(1,70) = 0.25, p = 0.6200; main effect of carrageenan: F_(1,70) = 0.08, p = 0.7733; AIE × carrageenan interaction: F_(1,70) = 0.24, p = 0.6239; AIE × sex interaction: F_(1,70) = 0.09, p = 0.7713; sex × carrageenan interaction: F_(1,70) = 3.62, p = 0.0613; AIE × sex × carrageenan interaction: F_(1,70) = 0.47, p = 0.4974; Figure 7B, C). Likewise, assessment of the aEPSC interevent interval revealed no significant effects of AIE, carrageenan, or sex (main effect of AIE: F_(1,70) = 0.52, p = 0.4731; main effect of carrageenan: F_(1,70) = 0.01, p = 0.9106; main effect of sex: F_(1,70) = 0.03, p = 0.8678; AIE × carrageenan interaction: F_(1,70) = 0.15, p = 0.7021; AIE × sex interaction: F_(1,70) = 0.65, p = 0.4224; sex × carrageenan interaction: F_(1,70) = 2.39, p = 0.1265; AIE × sex × carrageenan interaction: F_(1,70) = 0.02, p = 0.8815; Figure 7D, E).

Figure 7

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Numerical data characterizing optically evoked asynchronous excitatory postsynaptic currents (aEPSCs) at monosynaptic inputs from the basolateral amygdala (BLA) onto PrL^PAG neurons.

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AIE exposure decreased and carrageenan increased the intrinsic excitability of PrL PVINs

After observing altered inhibition of PrL^PAG neurons, the impact of AIE exposure- and carrageenan-induced hyperalgesia on PrL PVIN intrinsic excitability was evaluated through current-clamp recordings of current evoked firing obtained from mCherry-tagged cells in the PrL cortex. As expected, mCherry-tagged neurons were distributed throughout layers II/III and V/VI in the PrL, with the highest concentration observed in layer V. Table 2 contains a summary of the biophysical properties of the recorded PVINs. Analysis of these properties revealed a significant reduction in the AP threshold of PVINs from AIE exposed rats treated with carrageenan (AIE × carrageenan interaction: F_(1,72) = 4.56, p = 0.0361, partial η² = 0.0596 [0.0000, 0.1865]) as well as greater afterhyperpolarization in PVINs from male rats (main effect of sex: F_(1,72) = 14.51, p = 0.0003, partial η² = 0.1677 [0.0394, 0.3165]). No other significant differences between treatment conditions or sex were observed for these properties.

Table 2

Condition	Sex	Vrest (mV)	Rinput (MΩ)	APThresh (mV)	Sag ratio (%)	AHP (mV)
AIR: SAL	Male	–74.3 ± 1.9	145.1 ± 5.1	–42.4 ± 0.7	2.6 ± 0.8	18.0 ± 0.9
	Female	–72.6 ± 1.6	155.4 ± 8.8	–42.3 ± 1.6	2.7 ± 0.4	15.3 ± 0.2
AIR: CAR	Male	–72.5 ± 1.9	138.1 ± 7.4	–42.0 ± 1.4	1.3 ± 0.5	19.4 ± 1.3
	Female	–73.7 ± 1.6	135.4 ± 8.9	–41.2 ± 0.8	2.3 ± 0.6	12.0 ± 1.2
AIE: SAL	Male	–73.6 ± 1.3	155.7 ± 4.9	–40.4 ± 0.6	1.2 ± 0.5	16.0 ± 1.8
	Female	–74.6 ± 1.8	147.0 ± 5.2	–41.4 ± 1.2	3.2 ± 0.8	14.1 ± 1.0
AIE: CAR	Male	–71.6 ± 2.1	153.2 ± 8.3	–44.3 ± 1.6*	2.6 ± 0.7	16.1 ± 1.2
	Female	–74.0 ± 1.6	141.3 ± 5.9	–44.0 ± 2.0*	2.5 ± 0.7	14.6 ± 1.8
* Denotes a significant difference; p < 0.05.

Analysis of the firing of PrL PVINs revealed AIE reduced intrinsic excitability, with the magnitude of this reduction increasing with the amount of current injected (main effect of AIE: F_(1,72) = 5.41, p = 0.0228, partial η² = 0.0699 [0.0004, 0.2010]; AIE × current step interaction: F_(20,1440) = 4.77, p = 0.0098, partial η² = 0.0621 [0.0289, 0.0744]; Figure 8A–G). Additionally, carrageenan enhanced the number of evoked APs at large but not small current steps, although the main effect was not significant (main effect of carrageenan: F_(1,72) = 2.26, P = 0.1374; carrageenan × current step interaction: F_(20,1440) = 3.57, p = 0.0304, partial η² = 0.0472 [0.0170, 0.0566]). There were no significant effects of sex, or interactions between treatment conditions, sex, and current step (main effect of sex: F_(1,72) = 0.00, p = 0.9928; AIE × carrageenan interaction: F_(1,72) = 0.08, p = 0.7720; AIE × sex interaction: F_(1,72) = 0.18, p = 0.6685; sex × carrageenan interaction: F_(1,72) = 0.01, p = 0.9110; AIE × sex × carrageenan interaction: F_(1,72) = 0.00, p = 0.9700; sex × current step interaction: F_(20,1440) = 0.43, p = 0.6532; AIE × sex × current step interaction: F_(20,1440) = 0.80, p = 0.4510; AIE × carrageenan × current step interaction: F_(20,1440) = 0.63, p = 0.5367; sex × carrageenan × current step interaction: F_(20,1440)=0.27, p = 0.7654; AIE × sex × carrageenan × current step interaction: F_(20,1440) = 0.44, p = 0.6468). However, the number of current evoked APs increased with the amount of current injected (main effect of current step: F_(20,1440) = 181.06, p = 0.000, partial η² = 0.7155 [0.6903, 0.7307]).

Figure 8

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Numerical data for the intrinsic excitability of prelimbic (PrL) parvalbumin interneurons (PVINs).

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AIE exposure decreased and carrageenan increased the E/I balance at inputs from the BLA onto PrL PVINs

After assessing the intrinsic excitability of PVINs, the next set of experiments characterized the impact of AIE exposure- and carrageenan-induced hyperalgesia on synaptic function at BLA inputs to PrL PVINs. This involved recording from mCherry-tagged PVINs in the PrL cortex while optically stimulating terminals from the BLA (Figure 9A).

Figure 9

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Numerical data characterizing optically evoked postsynaptic excitatory and inhibitory currents onto prelimbic (PrL) parvalbumin interneurons (PVINs).

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To evaluate the E/I balance at BLA inputs to PVINs, voltage-clamp recordings of oEPSCs and oIPSCs were obtained from mCherry-tagged neurons in the PrL. Analysis revealed that AIE significantly reduced oEPSC amplitude (main effect of AIE: F_(1,72) = 14.80, p = 0.0003, partial η² = 0.1705 [0.0409, 0.3194]; Figure 9B, C), with larger oEPSCs in females than males (main effect of sex: F_(1,72) = 8.22, p = 0.0054, partial η² = 0.1025 [0.0094, 0.2429]). No significant effects of carrageenan, or interactions between AIE, carrageenan, or sex were observed (main effect of carrageenan: F_(1,72) = 1.33, p = 0.2520; AIE × carrageenan interaction: F_(1,72) = 0.08, p = 0.7839; AIE × sex interaction: F_(1,72) = 0.26, p = 0.6126; sex × carrageenan interaction: F_(1,72) = 0.41, p = 0.5254; AIE × sex × carrageenan interaction: F_(1,72) = 0.26, p = 0.6110).

Examination of oIPSC amplitudes uncovered a significant AIE × sex interaction (AIE × sex interaction: F_(1,72) = 4.48, p = 0.0377, partial η² = 0.0586 [0.0000, 0.1851]; Figure 9D, E), but no further significant effects of AIE, carrageenan, or sex (main effect of AIE: F_(1,72) = 0.00, p = 0.9927; main effect of carrageenan: F_(1,72) = 0.16, p = 0.6871; main effect of sex: F_(1,72) = 0.14, p = 0.7094; AIE × carrageenan interaction: F_(1,72) = 1.15, p = 0.2867; sex × carrageenan interaction: F_(1,72) = 0.19, p = 0.6673; AIE × sex × carrageenan interaction: F_(1,72) = 0.07, p = 0.7895). Subsequent post hoc analysis did not find significant effects of AIE in male (t = 1.49, p = 0.281) or female (t = –1.50, p = 0.274) rats nor did it find significant sex differences in Air (t = 1.23, p = 0.443) or AIE exposed (t = –1.76, p = 0.165) rats.

Comparisons of the oEPSC to oIPSC ratios indicated larger E/I ratios in females compared to males (main effect of sex: F_(1,72) = 12.12, p = 0.0009, partial η² = 0.1440 [0.0269, 0.2909]; Figure 9F, G). Additionally, AIE reduced the E/I ratio, with a more pronounced reduction observed in males than in females (main effect of AIE: F_(1,72) = 15.60, p = 0.0002, partial η² = 0.1781 [0.0453, 0.3273]; AIE × sex interaction: F_(1,72) = 4.64, p = 0.0345, partial η² = 0.0606 [0.0000, 0.1880]). In contrast, carrageenan augmented the E/I balance (main effect of carrageenan: F_(1,72) = 7.48, p = 0.0079, partial η² = 0.0941 [0.0067, 0.2326]). No further significant interactions between AIE, carrageenan, and sex were observed (AIE × carrageenan interaction: F_(1,72) = 1.48, p = 0.2278; sex × carrageenan interaction: F_(1,72) = 0.57, p = 0.4523; AIE × sex × carrageenan interaction: F_(1,72) = 0.12, p = 0.7347).

AIE exposure blunted carrageenan-induced increases in the AMPA/NMDA ratio at direct inputs from the BLA onto PrL PVINs

To assess the AMPA/NMDA ratio at monosynaptic BLA inputs onto PrL PVINs, voltage-clamp recordings of oAMPA and oNMDA currents were obtained from mCherry-tagged neurons in the PrL cortex during bath application of TTX and 4-AP (Figure 10A). Analysis revealed that AIE exposure attenuated oAMPA current amplitude, whereas carrageenan enhanced it (main effect of AIE: F_(1,72) = 5.76, p = 0.0190, partial η² = 0.0740 [0.0013, 0.2066]; main effect of carrageenan: F_(1,72) = 4.44, p = 0.0385, partial η² = 0.0581 [0.0000, 0.1844]; Figure 10B, C). No significant effects of sex or any interactions were observed (main effect of sex: F_(1,72) = 0.28, p = 0.5974; AIE × carrageenan interaction: F_(1,72) = 1.32, p = 0.2542; AIE × sex interaction: F_(1,72) = 0.37, p = 0.5458; sex × carrageenan interaction: F_(1,72) = 0.15, p = 0.7029; AIE × sex × carrageenan interaction: F_(1,72) = 0.22, p = 0.6428).

Figure 10

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Numerical data characterizing optically evoked AMPA and NMDA currents at monosynaptic inputs from the basolateral amygdala (BLA) onto prelimbic (PrL) parvalbumin interneurons (PVINs).

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Similarly, the amplitude of oNMDA currents was reduced by AIE (main effect of AIE: F_(1,72) = 7.32, p = 0.0085, partial η² = 0.0923 [0.0062, 0.2303]; Figure 10D, E). However, neither carrageenan nor sex, nor any interaction between AIE, carrageenan, or sex was observed to impact oNMDA currents (main effect of carrageenan: F_(1,72) = 0.08, p = 0.7814; main effect of sex: F_(1,72) = 1.69, p = 0.1971; interaction AIE × carrageenan: F_(1,72) = 2.37, p = 0.1279; interaction AIE × sex: F_(1,72) = 0.04, p = 0.8458; interaction sex × carrageenan: F_(1,72) = 1.80, p = 0.1836; interaction AIE × sex × carrageenan: F_(1,72) = 0.10, p = 0.7493).

After analyzing the oAMPA and oNMDA currents individually, ratios of oAMPA to oNMDA currents were compared. This revealed that carrageenan significantly enhanced the AMPA/NMDA ratio (main effect of carrageenan: F_(1,72) = 6.28, p = 0.0145, partial η² = 0.0802 [0.0029, 0.2148]; Figure 10F, G). The analysis also uncovered a significant interaction between AIE and carrageenan, reflecting a reduction in the effect of carrageenan on AIE exposed rats (AIE × carrageenan interaction: F_(1,72) = 6.47, p = 0.0131, partial η² = 0.0825 [0.0034, 0.2178]). No additional significant effects were detected (main effect of AIE: F_(1,72) = 0.16, p = 0.6888; main effect of sex: F_(1,72) = 2.21, p = 0.1411; AIE × sex interaction: F_(1,72) = 2.90, p = 0.0928; sex × carrageenan interaction: F_(1,72) = 1.00, p = 0.3203; AIE × sex × carrageenan interaction: F_(1,72) = 0.73, p = 0.3953).

AIE exposure reduced the amplitude of aEPSCs at direct inputs from the BLA onto PrL PVINs

To assess pre- and postsynaptic changes in glutamatergic neurotransmission at direct inputs from the BLA onto PVINs in the PrL cortex, voltage-clamp recordings were acquired from mCherry-tagged neurons during bath application of TTX and 4-AP (Figure 11A). Analysis revealed an AIE-induced reduction in amplitude (main effect of AIE: F_(1,71) = 17.60, p = 0.0001, partial η² = 0.1986 [0.0572, 0.3493]; Figure 11B, C). No additional effects of carrageenan, sex, or interactions between treatment condition and sex were detected (main effect of carrageenan: F_(1,71) = 0.15, p = 0.6961; main effect of sex: F_(1,71) = 1.38, p = 0.2441; AIE × carrageenan interaction: F_(1,71) = 0.10, p = 0.7556; AIE × sex interaction: F_(1,71) = 0.44, p = 0.5115; sex × carrageenan interaction: F_(1,71) = 1.89, p = 0.1733; AIE × sex × carrageenan interaction: F_(1,71) = 0.00, p = 0.9505). In contrast, evaluation of the interevent interval revealed female rats had smaller interevent intervals than male rats (main effect of sex: F_(1,71) = 8.44, p = 0.0049, partial η² = 0.1062 [0.0104, 0.2484]; Figure 11D, E), while finding no additional significant effects of AIE, carrageenan, or interactions between treatment conditions and sex (main effect of AIE: F_(1,71) = 0.51, p = 0.4795; main effect of carrageenan: F_(1,71) = 1.05, p = 0.3079; AIE × carrageenan interaction: F_(1,71) = 1.00, p = 0.3196; AIE × sex interaction: F_(1,71) = 0.46, p = 0.5009; sex × carrageenan interaction: F_(1,71) = 0.00, p = 0.9530; AIE × sex × carrageenan interaction: F_(1,71) = 0.29, p = 0.5943).

Figure 11

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Numerical data characterizing optically evoked asynchronous excitatory postsynaptic currents (aEPSCs) at monosynaptic inputs from the basolateral amygdala (BLA) onto prelimbic (PrL) parvalbumin interneurons (PVINs).

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Emerging evidence from preclinical rodent models indicates that AIE exposure induces long-lasting hyperalgesia (Bertagna et al., 2024; Kelley et al., 2024; Khan et al., 2023; Secci et al., 2024). While changes within the extended amygdala circuitry contribute to altered nociception in AIE exposed animals (Bertagna et al., 2024; Kelley et al., 2024; Secci et al., 2024), the impact on prefrontal nociceptive circuits remains unexplored. This study investigated the effects of AIE exposure and carrageenan-induced inflammatory paw pain on synaptic function and intrinsic excitability within a BLA–PrL–vlPAG circuit involved in modulating the descending pain pathway. The central finding was that AIE enhanced mechanical allodynia, and that this enhancement was accompanied by altered E/I balance and intrinsic excitability at PrL^PAG neurons and PVINs (Figure 12). Carrageenan-induced hyperalgesia was unaltered by AIE exposure while pain-induced plasticity at BLA inputs to PrL PVINs was reduced.

Figure 12

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In this study, mechanical and thermal sensitivity were assessed from adolescence to early adulthood using the electronic Von Frey and Hargreaves tests. Mechanical sensitivity was heightened in both male and female rats exposed to AIE, consistent with previous findings in rodents (Bertagna et al., 2024; Kelley et al., 2024; Khan et al., 2023; Secci et al., 2024). While AIE-induced significant mechanical hypersensitivity, thermal sensitivity remained unchanged. Although this contrasts with previous research (Khan et al., 2023; Secci et al., 2024), for female rats this difference may be due, at least in part, to different statistical approaches for analysis of the data. In the present study, sex was included as a between-subjects factor in the ANOVA, whereas the prior study analyzed for male and female rat data separately. Consistent with this, when we analyzed our male and female data separately, we did observe a transient increase in thermal sensitivity in female rats as was reported previously (Secci et al., 2024). Discrepancies regarding the impact of AIE on thermal sensitivity in males may stem from methodological differences such as the use of different strains of rats and differences in the ethanol exposure paradigms.

After assessing the effects of AIE exposure on mechanical sensitivity, patch-clamp slice electrophysiology was performed to assess intrinsic excitability and synaptic function within the BLA–PrL–vlPAG circuit. For PrL^PAG neurons, these experiments revealed that AIE increased intrinsic excitability. Previous research has shown both unchanged (Galaj et al., 2020; Obray et al., 2022; Trantham-Davidson et al., 2017) and increased intrinsic excitability of PrL pyramidal neurons following AIE (Galaj et al., 2020) or voluntary alcohol consumption during adolescence (Salling et al., 2018). As pyramidal neurons in the PrL cortex can be classified as intratelencephalic (IT) or extratelencephalic (ET) based on their projection targets (Anastasiades and Carter, 2021; Baker et al., 2018a) and as each of these populations display unique physiological properties (Baker et al., 2018b; Dembrow et al., 2010), one potential explanation for these results is that adolescent ethanol exposure differentially effects these populations. Consistent with this hypothesis, IT pyramidal neurons projecting from the PrL to the BLA and the nucleus accumbens do not display altered intrinsic excitability following AIE exposure (Obray et al., 2022) whereas PT PrL^PAG neurons do. Notably, PT neurons display greater I_h-dependent voltage sag than IT neurons, and adolescent alcohol exposure has been reported to reduce I_h current (Salling et al., 2018), suggesting a possible mechanism for greater ethanol effects on intrinsic excitability in ET neurons than in IT neurons. However, for PrL^PAG neurons in the present study there was not a significant AIE-induced reduction in voltage sag. This disparity could be related to several methodological differences between studies, including differences in the exposure method (passive vapor versus voluntary drinking), the age of the animals at testing, and the use of rats versus mice. Regardless, it remains possible AIE may differentially affect the intrinsic excitability of ET and IT pyramidal neurons in the PrL.

Increased intrinsic excitability of PrL^PAG neurons was accompanied by augmented E/I balance in AIE exposed rats. The increased E/I balance resulted from reduced oIPSC amplitude. This indicated that AIE exposure reduced BLA-driven feedforward inhibition of PrL^PAG neurons. This is an interesting observation as it suggests that AIE prevents the normal developmental shift toward greater inhibition at PrL pyramidal neurons that occurs as PVINs mature during adolescence (Caballero et al., 2014; Caballero et al., 2021; Du et al., 2018; Klune et al., 2021).

To investigate the change in feedforward inhibition, we conducted electrophysiological recordings from PrL PVINs. These recordings revealed significantly reduced intrinsic excitability in AIE exposed animals, consistent with previous reports (Trantham-Davidson et al., 2017). Notably, PVIN intrinsic excitability is developmentally regulated and increases during adolescence (Koppensteiner et al., 2019). Decreased intrinsic excitability coincided with reduced BLA-driven E/I balance at PrL PVINs in male AIE exposed rats, with an attenuated effect in females. The reduced E/I balance primarily stemmed from a decrease in oEPSC amplitude, although AIE did alter oIPSC amplitude in a sex-dependent manner. The sex-dependent effect of AIE on oIPSCs is difficult to interpret, as post hoc tests did not reveal any significant effects. However, visual inspection of the data suggests a potential trend toward increased oIPSC amplitude in male AIE exposed rats and reduced amplitude in females. Intriguingly, voluntary alcohol consumption during adolescence elicits sex-dependent effects on PrL somatostatin interneuron intrinsic excitability (Sicher et al., 2023). As these neurons receive inputs from the BLA (Cummings and Clem, 2020; McGarry and Carter, 2016) and project to PrL PVINs (Cummings and Clem, 2020), it is possible that somatostatin neurons are responsible for the sex-dependent effects of AIE on E/I balance and oIPSCs at PVINs.

To better characterize the reduction in glutamate signaling, we next measured monosynaptic oAMPA and oNMDA currents at BLA inputs onto PVINs. This revealed that AIE significantly decreased oAMPA and oNMDA currents without altering the overall AMPA/NMDA ratio. This agreed with prior research from our lab which found AIE reduced electrically evoked AMPA and NMDA currents onto PVINs (Trantham-Davidson et al., 2017). To determine whether this reduction resulted from pre- or postsynaptic changes, aEPSCs were recorded from PVINs in the PrL cortex. The amplitude but not the interevent interval of the aEPSCs was reduced in AIE exposed animals, indicating a reduction in postsynaptic receptor function. These findings provide strong evidence for reduced glutamate signaling efficacy at BLA inputs onto PVINs following AIE exposure. It was also revealed that following AIE exposure, BLA-dependent feedforward inhibition of PrL^PAG neurons was decreased. This reduction may have resulted, at least in part, from the observed decreases in PrL PVIN intrinsic excitability and postsynaptic glutamate receptor function at BLA inputs to PrL PVINs. Within the BLA–PrL–vlPAG circuit, PrL^PAG neuron activation is generally associated with antinociceptive effects (Drake et al., 2021; Gadotti et al., 2019; Gao et al., 2023; Huang et al., 2019; Yin et al., 2020), while PrL PVIN activation is associated with pronociceptive effects (Zhang et al., 2015). However, PrL^PAG neuron activation can be pronociceptive (Fan et al., 2018). Notably, chronic activation of a PrL nociceptive ensemble including PrL^PAG neurons has been shown to induce chronic pain-like behaviors (Qi et al., 2022). As PrL^PAG neurons project to both GABA and glutamate neurons in the vlPAG (Guo et al., 2023; Huang et al., 2019), and these populations have been shown to be pro- and antinociceptive, respectively (Samineni et al., 2017), the balance of PrL^PAG input onto these populations may determine the effect of PrL^PAG activation on nociception. While speculative, we hypothesize that AIE selectively strengthens the input from PrL^PAG neurons onto vlPAG GABA neurons, resulting in mechanical allodynia that could be alleviated by inhibiting PrL^PAG neurons or activating PrL PVINs. Alternatively, these changes could represent an antinociceptive compensatory response for pronociceptive changes in other nociceptive circuits. Future experiments could test these hypotheses.

The intrinsic excitability of PrL^PAG neurons was increased following a carrageenan-induced pain challenge, akin to the heightened excitability observed in AIE exposed rats. Intrinsic excitability is also enhanced in PrL pyramidal neurons 3–5 days after intraplantar injection with complete Freund’s adjuvant (Wu et al., 2016). In addition to enhancing the excitability of PrL^PAG neurons, we observed that carrageenan also increased the amplitude of oIPSCs in Air control rats, with a blunted effect in AIE exposed rats. Despite no change in glutamate transmission at BLA inputs onto PrL^PAG neurons, the E/I balance remained unaltered. These findings are similar to those seen in a kaolin-carrageenan arthritis model, where the amplitude of BLA-driven electrically evoked IPSCs (eIPSCs) onto PrL pyramidal neurons is significantly increased without a corresponding change in glutamate transmission (Ji et al., 2010). These findings indicate that enhanced inhibitory transmission at PrL pyramidal neurons is a hallmark of carrageenan-induced inflammatory pain.

As with PrL^PAG neurons, carrageenan elevated the intrinsic excitability of PVINs, albeit only at large current steps. Accompanying this elevation was enhanced E/I balance at PVINs. There was not a significant change in either oEPSC or oIPSC amplitude, suggesting subtle alterations in both excitatory and inhibitory neurotransmission. The monosynaptic oAMPA current was enhanced at PrL PVINs, leading to an increase in the AMPA/NMDA ratio of Air control rats, with a weakened effect observed in AIE exposed rats. Surprisingly, the increased oAMPA currents at PVINs did not correspond to changes in either aEPSC amplitude or interevent interval. This leaves it unclear whether the observed change in synaptic strength resulted from a modification in presynaptic release probability or postsynaptic receptor function.

The present study demonstrates that in carrageenan-induced inflammatory pain, BLA inputs onto PVINs are strengthened. This strengthening, alongside increased PVIN excitability, enhances inhibition of PrL^PAG neurons. Notably, PrL^PAG neuron intrinsic excitability is increased, possibly to compensate for increased inhibitory input. Remarkably, the effects of carrageenan on synaptic function were blunted in AIE exposed rats, suggesting that AIE not only induces PVIN hypofunction but also restricts pain-induced plasticity at PVINs. Intriguingly, AIE augments the number of perineuronal net (PNN) enwrapped PVINs in the PrL cortex (Dannenhoffer et al., 2022; Obray et al., 2025). As PNNs play a role in stabilizing synapses and restricting plasticity (Cornez et al., 2020; Pizzorusso et al., 2002), this may reduce synaptic plasticity at PrL PVINs. Surprisingly, despite restricted plasticity at BLA inputs to PVINs, there were no AIE-dependent changes in carrageenan-induced hyperalgesia.

In conclusion, the present study examined the effects of AIE exposure and a carrageenan pain challenge on a BLA–PrL–vlPAG circuit involved in modulating the descending pain pathway. Following AIE exposure, while rats displayed enhanced mechanical sensitivity, carrageenan-induced hyperalgesia was not altered by a history of AIE exposure. In AIE exposed rats, BLA inputs to the PrL were biased toward decreased feedforward inhibition of PrL^PAG neurons. A carrageenan pain challenge increased BLA-mediated inhibitory drive onto these neurons in Air control but not AIE exposed animals. These changes suggest that AIE induces long-lasting reductions in feedforward inhibition of PrL^PAG neurons which accompany increased mechanical sensitivity. In addition to reduced feedforward inhibition, AIE may also diminish plasticity at PrL PVINs. Beyond the implications for nociception, these AIE-induced alterations in BLA–PrL–vlPAG function may impact other PrL^PAG neuron-mediated behaviors such as context fear discrimination (Rozeske et al., 2018), passive avoidance (Johnson et al., 2022), and arousal (Guo et al., 2023).

Raw data are presented as individual data points in figures where feasible. Numerical raw data is included in source data files and has also been published at https://doi.org/10.5061/dryad.wwpzgmswd.

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Author details

1. J Daniel Obray

   Department of Neuroscience, Medical University of South Carolina, Charleston, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7526-4869

2. Erik T Wilkes

   Department of Neuroscience, Medical University of South Carolina, Charleston, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Mike Scofield

   1. Department of Neuroscience, Medical University of South Carolina, Charleston, United States
   2. Department of Anesthesia and Perioperative Medicine, Medical University of South Carolina, Charleston, United States

   Contribution

   Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2330-6999

4. L Judson Chandler

   Department of Neuroscience, Medical University of South Carolina, Charleston, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – review and editing

   For correspondence

   chandj@musc.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1468-7320

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