Introduction

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 & 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 & 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., 2023; 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 & 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 & 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 & 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, 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; Insel et al., 1990; Murphy et al., 2012), and increased innervation from the BLA (Cunningham et al., 2002, 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 adolescent intermittent ethanol (AIE) exposure disrupts the normative developmental trajectory of the mPFC, inducing persistent alterations in intrinsic excitability and synaptic function. Specifically, AIE 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.

Results

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 8 intermittent cycles of ethanol vapor from PD 28 to PD 54. Behavioral intoxication and 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 (rs = 0.499, p = 0.0001).

Adolescent Intermittent Ethanol Exposure Augments 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 four 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 AIE: F(1,115) = 12.81, p = 0.0005, partial η² = 0.1002 [0.0203, 0.2105]; Fig. 1 A-B).

Figure 1.

Additionally, female rats exhibited greater sensitivity to mechanical touch compared to male rats (main effect 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 x sex interaction: F(1,115) = 1.02, p = 0.3147). Mechanical touch sensitivity decreased with age (main effect 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 x age interaction: F(8,920) = 0.70, p = 0.6327; sex x age interaction: F(8,920) = 1.50, p = 0.1829; AIE x sex x 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 AIE: F(1,115) = 13.33, p = 0.0004, partial η² = 0.1039 [0.0221, 0.2149]; Fig. 1 C) and female rats were more sensitive to mechanical touch than male rats (main effect 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 x sex interaction: F(1,115) = 1.28, p = 0.2608).

For thermal sensitivity, analysis of data from the Hargreaves test indicated no significant effect of AIE on paw withdrawal latency (main effect AIE: F(1,115) = 1.21, p = 0.2738; Fig. 1 D-E). Additionally, there were no significant sex differences or interactions with AIE (main effect sex: F(1,115) = 0.59, p = 0.4446; AIE x sex interaction: F(1,115) = 1.85, p = 0.1767). Thermal sensitivity was found to decrease with age (main effect 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 x age interaction: F(8,920) = 1.11, p = 0.3567; sex x age interaction: F(8,920) = 2.02, p = 0.0546; AIE x sex x 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 AIE: F(1,115) = 1.09, p = 0.2977; main effect sex: F(1,115) = 1.57, p = 0.2128; AIE x sex interaction: F(1,115) = 2.20, p = 0.1408; Fig. 1 F).

Carrageenan-Induced Inflammatory Paw Pain is Unaltered by Adolescent Intermittent Ethanol 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 & Hall, 1959; Neves et al., 2020; Vazquez et al., 2015; Winter et al., 1962; Yang & 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 eight weeks after discontinuing ethanol vapor exposure (main effect AIE: F(1,109) = 5.50, p = 0.0209, partial η² = 0.0480 [0.0006, 0.1441]; Fig. 2 A). In addition, female rats continued to display greater mechanical sensitivity than males, with no significant interactions between AIE and sex (main effect sex: F(1,109) = 10.66, p = 0.0015, partial η² = 0.0891 [0.0139, 0.1998]; AIE x sex interaction: F(1,109) = 0.93, p = 0.3367).

Figure 2.

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 AIE: F(1,109) = 1.69, p = 0.1968; main effect sex: F(1,109) = 1.27, p = 0.2616; AIE x sex interaction: F(1,109) = 0.38, p = 0.5384; Fig. 2 B).

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 carrageenan: F(1,105) = 116.41, p = 0.0000, partial η² = 0.5258 [0.3934, 0.6201]; Fig. 2 C,D). 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 sex: F(1,105) = 6.22, p = 0.0142, partial η² = 0.0559 [0.0020, 0.1578]; main effect AIE: F(1,105) = 0.93, p = 0.3366; AIE x sex interaction: F(1,105) = 0.03, p = 0.8706; AIE x carrageenan interaction: F(1,105) = 0.59, p = 0.4441; sex x carrageenan interaction: F(1,105) = 1.72, p = 0.1925; AIE x sex x 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 carrageenan: F(1,105) = 132.70, p = 0.0000, partial η² = 0.5583 [0.4308, 0.6470]; Fig. 2 E,F). No significant effects of AIE, sex, or interactions between AIE, sex, and carrageenan were observed (main effect AIE: F(1,105) = 0.00, p = 0.9625; main effect sex: F(1,105) = 0.00, p = 0.9510; AIE x sex interaction: F(1,105) = 0.23, p = 0.6307; AIE x carrageenan interaction: F(1,105)

= 0.01, p = 0.9352; sex x carrageenan interaction: F(1,105) = 0.14, p = 0.7120; AIE x sex x carrageenan interaction: F(1,105) = 3.85, p = 0.0524).

Analysis of the Intrinsic Excitability of Prelimbic Neurons Projecting to the Ventrolateral Periaqueductal Gray

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 labelled cells in the PrL cortex. Labelling with green retrobeads indicated that the neuron projected ipsilateral from the left hemisphere of the PrL cortex to the vlPAG (Fig. 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.

Table 1.

Analysis of the firing data indicated that both AIE and carrageenan enhanced intrinsic excitability (main effect AIE: F(1,72) = 7.24, p = 0.0089, partial η² = 0.0914 [0.0059, 0.2292]; main effect carrageenan: F(1,72) = 4.07, p = 0.0474, partial η² = 0.0535 [0.0000, 0.1776]; Fig. 4 A-G). Moreover, the effect of AIE on intrinsic excitability became more pronounced with increasing current step size (AIE x 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 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 x carrageenan interaction: F(1,72) = 0.00, p = 0.9760; AIE x sex interaction: F(1,72) = 0.17, p = 0.6823; sex x carrageenan interaction: F(1,72) = 0.00, p = 0.9686; AIE x sex x carrageenan interaction: F(1,72) = 0.46, p = 0.4985; sex x current step interaction: F(20,1440) = 1.27, p = 0.2851; carrageenan x current step interaction: F(20,1440) = 1.13, p = 0.3377; AIE x sex x current step interaction: F(20,1440) = 0.66, p = 0.5694; AIE x carrageenan x current step interaction: F(20,1440) = 2.46, p = 0.0656; sex x carrageenan x current step interaction: F(20,1440) = 0.20, p = 0.8924; AIE x sex x carrageenan x current step interaction: F(20,1440) = 1.13, p = 0.3383).

Figure 4.

Analysis of the Excitatory/Inhibitory Balance at Inputs from the Basolateral Amygdala onto Prelimbic Neurons Projecting to the Ventrolateral Periaqueductal Gray

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 labelled pyramidal neurons in the PrL cortex while optically stimulating terminals from the BLA (Fig. 5 A).

Figure 5.

To evaluate the E/I balance at BLA inputs to PrL^PAG neurons, voltage-clamp recordings of oEPSCs and oIPSCs were obtained from green retrobead labelled cells in the PrL. Analysis of oEPSC peak amplitudes indicated no significant effects of AIE, carrageenan, or sex (main effect 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 x carrageenan interaction: F(1,69) = 0.00, p = 0.9757; AIE x sex interaction: F(1,69) = 1.45, p = 0.2334; sex x carrageenan interaction: F(1,69) = 0.00, p = 0.9617; AIE x sex x carrageenan interaction: F(1,69) = 0.76, p = 0.3851; Fig. 5 B,C).

Conversely, oIPSC amplitude was significantly reduced in AIE exposed rats (main effect AIE: F(1,69) = 37.66, p = 0.0000, partial η² = 0.3531 [0.1771, 0.4942]; Fig. 5 D,E). Additionally, carrageenan was found to enhance oIPSC amplitude (main effect 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 x 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 sex: F(1,69) = 0.83, p = 0.3644; AIE x sex interaction: F(1,69) = 2.90, p = 0.0929; sex x carrageenan interaction: F(1,69) = 0.97, p = 0.3287; AIE x sex x 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 AIE: F(1,69) = 52.48, p = 0.0000, partial η² = 0.4320 [0.2545, 0.5611]; Fig. 5 F,G) and female rats exhibited larger E/I ratios than male rats (main effect 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 carrageenan: F(1,69) = 0.96, p = 0.3307; AIE x carrageenan interaction: F(1,69) = 0.01, p = 0.9158; AIE x sex interaction: F(1,69) = 0.05, p = 0.8190; sex x carrageenan interaction: F(1,69) = 1.72, p = 0.1935; AIE x sex x carrageenan interaction: F(1,69) = 0.08, p = 0.7741).

Analysis of AMPA and NMDA Currents at Direct Inputs from the Basolateral Amygdala onto Prelimbic Neurons Projecting to the Ventrolateral Periaqueductal Gray

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 labelled cells in the PrL during application of TTX and 4-AP (Fig. 6 A). Analysis of oAMPA current amplitude revealed no significant effects of AIE, carrageenan, or sex (main effect AIE: F(1,72) = 0.50, p = 0.4817; main effect carrageenan: F(1,72) = 0.31, p = 0.5806; main effect sex: F(1,72) = 3.19, p = 0.0784; AIE x carrageenan interaction: F(1,72) = 0.03, p = 0.8652; AIE x sex interaction: F(1,72) = 0.20, p = 0.6576; sex x carrageenan interaction: F(1,72) = 0.29, p = 0.5889; AIE x sex x carrageenan interaction: F(1,72) = 0.26, p = 0.6122; Fig. 6 B,C). Likewise, evaluation of the amplitude of oNMDA currents revealed no effects of treatment condition or sex (main effect AIE: F(1,72) = 1.25, p = 0.2680; main effect carrageenan: F(1,72) = 0.29, p = 0.5932; main effect sex: F(1,72) = 2.86, p = 0.0952; AIE x carrageenan interaction: F(1,72) = 0.34, p = 0.5635; AIE x sex interaction: F(1,72) = 0.42, p = 0.5204; sex x carrageenan interaction: F(1,72) = 1.55, p = 0.2176; AIE x sex x carrageenan interaction: F(1,72) = 2.40, p = 0.1257; Fig. 6 D,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 AIE: F(1,72) = 1.34, p = 0.2513; main effect carrageenan: F(1,72) = 1.00, p = 0.3199; main effect sex: F(1,72) = 2.81, p = 0.0982; AIE x carrageenan interaction: F(1,72) = 0.25, p = 0.6173; AIE x sex interaction: F(1,72) = 0.75, p = 0.3907; sex x carrageenan interaction: F(1,72) = 0.01, p = 0.9328; AIE x sex x carrageenan interaction: F(1,72) = 1.30, p = 0.2582; Fig. 6 F,G).

Figure 6.

Analysis of Asynchronous Excitatory Postsynaptic Currents at Direct Inputs from the Basolateral Amygdala onto Prelimbic Neurons Projecting to the Ventrolateral Periaqueductal Gray

To examine pre- and postsynaptic alterations in glutamatergic neurotransmission at BLA inputs onto PrL^PAG neurons, voltage-clamp recordings of aEPSCs were obtained from green retrobead labelled cells while bath applying TTX and 4-AP (Fig. 7 A). Analysis showed that female rats exhibited larger aEPSCs than male rats (main effect 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 AIE: F(1,70) = 0.25, p = 0.6200; main effect carrageenan: F(1,70) = 0.08, p = 0.7733; AIE x carrageenan interaction: F(1,70) = 0.24, p = 0.6239; AIE x sex interaction: F(1,70) = 0.09, p = 0.7713; sex x carrageenan interaction: F(1,70) = 3.62, p = 0.0613; AIE x sex x carrageenan interaction: F(1,70) = 0.47, p = 0.4974; Fig. 7 B,C). Likewise, assessment of the aEPSC interevent interval revealed no significant effects of AIE, carrageenan, or sex (main effect 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 x carrageenan interaction: F(1,70) = 0.15, p = 0.7021; AIE x sex interaction: F(1,70) = 0.65, p = 0.4224; sex x carrageenan interaction: F(1,70) = 2.39, p = 0.1265; AIE x sex x carrageenan interaction: F(1,70) = 0.02, p = 0.8815; Fig. 7 D,E).

Figure 7.

Analysis of the Intrinsic Excitability of Prelimbic Parvalbumin Positive Interneurons

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. No significant differences between treatment conditions or sex were observed for these properties.

Table 2.

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 AIE: F(1,72) = 5.41, p = 0.0228, partial η² = 0.0699 [0.0004, 0.2010]; AIE x current step interaction: F(20,1440) = 4.77, p = 0.0098, partial η² = 0.0621 [0.0289, 0.0744]; Fig. 8 A-G). Additionally, carrageenan enhanced the number of evoked action potentials at large but not small current steps, although the main effect was not significant (main effect carrageenan: F(1,72) = 2.26, p = 0.1374; carrageenan x 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 sex: F(1,72) = 0.00, p = 0.9928; AIE x carrageenan interaction: F(1,72) = 0.08, p = 0.7720; AIE x sex interaction: F(1,72) = 0.18, p = 0.6685; sex x carrageenan interaction: F(1,72) = 0.01, p = 0.9110; AIE x sex x carrageenan interaction: F(1,72) = 0.00, p = 0.9700; sex x current step interaction: F(20,1440) = 0.43, p = 0.6532; AIE x sex x current step interaction: F(20,1440) = 0.80, p = 0.4510; AIE x carrageenan x current step interaction: F(20,1440) = 0.63, p = 0.5367; sex x carrageenan x current step interaction: F(20,1440) = 0.27, p = 0.7654; AIE x sex x carrageenan x current step interaction: F(20,1440) = 0.44, p = 0.6468). However, the number of current evoked action potentials increased with the amount of current injected (main effect current step: F(20,1440) = 181.06, p = 0.000, partial η² = 0.7155 [0.6903, 0.7307]).

Figure 8.

Analysis of the Excitation/Inhibition Balance at Inputs from the Basolateral Amygdala onto Prelimbic Parvalbumin Positive Interneurons

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 (Fig. 9 A).

Figure 9.

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 AIE: F(1,72) = 14.80, p = 0.0003, partial η² = 0.1705 [0.0409, 0.3194]; Fig. 9 B,C), with larger oEPSCs in females than males (main effect 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 x carrageenan interaction: F(1,72) = 0.08, p = 0.7839; AIE x sex interaction: F(1,72) = 0.26, p = 0.6126; sex x carrageenan interaction: F(1,72) = 0.41, p = 0.5254; AIE x sex x carrageenan interaction: F(1,72) = 0.26, p = 0.6110).

Examination of oIPSC amplitudes uncovered a significant AIE x sex interaction (AIE x sex interaction: F(1,72) = 4.48, p = 0.0377, partial η² = 0.0586 [0.0000, 0.1851]; Fig. 9 D,E), but no further significant effects of AIE, carrageenan, or sex (main effect AIE: F(1,72) = 0.00, p = 0.9927; main effect carrageenan: F(1,72) = 0.16, p = 0.6871; main effect sex: F(1,72) = 0.14, p = 0.7094; AIE x carrageenan interaction: F(1,72) = 1.15, p = 0.2867; sex x carrageenan interaction: F(1,72) = 0.19, p = 0.6673; AIE x sex x 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 oEPSCs to oIPSCs ratios to evaluate the E/I balance indicated larger ratios in females compared to males (main effects sex: F(1,72) = 12.12, p = 0.0009, partial η² = 0.1440 [0.0269, 0.2909]; Fig. 9 F,G). Additionally, AIE reduced the E/I ratio, with a more pronounced reduction observed in males than in females (main effect AIE: F(1,72) = 15.60, p = 0.0002, partial η² = 0.1781 [0.0453, 0.3273]; AIE x 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 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 x carrageenan interaction: F(1,72) = 1.48, p = 0.2278; sex x carrageenan interaction: F(1,72) = 0.57, p = 0.4523; AIE x sex x carrageenan interaction: F(1,72) = 0.12, p = 0.7347).

Analysis of AMPA and NMDA Currents at Direct Inputs from the Basolateral Amygdala onto Prelimbic Parvalbumin Positive Interneurons

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 (Fig. 10 A). Analysis revealed that AIE exposure attenuated oAMPA current amplitude, whereas carrageenan enhanced it (main effect AIE: F(1,72) = 8.67, p = 0.0043, partial η² = 0.1075 [0.0112, 0.2490]; main effect carrageenan: F(1,72) = 8.55, p = 0.0046, partial η² = 0.1062 [0.0107, 0.2474]; Fig. 10 B,C). No significant effects of sex or any interactions were observed (main effect sex: F(1,72) = 0.13, p = 0.7202; AIE x carrageenan interaction: F(1,72) = 1.33, p = 0.2533; AIE x sex interaction: F(1,72) = 0.56, p = 0.4549; sex x carrageenan interaction: F(1,72) = 0.31, p = 0.5783; AIE x sex x carrageenan interaction: F(1,72) = 0.09, p = 0.7650).

Figure 10.

Similarly, the amplitude of oNMDA currents was reduced by AIE (main effect AIE: F(1,72) = 7.32, p = 0.0085, partial η² = 0.0923 [0.0062, 0.2303]; Fig. 10 D,E). However, neither carrageenan nor sex, nor any interaction between AIE, carrageenan, or sex was observed to impact oNMDA currents (main effect carrageenan: F(1,72) = 0.08, p = 0.7814; main effect sex: F(1,72) = 1.69, p = 0.1971; interaction AIE x carrageenan: F(1,72) = 2.37, p = 0.1279; interaction AIE x sex: F(1,72) = 0.04, p = 0.8458; interaction sex x carrageenan: F(1,72) = 1.80, p = 0.1836; interaction AIE x sex x 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 carrageenan: F(1,72) = 6.70, p = 0.0116, partial η² = 0.0852 [0.0042, 0.2213]; Fig. 10 F,G). The analysis also uncovered a trend toward a significant interaction between AIE and carrageenan, reflecting a nearly significant reduction of the effect of carrageenan in AIE exposed rats (AIE x carrageenan interaction: F(1,72) = 3.97, p = 0.0501). No additional significant effects were detected (main effect AIE: F(1,72) = 0.39, p = 0.5330; main effect sex: F(1,72) = 0.19, p = 0.6635; AIE x sex interaction: F(1,72) = 0.50, p = 0.4814; sex x carrageenan interaction: F(1,72) = 2.49, p = 0.1191; AIE x sex x carrageenan interaction: F(1,72) = 0.00, p = 0.9547).

Analysis of Asynchronous Excitatory Postsynaptic Currents at Direct Inputs from the Basolateral Amygdala onto Prelimbic Parvalbumin Positive Interneurons

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 (Fig. 11 A). Analysis revealed an AIE-induced reduction in amplitude (main effect AIE: F(1,71) = 17.60, p = 0.0001, partial η² = 0.1986 [0.0572, 0.3493]; Fig. 11 B,C). No additional effects of carrageenan, sex, or interactions between treatment condition and sex were detected (main effect carrageenan: F(1,71) = 0.15, p = 0.6961; main effect sex: F(1,71) = 1.38, p = 0.2441; AIE x carrageenan interaction: F(1,71) = 0.10, p = 0.7556; AIE x sex interaction: F(1,71) = 0.44, p = 0.5115; sex x carrageenan interaction: F(1,71) = 1.89, p = 0.1733; AIE x sex x 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 sex: F(1,71) = 8.44, p = 0.0049, partial η² = 0.1062 [0.0104, 0.2484]; Fig. 11 D,E), while finding no additional significant effects of AIE, carrageenan, or interactions between treatment conditions and sex (main effect AIE: F(1,71) = 0.51, p = 0.4795; main effect carrageenan: F(1,71) = 1.05, p = 0.3079; AIE x carrageenan interaction: F(1,71) = 1.00, p = 0.3196; AIE x sex interaction: F(1,71) = 0.46, p = 0.5009; sex x carrageenan interaction: F(1,71) = 0.00, p = 0.9530; AIE x sex x carrageenan interaction: F(1,71) = 0.29, p = 0.5943).

Figure 11.

Discussion

Emerging evidence from preclinical rodent models indicates that AIE exposure induces long-lasting hyperalgesia (Bertagna et al., 2024; Kelley et al., 2023; 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., 2023; 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. Carrageenan-induced hyperalgesia was unaltered by AIE exposure while pain-induced plasticity at BLA inputs to PrL PVINs was reduced.

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., 2023; 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 either unchanged (Galaj et al., 2020; Obray et al., 2022; Trantham-Davidson et al., 2017) or increased intrinsic excitability of PrL pyramidal neurons following AIE (Galaj et al., 2020; Salling et al., 2018). Notably, the maximum current injected in the present study was larger than that used in studies that reported no effect of AIE on intrinsic excitability and closely matched that of studies that observed increased intrinsic excitability. Thus, it is possible that the size of the largest current steps is a key determinant as to whether changes in intrinsic excitability are detected. Alternatively, AIE may produce projection specific changes in intrinsic excitability.

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, PVIN mediated, 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, AIE exposure elicits sex-dependent effects on PrL somatostatin interneuron intrinsic excitability (Sicher et al., 2023). As these neurons receive inputs from the BLA (Cummings & Clem, 2020; McGarry & Carter, 2016) and project to PrL PVINs (Cummings & 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 is decreased. This reduction resulted from decreased PrL PVIN intrinsic excitability and reduced 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 PNN enwrapped PVINs in the PrL cortex (Dannenhoffer et al., 2022). 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 PVIN mediated 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 PVIN mediated 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).

Materials and methods

Animals

Parvalbumin-Cre rats on a Long-Evans background were obtained from the Rat Resource and Research Center (line #00773) and bred to establish a colony at the Medical University of South Carolina. Rats were genotyped at postnatal day (PD) 14, with only hemizygote rats included in this study. On PD 21, rat pups were weaned, same sex group-housed (2-3 per cage) and assigned to one of four treatment groups: Air-saline (n = 30), Air-carrageenan (n = 30), AIE-saline (n = 30), or AIE- carrageenan (n = 29). Rats were housed in a temperature and humidity-controlled environment on a 12hr/12hr light/dark cycle, with lights off from 09:00-21:00 each day. Teklad 2918 (Envigo, Indianapolis, IN) chow and water were provided to the rats ad libitum. All procedures were carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals (2011) and were approved by the Medical University of South Carolina Institutional Animal Care and Use Committee.

Adolescent Intermittent Ethanol Exposure

Adolescent intermittent ethanol exposure was carried out as previously described (Chandler et al., 2022; Gass et al., 2014). All rats underwent 8 cycles of intermittent ethanol vapor exposure beginning at PD 28 and continuing through PD 54. Each cycle consisted of two days of ethanol vapor exposure separated by two days of no ethanol exposure. The litter-matched Air rats received the same treatment except they were not exposed to ethanol vapor. On ethanol exposure days, rats were placed in the chambers at 18:00 and removed from the chambers at 08:00 on the following day. Upon removal from the chambers, the level of intoxication of each rat was rated on the following 5-point behavioral intoxication scale: 1 = no signs of intoxication; 2 = slightly intoxicated (slight motor impairment); 3 = moderately intoxicated (obvious motor impairment but able to walk); 4 = highly intoxicated (dragging abdomen, loss of righting reflex); 5 = extremely intoxicated (loss of righting reflex, and loss of eye blink reflex) (Barker et al., 2017; Gass et al., 2014; Glover et al., 2021). On the last day of each cycle, blood was collected from the tail vein and analyzed for blood ethanol concentration (BEC) using an Analox alcohol analyzer (AM1, Analox Instruments, Stourbridge, GBR). Following the last exposure cycle, the rats remained group housed in the vivarium until undergoing surgery at ∼ PD 80, after which they were single housed until being sacrificed to obtain slices for experimental use.

Stereotaxic Surgery

Rats undergoing stereotaxic surgery were induced and maintained under a surgical plane of anesthesia using isoflurane (2-3%). Intracranial injections were performed on a Kopf rat stereotaxic instrument (Kopf Instruments, Tujunga, CA, USA). A Micro4 (World Precision Instruments [WPI], Sarasota, FL, USA) controlled UMP3 microinjection pump (WPI) connected to a glass syringe (80100, Hamilton Company, Reno, NV) under stereotaxic control was used to inject 500 nl green retrobeads IX (Lumafluor Inc., Durham, NC) into the left vlPAG (from bregma: -8.4 mm AP, -0.7 mm ML, -6.3 mm DV), 500 nl of AAV5-hSyn-hChR2(H134R)-EYFP (26973-AAV5, Addgene, Watertown, MA) into the left BLA (from bregma: -2.7 mm AP, -5.1 mm ML, -8.8 mm DV), and 750 nl of AAV2-hSyn-DIO-mCherry (50459-AAV2, Addgene) into the left PrL cortex (from bregma: +2.8 mm AP, -0.6 mm ML, -3.8 mm DV). All injections occurred at 1 nl/s with the injector remaining in place for an additional 5 min following completion of the infusion. After surgery, a minimum of 4 weeks was given to allow the rats to recover and for retrograde transport and viral expression to occur.

Assessment of Mechanical and Thermal Sensitivity

Mechanical and thermal sensitivity were assessed using the electronic Von Frey and Hargreaves tests, respectively. Assessment began on PD 24 prior to initiation of ethanol vapor exposure on PD 28, and continued every 7 days until PD 80. Rats that subsequently underwent stereotaxic surgery followed by 4 wks of recovery were reassessed for pain sensitivity. This was followed by injection with 100 µl of carrageenan (C2871, Tokyo Chemical Industry, Tokyo, JPN; 1% w/v in saline) or saline in the right hindpaw under brief isoflurane anesthesia. Subsequent pain sensitivity tests were conducted at 2 hr, 6 hr, and 24 hr post-injection, after which rats were sacrificed to obtain slices for experimental use.

Electronic Von Frey test for mechanical allodynia

On pain sensitivity test days, rats were placed in a 17” x 8.5” x 10” Plexiglas enclosure with a metal mesh floor. After a 5 min acclimation period, the mechanical sensitivity of the right hindpaw was assessed using an electronic Von Frey unit (38450, Ugo Basile, Gemonio, ITA). This consisted of placing the electronic Von Frey filament perpendicular to the plantar surface of the hindpaw and applying force until a sharp withdrawal response was elicited. Mechanical sensitivity was assessed three times per session for each rat and the average gram-force required to elicit a withdrawal response was recorded.

Hargreaves test for thermal hyperalgesia

Following assessment of allodynia, rats were placed in a 4” x 8” x 5.5” plexiglass enclosure with a glass floor. After a 15 min habituation period, the right hindpaw was stimulated using an infrared emitter from Ugo Basile (37570; 60% maximum intensity) and the latency to hindpaw withdrawal was measured. A 30 s cutoff was used to prevent tissue damage in rats that were unresponsive to the thermal stimulus. In each session the paw withdrawal latency was measured three times per rat and the average score was used to quantify the level of thermal sensitivity.

Electrophysiological Recordings

Acute slices were obtained from rats for electrophysiological recordings beginning at PD 110. Current clamp experiments were performed as previously described(Trantham-Davidson et al., 2014; Trantham-Davidson et al., 2017). In brief, rats were anesthetized with isoflurane, and the brain was rapidly removed and placed into ice-cold cutting solution containing (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 10 d-glucose, 20 HEPES, 2.5 C5H9NO3S, 5 ascorbic acid, 15 sucrose, 10 MgCl2, and 0.5 CaCl2. Following sectioning using a Leica vibratome (VT 1200S, Wetzlar, DEU), 280 μM thick slices were incubated for at least 60 min at 34°C in artificial cerebrospinal fluid (aCSF) containing (in mM): 92 NaCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 10 d-glucose, 20 HEPES, 5 ascorbic acid, 10 MgCl2, and 0.5 CaCl2. After incubation, slices were transferred to a submerged recording chamber held at 34°C and constantly perfused with recording aCSF containing (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 10 d-glucose, 0.4 ascorbic acid, 1.3 MgCl2, and 2 CaCl2. Each of these solutions was pH adjusted (pH 7.3-7.43), with an osmolarity of 300-310 mOsm, and was continuously aerated with 95% O2/5% CO2.

Recordings were performed using a Multiclamp 700B amplifier (Molecular Devices, San Jose, CA) connected to a Windows-PC running Axograph X software through an ITC-18 digital to analog converter (HEKA Instruments, Holliston, MA). A Sutter Instruments P-1000 micropipette puller (Novato, CA) was used to pull borosilicate glass electrodes. Tip resistances ranged from 4 to 8 MΩ. All recordings were obtained from visually identified green retrobead labelled PrL^PAG neurons or mCherry tagged PVINs in the left PrL. Cells were identified using a Zeiss Axio Examiner.A1 microscope (Oberkochen, DEU) equipped with a DIC filter and a filter for visualizing green retrobeads and mCherry. All internal solutions were adjusted to pH 7.4 and 285 mOsm. Series and membrane resistance were measured at the beginning and end of each recording, and if the series resistance exceeded 20 MΩ or changed by more than 10% then the cell was excluded from the analysis.

Current clamp recordings

Electrodes were filled with an internal solution containing (in mM): 125 potassium gluconate, 20 KCl, 10 HEPES, 1 EGTA, 2 MgCl2, 2 Na2-ATP, 0.3 Tris-GTP, and 10 phosphocreatine. To assess intrinsic excitability, picrotoxin (100 µM) and kynurenic acid (2 mM) were added to the perfused aCSF and 1 s current steps were applied in 20 pA increments ranging from - 100 to +400 pA. Recordings were digitized at 10 kHz, filtered at 2 kHz, and analyzed for the number of action potentials elicited by each current step.

Voltage clamp recordings

Electrodes were filled with an internal solution containing (in mM): 125 cesium methanesulfonate, 10 CsCl, 4 NaCl, 10 HEPES, 1 EGTA, 2 MgCl2, 2 Na2-ATP, 0.5 Tris-GTP, 10 phosphocreatine, and 1 QX-314-Cl. All voltage clamp recordings were digitized at 10 kHz and filtered at 2 kHz. Postsynaptic events were evoked by optically stimulating channelrhodopsin expressing terminals from the BLA in the PrL cortex. This involved using Axograph to trigger a 5 ms pulse of light from an MDL-III-447 diode blue laser collimated to fit the microscope.

For experiments measuring the excitation/inhibition (E/I) balance of optically evoked postsynaptic currents onto PrL^PAG neurons and PrL PVINs, neurons were held at -70 mV for recordings of optically evoked excitatory postsynaptic currents (oEPSCs) and +10 mV for recordings of optically evoked inhibitory postsynaptic currents (oIPSCs). The peak amplitudes of the oEPSC and oIPSC events were analyzed, and their ratio (oEPSC/oIPSC) was computed.

For experiments measuring the AMPA/NMDA ratio at BLA inputs onto PrL^PAG and PrL PVINS, 1 µM tetrodotoxin (TTX) and 100 µM 4-aminopyridine (4-AP) were included in the aCSF to isolate monosynaptic transmission(Cho et al., 2013; Petreanu et al., 2009). Neurons were held at +40 mV and optically evoked AMPA (oAMPA) and NMDA (oNMDA) currents were isolated using the following procedure: first, a combined oAMPA and oNMDA current was recorded in aCSF containing 100 µM picrotoxin. Subsequently, 50 µM dl-APV was added to the recording aCSF to isolate the oAMPA current. Finally, to isolate the oNMDA current, the oAMPA current was subtracted from the combined oAMPA and oNMDA current. The AMPA/NMDA ratio was then computed based on the amplitude of each current.

For experiments measuring optically evoked asynchronous EPSCs (aEPSCs), TTX (1 µM) and 4-AP (100 µM) were added to the recording aCSF and SrCl2 (2 mM) was substituted for CaCl2. The substitution of strontium for calcium induces asynchronous neurotransmitter release after the initial release event. The resulting interevent interval and amplitude of the asynchronous events are commonly used to quantify pre- and postsynaptic function within defined circuits(Choi & Lovinger, 1997; Dodge et al., 1969; Xu-Friedman & Regehr, 2000). Recordings of aEPSCs were collected from neurons voltage clamped at -70 mV and analyzed within a 400 ms window beginning 50 ms poststimulation.

Statistical Analyses

Statistical analyses were performed using Stata 15.1 (StataCorp LLC, College Station, TX). Data were assessed for normality using the Wilks-Shapiro test and checked for outliers using the IQR rule. The experimental unit for this study was the individual animal. As such, while electrophysiological measures (e.g. E/I balance, intrinsic excitability) were obtained from multiple neurons within each animal, the data were averaged within each animal prior to analysis and reporting. Unless otherwise indicated, all data were analyzed using analysis of variance (ANOVA) models including all relevant factors. Repeated measures analyses used the Greenhouse-Geisser correction for sphericity. Post- hoc tests were corrected for multiple comparisons using the Bonferroni method. All values reported are mean ± SEM. For purposes of statistical significance, p < 0.05 was considered significant.

Acknowledgements

The authors would like to thank members of the Chandler Lab for their help carrying out the ethanol vapor exposure. This work was supported by NIH grants AA019967 (LJC), T32 AA007474 (JDO), and F32 AA030193 (JDO). The authors declare that the research was carried out in the absence of any commercial or financial relationships that could be construed as a conflict of interest. The artwork depicting electrophysiological recordings from PrL^PAG neurons and PVINs in figures 4-11 was drawn in part using images from Servier Medical Art. Servier Medical Art is licensed under a Creative Commons Attribution 4.0 Unported License (https://creativecommons.org/licenses/by/4.0/).