The main findings of the present study are that STZ animals show a persistent impairment in extinguishing a fear memory associated with delayed fear sensitization demonstrated by the increased anxiety-like response. Interestingly, a single injection of PGB in these STZ animals just before fear extinction training was able to facilitate fear extinction memory and alleviate anxiety-like behavior. These effects were observed in short and long-term (9 days later) periods. In addition, our data indicate that the beneficial effects induced by PGB may be related, at least partially, to its antioxidant activity demonstrated in the HIP and PFC.
As already observed (Ikeda et al. 2015, 2021; de Souza et al. 2019; Ribeiro et al. 2020), our data confirmed that STZ animals present a greater fear response and a difficulty in extinguishing fear memory when compared to NGL animals (Fig. 1), indicating an overconsolidation of this fear memory. However, this is the first study to show the persistence of this fear memory in animals with induced-T1DM when re-exposed in the same context after 1 week later (Fig. 1C) indicating how strong or dysfunctional are the mechanisms associated with this type of learning/memory. In this same direction, clinical studies point out a higher prevalence of PTSD in adults with T1DM (Renna et al. 2016), along with a positive association between the trauma of war and an increase in the incidence of T1DM in children and adolescents (Zung et al. 2012). The presence of severe and moderate PTSD symptoms has also been demonstrated in children (8-18 years) diagnosed with T1DM (Auxéméry 2012).
Regarding anxiety, STZ animals (not exposed to the same context of CFC) showed a more exacerbated anxiety-like response (decrease in the time spent and the number of entries in the open arms and decrease in the head dipping frequency) when compared to NGL animals - conditioned or not (Fig. 2A-C). These findings reinforce previous reports showing that these induced-T1DM animals present a more pronounced anxious-like behavior when compared to NGL animals (de Morais et al. 2014; Gambeta et al. 2016; Rebolledo-Solleiro et al. 2016; de Souza et al. 2019). Concerning NGL animals, even after acquiring extinction memory, they exhibited a more pronounced later anxiety-like behavior than non-conditioned NGL animals (Fig, 2A-D). This result is not surprising since other evidence show that previous stress may induce a response related to a fear sensitization or even to a generalization of fear memory, which depends on several aspects, such as external factors including the type and intensity of aversive stimulation, early-life stress, as well as the saliency of particular elements in the environment (Korte e De Boer 2003; Asok et al. 2019).
The next block of experiments was designed based on two main premises: 1. Induced-T1DM rats have an overconsolidation of fear memory, in addition to demonstrating the persistence of this memory and anxiety-like behavior, and 2. the use of PGB is approved for diabetic neuropathic pain, and its use has been successfully employed for treating more severe states of anxiety disorders and also PTSD (Pande et al. 2003; Pohl et al. 2005; Garakani et al. 2020). Our results revealed that all doses of PGB were able to decrease the freezing time when submitted to the extinction tests 1 and 2 (Fig. 3B-C), indicating that the drug facilitated the acquisition of this extinction memory, being this effect of long-lasting. Interestingly, the highest dose of PGB (300 mg/kg) was able to restore the behavior of the STZ animals, equating it with NGL animals (Fig. 3B-C). Regarding the delayed effect on the anxiety-like response, PGB (two highest doses) restored the most expressive anxious-like behavior of STZ animals, while only the highest dose (300 mg/kg) was able to induce a significant improvement in the behavior of head dipping (Fig. 4C). That is, the beneficial effect of PGB on fear memory seems not to be dissociated from its anxiolytic-like effect.
Although we found a sedative effect caused by the acute injection of PGB in the dose of 300 mg/kg (during extinction training) as demonstrated in the OFT (Fig. 5), the animals showed significant and consistent effects on fear extinction memory (Fig. 3B-C) and anxiety-like responses (Fig. 4A-C) in the subsequent tests in which they were not under this acute effect of PGB, once the half-life of PGB is around 6 hours (Buoli et al. 2017). Thus, this sedative effect during the extinction training session did not impair the acquisition of fear extinction memory and the anxiolytic-like effect. In addition, lower doses of PGB (30 mg/kg and 100 mg/kg) also showed effects in reducing freezing and improving anxiety, without inducing a sedative effect (Fig. 5).
At this point, we could mention as a limitation of the present study the fact that the best effects have been achieved after the administration of the highest dose of PGB. In this sense, it is known that the clinical efficacy dosage of PGB in GAD, for example, is 150-600 mg/day, and the frequently reported side effects (drowsiness and dizziness) are dose-dependent (Baldwin et al. 2015). In double-blind, placebo-controlled trials the adverse events during treatment with PGB were considered mild and moderate (Mann et al. 2014). However, in a recent meta-analysis study, Onakpoya et al. (2019) demonstrated that despite being effective for the treatment of diseases such as diabetic peripheral neuropathy and post-herpetic neuralgia, many patients discontinue the treatment due to adverse effects. Furthermore, some studies have reported cases of increased heart failure, peripheral edema, and also Addiction. All these effects seem to be related to the chronic use of PGB (Robert Lee Page et al. 2008; Grosshans et al. 2010; Dobrea et al. 2012; Gahr et al. 2013; Aldemir et al. 2015). According to Buoli et al. (2017) at the highest doses of PGB, there is an increase of the enzyme responsible for the synthesis of GABA (L-Glutamic acid decarboxylase), and although PGB does not directly bind to GABA-A or GABA-B receptors, these receptors may be more active due to increased GABA levels (Buoli et al. 2017). Also, at the highest dose, PGB may bind to α2δ-2 subunit, in addition to the α2δ-1 subunit, which in the brain is concentrated in the cerebellum and partially correlates with GABAergic neurons (Barclay et al. 2001; Li et al. 2011). Thus, the adverse effects observed at the highest doses of PGB may be related preferentially to the unspecific effects of PGB on other sites of action. Here, if we think in translational terms, it is important to bear in mind what would be the risk and benefit of the treatment for the patient, and this response should be linked to the type of treatment and the dosage used. For example, in the present study, despite the most effective dose being the highest dose, which could cause a series of unwanted effects in continuous treatment, the PGB application was unique and possibly not associated with the consequences of adverse effects.
The International guidelines consider PGB along with the selective serotonin reuptake inhibitors (paroxetine, sertraline, escitalopram), and serotonin noradrenaline reuptake inhibitors as first-line options for treating GAD (Pande et al. 2003; Buoli et al. 2017; Greenblatt e Greenblatt 2018). Moreover, both open-label and randomized, double-blind, placebo-controlled studies have demonstrated the efficacy of PGB, being in many of these studies superior to antidepressants and benzodiazepines depending on the parameter evaluated (for a review see Frampton 2014; Baldwin et al. 2015 and Buoli et al. 2017). Regarding treating PTSD with PGB, although studies are still limited, a retrospective clinical study of 290 burned service members revealed that PGB or gabapentin did not affect the development of PTSD (Fowler et al. 2012). However, in a randomized clinical trial, Baniasadi et al. (2014) demonstrated that PGB was able to reduce emotional symptoms of PTSD related to combat in a group of 18 male patients (non-diabetics) who received 300 mg/day of PGB for a period of 6 weeks.
In preclinical studies, it is evident that depending on the experimental protocol, such as the condition of the animal (including the animal´s condition or the type of stress), doses used, and the time of treatment and its duration, the results are quite different. For example, Zohar et al. (2008) showed, in non-diabetic animals, that PGB (at the same doses used in the present study) induced beneficial short-term effects on behavioral responses (anxiety-like response) in animals exposed to traumatic stimuli (predator urine scent). However, the anxiolytic-like effect of PGB was observed only in these animals pre-exposed to predator urine scent, and not in that one’s not pre-exposed. Also, these effects were not observed after 30 days of the PGB injection. Differently, Valdivieso et al. (2018) did not observe any beneficial effect after PGB treatment on anxiety-like behavior in previously stressed (restraint and tail shock) non-diabetic animals. Nevertheless, they used a smaller dose of PGB (10 mg/kg).
Of particular interest for the present study, all these changes related to an exacerbated behavioral response observed in induced-T1DM animals have been related to the diabetic encephalopathy (Gupta et al. 2014; Prabhakar et al. 2015; da Silva Dias et al. 2016; Zanoveli et al. 2016; Aswar et al. 2017; Wang et al. 2019; Chaves et al. 2020). Thus, in the following experiments (Experiment 4 and 5) we investigated whether PGB would present an antioxidant activity per se and whether this single injection of PGB, 1 hour before the fear extinction memory training, would exert an antioxidant action by improving indirect oxidative stress parameters in the HIP and PFC from STZ rats. Our data demonstrated that PGB (in all concentrations - Fig. 6) presented an antioxidant activity by showing a lower absorbance level compared to the negative control (water). Also, PGB was able to reduce the increased LPO levels of STZ animals (HIP and PFC - highest dose) and to increase the reduced GSH levels (PFC). These changes on reduced GSH and LPO levels in these brain regions of STZ animals (Fig. 7) have been previously demonstrated (Pitocco et al. 2010; Pereira et al. 2018; de Souza et al. 2019; Réus et al. 2019). Regarding LPO, it is known that high levels of LPO are attributed to the increase of reactive oxygen species (Siba et al. 2017); in addition, it affects cell integrity only when antioxidant mechanisms are no longer able to cope with the generation of free radicals (Anwer et al. 2012). The GSH is the main antioxidant in the brain (Kanazawa et al. 2016) and it protects the cellular system against the toxic effects of LPO. Thereby, according to Sharma et al. (2016), this decline in the level of GSH in induced-T1DM animals may be due to excessive free radical generation, exposure to high glucose levels. In addition, it has been proposed that antioxidant compounds may play an important role in improving the dysregulations reported here, especially in brain regions such as PFC and HIP, linked to memory/learning and emotional processing (Venturini et al. 2010; de Morais et al. 2014; Pereira et al. 2018; Banagozar Mohammadi et al. 2019; de Souza et al. 2019).
In this sense and reinforcing our data after PGB treatment, we have already observed in this animal model of T1DM that prolonged treatment with other antioxidant compounds, the vitamin E or gallic acid, improved the dysfunctional processing of fear memory and/or anxiety-like responses from these induced-T1DM animals along with an improvement on oxidative stress related-parameters in these same brain areas (de Morais et al. 2014; Pereira et al. 2018; de Souza et al. 2019). This antioxidant action of PGB has already been demonstrated in STZ animals (Sałat et al. 2016; Demir et al. 2021). For example, Sałat et al. (2016) evaluated the effects of the single injection of PGB in STZ mice on contextual memory (-not related to fear) and oxidative stress parameters. The authors reported that although PGB in a smaller dose (10 mg/kg) was not able to attenuate T1DM-induced memory impairments, the PGB did not aggravate learning deficits of these diabetic mice. However, this dose was able to disrupt some markers of oxidative stress. More recently, Demir et al. (2021) demonstrated that prolonged treatment with PGB (50 mg/kg/day for 8 weeks) was able to reduce LPO, improve antioxidant capacity with a significant increase in superoxide dismutase and protect cells against apoptosis.
It is important to note that in the present study, the lowest dose of PGB (30 mg/kg), which was the dose that changed more discreetly the behavioral parameters evaluated in the present study, was able to increase only the reduced level of GSH in the PFC. Thus, we can speculate that the antioxidant action of PGB may not be the only mechanism responsible for the beneficial action of PGB on fear memory and anxiety-like response in these induced-T1DM animals. In this regard, it is not surprising that PGB induces neuroprotective effects through other mechanisms, including an anti-inflammatory action by disrupting the expression of several inflammatory markers (Sałat et al. 2016; Aslankoc et al. 2018; Cruz-Álvarez et al. 2018; Ali et al. 2019).
Taken together, a single injection of PGB in induced-T1DM rats in a specific time window - before a fear memory extinction training session - facilitates the acquisition of fear extinction memory in the short- and long-term, being this effect additionally associated with late improvement on anxiety-like behavior. All these short- and long-term beneficial actions of PGB may be associated with neuroprotective mechanisms of PGB, including its antioxidant action in brain areas like PFC and HIP. Despite the need for further investigations, the data are quite interesting and highlight the potential of PGB for future translational investigations, taking into account the cost-benefit of PGB as a facilitator or adjuvant in the process of extinction of the traumatic memories, in addition to anxiolytic effects.