Ventral pallidum GABA neuron inhibition augments context-appropriate defensive responses to learned threat cues methods

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    "name": "Ventral pallidum GABA neuron inhibition augments context-appropriate defensive responses to learned threat cues methods",
    "description": "Protocol for Ventral pallidum GABA neuron inhibition augments context-appropriate defensive responses to learned threat cues methods",
    "step": [
      {
        "@type": "HowToStep",
        "position": 1,
        "name": "Stereotaxic surgery",
        "text": "GAD1:Cre and WT rats were anesthetized with ketamine (56.5 mg/kg), xylazine (8.7 mg/kg), and meloxicam (1 mg/kg), and an AAV2 vector containing a Cre-dependent, mCherry-tagged inhibitory DREADD (hSyn-DIO-hM4Di-mCherry; titer: 5 × 10 12 vg/mL; Addgene Cat. #:44362-AAV2) was injected bilaterally with a glass pipette into VP (∼300nl/hemisphere), at bregma-relative coordinates (mm): AP: 0.0, ML: ±2.0, DV: -8.0 ( ), as previously described ( ). Rats were given at least 21 days to recover from surgery and allow for viral expression to occur before commencing behavioral testing."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "CNO preparation and injection",
        "text": "Clozapine-N-oxide (CNO) was obtained from the NIDA Drug Supply Program, stored at 4 °C in powder aliquots with desiccant, and protected from light. CNO (5 mg/kg/ml) was dissolved daily for IP injection in 5 % dimethyl sulfoxide, then diluted in 0.9 % saline to dose. Vehicle (VEH) or CNO was injected IP 30min prior to commencement of all behavioral tests (,,;,;; )."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Behavioral methods",
        "text": "At least 4 weeks after surgery, rats were handled for 5min on 5 consecutive days prior to behavioral training. Male and female GAD1:Cre (n = 24) and WT rats (n = 11) first underwent testing for shock probe fear expression (2 expression tests, counterbalanced VEH and CNO), then they were tested for expression of freezing elicited by 0.75 mA foot shock-paired cues in either VP GABA -Inhibited (GAD1:Cre + CNO; n = 16) or Control-treated (GAD1:Cre + VEH: n = 8; or WT + CNO: n = 11) rats in a between-subjects design. Other male and female rats first underwent testing for expression of auditory cue fear elicited by 0.3 mA foot shock-paired cues after VP GABA inhibition (GAD1:Cre + CNO: n = 16) or Control treatment (GAD1:Cre + VEH: n = 11; WT + CNO..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "Shock probe test general methods",
        "text": "All shock probe sessions were videorecorded for later quantification of behaviors, as described in Supplemental Methods. Rats were first habituated for 30min on two days to a specific plexiglass tub cage filled with corncob bedding (5 cm height from cage bottom). They were then returned to the chamber on the following day for a shock probe fear acquisition session. The chamber now had a copper-wrapped plastic probe inserted through a 1 cm diameter hole in the wall located 8 cm above the cage floor. The probe delivered a 1.5 mA shock when touched (Coulbourn Precision Animal Shocker). Rats were placed in the opposite end of the cage from the shock probe, and the shock probe acquisition session continued for 20min after the first rat-initiated shock (;;; ). The following day, rats underwent a shock probe fear expression session. The chamber was configured id..."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "VP GABA inhibition effects on shock probe fear expression",
        "text": "To test effects of VP GABA inhibition on expression of defensive responses directed at a threatening probe which previously delivered shock, rats received VEH or CNO injection, then were placed into the chamber with the inactive probe for 20min. 48h later, these rats underwent a short (5min) shock probe fear \"re-acquisition\" session, where the probe was re-electrified, and on which all rats were shocked again at least once. The next day, they underwent a second 20min shock probe fear expression test, conducted identically to the first, other than that they received the alternative injection (VEH/CNO) prior to the session. Rats received VEH and CNO treatments in counterbalanced order in these two shock probe fear expression tests, and defensive burying behaviors were statistically equivalent in rats that received VEH on the first versus the second shock probe fear expressio..."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "VP GABA inhibition effects on shock probe fear acquisition",
        "text": "To test effects of VP GABA inhibition on initial learning about the shock-delivering probe, rats received either VEH or CNO injections prior to a shock probe fear acquisition session, following prior testing of auditory cue fear expression. 48hrs later, these rats were returned to the chamber with the now-inactive probe, without further injections, for a 20min shock probe fear expression test."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "VP GABA inhibition effects on auditory cue fear expression",
        "text": "All sessions were videorecorded for later quantification of behaviors, as described in Supplemental Methods. Auditory cue fear conditioning training and testing took place in Med Associates rat operant chambers (30.5x24 × 21cm; St. Albans, VT), housed within sound-attenuating boxes. Chambers had a peppermint scent (McCormick) during cue/shock training, and standard metal bars that could deliver foot shock composed the chamber floor. On these 8.5min training sessions, rats were placed into the novel chamber, and a house light turned on for a 3min baseline period. Then, a 20sec auditory cue (continuous white noise: 80 dB) was played, which was followed immediately by either a low (0.3 mA) or high (0.75 mA) intensity foot shock, delivered for 2sec. Three cue/shock pairings were made during this training session, with 90sec elapsing between each. 48hrs l..."
      },
      {
        "@type": "HowToStep",
        "position": 8,
        "name": "VP GABA inhibition effects on probe threat-elicited neural activity",
        "text": "Male GAD1:Cre rats with VP GABA DREADDs were habituated and trained as described in Section on the shock probe fear acquisition procedure, except that about half the rats underwent the 20min training session with a probe that did not deliver shock. The following day, rats were injected with either VEH or CNO, then they underwent a shock probe fear expression session as described in section (probes did not deliver shock on this session). Rats were returned to their home cages for 90min after the session, then they were perfused and brains were prepared for Fos staining and quantification. One rat in the No-Threat + VEH group did not express sufficient mCherry in VP GABA cells for quantification, so it was excluded from mCherry-based VP Fos analyses, but this rat's data was included in analyses of Fos in VP ChAT neurons, LHb, and MDT, since these analyses do not require identi..."
      }
    ],
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    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "Ventral pallidum GABA neuron inhibition augments context-appropriate defensive responses to learned threat cues",
      "datePublished": "2026",
      "author": [
        {
          "@type": "Person",
          "name": "Erica M. Ramirez"
        },
        {
          "@type": "Person",
          "name": "Maricela X. Martinez"
        },
        {
          "@type": "Person",
          "name": "Ryan K. Rokerya"
        },
        {
          "@type": "Person",
          "name": "Vanessa Alizo Vera"
        },
        {
          "@type": "Person",
          "name": "Christina M. Ruiz"
        },
        {
          "@type": "Person",
          "name": "Mitchell R. Farrell"
        },
        {
          "@type": "Person",
          "name": "Shreeya A. Walawalkar"
        },
        {
          "@type": "Person",
          "name": "Hazael Ramirez-Ramirez"
        },
        {
          "@type": "Person",
          "name": "Grace J. Kollman"
        },
        {
          "@type": "Person",
          "name": "Stephen V. Mahler"
        }
      ],
      "identifier": "10.1016/j.ynstr.2026.100781"
    }
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