Tumor necrosis factor-α modulates GABAergic and dopaminergic neurons in the ventrolateral periaqueductal gray of female mice

Abstract

Neuroimmune signaling is increasingly identified as a critical component of various illnesses, including chronic pain, substance use disorder, and depression. However, the underlying neural mechanisms remain unclear. Proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), may play a role by modulating synaptic function and long-term plasticity. The midbrain structure periaqueductal gray (PAG) plays a well-established role in pain processing, and although TNF-α inhibitors have emerged as a therapeutic strategy for pain-related disorders, the impact of TNF-α on PAG neuronal activity has not been thoroughly characterized. Recent studies have identified subpopulations of ventrolateral PAG (vlPAG) with opposing effects on nociception, with dopamine (DA) neurons driving pain relief in contrast to GABA neurons. Therefore, we used slice physiology to examine the impact of TNF-α on neuronal activity of both these subpopulations. We focused on female mice since the PAG is a sexually dimorphic region and most studies use male subjects, limiting our understanding of mechanistic variations in females. We selectively targeted GABA and DA neurons using transgenic reporter lines. Following exposure to TNF-α, there was an increase in excitability of GABA neurons along with a reduction in glutamatergic synaptic transmission. In DA neurons, TNF-α exposure resulted in a robust decrease in excitability along with a modest reduction in glutamatergic synaptic transmission. Interestingly, TNF-α had no effect on inhibitory transmission onto DA neurons. Collectively, these data suggest that TNF-α differentially affects the function of GABA and DA neurons in female mice and enhances our understanding of how TNF-α-mediated signaling modulates vlPAG function.

NEW & NOTEWORTHY This study describes the effects of TNF-α on two distinct subpopulations of neurons in the vlPAG. We show that TNF-α alters both neuronal excitability and glutamatergic synaptic transmission on GABA and dopamine neurons within the vlPAG of female mice. This provides critical new information on the role of TNF-α in the potential modulation of pain, since activation of vlPAG GABA neurons drives nociception, whereas activation of dopamine neurons drives analgesia.

INTRODUCTION

The midbrain periaqueductal gray area (PAG) is an evolutionarily conserved region that regulates a wide range of complex behaviors, including pain, arousal, and fight-or-flight behaviors (1–3). The ventrolateral column of the PAG (vlPAG) is a major site of endogenous opioid-induced antinociception mediated through its descending projections via the rostral ventromedial medulla (RVM) (4–6). The vlPAG comprises heterogeneous subpopulations of neurons that modulate divergent behaviors. For example, it has been shown that vlPAG glutamate neurons promote antinociception and escape behaviors in contrast to vlPAG GABA neurons (7–9). Another understudied subpopulation of neurons within the vlPAG/dorsal raphe are dopamine (DA) neurons. vlPAG DA neurons are a subset of glutamate neurons that corelease glutamate and have been implicated in antinociceptive effects (10–13), fear learning (8, 14), arousal (15, 16), and incentivized salience (17). Also, recent work from our laboratory (18) has shown that either chemogenetic activation of vlPAG DA cells or optogenetic stimulation of vlPAG terminals in the extended amygdala reduces nociceptive sensitivity during naïve and inflammatory pain states in males but not in females.

Recent evidence has implicated glial-mediated neuroinflammatory response in the pathogenesis of chronic pain and morphine tolerance. Repeated administration of morphine results in upregulated expression of proinflammatory cytokines (19, 20). In rats, withdrawal from chronic morphine results in the upregulation of a proinflammatory cytokine, tumor necrosis factor-α (TNF-α) in the vlPAG, and microinjection of recombinant TNF-α resulted in morphine withdrawal-like behavioral signs (21). Furthermore, there is evidence demonstrating that PAG TNF-α signaling decreases the efficacy of opioids by promoting neuroinflammation and disrupting glutamate homeostasis (22, 23). Work from the same group also suggests that increased activation of PAG microglia in females contributes to sex differences in morphine analgesia (24). These results support the hypothesis that proinflammatory cytokine signaling in the PAG may play an important role in the sex-dependent modulation of pain through opioid signaling (for an extensive review, see Ref. 25).

Although there have been studies looking at how TNF-α can regulate synaptic plasticity and neuronal excitability in different brain regions via modulation of AMPA receptor and glutamate homeostasis (26–29), there has been no direct evidence showing TNF-α-mediated changes in excitability and synaptic transmission in the vlPAG. Given that chronic pain affects women disproportionately (30), more insight into the synaptic effects of TNF-α in female subjects is warranted. To address this disparity, in the present study, we utilized whole cell patch clamping to investigate the role of TNF-α in the vlPAG of female mice. We provide a novel characterization of the synaptic effects of TNF-α signaling on two specific subpopulations of vlPAG neurons: DA and GABA neurons that play critical roles in eliciting a diverse set of nociceptive and aversive behaviors.

MATERIALS AND METHODS

Mice

All experiments were performed on adult female mice (2–5 mo) in accordance with the NIH guidelines for animal research and with the approval of the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill. Animals were group-housed in a ventilated and temperature-controlled vivarium on a standard 12-h cycle (lights on at 07:00) with ad libitum access to food and water. For all experiments, the estrus cycle of the mouse was not monitored. To visualize vesicular GABA transporter (vGAT)-expressing neurons, vGAT-ires-Cre mice were maintained on a C57BL6/J background and crossed with a Rosa26-floxed-stop-L10-GFP reporter line (31) to produce vGAT-L10A-GFP mice. Female TH-eGFP mice on a Swiss Webster background were used for visualizing dopamine neurons (13). In the TH-eGFP mouse line, the genome was modified to contain multiple copies of a modified BAC in which an enhanced green fluorescent protein (eGFP) reporter gene was inserted immediately upstream of the coding sequence of the gene for tyrosine hydroxylase (TH). Data presented here were obtained from transgenic mice maintained in-house.

Slice Electrophysiology

For ex vivo slice physiology, mice were anesthetized with isoflurane and were rapidly decapitated. Then, 250-μm-thick coronal sections through the PAG were prepared as previously described (32). Given the known heterogeneity in PAG across the rostral-caudal axis, all recordings were restricted between anterior-posterior (AP) axis −4.60 to −4.84 mm with respect to bregma. Briefly, brains were quickly extracted, and slices were made using a Leica VT 1200s vibratome (Leica Biosystems, Buffalo Grove, IL) in ice-cold, oxygenated sucrose solution containing in mM: 194 sucrose, 20 NaCl, 4.4 KCl, 1 MgCl₂, 1.2 NaH₂PO₄, 10 glucose, and 26 NaHCO₃ saturated with 95% O₂/5% CO₂. Slices were incubated for at least 30 min in normal artificial cerebral spinal fluid (ACSF) maintained at 32°C–35°C that contained in mM: 124 NaCl, 4.0 KCl, 1 NaH₂PO₄, 1.2 MgSO₄, 10 d-glucose, 2 CaCl₂, and 26 NaHCO₃, saturated with 95% O₂/5% CO₂ before transferring to a submerged recording chamber (Warner Instruments, Hamden, CT) for experimental use. For whole cell recordings, slices were continuously perfused at a rate of 2.0–3.0 mL/min with oxygenated ACSF maintained at 30 ± 2°C.

Neurons were identified using infrared differential interference contrast on a Scientifica Slicescope II (East Sussex, UK). Fluorescent cells were visualized using a 470-nm LED. Whole cell patch-clamp recordings were performed using micropipettes pulled from a borosilicate glass capillary tube using a Flaming/Brown electrode puller (Sutter P-97; Sutter Instruments, Novato, CA). Electrode tip resistance was between 3 and 6 MΩ. All signals were acquired using an Axon MultiClamp 700B (Molecular Devices, Sunnyvale, CA). Data were sampled at 10 kHz, low-pass filtered at 3 kHz. Access resistance was continuously monitored and changes greater than 20% from the initial value were excluded from data analyses. Two to four cells were recorded from each animal per set of experiments.

Excitability experiments were performed in current-clamp mode using a potassium gluconate-based intracellular solution (in mM: 135 K-gluconate, 5 NaCl, 2 MgCl₂, 10 HEPES, 0.6 EGTA, 4 Na₂ATP, 0.4 Na₂GTP, pH 7.3, 285–295 mosmol/kgH₂O). Input resistance was measured immediately after breaking into the cell. Following stabilization, current was injected to hold cells at a common membrane potential of −70 mV to account for intercell variability. Changes in excitability were evaluated by measuring rheobase (minimum current required to elicit an action potential), action potential (AP) threshold, and the frequency of action potentials fired at increasing 20 pA current steps (0 to 200 pA). Parameters related to AP kinetics, which included AP height, AP duration at half-maximal height (AP half-width), time to fire an AP (AP latency), and afterhyperpolarization amplitude (AHP), were calculated from the first action potential fired during the F-I plot (33). AP height was calculated as the difference between AP peak and AP threshold voltage. AP latency was defined as the duration to the first AP following a depolarizing current step of fixed amplitude. AP half-width was measured as the AP duration at the membrane voltage halfway between AP threshold and AP peak. AHP was calculated as the difference from the action potential threshold to the minimum voltage in the 25 ms following the action potential. AP depolarization and repolarization rates were calculated from the first derivative of its voltage recording with respect to time.

For the assessment of spontaneous synaptic activity, two different intracellular solutions were used. Spontaneous excitatory postsynaptic currents (sEPSCs) were assessed in voltage clamp using a potassium gluconate-based internal (in mM: 135 K-gluconate, 5 NaCl, 2 MgCl₂, 10 HEPES, 0.6 EGTA, 4 Na₂ATP, 0.4 Na₂GTP, pH 7.3, 285–290 mosmol/kgH₂O). Cells were voltage-clamped at −80 mV in the presence of 25 μM picrotoxin (GABA-A receptor antagonist) to pharmacologically isolate EPSCs. Spontaneous inhibitory postsynaptic currents (sIPSCs) were pharmacologically isolated by adding kynurenic acid (3 mM) to the ACSF to block AMPA and N-methyl-d-aspartate (NMDA) receptor-dependent postsynaptic currents. Cells were clamped at −70 mV and recorded using a potassium-chloride gluconate-based intracellular solution (in mM: 80 KCl, 70 K-gluconate, 10 HEPES, 1 EGTA, 4 Na₂ATP, 0.4 Na₂GTP, pH 7.2, 285–290 mosmol/kgH₂O with 1 mg/mL QX-314-bromide).

TNF-α Incubation

Slices were preincubated in either ACSF alone or ACSF + TNF-α (100 ng/mL) for 1 h before transferring to a submerged recording chamber, similar to that in Refs. 27 and 29. For whole cell recordings, slices were continuously perfused with either oxygenated ACSF (control group) or ACSF + TNF-α (TNF-α group).

Drugs

All chemicals used for slice electrophysiology were obtained from either Tocris Bioscience (Minneapolis) or Abcam (Cambridge, UK). Recombinant human TNF-α protein was purchased from R&D Systems (Minneapolis) and stored at −20°C.

Data and Statistical Analysis

No statistical method was used to predetermine sample size, and no blinding procedures were used. Age-matched mice were randomly assigned to both groups. Differences in various electrophysiological measures were analyzed using Clampfit 10.7 (Molecular Devices, Sunnyvale, CA; RRID:SCR_011323) and compared between the control and TNF-α groups. Frequency, amplitude, and kinetics of E/IPSCs were analyzed and visually confirmed using either Clampfit 10.7 or Easy Electrophysiology (RRID:SCR_021190, v2.3.2) (34). For comparisons between two groups, P values were calculated using a standard unpaired t test. If the condition of equal variances was not met, Welch’s correction was used. Grubbs’ test was used for identifying outliers. Distributions of sE/IPSC interevent intervals (IEIs) and amplitudes were statistically analyzed by binning events into 50 ms (interevent interval) and 5 pA (amplitude) bins (35). For both groups, cumulative frequency distributions were plotted for each neuron, and comparisons between control and TNF-α-treated cumulative distributions were made using the two-tailed Mann–Whitney test. Between-group differences in the spike frequency fired across a range of current steps were analyzed using repeated-measures ANOVAs (treatment × current injection) with the Geisser–Greenhouse correction. All data are expressed as means ± SE. P values ≤0.05 were considered significant. All statistical analysis was performed using GraphPad Prism v.9 (La Jolla, CA; RRID:SCR_002798).

RESULTS

TNF-α Increases the Excitability of vlPAG GABA Neurons

To determine the effects of TNF-α on the excitability of vlPAG GABA neurons, we conducted whole cell recordings from vGAT neurons in vGAT-L10a reporter mice (Fig. 1A; 4–5 mice per group). Intrinsic properties of vGAT neurons were calculated from data generated in voltage clamp using steps from −70 to −80 mV. The average capacitance was generally <30 pF and did not differ between the two groups(Fig. 1B; n = 12 cells in the control group; n = 8 cells in the TNF-α group; P = 0.2301; unpaired t test). We found that the average input resistance in the TNF-α group was slightly higher than the control group but was not statistically different (Fig. 1C; P = 0.0808; unpaired t test). Next, we performed voltage-clamp experiments to evaluate the relationship between holding current and command potential between −70 and −120 mV with 10 mV current steps (Fig. 1D). A two-way repeated-measures ANOVA with Geisser–Greenhouse correction revealed a significant group × voltage interaction [F(5,90) = 3.316; P = 0.0085], along with a main effect of voltage [F(1.068,19.22) = 57.43; P ≤ 0.0001] and a strong trend toward main effect of group [F(1,18) = 4.370; P = 0.0510]. To further investigate the effect of TNF-α on neuronal excitability, we assessed excitability parameters in current-clamp mode. All measures of excitability were taken at −70 mV to normalize for intercell variability in resting membrane potential (RMP). The action potential threshold and the amount of current required to fire an action potential (rheobase) were assessed through a ramp protocol of 120 pA/1 s. TNF-α significantly reduced the rheobase compared with the control group (Fig. 1, E and F; P = 0.0296; unpaired t test with Welch’s correction) with no change in the action potential threshold (Fig. 1G; P = 0.640; unpaired t test). Next, we measured the action potential frequency across a range of current steps (0–200 pA for 500 ms, at an increment of 20 pA). There was no significant interaction between the two groups and AP frequency as revealed by repeated-measures two-way ANOVA with Geisser–Greenhouse correction [Fig. 1, H and I; F(10,180) = 0.1885, P = 0.9969 for group × current interaction; F(1,18) = 0.3953, P = 0.5374 for main effect of group; F(1.848,33.26) = 44.49, P < 0.0001 for main effect of current]. In addition, TNF-α failed to alter action potential kinetics of vGAT neurons. Specifically, both groups were similar with respect to AP latency (Fig. 1J; P = 0.844; unpaired t test), AP height (Fig. 1K; P = 0.1528; unpaired t test), AP half-width (Fig. 1L; P = 0.2032; unpaired t test), AHP (Fig. 1M; P = 0.8328; unpaired t test), depolarization rate (Fig. 1N; P = 0.5875; unpaired t test), and repolarization rate (Fig. 1O; P = 0.1993; unpaired t test). Together, these results suggest that TNF-α incubation results in alterations of intrinsic properties of vGAT neurons, potentially increasing the excitability of these neurons.

Figure 1.

TNF-α incubation alters intrinsic properties of ventrolateral periaqueductal gray (vlPAG) GABA neurons. A: schematics for whole cell recordings from vlPAG vGAT neurons (n = 8–12 cells from 4 to 5 mice per group) following preincubation of slices in TNF-α (100 ng/mL). B and C: there was no change in cell capacitance or input resistance between the two groups. D: TNF-α resulted in a reduction in current density in response to linear step changes in voltage from −70 to −120 mV. E: representative data obtained from vGAT neurons in response to a 120 pA/s current ramp while injecting a constant current to hold the cells at −70 mV. The minimum current required to fire an action potential (rheobase) was reduced following preincubation in TNF-α (F) without any changes (G) in action potential (AP) threshold. H: representative traces of action potentials fired in response to a step protocol of increased current steps of 20 pA/500 ms while holding the cells at −70 mV. I: there was no significant interaction between the frequency of APs in response to a graded current injection. TNF-α did not alter action potential kinetics as measured by latency (J), average AP height (K), AP half-width (L), afterhyperpolarization (AHP; M), depolarization rate (N), and repolarization rate (O). Data are expressed as means ± SE.*P < 0.05. depol., depolarization; repol., repolarization.

TNF-α Decreases Neuronal Excitability of vlPAG Dopamine Neurons

We next asked whether TNF-α might also modulate the excitability of vlPAG DA neurons. vlPAG DA neurons were identified using TH-eGFP reporter mice (Fig. 2A; 4–6 mice per group). There were no significant differences between the two groups with regard to capacitance (Fig. 2B; n = 10 cells per group; P = 0.1384; unpaired t test with Welch’s correction), input resistance (Fig. 2C; P = 0.2339; unpaired t test), and current-voltage relationship {Fig. 2D; two-way repeated-measures ANOVA with Geisser–Greenhouse correction revealed a significant effect of voltage [F(1.136,20.44) = 89.17; P ≤ 0.0001] but no main effect of group [F(1,18) = 0.0018; P = 0.9668] or interaction [F(5,90) = 0.017; P = 0.9999]}. Interestingly, we observed a significant increase in rheobase (Fig. 2, E and F; P = 0.0291; unpaired t test) without any change in AP threshold (Fig. 2G; P = 0.0759; unpaired t test with Welch’s correction). Furthermore, we found a significant reduction in action potential frequency (Fig. 2, H and I). A two-way repeated-measures ANOVA with Geisser–Greenhouse correction revealed a significant interaction between the two groups and the AP frequency [F(9,162) = 2.217; P = 0.0234], main effect of group [F(1,18) = 5.675; P = 0.0284], and main effect of current [F(1.310,23.58) = 23.56; P ≤ 0.0001]. Similar to vGAT neurons, there was no impact of TNF-α on AP kinetics as measured by AP latency (Fig. 2J; P = 0.7193; unpaired t test), AP height (Fig. 2K; P = 0.1714; unpaired t test), AP half-width (Fig. 2L; P = 0.1805; unpaired t test), AHP (Fig. 2M; P = 0.4687; unpaired t test), depolarization rate (Fig. 2N; P = 0.2633; unpaired t test), and repolarization rate (Fig. 2O; P = 0.4493; unpaired t test). These results collectively show that TNF-α reduces the excitability of vlPAG DA neurons suggesting differential modulation of two distinct subpopulations in the vlPAG.

Figure 2.

TNF-α incubation decreases excitability of ventrolateral periaqueductal gray (vlPAG) dopamine (DA) neurons. A: schematics for whole cell recordings from vlPAG DA (n = 10 cells from 4 to 6 mice per group) following preincubation of slices in TNF-α (100 ng/mL). B–D: there was no change in cell capacitance, input resistance, or current-voltage relationship between the two groups. E: representative traces of vlPAG DA neurons in response to a 120 pA/s current ramp while injecting a constant current to hold the cells at −70 mV. F: TNF-α significantly increased the rheobase without altering the action potential (AP) threshold (G). H: representative traces of action potentials fired across a range of current steps while injecting a constant current to hold the cells at −70 mV. I: there was a significant interaction between the frequency of action potential fired and TNF-α exposure. Similar to vlPAG vGAT neurons, TNF-α did not alter action potential kinetics of vlPAG DA neurons (J–O). Data are expressed as means ± SE. *P < 0.05. AHP, afterhyperpolarization; depol., depolarization; repol., repolarization; TH, tyrosine hydroxylase.

TNF-α Reduces Excitatory Neurotransmission on vlPAG GABA Neurons

TNF-α is known to have direct effects on glutamate transmission and has been shown to alter the expression of AMPA receptors on synapses (26, 27, 36). Therefore, we evaluated whether TNF-α influences glutamatergic synaptic transmission on vGAT (Fig. 3) and DA neurons (Fig. 4) in the vlPAG. We found that TNF-α decreased spontaneous excitatory synaptic transmission onto vlPAG GABA neurons (n = 14 cells from 4 to 5 mice per group). Specifically, we observed a strong trend toward reduction in mean sEPSC frequency in the TNF-α group (Fig. 3C; P = 0.0531; unpaired t test). There was also a nonsignificant trend toward a rightward shift of the cumulative distribution of interevents (Fig. 3B; P = 0.0935; Mann–Whitney test). Similar to sEPSC frequency, there was a strong trend toward an increase in the mean amplitude of sEPSCs in the TNF-α group (Fig. 3E; P = 0.0671; unpaired t test with Welch’s correction). Distribution analysis of sEPSC amplitudes revealed a significant leftward shift in the TNF-α group (Fig. 3D; P = 0.0360; Mann–Whitney test), indicative of a reduction in the amplitude of events. Thus, TNF-α incubation reduces excitatory synaptic drive on vlPAG GABA neurons, potentially through both pre- and postsynaptic mechanisms.

Figure 3.

TNF-α incubation reduces spontaneous excitatory transmission on ventrolateral periaqueductal gray (vlPAG) GABA neurons. A: representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) from vlPAG vGAT neurons (n = 14 cells from 5 mice per group) recorded either in artificial cerebral spinal fluid (ACSF) or in the presence of TNF-α (100 ng/mL). sEPSCs were recorded in the presence of picrotoxin (25 μM). A reduction in sEPSC frequency (C) was accompanied by a trend toward rightward shift in the distribution of sEPSC interevent intervals (IEIs; B) following preincubation of slices in TNF-α. D and E: there was a decrease in sEPSC amplitude onto vlPAG vGAT neurons in the presence of TNF-α along with a significant leftward shift in the distribution of amplitude of events. Data are expressed as means ± SE. *P < 0.05.

Figure 4.

TNF-α incubation reduces spontaneous excitatory synaptic transmission in ventrolateral periaqueductal gray (vlPAG) dopamine (DA) neurons. A: representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) from vlPAG DA neurons (n = 9–11 cells from 4 to 5 mice per group) recorded either in artificial cerebral spinal fluid (ACSF) or in the presence of TNF-α (100 ng/mL). sEPSCs were recorded in the presence of picrotoxin (25 μM). B: TNF-α induced a rightward shift in the distribution of sEPSC interevent intervals (IEIs), suggesting a lower frequency of glutamatergic events without any differences in average sEPSC frequency (C). No change in either the distribution (D) or the mean amplitude of sEPSC events (E) in vlPAG DA neurons. Data are expressed as means ± SE.*P < 0.05. TH, tyrosine hydroxylase.

TNF-α Reduces Excitatory Neurotransmission on vlPAG Dopamine Neurons

We next examined whether TNF-α alters glutamatergic synaptic transmission onto vlPAG DA neurons (Fig. 4; n = 14 cells from 5 mice per group). Preincubation in TNF-α resulted in a significant rightward shift in the interevent distribution to longer intervals (Fig. 4B; P = 0.0001; Mann–Whitney test), suggesting that the reduced frequency of sEPSCs in TNF-α group is driven by a higher proportion of events occurring with long IEIs, although no differences were observed in mean sEPSC frequency (Fig. 4C; P = 0.2167; unpaired t test). In addition, no change was observed in either distribution (Fig. 4D; P = 0.7515; Mann–Whitney test) or the mean amplitude of sEPSCs (Fig. 4E; P = 0.2462; unpaired t test). These findings indicate that TNF-α has a modest but significant impact on spontaneous glutamatergic transmission on vlPAG DA neurons through presynaptic but not postsynaptic function.

TNF-α Has a Mild Effect on Inhibitory Neurotransmission on vlPAG GABA Neurons

Prior studies have shown that TNF-α can differentially regulate the trafficking of AMPA and GABA receptors (26). In the hippocampus, TNF-α decreases inhibitory synaptic strength via endocytosis of GABA-A receptors to modulate neuronal excitation-inhibition balance (26). Given this bidirectional effect of TNF-α on neuronal circuit homeostasis, we sought to determine whether TNF-α could also modulate GABAergic transmission within the vlPAG. We recorded from vlPAG GABA neurons in the presence of kynurenic acid to isolate spontaneous IPSCs (Fig. 5A; n = 12–13 cells from 3 to 4 mice per group). Surprisingly, TNF-α had no effect on mean sIPSC frequency (Fig. 5C; P = 0.5345; unpaired t test) and mean sIPSC amplitude (Fig. 5E; P = 0.5809; unpaired t test with Welch’s correction). Interestingly, we observed differences in the cumulative distribution of amplitudes, suggesting an increase in the percentage of larger amplitude events (Fig. 5D; P = 0.0083; Mann–Whitney test) but not in the distribution of IEIs (Fig. 5B; P = 0.1005; Mann–Whitney test). These findings exhibit that TNF-α differentially impacts excitatory and inhibitory transmission on vlPAG GABA neurons.

Figure 5.

TNF-α incubation has a mild effect on spontaneous inhibitory synaptic transmission on ventrolateral periaqueductal gray (vlPAG) GABA neurons. A: representative traces of spontaneous inhibitory postsynaptic currents (sIPSCs) from vlPAG GABA neurons (n = 12–13 cells from 3 to 4 mice per group) recorded either in artificial cerebral spinal fluid (ACSF) or in the presence of TNF-α (100 ng/mL). sIPSCs were recorded in the presence of kynurenic acid (3 mM). Cumulative probability plots showing no change in the distribution of sIPSC interevent intervals (IEIs, B) and mean frequency (C). There were no between-group differences in mean amplitude (E), but a significant rightward shift in the distribution of amplitude of sIPSC events (D), suggesting an increase in the number of large amplitude GABAergic events in the presence of TNF-α. Data are expressed as means ± SE. *P < 0.05.

TNF-α Has No Effect on Inhibitory Neurotransmission on vlPAG Dopamine Neurons

Evidence from the literature on synaptic connectivity within the PAG suggests that RVM-projecting PAG neurons are tonically inhibited by local GABA neurons (5, 37). Based on the effects of TNF-α incubation on both excitability and synaptic transmission onto vlPAG GABA neurons, we hypothesized that TNF-α would also alter inhibitory drive onto vlPAG DA neurons. To investigate this, we isolated spontaneous IPSCs onto DA neurons in the vlPAG (Fig. 6A; n = 9–12 cells from 3 to 4 mice per group). Contrary to our hypothesis, TNF-α did not affect inhibitory transmission onto DA neurons. Specifically, we observed no differences in either cumulative interevent distribution of sIPSCs (Fig. 6B; P = 0.1486; Mann–Whitney test) or mean frequency of sIPSCs (Fig. 6C; P = 0.2580; unpaired t test). Similarly, TNF-α had no effect on either distribution (Fig. 6D; P = 0.6366; Mann–Whitney test) or the mean sIPSC amplitude (Fig. 6E; P = 0.5828; unpaired t test). Taken together, our results suggest that TNF-α selectively modulates spontaneous glutamate release on DA neurons without affecting action potential-dependent GABA release.

Figure 6.

TNF-α incubation does not affect spontaneous inhibitory synaptic transmission on ventrolateral periaqueductal gray (vlPAG) dopamine (DA) neurons. A: representative traces of spontaneous inhibitory postsynaptic currents (sIPSCs) from vlPAG DA neurons (n = 9–12 cells from 3 to 4 mice per group) recorded either in artificial cerebral spinal fluid (ACSF) or in the presence of TNF-α (100 ng/mL). sIPSCs were recorded in the presence of kynurenic acid (3 mM). No change in cumulative distribution plots comparing interevent intervals (IEIs; B) and amplitude of events (D) between control and TNF-α mice. There were no between-group differences in sIPSC parameters: mean frequency (C) and mean amplitude (E) in the presence of TNF-α. Data are expressed as means ± SE. TH, tyrosine hydroxylase.

DISCUSSION

The present set of experiments tested the neuromodulatory effects of human recombinant TNF-α on two distinct neuronal populations in the ventrolateral division of PAG. To our knowledge, our work is the first to examine TNF-α-mediated synaptic plasticity in the ventrolateral PAG of female mice. Here, we report that incubation of slices in TNF-α altered the intrinsic properties of vlPAG GABA neurons resulting in increased excitability but reduced excitatory drive onto these neurons. Interestingly, TNF-α altered inhibitory transmission by increasing the amplitude of GABAergic events on vlPAG GABA neurons without impacting the frequency. Conversely, TNF-α significantly decreased the excitability of vlPAG DA neurons. In addition, TNF-α lowered excitatory drive onto vlPAG DA neurons without altering inhibitory transmission. Thus, our data highlight cell-type-specific synaptic alterations induced by TNF-α within the vlPAG, a critical nucleus implicated in encoding divergent behaviors required for survival.

TNF-α is a homotrimer protein of 17 kDa produced centrally during various inflammatory pathologies and can signal through TNF receptors 1 (TNF-R1) and 2 (TNF-R2) (38). In addition to its role in multiple pathologies, it can also modulate neuronal function at low physiological levels (28). Evidence from the literature suggests that TNF-α is capable of modulating both presynaptic and postsynaptic signaling in neurons. In both rat and mouse hippocampus, TNF-α (across a range of concentrations) increases AMPA/NMDA ratio and glutamate release probability through surface trafficking of AMPA receptors (26, 36, 39). Interestingly, there are known regional differences in TNF-α modulation of neuronal excitability, since in the dorsolateral striatum, TNF-α drives the internalization of AMPA receptors and reduces synaptic strength (27). Similarly, in lateral habenula, TNF-α mediates a reduction in AMPA/NMDA ratio that drives reduced sociability associated with morphine withdrawal (40). Here, we demonstrate that even within the same brain region, TNF-α differentially modulates neuronal function in a cell-type-specific manner.

We found incubation of vlPAG slices in TNF-α (100 ng/mL) resulted in decreased firing rate of DA neurons while lowering the rheobase of GABA neurons. The mechanisms through which TNF-α alters the intrinsic excitability of these neurons remain unknown. It is possible that TNF-α may influence a nonselective cation channel. Previous studies in dorsal root ganglion (41), cortical neurons (42), and subfornical organ (43) suggest a potential involvement of voltage-gated sodium and calcium channels in driving TNF-mediated neuronal excitability. A recent study in the cerebellum showed TNF-R1-mediated downregulation of SK channels as a potential mechanism of increased excitability (44). Although these studies have identified possible ion channels involved in TNF-α-mediated hyperexcitability, not much is known about the mechanism behind TNF-α-driven hypoexcitability. Future studies examining the mechanism behind the reduced excitability induced by TNF-α may provide insight into the complex role of central TNF-α signaling.

Our study also indicates a reduction in excitatory synaptic drive onto both GABA and DA neurons. Comparing cumulative probability plots of amplitudes and interevent intervals is a more rigorous approach than simply examining mean amplitude and frequency because it compares the entire distribution; therefore, we compared the mean values and the cumulative distribution of sE/IPSCs. In vlPAG GABA neurons, there was a strong trend toward a decrease in both sEPSC frequency and amplitude, along with a shift in the distribution of sEPSC amplitudes. Curiously, despite a reduction in glutamatergic transmission on GABA neurons, we observed an increase in excitability. One plausible explanation could be that TNF-α-mediated change in glutamatergic transmission induces a homeostatic shift in excitability. TNF-α also had a modest effect on sIPSC amplitude on vlPAG GABA neurons without affecting frequency. Effects on amplitude are generally attributed to postsynaptic mechanisms. Surprisingly, TNF-α did not affect inhibitory transmission onto DA neurons but lowered the percentage of glutamatergic events. Our findings are consistent with the known role of TNF-α in altering pre- and postsynaptic transmission, but it is beyond our scope to address it further.

Conclusions

These experiments describe the impact of exogenous TNF-α on PAG neuronal activity. Constitutive TNF-α levels in the brain are thought to be low (in the picomolar range) and controlled by local glial cells (39). As such, the use of a nonphysiological concentration of TNF-α (100 ng/mL) is a limitation of our study and should be considered while interpreting these findings. However, pathological brain states can induce massive production of proinflammatory cytokines (45–47); thus, incubation of slices in saturating levels of TNF-α could potentially reflect plasticity associated with diseased conditions. More in-depth work is needed to demonstrate whether these findings translate under physiological concentrations of TNF-α. In addition, it is crucial to note that in our study, we used recombinant human TNF-α rather than murine TNF-α. Although murine TNF-R1 has a similar affinity for both human and murine TNF-α, mouse TNF-R2 has a higher specificity for murine TNF-α (48).

We decided to focus on TNF-α-mediated synaptic alterations in female mice, since most studies use male subjects, limiting our understanding of mechanistic variations in females. Although it is beyond the scope of the present study, it will be intriguing to determine whether there are sex-related differences in TNF-α modulation of the PAG circuitry. Overall, our findings suggest a potential mechanism whereby pathophysiological concentrations of TNF-α could lead to altered PAG function. Given the known sex differences in pain sensitivity and accumulating evidence pointing toward activated microglia in female PAG as a contributor to sex differences in morphine analgesia, this study provides a mechanistic framework that may provide insight into sex-dependent modulation of pain. As work from our laboratory and others have demonstrated, these neurons play an opposing role in pain modulation. Either activation of GABA neurons or inhibition of DA neurons results in increased nociception. Therefore, it is plausible that in females, chronic pain results in increased activation of microglia and release of pathophysiological levels of TNF-α, which suppresses the neuronal activity of vlPAG DA neurons, with the result being increased nociception. However, chronic pain impacts both pro- and anti-inflammatory cytokines and can lead to structural and functional changes in neuroimmune signaling. As such, the effect of endogenous TNF-α on the vlPAG neuronal circuit could look very different from its acute effects. Future work is needed to determine the complex mechanisms by which TNF-α signaling may be upregulated in this region and the behavioral consequences of selective manipulation of PAG-specific TNF-α signaling.

GRANTS

This work was funded by National Institute on Alcohol Abuse and Alcoholism (NIAAA) Grants R01 AA019454 (to T.L.K.), U01 P60 AA011605 (to T.L.K.), and R21 AA027460 (to T.L.K.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

D.P. and T.L.K. conceived and designed research; D.P. performed experiments; D.P. analyzed data; D.P. and T.L.K. interpreted results of experiments; D.P. prepared figures; D.P. drafted manuscript; D.P. and T.L.K. edited and revised manuscript; D.P. and T.L.K. approved final version of manuscript.