In this study, we aimed to investigate whether oral administration of a full-spectrum CBD-rich oil could modulate memory performance and cholinergic function in a rat model of MSUD. Our behavioral findings show that neither dose affected locomotion or exploratory activity in open-field test, indicating no nonspecific motor effects. In the object recognition task, the 3.5 mg/kg dose facilitated STM formation in animals submitted to BCAA administration, whereas the 7.5 mg/kg dose preserved STM only in saline-treated animals, without reversing the BCAA-induced impairment. These results indicate that BCAA administration can alter memory in both the short and long term. However, the animals treated with CBD 7.5 did not show differences in STM or LTM, suggesting no memory impairment, unlike the BCAA group. Also, the CBD treatment only reverses the memory damage in STM at 3.5 mg/kg and does not improve the LTM. However, we need to observe that this is the first work using cannabis-related products in MSUD. Moreover, we selected 3.5 and 7.5 mg/kg based on a pilot study; both doses showed greater effects, but lower doses may be preferable, given that we are using neonates. At the neurochemical level, both doses decreased AChE and increased choline acetyltransferase activity in cerebral cortex, accompanied by reduced oxidative damage—as evidenced by lower TBARS and DCFH levels—and an attenuation of inflammatory markers. Collectively, these findings indicate that full-spectrum CBD exerts dose-dependent effects on memory and provides neurochemical protection in neonate rat MSUD model.

Studies have shown that the accumulation of leucine, isoleucine, and valine leads to many problems in the CNS due to increased lactate formation from impaired nicotinamide adenine dinucleotide (NAD) oxidation, resulting in reduced adenosine triphosphate (ATP) formation. Also, the reduction of glutamate concentrations in the brain by branched-chain amino acid transaminase activity depletes glutamate, leading to the formation of alpha-ketoglutarate; collectively, these changes can disturb plasticity, affect memory and learning in animal models of MSUD (Amaral et al. 2010; da Silva Lemos et al. 2022; Strauss et al. 2020). In addition to glutamate depletion in the brain, other neurotransmitters are present at low levels in MSUD, including dopamine, serotonin, and norepinephrine. Attributable to decreased essential amino acids by competition across the blood-brain barrier and related to the appearance of mood and cognition alterations. Thus, it is observed that neurotransmitters, receptors, and synaptic changes are associated with a high risk of developing neuropsychiatric diseases in MSUD patients (Muelly et al. 2013; Zinnanti et al. 2009). Characterizing these alterations in patients is essential to improving our understanding of the pathophysiology and clinical progression of MSUD, particularly the neurological and metabolic impairments associated with BCAA accumulation. However, because MSUD is a rare inherited disorder and studies involving human patients—especially neonates and pediatric populations—are inherently limited by ethical, logistical, and recruitment challenges, animal models remain fundamental tools in this field. In this context, experimental models of MSUD are widely used to recapitulate the neurochemical and behavioral damage caused by BCAA accumulation, enabling investigation of disease mechanisms and preclinical evaluation of novel therapeutic approaches to mitigate metabolic, inflammatory, oxidative, and cognitive dysfunctions associated with the disorder (Amaral and Wajner 2022; Lemos et al. 2024; Scaini et al. 2014a).

Furthermore, as cognitive impairment is one of the major outcomes associated with this disorder, the process that leads to memory involves encoding and consolidation; thus, ACh modulates learning in the hippocampus, and the cortex initiates neurotransmission to temporary memory in the hippocampus. After that, these inputs are transferred again to form LTM in cortex (Haam and Yakel 2017). Thus, changes in this neurotransmitter are associated with cognitive and memory damage (Huang et al. 2022). Regarding behavioral tests, our results show no differences in crossing and rearing counts, indicating that animals did not exhibit impaired locomotion or exploration. However, in recognition test, rats in the control group showed STM and LTM memory; in contrast, rats induced with BCAA showed no difference in STM or LTM, indicating memory impairment. According to previous studies, the MSUD model in rats is associated with deficits in memory acquisition in recognition tasks (Lemos et al. 2023) and in inhibitory avoidance (de Castro Vasques et al. 2004), without changes in locomotion or exploration (Scaini et al. 2014a). Herein, it presents like-depressive behavior in sweet food consumption and forced swimming test (Morais et al. 2022; Scaini et al. 2014a). In concordance, when leucine is administered in zebrafish, animals present anxiety-like behavior (Wessler et al. 2019), memory, and social changes (da Silva Lemos et al. 2022; Duarte et al. 2023), as well as a genetic model of MSUD in zebrafish presents motor dysfunction (Friedrich et al. 2012). The authors associated these behavioral alterations with disruptions in specific brain areas, mainly the hippocampus and cortex, and with changes in neurotransmitter levels, specifically glutamate and ACh (da Silva Lemos et al. 2022; Friedrich et al. 2012; Lemos et al. 2023, 2024). Although the open-field results indicate that locomotor and exploratory activity were not altered by the treatments, it is important to consider that behavioral changes in recognition memory tasks may also be influenced by factors such as anxiety-like behavior, motivational state, or subtle emotional alterations. CBD has been reported to exert anxiolytic and motivational effects in various experimental models (Blessing et al. 2015; Campos et al. 2012), which could influence performance in behavioral paradigms involving the exploration of novel objects. Therefore, although the present results are consistent with an effect on cognitive performance, alternative behavioral influences cannot be entirely ruled out and should be considered when interpreting the findings.

Interestingly, the full-spectrum CBD-rich oil at 3,5 mg/kg + saline preserved both STM and LTM, and when the BCAA group was treated with the same dose, it rescued STM memory. In contrast, the 7,5 mg/kg dose preserved STM only in saline-treated animals and did not reverse the BCAA-induced deficit. It is important to note that some studies often cited in the context of cannabinoid effects in developing brains used synthetic agonists rather than CBD. For example, Schneider et al. employed WIN 55,212-2—a potent cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) agonist pharmacologically closer to THC than to CBD—and reported behavioral impairments in pubertal rats. Because WIN produces robust CB1 activation, its effects cannot be extrapolated to our findings. In our study, the intervention consisted of a full-spectrum CBD-rich oil, a phytocomplex whose actions reflect the synergistic interplay among cannabinoids, terpenes, and flavonoids—commonly described as the entourage effect. However, because the present study did not include a comparison with purified CBD, it is not possible to determine whether the behavioral effects observed here are specific to the full-spectrum preparation or attributable to CBD alone. Therefore, the potential contribution of phytochemical interactions should be interpreted cautiously and treated as a plausible hypothesis based on prior literature, rather than as a direct demonstration from the present data. This putative synergism has been repeatedly associated with broader anti-inflammatory, antioxidant, and behavioral benefits than those of isolated CBD, as shown in several preclinical studies. Therefore, rather than contradicting our results, the Schneider study highlights that different classes of cannabinoids exert distinct neurodevelopmental effects, underscoring the importance of evaluating full-spectrum preparations separately from synthetic agonists (Schneider et al. 2008). Consistent with these findings, Huffstetler et al. (2023) demonstrated dose-dependent effects of CBD itself in male mice, with lower doses producing more favorable behavioral outcomes. They observed no STM differences between doses (10 and 20 mg/kg) in the recognition test but found reduced LTM performance at lower doses. Further, they found a decrease in LTM in lower doses in wild-type mice compared with vehicle-treated mice (Huffstetler et al. 2023). These findings reinforce the notion that CBD treatment is dose-dependent and that its actions in developing organisms may follow a narrow therapeutic window, which could help explain why the 3.5 mg/kg dose rescued STM in BCAA-treated neonates, whereas the 7.5 mg/kg dose did not yield the same effect (Kruk-Slomka et al. 2017).

Furthermore, a possible explanation for these effects is the relation between N-methyl-D-aspartate (NMDA) and CB1 receptors. A study demonstrates that CB1 receptors modulate synaptic plasticity, in which the endocannabinoid system (ECS) leads to depolarization and activation of NMDA receptors, increasing calcium influx. Given that MSUD pathology itself involves NMDA-mediated excitotoxicity and impaired glutamatergic balance, ECS-mediated modulation of these pathways may partially counteract BCAA-induced disruptions (Hillard 2015). The CB1 receptor is important for LTM, long-term plasticity, and long-term depression in the hippocampus, and is related to the NMDA receptor (Kano 2014). Thus, ECS can regulate NMDA receptors by decreasing receptor activity, suggesting one mechanism of modulation, reflected in glutamate inhibition. Moreover, these ECS are related to emotional homeostasis, acting in the hypothalamic axis and sympathetic system, affecting anxiety and stress. This emotional regulation may involve astroglial CB1 and glutamate signaling (Katona and Freund 2012; Lutz et al. 2015). Also, astroglial CB1 is important for LTM consolidation, acting via NMDA receptors. Thus, when activated, the CB1 receptor enhances long-term synaptic plasticity, dependent on astrocytic calcium signaling and adenosine receptors, which are implicated in memory formation (Gonçalves-Ribeiro et al. 2024; Goodman and Packard 2015). Moreover, CB2 may be involved in cognitive processes, and studies have shown that this receptor is a target for memory disorders associated with the cholinergic system. However, the mechanisms underlying these receptors remain poorly understood (Kruk-Slomka et al. 2022). In this regard, previous studies in our lab have shown changes in the cholinergic system following neonatal BCAA administration. Scaini and colleagues first demonstrated alterations in AChE activity and expression and observed different effects on AChE expression when BCAA was administered acutely (three administrations in rats at 10 and 28 days of life) or chronically (two administrations per day for 21 days, starting at 7 days old). In the acute protocol, increased AChE activity in the hippocampus, striatum, and cerebral cortex, and increased expression in the hippocampus, were observed. In the chronic protocol, alterations in AChE activity across all structures and in RNA expression in the striatum and cerebral cortex were observed, correlating with behavioral alterations in learning and memory observed in MSUD (Scaini et al. 2012a). Here, we observed that neonatal BCAA administration in rats produced results similar to those reported by Scaini et al., altering AChE activity and causing behavioral deficits, suggesting cognitive injury.

It is possible to believe that the decrease in ACh levels may be involved in the cognitive changes observed, since it is well predicted that AChE hydrolyzes ACh, and it is the key enzyme that terminates the action of ACh at cholinergic synapses and is highly efficient in modulating the levels of extracellular ACh and in regulating cholinergic neurotransmission (Scaini et al. 2012a; Tan et al. 2014; Zimmerman and Soreq 2006). In concordance, previous studies have shown increased AChE and decreased ChAT activity in rats and zebrafish induced by MSUD, suggesting that memory and learning impairment are related to cholinergic system changes (Lemos et al. 2023, 2024; Wessler et al. 2019). In this context, studies relating the role of ACh in the CNS participating in the communication by neurons as an excitatory mediator (Picciotto et al. 2012; Sam and Bordoni 2025; Trang and Khandhar 2025), acting in memory, learning, attention, and motivation, also ACh is associated with discriminatory learning in the neocortex and low levels in the hippocampus related to forgetfulness (Haam and Yakel 2017; Trang and Khandhar 2025). Given that CBD modulated both AChE and ChAT activities in this study, it is possible that part of its cognitive effects occur through the restoration of cholinergic homeostasis, a pathway already known to be disrupted in MSUD.

In this line, Abdel-Salam et al. (2016) show that cannabis resin increases AChE activity at 10 and 20 mg/kg in the serum and brain of rats. However, at 5 mg/kg, it did not differ from the saline group. These results may vary by the administration route and treatment time, as the differences between cannabis extract concentrations can be explained. Once Abdel-Salam presents 20% of THC subcutaneously, we use one extract with 0.2% of THC orally. In addition, the animals’ weights and lines were different. In the present study, Wistar rat neonates weighing 10–15 g at birth were used, whereas Abdel-Salam used Sprague-Dawley rats weighing 130–140 g (Abdel-Salam et al. 2016). In contrast, Puopolo et al. report inhibition of AChE activity in vitro at CBD concentrations of 25, 50, 100, and 150 µM (Puopolo et al. 2022). Taken together, these findings suggest that cannabinoid-related modulation of AChE activity may vary according to the phytochemical composition of the preparation, including the relative abundance of THC, CBD, and other constituents. However, because the present study did not include a direct comparison with purified CBD, it is not possible to determine whether the observed cholinergic effects are attributable specifically to the full-spectrum formulation or to CBD itself. Thus, more studies are necessary to evaluate the effects of CBD on AChE activity, while accounting for oil type, cannabinoid concentration, treatment duration, lineage, and animal age.

Using a neonatal animal model of MSUD, Lemos and colleagues demonstrated that memantine treatment improved oxidative stress, inflammation, the cholinergic system, and memory impairment (Lemos et al. 2023). Importantly, the relevance of this finding lies in the fact that cognitive dysfunction is a parameter altered in the MSUD model, as is observed in other neurological disorders. Memantine, an NMDA receptor antagonist commonly used in Alzheimer’s disease (AD), has been associated with neuroprotective effects, particularly because hyperstimulation of NMDA receptors has been linked to cholinergic neuron death and consequent memory impairment in AD (Parsons et al. 2013). Further, a recent study demonstrated that BCAAs in MSUD can form amyloid-like structures, thereby increasing inflammation and oxidative stress. Since amyloid plaque formation is a hallmark pathological feature of Alzheimer’s disease (AD) and is strongly associated with cognitive impairment, these findings suggest a potential mechanistic link between MSUD-related neurotoxicity and AD-like pathological processes (Kreiser et al. 2023). Thus, cytokines are essential for proliferation, differentiation, and immune cell survival. In this regard, cytokines can be inflammatory or anti-inflammatory; inflammatory cytokines can increase inflammation by activating macrophages, natural killer cells, and lymphocytes. The anti-inflammatory will attenuate inflammatory responses by reducing pro-inflammatory mediators and suppressing monocyte activity. The results presented here demonstrated higher levels of IL-1β and IL-6, with no alteration in TNF-α. According to these results, the increase in pro-inflammatory cytokines in BCAA group reinforces the finding that inflammation is a mechanism underlying the disease-related damage. Corroborating this, Rosa et al. (2016) reported alterations in inflammatory cytokines, including decreased anti-inflammatory cytokines, in an acute model of MSUD (Rosa et al. 2016). Further, Scaini et al. (2014) demonstrated in an animal model of MSUD that inflammation was associated with blood-brain barrier alterations, suggesting that alterations in inflammatory parameters and oxidative stress could increase blood-brain barrier permeability, thereby exacerbating brain dysfunction (Scaini et al. 2014b). Thus, to accelerate enzymatic defense and stimulate immune cells, the organism increases inflammatory responses, which may, in turn, increase reactive species and alter cerebral homeostasis, thereby compromising neuronal function (Scaini et al. 2018).

In this regard, studies have shown that neurological damage and oxidative stress are involved in MSUD, with excess reactive species and reduced antioxidant defenses contributing to tissue damage (Barschak et al. 2008b; Sitta et al. 2014). In the present study, increases in reactive species (measured by DCFH), lipid peroxidation (measured by TBARS levels), and protein damage (measured by sulfhydryl content) were observed. In this line, it was previously established that MSUD-generated metabolites contribute to oxidative damage and disorganized antioxidant defenses. To counter oxidative damage from reactive species, the organism has an antioxidant system comprising enzymes such as SOD and CAT. These enzymes were altered by BCAA administration, with increased SOD activity and decreased CAT activity. Importantly, the oxidative stress profile observed here suggests not only increased production of reactive species but also a dysregulated antioxidant response. The increase in SOD activity accompanied by a reduction in CAT activity may indicate a compensatory response to superoxide overproduction that is not followed by efficient hydrogen peroxide detoxification. Since SOD converts superoxide radicals into hydrogen peroxide, reduced CAT activity may favor hydrogen peroxide accumulation, thereby promoting secondary oxidative damage through lipid peroxidation and protein oxidation (Fukai and Ushio-Fukai 2011). This imbalance between antioxidant enzymes may help explain the differential alterations observed among DCFH, TBARS, and sulfhydryl measurements, suggesting that oxidative damage in the MSUD model involves a complex disruption of redox homeostasis rather than a uniform increase in oxidative markers. The distinct changes among oxidative markers may also reflect differential susceptibility of cellular components to oxidative injury (Muralidharan and Mandrekar 2013). Further, increased TBARS levels suggest enhanced membrane lipid damage, whereas changes in sulfhydryl content indicate oxidation of protein thiol groups, which may directly affect enzyme function and neuronal signaling pathways. Such redox imbalance may contribute to mitochondrial dysfunction, synaptic impairment, and neuronal vulnerability, which are consistent with the behavioral and cognitive alterations observed in the MSUD model (Lemos et al. 2024; Sitta et al. 2014). Moreover, these results were consistent with previous studies reporting oxidative stress and alterations in antioxidant levels after BCAA administration (da Silva Lemos et al. 2025; Lemos et al. 2024).

So, new approaches are essential since MSUD is a rare disease, based on diet restriction, and the discovery of new drugs could improve the neurochemical damage and help to understand the molecular changes in this disease. In this context, studies have shown that cannabinoids can act in many tissues and systems, affecting their pathways. For this reason, they have emerged as a research target for their analgesic effects and their modulation of the immune system, apoptosis, learning, and memory processes. Where some ligands of cannabinoid (CB) receptors can interact with many systems of the CNS, like dopaminergic, glutamatergic, serotonergic, and cholinergic systems (Hillard 2015; Pagano et al. 2022). Thus, CB ligands may act through CB1 and CB2 receptors, which are mainly expressed in neurons and immune system cells, respectively (Gobira et al. 2024; Hillard 2015). Studies have shown that CB1 receptors are related to the psychoactive effects of Cannabis (Alves et al. 2020) and also regulate cell differentiation and proliferation. In the brain, they mainly modulate synaptic plasticity and regulate neurotransmitter release (Bara et al. 2021; Freund et al. 2003). In addition to receptor-mediated actions, recent evidence highlights that full-spectrum CBD-rich preparations may exert broader neurobiological effects through the so-called “entourage effect.” This synergistic interaction among cannabinoids, terpenes, and flavonoids has been shown to enhance anti-inflammatory, antioxidant, and neuroprotective effects beyond those observed with purified CBD alone. Supporting this view, Ribeiro de Novais Junior et al. (2024) demonstrated that a full-spectrum extract more effectively reversed inflammation-induced depressive-like behavior and hypolocomotion than isolated CBD, potentially due to multi-target modulation of cytokine and redox systems. Similarly, Stork et al. (2025) reported that a full-spectrum Cannabis sativa extract improved gut–brain–peripheral organ integrity following ischemic injury, reinforcing the hypothesis that phytochemical synergism contributes to functional recovery (de Souza Stork et al. 2025). Considering these findings, the cholinergic, antioxidant, and anti-inflammatory improvements observed in the present MSUD model may, at least in part, reflect these synergistic interactions inherent to full-spectrum formulations.

Furthermore, the ECS is involved in calcium blockade via NMDA receptor antagonism, immune function, stress regulation, and cognitive processes. Also, studies have shown ECS signaling dysregulation may be linked with neurodegenerative and psychiatric processes (Hillard 2015; Katona and Freund 2012; Śmiarowska et al. 2022). Also, the CBD has immunosuppressive and anti-inflammatory effects by suppressing pro-inflammatory cytokines and enhancing anti-inflammatory responses (Jean-Gilles et al. 2015; Santiago et al. 2019). These results are observed in the full-spectrum CBD-rich oil treatment in this study, suggesting that CBD’s anti-inflammatory actions extend to conditions of metabolic neurotoxicity such as MSUD. CBD can decrease elevated levels of IL-1β, IL-6, and TNF-α, potentially reducing BCAA-induced inflammation, which may be related to microglial activation and the restoration of redox balance. The anti-inflammatory properties of CBD may be related to the cortical reduction of TNF-α, which inhibits its expression and reduces inflammatory parameters (Pereira et al. 2021). Also, the CBD could act as a reactive species in generation of TNF-α, either directly or indirectly. The activation of CB1 receptors could be associated with an increase in pro-inflammatory responses through TNF-α synthesis and the generation of reactive species; in contrast, the activation of CB2 receptors leads to an inverse effect, decreasing reactive species and TNF-α, which is essential for reducing inflammation and oxidative stress (Atalay et al. 2019). CBD also reduces reactive species production by altering redox balance, for example, by regulating transcription factors such as Nrf2 in microglia, reducing leukocyte metabolism, or upregulating enzymes such as SOD and components of the glutathione cycle (Jastrząb et al. 2019; Pereira et al. 2021).

This work presents results showing that both doses of a full-spectrum, CBD-rich oil improve the cholinergic system and reduce oxidative stress and inflammation. Still, these effects did not translate into behavioral tests or antioxidant activities. Also, the studies using CBD present discrepancies in the data related to route administration, THC concentration, treatment time, and, mainly, the dose. Worth highlighting that this is the first work that uses CBD treatment for MSUD in a neonate model; more studies are necessary to understand the effects of CBD in neonates, as well as the secure doses and time of treatment. Also, the study used a neonatal Wistar rat model with high-BCAA administration, which should be interpreted as a limitation when extrapolating to the chronic metabolic course of the disease. Also, as an animal model, we cannot observe genetic alterations, developmental progression, and clinical complexity as found in humans. An additional limitation of the present study is the use of only male animals. Sex-dependent differences have been described in neurodevelopmental processes, endocannabinoid signaling, and susceptibility to metabolic and neuroinflammatory insults. Therefore, the present findings should be interpreted within the context of this experimental design. Future studies including both sexes will be important to determine whether the neurochemical and behavioral effects observed here are sex dependent.